Elevated intracellular cyclic AMP levels, which suppress the proliferation of naive T cells and type 1 T helper (Th1) cells are a property of T helper 2 (Th2) cells and regulatory T cells. While cyclic AMP signals interfere with the IL-2 promoter induction, they support the induction of Th2-type genes, in particular of il-5 gene. We show here that cyclic AMP signals support the generation of three inducible DNase I hypersensitive chromatin sites over the il-5 locus, including its promoter region. In addition, cyclic AMP signals enhance histone H3 acetylation at the IL-5 promoter and the concerted binding of GATA-3 and NFATc to the promoter. This is facilitated by direct protein-protein interactions involving the C-terminal Zn(2+)-finger of GATA-3 and the C-terminal region of the NFATc1 DNA binding domain. Because inhibition of NFATc binding to the IL-5 promoter in vivo also affects the binding of GATA-3, one may conclude that upon induction of Th2 effector cells NFATc recruits GATA-3 to Th2-type genes. These data demonstrate the functional importance of cyclic AMP signals for the interplay between GATA-3 and NFATc factors in the transcriptional control of lymphokine expression in Th2 effector cells.
Insm1 (IA-1) encodes a Zn-finger factor that is expressed in the developing nervous system. We demonstrate here that the development of the sympatho-adrenal lineage is severely impaired in Insm1 mutant mice. Differentiation of sympatho-adrenal precursors, as assessed by the expression of neuronal subtype-specific genes such as Th and Dbh, is delayed in a pronounced manner, which is accompanied by a reduced proliferation. Sympathetic neurons eventually overcome the differentiation blockade and mature correctly, but sympathetic ganglia remain small. By contrast, terminal differentiation of adrenal chromaffin cells does not occur. The transcription factors Mash1 (Ascl1), Phox2a, Gata3 and Hand2 (previously dHand) control the differentiation of sympatho-adrenal precursor cells, and their deregulated expression in Insm1 mutant mice demonstrates that Insm1 acts in the transcriptional network that controls differentiation of this lineage. Pronounced similarities between Mash1 and Insm1 phenotypes are apparent, which suggests that Insm1 might mediate aspects of Mash1 function in the subtype-specific differentiation of sympatho-adrenal precursors. Noradrenaline is the major catecholamine produced by developing sympatho-adrenal cells and is required for fetal survival. We demonstrate that the fetal lethality of Insm1 mutant mice is caused by catecholamine deficiency, which highlights the importance of Insm1 in the development of the sympatho-adrenal lineage.
Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.
GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.
Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.
Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion.
GATA3 is a zinc-binding transcription factor that regulates the differentiation of many human tissue types, including the mammary gland. In surgical pathology, immunohistochemistry for GATA3 is largely used to support urothelial or breast origin in a carcinoma of unknown origin. GATA3 is sensitive but not entirely specific in this setting. Although GATA3 labeling is highest in estrogen receptor-positive carcinomas, it also labels estrogen receptor-negative carcinomas and thus has particular diagnostic utility in the setting of triple-negative breast carcinomas, which are typically negative for other mammary-specific markers.
GATA-3, a member of the GATA family of zinc-finger DNA binding proteins, and FOXA1, a member of the forkhead transcription factor family, are both associated with estrogen receptor expression. Both GATA-3 and FOXA1 are useful markers for breast carcinoma, but their expression in the different breast cancer subtypes and other neoplasms has not been thoroughly evaluated. We examined the expression of GATA-3 and FOXA1 in estrogen receptor-positive, Her2/neu-positive, and triple-negative breast carcinomas as well as in 10 other common carcinomas, including hepatocellular, colonic, pancreatic, gastric, endometrial (endometrioid), lung, prostatic, renal cell, urothelial, and ovarian serous carcinomas. Primary and metastatic melanomas and mesotheliomas were also evaluated. GATA-3 and FOXA1 staining of estrogen receptor-positive breast carcinomas was seen in 96.6% and 96.2%, respectively. In triple-negative breast carcinomas, GATA-3 and FOXA1 staining was seen in 21.6% and 15.9%, respectively. Among the other tumors, GATA-3 staining was only seen in urothelial carcinoma (70.9%) and FOXA1 staining was only seen in prostatic (87.5%), urothelial (5.1%) carcinomas, and mesotheliomas (40.0%). In conclusion, GATA-3 and FOXA1 are excellent breast carcinoma markers; however, their utility is limited in the triple-negative subtype. The utility of FOXA1 in diagnosing prostatic carcinoma and mesothelioma warrants further investigation.
Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is a rare autosomal dominant disorder caused by mutations in the zinc finger transcription factor gene, GATA3. GATA3 has 2 zinc finger domains, which play an important role in the increase in target gene transcription activity.
A 50-year-old woman and her 27-year-old daughter were followed up because of hypoparathyroidism. They had bilateral sensorineural deafness. Abdominal computed tomography scanning revealed renal dysplasia in the mother, but no renal anomaly in the daughter. Direct sequencing of GATA3 gene revealed a novel heterozygous missense mutation at codon 299 (p.R299Q) in exon 4. This mutation is located at the junction between the 2 zinc fingers. The structure prediction showed that it caused a conformation change in this junction area, affecting the spatial position of the zinc fingers. Additionally, a more marked conformation change was observed in the N-terminal zinc finger region compared to that in the C-terminal region. Functional analysis of this mutant protein using an in vitro luciferase reporter assay system confirmed that the mutation abolished the enhancing effects of wild-type GATA3 on the promoter activity of the consensus GATA responsive element and that of human PTH gene.
We identified a novel R299Q mutation in GATA3 in a Japanese family with HDR syndrome. We confirmed that R299Q is a loss-of-function mutation, due to the extensive conformational change in the zinc fingers of GATA3.
GATA transcription factors are zinc finger DNA binding proteins that regulate transcription during development and cell differentiation. The three important GATA transcription factors GATA1, GATA2 and GATA3 play essential roles in the development and maintenance of hematopoietic systems. GATA1 is required for the erythroid and megakaryocytic commitment during hematopoiesis. GATA2 is crucial for the proliferation and survival of early hematopoietic cells, and is also involved in lineage specific transcriptional regulation as the dynamic partner of GATA1. GATA3 plays an essential role in T lymphoid cell development and immune regulation. As a result, mutations in genes encoding the GATA transcription factors or alteration in the protein expression level or their function have been linked to a variety of human hematologic disorders. In this review, we summarized the current knowledge regarding the disrupted biologic function of GATA in various hematologic disorders.
The transcription factor GATA1 helps regulate the expression of thousands of genes involved in blood development, by binding to single or double GATA sites on DNA. An important part of gene activation is chromatin looping, the bringing together of DNA elements that lie up to many thousands of basepairs apart in the genome. It was recently suggested, based on studies of the closely related protein GATA3, that GATA-mediated looping may involve interactions of each of two zinc fingers (ZF) with distantly spaced DNA elements. Here we present a structure of the GATA1 ZF region bound to pseudopalindromic double GATA site DNA, which is structurally equivalent to a recently-solved GATA3-DNA complex. However, extensive analysis of GATA1-DNA binding indicates that although the N-terminal ZF (NF) can modulate GATA1-DNA binding, under physiological conditions the NF binds DNA so poorly that it cannot play a direct role in DNA-looping. Rather, the ability of the NF to stabilize transcriptional complexes through protein-protein interactions, and thereby recruit looping factors such as Ldb1, provides a more compelling model for GATA-mediated looping.
The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge.
Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.
Haploinsufficiency of the Gata3 gene, which encodes a zinc-finger transcription factor, is associated with the disorder hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome in humans. However, the roles of Gata3 in transcriptional regulation in the parathyroid glands are not well-understood. In this study, we show that Gata3 activates transcription of parathyroid hormone (PTH), which is secreted from parathyroid glands and is critical for regulating serum calcium and phosphate homeostasis. Gata3 interacted with Gcm2 and MafB, two known transcriptional regulators of parathyroid development, and synergistically stimulated the PTH promoter. An SP1-binding element (GC box) located within the PTH-promoter proximal region was critical for activating transcription by Gata3. In addition, the ubiquitous transcription factor SP1 also interacted with Gata3 as well as MafB and Gcm2, and HDR syndrome-associated Gata3 mutants were defective in activating the PTH promoter. These results suggest that Gata3 is a critical regulator of PTH gene expression.
GATA3 is a zinc-finger transcription factor that is important for trophoblast differentiation. GATA3 is sensitive for urothelial and breast carcinomas, but the specificity is low. The aim of this study was to investigate the expression of GATA3 in trophoblast-related tissues and neoplasia.
GATA3 immunohistochemistry was performed on 33 placentas, one atypical placental site nodule, 25 hydatidiform moles (HMs), and 13 gestational trophoblastic tumours (GTTs). One hundred and sixty endometrial adenocarcinomas were also stained. Western blotting was performed on trophoblastic cell lines and compared to other cancer cell lines. Immature placentas were characterized by strong, diffuse nuclear GATA3 staining. Mature placentas showed less expression with scattered positive cells in the villous cytotrophoblast. HMs showed diffuse expression in cytotrophoblast and implantation site trophoblast, and heterogeneous expression in extravillous trophoblast. All GTTs were positive for GATA3. All endometrial adenocarcinomas were GATA3-negative. Western blotting demonstrated GATA3 in choriocarcinoma, whereas the placenta, and cervical and endometrial cancer cell lines, were negative.
All trophoblast lineages were positive for GATA3. The extent of GATA3 expression varied between immature and mature placentas, suggesting a role in trophoblast maturation. GATA3 does not distinguish normal placenta, HMs, or GTTs. Nevertheless, GATA3 may help in distinguishing trophoblastic tumors from Mullerian epithelial malignancies and a subset of tumours of unknown origin.
Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact that MT1 encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQ in vivo.
The mammalian target of rapamycin (mTOR) senses and incorporates different environmental cues via the two signaling complexes mTOR complex 1 (mTORC1) and mTORC2. As a result, mTOR controls cell growth and survival, and also shapes different effector functions of the cells including immune cells such as T cells. We demonstrate in this article that invariant NKT (iNKT) cell development is controlled by mTORC2 in a cell-intrinsic manner. In mice deficient in mTORC2 signaling because of the conditional deletion of the Rictor gene, iNKT cell numbers were reduced in the thymus and periphery. This is caused by decreased proliferation of stage 1 iNKT cells and poor development through subsequent stages. Functionally, iNKT cells devoid of mTORC2 signaling showed reduced number of IL-4-expressing cells, which correlated with a decrease in the transcription factor GATA-3-expressing cells. However, promyelocytic leukemia zinc-finger (PLZF), a critical transcription factor for iNKT cell development, is expressed at a similar level in mTORC2-deficient iNKT cells compared with that in the wild type iNKT cells. Furthermore, cellular localization of PLZF was not altered in the absence of mTOR2 signaling. Thus, our study reveals the PLZF-independent mechanisms of the development and function of iNKT cells regulated by mTORC2.
CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.
Recent evidence suggests the involvement of sex hormone receptors in bladder cancer initiation, while precise functions of androgens and estrogens in the carcinogenesis step remain poorly understood. We recently found down-regulation of GATA3, a zinc-finger transcription factor and a new urothelial marker, in bladder cancer, which also correlated with expression status of androgen receptor (AR) and estrogen receptors (ERs). We here assessed whether GATA3 acted as a suppressor of bladder tumorigenesis and sex hormones exerted an influence on GATA3 in non-neoplastic urothelial cells. Androgen (R1881, dihydrotestosterone) treatment in SVHUC immortalized normal urothelial cells stably expressing AR (SVHUC-AR) decreased GATA3 expression at both mRNA and protein levels, which was abolished by anti-androgens. Conversely, 17β-estradiol treatment increased it in SVHUC-control endogenously expressing ERβ. GATA3 levels were also found to be higher in intact female mouse bladders compared with intact males, and orchiectomy/ovariectomy augmented/reduced GATA3 expression, respectively, which was at least partially restored by dihydrotestosterone/17β-estradiol supplement. Additionally, GATA3 silencing via short hairpin RNA (shRNA) promoted cell proliferation of SVHUC with exposure to a chemical carcinogen 3-methylcholanthrene. In vitro transformation assay with 3-methylcholanthrene then showed a significantly higher number of colonies in SVHUC-AR/GATA3-shRNA, compared with control SVHUC, and R1881 further induced colony formation. GATA3 knockdown also resulted in down-regulation of the molecules that play a protective role in bladder tumorigenesis (i.e. UGT1A, PTEN, p53, p21) and up-regulation of oncogenic genes (i.e. c-myc, cyclin D1, cyclin D3, cyclin E, FGFR3). Thus, GATA3 likely prevented neoplastic transformation of urothelial cells. Furthermore, sex hormone signals contrary regulated GATA3 in the bladder. These findings may offer not only a molecular basis for the gender-specific difference in bladder cancer incidence but also great potential for androgen deprivation as a chemopreventive option for tumor recurrence.
The zinc-finger transcription factor GATA-3 has received much attention as a master regulator of T helper 2 (Th2) cell differentiation, during which it controls interleukin-4 (IL-4), IL-5, and IL-13 expression. More recently, GATA-3 was shown to contribute to type 2 immunity through regulation of group 2 innate lymphoid cell (ILC2) development and function. Furthermore, during thymopoiesis, GATA-3 represses B cell potential in early T cell precursors, activates TCR signaling in pre-T cells, and promotes the CD4(+) T cell lineage after positive selection. GATA-3 also functions outside the thymus in hematopoietic stem cells, regulatory T cells, CD8(+) T cells, thymic natural killer cells, and ILC precursors. Here we discuss the varied functions of GATA-3 in innate and adaptive immune cells, with emphasis on its activity in T cells and ILCs, and examine the mechanistic basis for the dose-dependent, developmental-stage- and cell-lineage-specific activity of this transcription factor.
The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3.
Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis.
Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc finger domains.
We identified a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.
The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells.
ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival.
Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.
Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate limiting enzyme in the NAD(+) synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (KD) of NAMPT or the KD of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin beta 3 class III), CSN2 (casein beta), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT KD promoted while POU5F1 KD inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 KD), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD(+) biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.
While long intergenic noncoding RNAs (lincRNAs) and mRNAs share similar biogenesis pathways, these transcript classes differ in many regards. LincRNAs are less evolutionarily conserved, less abundant, and more tissue-specific, suggesting that their pre- and post-transcriptional regulation is different from that of mRNAs. Here, we perform an in-depth characterization of the features that contribute to lincRNA regulation in multiple human cell lines. We find that lincRNA promoters are depleted of transcription factor (TF) binding sites, yet enriched for some specific factors such as GATA and FOS relative to mRNA promoters. Surprisingly, we find that H3K9me3-a histone modification typically associated with transcriptional repression-is more enriched at the promoters of active lincRNA loci than at those of active mRNAs. Moreover, H3K9me3-marked lincRNA genes are more tissue-specific. The most discriminant differences between lincRNAs and mRNAs involve splicing. LincRNAs are less efficiently spliced, which cannot be explained by differences in U1 binding or the density of exonic splicing enhancers but may be partially attributed to lower U2AF65 binding and weaker splicing-related motifs. Conversely, the stability of lincRNAs and mRNAs is similar, differing only with regard to the location of stabilizing protein binding sites. Finally, we find that certain transcriptional properties are correlated with higher evolutionary conservation in both DNA and RNA motifs and are enriched in lincRNAs that have been functionally characterized.
The industrial hemp varieties 'Yunma 5' and 'Bamahuoma,' which demonstrate growth vigor and environmental adaptability, have been primarily cultivated in Yunnan and Guangxi, China, respectively, for fiber and seeds. The results of physiological measurements showed the phenotypic differences between the two varieties in response to salt stress. RNA-Seq analysis was first performed on leaves of both varieties sampled at four time intervals (0, 2, 4, 6 days) after treatment with salt (500 mM NaCl) We identified 220 co-up-regulated differentially expressed genes (DEGs) in the two varieties, while 26 up-regulated DEGs and 24 down-regulated DEGs were identified exclusively in the single varieties after 2 days of salt stress. Among the 220 DEGs, we identified 22 transcription factors, including key transcription factors involved in salt stress, such as MYB, NAC, GATA, and HSF. We applied gene expression profile analysis and found that 'Yunma 5' and 'Bamahuoma' have variety-specific pathways for resisting salt stress. The DEGs of 'Yunma 5' were enriched in spliceosome and amino acid metabolism genes, while the DEGs of 'Bamahuoma' were enriched in fatty acid metabolism, amino acid metabolism, and endoplasmic reticulum protein processing pathway. Although there were common DEGs, such as genes encoding cysteine protease and alpha/beta-hydrolase superfamily, the two varieties' responses to salt stress impacted different metabolic pathways. The DEGs that were co-expressed in both varieties under stress may provide useful insights into the tolerance of cultivated hemp and other bast fiber crops to saline soil conditions. These transcriptomes also represent reference sequences for industrial hemp.
BACKGROUND De novo renal neoplasia developing after kidney transplantation at Verona Kidney Transplant Center were reviewed according to new 2016 WHO Renal Tumor Classification. MATERIAL AND METHODS Primary renal tumors developed in native or transplanted kidneys de novo following renal transplantation were retrieved and histologically reviewed by three expert uropathologists. Immunoexpression of the diagnostic antigens CD13, CD10, CK7, CK34bE12, AMACR, CAIX, AE1/AE3, CK14, GATA-3, HMB-45, cathepsin-k, S100A1, and parvalbumin was assessed. Predictive antigens ph-mTOR and ph-p70S6k were also tested. RESULTS Two thousands and sixteen kidney transplantations have been carried out from 1968-2015. Follow-up was available per 1,646 patients (mean 8.4 years). We observed 16 cases of de novo renal neoplasia arising in patients 16 to 286 months post-transplantation. Nine clear cell, two papillary RCCs and a single case of the new WHO entity denominated "acquired cystic disease-associated RCC" were identified in native kidneys. Another new WHO tumor entity called "clear cell papillary RCC" was diagnosed and a new variant of papillary RCC with diffuse clear cytoplasm was also identified. The majority of tumors were low stage and low grade according to the new ISUP grading system. Seven patients were additionally treated with mTOR inhibitors. Post-cancer follow-up ranged from 62 to 281 months. One patient showed a recurrence (a lung metastases) and died. Of the remaining patients, three died of non-cancer-related causes. CONCLUSIONS The application of the new WHO 2016 classification has importance as it identifies new (18% of tumors) morphotypes that are likely to behave in a less aggressive fashion.
In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX [insulin, dexamethasone and IBMX (isobutylmethylxanthine)], oleate and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of PPARγ (peroxisome proliferator-activated receptor-γ) and AFABP (adipocyte fatty acid-binding protein), but decreasing level of GATA2 (GATA-binding protein 2). In contrast, for cells cultured in the basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes.
T cells have been identified as key players in the pathogenesis of type 1 diabetes. However, the exact role of T-cell subpopulations in this pathway is presently unknown. The purpose of this study was to assess the expression pattern of two lineage-specifying transcription factors GATA-3 and T-bet, which are important in T helper type 1 (Th1) and Th2 cell development, respectively. Gene expression analysis of peripheral blood mononuclear cells (PBMCs) was performed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Plasma levels of IFN-γ and IL-4 were also determined by ELISA. T-bet and IFN-γ gene expression was significantly lower in patients group compared with healthy controls (p<0.05). The expression of GATA-3 was relatively similar in patients and controls; however, IL-4 mRNAs were significantly increased in the PBMCs from patients as compared with normal controls (p<0.05). In addition, a marked increase in plasma IL-4 levels were observed in patient group compared with controls (p<0.001). To the contrary, IFN-γ protein levels were decreased in patients in comparison with controls (p<0.001). These data suggest additional implications of the role of Th1/Th2 imbalance for the immunopathogenesis of type 1 diabetes.
It is now well established that type-2 immune mechanisms drive the inflammation in about 50% of asthma patients. The major cellular and molecular players regulating this important network have been identified. In terms of therapeutic intervention, cytokine and cytokine-receptor pathways have been given major attention, since these molecules are relatively easily accessible for a blockade through monoclonal antibodies, and a number of positive clinical results support this concept. However, targeting events controlling the type-2 immunity network upstream of selective cytokine pathways would be equally attractive. Type-2 immunity is regulated through a delicate interplay of several transcription factors (including GATA-3, STAT-6, NFAT, IRF4, c-maf), with GATA-3 as master regulator in this regard. Since transcription factors are intracellularly located they cannot be directly targeted by monoclonal antibodies. For intracellular targets, antisense technologies such as antisense DNA and siRNA have been shown to be a promising approach, and have recently made major advances towards clinical application. Here we summarize the development of a GATA-3-specific DNAzyme - a molecule class that combines the superior specificity of antisense molecules with an inherent RNA-cleaving enzymatic activity - for the treatment of type-2-driven asthma from preclinical development towards a proof-of-concept clinical study. This article is protected by copyright. All rights reserved.
The HomyGene19+14Y System (HG19+14Y) is a PCR-based amplification kit that enables typing of 18 autosomal short tandem repeat (STR) loci (i.e., CSF1PO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta E, TPOX, TH01, vWA), 14 widely used Y chromosome STR (Y-STR) loci (Y_GATA_H4, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS439, DYS456, DYS458, DYS635), and amelogenin. This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation, single-source searches, kinship (including familial searching), mixture profiles, international data sharing, and other forensic applications. In this study, the multiplex system was validated for sensitivity, specificity, DNA mixtures, stability, precision, stutter, reproducibility, parallel tests, PCR-based conditions, and population analysis according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation guidelines. A total of 212 alleles were detected for the 18 autosomal STR loci among 528 Guangdong Han individuals, and 431 haplotypes were found for 14 Y-STRs among 452 unrelated males. The combined match probability (CMP) of the HG19+14Y System was calculated as 2.39 × 10(-29). All the validation results showed that the HG19+14Y System would be a robust, reliable, highly polymorphic, and informative forensic kit.
In Saccharomyces cerevisiae, when L-phenylalanine (L-Phe) is used as the sole nitrogen source, 2-phenylethanol (PE) is mainly synthesized via the Ehrlich pathway. General amino acid permease Gap1p is response of aromatic amino acids transportation, and GATA transcription factors Gln3p and Gat1p regulate the transcription of permease gene and catabolic enzyme genes for nitrogen sources and aromatic amino acids utilization. In this study, it was demonstrated that over-expressing GLN3 gene from industrial yeast strain MT2 or S. cerevisiae haploid strain YS58, 2-PE synthesis levels of recombinant strains increased 54% or 40% than that of the control strain, which suggested that higher Gln3p activity in yeast has positive regulation effect on 2-PE biosynthesis via Ehrlich pathway. The recombinant strains with over-expression of GAT1 gene from MT2 or YS58 also up-regulated Ehrlich pathway for 2-PE biosynthesis and increased 2-PE production. Similarly, when GAP1 gene respectively from MT2 or YS58 was over-expressed, 2-PE yield was improved obviously, suggesting that GAP1 over-expressing in yeast also promoted Ehrlich pathway to produce 2-PE. The synergistic regulation of GLN3/GAT1 or GLN3/GAP1 over-expression was similar to that of single factor over-expression. Among these regulatory factors, Gln3p of industrial yeast strain MT2 caused stronger regulation on target genes than that of haploid strain YS58, which might be due to the differences in translational efficiency or nuclear localization of each Gln3p, or due to their different spatial structures and binding domains. Further results showed that efficient Gln3p expression in MT2 brought about higher 2-PE, 3.59 gL(-1), which was of potential significant for commercial exploitation.
Pilomatricomas are benign follicular skin appendage tumors, commonly occurring in children and young adults. Most patients admit to dermatologists to seek treatment and are well known by them; however, dental professionals, especially pediatric dentists are not familiar with these tumors.
This report presents a 16-year-old female with preauricular pilomatricoma, located beneath the overlying skin of the temporomandibular region. Clinical examination revealed an asymptomatic lump, the overlying skin revealed no abnormalities. Patient was unaware of the lesion.
Pilomatricomas are commonly encountered in the maxillofacial region, although not considered in differential diagnosis by dental professionals. They usually present as, asymptomatic, subcutaneous masses; although symptomatic cases have been reported. In literature, common differential diagnosis for head and neck pilomatricoma includes sebaceous cyst, ossifying hematoma, giant cell tumor, chondroma, dermoid cyst, foreign body reaction, degenerating fibroxanthoma, metastatic bone formation, and osteoma cutis. We are of the opinion that temporomandibular joint disease should also be considered in differential diagnosis for preauricular pilomatricoma.
Pediatric dentists should be aware of the condition and consider it in the differential diagnosis of pediatric conditions involving the temporomandibular joint.
Parasitic diseases are among the major public health issues worldwide. A number of tests are available for diagnosis, but the sentivity and specifity of these tests are assumed to be insufficient. Nevertheless, the most common diagnostic method is microscopic examination. In this study, we aimed to introduce the distribution of parasites detected in stool samples of patients admitted to our laboratory on the basis of parameters such as, age, and gender during a 3-year period between 2012 and 2014.
In total, 6757 stool samples were included in the study. After macroscopic examination, wet mounts of all samples were examined under a light microscope using ×100 and ×400 magnification lenses. Wet mounts were prepared with physiological saline and Lugol's iodine.
Parasites were detected in 3.7% (252) of the samples, while no parasites were detected in 96.3% (6505) of the samples. The distribution of intestinal parasites was as follows: Blastocystis hominis (63.5%), Giardia intestinalis (26.2%), Taenia sp. (4.8%), Enterobius vermicularis (2.4%), Entamoeba histolytica/dispar (1.6%), and Hymenolepis nana (1.6%).
When the burden of intestinal parasites on public health is considered, they are still a major health issue in Turkey. The frequency of parasitic diseases can be reduced by the education of individuals and implementation of effective diagnostic methods, treatments, and preventive measures.
Non-Hodgkin's lymphomas arising from the tissues other than primary lymphatic organs are named primary extranodal lymphoma. Most of the studies evaluated metabolic tumor parameters in different organs and histopathologic variants of this disease generally for treatment response. We aimed to evaluate the prognostic value of metabolic tumor parameters derived from initial FDG-PET/CT in patients with a medley of primary extranodal lymphoma in this study.
There were 67 patients with primary extranodal lymphoma for whom FDG-PET/CT was requested for primary staging. Quantitative PET/CT parameters: maximum standardized uptake value (SUVmax), average standardized uptake value (SUVmean), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were used to estimate disease-free survival and overall survival.
SUVmean, MTV and TLG were found statistically significant after multivariate analysis. SUVmean remained significant after ROC curve analysis. Sensitivity and specificity were calculated as 88% and 64%, respectively, when the cut-off value of SUVmean was chosen as 5.15. After the investigation of primary presentation sites and histo-pathological variants according to recurrence, there is no difference amongst the variants. Primary site of extranodal lymphomas however, is statistically important (p = 0.014). Testis and central nervous system lymphomas have higher recurrence rate (62.5%, 73%, respectively).
High SUVmean, MTV and TLG values obtained from primary staging FDG-PET/CT are potential risk factors for both disease-free survival and overall survival in primary extranodal lymphoma. SUVmean is the most significant one amongst them for estimating recurrence/metastasis.
Platelet-rich plasma (PRP) is an autologous concentration of human platelets contained in a small volume of plasma and has recently been shown to accelerate rejuvenate aging skin by various growth factors and cell adhesion molecules.
This study was conducted to evaluate the efficacy and safety of intradermal injection of PRP in the human facial rejuvenation.
This study was a prospective, single-center, single-dose, open-label, non-randomized controlled clinical study. PRP injected to the upper site of this right infra-auricular area and all face. Saline was injected to the left infra-auricular area. Histopathological examinations were performed before PRP treatment, 28 days after the PRP, and saline (control) treatments.
Twenty women ranging in age from 40 to 49 years (mean age, 43.65±2.43 years) were enrolled in the study. The mean optical densities (MODs) of collagen in the pre-treatment, control, and PRP-treated area were measured. They were 539±93.2, 787±134.15, 1,019±178, respectively. In the MOD of PRP, 89.05 percent improvement was found when MOD of PRP was compared with MOD of pre-treatment. The mean MOD of collagen fibers was clearly highest on the PRP side (p<0.001). The PRP-to-saline improvement ratio (89.05% to 46.01%) was 1.93:1. No serious side effects were detected.
PRP increases dermal collagen levels not only by growth factors, but also by skin needling (the mesotherapy technique 'point by point'). PRP application could be considered as an effective (even a single application) and safety procedure for facial skin rejuvenation.
Histone gene expression is regulated in a cell cycle-dependent manner, with a peak at S phase, which is crucial for cell division and genome integrity. However, the detailed mechanisms by which expression of histone genes are tightly regulated remain largely unknown. Fission yeast Ams2, a GATA-type zinc finger motif-containing factor, is required for activation of S phase-specific core histone gene transcription. Here we report the molecular characterisation of Ams2. We show that the zinc finger motif in Ams2 is necessary to bind the histone gene promoter region and to activate histone gene transcription. An N-terminal region of Ams2 acts as a self-interaction domain. Intriguingly, N-terminally truncated Ams2 binds to the histone gene promoters, but does not fully activate histone gene transcription. These observations imply that Ams2 self-interactions are required for efficient core histone gene transcription. Moreover, we show that Ams2 interacts with Teb1, which itself binds to the core histone gene promoters. We discuss the relationships between Ams2 domains and efficient transcription of the core histone genes in fission yeast.
In the present study, gene expression profiles were analyzed to identify the molecular mechanisms underlying gastric cardia adenocarcinoma (GCA) and gastric non-cardia adenocarcinoma (GNCA). A gene expression dataset (accession number GSE29272) was downloaded from Gene Expression Omnibus, and consisted of 62 GCA samples and 62 normal controls, as well as 72 GNCA samples and 72 normal controls. The two groups of differentially-expressed genes (DEGs) were compared to obtain common and unique DEGs. A differential analysis was performed using the Linear Models for Microarray Data package in R. Functional enrichment analysis was conducted for the DEGs using the Database for Annotation, Visualization and Integrated Discovery. Protein-protein interaction (PPI) networks were constructed for the DEGs with information from the Search Tool for the Retrieval of Interacting Genes. Subnetworks were extracted from the whole network with Cytoscape. Compared with the control, 284 and 268 genes were differentially-expressed in GCA and GNCA, respectively, of which 194 DEGs were common between GCA and GNCA. Common DEGs [e.g., claudin (CLDN)7, CLDN4 and CLDN3] were associated with cell adhesion and digestion. GCA-unique DEGs [e.g., MAD1 mitotic arrest deficient like 1, cyclin (CCN)B1, CCNB2 and CCNE1] were associated with the cell cycle and the regulation of cell proliferation, while GNCA-unique DEGs (e.g., GATA binding protein 6 and hyaluronoglucosaminidase 1) were implicated in cell death. A PPI network with 141 nodes and 446 edges were obtained, from which two subnetworks were extracted. Genes [e.g., fibronectin 1, collagen type I α2 chain (COL1A2) and COL1A1] from the two subnetworks were implicated in extracellular matrix organization. These common DEGs could advance our understanding of the etiology of gastric cancer, while the unique DEGs in GCA and GNCA could better define the properties of specific cancers and provide potential biomarkers for diagnosis, prognosis or therapy.
Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.
There is still a debate about the exact measurement of the oesophagogastric junction and the diaphragmatic hiatus among clinicians. The aim of this study was to investigate the differences between landmark readings of gastroscopy on intubation and extubation, and to correlate these readings with a gastro-oesophageal reflux questionnaire.
116 cases who underwent diagnostic gastroscopy between January 2013 and June 2013 were included in this study. Landmark measurements were noted while withdrawing the endoscope and were also evaluated after the gastric air was fully emptied. We first used a frequency scale for the gastro-oesophageal reflux disease symptoms (FSSG) questionnaire in order to investigate dysmotility and acid reflux symptoms in the study population and correlated the FSSG questionnaire with intubation and extubation measurements at endoscopic examination.
Mean age of included subjects was 49.41±17.7 (19-82) years. Males and females were equally represented. On FSSG scores, the total dysmotility score was 7.99±5.06 and the total score was 15.18±10.11. The difference between intubation and extubation measurements ranged from -3cm to +2cm (mean: -0.4). When an FSSG score of 30 was accepted as a cut-off value, we detected a significant difference between the measurements (p<0.05; t: 0.048).
Accuracy of landmark measurements during gastroscopy is clearly affected from insertion or withdrawal of the endoscope. When differences in measurements between insertion and withdrawal were evident, comparable with the FSSG scores, the results became significantly different. In conclusion, according to FSSG scores, these measurements should be performed at the end of the endoscopy.
Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In the present study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response were evaluated in mice infected with Leishmania braziliensis.
Balb/c mice were infected with L. braziliensis and subsequently treated with 3 sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA -3, Foxp3 and T-bet) were analyzed by quantitative PCR.
The parasite load in the treated group was significantly lower than in the untreated group (p <0.0001); and in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (p=0.012 and p=0.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (p = 0.0001).
5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA. This article is protected by copyright. All rights reserved.
In 2009-2014, dose surveys aimed to collect adult patient data and parameters of most common radiographic examinations were performed in six Russian regions. Typical patient doses were estimated for the selected examinations both in entrance surface dose and in effective dose. 75%-percentiles of typical patient effective dose distributions were proposed as preliminary regional diagnostic reference levels (DRLs) for radiography. Differences between the 75%-percentiles of regional typical patient dose distributions did not exceed 30-50% for the examinations with standardized clinical protocols (skull, chest and thoracic spine) and a factor of 1.5 for other examinations. Two different approaches for establishing national DRLs were evaluated: as a 75%-percentile of a pooled regional sample of patient typical doses (pooled method) and as a median of 75%-percentiles of regional typical patient dose distributions (median method). Differences between pooled and median methods for effective dose did not exceed 20%. It was proposed to establish Russian national DRLs in effective dose using a pooled method. In addition, the local authorities were granted an opportunity to establish regional DRLs if the local radiological practice and typical patient dose distributions are significantly different.
Endometriosis, the hormone-dependent extrauterine dissemination of endometrial tissue outside the uterus, affects 5-15% of women of reproductive age. Pathogenesis remains poorly understood as well as the estrogen production by endometriotic tissue yielding autocrine growth. Estrogens (E2) are normally produced by the ovaries. We investigated whether aberrant "ovarian-like" differentiation occurred in endometriosis. 69 women, with (n = 38) and without (n = 31) histologically proven endometriosis were recruited. Comparative RT-qPCR was performed on 20 genes in paired eutopic and ectopic lesions, together with immunohistochemistry. Functional studies were performed in primary cultures of epithelial endometriotic cells (EEC). A broaden ovarian-like differentiation was found in half eutopic and all ectopic endometriosis with aberrant expression of transcripts and protein for the transcription factors GATA4 and GATA6 triggering ovarian differentiation, for the FSH receptor (FSHR) and the ovarian hormone INSL3. Like in ovaries the FSHR induced aromatase, the key enzyme in E2 production, and vascular factors in EEC. The LH receptor (LHR) was also aberrantly expressed in a subset of ectopic endometriosis (21%) and induced strongly androgen-synthesizing enzymes and INSL3 in EEC, as in ovaries, as well as endometriotic cell growth. The ERK pathway mediates signaling by both hormones. A positive feedback loop occurred through FSHR and LHR-dependent induction of GATA4/6 in EEC, as in ovaries, enhancing the production of the steroidogenic cascade. This work highlights a novel pathophysiological mechanism with a broadly ovarian pattern of differentiation in half eutopic and all ectopic endometriosis. This study provides new tools that might improve the diagnosis of endometriosis in the future.
Vasohibin 2 (VASH2) is identified as an angiogenic factor, and has been implicated in tumor angiogenesis, proliferation and epithelial-mesenchymal transition (EMT). To investigate the EMT role of VASH2 in breast cancer, we overexpressed or knocked down expression of VASH2 in human breast cancer cell lines. We observed that VASH2 induced EMT in vitro and in vivo. The transforming growth factor β1 (TGFβ1) pathway was activated by VASH2, and expression of a dominant negative TGFβ type II receptor could block VASH2-mediated EMT. In clinical breast cancer tissues VASH2 positively correlated with TGFβ1 expression, but negatively correlated with E-cadherin (a marker of EMT) expression. Under hypoxic conditions in vitro or in vivo, we found that down-regulation of estrogen receptor 1 (ESR1) in VASH2 overexpressing ESR1 positive cells suppressed E-cadherin. Correlation coefficient analysis indicated that VASH2 and ESR1 expression were negatively correlated in clinical human breast cancer tissues. Further study revealed that a transcription factor of ESR1, GATA-binding factor 3 (GATA3), was down-regulated by VASH2 under hypoxia or in vivo. These findings suggest that VASH2 drives breast cancer cells to undergo EMT by activation of the TGFβ1 pathway and hypoxia dependent repression GATA3-ESR1 pathway, leading to cancer metastasis.
Allergic asthma is a worldwide increasing chronic disease of the airways which affects more than 300 million people. It is associated with increased IgE, mast cell activation, airway hyperresponsiveness (AHR), mucus overproduction and remodeling of the airways. Previously, this pathological trait has been associated with T helper type 2 (Th2) cells. Recently, different CD4(+) T cell subsets (Th17, Th9) as well as cells of innate immunity, like mast cells and innate lymphoid cells type 2 (ILC2s), which are all capable of producing the rediscovered cytokine IL-9, are known to contribute to this disease. Regarding Th9 cells, it is known that naïve T cells develop into IL-9-producing cells in the presence of interleukin-4 (IL-4) and transforming growth factor beta (TGFβ). Downstream of IL-4, several transcription factors like signal transducer and activator of transcription 6 (STAT6), interferon regulatory factor 4 (IRF4), GATA binding protein 3 (GATA3), basic leucine zipper transcription factor, ATF-like (BATF) and nuclear factor of activated T cells (NFAT) are activated. Additionally, the transcription factor PU.1, which is downstream of TGFβ signaling, also seems to be crucial in the development of Th9 cells. IL-9 is a pleiotropic cytokine that influences various distinct functions of different target cells such as T cells, B cells, mast cells and airway epithelial cells by activating STAT1, STAT3 and STAT5. Because of its pleiotropic functions, IL-9 has been demonstrated to be involved in several diseases, such as cancer, autoimmunity and other pathogen-mediated immune-regulated diseases. In this review, we focus on the role of Th9 and IL-9-producing cells in allergic asthma.
Chronic immune thrombocytopenia purpura (ITP) is an autoimmune disease that exhibits an abnormally high Th1/Th2 ratio. Granulocyte colony-stimulating factor (G-CSF) has been shown to decrease the Th1/Th2 ratio in healthy donors. In this study, we investigated the effects of G-CSF treatment on the Th1/Th2 cells and the underlying mechanisms in patients with ITP in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from patients with ITP and healthy controls were treated with G-CSF. Expression levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, and IL-13 in supernatants were measured by enzyme-linked immunosorbent assays. The expression of IFN-γ, IL-4, and G-CSF receptor (G-CSFR) on Th1 and Th2 cells were examined by flow cytometry and confocal microscopy. The mRNA expression of IFN-γ, IL-2, IL-4, IL-13, and T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) in PBMCs was evaluated by reverse transcription polymerase chain reaction. The results showed that G-CSF could significantly reduce the Th1/Th2 ratio in PBMCs from patients with ITP in vitro. As the concentration of G-CSF increased, Th1/Th2 ([IFN-γ+IL-2]/[IL-4+IL-13]) cytokine ratios and T-bet/GATA-3 mRNA ratios decreased in a concentration-dependent manner. Th1 cells and Th2 cells both expressed G-CSFR. These results suggest that G-CSF could decrease the Th1/Th2 ratio in the context of ITP, and elucidate the direct and indirect immunomodulatory mechanisms underlying G-CSF functions in Th1/Th2 cells, thus supporting the therapeutic potential of G-CSF in the treatment of patients with ITP.
Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.Leukemia advance online publication, 4 November 2016; doi:10.1038/leu.2016.285.
Sex chromosome evolution involves the accumulation of repeat sequences such as multigenic families, noncoding repetitive DNA (satellite, minisatellite, and microsatellite), and mobile elements such as transposons and retrotransposons. Most species of Characidium exhibit heteromorphic ZZ/ZW sex chromosomes; the W is characterized by an intense accumulation of repetitive DNA including dispersed satellite DNA sequences and transposable elements. The aim of this study was to analyze the distribution pattern of 18 different tandem repeats, including (GATA)n and (TTAGGG)n, in the genomes of C. zebra and C. gomesi, especially in the C. gomesi W chromosome. In the C. gomesi W chromosome, weak signals were seen for (CAA)10, (CAC)10, (CAT)10, (CGG)10, (GAC)10, and (CA)15 probes. (GA)15 and (TA)15 hybridized to the autosomes but not to the W chromosome. The (GATA)n probe hybridized to the short arms of the W chromosome as well as the (CG)15 probe. The (GATA)n repeat is known to be a protein-binding motif. GATA-binding proteins are necessary for the decondensation of heterochromatic regions that hold coding genes, especially in some heteromorphic sex chromosomes that may keep genes related to oocyte development. The (TAA)10 repeat is accumulated in the entire W chromosome, and this microsatellite accumulation is probably involved in the sex chromosome differentiation process and crossover suppression in C. gomesi. These additional data on the W chromosome DNA composition help to explain the evolution of sex chromosomes in Characidium.
Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.
Dendritic cells are specialized antigen-presenting cells which link innate and adaptive immunity, through recognition and presentation of antigen to T cells. Although the importance of dendritic cells has been demonstrated in many animal models, their contribution to human immunity remains relatively unexplored in vivo.Given their central role in infection, autoimmunity, and malignancy, dendritic cell deficiency or dysfunction would be expected to have clinical consequences.
Human dendritic cell deficiency disorders, related to GATA binding protein 2 (GATA2) and interferon regulatory factor 8 (IRF8) mutations, have highlighted the importance of dendritic cells and monocytes in primary immunodeficiency diseases and begun to shed light on their nonredundant roles in host defense and immune regulation in vivo. The contribution of dendritic cell and monocyte dysfunction to the pathogenesis of primary immunodeficiency disease phenotypes is becoming increasingly apparent. However, dendritic cell analysis is not yet a routine part of primary immunodeficiency disease workup.
Widespread uptake of dendritic cell/monocyte screening in clinical practice will facilitate the discovery of novel dendritic cell and monocyte disorders as well as advancing our understanding of human dendritic cell biology in health and disease.
Notch signaling is involved in the early process of differentiation to determine the fate of stem cells. However, the precise role of Notch in human bone marrow‑derived mesenchymal stem cells (hBMSCs) remains unclear. The present study aimed to investigate the involvement of Notch signalling during the course of hBMSC differentiation into cardiomyocytes using hBMSCs, with multilineage differentiation ability, isolated and purified from human bone marrow. Flow cytometric analysis revealed that CD29, CD44 and CD90 were highly expressed on the surface of cells in their fifth passage, whereas detection of CD34, CD45, CD54 and HLA‑DR was negative. Visualization of morphological changes, western blotting, immunocytochemistry and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) demonstrated that hBMSCs differentiate into cardiomyocytes through treatment with 5‑azacytidine (5‑aza). Transmission electron microscopy revealed ultramicroscopic details of differentiated hBMSCs. Western blotting and immunocytochemistry demonstrated increased protein expression levels of α‑actin and cardiac troponin T expression, and RT‑qPCR revealed increased mRNA expression of Notch1 early in the process of differentiation (days 1, 4 and 7), and increased mRNA expression levels of the transcription factors GATA binding protein‑4 and NK2 homeobox 5 at day 28 day. In conclusion, differentiation of hBMSCs into cardiomyocytes was induced in vitro by 5‑aza, and was associated with upregulation of Notch1, GATA binding protein‑4 and Nkx2.5 expression. Overexpression of the Notch1 signaling pathway may represent a potential mechanism underlying the differentiation of hBMSCs.
Recently, interest in the beneficial role of probiotics in the protection and management of allergic diseases caused by immune disorders has been increasing. This study investigated the inhibitory effect of Lactobacillus plantarum L67 on induced allergic inflammatory response in bisphenol A-treated rat basophilic leukemia 2H3 (RBL-2H3) cells and mouse splenocytes. We also evaluated the applicability of L. plantarum L67 as a yogurt starter culture. We measured the ability of Lactobacillus strains to induce the production of IL-12 and IFN- γ in cultured splenocytes by ELISA. Bisphenol A (50μM)-treated RBL-2H3 cells were cotreated with a glycoprotein (18kDa) isolated from L. plantarum L67 (5-100µg/mL) for 30min. We measured the expression of mitogen-activated protein kinase (ERK and p38), AP-1 (c-Fos and c-Jun), T-bet, and GATA-binding protein 3 (GATA-3) using Western blotting to examine the differentiation of T helper cells. Furthermore, we evaluated the gene expression of IL-1β, IL-6, and IL-10 using real-time quantitative PCR. Finally, we evaluated the applicability of L. plantarum L67 as a yogurt starter by measuring pH, enumeration of bacteria, and sensory scores. Our results showed that L67 protein inhibited the phosphorylation of ERK and p38 mitogen-activated protein kinase through the transcriptional activation of AP-1 in bisphenol A-treated RBL-2H3 cells. During differentiation of T helper cells, the expression of transcription factor GATA-3 was significantly suppressed by L67 protein (100µg/mL) treatment, whereas expression of transcription factor T-bet was increased. In addition, the L67 protein significantly attenuated the expression of T helper 2-linked cytokines IL-1β, IL-6, and IL-10. These results indicate that L. plantarum L67, made available as yogurt starters and dietary supplements, has the potential to prevent allergy-related immune disorders.
Breast cancer metastases to the gastrointestinal tract are very rare occurrences. Among the histological subtypes of breast cancer, invasive lobular carcinomas have a high capacity of metastasis to uncommon sites including the stomach. Conversely, there has not been sufficient evidence supporting the gastric metastasis of invasive ductal carcinoma. Herein, we report a unique case of metastatic ductal breast carcinoma mimicking primary linitis plastica in a male patient, particularly focusing on the clinical and pathological features of presentation. Moreover, we propose a immunohistochemical panel of selected antibodies including those for cytokeratin 20, cytokeratin 7, estrogen receptor, progesterone receptor, E-cadherin, gross cystic disease fluid protein 15, and GATA binding protein 3 for an accurate differential diagnosis.
Accumulating evidence reveals that hormone leptin, mainly produced by adipocyte, plays a unique role in promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a key step in liver fibrosis and peroxisome-proliferator activated receptor γ (PPARγ) exerts a crucial role in inhibition of HSC activation. Our previous researches demonstrated that leptin reduced PPARγ1 (a major subtype of PPARγ in HSCs) expression through GATA binding protein 2 (GATA2) binding to a site around -2323 in PPARγ1 promoter. The present researches aimed to examine the effect of GATA3 on leptin-induced inhibition of PPARγ1 and elucidate the relationship between GATA3 and GATA2. Gene expressions were analysed by real-time PCR, western blot, luciferase assay and immunostaining. C57BL/6J ob/ob mouse model of thioacetamide-induced liver injury was used in vivo. Results demonstrate that leptin significantly induces GATA3 expression in HSCs by multiple signalling pathways including NADPH oxidase pathway. There exist crosstalks between NADPH oxidase pathway and the other pathways. GATA3 can bind to GATA2-binding site in PPARγ1 promoter and interacts with GATA2, contributing to leptin inhibition of PPARγ1 expression in HSCs. These data demonstrated novel molecular events for leptin inhibition of PPARγ1 expression in HSCs and thus might have potential implications for clarifying the detailed mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as non-alcoholic steatohepatitis in obese patients.
Current understanding of adaptive immune, particularly T cell, responses to human rhinoviruses (RV) is limited. Memory T cells are thought to be of a primarily T helper 1 type, but both T helper 1 and T helper 2 memory cells have been described, and heightened T helper 2/ lessened T helper 1 responses have been associated with increased RV-induced asthma exacerbation severity. We examined the contribution of T helper 1 cells to RV-induced airways inflammation using mice deficient in the transcription factor T-Box Expressed In T Cells (Tbet), a critical controller of T helper 1 cell differentiation. Using flow cytometry we showed that Tbet deficient mice lacked the T helper 1 response of wild type mice and instead developed mixed T helper 2/T helper 17 responses to RV infection, evidenced by increased numbers of GATA binding protein 3 (GATA-3) and RAR-related orphan receptor gamma t (RORγt), and interleukin-13 and interleukin-17A expressing CD4+ T cells in the lung. Forkhead box P3 (FOXP3) and interleukin-10 expressing T cell numbers were unaffected. Tbet deficient mice also displayed deficiencies in lung Natural Killer, Natural Killer T cell and γδT cell responses, and serum neutralising antibody responses. Tbet deficient mice exhibited pronounced airways eosinophilia and mucus production in response to RV infection that, by utilising a CD4+ cell depleting antibody, were found to be T helper cell dependent. RV induction of T helper 2 and T helper 17 responses may therefore have an important role in directly driving features of allergic airways disease such as eosinophilia and mucus hypersecretion during asthma exacerbations.
Langerhans cell histiocytosis (LCH) is a neoplasm of myeloid origin characterized by a clonal proliferation of CD1a(+)/CD207(+) dendritic cells. Recurrent BRAF V600E mutation has been reported in LCH. In the present report, we confirm the feasibility of the high-specificity monoclonal antibody VE1 for detecting BRAF V600E mutation in 36/97 (37.1%) retrospectively enrolled patients with LCH; concordant immunohistochemistry and Sanger sequencing results were seen in 94.8% of cases. We then assessed the tumor immune microenvironment status in LCH, and found that the GATA binding protein 3 (GATA3)(+)/T-bet(+) ratio could distinguish between clinical multi-system/single-system (SS) multifocal and SS unifocal LCH. Notably, we found that BRAF V600E mutation is significantly correlated with increased programmed cell death 1 ligand 1 (PDL1) expression and forkhead box protein 3 (FOXP3)(+) regulatory T cells (p < 0.001, 0.009, respectively). Moreover, Cox multivariate survival analysis showed that BRAF V600E mutation and PDL1 were independent prognostic factors of poor disease-free survival (DFS) in LCH (hazard ratio [HR] = 2.38, 95% confidence interval [CI] 1.02-5.56, p = 0.044; HR = 3.06, 95%CI 1.14-7.14, p = 0.025, respectively), and the superiority of PDL1 in sensitivity and specificity as biomarker for DFS in LCH was demonstrated by receiver operator characteristic (ROC) curves when compared with BRAF V600E and risk category. Collectively, this study identifies for the first time relationship between BRAF V600E mutation and a suppressive tumor immune microenvironment in LCH, resulting in disruption of host-tumor immune surveillance, which is DFS. Our findings may provide a rationale for combining immunotherapy and BRAF-targeted therapy for treating patients with BRAF V600E mutant LCH.
Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T‑box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V‑fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick‑end labeling system. Cell cycle analysis was achieved using fluorescence‑activated cell sorting, and, an RT‑qPCR array was used to profile the expression of TBX20‑related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin‑dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell proliferation and cell cycle arrest.
Little is known about the aminopeptidase CD13 in renal neoplasia according to the new 2016 World Health Organization renal tumor classification. We selected 175 cases, including 79 clear cell, 31 papillary, 24 chromophobe, 8 clear cell papillary renal cell carcinomas (RCCs), 21 oncoytomas, and 12 microphthalmia transcription factor family translocation RCCs: 4 t(6;11)/transcription factor EB (TFEB), 7 t(Xp11) with 2 cystic variants and 1 t(X;17). GATA binding protein 3 (GATA-3) was inserted as control. Expression of proximal antigen CD13 was observed in 63/79 (80%) clear cell, 25/31 (81%) papillary, 3/8 (37%) clear cell papillary, 1/4 (25%) t(6;11)/TFEB, 2/7 (28%) cystic t(Xp11), and in 1/1 t(X;17) RCCs. All chromophobe RCC (0/24) and all oncocytomas (0/21) resulted negative. CD10 was seen in 76/79 (96%) clear cell, 15/31 (48%) papillary, 10/24 (42%) chromophobe, 1/8 (12%) clear cell papillary RCCs, 4/21 (19%) oncocytomas, 1/4 (25%) t(6;11)/TFEB, 2/7 (29%) cystic t(Xp11), and in 1/1 t(X;17) RCCs. GATA-3 was positive in 3/7 (42%) clear cell papillary RCCs and negative in all remaining RCCs, except a single chromophobe RCC and a single oncocytoma. We concluded that: (1) CD13 and GATA-3 immunostains may serve as a diagnostic aid in differentiating subtypes of RCC; (2) CD13 is always absent in chromophobe RCC and oncocytomas, whereas CD10 can be immunoexpressed in both; (3) CD13 should be included in a panel of antibodies to distinguish "proximal renal tumors" from "distal renal tumors" and between clear cell RCC versus microphthalmia transcription factor family translocations RCCs; and (4) when present, GATA-3 is specific for clear cell papillary RCC.
Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.
Development of engineered site-specific endonucleases like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 has been revolutionizing genetic approaches in biomedical research fields. These new tools have opened opportunities to carry out targeted genome editing in mouse zygotes without the need for manipulating embryonic stem cells, which have a higher technical burden and many constraints in strain availability. Specific genetic modifications can be directly generated in working genetic backgrounds. This new approach saves time and costs associated with generation and backcrossing of genetically modified animals and will facilitate their use in various cancer research fields.
Hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is a rare autosomal dominant disorder, secondary to mutations in the GATA-3 gene. Due to its wide range of penetrance and expressivity, the disease may not always be recognized. We herein describe clinical and genetic features of patients with HDR syndrome, highlighting diagnostic clues.
Medical records of eight patients from five unrelated families exhibiting GATA-3 mutations were reviewed retrospectively, in conjunction with all previously reported cases.
HDR syndrome was diagnosed in eight patients between the ages of 18 and 60 years. Sensorineural deafness was consistently diagnosed, ranging from clinical hearing loss since infancy in seven patients to deafness detected only by audiometry in adulthood in one single patient. Hypoparathyroidism was present in six patients (with hypocalcaemia and inaugural seizures in two out of six). Renal abnormalities observed in six patients were diverse and of dysplastic nature. Three patients displayed nephrotic-range proteinuria and reached end-stage renal disease (ESRD) between the ages of 19 and 61 years, whilst lesions of focal and segmental glomerulosclerosis were histologically demonstrated in one of them. Interestingly, phenotype severity differed significantly between a mother and son within one family. Five new mutations of GATA-3 were identified, including three missense mutations affecting zinc finger motifs [NM_001002295.1: c.856A>G (p.N286D) and c.1017C>G (p.C339W)] or the conserved linker region [c.896G>A (p.R299G)], and two splicing mutations (c.924+4_924+19del and c.1051-2A>G). Review of 115 previously reported cases of GATA-3 mutations showed hypoparathyroidism and deafness in 95% of patients, and renal abnormalities in only 60%. Overall, 10% of patients had reached ESRD.
We herein expand the clinical and mutational spectrum of HDR syndrome, illustrating considerable inter- and intrafamilial phenotypic variability. Diagnosis of HDR should be considered in any patient with hypoparathyroidism and deafness, whether associated with renal abnormalities or not. HDR diagnosis is established through identification of a mutation in the GATA-3 gene.
Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site-specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site-specific genomic double-strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error-prone non-homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology-directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here, we summarize the role of three classes of nucleases; zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site-specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. J. Cell. Physiol. 231: 2380-2392, 2016. © 2016 Wiley Periodicals, Inc.
Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.
Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.
Zebrafish is a powerful model for the study of vertebrate development, being amenable to a wide range of genetic and other manipulations to probe the molecular basis of development and its perturbation in disease. Over recent years, genome editing approaches have become increasingly used as an efficient and sophisticated approach to precisely engineer the zebrafish genome, which has further enhanced the utility of this organism. This review provides a practical overview of genome editing and its application in zebrafish research, including alternate strategies for introducing and screening for specific genetic changes.
Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.
Targeted genome-editing technology using designed nucleases has been evolving rapidly, and its applications are widely expanding in research, medicine and biotechnology. Using this genome-modifying technology, researchers can precisely and efficiently insert, remove or change specific sequences in various cultured cells, micro-organisms, animals and plants. This genome editing is based on the generation of double-strand breaks (DSBs), repair of which modifies the genome through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). In addition, designed nickase-induced generation of single-strand breaks can also lead to precise genome editing through HDR, albeit at relatively lower efficiencies than that induced by nucleases. Three kinds of designed nucleases have been used for targeted DSB formation: zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system. A growing number of researchers are using genome-editing technologies, which have become more accessible and affordable since the discovery and adaptation of CRISPR-Cas9. Here, the repair mechanism and outcomes of DSBs are reviewed and the three types of designed nucleases are discussed with the hope that such understanding will facilitate applications to genome editing.
Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.
Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.
Homozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.
Taking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual's cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.
We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.
Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.
The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell's naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.
Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells.
Gene editing using engineered endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases, requires the creation of a targeted, chromosomal DNA double-stranded break (DSB). In mammalian cells, these DSBs are typically repaired by one of the two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair process that can result in a wide range of end-joining events that leads to somewhat random mutations at the site of DSB. HDR is a precise repair pathway that can utilize either an endogenous or exogenous piece of homologous DNA as a template or "donor" for repair. Traditional gene editing via HDR has relied on the co-delivery of a targeted, engineered endonuclease and a circular plasmid donor construct. More recently, it has been shown that single-stranded oligodeoxynucleotides (ssODNs) can also serve as DNA donors and thus obviate the more laborious and time-consuming plasmid vector construction process. Here we describe the use of ssODNs for making defined genome modifications in combination with engineered endonucleases.
The ability to edit the genome of cell lines has provided valuable insights into biological processes and the contribution of specific mutations to disease biology. These techniques fall into two categories based on the DNA repair mechanism that is used to incorporate the genetic change. Nuclease-based technologies, such as Zinc-Finger Nucleases, TALENS, and Crispr/Cas9, rely on non-homologous end-joining (NHEJ) and homology directed repair (HDR) to generate a range of genetic modifications. Adeno-Associated Virus (AAV) utilizes homologous recombination to generate precise and predictable genetic modifications directly at the target locus. AAV has been used to create over 500 human isogenic cell lines comprising a wide range of genetic alterations from gene knockouts, insertions of point mutations, indels, epitope tags, and reporter genes. Here we describe the generation and use of AAV gene targeting vectors and viruses to create targeted isogenic cell lines.
CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.
To report a rare case of vitreo-retinal metastasis from urothelial carcinoma of the bladder.
Case report.
A 55-year-old man with a history of bladder cancer developed atypical vitritis and a white fundus mass. Intravenous fluorescein angiography demonstrated connection between the retinal and tumor vasculature consistent with a retinal malignancy. Cytologic analysis of the vitreous sample revealed large, atypical cells with pleomorphic nuclei, mucin vacuoles, and rare mitotic figures. The cells were immunoreactive for cytokeratin markers AE1/AE3, CK7, and CK20 and the urothelial carcinoma marker GATA3. Review of the patient's initial bladder tumor revealed an anaplastic epithelial neoplasm with dyscohesive cells that appeared histologically identical to those in the vitreous biopsy. Despite external beam radiation therapy, the patient's vision declined and the eye became painful and was ultimately enucleated.
Retinal metastasis from systemic adenocarcinoma is an extremely rare occurrence with poor prognosis for vision.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
Neuroendocrine breast carcinomas (NBCs) account for 2-5% of all invasive breast cancers and are histologically similar to neuroendocrine tumours from other sites. They typically express oestrogen receptor (ER), are HER2-negative and of luminal subtype. Here we sought to define the mutational profile of NBCs, and to investigate whether NBCs and common forms of luminal (ER+/HER2-) breast cancer display distinct repertoires of somatic mutations. Eighteen ER+/HER2- NBCs, defined as harbouring >50% of tumour cells expressing chromogranin A and/or synaptophysin, and matched normal tissue were microdissected and subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in breast cancer and/or related to DNA repair. Their mutational repertoire was compared to that of ER+/HER2- (n = 240), PAM50-defined luminal breast cancers (n = 209 luminal A; n = 111 luminal B) and invasive lobular carcinomas (n = 127) from The Cancer Genome Atlas. NBCs were found to harbour a median of 4.5 (range 1-11) somatic mutations, similar to that of luminal B breast cancers (median = 3, range 0-17) but significantly higher than that of luminal A breast cancers (median = 3, range 0-18, p = 0.02). The most frequently mutated genes were GATA3, FOXA1, TBX3, ARID1A (3/18, 17%), and PIK3CA, AKT1, CDH1 (2/18, 11%). NBCs less frequently harboured PIK3CA mutations than common forms of ER+/HER2, luminal A and invasive lobular carcinomas (p < 0.05) and displayed a significantly higher frequency of somatic mutations affecting ARID1A (17% versus 2%, p < 0.05) and the transcription factors FOXA1 (17% versus 2%, p = 0.01) and TBX3 (17% versus 3%, p < 0.05) than ER+/HER2- breast cancers. No TP53 somatic mutations were detected in NBCs. Compared to common forms of luminal breast cancers, NBCs display a distinctive repertoire of somatic mutations featuring lower frequency of TP53 and PIK3CA mutations, and enrichment for FOXA1, TBX3, and akin to neuroendocrine tumours from other sites, ARID1A mutations.
Discrete bladder cancer molecular subtypes exhibit differential clinical aggressiveness and therapeutic response, which may have significant implications for identifying novel treatments for this common malignancy. However, research is hindered by the lack of suitable models to study each subtype. To address this limitation, we classified bladder cancer cell lines into molecular subtypes using publically available data in the Cancer Cell Line Encyclopedia (CCLE), guided by genomic characterization of bladder cancer by The Cancer Genome Atlas (TCGA). This identified a panel of bladder cancer cell lines which exhibit genetic alterations and gene expression patterns consistent with luminal and basal molecular subtypes of human disease. A subset of bladder cancer cell lines exhibit in vivo histomorphologic patterns consistent with luminal and basal subtypes, including papillary architecture and squamous differentiation. Using the molecular subtype assignments, and our own RNA-seq analysis, we found overexpression of GATA3 and FOXA1 cooperate with PPARɣ activation to drive transdifferentiation of a basal bladder cancer cells to a luminial phenotype. In summary, our analysis identified a set of human cell lines suitable for the study of molecular subtypes in bladder cancer, and furthermore indicates a cooperative regulatory network consisting of GATA3, FOXA1, and PPARɣ drive luminal cell fate.
We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3(+) cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67(+) proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2-58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses.
Helper T (Th) cell subsets direct immune responses by producing signature cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular analysis of Th2 cell differentiation and maintenance of function has led to recent discoveries that have refined our understanding of Th2 cell biology. Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell identity. In the context of allergic diseases, memory-type pathogenic Th2 cells have been identified in both mice and humans. To better understand these disease-driving cell populations, we have developed a model called the pathogenic Th population disease induction model. The concept of defined subsets of pathogenic Th cells may spur new, effective strategies for treating intractable chronic inflammatory disorders. Expected final online publication date for the Annual Review of Immunology Volume 35 is April 26, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Bilateral breast cancers (BBC) are currently treated as independent tumors arising in the same patient. Herein, we investigated whether BBC indeed evolve independently at the genomic level. We examined paired targeted next generation sequencing genotypes from 155 paraffin tumors corresponding to 76 BBC patients (75 women and one man; 52 concurrent and 24 metachronous), for coding mutations (amino acid changing, minor allele frequency <0.1%) and single nucleotide polymorphism (SNP) zygosity. Germline genotypes were available for 29 patients. Mutations were present in 80 tumors (54/76 patients; 71%), were mostly tumor-private (90%), more frequent in TP53 (19%), PIK3CA (14%), CDH1, GATA3, MLL3. TP53 mutations were more frequent in metachronous tumors (P<0.001); hormone receptor negative (P<0.001); with higher Ki-67 (P=0.002); and, in younger patients (P=0.01). Hypermutated tumors, all TP53 mutated, were diagnosed as the first incidence in 5 patients; their metachronous counterparts were mutation poor without TP53 involvement. Paired tumors shared common mutations at intratumoral frequency >20% in 10/54 comparable BBC (18.5%), 8/10 concurrent. SNP zygosity status was less preserved in metachronous, compared to concurrent disease. Pathogenic germline mutations were present in 10/29 patients, 9 in BRCA1 and one in TP53 (p.Phe341Val, first report in the germline). BBC demonstrated extensive inter- and intra-patient heterogeneity in the present thus far largest series of corresponding paired genotypes. The majority evolve independently and unpredictably, supporting current clinical practice. A considerable minority though, retains clonal origin and may be regarded as a distinct group for therapeutic interventions among concurrent BBC.
Identification of the tissue of origin in cancer of unknown primary (CUP) poses a diagnostic challenge and is critical for directing site-specific therapy. Currently, clinical decision-making in patients with CUP primarily relies on histopathology and clinical features. Comprehensive molecular profiling has the potential to contribute to diagnostic categorization and, most importantly, guide CUP therapy through identification of actionable lesions. We here report the case of an advanced-stage malignancy initially mimicking poorly differentiated soft-tissue sarcoma that did not respond to multiagent chemotherapy. Molecular profiling within a clinical whole-exome and transcriptome sequencing program revealed a heterozygous, highly amplified KRAS G12S mutation, compound-heterozygous TP53 mutation/deletion, high mutational load, and focal high-level amplification of Chromosomes 9p (including PDL1 [CD274] and JAK2) and 10p (including GATA3). Integrated analysis of molecular data and histopathology provided a rationale for immune checkpoint inhibitor (ICI) therapy with pembrolizumab, which resulted in rapid clinical improvement and a lasting partial remission. Histopathological analyses ruled out sarcoma and established the diagnosis of a poorly differentiated adenocarcinoma. Although neither histopathology nor molecular data were able to pinpoint the tissue of origin, our analyses established several differential diagnoses including triple-negative breast cancer (TNBC). We analyzed 157 TNBC samples from The Cancer Genome Atlas, revealing PDL1 copy number gains coinciding with excessive PDL1 mRNA expression in 24% of cases. Collectively, these results illustrate the impact of multidimensional tumor profiling in cases with nondescript histology and immunophenotype, show the predictive potential of PDL1 amplification for immune checkpoint inhibitors (ICIs), and suggest a targeted therapeutic strategy in Chromosome 9p24.1/PDL1-amplified cancers.
Although previous studies suggested that microRNA-506-3p (miR-506-3p) was frequently downregulated, and functioned as a tumor suppressor in several cancers, the biological role and intrinsic regulatory mechanisms of miR-506-3p in non-small cell lung cancer (NSCLC) remain elusive. The present study found miR-506-3p expression was downregulated in advanced NSCLC tissues and cell lines. The expression of miR-506-3p in NSCLC was inversely correlated with larger tumor size, advanced TNM stage and lymph node metastasis. In addition, we also found patients with lower expression of miR-506-3p had a poor prognosis than those patients with higher expression of miR-506-3p. Function studies demonstrated that aberrant miR-506-3p expression modulates tumor cell growth, cell mobility, cell migration and invasion in vitro and in vivo. Mechanistic investigations manifested that coactosin-like protein 1 (COTL1) was a direct downstream target of miR-506-3p. Knockdown of COTL1 mimicked the tumor-suppressive effects of miR-506-3p overexpression in A549 cells, whereas COTL1 overexpression enhanced the tumorigenic function in HCC827 cells. Importantly, we also found GATA3 transcriptionally actives miR-506-3p expression, and the long non-coding RNA urothelial carcinoma-associated 1 (UCA1) exerts oncogenic function in NSCLC by competitively 'sponging' miRNA-506. Together, our combined results elucidated genetic and epigenetic silencing of miR-506-3p enhances COTL1 oncogene expression to foster NSCLC progression.
Asthma is a Th2 cell-driven inflammatory disease and a major public health concern. The cis-acting element Rad50 hypersensitive site 6 (RHS6) in the Th2 locus control region is essential for regulation of the Th2 cytokine genes; however, its role in allergic airway inflammation and underlying molecular mechanisms of the regulation by RHS6 are poorly understood.
We sought to understand the role of RHS6 in the development of allergic airway inflammation and its molecular mechanism for Th2 cytokine expression.
We used an ovalbumin-induced allergic inflammation model with RHS6-deficient mice to examine the role of RHS6 in this process. To examine molecular mechanism of RHS6 for Th2 cytokine expression, we used DNA affinity chromatography and mass spectrometry, quantitative RT-PCR, ELISA, intracellular cytokine staining, chromatin immunoprecipitation, and co-immunoprecipitation.
Deletion of RHS6 caused a dramatic resistance to allergic airway inflammation. RHS6 recruited transcription factors GATA3, SATB1, and IRF4, which play important roles in expression of all three Th2 cytokine genes. RHS6 deficiency caused inhibition of transcription factor-induced Th2 cytokine gene expression.
RHS6 is a critical regulatory element for allergic airway inflammation and for coordinate regulation of Th2 cytokine genes by recruiting GATA3, SATB1, and IRF4.
Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFNγ in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.
To identify factors predicting mucosal healing in ulcerative colitis patients treated with anti-TNFα agents with or without azathioprine.
In a prospective, multicenter, one-year study biologic naïve patients aged 25-65 years, with corticosteroid-dependent or refractory colitis received combination treatment with anti-TNFα and azathioprine for 6 months followed by anti-TNFα monotherapy. Patients who denied combination therapy or were outside this age range received anti-TNFα monotherapy (controls). Before and at weeks 12 and 54 of treatment the total Mayo score was calculated. Mucosal healing was defined as endoscopic subscore of 0. Mucosal expression of T helper (Th) cell-lineage specific transcription factors (Tbet, Gata3, Rorc, FoxP3) before treatment was also associated with mucosal healing.
Of 67 patients, 58 (86.6%) received combination and 9 (13.4%) anti-TNFα monotherapy. Overall 29 (43.3%) patients achieved mucosal healing; rates were higher in patients receiving combination therapy vs. monotherapy (p=0.03) and in azathioprine naïve vs. exposed patients in the combination group (p=0.01). Mucosal healing was associated with lower pre-treatment mucosal expression of transcription factor Th1-Tbet (p<0.05) and higher expression of Th17-Rorc (p<0.05).
Mucosal healing was associated with combination therapy, especially in biologic and azathioprine-naïve patients and pre-treatment mucosal expression of specific Th specific transcripting factors (Tbet and Rorc).
The present study was designed to investigate the upstream transcription factors (TFs) and the signature genes in cholangiocarcinoma (CCA), providing better clues on the regulatory mechanisms and therapeutic applications. Gene expression data sets of CCA were searched in the Gene Expression Omnibus database for integrated analysis. Functional annotation of differently expressed genes (DEGs) was then conducted and the TFs were identified. Moreover, a global transcriptional regulatory network of TFs-targets was constructed. Integrated analysis of five eligible Gene Expression Omnibus data sets led to a set of 993 DEGs and 48 TFs in CCA. The constructed TFs-targets regulatory network consisted of 697 TF-target interactions between 41 TFs and 436 DEGs. The top 10 TFs covering the most downstream DEGs were NFATC2, SOX10, ARID3A, ZNF263, NR4A2, GATA3, EGR1, PLAG1, STAT3 and FOSL1, which may have important roles in the tumorigenesis of CCA. Supporting the fact that defects of cell-cycle surveillance mechanism were closely related to various cancers, we found that cell cycle was the most significantly enriched pathway. KCNN2 and ADCY6 were involved in the bile secretion. Thus, their aberrant expression may be closely related to the pathogenesis of CCA. Particularly, we found that upregulation of EZH2 in CCA is a powerful potential marker for CCA.Cancer Gene Therapy advance online publication, 18 November 2016; doi:10.1038/cgt.2016.64.
Distant metastases are relatively common in breast cancer, but spread to the head and neck region is uncommon and can be diagnostically challenging. Pathology databases of two academic hospitals were searched for patients. The diagnoses were by morphologic comparison with the primary breast specimen (when available) or through the use of immunohistochemical stains characteristic of breast carcinoma (cytokeratin 7, mammaglobin, GCDFP15, and/or GATA3 positive-excluding new primary tumors at the respective head and neck sites). Of the 25 patients identified, only 22 (88.0 %) had a known history of breast carcinoma. Time from primary diagnosis to head and neck metastasis was highly variable, ranging from 1 to 33 years (mean = 10.9 years). The most common locations were neck lymph nodes (8 cases), orbital soft tissue (5), oral cavity (3), skull base (3), mastoid sinus (2), nasal cavity (1), palatine tonsil (1), and facial skin (1). Clinical presentations were highly variable, ranging from cranial nerve palsies without a mass lesion to oral cavity erythema and swelling to bone pain. Histologically, two cases showed mucosal (or skin)-based mass lesions with associated pagetoid spread in the adjacent epithelium, a feature normally associated with primary carcinomas. Three tumors were misdiagnosed pathologically as new head and neck primary tumors. This series demonstrates the extreme variability in clinical and pathologic features of breast cancer metastatic to the head and neck, including long time intervals to metastasis.
Although technological advances now allow increased tumor profiling, a detailed understanding of the mechanisms leading to the development of different cancers remains elusive. Our approach toward understanding the molecular events that lead to cancer is to characterize changes in transcriptional regulatory networks between normal and tumor tissue. Because enhancer activity is thought to be critical in regulating cell fate decisions, we have focused our studies on distal regulatory elements and transcription factors that bind to these elements.
Using DNA methylation data, we identified more than 25,000 enhancers that are differentially activated in breast, prostate, and kidney tumor tissues, as compared to normal tissues. We then developed an analytical approach called Tracing Enhancer Networks using Epigenetic Traits that correlates DNA methylation levels at enhancers with gene expression to identify more than 800,000 genome-wide links from enhancers to genes and from genes to enhancers. We found more than 1200 transcription factors to be involved in these tumor-specific enhancer networks. We further characterized several transcription factors linked to a large number of enhancers in each tumor type, including GATA3 in non-basal breast tumors, HOXC6 and DLX1 in prostate tumors, and ZNF395 in kidney tumors. We showed that HOXC6 and DLX1 are associated with different clusters of prostate tumor-specific enhancers and confer distinct transcriptomic changes upon knockdown in C42B prostate cancer cells. We also discovered de novo motifs enriched in enhancers linked to ZNF395 in kidney tumors.
Our studies characterized tumor-specific enhancers and revealed key transcription factors involved in enhancer networks for specific tumor types and subgroups. Our findings, which include a large set of identified enhancers and transcription factors linked to those enhancers in breast, prostate, and kidney cancers, will facilitate understanding of enhancer networks and mechanisms leading to the development of these cancers.
Cancers are heterogeneous by nature. While traditional oncology screens commonly use a single endpoint of cell viability, altering the phenotype of tumor-initiating cells may reveal alternative targets that regulate cellular growth by processes other than apoptosis or cell division. We evaluated the impact of knocking down expression of 420 kinases in bi-lineage triple-negative breast cancer (TNBC) cells that express characteristics of both myoepithelial and luminal cells. Knockdown of ERN1 or ALPK1 induces bi-lineage MDA-MB-468 cells to lose the myoepithelial marker keratin 5 but not the luminal markers keratin 8 and GATA3. In addition, these cells exhibit increased β-casein production. These changes are associated with decreased proliferation and clonogenicity in spheroid cultures and anchorage-independent growth assays. Confirmation of these assays was completed in vivo, where ERN1- or ALPK1-deficient TNBC cells are less tumorigenic. Finally, treatment with K252a, a kinase inhibitor active on ERN1, similarly impairs anchorage-independent growth of multiple breast cancer cell lines. This study supports the strategy to identify new molecular targets for types of cancer driven by cells that retain some capacity for normal differentiation to a non-tumorigenic phenotype. ERN1 and ALPK1 are potential targets for therapeutic development.
To report a rare case of vitreoretinal metastasis from urothelial carcinoma of the bladder.
Case report.
A 55-year-old male with a history of bladder cancer developed atypical vitritis and a white fundus mass. IV fluorescein angiography demonstrated connection between the retinal and tumor vasculature consistent with a retinal malignancy. Cytologic analysis of the vitreous sample revealed large atypical cells with pleomorphic nuclei, mucin vacuoles, and rare mitotic figures. The cells were immunoreactive for cytokeratin markers AE1/AE3, CK7, and CK20 and urothelial carcinoma marker GATA3. Review of the patient's initial bladder tumor revealed an anaplastic epithelial neoplasm with dyscohesive cells that appeared histologically identical to those in the vitreous biopsy. Despite external beam radiation therapy, the patient's vision declined and the eye became painful and was ultimately enucleated.
Retinal metastasis from systemic adenocarcinoma is extremely rare occurrence with poor prognosis for vision.
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disorder caused by heterozygous point mutations or genomic deletions involving FOXF1 or its 60-kb tissue-specific enhancer region mapping 270 kb upstream of FOXF1 and involving fetal lung-expressed long non-coding RNA genes and CpG-enriched sites. Recently, we have proposed that the FOXF1 locus at 16q24.1 may be a subject of genomic imprinting.
Using custom-designed aCGH and Sanger sequencing, we have identified a novel de novo 104 kb genomic deletion upstream of FOXF1 in a patient with histopathologically verified full phenotype of ACDMPV. This deletion allowed us to further narrow the FOXF1 enhancer region and identify its critical 15-kb core interval, essential for lung development. This interval harbors binding sites for lung-expressed transcription factors, including GATA3, ESR1, and YY1, and is flanked by the lncRNA genes and CpG islands. Bisulfite sequencing of one of these CpG islands on the non-deleted allele showed that it is predominantly methylated on the maternal chromosome 16.
Substantial narrowing and bisulfite sequencing of the FOXF1 enhancer region on 16q24.1 provided new insights into its regulatory function and genomic imprinting.
Atrophying pityriasis versicolor (PV), first described in 1971, is a rare variant in which lesions appear atrophic.
We sought to determine the pathophysiology of atrophying PV.
A retrospective chart review identified 6 cases of atrophying PV. In all cases, routine light microscopy, an elastic tissue stain, and immunohistochemical assessment for the expression of CD3, CD4, CD8, GATA3 and CXCR3 was performed.
All cases demonstrated hyperkeratosis with intracorneal infiltration by pathogenic hyphal forms as well as epidermal attenuation and papillary dermal elastolysis. A supervening, mild-to-moderate, superficial lymphocytic infiltrate was noted and characterized by a focal CD8(+) T cell-mediated interface dermatitis along with a mixed T-cell infiltrate composed of GATA3(+) and CXCR3(+) T cells.
Small sample size and the loss of some patients to follow-up.
Atrophying PV represents the sequelae of a mixed helper T-cell (TH1 and TH2) idiosyncratic immune response to Malassezia and can present as a protracted dermatosis that may clinically mimic an atypical lymphocytic infiltrate. TH1 cytokines can recruit histiocytes, a source of elastases, and upregulate matrix metalloproteinase activity, which may contribute to epidermal atrophy.
Acute chorioamnionitis contributes to premature birth, and is associated with post-birth complications. How chorioamnionitis impacts neonate's developing immune system has not been well defined.
Blood from extremely preterm infants (=28 weeks gestation) was drawn at the 1(st), 2(nd), and 4(th) week of life. Blood was either left unstimulated or stimulated for 4 hours with PMA/ionomycin. mRNA expression of transcription factors in unstimulated cells (RORC, TBET, GATA3, FOXP3) and inflammatory cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-6) in unstimulated and stimulated cells were analyzed. Data were analyzed based on the diagnosis of chorioamnionitis, funisitis and bronchopulmonary dysplasia (BPD).
At 1 week of life, exposure to funisitis, but not maternal chorioamnionitis was associated with an increased expression of RORC and RORC/FOXP3 ratio. These increases in RORC and RORC/FOXP3 ration were sustained over the 4 weeks of follow-up. Leukocytes from infants who developed BPD had increased stimulated and unstimulated IL-4 at the 1(st) week of life, but these increases were not sustained over time. In contrast, infants with mild BPD had a sustained decrease in stimulated IL-2.
Chorioamnionitis exposure, in particular to funisitis, lead to enhanced Th17-like responses that persist for 4 weeks after birth. Infants who later developed BPD did not exhibit a strikingly distinct immune profile.
GATA3 and T-box (T-bet) expressed in T-cells are transcriptional factors that play a critical role in development of Th2 and Th1 immunity respectively. GATA3 is expressed during Th2 differentiation and T-bet is expressed exclusively in Th1 cells. Thus, a balance between GATA3 and T-bet is believed to control Th2/Th1 polarization. Therefore, the high expression of T-bet and low expression of GATA3 indicate the existence of Th1 polarization in children with acute immune thrombocytopenic purpura (ITP). This might be related to the regulation of T-bet and GATA3. The objective of this work was to study the expression of transcriptional factors T-bet and GATA3 m RNA in children with idiopathic thrombocytopenic purpura and correlate it with clinical findings, laboratory findings, and outcome of patients. In this study the expression of T-bet and GATA3 genes was analysed in 20 normal healthy individuals and 40 children with ITP (20 acute and 20 persistent) using reverse transcriptase polymerase chain reaction to investigate a possible relation, association or correlation with the type of ITP and prognosis. T-bet was expressed significantly in 60 % of acute ITP children (12/20) (P value 0.001) and not expressed in persistent ITP children (0/20), while GATA3 was expressed in 25 % of persistent ITP patients (5/20) (P value 0.017) and not expressed in acute ITP patients (0/20). Both genes were not detected in healthy controls. We concluded that the high expression of T-bet and the low expression of GATA3 indicate the existence of Th1 polarization in children with acute ITP. This might be related to the regulation of T-bet and GATA3. Intensive studies of abnormal cytokine profiles in ITP have led to cytokine therapies that exploit the effects of IFN-γ on Th2 cells, but such therapies are often ineffective to develop safe and effective therapeutic tools. Targeting specific molecules such as T-bet and GATA3 may be a novel therapeutic tool in ITP.
Psoriasis is a common inflammatory skin disease with complex etiology and chronic progression. To provide novel insights into the regulatory molecular mechanisms of the disease, we performed RNA sequencing analysis of 14 pairs of skin samples collected from patients with psoriasis. Subsequent pathway analysis and extraction of the transcriptional regulators governing psoriasis-associated pathways was executed using a combination of the MetaCore Interactome enrichment tool and the cisExpress algorithm, followed by comparison to a set of previously described psoriasis response elements. A comparative approach allowed us to identify 42 core transcriptional regulators of the disease associated with inflammation (NFκB, IRF9, JUN, FOS, SRF), the activity of T cells in psoriatic lesions (STAT6, FOXP3, NFATC2, GATA3, TCF7, RUNX1), the hyperproliferation and migration of keratinocytes (JUN, FOS, NFIB, TFAP2A, TFAP2C) and lipid metabolism (TFAP2, RARA, VDR). In addition to the core regulators, we identified 38 transcription factors previously not associated with the disease that can clarify the pathogenesis of psoriasis. To illustrate these findings, we analyzed the regulatory role of one of the identified transcription factors (TFs), FOXA1. Using ChIP-seq and RNA-seq data, we concluded that the atypical expression of the FOXA1 TF is an important player in the disease as it inhibits the maturation of naive T cells into the (CD4+FOXA1+CD47+CD69+PD-L1(hi)FOXP3-) regulatory T cell subpopulation, therefore contributing to the development of psoriatic skin lesions.
Breast cancer is a common malignancy among women with a rising incidence. Our intention was to detect transcription factors (TFs) for deeper understanding of the underlying mechanisms of breast cancer. Integrated analysis of gene expression datasets of breast cancer was performed. Then, functional annotation of differentially expressed genes (DEGs) was conducted, including Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Furthermore, TFs were identified and a global transcriptional regulatory network was constructed. Seven publically available GEO datasets were obtained, and a set of 1196 DEGs were identified (460 up-regulated and 736 down-regulated). Functional annotation results showed that cell cycle was the most significantly enriched pathway, which was consistent with the fact that cell cycle is closely related to various tumors. Fifty-three differentially expressed TFs were identified, and the regulatory networks consisted of 817 TF-target interactions between 46 TFs and 602 DEGs in the context of breast cancer. Top 10 TFs covering the most downstream DEGs were SOX10, NFATC2, ZNF354C, ARID3A, BRCA1, FOXO3, GATA3, ZEB1, HOXA5 and EGR1. The transcriptional regulatory networks could enable a better understanding of regulatory mechanisms of breast cancer pathology and provide an opportunity for the development of potential therapy.
γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells and detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.
To identify shared and differential molecular pathways in blood and affected muscle between adult dermatomyositis (DM) and juvenile DM, and their association with clinical disease activity measures.
Gene expression of transcription factors and cytokines involved in differentiation and effector function of T cell subsets, regulatory T cells and follicular Th cells, were analyzed in the blood from 21 newly diagnosed adult and 26 juvenile DM subjects and in 15 muscle specimens (7 adult and 8 juvenile DM) using a custom RT2 Profiler PCR Array. Disease activity was determined and measured by established disease activity tools.
The most prominent finding was the higher blood expression of Th17-related cytokines [retinoic acid-related orphan receptor-γ, interferon regulatory factor 4, interleukin (IL)-23A, IL-6, IL-17F, and IL-21] in juvenile DM at baseline. In contrast, adult patients with DM showed increased blood levels of STAT3 and BCL6 compared with juvenile DM. In muscle, GATA3, IL-13, and STAT5B were found at higher levels in juvenile patients with DM compared with adult DM. Among 25 patients (11 adult and 14 juvenile DM) who had blood samples at baseline and at 6 months, increased expression of IL-1β, STAT3, STAT6, STAT5B, and BCL6 was associated with an improvement in global extramuscular disease activity.
We observed differences in gene expression profiling in blood and muscle between new-onset adult and juvenile DM. Cytokine expression in the blood of juvenile patients with new-onset DM was dominated by Th17-related cytokines compared with adult patients with DM. This may reflect the activation of different Th pathways between muscle and blood.
Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. We show that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3, which are activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert(+) precursors and for the serotonergic identity of the Vglut3(+) precursors. Similar to the serotonergic neurons, the Vglut3-expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1 Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. Our study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation.
Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Although fusion genes generated by chromosomal rearrangements are the most frequent genetic alterations in pediatric ALL, fusions are insufficient for the development of this disease, and thus, cannot serve as therapeutic targets for ALL. Recently, integrated genetic analysis using next generation sequencing technology has revealed the genetic landscapes of pediatric ALL. These studies disclosed that in addition to fusion genes, aberrations of cell proliferation pathways and epigenetic regulations are also involved in the pathogenesis of pediatric ALL. On the other hand, more recently, abnormalities of supper enhancer regions of TAL1 have been detected as a novel oncogenic mechanism of pediatric T cell ALL. Furthermore, germline mutations of ARID5B, PAX5, and GATA3 have been found to be involved in the genetic risk of developing ALL. Therefore, currently, the molecular mechanisms of pediatric ALL have been fully disclosed.
We report a series of 7 unusual and morphologically distinct cervical or upper vaginal lesions in women aged 41 to 70 years. The lesions involved the cervix in 3 cases, the upper vagina in 2, the cervix and vagina in 1, and in 1 case the site of origin could not be determined. The lesions had a consistent morphologic appearance with a surface "plaque-like" or "stuck-on" configuration apparent in those cases where surrounding normal tissues were present. Broad coalescing solid sheets and interconnecting trabeculae of cytologically bland cells with a rather "basaloid" appearance emanated from the surface and there were scattered squamous eddies. Other features included peripheral palisading and a stroma containing hyalinized basement membrane-like material. Immunohistochemically, the lesions were diffusely positive with p63, CK5/6, and 34βE12 and focally positive with CK7, but largely negative with CK20, EMA, CEA, and BerEP4. p16 was negative or exhibited nonblock-type immunoreactivity and GATA3 was negative or weakly positive. Molecular testing detected human papillomavirus type 42 in 3 of 7 cases, with no virus detected in the remaining 4 cases. Rarely, similar cases have been reported previously as inverted transitional papilloma of the cervix or vagina, but based on the morphology and immunophenotype we do not feel these represent transitional lesions. We suggest the term seborrheic keratosis-like lesions to designate this new and rare entity, which may be associated with low-risk human papillomavirus infection. Limited follow-up in a small number of cases suggests that these lesions follow a benign clinical course.
BACKGROUND Specific T cell phenotype has been reported to potentially contribute to the development of angiotensin II (Ang II)-induced several vascular disorders. Type 2 diabetes mellitus (T2DM) is intimately associated with cardiovascular disease. The present study aimed to investigate the relationship between T cell phenotypes and Ang II in T2DM patients combined with carotid atherosclerosis (CA). MATERIAL AND METHODS This study was performed on 50 patients with T2DM in our hospital. Based on the presence of CA, they were divided into CA group (presence of CA, n=30) or T2DM group (absence of CA, n=20). Additionally, 10 healthy participants were selected as controls. Basic characteristics of all participants were collected and recorded. Peripheral blood mononuclear cells (PBMCs) isolated from patients and controls with or without Ang II and Ang II receptor blocker (ARB) treatment were used to detect Th1, Th2, and Th17 cell proportions, mRNA levels of T-bet, GATA3, and RORγt as well as the expression of IFN-γ, IL-4, and IL-17 by flow cytometry, ELISA, and Real-Time PCR. RESULTS Ang II levels were notably higher in patients in the CA group than those in the T2DM and control group (p<0.05). Th1 and Th17 positive cells, mRNA levels of T-bet and RORgt as well as the expression of IFN-γ and IL-17 were significantly increased in the CA group compared with the T2DM group and control group (p<0.05). Moreover, the activities of T cells and related cytokines were significantly increased of healthy controls after Ang II treatment (p<0.05), while these changes were notably weakened by ARB treatment (p<0.05). CONCLUSIONS Ang II promotes the development of CA in T2DM patients by regulating T cells activities.
It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers.
We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n=132), Lund (n=308), and The Cancer Genome Atlas (TCGA) (n=408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n=89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer.
Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy.
The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification.
U.S. National Cancer Institute and National Institute of Health.
Although Bacillus Calmette-Guérin (BCG) is the most successful immunotherapy for high-risk non-muscle-invasive bladder cancer, approximately 30% of patients are unresponsive to treatment. New biomarkers are important to identify patients who will benefit most from BCG during a worldwide BCG shortage. Local immune cell subsets were measured on formalin-fixed, paraffin-embedded tissue sections of bladder cancer by immunohistochemistry, using monoclonal antibodies to tumor-associated macrophages (TAMs; CD68, CD163), B-lymphocytes (CD20) and T-lymphocyte subsets (CD3, CD4, CD8, GATA3, T-bet, FOXP3 and CD25). Cell densities in the lamina propria without invasion, at the invasive front if present, in the papillary tumor stroma, and in the neoplastic urothelium were calculated. Twenty-nine (72.5%) of 40 patients were classified as BCG responders after a mean follow-up of 35.3 months. A statistically significant association was observed for BCG failure with low density of CD4+ and GATA3+ T-cells, and increased expression of FOXP3+ and CD25+ regulatory T-cells (Tregs) as well as CD68+ and CD163+ TAMs. Survival analysis demonstrated prolonged recurrence-free survival (RFS) in patients with an increased count of CD4+ and GATA3+ T-cells. TAMs, Tregs and T-bet+ T-cells were inversely correlated with RFS. Thus, the tumor microenvironment seems to influence the therapeutic response to BCG, permitting an individualized treatment.
Programmed cell death ligand 1 (PD-L1) expression on tumor cells can be upregulated via activation of CD8(+) cytotoxic T lymphocytes (CTLs) or the T helper cell (Th1) pathway, counterbalancing the CTL/Th1 microenvironment. However, PD-L1 expression in association with subtypes of tumor-associated lymphocytes and molecular alterations has not been well characterized in lung adenocarcinomas.
PD-L1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and various scoring systems, and was correlated with clinicopathologic/molecular features, including the extent/subtype of tumor-associated lymphocytes (i.e., CD8, T-bet [Th1 transcription factor], and GATA3 [Th2 transcription factor]), and patient outcomes.
PD-L1 expression was present in 129 (49%), 95 (36.5%), and 62 (24%) cases using cutoffs of ≥1%, ≥5%, and ≥50%, respectively, 98 (38%) by H score and 72 (28%) by immune score. PD-L1 expression was associated with abundant CD8(+) and/or T-bet(+) tumor-infiltrating lymphocytes and EGFR wild-type, significant smoking history, and aggressive pathologic features. In addition, concurrent PD-L1 expression and abundant CD8(+) tumor-associated lymphocytes were seen in 25% of KRAS mutants or cases with no alterations by clinical molecular testing as opposed to only 7.4% of EGFR mutants. PD-L1 expression was significantly associated with decreased progression-free and overall survival rates by univariate analysis, but not by multivariate analysis.
PD-L1 expression in resected lung adenocarcinomas is frequently observed in the presence of CTL/Th1 microenvironment, in particular in those with KRAS mutations or no common molecular alterations, suggesting that blockade of the PD-1/PD-L1 axis may be a promising treatment strategy to reinstitute active immune response for at least a subset of such patient populations.
During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34(+) HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3β inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.
In spite of hyporesponsivity to Mycobacterium leprae, borderline lepromatous (BL) patients show clinical and immunological instability, and undergo frequent acute inflammatory episodes such as type 1 reaction (T1R), which may cause nerve damages. This work focused on the participation of T cell subsets from blood and skin at T1R onset. We observed a significantly increased ex vivo frequency of both effector and memory CD4+ and CD8+ T cells in T1R group. Besides, ex vivo frequency of T cell homing receptor, the Cutaneous Leukocyte-associated Antigen (CLA) was significantly increased in T cells from T1R patients. M. leprae induced a higher frequency of CD4+ TEM and CD8+ TEF cells, as well as of CD8+/TEMRA (terminally differentiated effector T cells) subset, which expressed high CD69+. The presence of IFN-γ‒producing-CD4+ TEF and naïve and effector CD8+ T lymphocytes was significant in T1R. TBX21 expression was significantly higher in T1R, while BL showed increased GATA3 and FOXP3 expression. In T1R, TBX21 expression was strongly correlated with CD8+/IFN-γ‒ T cells frequency. The number of double positive CD8+/CLA+ and CD45RA+/CLA+ cells was significantly higher in skin lesions from T1R, in comparison with non-reactional BL group. The observed increase of ex vivo T cells at T1R onset suggests intravascular activation at the beginning of reactional episodes. The antigen-specific response in T1R group confirmed the higher number of CD8+/CLA+ and CD45RA+/CLA+ cells in T1R lesions suggests possible migration of these cells activated by M. leprae components inside the vascular compartment to skin and participation in T1R physiopathology.
We investigated the effect of DWac on the gut microbiota composition in mice with 2,3,6-trinitrobenzenesulfonic acid- (TNBS-) induced colitis. Treatment with DWac restored TNBS-disturbed gut microbiota composition and attenuated TNBS-induced colitis. Moreover, we examined the effect of DWac in mice with mesalazine-resistant colitis (MRC). Intrarectal injection of TNBS in MRC mice caused severe colitis, as well as colon shortening, edema, and increased myeloperoxidase activity. Treatment with mesalazine (30 mg/kg) did not attenuate TNBS-induced colitis in MRC mice, whereas treatment with DWac (30 mg/kg) significantly attenuated TNBS-induced colitis. Moreover, treatment with the mixture of mesalazine (15 mg/kg) and DWac (15 mg/kg) additively attenuated colitis in MRC mice. Treatment with DWac and its mixture with mesalazine inhibited TNBS-induced activation of NF-κB and expression of M1 macrophage markers but increased TNBS-suppressed expression of M2 macrophage markers. Furthermore, these inhibited TNBS-induced T-bet, RORγt, TNF-α, and IL-17 expression but increased TNBS-suppressed Foxp3 and IL-10 expression. However, Th2 cell differentiation and GATA3 and IL-5 expression were not affected. These findings suggest that DWac can ameliorate MRC by increasing the polarization of M2 macrophage and correcting the disturbance of gut microbiota and Th1/Th17/Treg, as well as additively attenuating MRC along with mesalazine.
Immune checkpoint inhibitors blocking the interaction between programmed death-1 (PD-1) and PD-1 ligand-1 (PD-L1) are revolutionizing the cancer immunotherapies with durable clinical responses. Although high expression of PD-L1 in tumor tissues has been implicated to correlate with the better response to the anti-PD-1 therapies, this association has been controversial. In this study, to characterize immune microenvironment in tumors, we examined mRNA levels of immune-related genes and characterized T cell repertoire in the tumors of 13 melanoma patients before and after nivolumab treatment. We found that, in addition to the PD-L1 (p = 0.03), expression levels of PD-1 ligand-2 (PD-L2), granzyme A (GZMA) and human leukocyte antigen-A (HLA-A) in the pre-treatment tumors were significantly higher (p = 0.04, p = 0.01 and p = 0.006, respectively) in responders (n = 5) than in non-responders (n = 8). With nivolumab treatment, tumors in responders exhibited a substantial increase of CD8, GZMA and perforin 1 (PRF1) expression levels as well as increased ratio of TBX21/GATA3, suggesting dominancy of helper T cell type 1 (Th1) response to type 2 (Th2) response. T cell receptor β (TCR-β) repertoire analysis revealed oligoclonal expansion of tumor-infiltrating T lymphocytes (TILs) in the tumor tissues of the responders. Our findings suggest that melanoma harboring high PD-1 ligands (PD-L1 and PD-L2), GZMA and HLA-A expression may respond preferentially to nivolumab treatment, which can enhance Th1-skewed cellular immunity with oligoclonal expansion of TILs.
CD4(+) T cell activation and adequate differentiation into effector T helper (Th) cells are crucial for mediating adaptive immune responses to cope with foreign pathogens. Despite the significant role of Th cells, excessive increases in their numbers result in inflammatory and autoimmune diseases. In this study, we investigated the effects of costunolide, a plant-derived natural compound with an anti-inflammatory activity, in regulating Th cells and the underlying mechanisms. Costunolide significantly decreased cell populations of differentiated Th1, Th2, and Th17 subsets under Th subset-polarizing conditions, while exerting statistically negligible effects on Treg cell differentiation. Furthermore, costunolide inhibited the expression level of Th subset-polarizing master genes such as T-bet, GATA3, and RORγt, indicating that costunolide inhibits the differentiation of CD4(+) T cells into Th subsets. Additionally, costunolide suppressed the proliferative activity of CD4(+) T cells and the expression of CD69 activation marker on CD4(+) T cells. When the molecular targets of costunolide were investigated, phosphorylation of ERK and p38 was found to be decreased under Th subset-polarizing conditions, whereas activity of JNK remained unchanged. U0126, an ERK inhibitor, and SB203580, a p38 inhibitor, decreased the expression of CD69 upon TCR stimulation and inhibited CD4(+) T cell differentiation, indicating that both ERK and p38 are suggested to be critical molecular targets of costunolide. Taken together, these results suggest that costunolide inhibits the differentiation of CD4(+) T cells by suppressing ERK and p38 activities and can be an effective therapeutic agent for T cell-mediated immune diseases.
Pregnancies resulting from fresh in vitro fertilization (IVF) cycles exposed to supraphysiologic estrogen levels have been associated with higher rates of low birth weight and small for gestational age babies. We identified GATA3, a transcription factor selectively expressed in the trophectoderm during the blastocyst stage of embryo development, in an upstream analysis of genes that were differentially methylated in chorionic villus samples between IVF and non-IVF infertility treatment pregnancies. In this study, we investigate the hypothesis that GATA3 is hormonally regulated and plays an important functional role in trophoblast migration, invasion, and placentation. We found that GATA3 expression was hormonally regulated by estradiol in HTR8/SVneo first trimester trophoblast cells; however, no change in expression was seen with progesterone treatment. Furthermore, GATA3 knockdown resulted in decreased HTR8/SVneo cell migration and invasion compared with controls. RNA sequencing of GATA3 knockdown cells demonstrated 96 differentially regulated genes compared with controls. Genes known to play an important role in cell-cell and cell-extracellular matrix interactions, cell invasion, and placentation were identified, including CTGF, CYR61, ADAMTS12, and TIMP3 Our results demonstrate estradiol down-regulates GATA3, and decreased GATA3 expression leads to impaired trophoblast cell migration and invasion, likely through regulation of downstream genes important in placentation. These results are consistent with clinical data suggesting that supraphysiologic estrogen levels seen in IVF pregnancies may play an important role in attenuated trophoblast migration, invasion, and impaired placentation. GATA3 appears to be an important regulator of placentation and may play a role in impaired outcomes associated with fresh IVF cycles.
Accumulating studies have indicated that vitiligo, especially non-segmental vitiligo (NSV), is one kind of autoimmune diseases and CD4(+) T cells play important roles in the pathogenesis. However, there have been very limited data on the detailed changes of each of the CD4(+) T cell subsets in periphery in active NSV. To clarify this issue, we collected the peripheral blood mononuclear cells (PBMCs) from 30 patients with active NSV and 30 age- and sex-matched healthy controls. The percentages of circulating Th1, Th2, Th17 and Tregs were evaluated using flow cytometry and the expressions of their specific transcription factors T-bet, GATA3, RORγt and FOXP3 at mRNA level and protein levels were qualified by qPCR and flow cytometry, respectively. Meanwhile, the expression levels of IFN-γ, IL-4, TGF-β, and IL-17A in serum were measured. We found that in patients with NSV, the percentages and absolute numbers of circulating Th1 and Th17 were both significantly higher than those of healthy controls, while the percentages of Th2 and Tregs and absolute numbers showed no significant difference compared to healthy controls. Moreover, the ratios of Th1/Tregs and Th17/Tregs in circulation were both statistically elevated in active NSV. Similar results were got in qualification of their corresponding transcription factors at mRNA level and protein levels. Compared with healthy controls, the expression level of IL-17A was significantly increased in serum of patients with NSV, while the productions of IFN-γ, IL-4, TGF-β had no significant change. These data suggested that in circulating CD4(+) T cell subsets, Th1 and Th17 played the major role in cellular immunity in the progression of vitiligo. The immune lever in circulation was inclined to effector CD4(+) T cells not suppressor CD4(+) T cells that may result in the loss of self-tolerance to melanocytes.
To investigate the possible role of GATA3 rs3824662 polymorphism as risk factor for the development of acute lymphoblastic leukemia (ALL) in a cohort of Egyptian children and to evaluate its prognostic role. Typing of GATA3 rs3824662 polymorphism was done using real-time PCR for 116 patients with ALL and 273 healthy controls. The A allele and AA genotype were significantly higher in ALL patients (p = .015 and .016, respectively) especially B-ALL (p = .014 and .01, respectively). The AA genotype was associated with shorter disease free survival (DFS) in univariate (p = .017) and multivariate cox regression analysis (p = .028), increased incidence of relapse (p = .008) and poor prognosis (p = .028) in pediatric ALL. The GATA3 rs3824662 A allele and AA genotype may be risk factors for the development of pediatric ALL especially B-ALL in the studied cohort of Egyptian patients. The AA genotype is associated with shorter DSF, increased incidence of relapse and poor prognosis in pediatric ALL.
Early-life wheezing-associated infections with rhinovirus (RV) have been associated with asthma development in children. We have shown that RV infection of six day-old mice induces mucous metaplasia and airways hyperresponsiveness which is dependent on IL-13, IL-25 and type 2 innate lymphoid cells (ILC2s). Infection of immature mice fails to induce lung IFN-γ expression, in contrast to mature 8 week-old mice with a robust IFN-γ response, consistent with the notion that deficient IFN-γ production in immature mice permits RV-induced type 2 immune responses. We therefore examined the effects of IFN-γ administration on RV-induced ILC2 expansion and IL-13 expression in six-day-old BALB/c and IL-13 reporter mice. Selected mice were treated with intranasal IFN-γ. Airway responses were assessed by histology, immunofluorescence microscopy, qPCR, ELISA and flow cytometry. Lung ILC2s were also treated with IFN-γ ex vivo. We found that, compared to untreated RV-infected immature mice, IFN-γ treatment attenuated RV-induced IL-13 and Muc5ac mRNA expression and mucous metaplasia. IFN-γ also reduced ILC2 expansion and the percentage of IL-13-secreting ILC2s. IFN-γ had no effect on the mRNA or protein expression of IL-25, IL-33 or TSLP. Finally, IFN-γ treatment of sorted ILC2s reduced IL-5, IL-13, IL-17RB, ST2 and GATA3 mRNA expression. We conclude that, in immature mice, IFN-γ inhibits ILC2 expansion and IL-13 expression in vivo and ex vivo, thereby attenuating RV-induced mucous metaplasia. These findings demonstrate the antagonistic function of IFN-γ on ILC2 expansion and gene expression, the absence of which may contribute to the development of an asthma-like phenotype after early-life RV infection.
Despite the fact that accessory spleen (also known as supernumerary spleen, splenunculus, or splenule) can be found in 10-30% of patients undergoing autopsies, metastatic disease occurring in this organ has been barely reported. A case of lobular breast carcinoma metastatic to the spleen and accessory spleen found incidentally at therapeutic splenectomy for severe anemia and thrombocytopenia is described. On microscopic examination both organs revealed severe fibrocongestive changes and extramedullary hematopoiesis with no obvious carcinomatous involvement. Cytokeratin 7, estrogen receptors, and GATA3 immunohistochemistry disclosed the presence of numerous metastatic breast carcinoma cells infiltrating the splenic parenchyma. This case demonstrates that metastatic carcinoma can be encountered, although rarely, in accessory spleens and that cytokeratin stain should be performed in sections of spleens and/or accessory spleens excised from cancer patients in which the presence of malignant epithelial cells is not recognized on routine sections.
A recent comprehensive whole genome analysis of a large breast cancer cohort was used to link known and novel drivers and substitution signatures to the transcriptome of 266 cases. Here, we validate that subtype-specific aberrations show concordant expression changes for, for example, TP53, PIK3CA, PTEN, CCND1 and CDH1. We find that CCND3 expression levels do not correlate with amplification, while increased GATA3 expression in mutant GATA3 cancers suggests GATA3 is an oncogene. In luminal cases the total number of substitutions, irrespective of type, associates with cell cycle gene expression and adverse outcome, whereas the number of mutations of signatures 3 and 13 associates with immune-response specific gene expression, increased numbers of tumour-infiltrating lymphocytes and better outcome. Thus, while earlier reports imply that the sheer number of somatic aberrations could trigger an immune-response, our data suggests that substitutions of a particular type are more effective in doing so than others.
GATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL).
We analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3(low) and GATA3(high) expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine.
In our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3(low). DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3(low) compared with GATA3(high) ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3(low) ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3(high) ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation.
We propose GATA3(low) ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies.
Breast cancer is a common malignancy in women and contribute largely to the cancer related death. The purpose of this study is to confirm the roles of GATA3 and identify potential biomarkers of breast cancer. Chromatin Immunoprecipitation combined with high-throughput sequencing (ChIP-Seq) (GSM1642515) and gene expression profiles (GSE24249) were downloaded from the Gene Expression Omnibus (GEO) database. Bowtie2 and MACS2 were used for the mapping and peak calling of the ChIP-Seq data respectively. ChIPseeker, a R bioconductor package was adopted for the annotation of the enriched peaks. For the gene expression profiles, we used affy and limma package to do normalization and differential expression analysis. The genes with fold change >2 and adjusted P-Value <0.05 were screened out. Besides, BETA (Binding and Expression Target Analysis) was used to do the combined analysis of ChIP-Seq and gene expression profiles. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for the functional enrichment analysis of overlapping genes between the target genes and differential expression genes (DEGs). What's more, the protein-protein interaction (PPI) network of the overlapping genes was obtained through the Human Protein Reference Database (HPRD). A total of 46,487 peaks were identified for GATA3 and out of which, 3256 ones were found to located at -3000 ~ 0 bp from the transcription start sites (TSS) of their nearby gene. A total of 236 down- and 343 up-regulated genes were screened out in GATA3 overexpression breast cancer samples compared with those in control. The combined analysis of ChIP-Seq and gene expression dataset showed GATA3 act as a repressor in breast cancer. Besides, 68 overlaps were obtained between the DEGs and genes included in peaks located at -3000 ~ 0 bp from TSS. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to cancer progression and gene regulation were found to be enriched in those overlaps. In the PPI network, NDRG1, JUP and etc. were found to directly interact with large number of genes, which might indicate their important roles in the progression of breast cancer.
In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.
Uroplakin II antibody is exclusively specific for urothelial carcinoma. Nonurothelial carcinoma has not been reported to be immunoreactive for uroplakin II. In the present study, we hypothesized that breast carcinoma showing apocrine differentiation, such as invasive pleomorphic lobular carcinoma (IPLC) and apocrine carcinoma (AC), stains positive for uroplakin II. We identified 6 cases of IPLC between 2000 and 2014 by searching a computerized pathological database. We randomly selected 10 cases of each classic invasive lobular carcinoma (cILC) and AC and five cases of apocrine metaplasia (AM) that coexisted in a surgically resected breast carcinoma specimen. Immunohistochemistry was performed for uroplakin II, GATA3, CK7, CK20, and other representative markers positive for urothelial carcinoma. All cases of IPLC, AC, and AM, except those of cILC, showed immunoreactivity for uroplakin II. Poorly differentiated urothelial carcinoma sometimes shows similar morphology to IPLC with the following immunophenotype: CK7+, CK20-, GATA3+, and uroplakin II+. In the present study, this immunophenotype was observed in all the cases of IPLC and AC. Therefore, when studying metastatic, poorly differentiated carcinoma showing the aforementioned immunophenotype, we should consider the possibility of it being IPLC in addition to metastatic urothelial carcinoma.
GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice.
We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice.
Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice.
Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.
GATA3 has been reported as a specific urothelial marker among organs in the pelvic region, and has been classified as highly sensitive and specific for urothelial and breast carcinomas. Our aim was to verify GATA3 expression in extramammary Paget disease, and to determine whether it can be use to differentiate primary vulvar Paget disease from pagetoid urothelial intraepithelial neoplasia (PUIN). We also analysed HER2 protein expression and HER2 gene amplification and their roles as prognostic factors in extramammary Paget disease.
We analysed GATA3 and HER2 expression in 11 primary vulvar Paget disease cases and two PUIN cases. All cases showed nuclear expression of GATA3. Of 13 cases, five were equivocal for HER2 expression (score 2+) and one was positive (3+). Fluorescence in-situ hybridization results showed amplification in two of these six cases. Both HER2-amplified cases were invasive.
GATA3 was positive in all extramammary Paget disease cases tested (13 cases), and it has no value for differentiating between primary and secondary vulvar Paget disease from the urological tract. HER2 amplification might confer an aggressive and invasive pattern in primary vulvar Paget disease, as both amplified cases showed an invasive pattern.
Estrogen is a neuro-protective hormone in various central nervous system (CNS) disorders. The present study evaluated the role of estrogen during experimental autoimmune encephalomyelitis (EAE) at doses selected to mimic any suppressive potential from the hormone during pregnancy. Here, mice were ovariectomized and then 2 weeks later treated with MOG antigen to induce EAE. Concurrently, mice then received (subcutaneously) an implanted pellet to deliver varying estrogen amounts over a 21-day period. Clinical scores and other parameters were monitored daily for the 21 days. At the end of the period, brain/spinal cord histology was performed to measure lymphocyte infiltration; T-cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed using real-time PCR; T-cell differentiation was evaluated via flow cytometry. The results demonstrated that estrogen inhibited development of EAE. Histological studies revealed limited leukocyte infiltration into the CNS. High and medium dose of estrogen increased TH2 and Treg cell production of interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-β, but concurrently resulted in a significant reduction in production of interferon (IFN)-γ, IL-17, and IL-6. Flow cytometry revealed there were also significant decreases in the percentages of TH1 and TH17 cells, as well as significant increase in percentages of Treg and TH2 cells in the spleen and lymph nodes. Real-time PCR results indicated that high- and medium-dose estrogen treatments reduced T-bet and ROR-γt factor expression, but enhanced Foxp3 and GATA3 expression. Collectively, these results demonstrated that a medium dose of estrogen - similar to a pregnancy level of estrogen - could potentially reduce the incidence and severity of autoimmune EAE and possibly other autoimmune pathologies.
With an objective to understand natural development of bovine neonatal immunity, we analyzed 18 RNA-seq libraries from peripheral blood lymphocytes of three neonatal calves pre- (day 0) and post-colostrum (7, 14 and 28) uptake as compared to their dams. A significant global shift in neonatal transcriptome occurs within first week post-birth, in contrast to dams, with an upregulation of 717 genes. Global pathway analysis of the transcriptome revealed 110 differentially expressed immune-related genes, such as, complement, MHCII, chemokine receptors, defensins and cytokines, at birth. The signaling molecules (LAX1, BLK) and transcription factors (GATA3, FOXP3) are expressed at high levels. High expression of GATA3 transcription factor at birth seems to skew the neonatal immune response towards TH2 type. The high levels of T-cell signaling molecules, CD3G and CD3D, at birth are important in neonatal T cell development. Unlike adults, IGKC expression is high in the neonates where IGKV12 is preferentially expressed at birth. But IGLC is predominant in both neonates and adult where IGLV3.4 is preferentially expressed in B cells at birth. Both IGHM and IGHD are expressed at birth and IGHM achieves adult levels by day 7. This is followed by IGHA and IGHG expression 14-28 days post-birth. Importantly, preferential expression of IGHV1S1(BF4E9) and longest IGHD2(DH2) genes that encode immunoglobulin with exceptionally long CDR3H at birth indicates their critical role, as B cell antigen receptor, in the B cell development via idiotype-anti-idiotype interactions. The transcriptome signatures described here permit assessment bovine neonatal immunocompetence. Bovine neonates acquire innate and IgM-mediated humoral immunocompetence within first week post-birth.
To investigate the relationship between the severity of allergic asthma and the levels of atrial natriuretic peptide (ANP), and to analyze the potential role of ANP signaling in the pathogenesis of asthma.

We recruited 96 subjects, including 23 healthy volunteers, 25 stable allergic asthmatics, 21 mild allergic asthmatics and 27 moderate allergic asthmatics, from the Affiliated Hospital of Guilin Medical University. ANP, IFN-γ and IL-4 levels in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the mRNA and protein expressions of natriuretic peptide receptor A (NPRA), transcription factor T-bet and GATA3 were measured by RT-PCR and Western blot.

The levels of ANP in serum and the expressions of NPRA mRNA and protein in the peripheral blood mononuclear cell (PBMC) from the mild asthma group or the moderate group were elevated compared with those in the stable asthma group or the mild group, respectively (P<0.05). Consistently, expressions of GATA3 and levels of IL-4 showed the same tendency (P<0.05). In addition, levels of ANP in serum were positively correlated with the severity of asthma, whereas negatively correlated with the ratio of T-bet/GATA3 and IFN-γ/IL-4 (r=-0.85, P<0.05; r=-0.88, P<0.05, respectively).

Levels of ANP signaling in serum were significantly increased with the severity of allergic asthma, suggesting a close relation with the pathogenesis of asthma; ANP signaling may play a role in the pathogenesis of allergic asthma through inducing the Th2-type immune response.
目的：观察不同程度过敏性哮喘患者外周血心房钠尿肽(atrial natriuretic peptide，ANP)信号的变化，探讨其与哮喘程度的关系及ANP信号参与哮喘发病的可能机制。方法：共收集桂林医学院附属医院96例患者外周血标本，其中正常组23例、过敏性哮喘稳定组25例、轻度急性发作组21例、中度急性发作组27例。ELISA检测血清ANP及IFN-γ，IL-4的含量，RT-PCR及Western印迹分别检测利钠肽受体A(natriuretic peptide receptor A，NPRA)及T盒21转录因子(T-box 21 transcription factor，T-bet)，GATA结合蛋白3(GATA3 binding protein 3，GATA3)的mRNA与蛋白表达变化。
结果：哮喘轻度发作组外周血ANP水平及单个核细胞(peripheral blood mononuclear cell，PBMC)中NPRA的mRNA及蛋白表达水平均高于稳定组(P<0.05)，中度发作组则高于轻度发作组(P<0.05)；GATA3的表达及IL-4的水平在各组间变化趋势亦一致，即轻度发作组高于稳定组(P<0.05)，中度发作组则高于轻度发作组(P<0.05)。而血清ANP水平随着哮喘严重程度加重而升高，与T-bet/GATA3和IFN-γ/IL-4值则呈负相关(r=–0.85，P<0.05；r=–0.88，P<0.05)。结论：外周血ANP信号水平随着过敏性哮喘严重程度加重而升高，提示与哮喘发病密切相关。ANP信号可能通过介导Th2型免疫应答优势化而参与过敏性哮喘发病。.
Vascular endothelial cell growth factor (VEGF) plays a pivotal role in promoting neovascularization. VEGF gene expression in vascular endothelial cells in normal tissues is maintained at low levels but becomes highly up-regulated in a variety of disease settings including cancers. Tumor necrosis factor superfamily 15 (TNFSF15; VEGI; TL1A) is an anti-angiogenic cytokine prominently produced by endothelial cells in a normal vasculature. We report here that VEGF production in mouse endothelial cell line bEnd.3 can be inhibited by TNFSF15 via microRNA-29b (miR-29b) that targets the 3'-UTR of VEGF transcript. Blocking TNFSF15 activity by using either siRNA against the TNFSF15 receptor known as death domain-containing receptor-3 (DR3; TNFRSF25), or a neutralizing antibody 4-3H against TNFSF15, led to inhibition of miR-29b expression and reinvigoration of VEGF production. In addition, we found that TNFSF15 activated the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. Treatment of the cells either with SP600125, an inhibitor of JNK, or with JNK siRNA, led to eradication of TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. These findings suggest that VEGF gene expression can be suppressed by TNFSF15-stimulated activation of the JNK-GATA3 signaling pathway which gives rise to up-regulation of miR-29b.
Peripheral T cell lymphoma (PTCL) is an aggressive form of non-Hodgkin's lymphoma characterized by a poor prognosis. In this study, we examined the prognostic value of two T-cell-specific transcription factors, GATA3 and T-bet, in PTCL, uncovered the pathogenesis of PTCL, and investigated new PTCL therapeutic targets. Samples from 109 PTCL patients were examined for expression of GATA3, T-bet and CD68. High GATA3 expression correlated with poor survival in PTCL patients and with tumor-associated macrophage (TAM) infiltration, as indicated by the presence of CD68-positive cells. Multivariate analysis further confirmed that high GATA3 expression and Eastern Cooperative Oncology Group (ECOG) scores higher than 2 were independent predictors of patient survival. Using lentiviral transfection to induce stable GATA3 knockdown in a PTCL cell line, we observed that GATA-3 knockdown in Hut78 cells decreased levels of IL4, IL5, IL13 and VEGF mRNA and reduced the number of co-cultured U937 cells that differentiated towards the M2 phenotype. These results suggest that high GATA3 expression is a predictor of a poor prognosis in PTCL, and that T lymphoma cells promote M2-type macrophage differentiation through a GATA3-dependent mechanism.
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
GATA3, a lineage specifier, controls lymphoid cell differentiation and its function in T cell commitment and development has been extensively studied. GATA3 promotes T cell specification by repressing B cell potential in pro T cells and decreased GATA3 expression is essential for early B cell commitment. Inherited genetic variation in GATA3 has been associated with lymphoma susceptibility. However, it remains elusive how the loss of function of GATA3 promotes B cell development and induces B cell lymphomas. In this study, we found that haploid loss of Gata3 by heterozygous germline deletion increased B cell populations in the bone marrow (BM) and spleen, and decreased CD4 T cell populations in the thymus, confirming that Gata3 promotes T and suppresses B cell development. We discovered that haploid loss of Gata3 reduced thymocyte proliferation with induction of p18Ink4c (p18), an inhibitor of CDK4 and CDK6, but enhanced B cell proliferation in the BM and spleen independent of p18. Loss of p18 partially restored Gata3 deficient thymocyte proliferation, but further stimulated Gata3 deficient B cell proliferation in the BM and spleen. Furthermore, we discovered that haploid loss of Gata3 in p18 deficient mice led to the development of B cell lymphomas that were capable of rapidly regenerating tumors when transplanted into immunocompromised mice. These results indicate that Gata3 deficiency promotes B cell differentiation and proliferation, and cooperates with p18 loss to induce B cell lymphomas. This study, for the first time, reveals that Gata3 is a tumor suppressor specifically in B cell lymphomagenesis.
Mesenchymal stem cells (MSCs) have strong immunomodulatory properties, however these properties may show some differences according to the tissue type of their isolate. In this study we investigated the paracrine interactions between human DP derived MSCs (hDP-MSCs) and the CD4(+) T helper cell subsets to establish their immunomodulatory mechanisms. We found that the CD4(+)-Tbet(+) (Th1) and CD4(+)-Gata3(+) (Th2) cells were suppressed by the hDP-MSCs, but the CD4(+)-Stat3(+) (Th17) and CD4(+)-CD25(+)-FoxP3(+) (Treg) cells were stimulated. The expressions of T cell specific cytokines interferon gamma (IFN-g), interleukin (IL)-4 and IL-17a decreased, but IL-10 and transforming growth factor beta-1 (TGF-b1) increased with the hDP-MSCs. The expressions of indoleamine-pyrrole 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), soluble human leukocyte antigen G (sHLA-G) derived from hDP-MSCs slightly increased, but hepatocyte growth factor (HGF) significantly increased in the co-culture groups. According to our findings, the hDP-MSCs can suppress the Th1 and Th2 subsets but stimulate the Th17 and Treg subsets. The Stat3 expression of Th17 cells may have been stimulated by the HGF, and thus the pro-inflammatory Th17 cells may have altered into the immunosuppressive regulatory Th17 cells. Further prospective studies are needed to confirm our findings.
Protection against helminths consists of adaptive responses by TH2 cells and innate responses by group 2 innate lymphoid cells (ILC2s), with these latter being well characterized in mice but less so in human subjects.
We sought to characterize human circulating ILC2s and compare their functional profile with that of autologous TH2 cells.
Circulating ILC2s and TH2 cells were isolated by means of fluorescence-activated cell sorting and magnetic cell sorting and expanded in vitro. ILC2s were then stimulated with phorbol 12-myristate 13-acetate plus ionomycin, IL-25 plus IL-33 (IL-25/IL-33), or a mixture of Toll-like receptor ligands to evaluate their ability to produce cytokines, express CD154, and induce IgE production by autologous B cells. Cytokines and transcription factor gene methylation were assessed.
ILC2s expressed GATA-3, retinoic acid orphan receptor (RORC) 2, and RORα; were able to produce IL-5, IL-13, and IL-4; and, accordingly, were characterized by demethylation of IL4, IL13, IL5, GATA3, and RORC2, whereas the IFNG, IFNG promoter, and TBX21 regions of interest were methylated. ILC2s expressed TLR1, TLR4, and TLR6, and TLR stimulation induced IL-5 and IL-13 production. Moreover, ILC2s expressed CD154 in response to phorbol 12-myristate 13-acetate plus ionomycin, IL-25/IL-33, or a mixture of TLR ligands. Stimulated ILC2s also induced IgM, IgG, IgA, and IgE production by B cells. Finally, circulating ILC2s from atopic patients were not different in numbers and frequency but expressed higher IL-4 levels than those from nonatopic subjects.
This study provides the first evidence that human ILC2s can express CD154 and stimulate the production of IgE by B lymphocytes through IL-25/IL-33 stimulation or TLR triggering.
Recently, formation of tertiary lymphoid structures was demonstrated and further characterized in the R161H mouse model of spontaneous autoimmune uveitis. In the horse model of spontaneous recurrent uveitis, intraocular lymphoid follicle formation is highly characteristic, and found in all stages and scores of disease, but in depth analyses of immunologic features of these structures are lacking to date.
Paraffin-embedded eye sections of cases with equine spontaneous recurrent uveitis (ERU) were characterized with immunohistochemistry to gain insight into the distribution, localization, and signaling of immune cells in intraocular tertiary lymphoid tissues.
Ectopic lymphoid tissues were located preferentially in the iris, ciliary body, and retina at the ora serrata of horses with naturally-occurring ERU. The majority of cells in the tertiary lymphoid follicles were T cells with a scattered distribution of B cells and PNA+ cells interspersed. A fraction of T cells was additionally positive for memory cell marker CD45RO. Almost all cells coexpressed CD166, a molecule associated with activation and transmigration of T cells into inflamed tissues. Several transcription factors that govern immune cell responses were detectable in the tertiary lymphoid follicles, among them Zap70, TFIIB, GATA3, and IRF4. A high expression of the phosphorylated signal transducers and activators of transcription (STAT) proteins 1 and 5 were found at the margin of the structures.
Cellular composition and structural organization of these inflammation-associated tertiary lymphoid tissue structures and the expression of markers of matured T and B cells point to highly organized adaptive immune responses in these follicles in spontaneous recurrent uveitis.
Triple-negative breast cancer is a highly aggressive tumor subtype that lacks effective therapeutic targets. Here, we show that ELK3 is overexpressed in a subset of breast cancers, in particular basal-like and normal-like/claudin-low cell lines. Suppression of ELK3 in MDA-MB-231 cells led to transdifferentiation from an invasive mesenchymal phenotype to a non-invasive epithelial phenotype both in vitro and in vivo. Suppression of ELK3 resulted in extensive changes in genome expression profiles. Among these, GATA3, a master suppressor of metastasis, was epigenetically activated. Also, suppression of GATA3 led to the restoration of migration and invasion. These results suggest that the ELK3-GATA3 axis is a major pathway that promotes metastasis of MDA-MB-231 cells.
Proper cell models for breast cancer primary tumors have long been the focal point in the cancer's research. The genomic comparison between cell lines and tumors can investigate the similarity and dissimilarity and help to select right cell model to mimic tumor tissues to properly evaluate the drug reaction in vitro. In this paper, a comprehensive comparison in copy number variation (CNV), mutation, mRNA expression and protein expression between 68 breast cancer cell lines and 1375 primary breast tumors is conducted and presented.
Using whole genome expression arrays, strong correlations were observed between cells and tumors. PAM50 gene expression differentiated them into four major breast cancer subtypes: Luminal A and B, HER2amp, and Basal-like in both cells and tumors partially. Genomic CNVs patterns were observed between tumors and cells across chromosomes in general. High C > T and C > G trans-version rates were observed in both cells and tumors, while the cells had slightly higher somatic mutation rates than tumors. Clustering analysis on protein expression data can reasonably recover the breast cancer subtypes in cell lines and tumors. Although the drug-targeted proteins ER/PR and interesting mTOR/GSK3/TS2/PDK1/ER_P118 cluster had shown the consistent patterns between cells and tumor, low protein-based correlations were observed between cells and tumors. The expression consistency of mRNA verse protein between cell line and tumors reaches 0.7076. These important drug targets in breast cancer, ESR1, PGR, HER2, EGFR and AR have a high similarity in mRNA and protein variation in both tumors and cell lines. GATA3 and RP56KB1 are two promising drug targets for breast cancer. A total score developed from the four correlations among four molecular profiles suggests that cell lines, BT483, T47D and MDAMB453 have the highest similarity with tumors.
The integrated data from across these multiple platforms demonstrates the existence of the similarity and dissimilarity of molecular features between breast cancer tumors and cell lines. The cell lines only mirror some but not all of the molecular properties of primary tumors. The study results add more evidence in selecting cell line models for breast cancer research.
The avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern T/PT development are not fully understood. Here we study the effects of Notch and Hedgehog (Hh) signalling modulation during common primordium development using in vitro, in vivo and in ovo approaches. The impairment of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated domains in the 3/4PP and further compromised the development of the parathyroid glands. When Hh signalling was abolished, we observed a reduction in the Gata3/Gcm2- and Lfng-expression domains at the median/anterior and median/posterior territories of the pouches, respectively. In contrast, the Foxn1 expression-domain at the dorsal tip of the pouches expanded ventrally into the Lfng-expression domain. This study offers novel evidence on the role of Notch signalling in T/PT common primordium development, in an Hh-dependent manner.
Invariant natural killer T cells (iNKTs) are important innate immune cells which get involved in various immune responses in both mice and humans. These immune reactions range from self-tolerance to development of autoimmunity and responses to pathogens and tumor development. In this study, we aimed to explore the effects of the novel immunostimulators (CH1b and CH2b) containing thiazolidin-4-one on the functions of human invariant natural killer T cells (iNKTs). First of all, iNKTs in peripheral blood mononuclear cells were expanded with α-Galactosylceramide (α-Galcer) in vitro. Then, the highly purified iNKTs were isolated from PBMCs using magnetic cells sorting (MACS). Next, we investigated the impacts of CH1b and CH2b on proliferation, cytokines production, cytotoxicity, and the associated signaling pathways in iNKT cells. Finally, we found that CH2b could significantly promote the activated iNKTs proliferation, increase the production of Th2 cytokines, and induce Th0 differentiation into Th2 subset via GATA 3 signaling pathway. Besides, CH2b could markedly enhance the cytotoxic ability of the activated iNKTs. Therefore, we concluded that CH2b, a promising candidate immunostimulator, might be used for the treatment of infections, tumors, autoimmune and allergic diseases, and for the correction of Th1/Th2 balance disorders in future.
Several natural compounds exhibit strong capacity for decreasing triglyceride accumulation, enhancing lipolysis and inducing apoptosis. The present study reports the anti-adipogenic effects of Silybum marianum (SL), Citrus aurantium (CA), Taraxacum officinale (TO), resveratrol (RE), Curcuma longa (CU), caffeine (CF), oleuropein (OL) and docosahexaenoic acid (DHA) in reducing differentiation and increasing lipolysis and apoptosis. Analyses were performed on human primary visceral pre-adipocytes after 10 (P10) and 20 (P20) days of treatment during differentiation and on mature adipocytes after 7 days of treatment (A7). The percentage of apoptosis induced by TO extract in P10 and P20 cells was significantly higher than that induced by all other compounds and in CTRL cells. Triglyceride accumulation was significantly lower in cells treated with DHA, CF, RE in comparison to cells treated with OL and in CTRL cells. Treatments with CF, DHA and OL significantly incremented lipolysis in P20 cells in comparison to other compounds and in CTRL cells. On the contrary, the treatment of A7 cells with OL, CA and TO compounds significantly increased cell lipolysis. The addition of CF in differentiating P20 pre-adipocytes significantly increased the expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT1, WNT3A, SFRP5, and DLK1. Genes involved in promoting adipogenesis such as CCND1, CEBPB and SREBF1 were significantly down-regulated by the treatment. The screening of bioactive compounds for anti-adipogenic effects showed that in differentiating cells TO extract was the most effective in inducing apoptosis and CF and DHA extracts were more efficient in inhibition of differentiation and in induction of cell lipolysis.
Human papillomavirus (HPV)-negative cervical carcinomas are uncommon and typically encompass unusual histologic subtypes. Mesonephric adenocarcinoma is one such subtype. Mesonephric tumors in the female genital tract are thought to arise from Wolffian remnants, and are extremely rare tumors with widely variable morphology. Sarcomatoid dedifferentiation has been previously described in a few cases, but other forms of dedifferentiation have not been reported. Neuroendocrine carcinoma of the cervix (e.g. small cell carcinoma) is associated with HPV infection, typically HPV 18. These tumors often arise in association with a conventional epithelial component such as squamous cell carcinoma or usual-type endocervical adenocarcinoma. We describe a case of mesonephric adenocarcinoma of the uterine cervix associated with an HPV-negative high-grade neuroendocrine carcinoma at the morphologic and immunophenotypic level, for which we performed targeted massively parallel sequencing analysis of the 2 elements. Both components shared identical mutations in U2AF1 p.R156H (c.467G>A) and GATA3 p.M422fs (c.1263dupG), as well as MYCN amplification. In addition, the neuroendocrine carcinoma harbored TP53 and MST1R mutations not present in the mesonephric carcinoma. Our data suggest a clonal origin of the 2 components of this rare entity, rather than a collision tumor.
Two different forms of clinical paratuberculosis in sheep are recognised, related to the level of bacterial colonization. Paucibacillary lesions are largely composed of lymphocytes with few bacteria, and multibacillary pathology is characterized by heavily-infected macrophages. Analysis of cytokine transcripts has shown that inflammatory Th1/Th17 T cells are associated with development of paucibacillary pathology and Th2 cytokines are correlated with multibacillary disease. The master regulator T cell transcription factors TBX21, GATA3, RORC2 and RORA are critical for the development of these T cell subsets. Sequence variations of the transcription factors have also been implicated in the distinct disease forms of human mycobacterial and gastrointestinal inflammatory diseases. Relative RT-qPCR was used to compare expression levels of each transcript variant of the master regulators in the ileo-caecal lymph nodes of uninfected controls and sheep with defined paucibacillary and multibacillary pathology. Low levels of GATA3 in multibacillary sheep failed to confirm that multibacillary paratuberculosis is caused simply by a Th2 immune response. However, high levels of TBX21, RORC2 and RORC2v1 highlights the role of Th1 and Th17 activation in paucibacillary disease. Increased RORAv1 levels in paucibacillary tissue suggests a role for RORα in Th17 development in sheep; while elevated levels of RORAv4 hints that this variant might inhibit RORα function and depress Th17 development in multibacillary sheep.
Decreased expression of NKG2D ligands on HBV-infected human hepatoma cells impairs NK cells lysis. However, which components of HBV exert this effect and the precise mechanisms need to be further investigated. In the present study, we observed that the HBx and HBc genes significantly down-regulated MICA expression. Through analysis with the chromatin immunoprecipitation assay, we found that HBV infection promotes the expression of transcription factors GATA-2 and GATA-3, which specifically suppressed MICA/B expression by directly binding to the promoter region of MICA/B. HBx protein, acting as a co-regulator, forms a tripolymer with GATA2 and GATA3, thus promotes the GATA-2 or GATA-3-mediated of MICA/B suppression. HBc protein inhibits MICA/B expression via directly binding to the CpG island in the MICA/B promoter. Thus, our study identified the novel role of transcription factors GATA-2 and GATA-3 in suppressing MICA/B expression and clarified the mechanisms of HBx and HBc in downregulation of MICA/B expression. These findings provide novel mechanisms for the contribution of HBV to hepatoma cells escape from NK cell surveillance.
Differential prognostic roles of Androgen Receptor (AR) have been proposed in breast cancer (BC) depending on tumour oestrogen receptor (ER) status. This study aimed to evaluate the prognostic and/or predictive significance of AR expression in invasive BC. In this study AR expression was studied on a large (n = 1141) consecutive series of early-stage (I-III) BC using tissue microarray and immunohistochemistry (IHC). AR mRNA expression was assessed in a subset of cases. The prognostic impact of AR mRNA expression was externally validated using the online BC gene expression data sets (n = 25 data sets, 4078 patients). Nuclear AR IHC expression was significantly associated with features of good prognosis including older age, smaller tumour size, lower grade and lobular histology particularly in the ER-positive tumours. AR was associated with ER-related markers GATA3, FOXa1, RERG and BEX1. Negative association was observed with HER2, p53, Ki67, TK1, CD71 and AGTR1. AR Overexpression was associated with longer survival (p < 0.001), independent of tumour size, grade, stage [p = 0.033, hazard ratio (HR) = 0.80 95 % CI = 0.64-0.98]. Similar associations were maintained in ER+ tumours in univariate and multivariate analysis (p < 0.01) both in patients with and without adjuvant endocrine or chemotherapy. AR mRNA expression showed significant association with tumour grade, molecular subtypes, and longer 10 and 15 years survival in luminal BC. In the external validation cohorts, AR gene expression data were associated with improved patients' outcome (p < 0.001, HR = 0.84, 95 % CI 0.79-0.90). AR is not only an independent prognostic factor in ER-positive luminal BC but is also expressed in ER-negative tumours. AR could act as a molecular target in patients with ER-positive disease predicting response to adjuvant therapy.
Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.
We hypothesised that epigenetic regulation of CD4(+) T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4(+) T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-β signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4(+) T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4(+) T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4(+) T cell response during mycobacterial infection in cattle.
Much research effort has been focused on investigating new compounds derived from low-cost sources, such as natural products, for treating leishmaniasis. Oleuropein derived from numerous plants, particularly from the olive tree, Olea europaea L. (Oleaceae), is a biophenol with many biological activities. Our previous findings showed that oleuropein exhibits leishmanicidal effects against three Leishmania spp. in vitro, and minimizes the parasite burden in L. donovani-infected BALB/c mice. The aim of the present study is to investigate the possible mechanism(s) that mediate this leishmanicidal activity.
We determined the efficacy of oleuropein in elevating ROS and NO production in L. donovani-infected J774A.1 macrophages and in explanted splenocytes and hepatocytes obtained from L. donovani-infected BALB/c mice. We also assessed the expression of genes that are related to inflammation, T-cell polarization and antioxidant defense, in splenocytes. Finally, we determined the ratios of specific IgG2a/IgG1 antibodies and DTH reactions in L. donovani-infected BALB/c mice treated with oleuropein.
Oleuropein was able to elevate ROS production in both in vitro and in vivo models of visceral leishmaniasis and raised NO production in ex vivo cultures of splenocytes and hepatocytes. The extensive oxidative stress found in oleuropein-treated mice was obviated by the upregulation of the host's antioxidant enzyme (mGCLC) and the simultaneous downregulation of the corresponding enzyme of the parasite (LdGCLC). Moreover, oleuropein was able to mount a significant Th1 polarization characterized by the expression of immune genes (IL-12β, IL-10, TGF-β1, IFN-γ) and transcription factors (Tbx21 and GATA3). Moreover, this immunomodulatory effect was also correlated with an inhibitory effect on IL-1β gene expression, rather than with the expression of IL-1α, IL-1rn and TNF-α. Furthermore, oleuropein-treated BALB/c mice mounted a delayed-type hypersensitivity (DTH) response and an elevated Leishmania-specific IgG2a/IgG1 ratio that clearly demonstrated an in vivo protective mechanism.
The ability of Oleuropein to promote a Th1 type immune response in L. donovani-infected BALB/c mice points towards the candidacy of this bioactive compound as an immunomodulatory agent that may complement therapeutic approaches to leishmaniasis.
Hematopoietic stem cells (HSCs) serve as a life-long reservoir for all blood cell types and are clinically useful for a variety of HSC transplantation-based therapies. Understanding the role of chromatin organization and regulation in HSC homeostasis may provide important insights into HSC development. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator that possesses 4 nucleosome-binding domains and activates 3 lysine acetyltransferases (KAT6A, KAT6B, and KAT7), suggesting that this protein has the potential to stimulate crosstalk between different chromatin modifications. Here, we investigated the function of BRPF1 in hematopoiesis by selectively deleting its gene in murine blood cells. Brpf1-deficient pups experienced early lethality due to acute bone marrow failure and aplastic anemia. The mutant bone marrow and fetal liver exhibited severe deficiency in HSCs and hematopoietic progenitors, along with elevated reactive oxygen species, senescence, and apoptosis. BRPF1 deficiency also reduced the expression of multipotency genes, including Slamf1, Mecom, Hoxa9, Hlf, Gfi1, Egr, and Gata3. Furthermore, BRPF1 was required for acetylation of histone H3 at lysine 23, a highly abundant but not well-characterized epigenetic mark. These results identify an essential role of the multivalent chromatin regulator BRPF1 in definitive hematopoiesis and illuminate a potentially new avenue for studying epigenetic networks that govern HSC ontogeny.
Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.
The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.
The pathologic distinction between high-grade prostate adenocarcinoma (PAC) involving the urinary bladder and high-grade urothelial carcinoma (UC) infiltrating the prostate can be difficult. However, making this distinction is clinically important because of the different treatment modalities for these two entities.
A total of 249 patient cases (PAC, 111 cases; UC, 138 cases) collected between June 1995 and July 2009 at Seoul St. Mary's Hospital were studied. An immunohistochemical evaluation of prostatic markers (prostate-specific antigen [PSA], prostate-specific membrane antigen [PSMA], prostate acid phosphatase [PAP], P501s, NKX3.1, and α-methylacyl coenzyme A racemase [AMACR]) and urothelial markers (CK34βE12, p63, thrombomodulin, S100P, and GATA binding protein 3 [GATA3]) was performed using tissue microarrays from each tumor.
The sensitivities of prostatic markers in PAC were 100% for PSA, 83.8% for PSMA, 91.9% for PAP, 93.7% for P501s, 88.3% for NKX 3.1, and 66.7% for AMACR. However, the urothelial markers CK34βE12, p63, thrombomodulin, S100P, and GATA3 were also positive in 1.8%, 0%, 0%, 3.6%, and 0% of PAC, respectively. The sensitivities of urothelial markers in UC were 75.4% for CK34βE12, 73.9% for p63, 45.7% for thrombomodulin, 22.5% for S100P, and 84.8% for GATA3. Conversely, the prostatic markers PSA, PSMA, PAP, P501s, NKX3.1, and AMACR were also positive in 9.4%, 0.7%, 18.8%, 0.7%, 0%, and 8.7% of UCs, respectively.
Prostatic and urothelial markers, including PSA, NKX3.1, p63, thrombomodulin, and GATA3 are very useful for differentiating PAC from UC. The optimal combination of prostatic and urothelial markers could improve the ability to differentiate PAC from UC pathologically.
In 2015, progress in understanding asthma ranged from insights to asthma inception, exacerbations, and severity to advancements that will improve disease management throughout the lifespan. 2015's insights to asthma inception included how the intestinal microbiome affects asthma expression with the identification of specific gastrointestinal bacterial taxa in early infancy associated with less asthma risk, possibly by promoting regulatory immune development at a critical early age. The relevance of epigenetic mechanisms in regulating asthma-related gene expression was strengthened. Predicting and preventing exacerbations throughout life might help to reduce progressive lung function decrease and disease severity in adulthood. Although allergy has long been linked to asthma exacerbations, a mechanism through which IgE impairs rhinovirus immunity and underlies asthma exacerbations was demonstrated and improved by anti-IgE therapy (omalizumab). Other key molecular pathways underlying asthma exacerbations, such as cadherin-related family member 3 (CDHR3) and orosomucoid like 3 (ORMDL3), were elucidated. New anti-IL-5 therapeutics, mepolizumab and reslizumab, were US Food and Drug Administration approved for the treatment of patients with severe eosinophilic asthma. In a clinical trial the novel therapeutic inhaled GATA3 mRNA-specific DNAzyme attenuated early- and late-phase allergic responses to inhaled allergen. These current findings are significant steps toward addressing unmet needs in asthma prevention, severity modification, disparities, and lifespan outcomes.
Several genes have been recognized to be associated with non-surgical hypoparathyroidism. Data about gene mutations in adult-onset hypoparathyroidism patients is lacking. This study was designed to screen gene mutation in adult-onset hypoparathyroidism in Chinese through the targeted next-generation sequencing (NGS).
We recruited 17 patients with adult-onset hypoparathyroidism who were regularly followed or newly diagnosed at our centre during the past one year. Nine of them developed hypercalciuria during the treatment with calcium and vitamin D. Eight of them were newly diagnosed with no treatment. Targeted NGS was performed to screen 11 related genes, including AIRE, AP2S1, CASR, CLDN16, FAM111A, GATA3, GCM2, PTH, TBCE, TBX1 and TRPM6.
A novel homozygosis mutation of GCMB gene[c.130G>A (p.G44S)]was identified which was predicted to be deleterious by PolyPhen2. The patient was a 36-year-old woman who suffered from paroxysmal carpopedal spasms for ten years. Before treatment, the serum calcium and phosphorus was 1.48 mmol/L and 2.29 mmol/L, respectively.Parathyroid hormonel (PTH) concentration was lower than 3.0 ng/L. Intracranial calcification and cataract were also identified. She developed hypercalciuria during treatment with calcium and vitamin D. She had no physical deformity or family history of hypoparathyroidism.
In this study, the genetic defect was only identified in 1 patient (5.9%). In adult-onset hypoparathyroidism without other diagnostic clues, the gene mutation screening as the first choice to clarify the etiology was not recommended.
Follicular helper T cells (Tfh) support high-affinity Ab production by germinal center B cells through both membrane interactions and secretion of IL-4 and -21, two major cytokines implicated in B-cell survival and Ab class switch. Tfh-2 cells recently emerged in humans as a strong IL-4 producer Tfh cell subset implicated in both autoimmune and allergic diseases. Although the molecular mechanisms governing Tfh cell differentiation from naive T cells have been widely described, much less is known about the regulation of cytokine secretion by mouse Tfh-2 cells. The purpose of our study was to evaluate the role of dendritic cell-derived IL-6 in fine-tuning cytokine secretion by Tfh cells. Our results demonstrate that priming of Th cells by IL-6-deficient antigen-presenting dendritic cells preferentially leads to accumulation of a subset of Tfh cells characterized by high expression of GATA3 and IL-4, associated with reduced production of IL-21. STAT3-deficient Tfh cells also overexpress GATA3, suggesting that early IL-6/STAT3 signaling during Tfh cell development inhibits the expression of a set of genes associated with the Th2 differentiation program. Overall, our data indicate that IL-6/STAT3 signaling restrains the expression of Th2-like genes in Tfh cells, thus contributing to the control of IgE secretion in vivo.
Estrogen receptor (ER)/GATA3/Forkhead box A1 (FOXA1) network is necessary for the ERα functional signature. High FOXA1 expression indicates a good prognosis in ER-positive breast cancer. However, little is known about the significance of FOXA1 and GATA3 expression in neoadjuvant endocrine therapy (NAE). The aim of this study is to investigate their predictive potential for NAE and their expression changes after NAE.
FOXA1 and GATA3 expression was evaluated using immunohistochemistry in 66 patients with ER-positive/HER2-negative breast cancer who had been treated with NAE. The association between biological marker expressions and the efficacy of NAE and their expression changes after NAE were analyzed.
The median pre-treatment FOXA1 and GATA3 expressions were 94.6 and 90 %. Pre-treatment FOXA1 expression was positively correlated with GATA3 (P = 0.0003) and progesterone receptor (PgR) (P = 0.0138). There was no correlation between pre- or post-treatment FOXA1 and GATA3 expressions and the efficacy of NAE. Post-treatment Ki67 expression was significantly lower in tumors with partial response (PR) (P = 0.0007). In terms of the changes of the expression, PgR, Ki67, and FOXA1 expression significantly decreased after NAE (P < 0.0001, P < 0.0001, and P < 0.0001, respectively).
FOXA1 and GATA3 expression was not correlated with the efficacy of NAE, but FOXA1 expression was significantly reduced after NAE.
The integration of multiple profiling data and the construction of a transcriptional regulatory network may provide additional insights into the molecular mechanisms of hepatocellular carcinoma (HCC). The present study was conducted to investigate the deregulation of genes and the transcriptional regulatory network in HCC.
An integrated analysis of HCC gene expression datasets was performed in Gene Expression Omnibus. Functional annotation of the differentially expression genes (DEGs) was conducted. Furthermore, transcription factors (TFs) were identified, and a global transcriptional regulatory network was constructed.
An integrated analysis of eight eligible gene expression profiles of HCC led to 1,835 DEGs. Consistent with the fact that the cell cycle is closely related to various tumors, the functional annotation revealed that genes involved in the cell cycle were significantly enriched. A transcriptional regulatory network was constructed using the 62 TFs, which consisted of 872 TF-target interactions between 56 TFs and 672 DEGs in the context of HCC. The top 10 TFs covering the most downstream DEGs were ZNF354C, NFATC2, ARID3A, BRCA1, ZNF263, FOXD1, GATA3, FOXO3, FOXL1, and NR4A2. This network will appeal to future investigators focusing on the development of HCC.
The transcriptional regulatory network can provide additional information that is valuable in understanding the underlying molecular mechanism in hepatic tumorigenesis.
The BCG vaccine induces a Th1 phenotype, which is essential for protection against Mycobacterium tuberculosis. However, the effects of BCG vaccination over time on the T helper subpopulation and the microRNAs involved in adulthood have not been studied. In the present study, we explored the involvement of microRNAs, transcription factors and multifunctional cytokines in BCG vaccination by examining their levels both before and after vaccination of healthy adults. Peripheral blood mononuclear cells were obtained at 0, 2 and 6 months after vaccination. Cells were cultured in the presence or absence of ESAT-6 and CFP-10 or M. tuberculosis filtrate. The expression levels of miRNAs and transcription factors were evaluated using qRT-PCR. Cytokine production in supernatants and serum samples was evaluated using ELISA. Multifunctional CD4+ T cells were analyzed using multiparametric flow cytometry. We observed a decrease in the expression levels of T-BET, GATA3 and FOXP3 at 2 months and miR-146a, miR-326 and miR-155 at 6 months after receiving the vaccine. In the supernatant, the production of IL-17 was increased after 6 months, with both stimuli. In contrast, IL-10, TNF-α and IFN-γ increased at 2 months. In the serum, high levels of IL-10 were found after 2 months compared to time 0 and 6 months. The production of multifunctional cells that expressed the cytokine profiles CD4+TNF-α+IFN-γ-IL-10-, CD4+TNF-α+IL-1IFN-γ-, CD4+IL-10+IFN-γ-TNF-α- and CD4+IL-17+IFN-γ- predominantly increased after 2 months with and without the stimulus. Correlation analysis revealed a negative association between FOXP3 and miR-155 (r=-0.5120, p=0.0176) and between IL-17 and miR-326 (r=-0.5832, p=0.0364). This study is the first to demonstrate roles for microRNAs, transcription factors and cytokines in the T helper differentiation lineage and to describe the possible mechanism by which their expression is modulated by the presence of the BCG vaccine in adulthood. In conclusion, our results suggest that the BCG vaccine induces a modulation in transcription factors and miRNAs with high production of multifunctional cells CD4+TNF-α+IL-10+IFN-γ-.
T lymphocyte development and differentiation is a multi-step process that begins in the thymus and completed in the periphery. Sequential development of thymocytes is dependent on T cell receptor (TCR) signaling and an array of transcription factors. In this study we show that special AT-rich binding protein 1 (SATB1), a T lineage-enriched chromatin organizer and regulator, is induced in response to TCR signaling during early thymocyte development. SATB1 expression profile coincides with T lineage commitment and upregulation of SATB1 correlates with positive selection of thymocytes. CD4 thymocytes exhibit a characteristic bimodal expression pattern that corresponds to immature and mature CD4 thymocytes. We also demonstrate that GATA3, the key transcriptional regulator of αβ T cells positively regulates SATB1 expression in thymocytes suggesting an important role for SATB1 during T cell development.
The aim of this study was to detect the therapeutic effect of dioscin on collagen-induced arthritis (CIA). Mice model of CIA was induced by chicken collagen II and arthritis index was assessed. After suspension of dioscin (100mg/kg/d) or triptolide was intragastrically administered, the left paw swelling and body weight of each mouse were measured. Then tissue samples were assayed by histopathological analysis. The levels of Th1 and Th2 were detected by flow cytometry. The expression of p-STAT1, p-STAT4 and p-STAT6 was demonstrated by western blot analysis, and T-bet and GATA-3 expression was detected by RT-PCR. The paw swelling and arthritis index were decreased and body weight was increased in the high dose of dioscin group compared to the model group (P<0.05). Histopathological analysis revealed that the damage of synovium tissue in dioscin and triptolide group alleviated. The ratio of Th1/Th2 in the dioscin group (0.82±0.24) and triptolide group (0.99±0.44) was lower than that in the model group (1.84±0.70, P<0.05). Additionally, p-STAT4 expression was decreased, and both p-STAT6 and GATA3 expression was increased in the dioscin group than that in the model group (P<0.05). Dioscin might have some therapeutic effects on CIA through regulating the proportion of Th1/Th2 cells, which could reduce the expression of p-STAT4, increase the expression of p-STAT6 and GATA3 in the synovial tissue.
Cancer of the male breast is an uncommon event with metastases to the breast occurring even less frequently. Prostate carcinoma has been reported as the most frequent primary to metastasize to the breast; however, the reverse has not been previously reported. Herein, we present, for the first time, a case of breast carcinoma metastasizing to the prostate gland. Prostate needle core biopsy revealed infiltrative nests of neoplastic epithelioid cells, demonstrated by immunohistochemistry (IHC) to be positive for GATA3 and ER and negative for PSA and P501S. A prostate cocktail by IHC study demonstrated lack of basal cells (p63 and CK903) and no expression of P501S. The patient's previous breast needle core biopsy showed strong ER positivity and negative staining for PR and HER2. Similar to the prostate, the breast was negative for CK5/6, p63, and p40. This case demonstrates the importance of considering a broad differential diagnosis and comparing histology and IHC to prior known malignancies in the setting of atypical presentation or rare tumors.
The induced expression of the transcription factors neurogenin1 (Neurog1) or neuronal differentiation 1 (NeuroD1) has previously been shown to initiate neuronal differentiation in embryonic stem cells (ESC). Human bone marrow-derived mesenchymal stem cells (hBMSCs) are ethically non-controversial stem cells. However, they are not pluripotent. In cochlear implantation, regeneration or replacement of lost spiral ganglion neurons may be a measure for the improvement of implant function. Thus, the aim of the study was to investigate whether the expression of Neurog1 or NeuroD1 is sufficient for induction of neuronal differentiation in hBMSCs.
Human BMSCs were transduced with lentivirus expressing NeuroD1 or Neuorg1. Transduced cells were then treated with small molecules that enhanced neuronal differentiation. Markers of neuronal differentiation were evaluated.
Using quantitative reverse transcription PCR, the up-regulation of transcription factors expressed by developing primary auditory neurons, such as BRN3a (POU4F1) and GATA3, was quantified after induction of Neurog-1 expression. In addition, the expression of the receptor NTRK2 was induced by treatment with its specific ligand BDNF. The induction of expression of the vesicular glutamate transporter 1 was identified on gene and protein level. NeuroD1 seemed not sufficient to induce and maintain neuronal differentiation.
Induction of neuronal differentiation by overexpression of Neurog1 initiated important steps for the development of glutamatergic neurons such as the spiral ganglion neurons. However, it seems not sufficient to maintain the glutamatergic spiral ganglion neuron-like phenotype.
Clear cell urothelial carcinoma (CCUC) is a rare variant of urothelial carcinoma (UC) and its clinical significance has not been well elucidated. Consecutive cases of UC over a period of 5 years were reviewed. Histopathological tumor parameters, including the proportion of tumor cells with clear cell change, and patient outcomes were recorded. Expression of the following immunohistochemical markers was investigated: CK7, CK20, CK5, CD44, and PAX8. We also conducted a review of the literature for case reports/series of CCUC. Ten CCUCs were identified out of a total of 872 cases of UC. The clear cell component was characterized by prominent cytoplasmic membranes and voluminous clear cytoplasm, and accounted for 30% to 90% of the invasive tumor component. Of all the non-CCUC cases reviewed, at least 50% (noninvasive or invasive UC) showed focal areas of clear cell change that accounted for less than 5% of the neoplastic cells. Immunohistochemically, CCUC exhibited positive reactivity for CK5/CD44 (n = 9); CK20 (n = 5), PAX8 (very focal to extensive) (n = 6), and GATA3/CK7 (n = 10). Eight of 10 CCUC were of advanced clinical stage (pT3/pT4) and 6 of 10 experienced tumor recurrence and/or death due to disease. In conclusion, CCUC can be distinguished from non-CCUC by the extensive clear cell change in more than 30% of cells. This variant is associated with rapid progression to muscle invasion and metastasis, with an aggressive clinical course. Expression of CK5/CD44 may represent basal cell features in most CCUC cases, while PAX8 expression is suggestive of mesonephric derivation.
The balance between Th17 cells and regulatory T (Treg) cells has been shown to play an important role in the development of rheumatoid arthritis (RA). Recent studies have shown that treatment with abatacept (ABT) or tocilizumab (TCZ) affects Th17 and Treg cell populations. Although not unanimously accepted, several reports have shown that Treg cells are decreased by ABT and increased by TCZ, and that Th17 cells are decreased by TCZ. To further investigate the effects of ABT and TCZ on the skewing of T cell populations, we analyzed the expression of master regulators genes of helper T cell lineages following ABT/TCZ treatment of RA patients.
Ten patients treated with ABT and 10 patients treated with TCZ were enrolled. Total RNA was extracted from peripheral blood cells at baseline, and after 12 and 24 weeks of therapy. The expression levels of T-bet, GATA3, Foxp3 and Ror-γt were semi-quantified using real-time PCR. The relative expression levels were expressed as the ratios of two genes (T-bet/GATA3, Foxp3/GATA3, Foxp3/T-bet, Foxp3/Ror-γt, Ror-γt/T-bet, Ror-γt/GATA3), and the changes in these ratios with treatment were determined.
The Foxp3/Ror-γt ratio was decreased after ABT therapy (0.67 ± 0.16 at 24 weeks, P = 0.0034) but was increased after TCZ therapy (2.00 ± 1.03 at 24 weeks, P = 0.0013). In addition, the Ror-γt/GATA3 ratio was decreased after TCZ therapy (0.78 ± 0.37 at 24 weeks, P = 0.0008). Except for these ratios, no significant skewing in the expression of these factors was detected. No significant relationship between clinical response to the treatment and change in the ratios of these factors was determined.
Treatment with TCZ or ABT differently affected the balance between Foxp3 and Ror-γt expression in the peripheral blood of patients with RA.
Data integration has become a useful strategy for uncovering new insights into complex biological networks. We studied whether this approach can help to delineate the signal transducer and activator of transcription 6 (STAT6)-mediated transcriptional network driving T helper (Th) 2 cell fate decisions. To this end, we performed an integrative analysis of publicly available RNA-seq data of Stat6-knockout mouse studies together with STAT6 ChIP-seq data and our own gene expression time series data during Th2 cell differentiation. We focused on transcription factors (TFs), cytokines, and cytokine receptors and delineated 59 positively and 41 negatively STAT6-regulated genes, which were used to construct a transcriptional network around STAT6. The network illustrates that important and well-known TFs for Th2 cell differentiation are positively regulated by STAT6 and act either as activators for Th2 cells (e.g., Gata3, Atf3, Satb1, Nfil3, Maf, and Pparg) or as suppressors for other Th cell subpopulations such as Th1 (e.g., Ar), Th17 (e.g., Etv6), or iTreg (e.g., Stat3 and Hif1a) cells. Moreover, our approach reveals 11 TFs (e.g., Atf5, Creb3l2, and Asb2) with unknown functions in Th cell differentiation. This fact together with the observed enrichment of asthma risk genes among those regulated by STAT6 underlines the potential value of the data integration strategy used here. Thus, our results clearly support the opinion that data integration is a useful tool to delineate complex physiological processes.
Background. Construction of the transcriptional regulatory network can provide additional clues on the regulatory mechanisms and therapeutic applications in gastric cancer. Methods. Gene expression profiles of gastric cancer were downloaded from GEO database for integrated analysis. All of DEGs were analyzed by GO enrichment and KEGG pathway enrichment. Transcription factors were further identified and then a global transcriptional regulatory network was constructed. Results. By integrated analysis of the six eligible datasets (340 cases and 43 controls), a bunch of 2327 DEGs were identified, including 2100 upregulated and 227 downregulated DEGs. Functional enrichment analysis of DEGs showed that digestion was a significantly enriched GO term for biological process. Moreover, there were two important enriched KEGG pathways: cell cycle and homologous recombination. Furthermore, a total of 70 differentially expressed TFs were identified and the transcriptional regulatory network was constructed, which consisted of 566 TF-target interactions. The top ten TFs regulating most downstream target genes were BRCA1, ARID3A, EHF, SOX10, ZNF263, FOXL1, FEV, GATA3, FOXC1, and FOXD1. Most of them were involved in the carcinogenesis of gastric cancer. Conclusion. The transcriptional regulatory network can help researchers to further clarify the underlying regulatory mechanisms of gastric cancer tumorigenesis.
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and other inflammatory diseases in infected individuals. However, a complete understanding of how HTLV-1 transforms T cells is lacking. Expression of the chemokine receptor CCR4 on ATL cells and HTLV-1-infected cells suggested the hypothesis that CCR4 may mediate features of ATL and inflammatory diseases caused by HTLV-1. In this study, we show that the constitutively expressed HTLV-1 bZIP factor (HBZ) encoded by HTLV-1 is responsible for inducing CCR4 and its ability to promote T-cell proliferation and migration. Ectopic expression of HBZ was sufficient to stimulate expression of CCR4 in human and mouse T cells. Conversely, HBZ silencing in ATL cell lines was sufficient to inhibit CCR4 expression. Mechanistic investigations showed that HBZ induced GATA3 expression in CD4(+) T cells, thereby activating transcription from the CCR4 promoter. In an established air pouch model of ATL, we observed that CD4(+) T cells of HBZ transgenic mice (HBZ-Tg mice) migrated preferentially to the pouch, as compared with those in nontransgenic mice. Migration of CD4(+) T cells in HBZ-Tg mice was inhibited by treatment with a CCR4 antagonist. Proliferating (Ki67(+)) CD4(+) T cells were found to express high levels of CCR4 and CD103. Further, CD4(+) T-cell proliferation in HBZ-Tg mice was enhanced by coordinate treatment with the CCR4 ligands CCL17 and 22 and with the CD103 ligand E-cadherin. Consistent with this finding, we found that ATL cells in clinical skin lesions were frequently positive for CCR4, CD103, and Ki67. Taken together, our results show how HBZ activates CCR4 expression on T cells to augment their migration and proliferation, two phenomena linked to HTLV-1 pathogenesis. Cancer Res; 76(17); 5068-79. ©2016 AACR.
The advent of Omics technologies has been key to the molecular subclassification of urothelial bladder cancer. Several groups have used different strategies to this aim, with partially overlapping findings. The meeting at the Spanish National Cancer Research Center-CNIO was held to discuss such classifications and reach consensus where appropriate. After updated presentations on the work performed by the teams attending the meeting, a consensus was reached regarding the existence of a group of Basal-Squamous-like tumors - designated BASQ - charaterized the high expression of KRT5/6 and KRT14 and low/undetectable expression of FOXA1 and GATA3. An additional tumor subgroup with urothelial differentiation features was recognized whose optimal molecular definition is required. For other subtypes described, more work is needed to determine how robust they are and how to best define them at the molecular level.
GATA transcription factors are essential in mammalian cell lineage determination and have a critical role in cancer development. In cultured prostate cancer cells, GATA2 coordinates with androgen receptor (AR) to regulate gene transcription. In the murine prostate, among six GATA members, GATA2 and GATA3 are expressed. Immunofluorescence staining revealed that both GATA factors predominantly localize in the nuclei of luminal epithelial cells. The pioneer factor FoxA1 is exclusively detected in the luminal cells, whereas AR is detected in both luminal and basal cells. Using genetic engineering, we generated prostate-specific GATA2 and GATA3 knockout (KO) mice. Ablation of single GATA gene had marginal effect on prostate morphology and AR target gene expression, likely due to their genetic compensation. Double KO mice exhibited PIN III to IV lesions, but decreased prostate to body weight ratio, altered AR target gene expression, and expansion of p63-positive basal cells. However, deletion of GATA2 and GATA3 did not reduce the mRNA or protein levels of AR or FoxA1, indicating that GATA factors are not required for AR or FoxA1 expression in adult prostate. Surprisingly, GATA2 and GATA3 exhibit minimal expression in the ventral prostatic (VP) lobe. In contrast, FoxA1 and AR expression levels in VP are at least as high as those in anterior prostatic (AP) and dorsal-lateral prostatic (DLP) lobes. Together, our results indicate that GATA2 and GATA3 are essential for adult murine prostate function and in vivo AR signaling, and the lack of the GATA factor expression in the VP suggests a fundamental difference between VP and other prostatic lobes.
An important step towards personalizing cancer treatment is to integrate heterogeneous evidences to catalog mutational hotspots that are biologically and therapeutically relevant and thus represent where targeted therapy would likely be beneficial. However, existing methods do not sufficiently delineate varying functionality of individual mutations within the same genes.
We observed a large discordancy of mutation rates across different mutation subtypes and tumor types, and nominated 702 hotspot mutations in 549 genes in the Catalog of Somatic Mutations in Cancer (COSMIC) by considering context specific mutation characteristics such as genes, cancer types, mutation rates, mutation subtypes and sequence contexts. We observed that hotspot mutations were highly prevalent in Non CpG-island C/G transition and transversion sequence contexts in 10 tumor types, and specific insertion hotspot mutations were enriched in breast cancer and deletion hotspot mutations in colorectal cancer. We found that the hotspot mutations nominated by our approach were significantly more conserved than non-hotspot mutations in the corresponding cancer genes. We also examined the biological significance and pharmacogenomics properties of these hotspot mutations using data in the Cancer Genome Atlas (TCGA) and the Cancer Cell-Line Encyclopedia (CCLE), and found that 53 hotspot mutations are independently associated with diverse functional evidences in 1) mRNA and protein expression, 2) pathway activity, or 3) drug sensitivity and 82 were highly enriched in specific tumor types. We highlighted the distinct functional indications of hotspot mutations under different contexts and nominated novel hotspot mutations such as MAP3K4 A1199 deletion, NR1H2 Q175 insertion, and GATA3 P409 insertion as potential biomarkers or drug targets.
We identified a set of hotspot mutations across 17 tumor types by considering the background mutation rate variations among genes, tumor subtypes, mutation subtypes, and sequence contexts. We illustrated the common and distinct mutational signatures of hotspot mutations among different tumor types and investigated their variable functional relevance under different contexts, which could potentially serve as a resource for explicitly selecting targets for diagnosis, drug development, and patient management.
Macrophages are highly plastic cells with the ability to differentiate into both M1- and M2-polarized phenotypes. As a distinct M2-polarized population, tumour-associated macrophages (TAMs) promote tumorigenesis owing to their pro-angiogenic and immune-suppressive functions in tumour microenvironment. In the present study, we found that the microRNA-720 (miR-720) was down-regulated in TAMs isolated from breast carcinomas and M2-polarization macrophages. Overexpression of miR-720 attenuated M2 phenotype expression and thus inhibited M2 polarization. We further identified GATA binding protein 3 (GATA3), a transcriptional factor that plays an important role in M2 macrophage polarization, was the downstream target of miR-720 Ectopic expression of GATA3 restored the M2 phenotype in miR-720 overexpressed macrophages. Importantly, overexpression of miR-720 inhibited pro-migration behaviour and phagocytic ability of M2-polarized macrophages. Thus, our data suggest that miR-720 plays an important role in regulating M2 macrophage polarization and function.
Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for ~80% of all kidney cancer cases. However, the pathogenesis of RCC has not yet been fully elucidated. To interpret the pathogenesis of RCC at the molecular level, gene expression data and bio-informatics methods were used to identify RCC associated genes. Gene expression data was downloaded from Gene Expression Omnibus (GEO) database and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in RCC patients compared with controls. In addition, a regulatory network was constructed using the known regulatory data between transcription factors (TFs) and target genes in the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) and the regulatory impact factor of each TF was calculated. A total of 258,0427 pairs of DCGs were identified. The regulatory network contained 1,525 pairs of regulatory associations between 126 TFs and 1,259 target genes and these genes were mainly enriched in cancer pathways, ErbB and MAPK. In the regulatory network, the 10 most strongly associated TFs were FOXC1, GATA3, ESR1, FOXL1, PATZ1, MYB, STAT5A, EGR2, EGR3 and PELP1. GATA3, ERG and MYB serve important roles in RCC while FOXC1, ESR1, FOXL1, PATZ1, STAT5A and PELP1 may be potential genes associated with RCC. In conclusion, the present study constructed a regulatory network and screened out several TFs that may be used as molecular biomarkers of RCC. However, future studies are needed to confirm the findings of the present study.
Cutaneous T-cell lymphomas (CTCL) are skin malignancies including mycosis fungoides (MF) and CD30(+) lymphoproliferative disorders (LPD). In early disease, CTCL can be difficult to diagnose, especially in MF for which there is no reliable diagnostic marker. MF/CTCL have increased expression of thymocyte selection-associated HMG box protein (TOX). Although TOX has been proposed to be a diagnostic marker for MF, further validation studies are needed. Moreover, it is unclear what drives TOX expression or its role in MF/CTCL.
We hypothesize evaluation of TOX levels across a spectrum of CTCL, including MF precursor (large plaque parapsoriasis, LPP), will help elucidate the implications of altered TOX expression.
TOX staining was performed in MF, CD30(+) LPD, LPP as well as benign inflammatory dermatoses (BID) and normal skin (NS). CTCL cell lines were utilized to evaluate the regulation of TOX.
Positive TOX expression was identified in 73.6% of MF cases and in 31.6% of BID/NS. TOX had a positive predictive value (PPV) for MF of 86.7% and a negative predictive value (NPV) of 48.1%. TOX expression in MF was detected more commonly in Black patients (P = 0.015) and less commonly in transformed MF (P = 0.045). LPP had positive TOX staining in 70.0%. In CTCL cells, GATA3 knockdown decreased TOX mRNA and protein expression. TOX expression also decreased in the presence of CTCL therapeutics.
Our data indicate that TOX is useful as a diagnostic marker in MF. Moreover, TOX expression was evident in LPP, indicating it may have a previously unappreciated role in the development of MF. Finally, our data suggest that GATA3 regulates TOX, revealing insight into TOX regulation.
A subset of obese individuals remains insulin sensitive by mechanisms as yet unclear. The hypothesis that maintenance of normal subcutaneous (SC) adipogenesis accounts, at least partially, for this protective phenotype and whether it can be abrogated by chronic exposure to IL-6 was investigated.
Adipose tissue biopsies were collected from insulin-sensitive (IS) and insulin-resistant (IR) individuals undergoing weight-reduction surgery. Adipocyte size, pre-adipocyte proportion of stromal vascular fraction (SVF)-derived cells, adipogenic capacity and gene expression profiles of isolated pre-adipocytes were determined, along with local in vitro IL-6 secretion. Adipogenic capacity was further assessed in response to exogenous IL-6 application.
Despite being equally obese, IR individuals had significantly lower plasma leptin and adiponectin levels and higher IL-6 levels compared with age-matched IS counterparts. Elevated systemic IL-6 in IR individuals was associated with hyperplasia of adipose tissue-derived SVF cells, despite higher frequency of hypertrophied adipocytes. SC pre-adipocytes from these tissues exhibited lower adipogenic capacity accompanied by downregulation of PPARγ (also known as PPARG) and CEBPα (also known as CEBPA) and upregulation of GATA3 expression. Impaired adipogenesis in IR individuals was further associated with increased adipose secretion of IL-6. Treatment of IS-derived SC pre-adipocytes with IL-6 reduced their adipogenic capacity to levels of the IR group.
Obesity-associated insulin resistance is marked by impaired SC adipogenesis, mediated, at least in a subset of individuals, by elevated local levels of IL-6. Understanding the molecular mechanisms underlying reduced adipogenic capacity in IR individuals could help target appropriate therapeutic strategies aimed at those at greatest risk of insulin resistance and type 2 diabetes mellitus.
Loss of the SWI/SNF chromatin remodeling complex has been recently implicated in the pathogenesis of dedifferentiated carcinomas from different organs, but its possible role in undifferentiated urothelial carcinoma (UC) has not been studied to date. In this study, we analyzed by immunohistochemistry 14 undifferentiated UCs (11 from bladder and 3 from renal pelvis) with a nondescript anaplastic or rhabdoid morphology, using commercially available antibodies against the SWI/SNF components SMARCB1 (INI1), SMARCA2, SMARCA4, SMARCC1, SMARCC2, and ARID1A. Patients were eight females and six males aged 40 to 84 years (median, 65). All tumors were muscle-invasive (9 were T3-4). A conventional UC component was seen in eight cases and varied from in situ to papillary. The undifferentiated component comprised 60-100 % of the tumors. Histologically, most tumors showed diffuse dyscohesive or pseudoalveolar growth of variably sized cells with frequent rhabdoid features. Transition from conventional to undifferentiated UC was abrupt, except in one case. The undifferentiated component almost always expressed pan-cytokeratin AE1/AE3 (13/14) and variably vimentin (8/14) and GATA3 (9/14). Complete loss of at least one SWI/SNF subunit limited to the undifferentiated component was detected in 10/14 cases (71 %). SMARCA2 was most frequently lost (six) followed by ARID1A (four), SMARCB1/INI1 (two), SMARCA4 (one), and SMARCC1 (one). This is the first study exploring SWI/SNF expression in undifferentiated UC of the urinary tract. Our results are in line with recent studies reporting involvement of the SWI/SNF complex in the dedifferentiation process of a variety of epithelial neoplasms in different organs, including the urinary tract, and association with aggressive clinical course.
GATA-3 is a transcription factor involved in human tissue growth and differentiation. It is a potential marker for breast carcinoma origin in metastasis and predictive of good prognosis. We aim to evaluate the role of GATA3 in determining the breast origin of metastatic adenocarcinoma in malignant effusions using immunohistochemistry on cell-block microarray in comparison with ER and PR results.
Cell blocks from 100 cases of malignant and reactive serous effusions with confirmed diagnosis were selected; 28 mammary carcinomas, 64 extra-mammary adenocarcinomas (gastrointestinal, pulmonary, gynecologic), and 8 reactive mesothelium proliferation as control. Immunohistochemistry on cell-block microarray was used. Microarray slides were stained for GATA-3, ER and PR. Nuclear staining of >1% was considered positive.
GATA3, ER and PR were positive in 25 (89%), 20 (71%) and 16 (57%) of breast carcinoma cases, respectively. All non-breast cancer cases were negative for GATA-3 with the exception of one Müllerian adenocarcinoma (1.6%). The calculated sensitivity, specificity and positive predictive value (PPV) of GATA3 reactivity in determining the breast origin of metastatic adenocarcinoma was 89.3% (95% CI: 71.7-97.7%), 98.6% (95% CI: 91.6-99.9%) and 96.2% (95% CI: 80.4-99.9%), respectively. GATA3 positivity was associated with ER or PR positivity in 84% of cases.
GATA3 is a useful marker in determining the breast origin of metastatic adenocarcinoma in malignant serous effusions. Reactivity to GATA3 may indicate good prognosis. Diagn. Cytopathol. 2016;44:731-736. © 2016 Wiley Periodicals, Inc.
Hearing impairment most often involves loss of sensory hair cells and auditory neurons. As this loss is permanent in humans, a cell therapy approach has been suggested to replace damaged cells. It is thus of interest to generate lineage restricted progenitor cells appropriate for cell based therapies. Human long-term self-renewing neuroepithelial stem (lt-NES) cell lines exhibit in vitro a developmental potency to differentiate into CNS neural lineages, and importantly lack this potency in vivo, i.e do not form teratomas. Small-molecules-driven differentiation is today an established route obtain specific cell derivatives from stem cells. In this study, we have investigated the effects of three small molecules SB431542, ISX9 and Metformin to direct differentiation of lt-NES cells into sensory neurons. Exposure of lt-NES cells to Metformin or SB431542 did not induce any marked induction of markers for sensory neurons. However, a four days exposure to the ISX9 small molecule resulted in reduced expression of NeuroD1 mRNA as well as enhanced mRNA levels of GATA3, a marker and important player in auditory neuron specification and development. Subsequent culture in the presence of the neurotrophic factors BDNF and NT3 for another seven days yielded a further increase of mRNA expression for GATA3. This regimen resulted in a frequency of up to 25-30% of cells staining positive for Brn3a/Tuj1. We conclude that an approach with ISX9 small molecule induction of lt-NES cells into auditory like neurons may thus be an attractive route for obtaining safe cell replacement therapy of sensorineural hearing loss.
Lung cancer is a leading cause of cancer-related mortality worldwide, and cigarette smoking is the major environmental hazard for its development. This study intended to examine whether smoking could alter methylation of genes at lung cancer risk loci identified by genome-wide association studies (GWASs). By systematic literature review, we selected 75 genomic candidate regions based on 120 single-nucleotide polymorphisms (SNPs). DNA methylation levels of 2854 corresponding cytosine-phosphate-guanine (CpG) candidates in whole blood samples were measured by the Illumina Infinium Human Methylation450 Beadchip array in two independent subsamples of the ESTHER study. After correction for multiple testing, we successfully confirmed associations with smoking for one previously identified CpG site within the KLF6 gene and identified 12 novel sites located in 7 genes: STK32A, TERT, MSH5, ACTA2, GATA3, VTI1A and CHRNA5 (FDR <0.05). Current smoking was linked to a 0.74% to 2.4% decrease of DNA methylation compared to never smoking in 11 loci, and all but one showed significant associations (FDR <0.05) with life-time cumulative smoking (pack-years). In conclusion, our study demonstrates the impact of tobacco smoking on DNA methylation of lung cancer related genes, which may indicate that lung cancer susceptibility genes might be regulated by methylation changes in response to smoking. Nevertheless, this mechanism warrants further exploration in future epigenetic and biomarker studies.
The aim of the present study was to investigate the overall clinical expression characteristics of the cluster of differentiation (CD)28 family receptors [CD28, inducible T-cell co-stimulator, programmed cell death protein 1 (PD‑1), cytotoxic T-lymphocyte-associated protein 4 and B‑ and T-lymphocyte attenuator] on T cells in patients with chronic hepatitis B (CHB), analyze the correlations among these receptors and the clinical parameters, and to investigate the effects of PD‑1 blockade on the receptor expression profiles, T‑cell function and other biological effects. The expression characteristics of the CD28 family of receptors, the effects of PD‑1 blockade on the receptor expression profiles and the levels of interferon (IFN)‑γ were investigated in the T cells of patients with CHB. In addition, the transcription factor, T‑box 21 (T‑bet) and GATA binding protein 3 (GATA‑3) mRNA expression levels were investigated in the peripheral blood mononuclear cells (PBMCs) of patients with CHB. The expression levels of the CD28 family receptors in the T cells of patients with CHB demonstrated distinct characteristics , for example levels of PD‑1 and CTLA‑4 on CD4 T cells and ICOS, PD‑1, and BTLA on CD8 T cells were increased in cells from patients with CHB compared with those from the healthy individuals. A significant positive correlation was demonstrated among the serum HBV DNA titers and the levels of PD‑1 on CD8+ T cells with the highest expression of PD‑1 corresponding to viral levels >106 IU/ml. A significant positive correlation was observed between the serum HBV DNA titers and the expression levels of BTLA on CD8+ T cells with the highest expression of BTLA corresponding to viral levels >106 IU/ml. PD‑1 blockade altered the expression profiles of CD28 family receptors in the T cells of patients with CHB, partly enhanced T cell function and increased the ratio of T‑bet/GATA‑3 mRNA in PBMCs. Thus, CD28 family receptors are potential clinical indicators for the rapid monitoring of changes in T cell function during CHB treatment. Furthermore, PD‑1 blockade has a therapeutic potential that may be enhanced by modulating the expression of co-stimulatory and -inhibitory receptors of the CD28 family.
Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO-; CD8CD45RO+ to CD8CD45RO-, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.
Donepezil (DNPZ) is a drug commonly used for Alzheimer's disease (AD) that may favour a T helper 2 phenotype leading to increased naturally occurring auto-antibodies (NAb) against beta-amyloid (Aβ). We hypothesized the involvement of the cholinergic receptors [α7-nicotnic acetylcholine receptor (α7nAChR)] expressed on peripheral blood mononuclear cells (PBMC).
Fifty patients with mild-to-moderate AD, DNPZ treated (DNPZ+, n = 25) or not (DNPZ-, n = 25), and 25 matched controls were enrolled and PBMC extracted for both in vitro cultures, and real-time polymerase chain reaction and chromatin immunoprecipitation assay. Plasma samples were also obtained for Aβ and NAb determination.
Donepezil increased in vitro the expression of the transcription factor GATA binding protein 3 (GATA3) through α7nAChR, because prevented by the specific antagonist methyllycaconitine. Ex vivo PBMC α7nAChR mRNA expression was increased in both AD groups, while GATA3 expression was not. A significant increase in the GATA3/interleukin 5 promoter association was found in DNPZ+ patients. Finally, DNPZ+ patients showed both significantly higher plasma levels of anti-Aβ NAb with respect to DNPZ- patients and Aβ 1-42 with respect to normal controls.
Donepezil might modulate a T helper 2 bias via α7nAChR leading to increased expression of NAb. Further studies on the role of the modulation of the immune response against Aβ may pave the way to innovative therapeutic strategies for AD. Copyright © 2016 John Wiley & Sons, Ltd.
In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800μg+oil group were significantly higher than in the other adjuvant groups (p<0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p<0.05) on day 37. The ECMS800μg+oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p<0.05). After Bb challenge, the ECMS800μg+oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p<0.05 and p<0.01). IgA cells in the ECMS800μg+oil group differed significantly from the IgA cells of other groups in the lungs (p<0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400μg+oil were higher than the number of cells for other groups except the ECMS(100μg/800μg)+oil groups (p<0.05). Intermediate cells in the ECMS(100μg/400μg)+oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p<0.05). Neutrophils in the ECMS(100μg/400μg/800μg)+oil groups were significantly higher than the ECMS 800μg and control using antigen alone groups (p<0.05). White blood cells in the ECMS100μg+oil group were significantly higher than the oil, ECMS800μg and control using antigen alone groups (p<0.05). IL-2 expression in the ECMS800μg+oil group was significantly higher than other groups, except for the ECMS400μg+oil group (p<0.05). IL-4 expression in the ECMS800μg+oil group was significantly higher than other groups (p<0.05). GATA3 in the ECMS800μg+oil groups was significantly higher than the oil, ECMS800μg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.
Hydroxytyrosol has various pharmacological properties, including anti-oxidative stress and anti-inflammatory activities, preventing hyperglycemia, insulin resistance, and the metabolic syndrome. The present study is focused on the anti-adipogenic and lipolytic activity of hydroxytyrosol on primary human visceral adipocytes. Pre-adipocytes were analyzed after 10 (P10) and 20 (P20) days of treatment during differentiation and after 7 (A7) days of treatment when they reached mature shape. The treatment with hydroxytyrosol extract significantly (P < 0.001) increased apoptosis in P10 and P20 cells in comparison to control and A7 cells; significantly (P < 0.001) reduced triglyceride accumulation in P20 cells compared to P10 and control cells; and significantly (P < 0.001) increased lipolysis in P20 cells in comparison to control cells and A7 mature adipocytes. Hydroxytyrosol-treated P20 cells significantly (P < 0.05) increased expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT3A, SFRP5, HES1, and SIRT1. In contrast, genes involved in promoting adipogenesis such as LEP, FGF1, CCND1, and SREBF1 were significantly down-regulated by hydroxytyrosol treatment. These data suggest that hydroxytyrosol promotes lipolysis and apoptotic activity in primary human visceral pre-adipocytes during differentiation and does not affect already mature adipocytes.
Histologic grade is one of the most important microscopic features used to predict the prognosis of invasive breast cancer and may serve as a marker for studying cancer driving genomic abnormalities in vivo. We analyzed whole genome sequencing data from 680 cases of TCGA invasive ductal carcinomas of the breast and correlated them to corresponding pathology information. Ten genetic abnormalities were found to be statistically associated with histologic grade, including three most prevalent cancer driver events, TP53 and PIK3CA mutations and MYC amplification. A distinct genetic interaction among these genomic abnormalities was revealed as measured by the histologic grading score. While TP53 mutation and MYC amplification were synergistic in promoting tumor progression, PIK3CA mutation was found to have alleviated the oncogenic effect of either the TP53 mutation or MYC amplification, and was associated with a significant reduction in mitotic activity in TP53 mutated and/or MYC amplified breast cancer. Furthermore, we discovered that different types of genetic abnormalities (mutation versus amplification) within the same cancer driver gene (PIK3CA or GATA3) were associated with opposite histologic changes in invasive breast cancer. In conclusion, our study suggests that histologic grade may serve as a biomarker to define cancer driving genetic events in vivo.
WDR77 (WD repeat domain 77) is expressed during earlier lung development when cells are rapidly proliferating, but is absent from adult lung. It is re-activated during lung tumorigenesis and is essential for lung cancer cell proliferation. Signalling pathways/molecules that control WDR77 gene expression are unknown. Promoter mapping, gel shift assay and ChIP revealed that the WDR77 promoter contains bona fide response elements for E2F and GATA transcriptional factors as demonstrated in prostate cancer, lung cancer and erythroid cells, as well as in mouse lung tissues. The WDR77 promoter is transactivated by E2F1, E2F3, GATA1 and GATA6, but suppressed by E2F6, GATA1 and GATA3 in prostate cancer PC3 cells. WDR77 expression is associated with E2F1, E2F3, GATA2 and GATA6 occupancy on the WDR77 gene, whereas, in contrast, E2F6, GATA1 and GATA3 occupancy is associated with the loss of WDR77 expression during erythroid maturation and lung development. More importantly, the loss of WDR77 expression that results from E2F and GATA switches is required for cellular differentiation of erythroid and lung epithelial cells. In contrast, lung cancer cells avoid post-mitotic differentiation by sustaining WDR77 expression. Altogether, the present study provides a novel molecular mechanism by which WDR77 is regulated during erythroid and lung development and lung tumorigenesis.
Th2-eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2-eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis, Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically, this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore, we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together, these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis.
Although increasing evidence demonstrated that deregulation of mircoRNA-503 (miRNA-503) contributes to tumorigenesis, little is known about the biological role and intrinsic regulatory mechanisms of miR-503 in prostate cancer (PCa). In present study, we found that miR-503 was significantly downregulated in advanced PCa tissues and cell lines. Downregulation of miR-503 was strongly associated with aggressive clinical-pathological features and poor prognosis in PCa patients. Ectopic expression of miR-503 significantly inhibited tumor cells growth, cell migration and invasion in vitro and in vivo. Mechanistic studies revealed that ZNF217 was a direct target downstream target of miR-503. Knockdown of ZNF217 mimicked the tumor-suppressive effects of miR-503 overexpression on PCa invasion, whereas ZNF217 overexpression attenuated the tumor-suppressive function of miR-503. Subsequently, miR-503 further modulated the activation of ZNF217-downstream epithelial-mesenchymal transition (EMT) genes. Besides, we also found that GATA3 directly increased miR-503 expression and thus decreased ZNF217 expression, indicating the involvement of GATA3/miR-503/ZNF217 signaling in EMT process. Collectively, our results demonstrated that GATA3-driven expression of miR-503 inhibits PCa progression by repressing ZNF217 expression, and also implicated the potential application of miR-503 in PCa therapy.
Dendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin(-)Sca-1(+)Kit(+) cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte-related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation.
Recent studies have investigated the significance of GATA3 expression in patients with various malignant tumors. However, no previous studies have evaluated the clinicopathological importance of GATA3 expression in soft tissue sarcomas (STS) patients.
We evaluated GATA3 expression in 76 STS cases using immunohistochemical analysis, and statistically compared clinicopathological characteristics between GATA3-positive and GATA3-negative cases.
GATA3-positive expression was significantly associated with a higher mitotic count (P < 0.0001). Disease-free survival (DFS) of GATA3-positive cases was significantly shorter than that of cases without GATA3 expression (P = 0.0104). Overall survival (OS) of GATA3-positive cases was significantly shorter than that of cases without GATA3 expression (P = 0.0006). GATA3-positive expression was significantly associated with shorter DFS in both univariate analysis (hazard ratio [HR], 2.719; P = 0.012) and multivariate analysis (HR, 2.711; P = 0.014). GATA3-positive expression was also significantly associated with worse OS in both univariate analysis (HR, 5.730; P = 0.0007) and multivariate analysis (HR, 5.789; P = 0.0008).
These results indicate that GATA3 is an independent prognostic factor and suggest that evaluation of GATA3 expression might enable more effective clinical follow-up using prognostic stratification of STS patients.
Distinction between 2 forms of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), can be challenging. Aberrant mucosal immunity suggests that CD is a T helper type 1 cell (Th1)-driven disease, whereas UC as Th2-driven response. However, whether this paradigm truly distinguishes CD from UC is controversial. We aimed to clarify the discriminating potential of lamina propria Th subsets in patients with IBD.
Biopsies from 79 patients with IBD and 20 healthy controls were collected for Th subsets analysis (Th1:interferon γ [IFN-γ], T-bet; Th2:interleukin 13 [IL-13], Gata3; Th17:IL-17, RORγt; Treg:FoxP3). The receiver-operating characteristic curves were constructed to assess the discriminating ability by calculating the area under the receiver-operating characteristic curve. The equation with the highest area under the receiver-operating characteristic curve was applied to newly diagnosed patients to evaluate discriminating ability.
Patients with CD showed increased IFN-γ or T-bet cells and decreased IL-13 or Gata3 cells compared with UC. A discriminant equation composed of 4 markers (IFN-γ, T-bet, IL-13, and Gata3) yielded the highest area under the receiver-operating characteristic curve. In 36 established CD or UC, the sensitivity, specificity, positive and negative predictive probabilities were 92.6%, 55.6%, 86.2%, and 71.4% and in 14 newly diagnosed patients were 100.0%, 42.9%, 63.6%, and 100.0%. Furthermore, Gata3 cells were increased in tumor necrosis factor inhibitor therapy nonresponders compared with responders in CD. IFN-γ cells were directly and inversely proportional to disease activity in patients with CD and UC, respectively.
The Th1/Th2 paradigm can distinguish CD from UC and may be further associated with response to tumor necrosis factor inhibitor in CD and disease activity in patients with IBD.
GATA binding protein 3 (GATA3) is a transcription factor, which belongs to a distinct family of tumor suppressor genes. It is involved in human cancer cell growth and differentiation, and plays an important role in cell proliferation and apoptosis. Although, its expression has been reported in various cancers, there are limited data in genitourinary malignancies. Recent studies found GATA3 to be a sensitive marker for urothelial carcinoma (UC) and associated with prognostic pathologic features. Its level of expression was found to be an independent factor predicting cancer recurrence.
In this article, immunohistochemical evaluation of GATA3 expression in genitourinary malignancies (invasive UC, renal cell carcinoma, and prostatic adenocarcinomas) was performed.
GATA3 was positive in 56/79 (70.8%) of invasive UC, and was negative in all renal cell carcinoma and prostatic adenocarcinomas. The pattern of GATA3 staining, when positive, was intensely nuclear within the clusters of malignant cells. No cytoplasmic staining was noted. Negative controls were all negative. High GATA3 expression was associated with larger tumor size in invasive UC (3.19cm vs. 1.65cm, P = 0.01). GATA3 expression did not correlate with other clinicopathologic parameters in UC.
This data suggest that GATA3 is a sensitive marker in confirming invasive UC, and may be helpful in differentiating it from metastatic tumors of renal and prostatic origin. Furthermore, strong GATA3 expression was noted to have an effect on tumor size in patients with UC.
Oxyphilic (oncocytic) parathyroid lesions are very uncommon and their cytological features are rarely described. Due to the similarities in anatomical location and indistinguishable cytomorphological features, these lesions are easily confused with neoplastic and non-neoplastic thyroid lesions on fine needle aspiration (FNA). The diagnosis becomes more challenging in cases of unusual intrathyroidal location of the parathyroid lesions in the absence of clinical evidence of hyperparathyroidism, which simulate thyroid nodules clinically. We describe a case of intrathyroidal oxyphilic parathyroid carcinoma in a 66-year-old female, who presented with a dominant left "thyroid" nodule. FNA smears were cellular, comprising predominantly of oxyphilic cells arranged in papillary-like architecture with occasional nuclear grooves, which was mistaken for oncocytic variant of papillary carcinoma of the thyroid. The histological diagnosis of oxyphilic parathyroid "adenoma" was made following total thyroidectomy. The tumor, unfortunately, recurred 7 years later with associated multiple lung metastases. When dealing with thyroid lesions comprising predominantly of oncocytic cells, one should consider oxyphilic parathyroid neoplasms as one of the differential diagnosis. In difficult equivocal cases, a panel of immunocytochemical stains (PTH, GATA3, TTF-1, PAX8, and thyroglobulin) can be helpful. In addition, a combination of valuable clinical, radiological, and laboratory data, including serum calcium and parathyroid hormone levels are key to arriving at an accurate cytological diagnosis. Diagn. Cytopathol. 2016;44:688-692. © 2016 Wiley Periodicals, Inc.
To report a clear cell renal cell carcinoma recipient of a metastasizing ductal carcinoma of the breast: A tumor-to-tumor metastasis.
A 71 year-old woman with a past history of breast carcinoma, diagnosed 12 years before, underwent a nephrectomy for an incidental kidney mass found in a routine imaging examination.
Histological examination revealed foci of ductal carcinoma of the breast in an otherwise typical clear cell renal cell carcinoma of the kidney. Immunohistochemical examination confirmed a metastasis of an infiltrating breast carcinoma to a clear cell renal cell carcinoma (positive to GATA3, hormonal receptors and mamoglobin) in a clear cell renal cell carcinoma (positive to PAX8, CD10 and vimentin).
Awareness of this phenomenon should always be kept in mind by urologist in patients with a known history of a previous malignancy and by pathologists when finding a renal tumor with an unusual or dimorphic morphology. Immunohistochemistry plays an important role to establish the exact diagnosis.
In contrast to other Bet v 1-related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process we sought to characterize the T cell response to Dau c 1 and its cross-reactivity with Bet v 1.
Dau c 1-specific T cell lines (TCL) and clones (TCC) established from PBMC of birch pollen-allergic patients with carrot allergy were analyzed for reactivity to Bet v 1, epitope-specificity, allergen-induced cytokine secretion and expression of integrins α4β7 and α4β1, critical for gut and lung homing, respectively. mRNA expression of GATA3 and Tbet was analyzed in sorted CD3(+) CD4(+) CFSE(low) cells proliferating upon stimulation of PBMC with Dau c 1 or Bet v 1. Dau c 1 was incubated with endolysosomal proteases and the resulting fragments were identified by mass spectrometry.
Among 14 distinct T cell-activating regions, Dau c 1139-153 was recognized by 55% of the patients. Only 6/15 (40%) Dau c 1-specific TCL and 9/21 (43%) TCC reacted with Bet v 1. Bet v 1-non-reactive TCC were mainly Th1-like and showed a higher expression of the integrin β7 and a significantly lower expression of the integrin β1 than Bet v 1-positive TCC. A Th1-like response was also detected in Dau c 1-reactive CD3(+) CD4(+) CFSE(low) cells. Full-length Dau c 1 was still detectable after 48 hours of endolysosomal degradation. Proteolytic fragments of Dau c 1 matched its T cell-activating regions.
Dau c 1 displays several characteristics of sensitizing allergens, namely a major T cell-activating region, low susceptibility to endolysosomal degradation and induction of a Bet v 1-independent T cell response. These cellular insights confirm that the major carrot allergen has a special status among Bet v 1-related food allergens. This article is protected by copyright. All rights reserved.
Autoimmune Addison's disease (AAD) is a complex disease that results from the interaction of a predisposing genetic background with still unknown environmental factors. Pathogenic variants in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrine syndrome type 1, of which AAD is a major disease component. Among the genetic factors for isolated AAD and autoimmune polyendocrine syndrome type 2, a key role is played by HLA class II genes: HLA-DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*04-DQA1*0301-DQB1*0302 are positively, and DRB1*0403 is negatively, associated with genetic risk for AAD. The MHC class I chain-related gene A (MICA) allele 5.1 is strongly and positively associated with AAD. Other gene polymorphisms contribute to the genetic risk for AAD, including CIITA (MHC class II transactivator), the master regulator of MHC class II expression, cytotoxic T-lymphocyte antigen-4 (CTLA-4), PTPN22, STAT4, PD-L1, NALP1, FCRL3, GPR174, GATA3, NFATC1, CYP27B1 and the vitamin D receptor.
Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation.
Less than 10% of registered drug intervention trials for pancreatic ductal adenocarcinoma (PDAC) include a biomarker stratification strategy. The ability to identify distinct mutation subsets via endoscopic ultrasound fine needle aspiration (EUS FNA) molecular cytology could greatly aid clinical trial patient stratification and offer predictive markers. We identified chemotherapy treatment naïve ampullary adenocarcinoma and PDAC patients who underwent EUS FNA to assess multigene mutational frequency and diversity with a surgical resection concordance assessment, where available.
Following strict cytology smear screening criteria, targeted next generation sequencing (NGS) using a 160 cancer gene panel was performed.
Complete sequencing was achieved in 29 patients, whereby 83 pathogenic alterations were identified in 21 genes. Cytology genotyping revealed that the majority of mutations were identified in KRAS (93%), TP53 (72%), SMAD4 (31%), and GNAS (10%). There was 100% concordance for the following pathogenic alterations: KRAS, TP53, SMAD4, KMT2D, NOTCH2, MSH2, RB1, SMARCA4, PPP2R1A, PIK3R1, SCL7A8, ATM, and FANCD2. Absolute multigene mutational concordance was 83%. Incremental cytology smear mutations in GRIN2A, GATA3 and KDM6A were identified despite re-examination of raw sequence reads in the corresponding resection specimens.
EUS FNA cytology genotyping using a 160 cancer gene NGS panel revealed a broad spectrum of pathogenic alterations. The fidelity of cytology genotyping to that of paired surgical resection specimens suggests that EUS FNA represents a suitable surrogate and may complement the conventional stratification criteria in decision making for therapies and may guide future biomarker driven therapeutic development.
Recurrences of diffuse large B-cell lymphomas (DLBCL) result in significant morbidity and mortality, but their underlying genetic and biological mechanisms are unclear. Clonal relationship in DLBCL relapses so far is mostly addressed by the investigation of immunoglobulin (IG) rearrangements, therefore, lacking deeper insights into genome-wide lymphoma evolution. We studied mutations and copy number aberrations in 20 paired relapsing and 20 non-relapsing DLBCL cases aiming to test the clonal relationship between primaries and relapses to track tumors' genetic evolution and to investigate the genetic background of DLBCL recurrence. Three clonally unrelated DLBCL relapses were identified (15%). Also, two distinct patterns of genetic evolution in clonally related relapses were detected as follows: (1) early-divergent/branching evolution from a common progenitor in 6 patients (30%), and (2) late-divergent/linear progression of relapses in 11 patients (65%). Analysis of recurrent genetic events identified potential early drivers of lymphomagenesis (KMT2D, MYD88, CD79B and PIM1). The most frequent relapse-specific events were additional mutations in KMT2D and alterations of MEF2B. SOCS1 mutations were exclusive to non-relapsing DLBCL, whereas primaries of relapsing DLBCL more commonly displayed gains of 10p15.3-p12.1 containing the potential oncogenes PRKCQ, GATA3, MLLT10 and ABI1. Altogether, our study expands the knowledge on clonal relationship, genetic evolution and mutational basis of DLBCL relapses.
The epidemiology of hypoparathyroidism (HP) is largely unknown. We aimed to determine prevalence, etiologies, health related quality of life (HRQOL) and treatment pattern of HP.
Patients with HP and 22q11 deletion syndrome (DiGeorge syndrome) were identified in electronic hospital registries. All identified patients were invited to participate in a survey. Among patients who responded, HRQOL was determined by Short Form 36 and Hospital Anxiety and Depression scale. Autoantibodies were measured and candidate genes (CaSR, AIRE, GATA3, and 22q11-deletion) were sequenced for classification of etiology.
We identified 522 patients (511 alive) and estimated overall prevalence at 102 per million divided among postsurgical HP (64 per million), nonsurgical HP (30 per million), and pseudo-HP (8 per million). Nonsurgical HP comprised autosomal dominant hypocalcemia (21%), autoimmune polyendocrine syndrome type 1 (17%), DiGeorge/22q11 deletion syndrome (15%), idiopathic HP (44%), and others (4%). Among the 283 respondents (median age, 53 years [range, 9-89], 75% females), seven formerly classified as idiopathic were reclassified after genetic and immunological analyses, whereas 26 (37% of nonsurgical HP) remained idiopathic. Most were treated with vitamin D (94%) and calcium (70%), and 10 received PTH. HP patients scored significantly worse than the normative population on Short Form 36 and Hospital Anxiety and Depression scale; patients with postsurgical scored worse than those with nonsurgical HP and pseudo-HP, especially on physical health.
We found higher prevalence of nonsurgical HP in Norway than reported elsewhere. Genetic testing and autoimmunity screening of idiopathic HP identified a specific cause in 21%. Further research is necessary to unravel the causes of idiopathic HP and to improve the reduced HRQOL reported by HP patients.
GATA-binding protein 3 (GATA3) is a diagnostically useful immunohistochemical marker of breast cancer. Because of its strong association with estrogen receptor expression, GATA3 has markedly reduced sensitivity in triple-negative breast cancer (TNBC). We constructed a tissue microarray using a large series of TNBCs and evaluated GATA3 expression by TNBC subtype as defined by surrogate immunohistochemical markers. A total of 205 TNBCs were classified into cancers of the molecular apocrine type (n=23, 11.2%), claudin-low type (n=21, 10.2%), basal-like type (n=91, 44.4%), mixed type (n=62, 30.2%), and null type (n=8, 3.9%). The GATA3 scores (staining intensity × proportion) were categorized as negative (0), focally positive (1-10), or positive (11-300). GATA3 staining was negative in 153 cancers (74.6%), focally positive in 11 (5.4%), and positive in 41 (20.0%). The rate of focal positivity or positivity for GATA3 was significantly higher in the molecular apocrine type (73.9%, 17/23) than in other types of TNBCs (P=.001). The mean GATA3 score of molecular apocrine-type TNBC was significantly higher than that of the other types (P=.001) and differed significantly between androgen receptor (AR)-positive and AR-negative TNBCs (P<.001). In conclusion, GATA3 expression was correlated strongly with AR-positive, molecular apocrine-type TNBCs. Co-expression of AR and GATA3 is a specific feature of molecular apocrine-type TNBC, which may serve as a diagnostic aid for cancer of unknown primary.
Two tubulosquamous polyps arising in the vagina are reported. Both were diffusely positive for GATA3 in the squamous component and focally positive for NKX3.1 in the glandular component, prostate acid phosphatase was focally positive in only 1 case in the glandular component. Both cases were negative for PAX2, PAX8, SALL4, and prostate-specific antigen. In addition, we included 3 cases of cervical squamous-lined cysts most likely representing ectopic prostatic tissue in the cervix and 1 case of paraurethral Skene-type glands to compare the immunophenotype. We analyze this immunoprofile, not previously reported. We also suggest unifying the nomenclature because vaginal Brenner tumors are most likely synonymous with tubulosquamous polyp (TSP) of the vagina. Our findings add support to the postulated origin of TSPs and cervical ectopic prostatic tissue from eutopic or misplaced Skene glands, equivalent of the prostate in the female. NKX3.1 seems a better marker to study and diagnose ectopic prostatic tissue in the cervix as well as TSPs.
T helper (Th) and regulatory T (Treg) cell-related cytokines are implicated in inflammatory bowel diseases, including ulcerative colitis (UC). While these cytokines are generally upregulated in inflamed mucosae, the key cytokine profile explaining disease severity has not been determined.
The Rachmilewitz endoscopic index (REI) was assessed in 61 UC patients undergoing colonoscopy. Biopsies obtained from inflamed (REI 3-12) and noninflamed (REI 0-2) areas were analyzed by quantitative PCR for expression of mRNAs encoding cytokines and transcription factors related to Th1 (TNF-α, IFN-γ, IL-12p35, IL-12p40, and T-bet), Th2 (IL-4, IL-13, IL-33, and GATA3), Th17 (IL-17A, IL-17F, IL-21, IL-22, IL-23p19, IL-6, and RORC), Th9 (IL-9, IRF4, and PU.1), and Treg (TGF-β and Foxp3). Expression patterns associated with higher REI were determined by univariate and multivariate analyses.
Despite general upregulation, none of these mRNAs showed univariate correlation with REI in inflamed samples. Multiple regression analysis, however, found that joint expression of IL-17A, IL-17F, IL-21, RORC, and TGF-β was significantly predictive of REI (P < 0.0002, R(2) = 0.380), with major individual contributions by IL-17A (P < 0.0001) and IL-17F (P < 0.0001), which were associated with increased and decreased REI, respectively. Partial correlation analysis, validating this model, indicated differences between IL-17A and IL-17F in correlating with other targets. The IL-17A/IL-17F ratio showed a significant correlation with REI (r = 0.5124, P < 0.0001), whereas no other mRNAs were essentially predictive of REI.
Mucosal IL-17A/IL-17F ratio significantly correlates with endoscopic score in UC patients, accompanied by their disparate interactions with other Th/Treg-related genes.
The expression of immune response gene mRNA in the umbilical and venous blood were compared in newborns of the first day of life with and without signs of infection. The expression of il1b, il6, il8, il10, il12a, il15, il18, tnfa, tgfb1, tbx21, gata3, foxp3, rorc2, cd45, cd68, cd69, tlr2, tlr4, tlr9, and mmp8 mRNA was evaluated in umbilical and venous blood cells of newborns by reverse transcription real time PCR. In full-term newborns without signs of infection, the expression of il8, tlr2, tlr4, and mmp8 in venous blood was higher than in umbilical blood, while in preterm newborns, the levels of mmp8 transcript were elevated while the levels of tlr9, cd45, and gata3 were reduced. The expression of some markers differed in the umbilical and venous blood and in newborns with congenital infectious disease and without signs of infection.
The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2- and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2- tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2- tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.
Various pivotal stages in smoking behavior can be identified, including initiation, conversion from experimenting to established use, development of tolerance, and cessation. Previous studies have shown high heritability for age of smoking initiation and cessation; however, time-to-event genome-wide association studies aiming to identify underpinning genes that accelerate or delay these transitions are missing to date.
We investigated which single nucleotide polymorphisms (SNPs) across the whole genome contribute to the hazard ratio of transition between different stages of smoking behavior by performing time-to-event analyses within a large Finnish twin family cohort (N = 1962), and further conducted mediation analyses of plausible intermediate traits for significant SNPs.
Genome-wide significant signals were detected for three of the four transitions: (1) for smoking cessation on 10p14 (P = 4.47e-08 for rs72779075 flanked by RP11-575N15 and GATA3), (2) for tolerance on 11p13 (P = 1.29e-08 for rs11031684 in RP1-65P5.1), mediated by smoking quantity, and on 9q34.12 (P = 3.81e-08 for rs2304808 in FUBP3), independent of smoking quantity, and (3) for smoking initiation on 19q13.33 (P = 3.37e-08 for rs73050610 flanked by TRPM4 and SLC6A16) in analysis adjusted for first time sensations. Although our top SNPs did not replicate, another SNP in the TRPM4-SLC6A16 gene region showed statistically significant association after region-based multiple testing correction in an independent Australian twin family sample.
Our results suggest that the functional effect of the TRPM4-SLC6A16 gene region deserves further investigation, and that complex neurotransmitter networks including dopamine and glutamate may play a critical role in smoking initiation. Moreover, comparison of these results implies that genetic contributions to the complex smoking behavioral phenotypes vary among the transitions.
Metastatic neuroendocrine neoplasms to the breast may show considerable morphologic overlap with primary mammary carcinomas, particularly those showing evidence of neuroendocrine differentiation, and may be misdiagnosed as such. Accurate distinction between these two entities is crucial for determination of appropriate clinical management. The histologic and immunohistochemical features of metastatic neuroendocrine neoplasms to the breast were studied and compared with the features of primary invasive mammary carcinomas with neuroendocrine differentiation, which served as controls. Of the metastatic neuroendocrine neoplasms, 15 were well-differentiated neuroendocrine tumors with carcinoid tumor-type morphology and 7 were poorly differentiated/high-grade neuroendocrine carcinomas with small-cell or large-cell neuroendocrine carcinoma morphology. The majority of the metastatic neoplasms originated in the lung and gastrointestinal tract. There were histologic similarities between metastatic neuroendocrine neoplasms and invasive mammary carcinomas with neuroendocrine differentiation, both of which exhibited neuroendocrine histologic features (nested and trabecular architecture, minimal tubular differentiation, and characteristic nuclear features). Only one case of the invasive mammary carcinomas with neuroendocrine differentiation was modified Bloom-Richardson grade 1 (largely due to minimal tubular differentiation on most such tumors), and the invasive mammary carcinomas with neuroendocrine differentiation were often associated with in situ carcinoma. Immunohistochemistry was helpful in distinguishing metastatic neuroendocrine neoplasms from invasive mammary carcinomas with neuroendocrine differentiation. Whereas the majority of invasive mammary carcinomas with neuroendocrine differentiation were positive for estrogen receptor and GATA3, metastatic neuroendocrine neoplasms were typically negative for estrogen receptor and GATA3, and metastatic well-differentiated neuroendocrine tumors often showed immunoreactivity for site-specific markers. Although the histologic and immunohistochemical features of a breast tumor may raise the suspicion of a metastatic neuroendocrine neoplasm, the pathologic findings should be interpreted in the context of the clinical history and imaging findings in order to establish an accurate diagnosis.
The intimate link between location of fat accumulation and metabolic disease risk and depot-specific differences is well established, but how these differences between depots are regulated at the molecular level remains largely unclear. Here we show that TRIP-Br2 mediates endoplasmic reticulum (ER) stress-induced inflammatory responses in visceral fat. Using in vitro, ex vivo and in vivo approaches, we demonstrate that obesity-induced circulating factors upregulate TRIP-Br2 specifically in visceral fat via the ER stress pathway. We find that ablation of TRIP-Br2 ameliorates both chemical and physiological ER stress-induced inflammatory and acute phase response in adipocytes, leading to lower circulating levels of inflammatory cytokines. Using promoter assays, as well as molecular and pharmacological experiments, we show that the transcription factor GATA3 is responsible for the ER stress-induced TRIP-Br2 expression in visceral fat. Taken together, our study identifies molecular regulators of inflammatory response in visceral fat that-given that these pathways are conserved in humans-might serve as potential therapeutic targets in obesity.
Inner ear hair cells are the sensory receptors that detect and convert sound vibrations and head movements into neural signals. However, in humans, these cells are unable to regenerate if they are damaged or lost. Over thepast decade,there has been an exponential increase in interest and progress in understanding of the development of the inner ear and of hair cells, aiming to gain insights into hair cell repair or even regeneration. In hair cell development, various transcription factors have been found to be involved in the processes of hair cell proliferation, differentiation and survival. Among these transcription factors, Math1, Gata3, Sox2 and Atoh1 have been highlighted for their crucial role in the fate of hair cells. In this article, we will summarize the current understanding of the role of transcription factors in hair cell development, focusing on the role and possible mechanisms of Math1, Gata3, Sox2 and Atoh1.
This article describes the case of a 70-year-old woman with an adnexal cystadenocarcinoma located in the right broad ligament and displaying a striking resemblance to a well-differentiated endometrioid adenocarcinoma. The uniqueness of this pseudoendometrioid carcinoma lies in the fact that its mesonephric nature is revealed by the origin from a papillary cystadenoma of the broad ligament, where remnants of the mesonephric duct are seated, and the immunohistochemical profile, particularly a uniform negativity for estrogen and progesterone receptors, apical-luminal positivity for CD10, and strong nuclear positivity for GATA3.
KLK6 is expressed in normal mammary tissues and is aberrantly regulated in breast cancer. At physiological levels of expression, i.e. those found in normal mammary tissues, KLK6 acts as a tumor suppressor in human breast cancer. However, aberrant overexpression of KLK6 (i.e. 50-100-fold higher than normal), a characteristic of a subset of human breast cancers is associated with increased tumorigenicity (Pampalakis et al. Cancer Res 69:3779-3787, 2009). Here, we stably transfected KLK6-non-expressing MDA-MB-231 breast cancer cells with the full-length KLK6 cDNA to overexpress KLK6 at levels comparable to those observed in patients, and investigated potential oncogenic miRNA networks regulated by these abnormally high KLK6 expression levels and increased activity of this serine protease. A number of miRNAs that are upregulated (e.g. miR-146a) or downregulated (e.g. miR-34a) via KLK6-induced alterations in the miRNA biogenesis machinery were identified. Integrated experimental and bioinformatics analyses identified convergent miRNA networks targeting the cell cycle, MYC, MAPK, and other signaling pathways. In large clinical datasets, significant correlations between KLK6 and downstream MAPK and MYC targets at both the RNA and protein levels was confirmed, as well as negative correlation with GATA3. It was also demonstrated that KLK6 overexpression and likely its proteolytic activity is associated with alterations in downstream miRNAs and their targets, and these differ with the molecular subtypes of breast cancer. The data partly explains the different characteristics of breast cancer subtypes. Importantly, we introduce a combined KLK6-CDKN1B+MYC+CDKN1C score for prediction of long-term patient survival outcomes, with higher scores indicating poor survival.
Acute anterior uveitis (AAU) is the most common form of uveitis and is a frequent ocular manifestation of ankylosing spondylitis (AS). Thymocyte CD4+ cells have been reported to play an important role in the pathogenesis of both AAU and AS. To test whether the copy number variations (CNVs) of CD4+ T cell transcription factor genes including T-bet, GATA binding protein 3 (GATA)-3, related orphan receptor C (RORC) and forkhead box protein 3 (FOXP3) are associated with acute anterior uveitis either in the presence or absence of ankylosing spondylitis (AAU+AS+; AAU+AS-).
The study included 676 patients with AAU, including 298 patients with AAU+AS+, 378 patients with AAU+AS-, and 596 unrelated healthy controls in a Chinese Han population. Copy number variations were examined by real-time PCR.
The frequency of a high copy number (CN) of T-bet was increased in AAU+AS+ as well as AAU+AS- patients when compared with controls (P value after Bonferroni correction [P(corr)] = 4.3 × 10(-5); odds ratio [OR] = 2.0 and P(corr) = 1.2 × 10(-8); OR = 2.3, respectively). The frequency of a high CN of GATA-3 was significantly higher in AAU+AS+ patients than in controls (P(corr) = 1.8 × 10(-7); OR = 4.9). A higher frequency of CN of FOXP3 was found in female AAU+AS+ patients and female AAU+AS- patients (P(corr) = 0.005, OR = 5.9 and P(corr) = 0.004, OR = 4.9, respectively ). No association was found between CNVs of RORC and AAU+AS- or AAU+AS+ patients.
A high copy number of T-bet and GATA-3 confers susceptibility to AAU and AS, and a high copy number of FOXP3 confers susceptibility to female patients with AAU either with or without AS.
Although the chlorinated flame retardant Dechlorane (Dec) 602 has been detected in food, human blood, and breast milk, there is limited information on potential health effects, including possible immunotoxicity.
We determined the immunotoxic potential of Dec 602 in mice by examining the expression of phenotypic markers on thymocyte and splenic lymphocyte subsets, Th1/Th2 transcription factors, and the production of cytokines and antibodies.
Adult male C57BL/6 mice were orally exposed to environmentally relevant doses of Dec 602 (1 and 10 μg/kg body weight per day) for 7 consecutive days. Thymocyte and splenic CD4 and CD8 subsets and splenocyte apoptosis were examined by flow cytometric analysis. Cytokine expression was measured at both the mRNA and the protein levels. Levels of the transcription factors Th1 (T-bet and STAT1) and Th2 (GATA3) were determined using quantitative real-time polymerase chain reaction (qPCR). Serum levels of immunoglobulins IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA).
Splenic CD4+ and CD8+ T cell subsets were decreased compared with vehicle controls, and apoptosis was significantly increased in splenic CD4+ T cells. Expression (mRNA and protein) of Th2 cytokines [interleukin (IL)-4, IL-10, and IL-13] increased, and that of Th1 cytokines [IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] decreased. The Th2 transcriptional factor GATA3 increased, whereas the Th1 transcriptional factors T-bet and STAT1 decreased. As additional indicators of the Th2-Th1 imbalance, production of IgG1 was significantly increased, whereas IgG2a was reduced.
To our knowledge, we are the first to report evidence of the effects of Dec 602 on immune function in mice, with findings indicating that Dec 602 exposure favored Th2 responses and reduced Th1 function.
Feng Y, Tian J, Xie HQ, She J, Xu SL, Xu T, Tian W, Fu H, Li S, Tao W, Wang L, Chen Y, Zhang S, Zhang W, Guo TL, Zhao B. 2016. Effects of acute low-dose exposure to the chlorinated flame retardant dechlorane 602 and Th1 and Th2 immune responses in adult male mice. Environ Health Perspect 124:1406-1413; http://dx.doi.org/10.1289/ehp.1510314.
Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is a receptor for PGD2 and expressed by T cells, eosinophils, basophils, and ILC2 cells. CRTh2 expression by CD4(+) T cells identifies the Th2 subset, and these cells have been characterized as allergen-specific central memory Th2 cells. Recently, activation of the PGD2 -CRTh2 pathway in the lungs was associated with severe asthma.
To assess circulating levels of Th2 cells and related mediators in severe asthma and those who experience asthma exacerbations.
Peripheral blood cells expressing CRTh2 were characterized by flow cytometry and qRT-PCR. Serum IL-13 and PGD2 were measured by ELISA and compared with asthma severity and tendency to exacerbate.
Severe asthmatics had more circulating CD4(+) CRTh2(+) T cells, CRTh2 and GATA3 mRNA, and a higher level of serum IL-13 compared to mild/moderate asthmatics. The proportion of CD4(+) CRTh2(+) T cells was associated with lower lung function and was highest in severe asthmatics that exacerbated in the last year. Circulating CD4(+) CRTh2(+) T cells, unlike eosinophils, were positively correlated with inhaled steroid dose.
Elevated levels of circulating CD4(+) CRTh2(+) T cells are a feature of severe asthma, despite high-dose corticosteroids. Tracking the systemic level of these cells may help identify type 2 severe asthmatics at risk of exacerbation.
We present the second reported mammary analog secretory carcinoma (MASC) apparently arising in the thyroid and propose a potential close relationship to ETV6-NTRK3 fusion papillary thyroid carcinoma. The patient, a 36 year old woman, presented with a neck mass of 1 year's duration. Imaging studies showed a tumor involving most of the thyroid with enlarged regional lymph nodes. FNA biopsy yielded a diagnosis of "papillary thyroid carcinoma". Resection revealed a 4.5 cm infiltrative tumor. Final diagnosis was "papillary thyroid carcinoma (PTC) consistent with diffuse sclerosing variant" with positive lymph nodes (2+/4) and margins. Histologic features included mixed microcystic, solid, follicular and papillary architecture, prominent nucleoli, abundant nuclear grooves and rare nuclear pseudo-inclusions. Despite radioactive iodine, radiotherapy and multiagent chemotherapy, the patient progressed over 6 years with local recurrence and additional lymph node involvement finally developing widespread distant metastases. Prompted by the breast carcinoma-like histopathology of a metastasis, immunohistochemical staining was performed and revealed strong expression of GATA3 and mammaglobin with no reactivity for thyroglobulin or TTF-1. The original tumor was then tested and showed the same immunoprofile. RT-PCR confirmed the presence of an ETV6-NTRK3 fusion consistent with a diagnosis of MASC. Our patient's clinical, imaging and morphologic features remarkably mimicked papillary thyroid carcinoma. At the molecular level, the ETV6-NTRK3 fusion in this patient involved exons reported in the rare "papillary thyroid carcinoma" with this translocation. Given the immunophenotype of this case, it is possible that at least some ETV6-NTRK3 fusion positive PTC are actually MASC masquerading as papillary thyroid carcinoma.
Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80(-)CD11b(+)Gr(+) cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80(-)CD11b(hi)Gr1(hi) cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4(+)GATA3(+) T cells and AAMØ in MLN and spleen. Purified CD11c(-)CD11b(+)Gr1(+) cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4(+) T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c(-)CD11b(+)Gr1(+) cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4(+) Th2 responses and promote chronic infection.Mucosal Immunology advance online publication 13 April 2016. doi:10.1038/mi.2016.36.
Infections with helminth parasites are endemic in the developing world and are a target for intervention with new therapies. Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic effects in inflammation and immune responses. We investigated the role of MIF in a naturally cleared model of helminth infection in rodents, Nippostrongylus brasiliensis. At day 7 postinfection, MIF-deficient (MIF(-/-)) mice had reduced parasite burden and mounted an enhanced type 2 immune response (Th2), including increased Gata3 expression and interleukin-13 (IL-13) production in the mesenteric lymph nodes (MLNs). Bone marrow reconstitution demonstrated that MIF produced from hematopoietic cells was crucial and Rag1(-/-) reconstitution provided direct evidence that MIF(-/-) CD4(+) T cells were responsible for the augmented parasite clearance. MIF(-/-) CD4(+) T cells produced less IL-6 postinfection, which correlated with enhanced Th2 responses. MIF(-/-) CD4(+) T cells exhibited lower nuclear factor-κB activation, potentially explaining the reduction in IL-6. Finally, we demonstrated enhanced clearance of the parasite and Th2 response in wild-type mice treated with the MIF tautomerase inhibitor, sulforaphane, a compound found naturally found in cruciferous vegetables. These results are the first to describe the importance of the tautomerase enzyme activity in MIF function in N. brasiliensis infection.Mucosal Immunology advance online publication, 6 April 2016; doi:10.1038/mi.2016.29.
The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment.
Estrogen receptor-α (ERα) mediates the essential biological function of estrogen in breast development and tumorigenesis. Multiple mechanisms, including pioneer factors, coregulators and epigenetic modifications have been identified as regulators of ERα signaling in breast cancer. However, previous studies of ERα regulation have focused on luminal and HER2-positive subtypes rather than basal-like breast cancer (BLBC), in which ERα is underexpressed. In addition, mechanisms that account for the decrease or loss of ER expression in recurrent tumors after endocrine therapy remain elusive. Here, we demonstrate a novel FOXC1-driven mechanism that suppresses ERα expression in breast cancer. We find that FOXC1 competes with GATA-binding protein 3 (GATA3) for the same binding regions in the cis-regulatory elements upstream of the ERα gene and thereby downregulates ERα expression and consequently its transcriptional activity. The forkhead domain of FOXC1 is essential for the competition with GATA3 for DNA binding. Counteracting the action of GATA3 at the ERα promoter region, overexpression of FOXC1 hinders recruitment of RNA polymerase II and increases histone H3K9 trimethylation at ERα promoters. Importantly, ectopic FOXC1 expression in luminal breast cancer cells reduces sensitivity to estrogen and tamoxifen. Furthermore, in breast cancer patients with ER-positive primary tumors who received adjuvant tamoxifen treatment, FOXC1 expression is associated with decreased or undetectable ER expression in recurrent tumors. Our findings highlight a clinically relevant mechanism that contributes to the low or absent ERα expression in BLBC. This study suggests a new paradigm to study ERα regulation during breast cancer progression and indicates a role of FOXC1 in the modulation of cellular response to endocrine treatment.
During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8-cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose-dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation.
-Sarcomatoid urothelial carcinoma (UCa) is a rare but aggressive variant of bladder cancer that can show diagnostic challenges even using ancillary techniques.
-To examine immunohistochemical markers in the context of sarcomatoid UCa, including those associated with epithelial-to-mesenchymal transition.
-Twenty-eight cases of sarcomatoid UCa were rereviewed. Clinical outcomes were obtained through database search. Immunohistochemistry for clinical and epithelial-to-mesenchymal transition markers was performed.
-All patients had biopsy-proven invasive UCa; 61% (17 of 28) had sarcomatoid UCa at initial diagnosis. A recognizable epithelial component(s) was present in 17 lesions. The sarcomatoid component accounted for 65% of the lesion (average), with heterologous elements present in 3 of 28 cases (11%). The morphologic spectrum of the sarcomatoid element included spindled not otherwise specified, myxoid, pseudoangiosarcomatous, and malignant fibrous histiocytoma-like undifferentiated features. The sarcomatoid component was immunoreactive for pancytokeratin (22 of 26; 85%), p63 (20 of 26; 77%), cytokeratin 903 (17 of 26; 65%), cytokeratin 7 (16 of 26; 62%), GATA3 (16 of 26; 62%), and cytokeratin 5/6 (16 of 26; 62%). STAT-6, CD31, CD34, and HMB45 were all nonreactive, whereas smooth muscle actin often showed at least focal immunoreactivity (22 of 26; 85%). Epithelial-to-mesenchymal transition markers were frequently expressed, including vimentin (26 of 26; 100%), FoxC2 (26 of 26; 100%), SNAIL (23 of 26; 88.5%), and ZEB1 (18 of 26; 69.2%). Follow-up was available for 24 patients (median, 7 months). Sixteen of 28 patients (57%) died of disease (overall mean survival, 9.1 months). The presence of myxoid or chordoid features was associated with reduced survival (P < .05).
-Sarcomatoid UCa is an aggressive form of UCa that frequently expresses epithelial-to-mesenchymal transition markers, suggesting a possible mechanism associated with aggressive tumor behavior.
In breast cancer, GATA3 mutations have been associated with a favorable prognosis and the response to neoadjuvant aromatase inhibitor treatment. Therefore, we investigated whether GATA3 mutations predict the outcome of tamoxifen treatment in the advanced setting. In a retrospective study consisting of 235 hormone-naive patients with ER-positive breast cancer who received tamoxifen as first-line treatment for recurrent disease, GATA3 mutations (in 14.0% of patients) did not significantly associate with either the overall response rate (ORR) or with the length of progression-free survival (PFS) after the start of tamoxifen therapy. Interestingly, among 148 patients for whom both mutation and mRNA expression data were available, GATA3 mutations associated with an increased expression of GATA3. However, only 23.7% of GATA3 high tumors had a mutation. Evaluation of the clinical significance of GATA3 mRNA revealed that it was associated with prolonged PFS, but not with the ORR, also in multivariate analysis. Thus, GATA3 mRNA expression, but not GATA3 mutation, is an independent predictor of prolonged PFS in ER-positive breast cancer patients who received first-line tamoxifen for recurrent disease. Besides GATA3 mutation, other mechanisms must exist that underlie increased GATA3 levels.
Delta-like ligand 4 (Dll4), one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti-VEGF therapy. Here, we examined the biologic effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biologic effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibit tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse-phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior antitumor effects in ovarian cancer models compared with either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel (P < 0.05 for the combination vs. aflibercept). The superior antitumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared with the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach. Mol Cancer Ther; 15(6); 1344-52. ©2016 AACR.
S100A8 and S100A9 are calcium-binding proteins that are secreted primarily by granulocytes and monocytes, and are upregulated during the inflammatory response. S100A8 and S100A9 have been identified to be expressed by epithelial cells involved in malignancy. In the present study, the transcriptional levels of S100A8 and S100A9 were investigated in various subtypes of breast cancer (BC), and the correlation with estrogen receptor 1 (ESR1) and GATA binding protein 3 (GATA3) gene expression was evaluated using microarray datasets. The expression of S100A8 and S100A9 in BC cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The regulation of ESR1 and GATA3 by administration of recombinant S100A8/A9 was examined in the BC MCF-7 cell line using quantitative (q)PCR. The association between S100A8 and S100A9 and overall survival (OS) was investigated in GeneChip® data of BC. The expression levels of S100A8 and S100A9 were higher in human epidermal growth factor receptor 2 (Her2)-amplified and basal-like BC. The messenger (m)RNA levels of S100A8 and S100A9 were inversely correlated with ESR1 and GATA3 expression. S100A8/A9 induced a 10-fold decrease in the mRNA levels of ESR1 in MCF-7 cells. Poor OS was associated with high expression levels of S100A9, but not with high expression levels of S100A8 in BC. In conclusion, strong expression and secretion of S100A8/A9 may be associated with the loss of estrogen receptor in BC, and may be involved in the poor prognosis of Her2+/basal-like subtypes of BC.
Different genes are associated with categorical classifications of asthma severity. However, continuous outcomes should be used to catch the heterogeneity of asthma phenotypes and to increase the power in association studies. Accordingly, the aim of this study was to evaluate the association between single nucleotide polymorphisms (SNPs) in candidate gene regions and continuous measures of asthma severity, in adult patients from the general population. In the Gene Environment Interactions in Respiratory Diseases (GEIRD) study (www.geird.org), 326 subjects (aged 20-64) with ever asthma were identified from the general population in Verona (Italy) between 2007 and 2010. A panel of 236 SNPs tagging 51 candidate gene regions (including one or more genes) was analysed. A symptom and treatment score (STS) and pre-bronchodilator FEV1% predicted were used as continuous measures of asthma severity. The association of each SNP with STS and FEV1% predicted was tested by fitting quasi-gamma and linear regression models, respectively, with gender, body mass index and smoking habits as potential confounders. The Simes multiple-test procedure was used for controlling the false discovery rate (FDR). SNP rs848 in the IL13 gene region (IL5/RAD50/IL13/IL4) was associated with STS (TG/GG vs TT genotype: uncorrected p-value = 0.00006, FDR-corrected p-value = 0.04), whereas rs20541 in the same gene region, in linkage disequilibrium with rs848 (r(2) = 0.94) in our sample, did not reach the statistical significance after adjusting for multiple testing (TC/CC vs TT: uncorrected p-value = 0.0003, FDR-corrected p-value = 0.09). Polymorphisms in other gene regions showed a non-significant moderate association with STS (IL12B, TNS1) or lung function (SERPINE2, GATA3, IL5, NPNT, FAM13A) only. After adjusting for multiple testing and potential confounders, SNP rs848 in the IL13 gene region is significantly associated with a continuous measure of symptom severity in adult subjects with ever asthma.
IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions.
We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites.
We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging.
CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs.
IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis.
High serum sCD30 levels are associated with inflammatory disorders and poor outcome in renal transplantation. The contribution to these phenomena of transcripts and proteins related to CD30-activation and -cleavage is unknown. We assessed in peripheral blood of end-stage renal disease patients (ESRDP) transcripts of CD30-activation proteins CD30 and CD30L, CD30-cleavage proteins ADAM10 and ADAM17, and Th1- and Th2-type immunity-related factors t-bet and GATA3. Additionally, we evaluated the same transcripts and release of sCD30 and 32 cytokines after allogeneic and polyclonal T-cell activation. In peripheral blood, ESRDP showed increased levels of t-bet and GATA3 transcripts compared to healthy controls (HC) (both P<0.01) whereas levels of CD30, CD30L, ADAM10 and ADAM17 transcripts were similar. Polyclonal and allogeneic stimulation induced higher levels of CD30 transcripts in ESRDP than in HC (both P<0.001). Principal component analysis (PCA) in allogeneic cultures of ESRDP identified two correlation clusters, one consisting of sCD30, the Th-1 cytokine IFN-γ, MIP-1α, RANTES, sIL-2Rα, MIP-1β, TNF-β, MDC, GM-CSF and IL-5, and another one consisting of CD30 and t-bet transcripts, IL-13 and proinflammatory proteins IP-10, IL-8, IL-1Rα and MCP-1. Reflecting an activated immune state, ESRDP exhibited after allostimulation upregulation of CD30 transcripts in T cells, which was associated with Th1 and proinflammatory responses.
Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers.
All male breast cancers were estrogen receptor-positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor-normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers.
Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A-like or luminal B-like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair-related genes.
Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045-56. ©2016 AACR.
Pathologists should be aware of the existence of a rare CK7-negative variant of breast carcinoma in general, and of Paget's disease in particular. Cytokeratin 7-negative Paget's disease and CK7-negative ductal intraepithelial neoplasia (ductal carcinoma in situ) present a major diagnostic challenge for pathologists since there is limited awareness of their existence. When there is classic Paget's morphology on H&E sections, GATA3 positivity should resolve any doubts about the diagnosis in the setting of a CK7-negative neoplastic cell population.
The incidence of acute lymphoblastic leukemia (ALL) is nearly 20% higher among Hispanics than non-Hispanic Whites. Previous studies have shown evidence for association between risk of ALL and variation within IKZF1, ARID5B, CEBPE, CDKN2A, GATA3, and BM1-PIP4K2A genes. However, variants identified only account for <10% of the genetic risk of ALL.
We applied pathway-based analyses to genome-wide association study (GWAS) data from the California Childhood Leukemia Study to determine whether different biologic pathways were overrepresented in childhood ALL and major ALL subtypes. Furthermore, we applied causal inference and data reduction methods to prioritize candidate genes within each identified overrepresented pathway, while accounting for correlation among SNPs.
Pathway analysis results indicate that different ALL subtypes may involve distinct biologic mechanisms. Focal adhesion is a shared mechanism across the different disease subtypes. For ALL, the top five overrepresented Kyoto Encyclopedia of Genes and Genomes pathways include axon guidance, protein digestion and absorption, melanogenesis, leukocyte transendothelial migration, and focal adhesion (PFDR < 0.05). Notably, these pathways are connected to downstream MAPK or Wnt signaling pathways which have been linked to B-cell malignancies. Several candidate genes for ALL, such as COL6A6 and COL5A1, were identified through targeted maximum likelihood estimation.
This is the first study to show distinct biologic pathways are overrepresented in different ALL subtypes using pathway-based approaches, and identified potential gene candidates using causal inference methods.
The findings demonstrate that newly developed bioinformatics tools and causal inference methods can provide insights to furthering our understanding of the pathogenesis of leukemia. Cancer Epidemiol Biomarkers Prev; 25(5); 815-22. ©2016 AACR.
Clear cell papillary renal cell carcinoma (CCPRCC) cases were evaluated for mutations on the following genes: KRAS, NRAS, BRAF, PIK3CA, ALK, ERBB2, DDR2, MAP2K1, RET and EGFR. Four male and three female patients of age 42-74 years were evaluated. All cases were incidentally detected by ultrasound and ranged 1.8-3.5 cm. Microscopic examination showed variably tubulopapillary, tubular acinar, cystic architecture and the characteristic linear arrangement of nuclei. The cells were reactive with CK7 (strong), CA IX (cup-shape) and 34 β E12. CD10, AMACR/RACEMASE and GATA3 were negative. There were no mutations on any of the investigated genes. This preliminary observation supports the concept that CCPRCC might be indeed an indolent tumour worth it to be named as clear cell papillary neoplasm of low potential.
Although kidney transplantation is the best therapy for end-stage renal disease, rejection remains a concern, and currently available immunosuppressive agents contribute to morbidity and mortality. Thus, novel immunosuppressive drugs are required. Dichloroacetate (DCA) is already used in the treatment of congenital lactic acidosis and characterized by limited toxicity. As DCA inhibits aerobic glycolysis, which is a prerequisite for CD4+ T-cell proliferation and differentiation into effector T-cells, its possible immunosuppressive role in mixed lymphocyte reaction (MLR), a model of alloreactivity, was investigated. Glucose and lactate concentrations were measured in the supernatants, and cell proliferation was assessed immunoenzymatically. CD4+ T‑cells were then isolated from the MLRs and the expression of cleaved caspase‑3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T‑cell subsets were evaluated by western blotting. In MLRs, DCA decreased glucose consumption and aerobic glycolysis, while it exerted a negligible effect on cell proliferation. In CD4+ T‑cells, DCA induced apoptosis, and decreased the expression of glucose trasporter‑1, hexokinase II, lactate dehydrogenase‑A and phosphorylated pyruvate dehydrogenase, while it increased total pyruvate dehydrogenase. In addition, DCA increased the expression of transcription factor forkhead box P3, whereas it decreased the expression of T‑box transcription factor TBX21, trans‑acting T-cell-specific transcription factor GATA‑3 and retinoic acid receptor related orphan receptor‑γt. In conclusion, in alloreactive CD4+ T‑cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T‑cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation.
HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets.
Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse.
Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET.
These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.
To investigate the role of ROG, GATA3 and T-bet in the progression of chronic hepatitis B (CHB).
The mRNA levels of ROG, GATA3 and T-bet in peripheral blood mononuclear cells (PBMCs) from 135 patients with CHB (including 45 mild cases, 42 moderate cases, and 48 severe cases) and 15 healthy control subjects were detected by real-time quantitative PCR.
The levels of T-bet mRNA in the PBMCs were significantly higher in CHB patients than in the healthy controls (P<0.05), and also differed significantly between the 3 groups of CHB patients (P<0.05). ROG mRNA levels were significantly higher in severe cases of CHB than in the healthy controls and mild and moderate CHB cases (P<0.05), but were similar among the latter 3 groups (P>0.05). The mRNA level of GATA3 in the PBMCs were significantly higher in moderate and severe CHB cases than in the healthy controls and mild CHB cases (P<0.05). The T-bet/GATA3 ratio was significantly greater in the 3 CHB groups than in the control group (P<0.05) but comparable between the 3 CHB groups (P>0.05). ROG levels were not correlated with GATA3 levels or T-bet/GATA3 ratio in the CHB cases.
The mRNA levels of ROG, GATA3 and T-bet in the PBMCs are obviously up-regulated in CHB patients and these 3 genes may participate in the progression of CHB. ROG plays an important role in correcting and maintaining the new balance of Th1/Th2.
Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae.
The current World Health Organization classification recognizes 3 subtypes of breast carcinomas with neuroendocrine features. Their reported prevalence is highly variable, ranging from <1% to up to 20% of all breast carcinomas. We report the case of a 73-year-old woman who underwent lumpectomy with a postoperative diagnosis of invasive ductal breast carcinoma. Six weeks after lumpectomy, pancreatic biopsies showed tumor cells with neuroendocrine features. The first immunohistochemical panel showed positivity for synaptophysin and cytokeratins, raising suspicion of a pancreatic neuroendocrine tumor. However, a second panel revealed positivity for estrogen receptors and GATA3. On review of the lumpectomy specimen, a significant neuroendocrine component was found, leading to the final diagnosis of breast carcinoma with neuroendocrine features metastasizing to the pancreas. Neuroendocrine markers are not routinely analyzed in breast tumors. Hence, metastases from breast carcinomas with unrecognized neuroendocrine features may lead to false diagnoses of primary neuroendocrine tumors at different metastatic sites, such as the pancreas.
Little is known about mutational landscape of rare breast cancer (BC) subtypes. The aim of the study was to apply next generation sequencing to three different subtypes of rare BCs in order to identify new genes related to cancer progression. We performed whole exome and targeted sequencing of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular BCs. Micropapillary BCs exhibit a profile comparable to common BCs: PIK3CA, TP53, GATA3, and MAP2K4 were the most frequently mutated genes. Metaplastic BCs presented a high frequency of TP53 (78 %) and PIK3CA (48 %) mutations and were recurrently mutated on KDM6A (13 %), a gene involved in histone demethylation. Pleomorphic lobular carcinoma exhibited high mutation rate of PIK3CA (30 %), TP53 (22 %), and CDH1 (41 %) and also presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. Next generation sequencing methods applied on rare cancer subtypes can serve as a useful tool in order to uncover new potential therapeutic targets. Sequencing of pleomorphic lobular carcinoma identified a high rate of alterations in PYGM. These findings emphasize the role of glycogen metabolism in cancer progression.
To study the expression levels of annexin A1 (ANXA1), GATA-3, and T-bet in T lymphocytes of peripheral blood in burned mice with sepsis at early stage, and to analyze their immune regulatory mechanisms.
Seven-hundred and eighty male mice of clean grade were divided into sham injury group (n=60, sham injured on the back by immersing in 37 ℃ warm water for 10 s), burn group (n=240, inflicted with 20% TBSA deep partial- thickness burn on the back by immersing in 100 ℃ hot water for 10 s), sepsis group (n=240, intraperitoneally injected with 6 mg/kg lipopolysaccharide), and burn+ sepsis group (n=240) according to the random number table. Mice of burn+ sepsis group were treated as that in burn group at first, and then they were treated as that in sepsis group. (1) Immediately after injury, six mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table. According to the random number table, 6 mice of each of the other three groups were respectively selected at post injury hour (PIH) 12, 24, 48, and 72 for the collection of lymphocyte suspension from peripheral blood (1 tube each mouse). Each tube of cell suspension was equally divided into two parts. Fluorescein isothiocyanate (FITC)-labeled human anti-mouse CD4 monoclonal antibody and phycoerythrin (PE)-labeled human anti-mouse interferon-γ monoclonal antibody were added to one part of cell suspension to mark helper T lymphocyte 1 (Th1). FITC-labeled human anti-mouse CD4 monoclonal antibody and PE-labeled human anti-mouse interleukin-4 (IL-4) monoclonal antibody were added to the other part of cell suspension to mark Th2. The percentages of Th1 and Th2 were determined with flow cytometer, and the ratio of Th1 to Th2 was calculated. (2) According to the random number table, 18 mice in sham injury group were selected immediately after injury for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and 18 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 to collect the lymphocyte suspension of peripheral blood (1 tube each mouse). The mRNA expression levels of ANXA1, GATA-3, and T-bet were determined by real-time fluorescent quantitative reverse transcription-PCR. (3) Immediately after injury, 36 mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table, and then 36 tubes of cell suspension were divided into 6 batches (6 tubes each batch). Each one of 6 kinds of antibody combinations: antibodies for labeling Th1 and Th2 in combination with PE-anthocyanin 7 labeled human anti-mouse ANXA1 monoclonal antibody, PE-anthocyanin 7 labeled human anti-mouse GATA-3 monoclonal antibody, and PE-anthocyanin 7 labeled human anti-mouse T-bet monoclonal antibody was added to 1 tube of cell suspension at each batch. According to the random number table, 36 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and then 36 tubes of cell suspension at each time point were divided into 6 batches for marking with 3 kinds of surface markers of Th1 and Th2 (6 tubes each batch). Each one of above-mentioned 6 kinds of antibodies was added to 1 tube of cell suspension at each time point for each batch. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 were determined with flow cytometer. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test. The relationship between the percentages of ANXA1 positive cell and the percentages of GATA-3 positive cell in Th1 and Th2, and mRNA expression level of ANXA1 and mRNA expression level of GATA-3 in lymphocytes were assessed by linear correlation analysis.
(1) Compared with those in sham injury group immediately after injury, the percentages of Th1 and Th2 and the ratio of Th1 to Th2 of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the percentages of Th1 and Th2 and the ratios of Th1 to Th2 of mice in sepsis group and burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05. (2) Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocyte of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the mRNA expression level of T-bet was significantly decreased at PIH 24 but significantly increased at PIH 48 and 72, with P values below 0.05. Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocytes of mice in sepsis group were significantly decreased from PIH 12 on, and the mRNA expression level of T-bet was increased significantly from PIH 12 on, with P values below 0.05; the mRNA expression levels of ANXA1, GATA-3, and T-bet in lymphocytes of mice in burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05, reaching the nadir at PIH 72 (0.50±0.04, 0.45±0.03, 0.21±0.05, respectively). (3) A significant positive correlation was observed between ANXA1 mRNA expression level and GATA-3 mRNA expression level in lymphocytes of peripheral blood (r=0.862, P<0.05). (4) Compared with those in sham injury group immediately after injury, the percentages of ANXA1 and GATA-3 positive cellsin Th1 and Th2 of mice in burn group were significantly lowered from PIH 24 on, and the percentage of T-bet positive cells was significantly decreased at PIH 24, but it was increased from PIH 48 on, with P values below 0.05. The percentages of ANXA1 and GATA-3 positive cells in Th1 and Th2 of mice in sepsis group were continuously decreased from PIH 12 on, which were lower at most time points than those in sham injury group immediately after injury, with P values below 0.05. The percentages of T-bet positive cells in Th1 and Th2 of mice in sepsis group were significantly increased since PIH 12 as compared with those in sham injury group immediately after injury, with P values below 0.05. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 of mice in burn+ sepsis group were continuously lowered from PIH 12, with significantly statistical differences at most time points as compared with those in sham injury group immediately after injury, with P values below 0.05. (5) The percentages of GATA-3 positive cells in Th1 and Th2 were significantly positively correlated with those of ANXA1 (with r values respectively 0.747 and 0.787, P values below 0.05).
The expression levels of ANXA1, GATA-3, and T-bet were continuously lowered in burned mice with sepsis, and it may play an important role in Th1/Th2 balance switching to Th2 bias and immunosuppressive process.
This study used selected lambs that varied in their resistance to the gastrointestinal parasite Teladorsagia circumcincta. Infection over 12 weeks identified susceptible (high adult worm count, AWC; high fecal egg count, FEC; low body weight, BW; low IgA) and resistant sheep (no/low AWC and FEC, high BW and high IgA). Resistance is mediated largely by a Th2 response and IgA and IgE antibodies, and is a heritable characteristic. The polarization of T cells and the development of appropriate immune responses is controlled by the master regulators, T-bet (TBX21), GATA-3 (GATA3), RORγt (RORC2) and RORα (RORA); and several inflammatory diseases of humans and mice are associated with allelic or transcript variants of these transcription factors. This study tested the hypothesis that resistance of sheep to T. circumcincta is associated with variations in the structure, sequence or expression levels of individual master regulator transcripts. We have identified and sequenced one variant of sheep TBX21, two variants of GATA3 and RORC2 and five variants of RORA from lymph node mRNA. Relative RT-qPCR analysis showed that TBX21, GATA3 and RORC2 were not significantly differentially-expressed between the nine most resistant (AWC, 0; FEC, 0) and the nine most susceptible sheep (AWC, mean 6078; FEC, mean 350). Absolute RT-qPCR on 29 all 45 animals identified RORAv5 as being significantly differentially-expressed (p = 0.038) 30 between resistant, intermediate and susceptible groups; RORAv2 was not differentially- 31 expressed (p = 0.77). Spearman’s rank analysis showed that RORAv5 transcript copy number 32 was significantly negatively correlated with parameters of susceptibility, AWC and FEC; and 33 was positively correlated with BW. RORAv2 was not correlated with AWC, FEC or BW but 34 was significantly negatively correlated with IgA antibody levels [corrected]. This study identifies the full length RORA variant (RORAv5) as important in controlling the protective immune response to T. circumcincta infection in sheep.
Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC).
All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge.
In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively).
The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response.
Craniofacial microsomia (CFM) is a rare congenital anomaly that involves immature derivatives from the first and second pharyngeal arches. The genetic pathogenesis of CFM is still unclear. Here we interrogate 0.9 million genetic variants in 939 CFM cases and 2,012 controls from China. After genotyping of an additional 443 cases and 1,669 controls, we identify 8 significantly associated loci with the most significant SNP rs13089920 (logistic regression P=2.15 × 10(-120)) and 5 suggestive loci. The above 13 associated loci, harboured by candidates of ROBO1, GATA3, GBX2, FGF3, NRP2, EDNRB, SHROOM3, SEMA7A, PLCD3, KLF12 and EPAS1, are found to be enriched for genes involved in neural crest cell (NCC) development and vasculogenesis. We then perform whole-genome sequencing on 21 samples from the case cohort, and identify several novel loss-of-function mutations within the associated loci. Our results provide new insights into genetic background of craniofacial microsomia.
The frequency of breast cancer in men is extremely rare, reported to be less than 1% and there is currently no available animal model for male mammary tumors. We compared the characteristics of various immunohistochemical markers in N-methyl-N-nitrosourea (MNU)-induced mammary adenocarcinomas in male and female Crj:CD(SD)IGS rats including: estrogen receptor α (ER), progesterone receptor (PgR), androgen receptor (AR), receptor tyrosine-protein kinase erbB-2 (HER2), GATA binding protein 3 (GATA3), and proliferating cell nuclear antigen (PCNA). Female mammary adenocarcinomas were strongly positive in the nuclei of tumor cells for PCNA and ER (100%) with only 60% and 53% expressing PgR and GATA3, respectively. 100% of male adenocarcinomas also exhibited strongly positive expression in the nuclei of tumor cells for PCNA, with 25% expressing AR and only 8% showing positivity for ER. Male carcinomas did not express PgR or GATA3 and none of the tumors, male or female, were positive for HER2. Based on the observed ER and PgR positivity and HER2 negativity within these tumors, MNU-induced mammary adenocarcinomas in female rats appear to be hormonally dependent, similar to human luminal A type breast cancer. In contrast, MNU-induced mammary adenocarcinomas in male rats showed no reactivity for ER, PgR, HER2 or GATA3, suggesting no hormonal dependency. Both male and female adenocarcinomas showed high proliferating activity by PCNA immunohistochemistry. Based on our literature review, human male breast cancers are mainly dependent on ER and/or PgR, therefore the biological pathogenesis of MNU-induced male mammary cancer in rats may differ from that of male breast cancer in humans.
Penile clear cell carcinoma originating in skin adnexal glands has been previously reported. Here, we present 3 morphologically distinctive penile tumors with prominent clear cell features originating not in the penile skin but in the mucosal tissues of the glans surface squamous epithelium. Clinical and pathologic features were evaluated. Immunohistochemical stains were GATA3 and p16. Human papilloma virus (HPV) detection by in situ hybridization was performed in 3 cases, and whole-tissue section-polymerase chain reaction was performed in 1 case. Patients' ages were 52, 88, and 95 years. Tumors were large and involved the glans and coronal sulcus in all cases. Microscopically, nonkeratinizing clear cells predominated. Growth was in solid nests with comedo-like or geographic necrosis. Focal areas of invasive warty or basaloid carcinomas showing in addition warty or basaloid penile intraepithelial neoplasia were present in 2 cases. There was invasion of corpora cavernosa, lymphatic vessels, veins, and perineural spaces in all cases. p16 was positive, and GATA3 stain was negative in the 3 cases. HPV was detected in 3 cases by in situ hybridization and in 1 case by polymerase chain reaction. Differential diagnoses included other HPV-related penile carcinomas, skin adnexal tumors, and metastatic renal cell carcinoma. Features that support primary penile carcinoma were tumor location, concomitant warty and/or basaloid penile intraepithelial neoplasia, and HPV positivity. Clinical groin metastases were present in all cases, pathologically confirmed in 1. Two patients died from tumor dissemination at 9 and 12 months after penectomy. Clear cell carcinoma, another morphologic variant related to HPV, originates in the penile mucosal surface and is probably related to warty carcinomas.
The T helper type 2 (Th2) locus control region (LCR) regulates Th2 cell differentiation. Several transcription factors bind to the LCR to modulate the expression of Th2 cytokine genes, but the molecular mechanisms behind Th2 cytokine gene regulation are incompletely understood. Here, we used database analysis and an oligonucleotide competition/electrophoretic mobility shift assays to search for transcription factors binding to RHS5, a DNase I hypersensitive site (DHS) within the Th2 LCR. Consequently, we demonstrated that GATA-binding protein-3 (GATA-3), E26 transformation-specific protein 1 (Ets-1), octamer transcription factor-1 (Oct-1), and Oct-2 selectively associate with RHS5. Furthermore, chromatin immunoprecipitation and luciferase reporter assays showed that Oct-1 and Oct-2 bound within the Il4 promoter region and the Th2 LCR, and that Oct-1 and GATA-3 or Oct-2 synergistically triggered the transactivational activity of the Il4 promoter through RHS5. These results suggest that Oct-1 and GATA-3/Oct-2 direct Th2 cytokine gene expression in a cooperative manner.
Considerable progress has been made in understanding the mechanisms that control the production of specialized neuronal types. However, how the timing of differentiation contributes to neuronal diversity in the developing spinal cord is still a pending question. In this study, we show that cerebrospinal fluid-contacting neurons (CSF-cNs), an anatomically discrete cell type of the ependymal area, originate from surprisingly late neurogenic events in the ventral spinal cord. CSF-cNs are identified by the expression of the transcription factors Gata2 and Gata3, and the ionic channels Pkd2l1 and Pkd1l2. Contrasting with Gata2/3(+) V2b interneurons, differentiation of CSF-cNs is independent of Foxn4 and takes place during advanced developmental stages previously assumed to be exclusively gliogenic. CSF-cNs are produced from two distinct dorsoventral regions of the mouse spinal cord. Most CSF-cNs derive from progenitors circumscribed to the late-p2 and the oligodendrogenic (pOL) domains, whereas a second subset of CSF-cNs arises from cells bordering the floor plate. The development of these two subgroups of CSF-cNs is differentially controlled by Pax6, they adopt separate locations around the postnatal central canal and they display electrophysiological differences. Our results highlight that spatiotemporal mechanisms are instrumental in creating neural cell diversity in the ventral spinal cord to produce distinct classes of interneurons, motoneurons, CSF-cNs, glial cells and ependymal cells.
Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, we show that the miR-23∼27∼24 clusters regulate multiple aspects of T cell biology, particularly helper T (Th) 2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23∼27∼24 clusters revealed that miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, although overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17, and induced regulatory T cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest that tight regulation of miR-23∼27∼24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.
Calcitonin gene-related peptide (CGRP) is a neuropeptide with well-established immunomodulatory functions. CGRP-containing nerves innervate dermal blood vessels and lymph nodes. We examined whether CGRP regulates the outcome of Ag presentation by Langerhans cells (LCs) to T cells through actions on microvascular endothelial cells (ECs). Exposure of primary murine dermal microvascular ECs (pDMECs) to CGRP followed by coculture with LCs, responsive CD4(+) T cells and Ag resulted in increased production of IL-6 and IL-17A accompanied by inhibition of IFN-γ, IL-4, and IL-22 compared with wells containing pDMECs treated with medium alone. Physical contact between ECs and LCs or T cells was not required for this effect and, except for IL-4, we demonstrated that IL-6 production by CGRP-treated pDMECs was involved in these effects. CD4(+) cells expressing cytoplasmic IL-17A were increased, whereas cells expressing cytoplasmic IFN-γ or IL-4 were decreased by the presence of CGRP-treated pDMECs. In addition, the level of retinoic acid receptor-related orphan receptor γt mRNA was significantly increased, whereas T-bet and GATA3 expression was inhibited. Immunization at the site of intradermally administered CGRP led to a similar bias in CD4(+) T cells from draining lymph node cells toward IL-17A and away from IFN-γ. Actions of nerve-derived CGRP on ECs may have important regulatory effects on the outcome of Ag presentation with consequences for the expression of inflammatory skin disorders involving Th17 cells.
Breast cancer, one of the leading causes of mortality and morbidity among females, is regulated in part by diverse classes of adhesion molecules one of which is known as cadherins. Located at adherens junctions, the members of this superfamily are responsible for upholding proper cell-cell adhesion. Cadherins possess diverse structures and functions and any alteration in their structures or functions causes impeding of normal mammary cells development and maintenance, thus leading to breast malignancy. E-, N-, P-, VE-, Proto-, desmosomal and FAT cadherins have been found to regulate breast cancer in positive as well as negative fashion, whereby both Ecadherin (CDH1) and N-cadherin (CDH2) contribute significantly towards transitioning from epithelial state to mesenchymal state (EMT) and enacting the abnormal cells to invade and metastasize nearby and distant tissues. Aberration in gene expression of cadherins can be either due to somatic or epigenetic silencing or via transcriptional factors. Besides other cadherins, E-cadherin which serves as hallmark of EMT is associated with several regulatory factors such as Snail, Slug, Twist, Zeb, KLF4, NFI, TBX2, SIX, b-Myb, COX-2, Arf6, FOXA2, GATA3 and SMAR1, which modulate E-cadherin gene transcription to promote or represses tumor invasion and colonization. Signaling molecules such as Notch, TGF-β, estrogen receptors, EGF and Wnt initiate numerous signaling cascades via these vital factors of cell programming, controlling expression of E-cadherin at transcriptional (mRNA) and protein level. Thus, interactions of cadherins with their roles in tumor suppression and oncogenic transformation can be beneficial in providing valuable insights for breast cancer diagnosis and therapeutics development.
The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on MycN have been conducted in human tumor cells, thus, the role of MycN in normal human neural crest development is completely unknown. In the present study, we determined the role of MycN in hNCSCs isolated from in vitro-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of MycN in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of MycN in hNCSCs increases the expression of Cdkn1a, Cdkn2a and Cdkn2b, which encodes the cyclin-dependent kinases p21CIP1, p16 INK4a and p15INK4b. In addition, MycN is involved in the regulation of human sympathetic neurogenesis, as knockdown of MycN enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including Phox2a, Phox2b, Mash1, Hand2 and Gata3. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.
The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.
Although the skin constitutes the first line of defense against waterborne pathogens, there is a great lack of information regarding the skin associated lymphoid tissue (SALT) and whether immune components of the skin are homogeneously distributed through the surface of the fish is still unknown. In the current work, we have analyzed the transcription of several immune genes throughout different rainbow trout (Oncorhynchus mykiss) skin areas. We found that immunoglobulin and chemokine gene transcription levels were higher in a skin area close to the gills. Furthermore, this skin area as well as other anterior sections also transcribed significantly higher levels of many different immune genes related to T cell immunity such as T cell receptor α (TCRα), TCRγ, CD3, CD4, CD8, perforin, GATA3, Tbet, FoxP3, interferon γ (IFNγ), CD40L and Eomes in comparison to posterior skin sections. In agreement with these results, immunohistochemical analysis revealed that anterior skin areas had a higher concentration of CD3(+) T cells and flow cytometry analysis confirmed that the percentage of CD8(+) T lymphocytes was also higher in anterior skin sections. These results demonstrate for the first time that T cells are not homogeneously distributed throughout the teleost skin. Additionally, we studied the transcriptional regulation of these and additional T cell markers in response to a bath infection with viral hemorrhagic septicemia virus (VHSV). We found that VHSV regulated the transcription of several of these T cell markers in both the skin and the spleen; with some differences between anterior and posterior skin sections. Altogether, our results point to skin T cells as major players of teleost skin immunity in response to waterborne viral infections.
Leprosy outcome is a complex trait and the host-pathogen-environment interaction defines the emergence of the disease. Host genetic risk factors have been successfully associated to leprosy. The 10p13 chromosomal region was linked to leprosy in familial studies and GATA3 gene is a strong candidate to be part of this association. Here, we tested tag single nucleotide polymorphisms at GATA3 in two case-control samples from Brazil comprising a total of 1633 individuals using stepwise strategy. The A allele of rs10905284 marker was associated with leprosy resistance. Then, a functional analysis was conducted and showed that individuals carrying AA genotype express higher levels of GATA-3 protein in lymphocytes. So, we confirmed that the rs10905284 is a locus associated to leprosy and influences the levels of this transcription factor in the Brazilian population.
A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20 °C-35 °C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25 °C and 30 °C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.
Hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder. We report the first detailed case of hypoparathyroidism complicated by biliary atresia.
A 1-year-old Japanese girl was admitted to our hospital for living donor liver transplantation. She suffered from obstructive jaundice owing to biliary atresia. She also had persistent hypocalcemia. Despite oral calcium and abundant vitamin D supplementation, a laboratory test showed hypocalcemia (1.4 mmol/l) and hyperphosphatemia (2.6 mmol/l). The intact parathyroid hormone level was normal (66 ng/l) with severe vitamin D deficiency (25-hydroxy vitamin D: undetectable levels). There were no rachitic changes in metaphysis on X-rays. Her family history showed that her mother had sensorineural deafness, a low serum calcium level (2.1 mmol/l), hypoplastic left kidney, and a past history of an operation for right vesicoureteral reflux. We suspected that this patient and her mother have hypoparathyroidism, sensorineural deafness, and renal dysplasia syndrome. A heterozygous GATA3 gene mutation (c.736delGinsAT) was found in this patient and her mother, but not in her father.
This familial case confirms the importance of family history in the diagnosis of HDR syndrome. Regardless of marked vitamin D deficiency, the complication of hypoparathyroidism prevented the onset of vitamin D deficiency rickets in our patient.
The purpose of the current study was to investigate the anti-asthmatic effect of esculetin (ES) and explore its potential mechanism with a mouse model of allergic asthma. A total number of 50 mice were randomly assigned to five groups: control, model, dexamethasone (Dex, 2 mg/kg), and ES (20 mg/kg, 40 mg/kg). Mouse asthma model was developed with the sensitization and challenge of ovalbumin (OVA). The levels of IgE in serum, eosinophilia infiltration, Th2/Th17 cytokines, Th17 cell frequency, histological condition, and the protein expressions of RORγt, GATA3 were detected. Our study demonstrated that ES inhibited, OVA-induced eosinophil count, interleukin-4 (IL-4), IL-5, IL-13, and IL-17A levels were recovered in bronchoalveolar lavage fluid. Flow cytometry (FCM) studies revealed that ES substantially inhibited Th17 cells' percentage. Western blot study also indicated that ES downregulated RORγt and GATA3 expressions. Meanwhile, ES had beneficial effects on the histological alteration. These findings suggested that ES might effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.
Although many studies have implicated the crosstalk between the Wnt and PKC signaling pathways in tumor initiation and progression, the molecular roles of PKC isoforms in the Wnt signaling pathway remain poorly understood. In this study, we explored the contribution of PKC isoforms to canonical and noncanonical Wnt signaling pathway in mediating cell migration and an epithelial-mesenchymal transition (EMT). When MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 3 weeks, the effect of TPA on Wnt signaling pathway was dramatically different depending on the exposure time. The short term exposure (3 days) of MCF-7 cells to TPA exhibited significant induction of Wnt5a expression, along with the enhanced expression of PKC-α, to promote cell migration, which suggested that activation of noncanonical Wnt signaling pathway is associated with PKC-α. However, the chronic exposure (3 weeks) of cells to TPA completely suppressed Wnt5a expression and the expression of PKC-η and PKC-δ, whereas the expression of Wnt3a and PKC-θ were up-regulated to activate the canonical Wnt signaling pathway. Moreover, the loss of epithelial markers, including E-cadherin and GATA-3, suggested that chronic exposure of TPA stimulates EMT. Taken together, our data suggest that PKC-θ positively regulates the canonical Wnt signaling pathway, and that PKC-η and PKC-δ negatively modulate this signaling pathway.
Chronic inflammatory diseases including allergies and asthma are the result of complex interactions between genes and environmental factors. Epigenetic mechanisms comprise a set of biochemical reactions that regulate gene expression. In order to understand the cause-effect relationship between environmental exposures and disease development, methods capable of assessing epigenetic regulation (also) in large cohorts are needed.
For this purpose, we developed and evaluated a miniaturized chromatin immunoprecipitation (ChIP) assay allowing for a cost-effective assessment of histone acetylation of candidate genes in a quantitative fashion. This method was then applied to assess H3 and H4 histone acetylation changes in cord blood (CB) samples from an established cohort of Australian children exposed in the fetal period to either very low or very high levels of maternal folate.
Our ChIP assay was validated for a minimum requirement of 1 × 105 target cells (e.g. CD4+ T cells). Very high levels of maternal folate were significantly associated with increased H3/H4 acetylation at GATA3 and/or IL9 promoter regions in CD4+ T cells in CB.
We developed a ChIP method allowing reliable assessment of H3/H4 acetylation using 1 × 105 cells only. Practical application of this assay demonstrated an association between high maternal folate exposure and increased histone acetylation, corresponding to a more transcriptionally permissive chromatin status in the promoter regions of some Th2-related genes.
Hypoparathyroidism, deafness, and renal dysplasia (HDR; OMIM 146255) syndrome is a rare disease, inherited dominantly and found to be related with GATA3 (GATA binding protein 3) gene mutations. A 13-year and 8-month-old boy who presented with hypocalcemia was diagnosed with hypoparathyroidism. He also had dysmorphic facial features, renal anomaly (pelvic kidney), and mild sensorineural hearing loss. His cranial computed tomography revealed multiple calcifications in bilateral centrum semiovale, corona radiata, and basal ganglions suggesting a persistent hypoparathyroidism. Thus, the presence of triad of HDR syndrome was considered, and genetic analysis using a next-generation sequencer identified a novel de novo missense mutation in exon 4 p.R276Q (c.827G>A) of GATA3 gene. This is the second patient who was reported to have a mutation in GATA3 gene from Turkey. In conclusion, although HDR syndrome is a rare condition, it should be kept in mind in patients with hypoparathyroidism. Classical triad can easily be identified if patients diagnosed with hypoparathyroidism are also evaluated with a urinary tract ultrasound and an audiometer.
Bone marrow-derived monocyte-to-fibroblast transition is a key step in renal fibrosis pathogenesis, which is regulated by the inflammatory microenvironment. However, the mechanism by which the inflammatory microenvironment regulates this transition is not fully understood. In this study, we examined how the CD8(+) T cell/IFN-γ microenvironment regulates the monocyte-to-fibroblast transition in renal fibrosis. Genetic ablation of CD8 promoted a monocyte-to-fibroblast transition and increased renal interstitial fibrosis, whereas reconstitution of CD8 knockout (KO) mice with CD8(+) T cells decreased fibrosis. However, depletion of CD4(+) T cells in CD8 KO mice also reduced fibrosis. To elucidate the role of CD4(+) T cells in mediating CD8-regulated monocyte-to-fibroblast transition, CD4(+) T cells were isolated from obstructed kidneys of CD8 KO or wild-type mice. CD4(+) T cells isolated from CD8 KO obstructed kidney expressed more IL-4 and GATA3 and less IFN-γ and T-bet and showed increased monocyte-to-fibroblast transition in vitro compared with those isolated from wild-type obstructed kidney. To examine the role of IFN-γ-expressing CD8(+) T cells, we reconstituted CD8 KO mice with CD8(+) T cells isolated from IFN-γ KO mice. The IFN-γ KO CD8(+) cells had no effect on IL-4, GATA3, IFN-γ, and T-bet mRNA expression in obstructed kidneys or renal fibrosis. Taken together, our findings identify the axis of CD8(+) T cells and IFN-γ-CD4(+) T cells as an important microenvironment for the monocyte-to-fibroblast transition, which negatively regulates renal fibrosis.
NK cells are innate lymphocytes capable of eliciting an innate immune response to pathogens. NK cells develop and become mature in the bone marrow (BM) before they migrate out to peripheral organs. Although the developmental program leading to mature NK cells has been studied in the context of several transcription factors, the stage-specific role of GATA3 in NK cell development has been incompletely understood. Using NKp46-Cre-Gata3(fl/fl) mice in which Gata3 deficiency was induced as early as the immature stage of NK cell differentiation, we demonstrated that GATA3 is required for the NK cell maturation beyond the CD27 single-positive stage and is indispensable for the maintenance of liver-resident NK cells. The frequencies of NK cells from NKp46-Cre-Gata3(fl/fl) mice were found higher in the BM but lower in peripheral organs compared with control littermates, indicating that GATA3 controls the maturation program required for BM egress. Despite the defect in maturation, upon murine CMV infection, NK cells from NKp46-Cre-Gata3(fl/fl) mice expanded vigorously, achieving NK cell frequencies surpassing those in controls and therefore provided comparable protection. The heightened proliferation of NK cells from NKp46-Cre-Gata3(fl/fl) mice was cell intrinsic and associated with enhanced upregulation of CD25 expression. Taken together, our results demonstrate that GATA3 is a critical regulator for NK cell terminal maturation and egress out of the BM and that immature NK cells present in the periphery of NKp46-Cre-Gata3(fl/fl) mice can rapidly expand and provide a reservoir of NK cells capable of mounting an efficient cytotoxic response upon virus infection.
We identified 109 testicular tumors, including pure and mixed germ cell tumors and sex cord-stromal tumors, and conducted immunohistochemical staining for CDX2, DOG1, and GATA3 to address the potential utility of these readily available and commonly used markers in the evaluation of testicular tumors. Their expression has not been previously thoroughly examined in testicular germ cell tumors. The distribution, percentage, and intensity of positivity were assessed. CDX2 was positive in all yolk sac tumors, 25% of choriocarcinomas, 9% of seminomas, and 4% of embryonal carcinomas (sensitivity for yolk sac tumor, 100%; specificity, 89% [teratomas excluded]). CDX2 also stained glandular components within teratomas and identified inconspicuous yolk sac tumor components in 3 cases previously diagnosed as pure embryonal carcinoma. GATA3 was positive in all choriocarcinomas (sensitivity, 100%). Weak GATA3 immunostaining was also seen in 12% of yolk sac tumors and 2 of 2 primitive neuroectodermal tumors. DOG1 was negative in all tumors, but stained spermatocytes and spermatids and the luminal borders of the epididymis and rete testis of nonneoplastic testis. We conclude that CDX2 is a sensitive and relatively specific marker for yolk sac tumor among the nonteratomatous germ cell tumors. It may serve to screen for yolk sac tumor components often overlooked on hematoxylin and eosin-stained slides. GATA3 is helpful in the recognition of trophoblastic cells, especially of intermediate type. DOG1 is a sensitive marker for spermatocytes and needs to be further studied for its significance.
This review summarizes the three major breast-associated markers that can be of assistance in evaluating metastatic carcinomas for which a breast primary diagnosis is entertained. These markers include gross cystic disease fluid protein-15 (GCDFP-15), mammaglobin, and GATA3. The first two are cytoplasmic markers that show comparable sensitivities for breast cancer, although relatively few of the published studies have employed the same antibodies against the target molecule, making direct comparisons challenging. GATA3 is a nuclear transcription factor that shows superior sensitivity to GCDFP-15 and mammaglobin. However, the specificity of GATA3 can pose challenges, inasmuch as carcinomas of the bladder and other sites can show significant levels of positivity. Determination of the optimal panel of antibodies employed in a given clinical setting will thus depend on the non-breast tumours included in the differential diagnosis.
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a severe autoimmune disease that is caused by regulatory T cell deficiency due to FOXP3 gene mutations. The long-term outcome can be variable depending on the extent of tissue damage caused by autoimmunity and infections, the use of immunosuppressive treatment or sequela of bone marrow transplantation.
We used immunohistochemical staining to analyze cell types infiltrating the tissue of affected organs from a classic IPEX patient with a splicing mutation (c.736-2A>C) in the FOXP3 gene. Expression of transcription factors that are critical for immune responses including T-bet, GATA-3, RORγt, and FOXP3 were evaluated in various tissue samples. For objective analysis of the distribution of different cell types in tissues, we used an automated microscope-based image acquiring system to assess quantitatively the different cell types by investigating the histopathological changes in the patient's biopsy samples obtained from the intestine and the kidneys before and after treatment.
The percentages of cells expressing the TH2-associated transcription factor GATA3 were higher in the IPEX patient before treatment than in controls, suggesting that TH2-type cells contribute to the tissue inflammation of the gut and kidneys in IPEX syndrome. Immunosuppressive treatment effectively decreased the number of effector cells in the kidneys and intestine of the IPEX patient.
This study provides quantitative evidence that the inflamed intestinal and renal tissues of the IPEX patient contain TH2-type immune effector cells, which decreased in number after immunosuppressive treatment was initiated and the clinical symptoms had improved.
Protection of epithelial and mucosal surfaces is required for survival. The recent discovery of a diverse array of innate lymphoid cells that lie immediately beneath these surfaces has unexpectedly uncovered an entire defense system distinct from the adaptive system essential to protect these barriers. This multilayered design provides a robust system through coupling of two highly complementary networks to ensure immune protection. Here, we discuss the similarities in the hardwiring and diversification of innate lymphoid cells and T cells during mammalian immune responses.
Human CRTh2(+) Th2 cells express IL-25 receptor (IL-25R) and IL-25 has been shown to potentiate production of Th2 cytokines. However, regulation of IL-25R and whether it participates in Th2 differentiation of human cells have not been examined. We sought to characterize IL-25R expression on CD4(+) T cells and determine whether IL-25 plays a role in Th2 differentiation. Naïve human CD4(+) T cells were activated in the presence of IL-25, IL-4 (Th2 conditions) or both cytokines to assess their relative influence on Th2 differentiation. For experiments with differentiated Th2 cells, CRTh2-expressing cells were isolated from differentiating cultures. IL-25R, GATA3, CRTh2 and Th2 cytokine expression were assessed by flow cytometry, qRT-PCR and ELISA. Expression of surface IL-25R was induced early during Th2 differentiation (2 days). Addition of IL-25 to naïve CD4(+) T cells revealed that it induces expression of its own receptor, more strongly than IL-4. IL-25 also increased the proportions of IL-4-, GATA3- and CRTh2-expressing cells and expression of IL-5 and IL-13. Activation of differentiated CRTh2(+) Th2 cells through the TCR or by CRTh2 agonist increased surface expression of IL-25R, though re-expression of CRTh2 following TCR downregulation was impeded by IL-25. These data suggest that IL-25 may play various roles in Th2 mediated immunity. We establish here it regulates expression of its own receptor and can initiate Th2 differentiation, though not as strongly as IL-4.
Atypical presentations of complex multisystem disorders may elude diagnosis based on clinical findings only. Appropriate diagnostic tests may not be available or available tests may not provide appropriate coverage of relevant genomic regions for patients with complex phenotypes. Clinical whole-exome/-genome sequencing is often considered for complex patients lacking a definitive diagnosis.
A boy who is now 7 years old presented as a newborn with congenital ichthyosis. At 6 weeks of age, he presented with failure to thrive and hypoparathyroidism. At 4 years of age, he was diagnosed with sensorineural hearing loss. Whole-genome sequencing identified novel mutations in GATA3, which causes HDR syndrome (hypoparathyroidism and deafness), and STS, which causes X -linked congenital ichthyosis.
Whole-genome sequencing led to a definitive clinical diagnosis in a case where no other clinical test was available for GATA3, and no sequencing panel would have included both genes because they have disparate phenotypes. This case demonstrates the power of whole-genome (or exome) sequencing for patients with complex clinical presentations involving endocrine abnormalities.
Breast cancer is one of the most frequently occurring cancers in women. In recent years, Dendrobium candidum has played a part in antihyperthyroidism and anticancer drugs. This study aims to examine the antitumor effect of D. candidum on breast cancer.
Human breast cancer cell line MCF-7 and normal breast epithelial cell line MCF10A were used to observe the effects of D. candidum treatment on human breast cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to examine the cell proliferation of the MCF-7 and MCF10A cells. Western blot analysis and reverse transcription polymerase chain reaction were used to detect the key molecules and biomarkers in breast cancer pathology. Cell cycle was analyzed by using Becton Dickinson FACScan cytofluorometer.
The results indicated that D. candidum significantly decreased cell viability at different concentrations compared to the control group (P<0.05). D. candidum-treated MCF-7 cells in the G2/M phase was significantly increased compared to the control group (P<0.05). The messenger RNA levels of estrogen receptor alpha, IGFBP2, IGFBP4, and GATA3 were significantly decreased, and the messenger RNA and protein levels of ELF5, p53, p21, p18, CDH1, CDH2, and p12 were significantly increased, compared to the control group (P<0.05). The protein levels of estrogen receptor alpha, PGR, GATA3, and Ki67 were significantly decreased and the protein levels of p53 and ELF5 were significantly increased compared to the control group (P<0.05). The general apoptosis biomarker, Bcl-2, was significantly decreased and the Bax was significantly increased compared to the control group (P<0.05). In contrast to that in MCF-7, D. candidum does not affect cell proliferation at any concentration and any time points in normal breast epithelial cells, MCF10A cells.
D. candidum could decrease the cell viability of MCF-7 cells by inducing cell cycle arrest at the G2/M phase and regulating the key biomarkers in breast cancer cells.
Genetic selection for enhanced immune response has been shown to decrease disease occurrence in dairy cattle. Cows can be classified as high (H), average, or low responders based on antibody-mediated immune response (AMIR), predominated by type-2 cytokine production, and cell-mediated immune response (CMIR) through estimated breeding values for these traits. The purpose of this study was to identify in vitro tests that correlate with in vivo immune response phenotyping in dairy cattle. Blood mononuclear cells (BMC) isolated from cows classified as H-AMIR and H-CMIR through estimated breeding values for immune response traits were stimulated with concanavalin A (ConA; Sigma Aldrich, St. Louis, MO) and gene expression, cytokine production, and cell proliferation was determined at multiple time points. A repeated measures model, which included the effects of immune response group, parity, and stage of lactation, was used to compare differences between immune response phenotype groups. The H-AMIR cows produced more IL-4 protein than H-CMIR cows at 48 h; however, no difference in gene expression of type-2 transcription factor GATA3 or IL4 was noted. The BMC from H-CMIR cows had increased production of IFN-γ protein at 48, 72, and 96 h compared with H-AMIR animals. Further, H-CMIR cows had increased expression of the IFNG gene at 16, 24, and 48 h post-treatment with ConA, although expression of the type-1 transcription factor gene TBX21 did not differ between immune response groups. Although proliferation of BMC increased from 24 to 72 h after ConA stimulation, no differences were found between the immune response groups. Overall, stimulation of H-AMIR and H-CMIR bovine BMC with ConA resulted in distinct cytokine production profiles according to genetically defined groups. These distinct cytokine profiles could be used to define disease resistance phenotypes in dairy cows according to stimulation in vitro; however, other immune response phenotypes should be assessed.
Skin tumor susceptibility 5 (Skts5) was previously mapped to mouse chromosome 12 through linkage analysis of skin tumor susceptible Mus musculus (NIH/Ola-S) and skin tumor resistant outbred Mus spretus (SPRET/Out-R) mice. Hdac9 was identified as a potential candidate for Skts5 based on conserved non-synonymous sequence variants and expression analyses. Studies by others identified an enhancer in human HDAC9 that correlated with TWIST1 expression. We identified 45 sequence variants between NIH/Ola-S and SPRET/Out-R mice from the orthologous region of the human HDAC9 enhancer. Variants mapping to intron 18 differentially affected luciferase expression in vitro. NIH/Ola-S clones showed an approximate 1.7-fold increased luciferase expression relative to vector alone or the equivalent clones from SPRET/Out-R-R. Furthermore, cells transfected with a portion of the NIH/Ola-S intron induced 2.2-fold increases in Twist1 expression, but the same region from SPRET/Out-R mice resulted in no up-regulation of Twist1. In silico transcription factor analyses identified multiple transcription factors predicted to differentially bind NIH/Ola-S and SPRET/Out-R polymorphic sites. Chromatin immunoprecipitation studies of two transcription factors, Gata3 and Oct1, demonstrated differential binding between NIH/Ola-S and SPRET/Out-R plasmids that corroborated the in silico predictions. Together these studies provide evidence that the murine orthologous region to a human HDAC9 enhancer also acts as a transcriptional enhancer for mouse Twist1. As ectopic sequence variants between NIH/Ola-S and SPRET/Out-R differentially impacted luciferase expression, correlated with Twist1 expression in vitro, and affected Gata3 and Oct1 binding, these variants may explain part of the observed differences in skin tumor susceptibility at Skts5 between NIH/Ola-S and SPRET/Out-R.
GATA3 and fascin proteins are known prognostic markers in several cancers. GATA3 is a key regulator of mammary gland morphogenesis and luminal cell differentiation, whereas fascin is a pro-metastatic actin-bundling protein. In this study, we analyzed and compared the predictive abilities of GATA3 and fascin for clinical outcomes of patients with breast cancer. The combined expression pattern based on GATA3-/+ and fascin-/+ was evaluated by immunostaining using a tissue microarray, and relationships between protein expression and several clinicopathological parameters were analyzed. GATA3 expression was associated with good prognostic parameters, but fascin was correlated with poor prognostic parameters. On comparing GATA3 and fascin, we found an inverse relationship between fascin and GATA3 expressions. On analysis of combined markers, GATA3+/fascin- was correlated with improved clinical outcomes compared to GATA3-/fascin+. Univariate and multivariate analyses revealed significant differences in relapse-free and overall survival between GATA3+/fascin- and GATA3-/fascin+. Combined marker analysis of GATA3/fascin showed an inverse association and improved prognostic information for patients with breast cancer.
We examined the pathogenesis of glomerular damage in Th2 type-dependent GATA-3 transgenic (GATA-3 Tg) mice with IgA nephropathy (IgAN). GATA-3 Tg mice were immunized orally using OVA plus cholera toxin B (CTB), and measurement of the serum IgA antibody level and histopathological examination were performed. Marked increases in the serum levels of OVA-specific IgA antibody, IgA and IgG, C3 deposits analogous to those seen in IgAN, and expansion of the matrix in association with mesangial cell proliferation were observed. Furthermore, glomerular IgA deposits were co-localized with mannan-binding lectin (MBL) deposits, which might actually have been abnormal IgA deposits. In GATA-3/TCR-Tg mice that had been orally sensitized with CTB plus OVA and were re-stimulated with OVA in vitro, cultured Peyer's patch cells showed the enhanced production of IL-5 and supernatants from cultures of spleen cells showed a reduction of TGF-β production with a simultaneous increase in IL-2 production and the recovery of IFN-γ formation. The amount of TGF-β produced by the spleen cells was found to be correlated with the amount of IFN-γ and IL-IL-2 produced by the cells. Also, the percentage of regulatory T cells (Treg) in the spleens of mice sensitized with OVA plus CTB was lower than that in mice orally sensitized with OVA alone. These results suggest that the increased production of IL-5 from Peyer's patch cells (PPc) and the restored Th1-type immune response might cause the production of abnormal IgA and might induce the deposition of IgA in glomeruli.
Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits.
The syndrome of familial hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR syndrome) is inherited as an autosomal dominant trait, caused by haploinsufficiency of the GATA3 gene in chromosome 10p. Although first described years ago, but the disease is considered to be very rare. Patients usually present with hypocalcemia, tetany, or afebrile convulsions at any age. Hearing loss is usually bilateral, range from mild to profound impairment. Renal disease includes dysplasia, hypoplasia or aplasia.
Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.
The plant protein trichosanthin (Tk) and its derived peptide tetramer Tk-tPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7.7 ± 0.3 days (control) to 22.7 ± 3.9 days (P < 0.01) or 79.1 ± 19.2 days (P < 0.0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/transcription factor (IL-2(+) IFN-γ(+) T-bet(+)), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL-4(+) IL-10(+) Gata3+), together with a selective expansion of the IL-4/IL-10-producing CD8+ CD28- regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28- regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation.
Methamphetamine (METH) is an addictive psychostimulant and has been shown to induce oxidative stress and inflammation in various tissues. Thioredoxin-1 (Trx-1) plays the roles in regulating redox and inhibiting inflammation. Whether Trx-1 is involved in METH-induced inflammation is still unknown.
The present study was designed to investigate inflammatory factors in spleen of wild type and Trx-1 overexpression transgenic mice after METH treatment.
We found the mRNA level of Trx-1 was decreased and mRNA level of Trx-1 binding protein-2 (TBP-2) was increased. The mRNA levels of tumor necrosis factor-α (TNF-α), interferon-γ(IFN-γ), interleukin-2 (IL-2), T-bet and signal transducer and activators of transcription 4 (STAT 4) were increased and the mRNA levels of IL-10, GA-TA-binding protein-3 (GATA-3) and STAT 6 were decreased. Overexpression of Trx-1 reversed the above effects induced by METH.
The present study showed for the first time that Trx-1 overexpression suppressed the inflammation induced by METH.
Previously, we observed that wild yam (Dioscorea villosa) root extract (WYRE) was able to activate GATA3 in human breast cancer cells targeting epigenome. This study aimed to find out if dioscin (DS), a bioactive compound of WYRE, can modulate GATA3 functions and cellular invasion in human breast cancer cells. MCF-7 and MDA-MB-231 cells were treated in the absence/presence of various concentrations of DS and subjected to gene analysis by RT-qPCR, immunoblotting, and immunocytochemistry. We determined the ability of MDA-MB-231 cells to migrate into wound area and examined the effects of DS on cellular invasion using invasion assay. DS reduced cell viability of both cell lines in a concentration and time-dependent manner. GATA3 expression was enhanced by DS (5.76 μM) in MDA-MB-231 cells. DS (5.76 μM)-treated MDA-MB-231 cells exhibited the morphological characteristic of epithelial-like cells; mRNA expression of DNMT3A, TET2, TET3, ZFPM2 and E-cad were increased while TET1, VIM and MMP9 were decreased. Cellular invasion of MDA-MB-231 was reduced by 65 ± 5% in the presence of 5.76 μM DS. Our data suggested that DS-mediated pathway could promote GATA3 expression at transcription and translation levels. We propose that DS has potential to be used as an anti-invasive agent in breast cancer.
Interleukin (IL)-10, a non-redundant anti-inflammatory cytokine is produced by different cells and its production involves activation of cell-specific transcriptional regulatory machinery in response to specific pathogen. We have previously demonstrated downregulated levels of IL-10 in severe falciparum malaria. The present study investigated transcriptional regulation of IL-10 in severe malaria. Comparative expression analysis of cell-specific signalling proteins and transcription factors for IL-10 production during the stage of active infection and with resolution of parasitaemia was performed. Interestingly, T-bet and GATA3, the Th1 and Th2 transcription factors, respectively, were downregulated in severe malaria with fold change values of 0.59 and 0.86. Increase in the levels of both the factors with resolution of parasitaemia implicated a role for parasite in depressed levels of these factors. Further support for probable parasite manipulation of GATA3 was obtained from negative correlation of GATA3 with parasitaemia. In addition, a role for interferon-α in suppressing IL-10 transcription was evident from its negative correlation with GATA3 and IL-10 levels. In summary, IL-10 transcription in Th1 and Th2 is defective and appears to have major contribution to low levels in severe malaria.
The natural progression of HIV-1 begins with a short acute retroviral syndrome which typically transit to chronic and clinical latency stages and subsequently progresses to a symptomatic, life-threatening immunodeficiency disease known as AIDS. Microarray analysis based on gene coexpression is widely used to investigate the coregulation pattern of a group (or cluster) of genes in a specific phenotype. Moreover, an investigation on the topological patterns across multiple phenotypes can facilitate the understanding of stage specific infection pattern of HIV-1 virus. Here, we develop a novel framework to identify topological patterns of gene coexpression network and detect changes of modular structure across different stages of HIV progression. This is achieved by comparing the topological and intramodular properties of HIV infection modules. To capture the diversity in modular structure, some topological, correlation based and eigengene based measures are utilized here. We have applied a rank aggregation scheme to rank all the modules to provide a good agreement between these measures. Some novel transcription factors like 'FOXO1','GATA3', 'GFI1','IRF1', 'IRF7', 'MAX', 'STAT1', 'STAT3', 'XBP1' and 'YY1' that emerge from the modules show significant change in expression pattern over HIV progression stages. Moreover, we have performed an eigengene based analysis to reveal the perturbation in modular structure across three stages of HIV-1 progression.
Complex insertions and deletions (indels) are formed by simultaneously deleting and inserting DNA fragments of different sizes at a common genomic location. Here we present a systematic analysis of somatic complex indels in the coding sequences of samples from over 8,000 cancer cases using Pindel-C. We discovered 285 complex indels in cancer-associated genes (such as PIK3R1, TP53, ARID1A, GATA3 and KMT2D) in approximately 3.5% of cases analyzed; nearly all instances of complex indels were overlooked (81.1%) or misannotated (17.6%) in previous reports of 2,199 samples. In-frame complex indels are enriched in PIK3R1 and EGFR, whereas frameshifts are prevalent in VHL, GATA3, TP53, ARID1A, PTEN and ATRX. Furthermore, complex indels display strong tissue specificity (such as VHL in kidney cancer samples and GATA3 in breast cancer samples). Finally, structural analyses support findings of previously missed, but potentially druggable, mutations in the EGFR, MET and KIT oncogenes. This study indicates the critical importance of improving complex indel discovery and interpretation in medical research.
Atopic dermatitis (AD) is a skin condition caused by an imbalance of distinct subsets of T helper cells. Previously, we showed that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) inhibits T cell activation but does not induce apoptosis. Here, we examined the mechanism underlying the inhibitory effect of 4H3MC on AD both in vivo and in vitro. We sought to test the pharmacological effects of 4H3MC using a mouse model of 2, 4-'2,4-dinitrocholorobenzene' (DNCB)- and mite-induced AD. Also, we determined whether 4H3MC affects T cell differentiation and proliferation. Oral administration of 4H3MC attenuated the symptoms of DNCB- and mite-induced AD, including increased ear thickness, serum IgE levels, immune cell infiltration into inflammatory lesions, and pathogenic cytokine expression in ear tissues. In vitro, 4H3MC blocked T cell differentiation into Th1 and Th2 subtypes, as reflected by suppression of T-bet and GATA3, which are key transcription factors involved in T cell differentiation. In addition, 4H3MC downregulated T cell proliferation during Th1 and Th2 differentiation and keratinocyte activation. Collectively, these findings suggest that 4H3MC ameliorates AD symptoms by modulating the functions of effector T cells and keratinocytes.
Chronic rhinosinusitis (CRS) affects more than 10% of the population in the United States and Europe. Recent findings point to a considerable variation of inflammatory subtypes in patients with CRS with nasal polyps and patients with CRS without nasal polyps. According to current guidelines, glucocorticosteroids and antibiotics are the principle pharmacotherapeutic approaches; however, they fail in a group of patients who share common clinical and laboratory markers. Several clinical phenotypes often leading to uncontrolled disease, including adult nasal polyposis, aspirin-exacerbated respiratory disease, and allergic fungal rhinosinusitis, are characterized by a common endotype: a TH2 bias is associated with a higher likelihood of comorbid asthma and recurrence after surgical treatment. As a consequence, several innovative approaches targeting the TH2 bias with humanized mAbs have been subjected to proof-of-concept studies in patients with CRS with nasal polyps with or without comorbid asthma: omalizumab, reslizumab, mepolizumab, and recently dupilumab. Future concepts using upstream targets, such as GATA-3, also focus on this endotype. This current development might result in advantages in the treatment of patients with the most severe CRS.
The effects of Lactobacillus plantarum (L. plantarum) NCU116 isolated from pickled vegetables on intestine mucosal immunity in cyclophosphamide treated mice were investigated. Animals were divided into six groups: normal group (NIM), immunosuppression group (IM), immunosuppression plus L. plantarum NCU116 groups with three different doses (NCU-H, NCU-M, and NCU-L), and plus Bifidobacterium BB12 as positive control group (BB12). Results showed that the thymus indexes of the four treatment groups were significantly higher than that of the IM group (2.02 ± 0.16) (p < 0.05) and close to the index of the NIM group (2.61 ± 0.37) at 10 days. The level of immune factor IL-2 notably increased (IM, 121 ± 9.0) (p < 0.05) and was close to 65% of NIM group's level (230 ± 10.7). The levels of other immune factors (IFN-γ, IL-10, IL-12p70, and sIgA), the gene expression levels of IL-2 and IFN-γ, and the number of IgA-secreting cells showed similar patterns (p < 0.05). However, the level of immune factor IL-4 remarkably decreased (IM, 128 ± 10.2) (p < 0.05) and was only approximately 50% of the NIM group (154 ± 18.2). The levels of other immune factors (IL-6 and IgE) and the gene expression level of IL-6 at 10 days exhibited similar changes (p < 0.05) but showed a slight recovery at 20 days, accompanied by the altered protein expression levels of T-bet and GATA-3 in the small intestine. These findings suggest that L. plantarum NCU116 enhanced the immunity of the small intestine in the immunosuppressed mice.
The objective of the present study was to validate prognostic gene signature for estrogen receptor alpha-positive (ER03B1+) and lymph node (+) breast cancer for improved selection of patients for adjuvant therapy. In our previous study, we identified a group of seven genes (GATA3, NTN4, SLC7A8, ENPP1, MLPH, LAMB2, and PLAT) that show elevated messenger RNA (mRNA) expression levels in ERα (+) breast cancer patient samples. The prognostic values of these genes were evaluated using gene expression data from three public data sets of breast cancer patients (n = 395). Analysis of ERα (+) breast cancer cohort (n = 195) showed high expression of GATA3, NTN4, and MLPH genes significantly associated with longer relapse-free survival (RFS). Next cohort of ERα (+) and node (+) samples (n = 109) revealed high mRNA expression of GATA3, SLC7A8, and MLPH significantly associated with longer RFS. Multivariate analysis of combined three-gene signature for ERα (+) cohort, and ERα (+) and node (+) cohorts showed better hazard ratio than individual genes. The validated three-gene signature sets for ERα (+) cohort, and ERα (+) and node (+) cohort may have potential clinical utility since they demonstrated predictive and prognostic ability in three independent public data sets.
The present study shows that an application of cyclophosphamide (CY) supported by dendritic cell (DC)-based vaccines affected differentiation of the activity of CD4+ T cell subpopulations accompanied by an alteration in CD8+ cell number. Vaccines were composed of bone marrow-derived DCs activated with tumor cell lysate (BM-DC/TAgTNF-α) and/or genetically modified DCs of JAWS II line (JAWS II/Neo or JAWS II/IL-2 cells). Compared to untreated or CY-treated mice, the combined treatment of MC38 colon carcinoma-bearing mice resulted in significant tumor growth inhibition associated with an increase in influx of CD4+ and CD8+ T cells into tumor tissue. Whereas, the division of these cell population in spleen was not observed. Depending on the nature of DC-based vaccines and number of their applications, both tumor infiltrating cells and spleen cells were able to produce various amount of IFN-γ, IL-4 and IL-10 after mitogenic ex vivo stimulation. The administration of CY followed by BM-DC/TAgTNF-α and genetically modified JAWS II cells, increased the percentage of CD4+T-bet+ and CD4+GATA3+ cells and decreased the percentage of CD4+RORγt+ and CD4+FoxP3+ lymphocytes. However, the most intensive response against tumor was noted after the ternary treatment with CY + BM-DC/TAgTNF-α + JAWS II/IL-2 cells. Thus, the administration of various DC-based vaccines was responsible for generation of the diversified antitumor response. These findings demonstrate that the determination of the size of particular CD4+ T cell subpopulations may become a prognostic factor and be the basis for future development of anticancer therapy.
GATA3 is a highly conserved, essential transcription factor expressed in a number of tissues, including the mammary gland. GATA3 expression is required for normal development of the mammary gland where it is estimated to be the most abundant transcription factor in luminal epithelial cells. In breast cancer, GATA3 expression is highly correlated with the luminal transcriptional program. Recent genomic analysis of human breast cancers has revealed high-frequency mutation in GATA3 in luminal tumors, suggesting "driver" function(s). Here we discuss mutation of GATA3 in breast cancer and the potential mechanism(s) by which mutation may lead to a growth advantage in cancer.
Previous studies have shown that BMP4 may play an important part in the development of auditory neurons (ANs), which are degenerated in sensorineural hearing loss. However, whether BMP4 can promote sensory fate specification from mesenchymal stromal cells (MSCs) is unknown so far.
MSCs isolated from Sprague-Dawley (SD) rats were confirmed by expression of MSC markers using flow cytometry and adipogenesis/osteogenesis using differentiation assays. MSCs treated with a complex of neurotrophic factors (BMP4 group and non-BMP4 group) were induced into auditory neuron-like cells, then the differences between the two groups were analyzed in morphological observation, cell growth curve, qRT-PCR, and immunofluorescence.
Flow cytometric analysis showed that the isolated cells expressed typical MSC surface markers. After adipogenic and osteogenic induction, the cells were stained by oil red O and Alizarin Red. The neuronal induced cells were in the growth plateau and had special forms of neurons. In the presence of BMP4, the inner ear genes NF-M, Neurog1, GluR4, NeuroD, Calretinin, NeuN, Tau, and GATA3 were up-regulated in MSCs.
MSCs have the capacity to differentiate into auditory neuron-like cells in vitro. As an effective inducer, BMP4 may play a key role in transdifferentiation.
Allergic contact dermatitis (ACD) remains a major skin disease in many countries, necessitating the discovery of novel and effective anti-ACD agents. In this study, we investigated the preventive effects of Achyranthis radix extract (AcRE) on trimellitic anhydride (TMA)-induced dermatitis and the potential mechanism of action involved. Oral administration of AcRE and prednisolone (PS) significantly suppressed TMA-induced increases in ear and epidermal thickness, and IgE expression. In addition, abnormal expression of IL-1β and TNF-α protein and mRNA was also significantly attenuated by oral administration of AcRE. Treatment with AcRE also significantly suppressed TMA-induced IL-4 and IL-13 cytokines and mRNA expression in vivo. Moreover, AcRE strongly suppressed TMA-induced IL-4 and IL-5 production in draining lymph nodes, as well as OVA-induced IL-4 and IL-5 expression in primary cultured splenocytes. Interestingly, AcRE suppressed IL-4-induced STAT6 phosphorylation in both primary cultured splenocytes and HaCaT cells, and TMA-induced GATA3 mRNA expression ex vivo. AcRE also suppressed TMA-mediated CCL11 and IL-4-induced CCL26 mRNA expression and infiltration of CCR3 positive cells. The major compounds from AcRE were identified as gentisic acid (0.64 ± 0.2 μg/g dry weight of AcRE), protocatechuic acid (2.69 ± 0.1 μg/g dry weight of AcRE), 4-hydroxybenzoic acid (5.59 ± 0.3 μg/g dry weight of AcRE), caffeic acid (4.21 ± 0.1 μg/g dry weight of AcRE), and ferulic acid (14.78 ± 0.4 ± 0.3 μg/g dry weight of AcRE). Taken together, these results suggest that AcRE has potential for development as an agent to prevent and treat allergic contact dermatitis.
Placenta creta is characterized by invasion of placental villi into the myometrium in the setting of a dysfunctional or absent decidua. Histopathologic diagnosis of placenta creta is important, particularly in cases of hysterectomy because of unanticipated intractable postpartum hemorrhage. Previous studies have documented a higher amount and depth of myometrial infiltration by the implantation site intermediate trophoblast compared with controls. In addition, we have anecdotally observed chorionic villi in myometrial vascular spaces in specimens with placenta creta. The aim of this study was to explore the prevalence and specificity of these features. Sixty-one postpartum hysterectomies, 44 with placenta creta and 17 without were reviewed. Villous intrusion into vascular spaces was recorded. Using immunohistochemistry for GATA3, the amount of intermediate trophoblast (number of positive cells in five 40× fields) and depth of trophoblast myometrial infiltration were assessed. Mean gestational ages of the creta group (34.4 yr; range, 20-43 yr) and control group (35 yr; range, 25-51 yr) were comparable. Presence of chorionic villi in myometrial vascular spaces was frequent in placenta creta: 31/44 versus 1/17 controls (70.4% vs. 5.8%, P<0.0001). This finding was more common in the percreta (87.5%) and increta (84%) than in the accreta (27.2%, P=0.0008). Mean depth of trophoblast myometrial invasion was greater in cretas (47.9%) than in controls (14.5%, P=0.004). Likewise, mean distance of deepest trophoblast to serosa was shorter in the cretas (7.3 mm) than in controls (23.8 mm, P<0.0001). These differences were, however, attributable to placentas increta and percreta. When only accretas and controls were compared, the myometrial depth of trophoblast was similar. The mean intermediate trophoblast cell count in the placental bed was greater in cretas (664) than in controls (288, P<0.0001). Such difference was seen in all creta cases despite the type (accreta 639, increta 676, percreta 661). A trophoblast count of ≥100 cells/high-power field was seen in 75.8% of cretas and 11.1% of controls (P=0.0009). For the first time, we document the finding of chorionic villi intrusion into myometrial vascular spaces, which is highly specific of placenta creta. In addition, assessment of the amount of intermediate trophoblast using GATA3 immunohistochemistry can assist in the diagnosis. We hypothesize that placental invasion in placenta creta is due, at least partially, to transformation of low-resistance myometrial vessels leading to subsequent protrusion of villi into their lumens, in the context of absent decidua.
High mobility group box 1 (HMGB1) is an inflammatory mediator involved into the advanced stage of systemic inflammatory response syndrome (SIRS), and is over-expressed in bacterial sepsis and hemorrhagic shock. Recently, it has been found that the HMGB1 was abnormally expressed in induced sputum and plasma of asthmatic patients. However, the precise role of HMGB1 in the acute allergic asthma is unclear. Therefore, we aim to investigate the role HMGB1 in regulating airway inflammation of acute allergic asthma and its possible mechanism in this study.
Forty-eight BALB/c female mice were randomly divided into four groups: control group (Control), asthma group (Asthma), HMGB1 group (HMGB1) and anti-HMGB1 (HMGB1 monoclonal antibody of mice) group (Anti-HMGB1). Acute allergic asthma mice models were established by ovalbumin (OVA)-challenge. Then, we measured the levels of HMGB1 in bronchoalveolar lavage fluid (BALF) and lung tissue of mice. Finally, after exogenous HMGB1 and/or anti-HMGB1 administration, pulmonary function test, histological analysis, Western blot, cytological analysis and ELISA assay were performed to explore the effect of HMGB1 in acute allergic asthma.
The levels of HMGB1 in BALF and lung tissue and the expression of HMGB1 protein in the lung tissue of asthma group were significantly higher than those in control group, respectively (P<0.01). Moreover, the HMGB1 group was showed an increased mucus secretion and infiltration of eosinophils and neutrophils in the airway of asthma mice, and a decrease of pulmonary function, compared to control group (P<0.01, respectively). Meanwhile, exogenous HMGB1 could increase the levels of IL-4, IL-5, IL-6, IL-8 and IL-17, whereas could reduce the IFN-γ in the BALF and lung tissue (P<0.05, respectively). Exogenous HMGB1 could enhance GATA3 expression of Th2 cells and attenuate the T-bet expression of Th1 cells (P<0.05, respectively), which could be abrogated after inhibiting HMGB1.
HMGB1 could aggravate eosinophilic inflammation in the airway of acute allergic asthma through inducing a dominance of Th2-type response and promoting the neutrophilic inflammation.
To determine the role of regulatory T/type 2 helper cell-mediated immune imbalance in the pathogenesis of allergic rhinitis and examine the association between clinical severity and regulatory T/type 2 helper cell-mediated immune imbalance.
Levels of interleukins 4 and 5 and transforming growth factor β1, and expression of FOXP3 and GATA3 (which are functionally related to regulatory T and type 2 helper cells, respectively), were evaluated in 46 allergic rhinitis patients and 42 healthy subjects.
Compared to controls, allergic rhinitis patients showed significantly higher interleukin 4 and 5 levels, but lower transforming growth factor β1 levels. Furthermore, FOXP3 messenger RNA expression was lower in allergic rhinitis patients, while GATA3 messenger RNA and protein expression was significantly higher. Regulatory T/type 2 helper cell ratio was inversely correlated with clinical symptom scores.
Regulatory T/type 2 helper cell immune imbalance may contribute to allergic rhinitis development. These findings provide a new insight into disease pathogenesis and potential therapeutic approaches.
our aim was to evaluate whether somatic mutations in five genes were associated with an early age at presentation of breast cancer (BC) or serous ovarian cancer (SOC).
COSMIC database was searched for the five most frequent somatic mutations in BC and SOC. A systematic review of PubMed was performed. Young age for BC and SOC patients was set at ≤ 35 and ≤ 40 years, respectively. Age groups were also classified in < 30 years and every 10 years thereafter.
twenty six (1,980 patients, 111 younger) and 16 studies (598, 41 younger), were analyzed for BC and SOC, respectively. In BC, PIK3CA wild type tumor was associated with early onset, not confirmed in binary regression with estrogen receptor (ER) status. In HER2-negative tumors, there was increased frequency of PIK3CA somatic mutation in older age groups; in ER-positive tumors, there was a trend towards an increased frequency of PIK3CA somatic mutation in older age groups. TP53 somatic mutation was described in 20% of tumors from both younger and older patients; PTEN, CDH1 and GATA3 somatic mutation was investigated only in 16 patients and PTEN mutation was detected in one of them. In SOC, TP53 somatic mutation was rather common, detected in more than 50% of tumors, however, more frequently in older patients.
frequency of somatic mutations in specific genes was not associated with early-onset breast cancer. Although very common in patients with serous ovarian cancer diagnosed at all ages, TP53 mutation was more frequently detected in older women.
Tumor collision is the encounter of two tumors from two different topographical sites. Cases of metastatic lymph node collision are exceptional. We report the case of a metastatic lymph node collision of an urothelial carcinoma and a prostatic adenocarcinoma.
A 61-year-old man was hospitalized for a right nephroureterectomy with peri-ureteral lymph node dissection. He was followed since 2004 for prostatic adenocarcinoma and treated with radical prostatectomy then radiation therapy 4 years later due to a new increase of PSA. In the follow-up, an urothelial carcinoma of the lower right ureter was discovered in 2014. Histological analysis of a peri-ureteral lymph node showed a double metastasis of urothelial and prostatic origin. The prostatic adenocarcinoma was composed of acinar and ductal subtypes. Immunohistochemical study including CK7, CK20, PSA, GATA3, P63 antibodies confirmed the distinct phenotype of the 2 tumors.
Metastatic collision of urothelial carcinoma and prostatic adenocarcinoma has been reported in 4 cases only. Our review of literature shows that prostatic adenocarcinoma always precedes the urothelial carcinoma. Immunohistochemical study, when carried out for distinguishing both tumors, should include CK7, CK20 and PSA. GATA3, androgen receptor and P63 could be added in a second time.
The transcription factor GATA-3 is indispensable for the development of all innate lymphoid cells (ILCs) that express the interleukin 7 receptor α-chain (IL-7Rα). However, the function of low GATA-3 expression in committed group 3 ILCs (ILC3 cells) has not been identified. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served a critical function in the development of the NKp46(+) ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Among NKp46(+) ILC3 cells, although GATA-3 positively regulated genes specific to the NKp46(+) ILC3 subset, it negatively regulated genes specific to lymphoid tissue-inducer (LTi) or LTi-like ILC3 cells. Furthermore, GATA-3 was required for IL-22 production in both ILC3 subsets. Thus, despite its low expression, GATA-3 was critical for the homeostasis, development and function of ILC3 subsets.
Initial investigations reported GATA3 to be a sensitive and relatively specific marker for mammary and urothelial carcinomas. Recently, GATA3 expression has been described in several other epithelial tumors. However, there has been only limited investigation of GATA3 expression in cutaneous epithelial tumors. The objective of this study was to examine the immunohistochemical expression of GATA3 in a wide variety of cutaneous epithelial neoplasms. GATA3 expression was evaluated in 99 benign and 63 malignant cutaneous epithelial tumors. GATA3 was consistently and usually strongly expressed in clear cell acanthoma, trichofolliculoma, trichoepithelioma, trichilemmoma, sebaceous adenoma, sebaceoma, apocrine hidrocystoma, apocrine tubular papillary adenoma, hidradenoma papilliferum, and syringocystadenoma papilliferum. Hidradenomas exhibited variable positive staining. Most poromas, syringomas, chondroid syringomas, cylindromas, and spiradenomas were negative or only focally and weakly positive. Focal staining was present in all pilomatrixomas. Thirteen of 14 basal cell carcinomas, 21 of 24 squamous carcinomas, and all 6 sebaceous carcinomas exhibited positive staining. The 1 apocrine carcinoma, both mucinous carcinomas, and 2 of 3 microcystic adnexal carcinomas also exhibited positive staining, whereas the 1 eccrine porocarcinoma and the 1 adenoid cystic carcinoma were negative. One of 11 Merkel cell carcinomas exhibited focal weak staining. Our findings demonstrate that GATA3 is expressed in a wide variety of benign and malignant cutaneous epithelial neoplasms. In addition to carcinomas of breast and urothelial origin and other more recently described GATA3-positive tumors, the differential diagnosis of a metastatic tumor of unknown primary origin that expresses GATA3 should also include a carcinoma of cutaneous epithelial origin.
Chronic rhinosinusitis with nasal polyps is characterized by local inflammation and is categorized into two subtypes in Japan: eosinophilic chronic rhinosinusitis, and non-eosinophilic chronic rhinosinusitis. The objective of this study was to investigate the expression of key transcription factors for Treg and Th1/Th2/Th17 cells, in relation to the mRNA expression of representative cytokines in these two subtypes of chronic rhinosinusitis with nasal polyps.
The expression of forkhead box P3 (FOXP3), T-box transcription factor (T-bet), GATA3, retinoid acid-related orphan receptor C (RORc), the suppressive cytokines TGF-β1 and IL-10, and Th1/Th2/Th17 cytokines (IFN-γ, IL-4, IL-5, IL-13, IL-17) were analyzed by means of RT-PCR in eosinophilic polyps. Eosinophilic polyps were defined as having an eosinophil count of more than 50 per microscopic field (×400 magnification) using five fields located in the subepithelial area of the polyps, while the non-eosinophilic polyps and controls did not fulfill this criteria. The numbers of T cells, CD4+ T cells, CD8+ T cells and Treg were histologically counted using sections that were immunostained for CD3, CD4, CD8, and FOXP3, respectively.
In eosinophilic polyps, we observed significantly fewer CD4+ T cells and CD8+ T cells, and lower GATA3, RORc and IL-10 mRNA expression, but a significantly higher IL-5, and IL-13 mRNA expression compared with controls, whereas FOXP3 and T-bet mRNA expression were not significantly different compared with controls. In non-eosinophilic polyps, FOXP3, IL-10, IL-17A, TGFβ1 and IFNγ mRNA expression was significantly higher compared with controls, whereas IL-4, 5 and 13 expression was not significantly different from controls.
We showed a reduction of GATA3 and RORc mRNA, low Treg-related cytokines and elevated Th2 cytokine levels in eosinophilic chronic rhinosinusitis, whereas we demonstrated the upregulation of Treg cells and increases of Th1 and Th17 cytokines in non-eosinophilic chronic rhinosinusitis in the Japanese population. The different mRNA expression profiles of Treg and Th1/Th2/Th17 signature transcription factors and cytokines between eosinophilic chronic rhinosinusitis and non-eosinophilic chronic rhinosinusitis suggests heterogeneity in the pathogenesis of chronic rhinosinusitis with nasal polyps.
Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are key effector cytokines for the differentiation of T helper type 1 and 2 (Th1 and Th2) cells. Both cytokines induce fate-decisive transcription factors such as GATA3 and TBX21 that antagonize the polarized development of opposite phenotypes by direct regulation of each other's expression along with many other target genes. Although it is well established that mesenchymal cells directly respond to Th1 and Th2 cytokines, the nature of antagonistic differentiation programs in airway epithelial cells is only partially understood. In this study, primary normal human bronchial epithelial cells (NHBEs) were exposed to IL-4, IFN-γ, or both and genome-wide transcriptome analysis was performed. The study uncovers an antagonistic regulation pattern of IL-4 and IFN-γ in NHBEs, translating the Th1/Th2 antagonism directly in epithelial gene regulation. IL-4- and IFN-γ-induced transcription factor hubs form clusters, present in antagonistically and polarized gene regulation networks. Furthermore, the IL-4-dependent induction of IL-24 observed in rhinitis patients was downregulated by IFN-γ, and therefore IL-24 represents a potential biomarker of allergic inflammation and a Th2 polarized condition of the epithelium.
The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a matching tumor type, and which represent candidate PAI regions warranting further investigation.
The Th2 pathway starts with the binding of IL-4 to the IL-4 receptor followed by the phosphorylation of signal transducer and activator of transcription 6 and the activation of GATA3. The most important question relates to the sources of IL-4 and IL-4 related inflammation. Which cells other than Th2 cells are responsible for airway inflammation in asthma?
Accumulating data indicate that basophils contribute to endothelium-related IL-4-dependent inflammation. There is also a dendritic cell-related alternative for the induction of Th2 cells via Notch signalling. GATA3 deoxyribozyme improves asthma that is not clearly related to T-cells. The innate immune response in allergy is linked to mast cells, basophils, and the innate lymphoid cell type 2 (ILC2). ILC2s respond to IL-25, IL-33, thymic stromal lymphopoietin, and leukotrienes by producing IL-4, IL-5, and IL-13. In addition to all this inflammatory-cell-driven asthma, increasing evidence has emerged relating to smooth muscle cell activation, the endothelial and epithelial barrier functions, and improvements in the barrier function. The elevation of intracellular cyclic adenosine monophosphate because of the use of phosphodiesterase inhibitors adds to the prevention of epithelial-endothelial leakage, supports airway smooth muscle relaxation, and is immunosuppressive.
IL-4 is the predominant Th2 cell cytokine. Many more cells, including eosinophils, basophils, mast cells, and ILC2, contribute to the production of IL-4 in the airways. Epithelial cells and endothelial cells lose barrier function in the context of allergic airway inflammation, and this could be at least partially remedied by increasing the intracellular cyclic adenosine monophosphate levels through phosphodiesterase inhibition.
Early regulators of disease may increase understanding of disease mechanisms and serve as markers for presymptomatic diagnosis and treatment. However, early regulators are difficult to identify because patients generally present after they are symptomatic. We hypothesized that early regulators of T cell-associated diseases could be found by identifying upstream transcription factors (TFs) in T cell differentiation and by prioritizing hub TFs that were enriched for disease-associated polymorphisms. A gene regulatory network (GRN) was constructed by time series profiling of the transcriptomes and methylomes of human CD4(+) T cells during in vitro differentiation into four helper T cell lineages, in combination with sequence-based TF binding predictions. The TFs GATA3, MAF, and MYB were identified as early regulators and validated by ChIP-seq (chromatin immunoprecipitation sequencing) and small interfering RNA knockdowns. Differential mRNA expression of the TFs and their targets in T cell-associated diseases supports their clinical relevance. To directly test if the TFs were altered early in disease, T cells from patients with two T cell-mediated diseases, multiple sclerosis and seasonal allergic rhinitis, were analyzed. Strikingly, the TFs were differentially expressed during asymptomatic stages of both diseases, whereas their targets showed altered expression during symptomatic stages. This analytical strategy to identify early regulators of disease by combining GRNs with genome-wide association studies may be generally applicable for functional and clinical studies of early disease development.
Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 × 10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P < 0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P < 0.0001) and reporter assays (P = 0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells.
Studies have examined gene expression changes in Sézary syndrome (SS), but disease pathogenesis remains largely unknown, and diagnosis and treatment are difficult. TOX is a transcription factor involved in CD4+ T-cell development with downstream effects on RUNX3, a known tumor suppressor gene. We sought to identify genes involved in SS disease pathogenesis with the potential to enable diagnosis and treatment. We utilized previously reported transcriptome sequencing data to construct a list of candidate genes, which was narrowed using pathway analysis. qRT-PCR confirmed TOX upregulation (>7 fold increase) in SS (n = 5), as well as two established markers, PLS3 and KIRD3DL2. We also evaluated expression of members of the TOX-RUNX3 pathway and confirmed downregulation of RUNX3 (0.59 fold decrease) and upregulation of GATA3 (2 fold increase). Moreover, TOX and RUNX3 expression were significantly inversely proportional. Using siRNA to suppress TOX, we demonstrated that TOX knockdown rescues RUNX3 expression and reduces cell viability. We evaluated TOX protein expression in paraffin-embedded skin biopsies with immunohistochemistry, showing nuclear staining of CTCL infiltrates, suggesting it is a candidate diagnostic biomarker. Further studies validating our findings and evaluating the TOX-RUNX3 pathway and the role of TOX as a disease marker and therapeutic target are warranted.
Allergic airway inflammation (AAI) in response to environmental antigens is an increasing medical problem, especially in the Western world. Type 2 interleukins (IL) are central in the pathological response but their importance and cellular source(s) often rely on the particular allergen. Here, we highlight the cellular sources and regulation of the prototypic type 2 cytokine, IL-13, during the establishment of AAI in a fungal infection model using Cryptococcus neoformans. IL-13 reporter mice revealed a rapid onset of IL-13 competence within innate lymphoid cells type 2 (ILC2) and IL-33R(+) T helper (Th) cells. ILC2 showed IL-33-dependent proliferation upon infection and significant IL-13 production. Th cells essentially required IL-33 to become either GATA3(+) or GATA3(+)/Foxp3(+) hybrids. GATA3(+) Th cells almost exclusively contributed to IL-13 production but hybrid GATA3(+)/Foxp3(+) Th cells did not. In addition, alveolar macrophages upregulated the IL-33R and subsequently acquired a phenotype of alternative activation (Ym1(+), FIZZ1(+), and arginase-1(+)) linked to type 2 immunity. Absence of adaptive immunity in rag2(-/-) mice resulted in attenuated AAI, revealing the need for Th2 cells for full AAI development. Taken together, in pulmonary cryptococcosis ILC2 and GATA3(+) Th2 cells produce early IL-13 largely IL-33R-dependent, thereby promoting goblet cell metaplasia, pulmonary eosinophilia, and alternative activation of alveolar macrophages.
Primary mucinous adenocarcinoma of the female urethra is very rare and may lead to both diagnostic and therapeutic challenges. Although primary mucinous adenocarcinoma of the prostate and prostatic urethra has been well characterized in men, this is the largest clinicopathologic study to date of primary mucinous adenocarcinoma of the female urethra. A search was made through the files of 2 major academic institutions for cases of confirmed primary mucinous adenocarcinoma arising from the female urethra. Tumors arising from adjacent organs were excluded both clinically and pathologically in all cases. Five cases were identified. The mean patient age was 67 years (range, 54-74 years). All patients presented with a polypoid/papillary mass arising from the urethra. Pathologic stages were as follows: pT4 3 (60%) of 5 cases; pT3 1 (20%) of 5 cases, and pT2 1 (20%) of 5 cases. Immunohistochemical stains for GATA3, p63, CK7, CK20, CDX2, ER, PAX8, and β-catenin were performed on all cases. Immunohistochemical stains were positive in the tumor cells for CDX2 in 4/5 (80%) cases; focally positive for CK20 in 4/5 (80%) cases; focally positive for CK7 in 4/5 cases (80%); and negative for p63, GATA3, ER, PAX8 and β-catenin in all cases. In the 4 patients with available follow-up data, mean follow-up was 25 months (range, 4-54 months). It is critical for pathologists to be aware of this entity in light of potential diagnostic pitfalls and therapeutic implications.
Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.
Alopecia areata represents an autoimmune pathological process driven primarily by cellular aberrations contained within the immune system, which activates various humoral and cellular elements of the immune response. The aim of this study was to determine the mRNA expression levels of T-bet and GATA-3 as potential inducers of T helper (Th)1 and Th2 differentiation, respectively, as well as Th1(IFN-γ) and Th2(IL-4) cytokine mRNA expression in patients with alopecia areata. Using real-time reverse transcriptase PCR (RT-PCR), the relative amounts of T-bet, GATA-3, IFN-γ, and IL-4 mRNA transcripts were determined in PBMCs from 20 Iranian patients with alopecia areata and compared with those of 20 healthy control subjects. In comparison with the normal group, T-bet and IFN-γ mRNA expression levels were significantly up-regulated in the alopecia areata patients, while GATA-3 and IL-4 mRNA expression levels were down-regulated. Notably, positive correlation (P < 0.05) was found between IFN-γ and T-bet levels in patients and controls. In addition, significant positive correlations existed between GATA-3 and IL-4 (P < 0.05). These results indicate that a Th1/Th2 imbalance exists in alopecia areata, and it may be implicated in the pathogenesis of disease.
The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3-Gata3, Six1 and Eya1-are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.
Regulatory dendritic cell (DC) markers, such as C1Q, are upregulated in PBMCs of patients with grass pollen allergy exhibiting clinical benefit during allergen immunotherapy (AIT).
We sought to define markers differentially expressed in human monocyte-derived DCs differentiated toward a proallergic (DCs driving the differentiation of TH2 cells [DC2s]) phenotype and investigate whether changes in such markers in the blood correlate with AIT efficacy.
Transcriptomes and proteomes of monocyte-derived DCs polarized toward DCs driving the differentiation of TH1 cells (DC1s), DC2s, or DCs driving the differentiation of regulatory T cells (DCreg cells) profiles were compared by using genome-wide cDNA microarrays and label-free quantitative proteomics, respectively. Markers differentially regulated in DC2s and DCreg cells were assessed by means of quantitative PCR in PBMCs from 80 patients with grass pollen allergy before and after 2 or 4 months of sublingual AIT in parallel with rhinoconjunctivitis symptom scores.
We identified 20 and 26 new genes/proteins overexpressed in DC2s and DCreg cells, respectively. At an individual patient level, DC2-associated markers, such as CD141, GATA3, OX40 ligand, and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), were downregulated after a 4-month sublingual AIT course concomitantly with an upregulation of DCreg cell-associated markers, including complement C1q subcomponent subunit A (C1QA), FcγRIIIA, ferritin light chain (FTL), and solute carrier organic anion transporter family member 2B1 (SLCO2B1), in the blood of clinical responders as opposed to nonresponders. Changes in such markers were better correlated with clinical benefit than alterations of allergen-specific CD4(+) T-cell or IgG responses.
A combination of 5 markers predominantly expressed by blood DCs (ie, C1Q and CD141) or shared with lymphoid cells (ie, FcγRIIIA, GATA3, and RIPK4) reflecting changes in the balance of regulatory/proallergic responses in peripheral blood can be used as early as after 2 months to monitor the early onset of AIT efficacy.
To investigate the level of interleukin-19 (IL-19) in the peripheral blood of the patients with allergic rhinitis (AR) and reveal the possible role of IL-19 in the development of disease.
The levels of IL-19 mRNA and protein in the peripheral blood mononuclear cell (PBMCs) of AR patients and healthy controls were determined by real-time quantitative PCR (qRT-PCR) and ELISA. The clinical severity in AR group was evaluated by the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) and the Visual Analog Scale (VAS) scores. Moreover, PBMCs from the AR group were stimulated by recombinant IL-19 or PBS, and then the frequency of Th2 cells, levels of GATA binding protein-3 (GATA-3) mRNA and IL-4 protein in PBMCs were detected by flow cytometry, qRT-PCR and ELISA, respectively.
Compared with the control group, the level of IL-19 in the AR group was significantly elevated. In addition, RQLQ and VAS scores in the AR group were also significantly higher than those in the controls, and they were positively correlated with the levels of IL-19 in the AR group (rmRNA-RQLQ=0.673, rmRNA-VAS=0.619, protein-RQLQ=0.669, protein-VAS=0.662, P<0.01).
The level of IL-19 in the peripheral blood of AR patients increased and was related with the clinical severity of AR.
Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression by virus-specific CTLs was associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependent regulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.
Mesonephric adenocarcinoma is a rare tumor type that is usually found in areas where the Wolffian duct was present during the fetal period. We report a case of mesonephric adenocarcinoma of the uterine corpus in a 66-year-old woman who presented with vaginal bleeding. Pelvic magnetic resonance imaging revealed a 2.7-cm-sized irregular thickening and enhancement of the uterine body. The diagnosis following endometrial curettage biopsy was endometrioid adenocarcinoma, and the patient underwent a total hysterectomy with bilateral salpingo-oophorectomy. The tumor was composed of small tubular and ductal components, and a retiform appearance was also observed in the deeper areas. The tumor cells were immunopositive for cytokeratin, vimentin, CD10 with a luminal staining pattern, PAX2, and PAX8, and immunonegative for estrogen receptor and progesterone receptor, which was consistent with tumor of mesonephric origin. Mesonephric neoplasms reveal relatively low-grade nuclear feature, characteristic immunoprofiles (immunonegative for ER and PR, and immunopositive for CD10, PAX2, PAX8, and GATA3), and unique tumor location (myometrium), whereas Müllerian neoplasms such as endometrial adenocarcinoma show various morphology, immunopositivity for ER and PR, and primarily endometrial location. As described above, an integration of the clinical features, morphologic characteristics, and immunohistochemical profiles is needed to make a diagnosis.
Molecular heterogeneity in human breast cancer has challenged diagnosis, prognosis, and clinical treatment. It is well known that molecular subtypes of breast tumors are associated with significant differences in prognosis and survival. Assuming that the differences are attributed to subtype-specific pathways, we then suspect that there might be gene regulatory mechanisms that modulate the behavior of the pathways and their interactions. In this study, we proposed an integrated methodology, including machine learning and information theory, to explore the mechanisms. Using existing data from three large cohorts of human breast cancer populations, we have identified an ensemble of 16 master regulator genes (or MR16) that can discriminate breast tumor samples into four major subtypes. Evidence from gene expression across the three cohorts has consistently indicated that the MR16 can be divided into two groups that demonstrate subtype-specific gene expression patterns. For example, group 1 MRs, including ESR1, FOXA1, and GATA3, are overexpressed in luminal A and luminal B subtypes, but lowly expressed in HER2-enriched and basal-like subtypes. In contrast, group 2 MRs, including FOXM1, EZH2, MYBL2, and ZNF695, display an opposite pattern. Furthermore, evidence from mutual information modeling has congruently indicated that the two groups of MRs either up- or down-regulate cancer driver-related genes in opposite directions. Furthermore, integration of somatic mutations with pathway changes leads to identification of canonical genomic alternations in a subtype-specific fashion. Taken together, these studies have implicated a gene regulatory program for breast tumor progression.
GATA3 is a transcription factor regulating luminal cell differentiation in the mammary glands and has been implicated in the luminal types of breast carcinoma. The prognostic significance of GATA3 in breast cancer remains controversial.
In this study, we assessed the prognostic value of the molecule in a subset of 62 advanced breast cancers and 10 control breast cancers (no metastasis after follow-up).
GATA3 expression levels in luminal tumors of advanced stage were significantly higher than that of the human epidermal growth factor receptor 2 (HER2) subtype and triple-negative carcinomas, as expected, but were similar to those of the luminal controls. Furthermore, 88% of nonluminal tumors showed variable GATA3 expression, for which the HER2 subtype had significantly higher GATA3 expression than that of the triple-negative carcinomas. Interestingly, GATA3 levels were significantly lower in carcinomas with lung relapse compared to those with metastatic recurrence to other organs, thus reflecting the findings in animal models. No significant difference was observed between tumors with bone relapse and those metastasized to nonskeletal sites. Moreover, high GATA3 expression was significantly associated with favorable relapse-free survival and overall survival.
These findings suggest that GATA3 may not act solely as a luminal differentiation marker, and further uncovering the molecular pathways by which GATA3 regulates the downstream targets will be crucial to our understanding of breast cancer dissemination.
To report a series of uterine corpus (n = 7) and ovarian (n = 5) neoplasms which we believe probably represent mesonephric adenocarcinomas based on their characteristic morphology and immunophenotype.
All neoplasms exhibited a relatively constant and characteristic morphological appearance with an admixture of architectural patterns with small glands or tubules, some containing luminal eosinophilic colloid-like material, typically predominating. Solid and papillary architectures were also often present. The nuclear features were characteristic with atypical angulated clear vesicular nuclei which often exhibited overlapping. All the tumours were 'flat' negative with oestrogen receptor and progesterone receptor and all except one exhibited nuclear staining with thyroid transcription factor 1 (TTF1), which was often diffuse. All tumours exhibited wild-type staining with p53. CD10, calretinin and GATA binding protein 3 (GATA3) were positive in a variable proportion of the neoplasms.
We believe these neoplasms to represent mesonephric adenocarcinomas which have only rarely been reported to arise in the uterine corpus and never in the ovary. We recommend they be termed mesonephric-like adenocarcinomas until their histogenesis is firmly established.
We have recently demonstrated that Caerulomycin A induces regulatory T cells differentiation by suppressing Th1 cells activity. The role of regulatory T cells is well established in suppressing the function of Th2 cells. Th2 cells are known to inflict the induction of the activation of asthma. Consequently, in the present study, we monitored the influence of Caerulomycin A in inhibiting the activity of Th2 cells and its impact in recuperating asthma symptoms. Interestingly, we observed that Caerulomycin A significantly suppressed the differentiation of Th2 cells, as evidenced by downregulation in the GATA-3 expression. Further, decline in the levels of IL-4, IL-5 and IL-13 cytokines and IgE was noted in the animals suffering from asthma. Furthermore, we noticed substantial suppression in the inflammatory response and number of eosinophils in the lungs. In essence, this study signifies an important therapeutic role of Caerulomycin A in asthma.
Inefficient T-cell reconstitution from x-ray-induced immune damage reduces antitumor response. To understand the profile of T-cell reconstitution after irradiation will overcome the barrier of antitumor immunity. This study aimed to identify the recovery profile of T-cell subsets following x-ray irradiation and to highlight the role of cinnamon on efficient T-cell restoration postexposure in the antitumor response.
CD3(+), CD8(+), and CD4(+) T cells and Th1, Th2, Th17, and regulatory T (Treg) cells were evaluated at different time points after single low-dose total body irradiation (SLTBI) with or without cinnamon treatments. T-bet, GATA3, RORγt, and Foxp3 signaling specific for Th1, Th2, Th17, and Treg were also analyzed by RT-PCR assay. The effects of cinnamon on efficient T-cell subset reconstitution was confirmed in a lung melanoma model in irradiated mice.
Reconstitution of CD4(+) T cells was delayed more than that of CD8(+) T cells in T-cell restoration after SLTBI. The production of IFNγ by Th1 or Tc1 cells was sharply decreased and was accompanied by reduced T-bet mRNA, even when total T-cell numbers had recovered; the frequencies of Th17 and Treg cells and their specific transcription factors (RORγt and Foxp3, respectively) were obviously increased. Irradiation-induced inefficient T-cell reconstitution impaired the antitumor capacities in the lung melanoma model. Pretreatment with cinnamon in irradiated mice accelerated the generation of Th1 and reduced the differentiation of Treg cells by activating T-bet and limiting transcriptions of Foxp3. Improvement resulting from cinnamon pretreatment on the efficient T-cell recovery profile from SLTBI promoted antitumor immunity in the lung melanoma model.
T-cell reconstitution from SLTBI was characterized by impaired Th1 and elevated Th17 and Treg cells. Cinnamon effectively improved the imbalance of T-cell subsets by promoting the proliferation of Th1 and by suppressing expansions of Th17 and Tregs. The role of cinnamon in efficient T-cell reconstitution from SLTBI is effective in antitumor immunity.
In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox genes MSX1 and OTX2 which are physiologically involved in development of the embryonal neural plate border region. Here, we examined in HL homeobox gene SIX1 an additional regulator of this embryonal region mediating differentiation of placodal precursors. SIX1 was aberrantly activated in 12 % of HL patient samples in silico, indicating a pathological role in a subset of this B-cell malignancy. In addition, SIX1 expression was detected in HL cell lines which were used as models to reveal upstream factors and target genes of this basic developmental regulator. We detected increased copy numbers of the SIX1 locus at chromosome 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the WNT-signalling pathway and transcription factor MEF2C regulate SIX1 expression. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL. In addition, cofactors EYA1 and TLE4, respectively, contrastingly mediated activation and suppression of SIX1 target gene expression. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets. Collectively, our data reveal a gene regulatory network with SIX1 centrally deregulating lymphoid differentiation and support concordance of lymphopoiesis/lymphomagenesis and developmental processes in the neural plate border region.
Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP).
In this study 15 AgP patients (53.3% males, 41.4±10.5 years) and 10 controls (40.0% males, 36.9±17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTect® Methyl Signature PCR Array Human Inflammatory Response.
In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p=0.015) and IL17C (6.89% vs. 19.27%, p=0.002) was significantly reduced as compared with periodontally healthy tissues.
We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment.
Age at breast cancer diagnosis is a known prognostic factor. Previously, several groups including ours have shown that young age at diagnosis is associated with higher prevalence of basal-like tumors and aggressive tumor phenotypes. Yet the impact of age at diagnosis on the genomic landscape of breast cancer remains unclear. In this study, we examined the pattern of somatic mutations, chromosomal copy number variations (CNVs) and transcriptomic profiles in young and elderly breast cancer patients.
Analyses were performed on The Cancer Genome Atlas (TCGA) dataset. Patients with metastatic disease at diagnosis, classified as normal-like by PAM50 or had missing clinical information were excluded. Young patients were defined as ≤45 years of age, while elderly patients were those ≥70 years of age at breast cancer diagnosis. The remaining patients were classified as "intermediate". We evaluated the association between age at diagnosis and somatic mutations, CNV and gene expression in a logistic regression model adjusting for tumor size, nodal status, histology and breast cancer subtype. All analyses were corrected for multiple testing using the Benjamini-Hochberg approach.
In this study, 125, 486 and 169 patients were ≤45, 46-69 and ≥70 years of age, respectively. Older patients had more somatic mutations (n = 44 versus 35 versus 31; P = 0.0009) and more CNVs, especially in ductal tumors (P = 0.02). Eleven mutations were independently associated with age at diagnosis, of which only GATA3 was associated with young age (15.2% versus 8.2% versus 9%; P = 0.003). Only two CNV events were independently associated with age, with more chr18p losses in older patients and more chr6q27 deletions in younger ones. Younger age at diagnosis was associated with higher expression of gene signatures related to proliferation, stem cell features and endocrine resistance.
Age adds a layer of biological complexity beyond breast cancer molecular subtypes, classic pathological and clinical variables, worthy of further consideration in future drug development as we seek to refine therapeutic strategies in the era of personalized medicine.
It is not uncommon to encounter challenges in the immunohistochemical confirmation of metastatic breast cancer given the limited sensitivities of mammaglobin and gross cystic disease fluid protein 15 (GCDFP-15/BRST-2) and the significant proportion of triple-negative breast carcinomas (ie, tumors that are negative for estrogen receptor [ER], and progesterone receptor [PgR], and human epidermal growth factor 2 [HER2]). GATA binding protein 3 (GATA3) has emerged as a potentially useful immunohistochemical adjunct during the evaluation of metastatic breast carcinomas in cytology specimens. The objective of the current study was to examine GATA3 expression in the context of malignant effusions secondary to both mammary and extramammary malignancies.
In total, 306 malignant effusions (from 62 metastatic breast carcinomas and 244 extramammary malignancies) were examined using GATA3 immunohistochemistry. Effusions with metastatic breast carcinoma were also examined using immunohistochemistry for additional breast markers (ER, PgR, HER2, mammaglobin, and GCDFP-15/BRST-2).
GATA3 immunohistochemistry highlighted the tumor cells in 58 of the 62 samples (93.5%) from patients with metastatic breast carcinoma, which was higher than the observed sensitivity of immunohistochemistry for ER (63.8%), PgR (41.4%), HER2 (15.5%), mammaglobin (22.4%), and GCDFP-15/BRST-2 (5.2%). GATA3 expression also was observed in a subset of malignant effusions secondary to extramammary primaries, specifically, in 28 of 244 specimens (11.5%).
GATA3 is a highly sensitive marker for the detection of metastatic breast carcinomas in effusion specimens. However, this marker is not entirely specific for malignancies of breast origin. Thus, GATA3 should be used in conjunction with additional immunohistochemical markers during the cytologic evaluation of malignant effusions.
This study evaluates immunohistochemical markers for the differential diagnosis of primary bladder adenocarcinoma (BAC) from secondarily involving colorectal adenocarcinoma (CAC). Additional staining of putative precursor lesions (cystitis cystica et glandularis (CC) and intestinal metaplasia (IM)) supports insights into metaplastic cell development and aberrant differentiation in tumours.
Tissue microarray sections of formalin-fixed, paraffin-embedded tissues from clinically verified 11 BAC, 11 CAC, 18 invasive urothelial carcinomas (UCs), 22 normal urothelium samples, 25 CC and 15 IM were stained for keratin 7, 5/6, 5/14 and 20, ß-catenin, e-cadherin, cadherin 17, cdx2, uroplakin II and III, CD10, androgen receptor (AR), S100P, MUC2, MUC5AC and GATA3 expression. Data were analysed using Kruskal-Wallis/Dunn's multiple comparison test and Fisher's exact test.
Significant difference (p<0.05) between all three tumour groups was observed for keratin 7 only. Further significant difference between BAC and CAC was found for GATA3 and nuclear ß-catenin staining. BAC-positive/CAC-negative markers without significance were: p63, keratin 5/6, 5/14, uroplakins II/III and AR. CC showed a urothelial phenotype (p63+, GATA3+, S100P+, uroplakin+ in single cells) with initial signs of intestinal differentiation (single cells cdx2+ or cadherin 17+). IM displayed a full intestinal phenotype (p63-, all urothelial markers-, cdx2/MUC2/MUC5AC+, cadherin17+).
Differential diagnosis of BAC and CAC remains difficult, but positive staining for keratin 7 in nuclear ß-catenin-negative tumours argues for BAC. Additional markers like GATA3 and p63 may be added, as positivity in some cases may be helpful. However, for reliable histological diagnosis, knowledge of comprehensive clinical data is still essential.
Dendritic cells (DCs) are key initiators and regulators of the immune response. The development of the DC lineage and their subsets requires an orchestrated regulation of their transcriptional program. Gata1, a transcription factor expressed in several hematopoietic cell lineages, has been recently reported to be required for mouse DC development and function. In humans, GATA1 is involved in the lineage separation between monocyte-derived DCs and Langerhans cells (LC) and loss of GATA1 results in differentiation arrest at the monocyte stage. The hematopoietic GATA factors (i.e. Gata1, Gata2, Gata3) are known to regulate each other's expression and to function consecutively throughout lineage commitment (so-called GATA switch). In humans, mutations in GATA2 are causative of MonoMAC disease, a human immunodeficiency syndrome characterized by loss of DCs, monocytes, B and NK cells. However, additional data on the expression of hematopoietic GATA factors in the DC lineage is missing. In this study, we have characterized the expression of hematopoietic GATA factors in murine and human DCs and their expression dynamics upon TLR stimulation. We found that all hematopoietic GATA factors are expressed in DCs, but identified species-specific differences in the relative expression of each GATA factor, and how their expression fluctuates upon stimulation.
Tumors of trophoblastic derivation other than choriocarcinoma are very rare in the testis but have been reported on occasion in association with other germ cell tumors. Their morphologic spectrum is analogous to the trophoblastic tumors of the female genital tract including epithelioid trophoblastic tumor (ETT) and placental site trophoblastic tumor (PSTT). Herein we report our experience with 8 cases of trophoblastic tumors of testicular origin that lacked the features of choriocarcinoma; these included 4 ETTs, 1 PSTT, 1 unclassified trophoblastic tumor (UTT), 1 partially regressed choriocarcinoma with a monophasic morphology, and 1 hybrid tumor showing a mixture of adenocarcinoma and a UTT. All tumors occurred in young men 19 to 43 years old. Five arose de novo within the testis (2 ETTs, 1 UTT, 1 regressing choriocarcinoma, and the hybrid tumor) as a component of mixed germ cell tumors, and 3 (2 ETTs and 1 PSTT) were found in metastatic sites after chemotherapy. The trophoblastic component was minor (5% to 10%) in 6 tumors but was 95% of 1 metastatic tumor (ETT) and 50% of the hybrid tumor. Other germ cell tumor elements were identified in all cases, most commonly teratoma. The ETTs consisted of nodules and nests of squamoid trophoblast cells showing abundant eosinophilic cytoplasm, frequent apoptotic cells, extracellular fibrinoid material, and positivity for p63 and negativity for human placental lactogen (HPL). The PSTT showed sheets of discohesive, pleomorphic, mononucleated trophoblast cells that invaded blood vessels with fibrinoid change and were p63 negative and HPL positive. The UTT showed a spectrum of small and large trophoblast cells, some multinucleated but lacking distinct syncytiotrophoblasts, and was patchily positive for both p63 and HPL. The hybrid tumor had ETT-like and adenocarcinomatous areas that coexpressed inhibin and GATA3 but were negative for p63 and HPL, leading to classification of the trophoblastic component as UTT. Seven of the patients were alive and well on follow-up (8 to 96 mo; median, 39 mo), whereas the patient with the hybrid tumor died of liver metastases at 2 years. Our study verifies that trophoblastic neoplasms often having the features of nonchoriocarcinomatous gestational trophoblastic tumors may arise from the testis, occur either in the untreated primary tumor or in metastases after chemotherapy, and should be distinguished from choriocarcinoma given what appears to be a less aggressive clinical course.
The metastastic cascade is a complex process that is regulated at multiple levels in prostate cancer (PCa). Recent evidence suggests that microRNAs (miRNAs) are involved in PCa metastasis and hold great promise as therapeutic targets. In this study, we found that miR-573 expression is significantly lower in metastatic tissues than matched primary PCa. Its downregulation is correlated with high Gleason score and cancer-related mortality of PCa patients (P = 0.041, Kaplan-Meier analysis). Through gain- and loss-of function experiments, we demonstrated that miR-573 inhibits PCa cell migration, invasion and TGF-β1-induced epithelial-mesenchymal transition (EMT) in vitro and lung metastasis in vivo. Mechanistically, miR573 directly targets the fibroblast growth factor receptor 1 (FGFR1) gene. Knockdown of FGFR1 phenocopies the effects of miR-573 expression on PCa cell invasion, whereas overexpression of FGFR1 partially attenuates the functions of miR-573. Consequently, miR-573 modulates the activation of FGFR1-downstream signaling in response to fibroblast growth factor 2 (FGF2). Importantly, we showed that GATA3 directly increases miR-573 expression, and thus down-regulates FGFR1 expression, EMT and invasion of PCa cells in a miR-573-dependent manner, supporting the involvement of GATA3, miR-573 and FGFR1 in controlling the EMT process during PCa metastasis. Altogether, our findings demonstrate a novel mechanism by which miR-573 modulates EMT and metastasis of PCa cells, and suggest miR-573 as a potential biomarker and/or therapeutic target for PCa management.
Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.
Adult T cell leukemia/lymphoma (ATL) is a peripheral T cell neoplasm of largely unknown genetic basis, associated with human T cell leukemia virus type-1 (HTLV-1) infection. Here we describe an integrated molecular study in which we performed whole-genome, exome, transcriptome and targeted resequencing, as well as array-based copy number and methylation analyses, in a total of 426 ATL cases. The identified alterations overlap significantly with the HTLV-1 Tax interactome and are highly enriched for T cell receptor-NF-κB signaling, T cell trafficking and other T cell-related pathways as well as immunosurveillance. Other notable features include a predominance of activating mutations (in PLCG1, PRKCB, CARD11, VAV1, IRF4, FYN, CCR4 and CCR7) and gene fusions (CTLA4-CD28 and ICOS-CD28). We also discovered frequent intragenic deletions involving IKZF2, CARD11 and TP73 and mutations in GATA3, HNRNPA2B1, GPR183, CSNK2A1, CSNK2B and CSNK1A1. Our findings not only provide unique insights into key molecules in T cell signaling but will also guide the development of new diagnostics and therapeutics in this intractable tumor.
Encysted cyathostomin larvae are ubiquitous in grazing horses. Arrested development occurs in this population and can lead to an accumulation of encysted larvae. Large numbers of tissue larvae place the horse at risk for developing larval cyathostominosis. This disease complex is caused by mass emergence of these larvae and is characterized by a generalized acute typhlocolitis and manifests itself as a profuse protein-losing watery diarrhea with a reported case-fatality rate of about 50%. Two anthelmintic formulations have a label claim for larvicidal therapy of these encysted stages; moxidectin and a five-day regimen of fenbendazole. There is limited knowledge about inflammatory and immunologic reactions to larvicidal therapy. This study was designed to evaluate blood acute phase reactants as well as gene expression of pro-inflammatory cytokines, both locally in the large intestinal walls and systemically. Further, mucosal tissue samples were evaluated histopathologically as well as analyzed for gene expression of pro- and anti-inflammatory cytokines, cluster of differentiation (CD) cell surface proteins, and select transcription factors. Eighteen juvenile horses with naturally acquired cyathostomin infections were randomly assigned to three treatment groups; one group served as untreated controls (Group 1), one received a five-day regimen of fenbendazole (10mg/kg) (Group 2), and one group received moxidectin (0.4mg/kg) (Group 3). Horses were treated on day 0 and euthanatized on days 18-20. Serum and whole blood samples were collected on days 0, 5, and 18. All horses underwent necropsy with collection of tissue samples from the ventral colon and cecum. Acute phase reactants measured included serum amyloid A, iron and fibrinogen, and the cytokines evaluated included interferon γ, tumor necrosis factor α, transforming growth factor (TGF)-β, and interleukins 1β, 4, 5, 6, and 10. Transcription factors evaluated were FoxP3, GATA3 and tBet, and CD markers included CD163, CD3z, CD4, CD40, and CD8b. Histopathology revealed an inflammatory reaction with higher levels of lymphocytes, T cells, B cells, eosinophils and fibrous tissue in the moxidectin-treated group compared to controls or horses treated with fenbendazole. No apparent systemic reactions were observed. Expression of IL-5 and TGF-β in intestinal tissues was significantly lower in Group 3 compared to Group 1. This study revealed a subtle inflammatory reaction to moxidectin, which is unlikely to cause clinical issues.
PELP1 is a novel coregulator of nuclear hormone receptors and is implicated in playing a role in driving breast cancer and enhancing metastatic potential. The PELP1 protein expression and potential role of PELP1 in triple-negative breast carcinoma (TNBC) have not been well characterized. We investigated PELP1 expression by immunohistochemistry in primary and metastatic triple-negative tumors in human tissues and compared its expression with GATA-binding protein 3 (GATA3), a novel diagnostic marker for TNBC. We examined the expression of PELP1 and GATA3 in 70 primary TNBC cases and found that PELP1 had a significantly higher frequency of expression compared to GATA3 (96% versus 46%; P < .0001). The mean extent score of expression of PELP1 was also significantly higher than GATA3's expression (3.87 ± 0.07 versus 0.91 ± 0.15; P < .0001). PELP1 had stronger staining intensity than GATA3. Furthermore, PELP1 immunoreactivity was consistently maintained in paired primary and metastatic TNBC cases (100%). The frequency of PELP1 expression (100%) in metastatic triple-negative tumors was higher than that of GATA3 (40%) in the same tumors (P < .0001). These findings indicate that PELP1 is a much more sensitive marker than GATA3 for TNBCs. PELP1 may have diagnostic utility for metastatic TNBC in appropriate settings, such as history of primary TNBC in cases where the primary is negative for GATA3, mammaglobin, and GCDFP-15. The diffuse and strong nuclear immunoreactivity of PELP1 in most cases suggests that PELP1 may be a molecular target for the treatment of TNBC. We hope that this study will provide insights into the role of PELP1 in TNBC.
The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.
Uterine cervical carcinoma secondarily involving the genitourinary tract is rarely documented histologically. These tumors present a unique diagnostic challenge as they can appear morphologically similar to urothelial carcinoma as well as primary squamous cell carcinoma and primary adenocarcinoma of the bladder. Genitourinary consult cases at the Johns Hopkins Hospital from 1984 to the present were searched for cases in which the differential diagnosis was primary bladder carcinoma versus secondary involvement by cervical carcinoma. We identified 10 cases that met these criteria and evaluated them by immunohistochemistry for p16 and GATA3 and in situ hybridization for human papillomavirus (HPV). Six cases were received with a gynecologic history. Four cases had been misdiagnosed as urothelial carcinoma, and 1 case was favored to be cystitis cystica et glandularis by the submitting institutions. Morphologically, the majority of cases showed basaloid nests of tumor cells infiltrating muscle bundles, with several having foci that mimicked urothelial carcinoma in situ. Six tumors were found to be diffusely positive with p16, 1 tumor was patchy, 1 was weak, and 2 were negative. GATA3 staining was negative in 6 cases, and 4 showed weak to strong positivity. Eight cases were positive for high-risk HPV (6 were positive for HPV 16, and 1 was positive for HPV 18). In the 2 cases that were negative for HPV by in situ hybridization, characteristic morphologic features of HPV-unrelated type of endocervical adenocarcinoma were present. On the basis of our findings we advocate a multifaceted approach, combining morphologic evaluation with ancillary studies including immunohistochemistry and in situ hybridization in the evaluation of genitourinary specimens for secondary involvement by cervical carcinoma. Furthermore, gynecologic clinical history is absolutely critical and most important to the evaluation and diagnosis of these specimens, as these ancillary studies are not completely sensitive or specific.
The transcription factor FOXP3 is essential for the differentiation and function of regulatory T cells (Treg). It is established that the transcription factor GATA-3 is induced in Treg cells under inflammatory conditions. GATA-3 stabilizes FOXP3 levels to avoid the differentiation of Treg cells into inflammatory-like T cells. The IL-6 signal pathway influences the sensitivity of Treg cells towards instability. The mechanism of GATA-3 in regulating FOXP3 and its relation to the IL-6 pathway remains unclear. Here we report how miR-125a-5p plays an important role in regulating the conversion of Treg cells by IL-6. miR-125a-5p expression is low in Treg cells under steady state conditions and can be induced by GATA-3 to inhibit the expression of IL-6R and STAT3. This finding reveals a GATA3/miR-125a-5p/IL-6R and STAT3/FOXP3 regulatory pathway, which determines how Treg cells respond to inflammatory IL-6-rich conditions.
An inhibitor of 3T3-L1 adipocyte differentiation was isolated from Streptomyces sp. TK08330 and identified by spectroscopy as the 18-membered macrolide borrelidin. Treatment with 1.0 μM borrelidin suppressed intracellular lipid accumulation by 80% and inhibited the expression of adipocyte-specific genes. Borrelidin suppressed the mRNA expression of two master regulators of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein (C/EBPα). Studies on well-known upstream regulators of PPARγ revealed that borrelidin down-regulated C/EBPδ mRNA expression but did not affect expression of C/EBPβ. Borrelidin increased mRNA expression of negative regulators of differentiation such as GATA-binding protein (GATA) 3, Krüppel-like factor (KLF) 3 and KLF7, as well as positive regulators, KLF4, KLF6 and KLF15, at early stages of differentiation. To elucidate a primary mediator of borrelidin differentiation inhibitory activity, small interfering RNA (siRNA) transfection experiments were performed. The mRNA expression of PPARγ, which was down-regulated by borrelidin, was not changed by KLF3 and KLF7 siRNA treatment. In contrast, expression of PPARγ in GATA-3 siRNA-treated cells was not significantly different from that of control siRNA-treated cells. Borrelidin significantly inhibited lipid accumulation in control siRNA-treated cells, and treatment with GATA-3 siRNA slightly reduced the inhibitory effect of borrelidin. These results indicate that borrelidin inhibited adipocyte differentiation partially via GATA-3.
Mast cell (MC) degranulation is the foundation of the acute phase of allergic rhinitis (AR). Previously, downregulation of GATA binding protein 3 (GATA-3) was shown to suppress MC activation in an AR mouse model. Binding of microRNA-135a (miR-135a) to GATA-3 was also observed, and overexpression of this miRNA decreased GATA-3 mRNA and protein expression. However, the effects of miR-135a on MCs during AR are currently unknown. In the present study, we utilized a lentiviral (LV) vector to intranasally administer miR-135a to ovalbumin (OVA)-sensitized AR mice. Following miR-135a treatment, the total serum IgE concentration observed during AR was significantly reduced. In the nasal mucosa, the expression of T-box expressed in T cells (T-bet) was higher, whereas that of GATA-3 was lower in the AR mice following miRNA treatment. Notably, during AR, the ratio of type 1 T-helper cells (Th1) to type 2 (Th2) cells in the spleen is unbalanced, favoring Th2. However, administering miR-135a to the AR mice appeared to balance this ratio by increasing and decreasing the percentage of Th1 and Th2 cells, respectively. MiR-135a also appeared to strongly suppress the infiltration of eosinophils and MCs into the nasal mucosa, and it was specifically localized in the MCs, suggesting that its influence is modulated through regulation of GATA-3 in these cells. Additional work identifying the full therapeutic potential of miR-135a in the treatment of AR and diseases involving allergen-induced inflammation is warranted.
Invasive lobular carcinoma of the breast is a relatively common diagnosis. However, other carcinomatous as well as noncarcinomatous neoplasms, either primary or metastatic to the breast, may mimic invasive lobular carcinoma. As treatment may differ, establishing the correct diagnosis is paramount to providing the appropriate care for these patients. This review outlines important mimics of invasive lobular carcinoma and the key clinicopathologic and immunohistochemical features as well as additional studies helpful in establishing their diagnoses.
Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.
Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.
Genome-wide association studies and other genetic analyses have identified a large number of genes and variants implicating a variety of disease etiological mechanisms. It is imperative for the study of human diseases to put these genetic findings into a coherent functional context. Here we use system biology tools to examine disease connections of five master genes for CD4+ T cell subtypes (TBX21, GATA3, RORC, BCL6, and FOXP3). We compiled a list of genes functionally interacting (protein-protein interaction, or by acting in the same pathway) with the master genes, then we surveyed the disease connections, either by experimental evidence or by genetic association. Embryonic lethal genes (also known as essential genes) are over-represented in master genes and their interacting genes (55% versus 40% in other genes). Transcription factors are significantly enriched among genes interacting with the master genes (63% versus 10% in other genes). Predicted haploinsufficiency is a feature of most these genes. Disease-connected genes are enriched in this list of genes: 42% of these genes have a disease connection according to Online Mendelian Inheritance in Man (OMIM) (versus 23% in other genes), and 74% are associated with some diseases or phenotype in a Genome Wide Association Study (GWAS) (versus 43% in other genes). Seemingly, not all of the diseases connected to genes surveyed were immune related, which may indicate pleiotropic functions of the master regulator genes and associated genes.
Graves' disease (GD) is a common autoimmune disorder that is primarily driven by malfunction of T lymphocytes, which influences the production of antibodies reacting with cellular and tissue components of the thyroid gland. The aim of this study was to determine the level of mRNA expression for the genes encoding T-bet and GATA3, as the master regulators of the T helper (Th)1 and Th2 differentiation, respectively together with Th1 (IFN-γ) and Th2(IL-4) cytokine mRNA expression. Relative quantification of T-bet, GATA3, IFN-γ and IL-4 transcripts in peripheral blood mononuclear cell (PBMCs) was conducted by real-time reverse transcriptase PCR (RT-PCR). Serum levels of IFN-γ and IL-4 were also determined by Enzyme-linked immunosorbent assay (ELISA). In comparison with the normal group, T-bet and IFN-γ mRNA expression levels were significantly up-regulated in the GD patients, while GATA3 and IL-4 mRNA expression levels were downregulated. In addition, a marked decrease in plasma IL-4 levels was observed in the GD group. IFN-γ levels were also higher in patients in comparison to the controls. Furthermore, a clear correlation between increased IFN-γ mRNA expression and IFN-γ (P<0.05) was revealed. These results indicate that a Th1/Th2 imbalance exists in GD, and it may be implicated in the pathogenesis of disease.
The hormone-sensing mammary epithelial cell (HS-MEC-expressing oestrogen receptor-alpha (ERα) and progesterone receptor (PGR)) is often represented as being terminally differentiated and lacking significant progenitor activity after puberty. Therefore while able to profoundly influence the proliferation and function of other MEC populations, HS-MECs are purported not to respond to sex hormone signals by engaging in significant cell proliferation during adulthood. This is a convenient and practical simplification that overshadows the sublime, and potentially critical, phenotypic plasticity found within the adult HS-MEC population. This concept is exemplified by the large proportion (~80 %) of human breast cancers expressing PGR and/or ERα, demonstrating that HS-MECs clearly proliferate in the context of breast cancer. Understanding how HS-MEC proliferation and differentiation is driven could be key to unraveling the mechanisms behind uncontrolled HS-MEC proliferation associated with ERα- and/or PGR-positive breast cancers. Herein we review evidence for the existence of a HS-MEC progenitor and the emerging plasticity of the HS-MEC population in general. This is followed by an analysis of hormones other than oestrogen and progesterone that are able to influence HS-MEC proliferation and differentiation: androgens, prolactin and transforming growth factor-beta1.
Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.
To identify differentially expressed T cells-related genes in peripheral blood mononuclear cells and compare their differences in T cell activation and subset functions in different stages of coronary atherosclerosis disease (CAD).
20 patients with acute myocardial infarction patients (AMI), 20 patients with stable angina pectoris (SA) and 20 healthy volunteers were recruited into the study. Whole human genome microarray analysis was used to detect the expression of T cell related genes among three groups.
mRNA expression of 68 genes involved in T cell was detected. 1) Antigen recognition: in the AMI patients 12 genes were down-regulated and 9 were significantly down-regulated among all 13 genes, compared with those of the SA and the control group, respectively. 2) Co-stimulators and regulators of T cell activation: among 16 genes in the AMI patients, 15 genes were lower and 8 were significantly lower than the other two groups. 3) T cell subsets, CTL: all 11 genes in the AMI patients were down-regulated, particularly GZMM and CASP8 were significantly down-regulated compared with the SA patients and controls. Th1/Th2: in the AMI patients, gene expressions including IL1 and IL18 were significantly higher, whereas GATA3 mRNA was significantly lower than those in other two groups. Th17/Treg: in the AMI group, RORC and CCR6 mRNAs were significantly down-regulated compared with the control group, while CD25 and CD127 expressions were significantly lower than SA group. There was no difference in T cell related genes between the SA patients and the controls.
In the AMI patients, the mRNA expression of T cell antigen recognition, activation and subset functions was imbalanced or decreased, indicating the dysfunction of cellular immunity in patients with AMI. Then improving T cell mediated cellular immunity may be considered as a potential target for medical interventions in the patients with AMI.
The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional activator but also as a repressor.
The shoot apical meristem (SAM) is essential for continuous organogenesis in higher plants, while the leaf is the primary source organ and the leaf shape directly affects the efficiency of photosynthesis. HANABA TARANU (HAN) encodes a GATA3-type transcription factor that functions in floral organ development, SAM organization, and embryo development in Arabidopsis, but is involved in suppressing bract outgrowth and promoting branching in grass species. Here the function of the HAN homologue CsHAN1 was characterized in cucumber, an important vegetable with great agricultural and economic value. CsHAN1 is predominantly expressed at the junction of the SAM and the stem, and can partially rescue the han-2 floral organ phenotype in Arabidopsis. Overexpression and RNAi of CsHAN1 transgenic cucumber resulted in retarded growth early after embryogenesis and produced highly lobed leaves. Further, it was found that CsHAN1 may regulate SAM development through regulating the WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) pathways, and mediate leaf development through a complicated gene regulatory network in cucumber.
Effector CD4 T cell lineages have been implicated as potent inducers of autoimmune diseases. Tbet, Gata3 and Rorgt are master transcriptional regulators of Th1, Th2 and Th17 lineages respectively and promote the distinct expression of signature cytokines. Significant progress has been made in understanding the transcriptional network that drives CD4 T cell differentiation, revealing novel points of regulation mediated by transcription factors, cell surface receptors, cytokines and chemokines. Epigenetic modifications and metabolic mediators define the transcriptional landscape in which master transcription factors operate and collaborate with a network of transcriptional modifiers to guide lineage specification, plasticity and function.
The transcription factor GATA3 is a recently described biomarker that is highly expressed in bladder and breast carcinomas. Although it has shown sensitivity as a marker of primary bladder carcinomas with purely urothelial differentiation, the ability of GATA3 to label primary bladder carcinomas with variant morphologic patterns has been incompletely assessed to date. The current study was designed to determine whether GATA3 staining is retained in "unconventional" bladder carcinomas. Eighty-eight cases of primary bladder cancers were retrieved from the authors' institutional archive, and they included the following histomorphologic types: 6 small cell carcinomas, 12 sarcomatoid carcinomas, 17 adenocarcinomas (both primary and urothelial variants with glandular differentiation), 24 micropapillary carcinomas, and 27 squamous cell carcinomas (both primary and urothelial variants with squamous differentiation). A tissue microarray was constructed and automated immunostaining for GATA3 (Clone L50-823, Biocare Medical, Concord, CA) was performed using standard technique. Among the 5 variants of unconventional bladder carcinoma, only the micropapillary and sarcomatoid forms exhibited consistent and strong immunolabeling for GATA3. Hence, the sensitivity of this determinant is diminished in several histologic forms of primary bladder carcinoma. That fact will affect the interpretation of GATA3 stains in the context of possible metastasis from primary bladder carcinomas with variant morphologic patterns, as well as their distinction from secondary bladder involvement by tumors of nonurothelial origin.
Hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant genetic disorder characterized by hypoparathyroidism, sensorineural deafness and renal dysplasia. We herein present the first Turkish patient with HDR syndrome, who has a p.R367X mutation. This report indicates that p.R367X is not a mutation specific for the Far Eastern populations and also that urological findings in infants with hypoparathyroidism should be carefully examined because clinical findings relating to the p.R367X mutation may show a variable age of onset.
β1,4-Galactosylransferases are a family of enzymes encoded by seven B4GALT genes and are involved in the development of anticancer drug resistance and metastasis. Among these genes, the B4GALT1 shows significant variations in the transcript origination sites in different cell types/tissues and encodes an interesting dually partitioning β-1, 4-galactosyltransferase protein. We identified at 5'-end of B4GALT1 a 1.454 kb sequence forming a transcription regulatory region, referred to by us as the TR1-PE1, had all characteristics of a bidirectional promoter directing the transcription of B4GALT1 in a divergent manner along with its long non-coding RNA (lncRNA) antisense counterpart B4GALT1-AS1. The TR1-PE1 showed unique dinucleotide base-stacking energy values specific to transcription factor binding sites (TFBSs), INR and BRE, and harbored CpG Island (CGI) that showed GC skew with potential for R-loop formation at the transcription starting sites (TSSs). The 5'-regulatory axis of B4GALT1 also included five more novel TFBSs for CTCF, GLI1, TCF7L2, GATA3 and SOX5, in addition to unique (TG)18 repeats in conjunction with 22 nucleotide TG-associated sequence (TGAS). The five lncRNA B4GALT1-AS1 transcripts showed significant complementarity with B4GALT1 mRNA. In contrast, the rest of B4GALT genes showed fewer lncRNAs, and all lacked the (TG)18 and TGAS. Our results are strongly supported by the FANTOM5 study which showed tissue-specific variations in transcript origination sites for this gene. We suggest that the unique expression patterns for the B4GALT1 in normal and malignant tissues are controlled by a differential usage of 5'-B4GALT1 regulatory units along with a post-transcriptional regulation by the antisense RNA, which in turn govern the cell-matrix interactions, neoplastic progression, anticancer drug sensitivity, and could be utilized in personalized therapy.
GATA3 is a critical transcription factor in the development of various human systems. The notion that GATA3 expression is required for the differentiation and maintenance of normal breast tissue has been well established. Recently, GATA3 is found to actively participate in the multistep process leading breast cancer pathogenesis, including tumorigenesis, tumor differentiation, epithelial mesenchymal transition, and metastasis through regulation of various target genes. On the other hand, several studies have raised questions and highlighted the role of GATA3-low or GATA3-negative cells during the malignant development of breast cancer. In addition to gene expression, GATA3 mutations provide another dimension of complexity. As one of the most frequently mutated genes in breast cancer, GATA3 mutations may have an effect on DNA-binding ability, protein production, and transactivation activity. Recognition of the multiple function of GATA3 in breast cancer will serve to deepen our understanding of the nature of this disease and develop novel therapeutic approaches.
Estradiol (E2 ) deficiency can cause bone loss and the skew of Th1/Th2 cells. However, the correlation between the Th1/Th2 cells and the bone loss induced by estrogen deficiency remains unclear. Our aim was to investigate the role of Th1/Th2 in bone loss induced by estrogen deficiency and elucidated the therapeutical effect of catalpol in this condition.
Young, sham-operated (Sham), ovariectomized (Ovx), and naturally aged mice, treated with catalpol at different doses or control vehicle, were used in this study as indicated in each experiment. ELISA assay, dual-energy X-ray absorptiometry, and flow cytometry were used to analyze E2 , C-terminal telopeptides of type I collagen (CTx-I), bone mineral density (BMD), and Th1/Th2 subsets, respectively. The mRNA and protein expressions of specific transcription factors for Th1/Th2 cells (T-bet and GATA-3) were analyzed using real-time quantitative PCR and Western blot, respectively.
Bone mineral density and E2 levels positively correlated with the proportion of Th2 subset while negatively correlated with that of Th1 subset and the ratio of Th1/Th2. Catalpol alleviated bone loss effectively by regulating Th1/Th2 polarization. Catalpol promoted the expression of Th2-specific transcription factors while inhibited that associated with Th1.
Th1/Th2 skew is involved in bone loss induced by estrogen deficiency. Catalpol alleviates bone loss effectively by regulating Th1/Th2 paradigm.
Transcription factors (TFs) regulate cell-type-specific gene expression programs by combinatorial binding to cis-genomic elements, particularly enhancers, subsequently leading to the recruitment of cofactors, and the general transcriptional machinery to target genes. Using data integration of genome-wide TF binding profiles, we defined regions with combinatorial binding of lineage-specific master TFs (T-BET, GATA3, and ROR-γt) and STATs (STAT1 and STAT4, STAT6, and STAT3) in murine T helper (Th) 1, Th2, and Th17 cells, respectively. Stringently excluding promoter regions, we revealed precise genomic elements which were preferentially associated with the enhancer marks p300 and H3K4me1. Furthermore, closely adjacent TF co-occupied regions constituted larger enhancer domains in the respective Th-cell subset (177 in Th1, 141 in Th2, and 266 in Th17 cells) with characteristics of so-called super-enhancers. Importantly, 89% of these super-enhancer regions were Th-cell subtype-specific. Genes associated with super-enhancers, including relevant Th-cell genes (such as Ifng in Th1, Il13 in Th2, and Il17a in Th17 cells), showed strong transcriptional activity. Altogether, the discovered catalog of enhancers provides information about crucial Th-cell subtype-specific regulatory hubs, which will be useful for revealing cell-type-specific gene regulation processes.
Although numerous efforts have been made, the pathogenesis underlying lung squamous-cell carcinoma (SCC) remains unclear. This study aimed to identify the CNV-driven genes by an integrated analysis of both the gene differential expression and copy number variation (CNV).
A higher burden of the CNVs was found in 10-50 kb length. The 16 CNV-driven genes mainly located in chr 1 and chr 3 were enriched in immune response [e.g. complement factor H (CFH) and Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A)], starch and sucrose metabolism [e.g. amylase alpha 2A (AMY2A)]. Furthermore, 38 TFs were screened for the 9 CNV-driven genes and then the regulatory network was constructed, in which the GATA-binding factor 1, 2, and 3 (GATA1, GATA2, GATA3) jointly regulated the expression of TP63.
The above CNV-driven genes might be potential contributors to the development of lung SCC.
Interleukin (IL)-35 is a novel regulatory cytokine primarily produced by regulatory T cells. Accumulating evidence has established that IL-35 plays an important role in the regulation of immune homeostasis, but little is known regarding the function of IL-35 in acquired aplastic anemia (AA). The aim of the present study was to investigate the expression of IL-35 and its effects on T cell response in AA. Our study demonstrated that significantly decreased plasma levels of IL-35 in AA were closely correlated with disease severity. In vitro stimulation experiment further confirmed the anti-inflammatory effects of IL-35, including suppressing the proliferation of CD4(+) and CD8(+) effector T cells, inhibiting the secretion of interferon-γ, tumor necrosis factor-α and IL-17 and promoting the production of transforming growth factor-β by peripheral blood mononuclear cells from patients with AA. Furthermore, we established that IL-35 inhibited the differentiation of type 1 T cells and T helper 17 cells but promoted the differentiation of type 2 T cells. Accordingly, the expression of T-bet and RORγt was inhibited while the expression of GATA3 was induced after IL-35 treatment. In summary, our findings suggested that decreased IL-35 might contribute to the loss of immune-tolerance and be critically involved in the pathogenesis of AA.
The distinction between metastatic breast carcinomas (BCs) and primary lung carcinomas (PLCs) can be difficult. This study tested the utility of trefoil factor 1 (TFF1) for this purpose and compared it with mammaglobin and GATA protein binding 3 (GATA-3).
Tissue microarrays containing 365 BCs and 338 PLCs were stained with TFF1, mammaglobin, and GATA-3, and an H-score was calculated. Sensitivity, specificity, and accuracy were calculated, and logistical regression analysis was performed.
Accuracy of correctly classifying the tumor type was 81.9%, 71.3%, and 64.0% for GATA-3, mammaglobin, and TFF1, respectively. Odds ratios for selecting BCs were 25.69, 93.15, and 4.17, respectively, with P values less than .001. With a single exception, the best immunopanel included GATA-3 and mammaglobin in all comparisons.
TFF1 demonstrated breast specificity but was inferior to mammaglobin and GATA-3. Therefore, its routine clinical use may not be justified. TFF1 showed little benefit when added to an immunopanel.
GATA3 plays a role in cell proliferation and differentiation in many tissues, including breast, and it has been suggested that GATA3 expression correlates with ER expression. However, little is known on GATA3 expression in various subtypes of breast carcinoma, its utilization in cytology, and on how GATA3 performs in comparison with GCDFP-15 and mammaglobin. Eighty-four histology cases of breast carcinoma of various subtypes, including 28 triple-negative breast carcinomas, along with 20 cytology cases of metastatic breast carcinoma and 12 cytology cases of ER-positive metastatic gynecologic malignancies, were stained for GATA3, GCDFP-15, and mammaglobin. In non-triple-negative breast carcinomas (n=56), GATA3 showed 100% sensitivity, higher than GCDFP-15 (42.8%; P<0.0001) and mammaglobin (58.9%; P<0.0001), whereas staining patterns were similar for all the histologic subtypes examined. Staining scores were determined by multiplying the percentage of cancer cells staining with an intensity score of 1+, 2+, or 3+ (range, 0 to 300). In non-triple-negative carcinomas, GATA3 showed a mean score of 273.7, higher than GCDFP-15 (107.5; P<0.0001) and mammaglobin (147.7; P<0.0001). In triple-negative breast carcinomas (n=28), GATA3 showed a sensitivity of 60.7%, greater than GCDFP-15 (17.9%; P=0.0022) and mammaglobin (7.1%; P<0.0001). These results were consistent irrespective of the subtype examined. In breast carcinoma cytology cases, 100% stained with GATA3, higher than GCDFP-15 (20%; P<0.0001) and mammaglobin (45%; P<0.0001). None of the metastatic endometrial or ovarian carcinomas were positive for GATA3. Although GATA3 expression correlates with ER expression in breast, no correlation is observed in gynecologic malignancies. Thus, in working up ER-positive metastatic malignancies GATA3 demonstrates specificity for breast.
We describe the case of a 21-year-old male with hypocalcaemia, hyperphosphataemia, recurrent limb twitch, deafness, proteinuria, increased serum creatinine and urea nitrogen levels, and shrinkage of both kidneys. Brain computed tomography showed intracranial calcifications. The patient was diagnosed with hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. DNA sequence analysis of the GATA3 gene showed a novel de novo mutation, c. 529dupC (p. Arg177profs*126), in exon 2, resulting in a frameshift mutation with a premature stop codon after a new 126 amino acid sequence. We provide further evidence that HDR syndrome is caused by haploinsufficiency of GATA3.
Sinonasal adenocarcinoma is a rare tumor of the head and neck accounting for 10% to 20% of all primary malignancies of the nose and paranasal sinuses. There tumors are classified as salivary, intestinal and nonintestinal, nonsalivary. Low-grade nonintestinal nonsalivary are rare tumors whose diagnosis is essentially that of exclusion. Here we present the first case of one such tumor associated with an exophyic Schneiderian papilloma. A 71-year-old retired aerospace engineer presented with a 1-year history of severe nasal obstruction. Endoscopy and compted tomography imaging demonstrated a polypoid lesion occupying his entire right nasal cavity extending into and filling the nasopharynx. Biopsy suggested adenocarcinoma, at least in situ and the patient subsequently underwent complete resection. Pathologic evaluation demonstrated polypoid tumor consistent with a low-grade papillary adenocarcinoma with micropapillary architecture associated with a small amount of residual exophytic Schneiderian papilloma. Immunohistochemistry revealed diffuse expression of CK7, CK 5/6, and S100 protein in tumor cells. Expression of p63 was seen in basal cells only. Tumor cells did not show expression of CK20, CDX2 (intestinal markers), mammaglobin, GATA3 (salivary markers), PAX8, WT1, nor estrogen, progesterone, or androgen receptors confirming its nonintestinal nonsalivary differentiation.
Ductal carcinoma in situ (DCIS) is a noninvasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer whereas others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome, and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty-two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM, and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor-intrinsic subtypes, proliferative, immune scores, and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like, or ERBB2(+)) displayed signatures characteristic of activated Treg cells (CD4(+)/CD25(+)/FOXP3(+)) and CTLA4(+)/CD86(+) complexes indicative of a tumor-associated immunosuppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly, ncRNA and methylation profiles reproduce changes observed postinvasion. Among the most significant findings, we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a preinvasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer.
Group 2 innate lymphoid cells (ILC2s) are innate effectors playing an important role in the defense against helminthic infections and in the pathogenesis of allergic inflammation. Cytokines have been identified as the major stimuli driving ILC2 activation and expansion. Conversely, it is unclear whether costimulatory molecules contribute to regulation of ILC2 functions. ILC2s display high expression of inducible T-cell costimulator (ICOS), which belongs to the CD28 superfamily, and which has been shown to control late effector T-cell functions, and is of utmost importance for the humoral immune response. However, the biological function of ICOS expression on ILC2s is unknown. Here, we show that ICOS signaling in mice regulates ILC2 homeostasis independently of T cells and B cells, by promoting proliferation and accumulation of mature ILC2s in lung and intestine. In a model of IL-33-induced airway inflammation, ICOS controls ILC2 activation and eosinophil infiltration in the lung. Our data identify a role of ICOS in innate immunity and indicate that not only cytokines, but also costimulatory pathways such as those involving ICOS, can contribute to regulate the ILC2 pool. Thus, ICOS costimulation blockade, which is currently under clinical evaluation for inhibiting the humoral immune response, could also target innate inflammatory circuits.
Metaplastic squamous cell carcinoma (SCC) of the breast is a rare type of breast cancer. Metastases to the lung, which can be a major site of second primary tumor development among breast cancer patients, are difficult to distinguish from primary SCC of the lung and present a unique challenge for pathologists. There are few available discriminating immunohistochemical markers as squamous differentiation typically leads to loss of expression of characteristic primary epithelial cell markers of both breast and lung origin. GATA protein binding 3 (GATA-3) is a useful marker of breast origin in metastatic ductal and lobular carcinomas including poorly differentiated triple-negative carcinomas and some metaplastic carcinomas. Here, we present a case of metastatic SCC presenting as a solitary lung mass with regional lymph node metastases and a single satellite lesion in a patient with a history of metaplastic SCC of the breast. In addition to the routine markers of squamous differentiation, the metastases were also positive for estrogen receptor (ER) and GATA-3 on cytologic material obtained by transbronchial FNA. This suggests that immunoreactivity for ER and GATA-3 may support a diagnosis of metastatic SCC in the context of a prior metaplastic SCC of the breast.
We discuss the histological and immunohistochemical features of 6 cases of urothelial carcinomas of lipid cell variant and 4 cases with shadow cell differentiation, one of which showed additionally sebaceous differentiation, one of which shows additional sebaceous differentiation, from our archive cases from the last 15 years. Conventional urothelial carcinoma (UC) was seen in all lipid cell variant cases, and micropapillary carcinoma was seen in 3. The ratio of the lipid cell component was between 10% and 40% in these 6 cases. Typical histologic features of the lipid cell variant include lipoblast-like cells with a notched nuclear appearance, abundant vacuoles, an eccentric nucleus, and pagetoid spread in some areas. GATA3 and pancytokeratin AE1/AE3 immunohistochemical staining were positive in all cases. Adipophilin was positive in various degrees in 5 of the 6 lipid cell variant cases but was also positive in the case with sebaceous differentiation. α-methylacyl-CoA racemase was positive in the lipid cell areas and negative or focal weakly positive in the conventional UC areas in 4 of the 6 cases. Vimentin, S-100 protein, and PAX8 were negative in the lipid cell component. Follow-up information was available for all cases with follow-up ranging from 6 to 84 months (mean, 34 months). Four patients died of the disease. One pT4 patient who had been followed up for 6 months lives with the disease, whereas another is disease free. In conclusion, the lipid cell variant is a rare UC variant that usually presents at an advanced stage, and tumor cells are histologically similar to lipoblasts, resemble sebaceous differentiation, and show positive immunohistochemical staining with adipophilin.
Type 2 innate lymphoid cells (ILC2s) promote anti-helminth responses and contribute to allergies. Here, we report that Bcl11b, previously considered a T-cell-specific transcription factor, acted directly upstream of the key ILC2 transcription factor Gfi1 to maintain its expression in mature ILC2s. Consequently, Bcl11b(-/-) ILC2s downregulated Gata3 and downstream genes, including Il1rl1 (encoding IL-33 receptor), and upregulated Rorc and type 3 ILC (ILC3) genes. Additionally, independent of Gfi1, Bcl11b directly repressed expression of the gene encoding the ILC3 transcription factor Ahr, further contributing to silencing of ILC3 genes in ILC2s. Thus, Bcl11b(-/-) ILC2s lost their functions and gained ILC3 functions, and although they expanded in response to the protease allergen papain, they produced ILC3 but not ILC2 cytokines and caused increased airway infiltration of neutrophils instead of eosinophils. Our results demonstrate that Bcl11b is more than just a T-cell-only transcription factor and establish that Bcl11b sustains mature ILC2 genetic and functional programs and lineage fidelity.
Transforming growth factor (TGF)-β and interleukin (IL)-13 play a crucial role in the pathogenesis of systemic sclerosis (SSc), partly through activation of collagen production that leads to fibrosis. The aim of the present study was to determine whether TFG-β alters IL-13 production in T lymphocytes from patients with SSc from that seen in those of healthy donors.
IL-13 mRNA and protein synthesis under TFG-β exposure was measured in circulating T lymphocytes from healthy donors and patients with SSc and also in the Jurkat Th2 T-cell line, using quantitative real-time PCR and fluorescence-activated cell sorting analysis, respectively. The involvement of Smad and GATA-3 transcription factors was assessed by using specific inhibitors and small interfering RNA, and the binding capacity of GATA-3 to the IL-13 gene promoter was evaluated by chromatin immunoprecipitation assay.
TGF-β induced a significant decrease in IL-13 mRNA and protein levels in lymphocytes from healthy donors (mean [±SD] inhibition of 30% ± 10% and 20% ± 7%, respectively; p < 0.05). In contrast, TGF-β promoted a significant increase in IL-13 mRNA levels and IL-13 synthesis by CD4(+) and CD8(+) T-cell subtypes from patients with SSc, with respective increases of 2.4 ± 0.3-fold, 1.6 ± 0.05-fold and 2.7 ± 0.02-fold. The involvement of the Smad signaling pathway and upregulation of GATA-3 binding capacity on the IL-13 promoter in lymphocytes from patients with SSc contributed to the effect of TGF-β on IL-13 production.
These results demonstrate that TGF-β upregulates IL-13 synthesis through GATA-3 expression in the T lymphocytes of patients with SSc, confirming that the GATA-3 transcription factor can be regarded as a novel therapeutic target in patients with SSc.
CD4+ T cells and many cytokines play critical roles in the pathogenesis of Guillain-Barré Syndrome (GBS), an immune-mediated inflammatory disease. However, the role of IL-35, a novel member of the IL-12 cytokine family, in this kind of disease has not yet been elucidated. In this study, we investigated the functional changes of CD4+ T cells from GBS patients with IL-35 treatment in vitro. This study involved 21 GBS patients and an equal number of healthy controls (HCs). The results indicated that the average concentration of IL-35 in the plasma of GBS patients was lower than that of healthy controls (HCs). Increased levels of STAT1, STAT3 and STAT4 proteins and T-bet, ROR γt, IFN-γ and IL-17A mRNA were observed in CD4+ T cells from GBS patients. In contrast, the levels of STAT5 and STAT6 proteins and GATA3, Foxp3, IL-4 and TGF-β1 mRNAs were decreased in GBS patients in comparison with those of HCs. In addition, treatment of CD4+ T cells from GBS patients with IL-35 upregulated IL-35, STAT5 and STAT6 protein and T-bet, GATA3, Foxp3, IFN-γ, IL-4, IL-17A and TGF-β1 mRNA while inhibited levels of STAT3 and STAT4 protein and RORγt and IL-17A mRNA. These results indicate that IL-35 might play a potential role in GBS pathogenesis. Further studies are required in order to evaluate its role in GBS.
Tim-3 is considered as one of the T-cell immunoglobulin mucin (TIM) gene family members, which contributes to the activating or silencing genes, but the mechanism of Tim-3 function in mediating SLE or tumor metastasis has not been well explored. Here, we reported Tim-3 was high expressed in the peripheral blood mononuclear cells (PBMCs) of patients with SLE, detected by RT-PCR, significantly, GATA-3 mRNA expression also increased in patients with SLE, compared with the healthy control groups. The bioinformatics used to detect the TCGA database indicated the abnormal expression of Tim-3 was involved in several different cancer types. Further, the higher expression of Tim-3 in kidney renal clear cell carcinoma TCGA database indicated it was a marker for worse 5-year survival. The high expression of Tim-3 in different ccRCC cell lines was detected in both RNA level and protein level. Further, two kinds of relative Tim-3 siRNAs in ccRCC cell lines inhibit cell migration and invasion in vitro, However, the inhibition could be partially rescued by the additional GATA3 knockdown. Further, the down regulation in the RNA and protein levels of GATA3, and the negative correlation between Tim-3 and GATA3 implied that suppression of downstream GATA3 was an important mechanism by which Tim-3 triggered metastasis in ccRCC cell lines. Together, our experiments reveal the role for Tim-3 in facilitating SLE or invasive potential of ccRCC cells by either activating GATA3 or inhibiting GATA3, suggesting that Tim-3 might be a potential therapeutic target for treating SLE or clear cell renal cell carcinoma.
T cells play a critical role in acute allograft rejection. TGF-β/Smad3 signaling is a key pathway in regulating T cell development. We report here that Smad3 is a key transcriptional factor of TGF-β signaling that differentially regulates T cell immune responses in a mouse model of cardiac allograft rejection in which donor hearts from BALB/c mice were transplanted into Smad3 knockout (KO) and wild type (WT) mice. Results showed that the cardiac allograft survival was prolonged in Smad3 KO recipients. This allograft protection was associated with a significant inhibition of proinflammatory cytokines (IL-1β, TNF-α, and MCP-1) and infiltration of neutrophils, CD3+ T cells, and F4/80+ macrophages. Importantly, deletion of Smad3 markedly suppressed T-bet and IFN-γ while enhancing GATA3 and IL-4 expression, resulting in a shift from the Th1 to Th2 immune responses. Furthermore, mice lacking Smad3 were also protected from the Th17-mediated cardiac injury, although the regulatory T cell (Treg) response was also suppressed. In conclusion, Smad3 is an immune regulator in T cell-mediated cardiac allograft rejection. Loss of Smad3 results in a shift from Th1 to Th2 but suppressing Th17 immune responses. Thus, modulation of TGF-β/Smad3 signaling may be a novel therapy for acute allograft rejection.
Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations in OPG are involved in a variety of human diseases. We have shown that cochlear spiral ganglion cells secrete OPG at high levels and lack of OPG causes sensorineural hearing loss in addition to the previously described conductive hearing loss. In order to study the regulation of OPG expression, we conducted a database search on regulatory elements in the promoter region of the OPG gene, and identified two potential GATA-3 binding sites. Using luciferase assays and site directed mutagenesis, we demonstrate that these two elements are GATA-3 responsive and support GATA-3 transactivation in human HEK and HeLa cells. The expression of wild type GATA-3 activated OPG mRNA and protein expression, while the expression of a dominant negative mutant of GATA-3 or a GATA-3 shRNA construct reduced OPG mRNA and protein levels. GATA-3 deficient cells generated by expressing a GATA-3 shRNA construct were sensitive to apoptosis induced by etoposide and TNF-α. This apoptotic effect could be partly prevented by the co-treatment with exogenous OPG. Our results suggest new approaches to rescue diseases due to GATA-3 deficiency - such as in hypoparathyroidism, sensorineural deafness, and renal (HDR) syndrome - by OPG therapy.
To study the role of boswellic acid in reducing asthma phenotype severity and the relationship between the expression of pSTAT6 and GATA3, thirty-six mice were randomly divided into normal control group, asthma group, and boswellic acid group (treatment group). The asthma model was established through an intraperitoneal injection of sensitization liquid (0.15 mL aluminum hydroxide gel at 88.67 mg/mL and 0.05 mg ovalbumin). pSTAT6 and GATA3 expression levels in peripheral blood were detected by reverse transcription-polymerase chain reaction and Western blot analysis. pSTAT6 and GATA3 gene expressions in the asthmatic group were significantly higher than in the normal control group; they were markedly lower in the treatment group than the asthma group, and there was no significant difference when compared with the normal control group. The pSTAT6 expressions in the asthma, control and treatment groups were 2.256 ± 0.125, 0.524 ± 0.210, and 0.897 ± 0.134 at gray level, respectively. The GATA3 expressions in the asthma, control, and treatment groups were 3.521 ± 0.631, 0.435 ± 0.136, and 0.743 ± 0.149 at gray level, respectively. pSTAT6 and GATA3 expression levels were similar in the treatment and control groups. GATA3 expression had a positive correlation with pSTAT6 expression. Boswellic acid may improve asthma symptoms by inhibiting pSTAT6 expression, which consequently reduces GATA3 expression.
Vesical pleomorphic giant cell carcinoma (PGCC) is a variant of urothelial carcinoma (UC) characterized by highly pleomorphic tumour with giant cells. Fewer than 10 cases have been reported, and our aim was to determine the clinical and pathological features of a series of tumours from a specialized uropathology laboratory.
Thirteen cases of PGCC of the bladder were identified. There were nine males and four females, ranging in age from 53 to 92 years (mean 72 years). Associated conventional high-grade UC was seen in eight cases, while three cases also had micropapillary UC and one plasmacytoid UC. UC in situ (CIS) was present in five cases and occasional bizarre cells were seen in both UC and CIS. The proportion of PGCC present varied from 40% to 100% of tumour. Immunostaining performed on 10 cases showed uniform positivity for CK 8/18 and AE1/AE3, while most tumours were positive for CK7, CK20, uroplakin III and GATA binding protein 3 (GATA3). β-human chorionic gonadotrophin (β-hCG) was negative. Of 10 patients with follow-up, five died within 1 year and four are alive with tumour.
The association of PGCC with UC and an overlap in immunoexpression suggests that PGCC represents an extreme form of UC de-differentiation.
Clinical improvement of psoriasis induced by IL-4 treatment has been ascribed to changes in dermal inflammatory cells, such as activation of Th2 cells and tolerization of dendritic cells by suppressing IL-23 production. The pathologic epidermal alterations in psoriatic lesional skin include increased epidermal expression of IL-1β, IL-6, S100A7, and human β-defensin 2 (hBD2) and a downregulated expression of the epidermal transcription factor GATA3. Effects of IL-4 on the epidermal compartment of psoriasis lesions were not previously investigated. Therefore, we investigated whether IL-4 directly affects abovementioned psoriatic markers in the epidermal compartment. We cultured freshly isolated psoriatic epidermal cells, whole psoriatic and healthy skin biopsies, human keratinocytes and Langerhans cells with IL-4. The secretion of IL-1β and IL-6 by psoriatic epidermal cells was inhibited by IL-4 via transcriptional and posttranscriptional mechanisms, respectively. In normal skin, IL-4 inhibited IL-1β- and IL-17A-induced hBD2 expression in vitro. In addition, IL-4 reduced the protein expression of hBD2 in psoriatic skin biopsies and induced phospho-STAT6 protein. Epidermal GATA3 mRNA and protein were significantly upregulated by IL-4 in epidermal cells and keratinocytes. Our data argue that IL-4 improves psoriasis not only via modification/induction of Th2 cells and type II dendritic cells, but also via direct inhibition of inflammatory cytokines in resident IL-4R-expressing epidermal cells and thereby alters the psoriatic skin phenotype toward a healthy skin phenotype.
An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease.
We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge.
Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells.
Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.
Recurrence and death in a significant number of patients with ERα-positive breast cancer occurs 10-20 years after diagnosis. Prognostic markers for late events have been more elusive. TFAP2C (AP2γ) regulates the expression of ERα, the ERα pioneer factors FOXA1 and GATA3, and controls ERα-dependent transcription. The purpose of this investigation is to determine the long-term prognostic value of TFAP2C. A tissue microarray (TMA) consisting of breast tumors from 451 patients with median follow-up time of 10.3 years was created and tested for the expression of TFAP2C by immunohistochemistry. Wilcoxon Rank-Sum and Kruskal-Wallis tests were used to determine if TFAP2C H-scores correlate with other tumor markers. Cox proportional hazards regression models were used to determine whether TFAP2C H-scores and other tumor markers were related to overall and disease-free survival in univariate and multivariable models. TFPAC2 overexpression did not impact overall survival during the first 10 years after diagnosis, but was associated with a shorter survival after 10 years (HR 3.40, 95 % CI 1.58, 7.30; p value = 0.002). This late divergence persisted in ER-positive (HR 2.86, 95 % CI 1.29, 6.36; p value = 0.01) and endocrine therapy-positive subgroups (HR 4.19, 95 % CI 1.72, 10.23; p value = 0.002). For the ER+ and endocrine therapy subgroup, the HR was 3.82 (95 % CI 1.53, 9.50; p value = 0.004). TFAP2C H-scores were not correlated with other tumor markers or related to disease-free survival. In this hypothesis-generating study, we show that higher TFAP2C scores correlate with poor overall survival after 10 years of diagnosis in ERα-positive and endocrine therapy-treated subgroups.
Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgi-gata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes.
Encapsulating peritoneal sclerosis is one of the most serious complications of long-term peritoneal dialysis. The pathogenesis of encapsulating peritoneal sclerosis has not been elucidated, but several putative factors necessary for the development of peritoneum fibrosis (PF) have been reported. However, the roles of T helper (Th) cells in the progression of PF are unknown. The purpose of this study was to clarify the roles of Th1, Th2, and Th17 cells in the progression of PF. T-bet, GATA-3, and RORγt are Th1, Th2, and Th17 lineage commitment transcription factors, respectively. We previously generated Th1-biased (T-bet transgenic (Tg)) mice, Th2-biased (GATA-3 Tg) mice, and Th17-biased (RORγt Tg) mice. In this study, Th1, Th2, Th17-biased, and wild-type mice were administered chlorhexidine gluconate (CG) intraperitoneally and analyzed on day 21. CG-injected GATA-3 Tg mice showed a distended intestinal tract and developed marked thickening of the submesothelial space compared with the other groups. CG-injected GATA-3 Tg mice also showed significant expression of α-SMA positive cells, macrophages, and collagen III in the submesothelium. In contrast, CG-injected T-bet Tg mice only developed mild peritoneal fibrosis. Cytokines analysis in peritoneal fluid showed that IFN-γ was significantly increased in CG-injected T-bet Tg mice and that IL-13 was significantly increased in CG-injected GATA-3 Tg mice. Moreover, intraperitoneal administration of IFN-γ improved PF in GC-injected wild-type mice. Our results suggest that Th2 cells may play roles in the development of experimental PF and that Th1 cells may alleviate the severity of experimental PF.
GATA3 is a transcription factor critical for embryogenesis, development, and cell differentiation. Recent studies have suggested that GATA3 is a sensitive and relatively specific biomarker for urothelial and breast carcinomas, with most Müllerian carcinomas being negative. We investigated GATA3 expression in mesonephric/Wolffian remnants and tumors in the female genital tract. A western blot was performed to assess specificity for the GATA3 antibody. GATA3 immunohistochemistry was performed on 59 formalin-fixed paraffin-embedded mesonephric samples, including 17 mesonephric remnants (MR; 11 cervical and 6 fallopian tube), 15 mesonephric hyperplasias, 21 mesonephric carcinomas, and 6 female adnexal tumors of probable Wolffian origin. Thirty conventional endocervical adenocarcinomas (ENDO-CA), 9 gastric-type cervical adenocarcinomas, and 165 endometrial adenocarcinomas (EM-CA) were also evaluated. GATA3 nuclear intensity and extent of staining was evaluated. The western blot revealed GATA3 expression in seminal vesicle and cell lines derived from breast and urothelial carcinomas, but not in other cell lines including ovarian, cervical, and endometrial cancers. All cervical MRs and mesonephric hyperplasias, 5/6 (83%) fallopian tube MRs, and 20/21 (95%) mesonephric carcinomas were GATA3 positive, although with great variability in both intensity (weak to strong) and extent (1+ to 3+) of staining. Only 1/6 (17%) female adnexal tumors of probable Wolffian origin showed weak multifocal staining. One of 30 (3%) usual-type ENDO-CAs and 3/165 EM-CAs exhibited weak-moderate GATA3 immunoreactivity; all gastric-type cervical adenocarcinomas were negative. GATA3 is a highly sensitive and specific marker for mesonephric lesions in the lower genital tract; however, its utility in the upper genital tract may be more limited. In addition, GATA3 can aid in distinguishing lower genital mesonephric lesions from usual-type and gastric-type ENDO-CAs and uterine EM-CAs.
Although atrial natriuretic peptide (ANP) has been well recognized for its role in the regulation of volume-pressure homeostasis in cardiovascular system, its impact on respiratory system, particularly on the pathogenesis of acute allergic asthma, is yet to be elucidated. In the present report, we induced mice with OVA for onset of acute allergic asthma along with the administration of recombinant ANP or A71915 (an antagonist for ANP/natriuretic peptide receptor A, NPRA). It was noted that treatment of mice with ANP significantly promoted inflammatory infiltration in the airway and the production of inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) and lung homogenates, and the number of inflammatory cells in the BALF was significantly higher as compared with that of PBS treated asthmatic mice. Moreover, blockade of ANP/NPRA signaling by A71915 almost completely attenuated the effect of ANP administration. Mechanistic studies revealed that ANP repressed the expression of Th1 transcription factor T-bet, but enhanced Th2 transcription GATA3 expression. Together, our data provided feasible evidence suggesting that ANP/NPRA signaling predominantly induces a Th2-type response in favor of pathological processes during the course of acute allergic asthma.
Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1(-/-) ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.
A better understanding of the mechanism and manipulation of the tightly regulated cellular differentiation process of adipogenesis may contribute to a reduction in obesity and diabetes. Multiple transcription factors and signaling pathways are involved in the regulation of adipogenesis. Here, we report that the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) can activate AMPK in preadipocytes and thus increase the expression of GATA3, an anti-adipogenic factor. However, AICAR-increased GATA3 is mediated by the stimulation of Wnt/β-catenin signaling in preadipocytes. Mechanistically, AICAR-activated AMPK inhibits GSK3β through a phosphorylation process that stabilizes β-catenin. This stabilized β-catenin then translocates into nucleus where it interacts with T-cell factors (TCF), leading to the increased β-catenin/TCF transcriptional activity that induces GATA3 expression. In addition, AICAR also relieves the repressing effect of the C-terminal-binding protein (CtBP) co-repressor by diverting CtBP away from the β-catenin·TCF complex at the GATA3 promoter. The anti-adipogenic effect of GATA3 and AICAR is consistently attenuated by the disruption of Wnt/β-catenin signaling. Furthermore, GATA3 suppresses key adipogenic regulators by binding to the promoters of these regulators, such as the peroxisome proliferator-activated receptor-γ (PPARγ) gene, and the disruption of Wnt/β-catenin signaling reduces the GATA3 binding at the PPARγ promoter. In differentiated adipocytes, GATA3 expression inhibition is facilitated by the down-regulation of β-catenin levels, the reduction in β-catenin binding, and the increase in CtBP binding at the GATA3 promoter. Our findings shed light on the molecular mechanism of adipogenesis by suggesting that different regulation pathways and adipogenic regulators collectively modulate adipocyte differentiation through cross-talk.
WNT/β-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed β-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3 inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (Days 3-5/6). β-Catenin was displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markers NANOG, POU5F1, SOX2 and SALL4 in these functional experiments. However, both β-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specific WNT3 protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane β-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMES expression specific for trophoblast development. In both studies, the canonical WNT pathway target gene, TCF1, was not affected. Therefore, we conclude that WNT3 and membrane-associated β-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology.
A key question in cancer systems biology is how to use molecular data to predict the biological behavior of tumors from individual patients. While genomics data have been heavily used, protein signaling data are more directly connected to biological phenotype and might predict cancer phenotypes such as invasion, metastasis, and patient survival. In this study, we mined publicly available data for colorectal adenocarcinoma from the Cancer Genome Atlas and identified protein expression and signaling changes that are statistically associated with patient outcome. Our analysis identified a number of known and potentially new regulators of colorectal cancer. High levels of insulin growth factor binding protein 2 (IGFBP2) were associated with both recurrence and death, and this was validated by immunohistochemical staining of a tissue microarray for a secondary patient dataset. Interestingly, GATA binding protein 3 (GATA3) was the protein most frequently associated with death in our analysis, and GATA3 expression was significantly decreased in tumor samples from stage I-II deceased patients. Experimental studies using engineered colon cancer cell lines show that exogenous expression of GATA3 decreases three-dimensional colony growth and invasiveness of colon cancer cells but does not affect two-dimensional proliferation. These findings suggest that protein data are useful for biomarker discovery and identify GATA3 as a regulator of colorectal cancer  aggressiveness.
Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.
Tfap2c is required for placental development and trophoblast stem cell maintenance. Deletion of Tfap2c results in early embryonic loss because of failure in placental development. We evaluated the effect of reduced Tfap2c expression on fetal outcome and placental development. Sixty percent of the heterozygous mice were lost directly after birth. Labyrinthine differentiation was impaired, as indicated by enhanced proliferation and inclusions of cobblestone-shaped cell clusters characterized by expression of Tfap2c and glycogen stores. Moreover, expression of marker genes such as Cdx2, Eomes, Gata3, and Ascl2 are decreased in the spongiotrophoblast and indicate a lowered stem cell potential. On Day 18.5 postcoitum, the labyrinth layer of Tfap2c(+/-) placentas exhibited massive hemorrhages in the maternal blood spaces; these hemorrhages might have contributed to the significantly reduced number of live-born pups. These morphological alterations were accompanied by a shift toward sinusoidal trophoblast giant cells as the cell subpopulation lining the maternal sinusoids and toward reduction in expression of the prolactin gene family member Prl2c2, a finding characteristic of the spiral arteries lining trophoblast cells. The trophoblast stem cells heterozygous for Tfap2c exhibited a reduction in the expression level of stem cell markers and in their proliferation and differentiation capacity but did not exhibit changes in marker genes of the trophoblast giant cell lineage. Taken together, these findings indicate that a reduction in the gene dosage of placental Tfap2c leads to morphological changes in the labyrinth at midgestation and in the maternal blood spaces during late pregnancy.
To explore the mechanism of beta2 glycoprotein I (β2GPI)-caused T helper cell subset differentiation and anti-β2GPI antibody (aβ2GPI Ab) production in mice.
The healthy BALB/c mice were randomly divided into two groups: normal saline (NS) group and β2GPI group. The mice were immunized i.v. with either human β2GPI or equal amount of NS for a total of two or four times at 2-week intervals. The titer of aβ2GPI Ab in mice serum was evaluated by indirect ELISA. The expressions of GATA binding protein 3 (GATA3), interleukin 4 (IL-4), T-box expressed in T cells (T-bet), interferon-γ (IFN-γ) and Foxp3 mRNAs in mouse splenic cells were detected by real-time quantitative PCR (qRT-PCR), and the percentage of GATA3+ or Foxp3+ cells in lymphocytes of mouse spleen was detected by flow cytometry.
The high titer (1:100,000) of aβ2GPI Ab was detected in mouse serum following the two times of immunization with β2GPI. The expressions of Th2 specific markers GATA3 and IL-4 mRNAs and the percentage of GATA3+ cells in lymphocytes were higher than those of NS treated group. After four times of immunization with β2GPI, the titer of aβ2GPI Ab in mouse serum was elevated to a higher level (>1:100,000). The expressions of GATA3, Th1 specific markers T-bet and IFN-γ, and regulatory T cell (Treg) specific marker Foxp3 mRNAs in mouse spleen cells, but no significant change was found in IL-4 mRNA expression. Furthermore, the percentage of GATA3+ cells in mouse spleen increased, while the percentage of Foxp3+ cells decreased.
β2GPI immunization could induce Th2 cell bias polarization and Th1 cell and Treg suppression in mice, which might contribute to the production of aβ2GPI Ab.
Interleukin-4 (IL-4), IL-5 and IL-13, the signature cytokines that are produced during type 2 immune responses, are critical for protective immunity against infections of extracellular parasites and are responsible for asthma and many other allergic inflammatory diseases. Although many immune cell types within the myeloid lineage compartment including basophils, eosinophils and mast cells are capable of producing at least one of these cytokines, the production of these "type 2 immune response-related" cytokines by lymphoid lineages, CD4 T helper 2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) in particular, are the central events during type 2 immune responses. In this review, I will focus on the signaling pathways and key molecules that determine the differentiation of naïve CD4 T cells into Th2 cells, and how the expression of Th2 cytokines, especially IL-4 and IL-13, is regulated in Th2 cells. The similarities and differences in the differentiation of Th2 cells, IL-4-producing T follicular helper (Tfh) cells and ILC2s as well as their relationships will also be discussed.
How loss-of-function of GATA3 contributes to the development of breast cancer is poorly understood. Here, we report that GATA3 nucleates a transcription repression program composed of G9A and MTA3-, but not MTA1- or MTA2-, constituted NuRD complex. Genome-wide analysis of the GATA3/G9A/NuRD(MTA3) targets identified a cohort of genes including ZEB2 that are critically involved in epithelial-to-mesenchymal transition and cell invasion. We demonstrate that the GATA3/G9A/NuRD(MTA3) complex inhibits the invasive potential of breast cancer cells in vitro and suppresses breast cancer metastasis in vivo. Strikingly, the expression of GATA3, G9A, and MTA3 is concurrently downregulated during breast cancer progression, leading to an elevated expression of ZEB2, which, in turn, represses the expression of G9A and MTA3 through the recruitment of G9A/NuRD(MTA1).
IFN-α/β was first described as a potent inhibitor of viral replication, but it is now appreciated that IFN signaling plays a pleiotropic role in regulating peripheral T cell functions. Recently, IFN-α/β was shown to block human Th2 development by suppressing the transcription factor GATA3. This effect is consistent with the role for IFN-α/β in suppressing allergic inflammatory processes by blocking granulocyte activation and IL-4-mediated B cell isotype switching to IgE. With the consideration of recent studies demonstrating a defect in IFN-α/β secretion in DCs and epithelial cells from individuals with severe atopic diseases, there is an apparent reciprocal negative regulatory loop in atopic individuals, whereby the lack of IFN-α/β secretion by innate cells contributes to the development of allergic Th2 cells. Is it possible to overcome these events by treating with IFN-α/β or by inducing its secretion in vivo? In support of this approach, case studies have documented the therapeutic potential of IFN-α/β in treating steroid-resistant allergic asthma and other atopic diseases. Additionally, individuals with asthma who are infected with HCV and respond to IFN therapy showed a reduction in symptoms and severity of asthma attacks. These findings support a model, whereby allergic and antiviral responses are able to cross-regulate each other, as IgER cross-linking of pDCs prevents IFN-α/β production in response to viral infection. The clinical importance of upper-respiratory viruses in the context of allergic asthma supports the need to understand how these pathways intersect and to identify potential therapeutic targets.
Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.
Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity.
IL-25 a 2o KDa protein mostly known as IL-17E, encoded by chromosome 14, and containing 117 amino acids. Cytokine IL-17 family consists of 6 members; IL-17A to IL-17F, among which IL-25 has a unique structure and function. The receptor of IL-25 (IL-17BR) is highly expressed in the main Th2 cells. IL-25 regulates the internal safety of adaptive immune responses which leads to begin allergic diseases and plays a role in stimulation of pulmonary mucosal cells and fibroblasts. IL-25 can also have some effects on production of other cytokines. For instance, production of IL-25 in human and mice or injection of IL-25 to animals has resulted in production of high concentrations of Th2 cytokines, including IL-4, IL-5, and IL-13. Pilot studies have shown that mRNA of IL-25 has a high expression in Th2 cells. However, the mechanism through which IL-25 leads to Th2 immune response is still unknown. Reaction between IL-25 and IL-17BR leads to activation of transcription factors, such as NF-KB, STAT6, GATA3, NF-ATC1, JUNNB, MAPK, and JNK. IL-25 has been used against the kidney damage in mice. A large number of researchers in various countries, including the U.S. and Taiwan, have stated that IL-25 is a strong inflammatory cytokine protein which is involved in allergic inflammations.
Molecular stratification of bladder cancer has revealed gene signatures differentially expressed across tumor subtypes. While these signatures provide important insights into subtype biology, the transcriptional regulation that governs these signatures is not well characterized.
In this study, we use publically available ChIP-Seq data on regulatory factor binding in order to link transcription factors to gene signatures defining molecular subtypes of urothelial carcinoma.
We identify PPARG and STAT3, as well as ADIRF, a novel regulator of fatty acid metabolism, as putative mediators of the SCC-like phenotype. We link the PLK1-FOXM1 axis to the rapidly proliferating Genomically Unstable and SCC-like subtypes and show that differentiation programs involving PPARG/RXRA, FOXA1/GATA3 and HOXA/HOXB are differentially expressed in UC molecular subtypes. We show that gene signatures and regulatory systems defined in urothelial carcinoma operate in breast cancer in a subtype specific manner, suggesting similarities at the gene regulatory level of these two tumor types.
At the gene regulatory level Urobasal, Genomically Unstable and SCC-like tumors represents three fundamentally different tumor types. Urobasal tumors maintain an apparent urothelial differentiation axis composed of PPARG/RXRA, FOXA1/GATA3 and anterior HOXA and HOXB genes. Genomically Unstable and SCC-like tumors differ from Urobasal tumors by a strong increase of proliferative activity through the PLK1-FOXM1 axis operating in both subtypes. However, whereas SCC-like tumors evade urothelial differentiation by a block in differentiation through strong downregulation of PPARG/RXRA, FOXA1/GATA3, our data indicates that Genomically Unstable tumors evade differentiation in a more dynamic manner.
Recent studies indicate that DNA methylation can be used to identify transcriptional enhancers, but no systematic approach has been developed for genome-wide identification and analysis of enhancers based on DNA methylation. We describe ELMER (Enhancer Linking by Methylation/Expression Relationships), an R-based tool that uses DNA methylation to identify enhancers and correlates enhancer state with expression of nearby genes to identify transcriptional targets. Transcription factor motif analysis of enhancers is coupled with expression analysis of transcription factors to infer upstream regulators. Using ELMER, we investigated more than 2,000 tumor samples from The Cancer Genome Atlas. We identified networks regulated by known cancer drivers such as GATA3 and FOXA1 (breast cancer), SOX17 and FOXA2 (endometrial cancer), and NFE2L2, SOX2, and TP63 (squamous cell lung cancer). We also identified novel networks with prognostic associations, including RUNX1 in kidney cancer. We propose ELMER as a powerful new paradigm for understanding the cis-regulatory interface between cancer-associated transcription factors and their functional target genes.
The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA).
We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1).
After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo.
Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).
Innate lymphoid cells (ILCs) function in producing effector cytokines in response to pathogenic infections. However, the roles and related mechanisms of the ILC subpopulations, ILC1 and ILC2, which mirror Th1 and Th2 in adaptive immunity, remain unclear. In this study, we found the markedly elevated levels of the ILC1 transcription factor T-bet, the effector cytokine IFN-γ and the IL/receptor signaling molecules IL-12/IL-12R, which are indispensable for ILC1 differentiation, in the helper ILCs of chronic hepatitis B (CHB) patients. The elevated level of the ILC1 population was significantly associated with hepatic damage in CHB patients, and was not related to telbivudine treatment. In contrast, although we also observed elevated levels of ILC2-related factors, including IL-33, ST2, GATA3 and IL-13 in helper ILCs, the extent of elevation shown by each was lower than that shown by the ILC1-related factors. Furthermore, the activity of the ILC2s did not correlate with either HBV copies or liver damage. The findings of this study suggest potential pro-inflammatory roles for ILC1s in CHB pathogenesis, potentiating these cells and their related molecules as targets of diagnostic, prognostic and/or therapeutic strategies for hepatitis B.
Paeoniflorin has been demonstrated to exert anti-inflammatory and immunomodulatory effects in the animal study. In this study, we investigated immunoregulatory effects of paeoniflorin on anti-asthmatic effects and underlying mechanisms. Asthma model was established by ovalbumin-induced. A total of 50 mice were randomly assigned to five experimental groups: control, model, dexamethasone (2 mg/kg), and paeoniflorin (10 and 20 mg/kg). Airway resistance (Raw) were measured by the forced oscillation technique; histological studies were evaluated by the hematoxylin and eosin (HE) staining; Th1/Th2 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA); Th1/Th2 cells were evaluated by flow cytometry (FCM); and GATA3 and T-bet were evaluated by Western blot. Our study demonstrated that, compared with model group, paeoniflorin inhibited ovalbumin (OVA)-induced increases in Raw and eosinophil count; interleukin (IL)-4, IgE levels were recovered in bronchoalveolar lavage fluid compared; increased IFN-γ level in bronchoalveolar lavage fluid; histological studies demonstrated that paeoniflorin substantially inhibited OVA-induced eosinophilia in lung tissue and lung tissue compared with model group. Flow cytometry studies demonstrated that paeoniflorin can regulate Th1/Th2 balance. These findings suggest that paeoniflorin may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.
Rhodiola rosea (Rro) has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS) or 1% salidroside and 3% rosavines (RR) on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation.
Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.
The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1-p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen- induced, Cre-mediated recombination of the ROSA(DTA) allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate-deficient embryos, the p1 alar plate exhibited reduced expression of subtype-specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments.
Atopic dermatitis (AD) is a heterogeneous chronic inflammatory skin disease. Most AD during infancy resolves during childhood, but moderate-to-severe AD with allergic sensitization is more likely to persist into adulthood and more often occurs with other allergic diseases.
We sought to find susceptibility loci by performing the first genome-wide association study (GWAS) of AD in Korean children with recalcitrant AD, which was defined as moderate-to-severe AD with allergic sensitization.
Our study included 246 children with recalcitrant AD and 551 adult control subjects with a negative history of both allergic disease and allergic sensitization. DNA from these subjects was genotyped; sets of common single nucleotide polymorphisms (SNPs) were imputed and used in the GWAS after quality control checks.
SNPs at a region on 13q21.31 were associated with recalcitrant AD at a genome-wide threshold of significance (P < 2.0 × 10(-8)). These associated SNPs are more than 1 Mb from the closest gene, protocadherin (PCDH)9. SNPs at 4 additional loci had P values of less than 1 × 10(-6), including SNPs at or near the neuroblastoma amplified sequence (NBAS; 2p24.3), thymus-expressed molecule involved in selection (THEMIS; 6q22.33), GATA3 (10p14), and S-phase cyclin A-associated protein in the ER (SCAPER; 15q24.3) genes. Further analysis of total serum IgE levels suggested 13q21.31 might be primarily an IgE locus, and analyses of published data demonstrated that SNPs at the 15q24.3 region are expression quantitative trait loci for 2 nearby genes, ISL2 and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1), in immune cells.
Our GWAS of recalcitrant AD identified new susceptibility regions containing genes involved in epithelial cell function and immune dysregulation, 2 key features of AD, and potentially extend our understanding of their role in pathogenesis.
Primary angiosarcoma of the bladder is very rare, with approximately 30 cases reported in the literature. Those with epithelioid morphology are even rarer, with only single-case reports published. We describe the histopathologic features and clinical follow-up of 9 patients with epithelioid angiosarcoma (EA) of the bladder retrieved from our Surgical Pathology files from 1998 to 2014. Eight cases were consults. The mean age at presentation was 65 years (range, 39 to 85 y). The M:F ratio was 8:1. The clinical presentation was hematuria and bladder mass in all cases. Six patients had a history of radiotherapy to the pelvis, 5 to treat prostate cancer and 1 to treat uterine cervical cancer. The time from radiotherapy to the diagnosis of EA ranged from 6 to 15 years. The average size of the tumor was 4 cm. (range, 1 to 8 cm.). The submitting diagnoses were poorly differentiated carcinoma (n=5), high-grade invasive urothelial carcinoma (n=3), and atypical vascular proliferation (n=1). Morphologically, the tumors were composed of nests and sheets of highly atypical cells with high nuclear to cytoplasmic ratio, occasional intracytoplasmic lumens, and a hemorrhagic background. None of the cases showed any urothelial carcinoma component. Three patients showed in addition usual angiosarcoma in the resection specimen. By immunohistochemistry, 5/9 cases were positive for cytokeratins, including CK7 (n=3), AE1/AE3 (n=3), and Cam5.2 (n=1). All cases were positive for at least 1 endothelial marker, including CD31 (n=7), CD34 (n=2), FVIII (n=3), and ERG (n=2). Urothelial markers (p63 and GATA3) were consistently negative. Surgical treatment included transurethral resection of the bladder (TURB) only (n=5), TURB followed by cystoprostatectomy (n=2), TURB followed by partial cystectomy (n=1), and cystoprostatectomy only (n=1). The tumor involved the muscularis propria in 5/9 patients, the periureteric adipose tissue in 1 patient, and the prostate and seminal vesicles in 1 patient. Five of 9 patients died of disease, with a median survival of 7 months (range, 6 to 14 mo). Two patients were alive with disease at 3 and 6 months of follow-up. One patient who underwent radical cystoprostatectomy was alive with no evidence of disease 12 months after surgery. EA of the bladder is a rare malignancy that is frequently misdiagnosed as high-grade carcinoma, especially due to positive immunostaining for cytokeratins. This tumor is more frequent in older men with a history of radiotherapy to the pelvis. Morphologic features that should suggest the vascular origin of the tumor include highly atypical nuclei with interspersed erythrocytes, hemorrhagic background, and occasional intracytoplasmic lumens. Patients usually present with muscle invasive disease, and the prognosis is dismal.
The present work aimed to investigate the cellular and immunochemical pattern of T cells population in biopsy material from chronic schistosomiasis haematobium Egyptian patients complicated with bladder cancer. Digital real-time quantitative photocytometry was applied to auto-analyze 29 stained tissue sections from cases and 17 controls using STAT4, GATA3, FOXP3, and CD8 markers specific for Th1, Th2, T regulatory, and T cytotoxic cells, respectively. Area percentage showed significant high level of GATA, followed by FOXP3 and low level of both STAT and CD8 was reported. Tissue samples from five healthy bladder tissues showed significant lower optical density (OD) values. Tissue samples from 12 non-bilharzial bladder cancers showed variable OD values, reflecting wide disparity in the control group.Our results hypothesized an exclusive pattern of T population in long standing complicated schistosomiasis haematobium. Our cases were poorly controlled by unbalanced Th1/Th2 in which Th2 was dominated. FOXP3 increased significantly, however, failed to downregulate Th2, instead, the relation between Th1 and T cytotoxic was forcibly limited by the high level of FOXP3, resulting in loss of their power in defending the host against both parasite and carcinogenic changes. These results provide more clarification for the immune evasion process played by the parasite and tumor cells under the supervision of T regulatory cells. Additionally a critical role of FOXP3 is suggested in manipulating STAT4 and CD8 in favor of malignant transformation in this life-threatening parasite.
There is accumulating evidence that aberrant T helper (Th)1 and Th2 cell responses play critical roles in the pathogenesis of autoimmune diseases. However, their importance in the pathobiology of vitiligo have yet to be elucidated.
To evaluate the expression profile of two transcription factors, namely, T-bet, a Th1-specific T box transcription factor and GATA binding protein 3 (GATA-3), a Th2-specific transcription factor, and to measure expression levels of interferon (IFN)-γ and interleukin (IL)-4 mRNAs as the signature cytokines of Th1 and Th2 cells, respectively.
Gene expression analysis of peripheral blood mononuclear cells (PBMCs) was performed using real-time reverse transcriptase PCR.
In patients with vitiligo compared with controls, mRNA expression was significantly higher for T-bet and IFN-γ, and significantly lower for GATA-3 and IL-4 mRNAs (P ≤ 0.05).
These data suggest additional implications for the role of Th1/Th2 balance in the immunopathogenesis of vitiligo.
Traditional markers mammaglobin and GCDFP15 show good specificity but lack sensitivity and can be difficult to interpret in small tissue samples. We undertook a comparative study of the novel nuclear marker GATA3 (expression typically restricted to breast and urothelial carcinomas) and GCDFP15 and mammaglobin. We first compared quantitative mRNA expression levels of these 3 markers across a diverse set of over 6000 tumors and 500 normal samples from The Cancer Genome Atlas which showed dramatically higher GATA3 expression (>10-fold higher) in breast cancer as compared with GCDFP15 or mammaglobin (both P<2.2e-16), suggesting that GATA3 may represent a more sensitive marker of breast cancer than GCDFP15 or mammaglobin. We next examined protein expression by immunohistochemistry in 166 cases (including surgical and cytology specimens) of metastatic breast carcinoma and 54 cases with available matched primaries. One whole-slide section from each case was stained for monoclonal GATA3 (L50-823), monoclonal mammaglobin (31A5), and monoclonal GCDFP15 (EP1582Y). Staining intensity (0 to 3+) and extent (0% to 100%) were scored with an H-score calculated (range, 0 to 300). Sensitivities by varying H-score cutoffs for a positive result in metastatic breast carcinoma among GATA3/GCDFP15/mammaglobin, respectively, were as follows: any H-score=95%/65%/78%, H-score>50=93%/37%/47%, H-score>100=90%/25%/27%, H-score>150=86%/21%/19%, H-score>200=73%/18%/9%, H-score>250=66%/14%/6%. Significant staining differences by specimen type, tumor subtype/grade, or ER/PR/HER2 status were not identified. Significantly stronger correlation was observed between primary/metastatic GATA3 expression [Pearson's correlation=0.81 (0.68-0.89)] as compared with the primary/metastatic correlations of GCDFP15 [Pearson's correlation=0.57 (0.33-0.74)] and mammaglobin [Pearson's correlation=0.50 (0.24-0.70)] (both P<0.05). In conclusion, the novel marker GATA3 stains a significantly higher proportion of both primary and metastatic breast carcinomas than GCDFP15 or mammaglobin with stronger and more diffuse staining, helpful in cases with small tissue samples. The matched primary/metastatic expression of GATA3 is also more consistent. We propose that GATA3 be included among a panel of confirmatory markers for metastatic breast carcinoma.
Helicobacter pylori (H. pylori) is a risk factor of gastric carcinoma, and inflammation with H.pylori infection has widely been suggested to trigger gastric carcinogenesis through "inflammation-carcinoma chain" (non-atrophic gastritis (NAG) → chronic atrophic gastritis (CAG) → intestinal metaplasia (IM) → dysplasia (DYS) and gastric carcinoma (GC)). Connexin43 (Cx43) is a major constituent of gap junction in normal gastric mucosa (NGM) and it is continuously down-regulated from normal gastric mucosa to precancerous lesions or ultimate gastric carcinoma, which shows novel target against gastric carcinoma by preventing the Cx43 decline. Our previous studies demonstrated that H. pylori infection in gastric mucosa down-regulates Cx43 expression, but its mechanism remains unknown. The transcriptional factor, GATA binding protein 3 (GATA-3) is the key to regulate adaptive immune response, which possibly relates to inflammation toward malignant transformation. Here the substantial rising of GATA-3 was screened by transcriptional factor microarray along the developmental stages of H. pylori associated gastric carcinoma. Moreover, the increased GATA-3 and inhibited Cx43 were confirmed in clinical specimens, Mongolian gerbils and normal gastric epithelial cell line GES-1 with H. pylori infection. GATA-3 silencing generated the Cx43 restoration both in intermediate differentiation gastric cancer cells BGC-803 and in H. pylori infected GES-1 cells. Dual-luciferase reporter assay further revealed the GATA-3 as one of Cx43 down-regulators by directly binding to its promoters. Together, the incremental GATA-3 is found in H. pylori associated gastric carcinogenesis, which is responsible for Cx43 inhibition as well.
Invariant natural killer T cells (iNKT cells) are innate-like T cells that rapidly produce cytokines that impact antimicrobial immune responses, asthma, and autoimmunity. These cells acquire multiple effector fates during their thymic development that parallel those of CD4(+) T helper cells. The number of Th2-type effector iNKT cells is variable in different strains of mice, and their number impacts CD8 T, dendritic, and B cell function. Here we demonstrate a unique function for the transcription factor lymphoid enhancer factor 1 (LEF1) in the postselection expansion of iNKT cells through a direct induction of the CD127 component of the receptor for interleukin-7 (IL-7) and the transcription factor c-myc. LEF1 also directly augments expression of the effector fate-specifying transcription factor GATA3, thus promoting the development of Th2-like effector iNKT cells that produce IL-4, including those that also produce interferon-γ. Our data reveal LEF1 as a central regulator of iNKT cell number and Th2-type effector differentiation.
Hyperglycemia represents one of possible mediators for activation of immune system and may contribute to worsening of inflammatory state associated with obesity. The aim of our study was to investigate the effect of a short-term hyperglycemia (HG) on the phenotype and relative content of immune cells in circulation and subcutaneous abdominal adipose tissue (SAAT) in obese women without metabolic complications.
Three hour HG clamp with infusion of octreotide and control investigations with infusion of octreotide or saline were performed in three groups of obese women (Group1: HG, Group 2: Octreotide, Group 3: Saline, n=10 per group). Before and at the end of the interventions, samples of SAAT and blood were obtained. The relative content of immune cells in blood and SAAT was determined by flow cytometry. Gene expression analysis of immunity-related markers in SAAT was performed by quantitative real-time PCR.
In blood, no changes in analysed immune cell population were observed in response to HG. In SAAT, HG induced an increase in the content of CD206 negative monocytes/macrophages (p<0.05) and T lymphocytes (both T helper and T cytotoxic lymphocytes, p<0.01). Further, HG promoted an increase of mRNA levels of immune response markers (CCL2, TLR4, TNFα) and lymphocyte markers (CD3g, CD4, CD8a, TBX21, GATA3, FoxP3) in SAAT (p<0.05 and 0.01). Under both control infusions, none of these changes were observed.
Acute HG significantly increased the content of monocytes and lymphocytes in SAAT of healthy obese women. This result suggests that the short-term HG can modulate an immune status of AT in obese subjects.
Calcium, magnesium, potassium, sodium, chloride and phosphorus are the major dietary minerals involved in various biological functions and are commonly measured in the blood serum. Sufficient mineral intake is especially important for children due to their rapid growth. Currently, the genetic mechanisms influencing serum mineral levels are poorly understood, especially for children. We carried out a genome-wide association (GWA) study on 5,602 European-American children and 4,706 African-American children who had mineral measures available in their electronic medical records (EMR). While no locus met the criteria for genome-wide significant association, our results demonstrated a nominal association of total serum calcium levels with a missense variant in the calcium -sensing receptor (CASR) gene on 3q13 (rs1801725, P = 1.96 × 10(-3)) in the African-American pediatric cohort, a locus previously reported in Caucasians. We also confirmed the association result in our pediatric European-American cohort (P = 1.38 × 10(-4)). We further replicated two other loci associated with serum calcium levels in the European-American cohort (rs780094, GCKR, P = 4.26 × 10(-3); rs10491003, GATA3, P = 0.02). In addition, we replicated a previously reported locus on 1q21, demonstrating association of serum magnesium levels with MUC1 (rs4072037, P = 2.04 × 10(-6)). Moreover, in an extended gene-based association analysis we uncovered evidence for association of calcium levels with the previously reported gene locus DGKD in both European-American children and African-American children. Taken together, our results support a role for CASR and DGKD mediated calcium regulation in both African-American and European-American children, and corroborate the association of calcium levels with GCKR and GATA3, and the association of magnesium levels with MUC1 in the European-American children.
Hypoparathyroidism, sensorineural deafness, and renal dysgenesis syndrome is an autosomal dominant disease caused by mutations in the GATA3 gene on chromosome 10p15. We identified a patient diagnosed with hypoparathyroidism who also had a family history of hypoparathyroidism and sensorineural deafness, present in the father. The patient was subsequently diagnosed and found to be a heterozygote for an insertion mutation c.255_256ins4 (GTGC) in exon 2 of GATA3. His father was also confirmed to have the same mutation in GATA3.
Psoriasis is a cytokine-mediated skin disease that can be treated effectively with immunosuppressive biologic agents. These medications, however, are not equally effective in all patients and are poorly suited for treating mild psoriasis. To develop more targeted therapies, interfering with transcription factor (TF) activity is a promising strategy.
Meta-analysis was used to identify differentially expressed genes (DEGs) in the lesional skin from psoriasis patients (n = 237). We compiled a dictionary of 2935 binding sites representing empirically-determined binding affinities of TFs and unconventional DNA-binding proteins (uDBPs). This dictionary was screened to identify "psoriasis response elements" (PREs) overrepresented in sequences upstream of psoriasis DEGs.
PREs are recognized by IRF1, ISGF3, NF-kappaB and multiple TFs with helix-turn-helix (homeo) or other all-alpha-helical (high-mobility group) DNA-binding domains. We identified a limited set of DEGs that encode proteins interacting with PRE motifs, including TFs (GATA3, EHF, FOXM1, SOX5) and uDBPs (AVEN, RBM8A, GPAM, WISP2). PREs were prominent within enhancer regions near cytokine-encoding DEGs (IL17A, IL19 and IL1B), suggesting that PREs might be incorporated into complex decoy oligonucleotides (cdODNs). To illustrate this idea, we designed a cdODN to concomitantly target psoriasis-activated TFs (i.e., FOXM1, ISGF3, IRF1 and NF-kappaB). Finally, we screened psoriasis-associated SNPs to identify risk alleles that disrupt or engender PRE motifs. This identified possible sites of allele-specific TF/uDBP binding and showed that PREs are disproportionately disrupted by psoriasis risk alleles.
We identified new TF/uDBP candidates and developed an approach that (i) connects transcriptome informatics to cdODN drug development and (ii) enhances our ability to interpret GWAS findings. Disruption of PRE motifs by psoriasis risk alleles may contribute to disease susceptibility.
This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10(-8), OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10(-5), OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.
Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)-localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10-expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2(+) Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2(-) Tregs. The proportion of ST2(+) Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2(+) Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance.
This is a follow-up study of our previous work in which we screened a series of Vasicine analogues for their anti-inflammatory activity in a preventive OVA induced murine model of asthma. The study demonstrated that R8, one of the analogues, significantly suppressed the Th2 cytokine production and eosinophil recruitment to the airways. In the present study, we have been using two standard experimental murine models of asthma, where the mice were treated with R8 either during (preventive use) or after (therapeutic use) the development of asthma features. In the preventive model, R8 reduced inflammatory cell infiltration to the airways, OVA specific IgE and Th2 cytokine production. Also, the R8 treatment in the therapeutic model decreased methacholine induced AHR, Th2 cytokine release, serum IgE levels, infiltration of inflammatory cells into the airways, phosphorylation of STAT6 and expression of GATA3. Moreover, R8 not only reduced goblet cell metaplasia in asthmatic mice but also reduced IL-4 induced Muc5AC gene expression in human alveolar basal epithelial cells. Further, R8 attenuated IL-4 induced differentiation of murine splenocytes into Th2 cells in vitro. So, we may deduce that R8 treatment profoundly reduced asthma features by attenuating the differentiation of T cells into Th2 cells by interfering with the binding of IL-4 to its receptor in turn decreasing the phosphorylation of STAT6 and expression of GATA3 in murine model of asthma. These preclinical findings suggest a possible therapeutic role of R8 in allergic asthma.
Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3.
In an ongoing screen for DNA sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conduct a genome-wide association study (GWAS) of 24,988,228 SNPs and small indels detected through whole-genome sequencing of 2,636 Icelanders and imputed into 4,572 BCC patients and 266,358 controls. Here we show the discovery of four new BCC susceptibility loci: 2p24 MYCN (rs57244888[C], OR=0.76, P=4.7 × 10(-12)), 2q33 CASP8-ALS2CR12 (rs13014235[C], OR=1.15, P=1.5 × 10(-9)), 8q21 ZFHX4 (rs28727938[G], OR=0.70, P=3.5 × 10(-12)) and 10p14 GATA3 (rs73635312[A], OR=0.74, P=2.4 × 10(-16)). Fine mapping reveals that two variants correlated with rs73635312[A] occur in conserved binding sites for the GATA3 transcription factor. In addition, expression microarrays and RNA-seq show that rs13014235[C] and a related SNP rs700635[C] are associated with expression of CASP8 splice variants in which sequences from intron 8 are retained.
GATA binding protein 3 (GATA3) is a recently described immunohistochemical marker that has proven useful in the characterization of breast and urothelial carcinomas. However, the expression pattern of GATA3 in mesonephric proliferations is largely unknown. The aim of this study was to examine the immunohistochemical expression of GATA3 in cervicovaginal mesonephric lesions and compare it to its expression in endocervical and endometrial adenocarcinomas and cervicovaginal endometriosis. A cohort of 107 cases, including 33 cases of mesonephric lesions and 74 cases of nonmesonephric lesions, was selected for the study. Of 33 mesonephric lesions, 31 (94%) cases (16 remnants, 12 hyperplasias, and 3 adenocarcinomas) were strongly and diffusely positive in tumor cell nuclei for GATA3. The remaining 2 mesonephric carcinosarcomas showed focal nuclear staining and rare nuclear positivity, respectively. Of 36 endocervical adenocarcinomas, 33 (92%) were negative for GATA3 and the remaining revealed focal weak nuclear staining. Of 34 endometrial adenocarcinomas, 32 (94%) were negative, whereas 2 showed rare nuclear positivity. All 4 cases of endometriosis were negative. The benign endocervical epithelium and the benign endometrium in most cases lacked GATA3 expression, whereas the benign squamous epithelium in the majority exhibited nuclear basal and parabasal staining pattern. Our study demonstrates that GATA3 protein is expressed in most mesonephric lesions, regardless of them being benign or malignant. In contrast, GATA3 is absent in the majority of endometrial and endocervical adenocarcinomas. These results support that GATA3 immunostain can be a useful tool in differentiating mesonephric lesions from endocervical and endometrial adenocarcinomas.
Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers.
Breast carcinomas rarely metastasize to the ovary and are even more rarely present clinically as primary ovarian tumors. However, patients with breast cancer not infrequently develop independent primary ovarian carcinomas. In these cases, distinction between independent primaries and metastatic tumors is crucial. Several comparative immunohistochemical studies have been reported, but few included significant clinicopathologic data and none investigated cases of ovarian and breast carcinomas from the same patients. In this study, we compared 18 cases of patients with bona fide independent breast and ovarian carcinomas (15 high-grade serous and 3 clear cell carcinomas), with 9 cases of patients with known mammary carcinomas (7 lobular and 2 ductal carcinomas) metastatic to the ovary. Immunohistochemical stains for Pax-8, WT-1, and GATA3 were carried out on tissue microarrays (TMA). Most primary ovarian carcinomas were larger than the metastatic tumors (P=0.001) and were diagnosed at an advanced stage. All primary ovarian tumors showed marked nuclear pleomorphism, whereas only 2 metastatic breast carcinomas had Grade 3 nuclei (P=0.000). The vast majority of ovarian metastases (7/9) showed the typical pattern of lobular breast carcinoma. Pax-8 and WT-1 expression were found in 16 of 18 (88%) and 13 of 18 (72%) primary ovarian carcinomas, respectively. In contrast, all primary ovarian carcinomas were negative for GATA3. The 2 Pax-8-negative ovarian carcinomas were also negative for WT-1. With the exception of 3 triple-negative carcinomas, all primary breast carcinomas were positive for GATA3. All metastatic breast carcinomas were positive for GATA3 and negative for Pax-8. WT-1 expression was seen in only 1 of 9 metastatic breast carcinomas (11%). Patients with ovarian metastases had worse prognosis than patients with independent breast and ovarian carcinomas (P=0.000). Pax-8, WT-1, and GATA3 immunoreactions are useful in the distinction between independent primaries and metastatic mammary carcinomas to the ovary in the light of clinicopathologic findings.
The aim of the study was to investigate the anti-asthma effects of ginsenoside Rb1 (Rb1) and its possible mechanisms. A total of 50 mice were randomly assigned to five experimental groups: control, model, dexamethasone (2 mg/kg), and Rb1 (10 and 20 mg/kg). Airway resistance (RI) was measured; histological studies were evaluated by the hematoxylin and eosin (HE) staining; Th1/Th2, ovalbumin (OVA)-specific serum, and bronchoalveolar lavage fluid (BALF) IgE levels were evaluated enzyme-linked immunosorbent assay (ELISA); and T-bet/GATA3 proteins were evaluated by Western blot. Our study demonstrated that Rb1 inhibited OVA-induced increases in RI and eosinophil counts; interleukin (IL)-4 was recovered, and IFN-γlevel increased in bronchoalveolar lavage fluid. Histological studies demonstrated that Rb1 substantially inhibited OVA-induced eosinophilia in lung tissue. Western blot studies demonstrated that Rb1 substantially inhibited GATA3 and increased T-bet. These findings suggest that Rb1 may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.
T cell differentiation into distinct T helper (Th) subpopulations is crucial in governing acquired immune responses as well as some inflammatory and autoimmune disorders. This study investigated potential of the novel neutral binuclear ruthenium(II) complexes 1-8 with general formula [{RuCl2(η(6)-p-cym)}2μ-(N(∩)N)] (N(∩)N = bis(nicotinate)- and bis(iso-nicotinate)-polyethylene glycol esters; (3-py)COO(CH2CH2O) n CO(3-py) and (4-py)COO(CH2CH2O) n CO(4-py); n = 1-4), as well as [RuCl2(η(6)-p-cym)(nic)] (R1, nic = nicotinate) and [RuCl2(η(6)-p-cym)(inic)] (R2, inic = isonicotinate) as an immunomodulatory agents capable to direct Th cell differentiation. From all investigated complexes, [{RuCl2(η(6)-p-cym)}2μ-{(3-py)COO(CH2CH2O)4CO(3-py)}] (4) was selected for further study because it did not affect splenocyte viability (in concentration up to 50 μM), but significantly reduced secretion of representative Th1 cytokine, IFN-γ induced by T cell mitogen. Besides IFN-γ, 4 inhibited dose dependently expression and production of representative Th17 cytokine, IL-17, in these cells. Otherwise, the production of anti-inflammatory cytokines IL-4 and IL-10 was upregulated. Also, 4 significantly increased CD4(+)CD25(+)FoxP3(+) Treg cell frequency in the activated splenocytes. Moreover, ConA-induced expression of Th1 transcription factors, T-bet and STAT1, as well as of Th17-related protein STAT3 was attenuated upon exposure to 4, while the expression of Th2-related transcription factor GATA3 remained stable. In conclusion, ruthenium(II) complex 4 modulates immune system cell functions in vitro by inhibiting T cell differentiation towards pathogenic Th1/Th17 phenotype and inducing a regulatory phenotype characterized by IL-10 and IL-4 production, which may provide novel therapeutic opportunities for immune-inflammatory and/or autoimmune disorders.
Mycobacterial antigens are not exclusively presented to T-cells by classical HLA-class Ia and HLA-class II molecules, but also through alternative antigen presentation molecules such as CD1a/b/c, MR1 and HLA-E. We recently described mycobacterial peptides that are presented in HLA-E and recognized by CD8+ T-cells. Using T-cell cloning, phenotyping, microbiological, functional and RNA-expression analyses, we report here that these T-cells can exert cytolytic or suppressive functions, inhibit mycobacterial growth, yet express GATA3, produce Th2 cytokines (IL-4,-5,-10,-13) and activate B-cells via IL-4. In TB patients, Mtb specific cells were detectable by peptide-HLA-E tetramers, and IL-4 and IL-13 were produced following peptide stimulation. These results identify a novel human T-cell subset with an unorthodox, multifunctional Th2 like phenotype and cytolytic or regulatory capacities, which is involved in the human immune response to mycobacteria and demonstrable in active TB patients' blood. The results challenge the current dogma that only Th1 cells are able to inhibit Mtb growth and clearly show that Th2 like cells can strongly inhibit outgrowth of Mtb from human macrophages. These insights significantly expand our understanding of the immune response in infectious disease.
Determining histologic types of lung cancer on biopsies can be difficult. This study addresses the role of immunohistochemistry in histologic typing, using a tissue microarray (TMA) as "model biopsies," and presents a classification generated by an unsupervised hierarchical cluster analysis. A TMA was made from resection specimens of a consecutive series of 165 lung tumors. In a "tissue-spot review" with hematoxylin and eosin sections all the large cell carcinomas (N=22) were assigned to the noncommittal class of non-small cell lung cancer (NSCLC), as were an additional 37 tumors of defined histologic types. Adenocarcinomas and squamous cell carcinomas included with these NSCLC could be diagnosed by immunohistochemistry with antibodies against TTF-1, Napsin A, cytokeratin (CK)7, p40, p63, and CK5/6 with moderate to good sensitivities and specificities. Unsupervised hierarchical clustering was done with these data and additional high-molecular-weight cytokeratins, CD56, synaptophysin, and chromogranin immunohistochemistry. This delineated separate clusters for adenocarcinomas, large cell carcinomas, neuroendocrine tumors, and squamous cell carcinomas. Notably, adenocarcinoma and large cell carcinoma clusters were closely related and clearly set off from the squamous cell carcinoma cluster. As would be expected for a clinically well-staged series CDX2, GATA3, estrogen, and progesterone receptor immunohistochemistry remained negative in the vast majority of the tumors and, if positive, were restricted to very few cells. These results, the clustering data in particular, underpin the pragmatic recommendation canvassed with the IASLC/ATS/ERS classification of lung cancers that adenocarcinoma-type molecular studies should include NSCLC with a nonsquamous cell carcinoma immunophenotype.
Allergic reactions to walnut can be life-threatening. Although IgE epitopes of walnut have been studied, CD4(+) T cell-specific epitopes for walnut remain uncharacterized. In particular, the relationship of both phenotype and frequency of walnut-specific T cells to the disease have not been examined.
We sought to provide a thorough phenotypic analysis for walnut-reactive T cells in allergic and nonallergic subjects, particularly the relationship of phenotypes and frequencies of walnut-specific T cells with the disease.
The CD154 upregulation assay was used to examine CD4(+) T-cell reactivity toward the walnut allergens Jug r 1, Jug r 2, and Jug r 3. A tetramer-guided epitope mapping approach was used to identify HLA-restricted CD4(+) T-cell epitopes in Jug r 2. Direct ex vivo staining with peptide-major histocompatibility complex class II tetramers enabled comparison of the frequency and phenotype of Jug r 2-specific CD4(+) T cells between allergic and nonallergic subjects. Jug r 2-specific T-cell clones were also generated, and mRNA transcription factor levels were assessed by using quantitative RT-PCR. Intracellular cytokine staining assays were performed for further phenotypic analyses.
Jug r 2 was identified as the major allergen that elicited CD4(+) T-cell responses. Multiple Jug r 2 T-cell epitopes were identified. The majority of these T cells in allergic subjects have a CCR4(+) phenotype. A subset of these T cells express CCR4(+)CCR6(+) irrespective of the asthmatic status of the allergic subjects. Intracellular cytokine staining confirmed these TH2-, TH2/TH17-, and TH17-like heterogenic profiles. Jug r 2-specific T-cell clones from allergic subjects mainly expressed GATA3, nonetheless, a portion of T-cell clones both GATA3 and RAR-related orphan receptor C (RORC) or RORC alone, confirming the presence of TH2, TH2/TH17, and TH17 cells.
Jug r 2-specific responses dominate walnut T-cell responses in patients with walnut allergy. Jug r 2 central memory CD4(+) cells and terminal effector T cells were detected in peripheral blood, with the central memory phenotype as the most prevalent phenotype. In addition to conventional TH2 cells, TH2/TH17 and TH17 cells were also detected in nonasthmatic and asthmatic patients with walnut allergy. Understanding this T-cell heterogeneity might render better understanding of the disease manifestation.
Recent studies have confirmed the utility of massively parallel sequencing (MPS) in addressing genetically heterogeneous hereditary hearing impairment. By applying a MPS diagnostic panel targeting 129 known deafness genes, we identified a novel frameshift GATA3 mutation, c.149delT (p.Phe51LeufsX144), in a hearing-impaired family compatible with autosomal dominant inheritance. The GATA3 haploinsufficiency is thought to be associated with the hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome. The pathogenicity of GATA3 c.149delT was supported by its absence in the 5400 NHLBI exomes, 1000 Genomes, and the 100 normal hearing controls of the present study; the co-segregation of c.149delT heterozygosity with hearing impairment in 9 affected members of the family; as well as the nonsense-mediated mRNA decay of the mutant allele in in vitro functional studies. The phenotypes in this family appeared relatively mild, as most affected members presented no signs of hypoparathyroidism or renal abnormalities, including the proband. To our knowledge, this is the first report of genetic diagnosis of HDR syndrome before the clinical diagnosis. Genetic examination for multiple deafness genes with MPS might be helpful in identifying certain types of syndromic hearing loss such as HDR syndrome, contributing to earlier diagnosis and treatment of the affected individuals.
We describe a hemodialysis patient with hypoparathyroidism due to HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome caused by GATA3 mutation. She presents tumoral calcinosis which is a rare complication of end-stage renal failure. A novel mutation of GATA3 is identified in this patient.
The mechanistic target of rapamycin (mTOR) signaling integrates diverse environmental cues, including growth factors, nutrients and immunological signals. Activation of mTOR signaling stimulates protein synthesis and anabolic metabolism and coordinates cell growth, proliferation and fate decisions. In recent years, mTOR signaling has been linked to the entire spectrum of T cell biology, ranging from T cell development and activation to lineage specification and memory formation. Mechanistically, mTOR activation profoundly affects the expression and activity of many immunologically relevant transcription factors to propagate immune signaling and mediate effector functions. These transcription factors orchestrate cell metabolism (MYC, SREBPs and HIF1), lineage differentiation (T-bet, GATA3, RORγt, FOXP3 and Eomesodermin) and immune activation and functions (NF-κB, FOXOs, IRF4, STATs and GFI-1). This review discusses how mTOR signaling, through impinging upon transcriptional factors, regulates T cell development, activation, and effector and memory differentiation.
Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process.
The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.
Psoriasis and atopic dermatitis (AD) are inflammatory diseases thought to be mediated by helper T-cell subtypes 1 and 2 (TH1 and TH2), respectively. Although psoriasis and AD show histopathologic differences during chronic disease, they are difficult to distinguish histologically during erythrodermic exacerbations.
To determine whether the immune phenotype of helper T cells can differentiate erythrodermic psoriasis and erythrodermic AD by studying skin biopsy specimens of patients with psoriasis and AD during erythrodermic and chronic disease phases.
We conducted a retrospective study using biopsy samples of psoriasis, AD, and erythroderma belonging to the surgical pathology files of the James Homer Wright Pathology Laboratories, Massachusetts General Hospital, and collected from January 1, 2004, through December 31, 2011. Samples were obtained from patients with chronic psoriasis (n = 20), chronic AD (n = 20), erythroderma subsequently diagnosed as psoriasis (n = 7), and erythroderma subsequently diagnosed as AD (n = 5). We evaluated immunohistochemical stains for CD3 and dual stains for CD4 and T-bet, GATA binding protein 3 (GATA3), signal transducer and activator of transcription 3 (STAT3), or basonuclin 2 (BNC2), which are transcription factors reported to be specific and mutually exclusive for TH1, TH2, TH17, and TH22 cells, respectively. Two investigators independently counted CD3+ cells and dual-labeled CD4+/T-bet+, CD4+/GATA3+, CD4+/STAT3+, and CD4+/BNC2+ cells in 5 consecutive high-power fields.
We evaluated the percentage of TH1, TH2, TH17, and TH22 cells in CD3+ T cells and the TH1:TH2 ratio in chronic psoriasis, chronic AD, erythrodermic psoriasis, and erythrodermic AD.
We found a significant difference in the TH1:TH2 ratio between chronic psoriasis and chronic AD (0.26 and 0.09, respectively; P = .005). However, we detected no significant difference in the percentage of TH1 (6.5% and 4.8%), TH2 (55.2% and 64.6%), TH17 (14.7% and 30.4%), and TH22 (3.8% and 3.3%) cells of CD3+ T cells or in the TH1:TH2 ratio (0.16 and 0.07) within biopsy specimens from patients with erythrodermic psoriasis and AD, respectively.
This study confirms the TH1- and TH2-skewed phenotype of chronic psoriasis and chronic AD, respectively. However, the immune phenotype, as determined by immunohistochemical analysis, cannot discriminate between these inflammatory diseases in the erythrodermic phase. These findings advance our understanding of the pathophysiological characteristics of erythroderma, psoriasis, and AD and may influence therapeutic decisions.
To examine whether Shenfu injection (SFI) reduces post-resuscitation myocardial dysfunction in a pig model by modulating expression imbalance of transcription factors of regulatory T cell, namely GATA-3 and T-bet.
Thirty pigs were randomly divided into sham group (n = 6) and cardiopulmonary resuscitation (CPR) group (n = 24) according to the random number table method, and the pigs in the CPR group were randomly subdivided into normal saline (NS) group, epinephrine (EP) group, and SFI group (n = 8 per group). After 8 minutes of untreated ventricular fibrillation (VF) followed by 2 minutes of CPR, animals in three groups respectively received central venous injection of either 20 mL SFI (1.0 mL/kg, SFI group), EP (0.02 mg/kg, EP group) or NS (NS group). Blood samples were obtained before VF and 0.5, 2, 6 hours after restoration of spontaneous circulation (ROSC), and the parameters of hemodynamics and oxygen metabolism were determined. Surviving pigs were sacrificed at 24 hours after ROSC, the pathological changes in myocardium were observed, the levels of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) were measured by enzyme linked immunosorbent assay (ELISA), and expressions of protein and mRNA of GATA-3 and T-bet were determined by Western Blot and quantitative real-time polymerase chain reaction (RT-qPCR), respectively.
Six pigs of three resuscitation groups were successfully resuscitated. The CPR time, number of defibrillation, defibrillation energy, and ROSC time were significantly decreased in the EP and SFI groups compared with those in the NS group. Compared with the sham group, the parameters of left ventricular systolic function and oxygen metabolism were significantly decreased, myofibril organelles were extensively damaged, and progressive and severe deterioration of the myocardium was found, and mitochondrial structure was not recognizable in the NS group; the level of IL-4 in myocardium were markedly decreased, while that of TNF-α, IFN-γ and IFN-γ/ IL-4 [reflecting helper T cell 1/2 (Th1/Th2)] were significantly increased. Protein and mRNA expressions of GATA-3 were markedly reduced in the myocardium of pigs in the NS group compared with that of the sham group at 24 hours after ROSC, while T-bet was significantly increased. Compared with the NS group, animals treated with SFI had minimal myocardial intracellular damage, with decreased heart rate (HR, bpm: 90.33 ± 3.79 vs. 106.83 ± 5.36) and increased mean arterial pressure (MAP), cardiac output (CO), oxygen delivery (DO₂), and oxygen consumption (VO₂) at 6 hours after ROSC [MAP (mmHg, 1 mmHg = 0.133 kPa): 107.67 ± 1.96 vs. 86.83 ± 1.85, CO (L/min): 2.47 ± 0.08 vs. 2.09 ± 0.04, DO₂ (mL/min): 364.31 ± 4.21 vs. 272.33 ± 3.29, VO₂(mL/min): 95.00±2.22 vs. 82.50 ±2 .28, all P < 0.05]. Compared with the NS groups at 24 hours after ROSC, level of IL-4 was markedly increased in myocardial cells (ng/L: 33.80 ± 3.06 vs. 16.15 ± 1.34, P < 0.05), while the levels of TNF-α, IFN-γ and IFN-γ/IL-4 were lowered significantly [TNF-α (ng/L): 18.16 ± 0.71 vs. 29.64 ± 1.89, IFN-γ (ng/L): 373.75 ± 18.36 vs. 512.86 ± 27.86, IFN-γ/IL-4: 16.15 ± 1.34 vs. 33.80 ± 3.06, all P < 0.05], and myocardial T-bet protein and mRNA expressions were reduced [T-bet protein (gray value): 0.41 ± 0.07 vs. 0.59 ± 0.11, T-bet mRNA (2(-ΔΔCt)): 4.37 ± 0.21 vs. 7.57 ± 0.55, both P < 0.05], furthermore, myocardial GATA-3 protein and mRNA expressions were significantly up-regulated in SFI group [GATA-3 protein (gray value): 0.25 ± 0.07 vs. 0.16 ± 0.07, GATA-3 mRNA (2(-ΔΔCt)): 0.63 ± 0.07 vs. 0.34 ± 0.05, both P < 0.05]. The parameters in SFI group were significantly improved compared with those of the EP group.
Myocardial immune dysfunction is induced by Th1/Th2 imbalance following myocardial injury subsequent to CPR in pigs. SFI can attenuate myocardial injury and regulate myocardial immune disorders, protect post-resuscitation myocardial injury by modulating expression imbalance of transcription factors GATA-3 and T-bet.
Asthma is a serious global health problem characterised by airway inflammation, airway epithelial wall shedding, enhanced mucus production, increased IgE levels and airway hyperresponsiveness. The pathophysiology of asthma is mediated by Th2 cells which produce Th2 cytokines like interleukin-4, interleukin-5, interleukin-13 and interleukin-9. The differentiation of Th2 cells is induced by the transcription factor GATA3 which is activated by pSTAT6 via IL-4 signalling. To investigate the anti-asthmatic potential of Boswellic acid, as well as the underlying mechanism involved, we studied its anti-asthmatic potential in a murine model of asthma. In this study, BALB/c mice were systemically sensitized by ovalbumin (OVA) followed by aerosol allergen challenges. We investigated the effect of Boswellic acid on airway hyperresponsiveness, inflammatory cell infiltration, Th2 cytokine and OVA-specific IgE production in a mouse model of asthma. We found that Boswellic acid treated groups suppressed allergic airway inflammation, AHR, OVA-specific IgE and Th2 cytokines secretion. It also suppressed the expression of pSTAT6 and GATA3 in a dose dependent manner. Our data suggest that the mechanism by which Boswellic acid effectively treats asthma is based on reductions of Th2 cytokines via inhibition of pSTAT6 and GATA-3 expression.
Asthma is a serious health problem causing significant mortality and morbidity globally. Persistent airway inflammation, airway hyperresponsiveness, increased immunoglobulin E (IgE) levels and mucus hypersecretion are key characteristics of the condition. Asthma is mediated via a dominant T-helper 2 (Th2) immune response, causing enhanced expression of Th2 cytokines. These cytokines are responsible for the various pathological changes associated with allergic asthma. To investigate the anti-asthmatic potential of afzelin, as well as the underlying mechanisms involved, its anti-asthmatic potential were investigated in a murine model of asthma. In the present study, BALB/c mice were systemically sensitized using ovalbumin (OVA) followed by aerosol allergen challenges. The effect of afzelin on airway hyperresponsiveness, eosinophilic infiltration, Th2 cytokine and OVA‑specific IgE production in a mouse model of asthma were investigated. It was found that afzelin‑treated groups suppressed eosinophil infiltration, allergic airway inflammation, airway hyperresponsiveness, OVA-specific IgE and Th2 cytokine secretion. The results of the present study suggested that the therapeutic mechanism by which afzelin effectively treats asthma is based on reduction of Th2 cytokine via inhibition of GATA-binding protein 3 transcription factor, which is the master regulator of Th2 cytokine differentiation and production.
Differentiating metastatic breast carcinoma in the lungs from primary lung tumors and mesotheliomas is important for determining prognosis and treatment. We evaluated novel breast specific markers, androgen receptor (AR) and GATA binding protein 3 (GATA3) immunohistostaining, for this differential, and compare to other traditional markers. The specimens comprised 33 metastatic breast carcinomas to the lung, 566 primary lung tumors (170 adenocarcinomas, 157 squamous cell carcinomas, 31 pleomorphic carcinomas, 115 large cell neuroendocrine carcinomas, 43 small cell carcinomas, and 49 typical carcinoids) and 42 malignant mesotheliomas. They were analyzed by immunohistochemistry using antibodies to AR, GATA3, estrogen receptor (ER), progesterone receptor (PgR), mammaglobin, gross cystic disease fluid protein-15 (GCDFP-15). Of the metastatic breast carcinomas, immunohistostaining of AR, GATA3, ER, PgR, mammaglobin, GCDFP-15 were positive in 27 cases (81.8%), 24 cases (72.7%), 26 cases (78.8%), 13 cases (39.4%), 12 cases (36.4%), 9 cases (27.3%), respectively. Of primary lung tumors and mesotheliomas, staining of AR, GATA3, ER, PgR, mammaglobin, GCDFP-15 were positive in 18 cases (3%), 3 cases (0.5%), 4 cases (0.7%), 2 cases (0.3%), 0 case (0%), 2 cases (0.3%), respectively. Immunohistochemistry of AR and GATA3 are reliable for differentiating metastatic breast carcinoma from primary lung tumors and mesotheliomas.
Chronic rhinosinusitis (CRS) with nasal polyps (NPs) in Western populations is associated with TH2 cytokine polarization. IL-25, an IL-17 family cytokine, was recently reported to induce TH2-type immune responses and to contribute to several allergic diseases, such as atopic dermatitis and asthma. However, the role of IL-25 in Asian patients with nasal polyposis remains unclear.
We sought to determine the role of IL-25 in Asian patients with nasal polyposis and CRS.
We investigated IL-25 expression and its cellular origins in NPs of human subjects using immunohistochemistry (IHC), quantitative RT-PCR, and ELISA of NP tissues. Correlations between IL-25 expression and expression of other inflammatory markers in NP tissues were also explored. Anti-IL-25 neutralizing antibody was administered in an ovalbumin- and staphylococcal enterotoxin B-induced murine NP model to confirm the function of IL-25 during nasal polypogenesis.
IL-25 expression was upregulated in NP mucosa from patients with CRS with NPs compared with uncinate process tissue from control subjects and those with CRS without NPs. Overexpression of epithelial IL-25 was confirmed by using IHC, and double IHC staining showed that tryptase-positive cells were one of the main sources of IL-25 among immune cells. Furthermore, IL-17 receptor B levels were also increased in immune cells of patients with NPs compared with those in control subjects. In NPs IL-25 mRNA expression positively correlated with the expression of several inflammatory markers, including T-box transcription factor, RAR-related orphan receptor C, GATA3, eosinophil cationic protein, TGF-β1, and TGF-β2. IL-25 was more abundant in the murine NP model compared with control mice, and similar correlations between IL-25 and inflammatory markers were observed in murine models. Anti-IL-25 treatment reduced the number of polyps, mucosal edema thickness, collagen deposition, and infiltration of inflammatory cells, such as eosinophils and neutrophils. This treatment also inhibited expression of local inflammatory cytokines, such as IL-4 and IFN-γ. Furthermore, expression of CCL11, CXCL2, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in the nasal mucosa was suppressed in the anti-IL-25-treated group.
Our results suggest that IL-25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the pathogenesis of CRS with NPs in Asian patients. In addition, our results suggest the novel possibility of treating nasal polyposis with anti-IL-25 therapy.
Exercise increases the levels of circulating inflammatory mediators.
Does an acute bout of exercise affect the mRNA gene expression level of inflammatory markers in peripheral blood mononuclear cells (PBMCs) and contribute to the circulating levels of inflammatory mediators?
Ten healthy, non-smoking men (22-28 years old) performed 1-hour cycling at 70% of VO2 max.
The gene transcripts of CXCL16, IL-1β, IL-8, COX-2, TXB21 and GATA3 were significantly up-regulated in PBMCs. Serum levels of CXCL16, IL-6, TNFα and IL-10 were also significantly increased after exercise.
Increased mRNA transcription of inflammatory genes in PBMCs may contribute to increased level of inflammatory markers after an acute bout of exercise. The increased mRNA levels of GATA-3 and TXB21 may indicate that T cell lymphocytes are activated and secrete cytokines into the circulation. It needs to be further investigated if exercise changes the Th1/Th2 balance.
Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells.
We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.
Rapid advancements in massively parallel sequencing methods have enabled the analysis of breast cancer genomes at an unprecedented resolution, which have revealed the remarkable heterogeneity of the disease. As a result, we now accept that despite originating in the breast, estrogen receptor (ER)-positive and ER-negative breast cancers are completely different diseases at the molecular level. It has become apparent that there are very few highly recurrently mutated genes such as TP53, PIK3CA, and GATA3, that no two breast cancers display an identical repertoire of somatic genetic alterations at base-pair resolution and that there might not be a single highly recurrently mutated gene that defines each of the "intrinsic" subtypes of breast cancer (ie, basal-like, HER2-enriched, luminal A, and luminal B). Breast cancer heterogeneity, however, extends beyond the diversity between tumors. There is burgeoning evidence to demonstrate that at least some primary breast cancers are composed of multiple, genetically diverse clones at diagnosis and that metastatic lesions may differ in their repertoire of somatic genetic alterations when compared with their respective primary tumors. Several biological phenomena may shape the reported intratumor genetic heterogeneity observed in breast cancers, including the different mutational processes and multiple types of genomic instability. Harnessing the emerging concepts of the diversity of breast cancer genomes and the phenomenon of intratumor genetic heterogeneity will be essential for the development of optimal methods for diagnosis, disease monitoring, and the matching of patients to the drugs that would benefit them the most.
The yolk sac is an extra-embryonic membrane that plays an important role in early embryonic survival. It is the production site for blood cells during embryonic mammalian development and is a likely source of stem cells. The aim of this study was to identify and characterize the putative haematopoietic cells from the yolk sac of bovine embryos at different stages of gestation. The yolk sac regresses according to gestational age and embryos are characterized into groups (I-V) according to the crown-rump measurement. Groups I-III survived in culture longer and exhibited the formation of cell clusters, whereas groups IV and V could not be maintained in culture for an extended period of time. Flow-cytometry analysis revealed that groups I-III had similar characteristics, including high expression levels of the haematopoietic markers CD34, CD90 and CD117. In groups IV and V, decreases were observed in the expression levels of CD117 and CD34. Cells were found to be capable of survival post-cryopreservation and exhibited varying abilities to form colonies in a methylcellulose matrix, depending on gestational age. Cytological analysis revealed the presence of blood cells (lymphocytes and monocytes). Quantitative PCR analysis demonstrated the presence of the haematopoietic progenitor genes GATA3 and LMO2, but not RUNX1. Thus, we have successfully isolated and characterized haematopoietic cells from the bovine embryo yolk sac at varying gestational ages. This study is crucial for the understanding of the development of the haematopoietic system and the embryonic function of this organ. Copyright © 2015 John Wiley & Sons, Ltd.
Atoh1 (Math1) was the first gene discovered in ear development that showed no hair cell (HC) differentiation when absent and could induce HC differentiation when misexpressed. These data implied that Atoh1 was both necessary and sufficient for hair cell development. However, other gene mutations also result in loss of initially forming HCs, notably null mutants for Pou4f3, Barhl1, and Gfi1. HC development and maintenance also depend on the expression of other genes (Sox2, Eya1, Gata3, Pax2) and several genes have been identified that can induce HCs when misexpressed (Jag1) or knocked out (Lmo4). In the ear Atoh1 is not only expressed in HCs but also in some supporting cells and neurons that do not differentiate into HCs. Simple removal of one gene, Neurod1, can de-repress Atoh1 and turns those neurons into HCs suggesting that Neurod1 blocks Atoh1 function in neurons. Atoh1 expression in inner pillar cells may also be blocked by too many Hes/Hey factors but conversion into HCs has only partially been achieved through Hes/Hey removal. Detailed analysis of cell cycle exit confirmed an apex to base cell cycle exit progression of HCs of the organ of Corti. In contrast, Atoh1 expression progresses from the base toward the apex with a variable delay relative to the cell cycle exit. Most HCs exit the cell cycle and are thus defined as precursors before Atoh1 is expressed. Atoh1 is a potent differentiation factor but can differentiate and maintain HCs only in the ear and when other factors are co-expressed. Upstream factors are essential to regulate Atoh1 level of expression duration while downstream, co-activated by other factors, will define the context of Atoh1 action. We suggest that these insights need to be taken into consideration and approaches beyond the simple Atoh1 expression need to be designed able to generate the radial and longitudinal variations in hair cell types for normal function of the organ of Corti.
Even with adequate history, the distinction of cutaneous metastatic breast carcinoma from primary sweat gland carcinoma can be difficult. Although previous studies have attempted to separate these tumors with various immunohistochemical panels, those series have been limited by small numbers of patients as well as the inclusion of benign sweat gland tumors.
In this analysis, stains for p63, CK5/6, and D2-40 were included, as well as GATA3 and mammaglobin, in an evaluation of 21 primary sweat gland carcinomas and 33 examples of cutaneous metastatic breast carcinoma.
Immunoreactivity for p63, CK5/6, D2-40, GATA3, and mammaglobin was respectively observed in 81%, 71%, 52%, 71%, and 5% of sweat gland carcinomas compared with 6%, 6%, 6%, 91%, and 45% of metastatic breast carcinomas. These differences were statistically significant for p63, CK5/6, and D2-40. For the diagnosis of metastatic breast carcinoma, GATA3 was the most sensitive marker (91%), but its sensitivity was substantially lower. Mammaglobin was 95% specific for breast carcinoma but again suffered from limited sensitivity (45%) in this context.
These data suggest that p63 and CK5/6 are specific determinants for sweat gland carcinoma in the stated setting. In the absence of those analytes, metastatic breast carcinoma cannot always be identified to the exclusion of a primary tumor. This diagnostic scenario continues to require the procurement of a detailed clinical history regarding the number and duration of skin lesions in any given case.
T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.
Ideal potential vaccine adjuvants to stimulate a Th1 immune response are urgently needed to control intracellular infections in clinical applications. Telocinobufagin (TBG), an active component of Venenum bufonis, exhibits immunomodulatory activity. Therefore, we investigated whether TBG enhances the Th1 immune response to ovalbumin (OVA) and formalin-inactivated Salmonella typhimurium (FIST) in mice. TBG augmented serum OVA- and FIST-specific IgG and IgG2a and the production of IFNγ by antigen-restimulated splenocytes. TBG also dramatically enhanced splenocyte proliferative responses to concanavalin A, lipopolysaccharide, and OVA and substantially increased T-bet mRNA levels and the CD3(+)/CD3(+)CD4(+)/CD3(+)CD8(+) phenotype in splenocytes from OVA-immunized mice. In in vivo protection studies, TBG significantly decreased the bacterial burdens in the spleen and prolonged the survival time of FIST-immunized mice challenged with live S. typhimurium. In vivo neutralization of IFNγ with anti-IFNγ mAbs led to a significant reduction in FIST-specific IgG2a and IFNγ levels and in anti-Salmonella effect in TBG/FIST-immunized mice. In conclusion, these results suggest that TBG enhances a Th1 immune response to control intracellular infections.
This study was to investigate the mRNA expression of T-bet, GATA-3, ROR γt and Foxp3 mRNA in peripheral blood of patients with chronic lymphocytic leukemia (CLL) in different stages and explore their potential role in the pathogenesis and clinical diagnosis.
A total of 46 newly diagnosed and untreated patients with CLL was chosen as patient group, including 16 patients in the stage of Binet A, 15 in the stage of Binet B, and 15 in the stage of Binet C; 20 healthy persons were selected as controls. The quantitative fluorescence PCR was adopted to detect the mRNA expression of T-bet, GATA-3, RORγt and Foxp3 in peripheral blood mononuclear cell (PBMNC).
(1) The expression of T-bet mRNA in patient group was lower than that in normal controls (P < 0.05), while the mRNA expression of GATA-3 mRNA, ROR γt, Foxp3 in CLL patients group were higher than that in normal controls (P < 0.05), and the ratio of T-bet/GATA-3 and RORγt/Foxp3 in CLL in patient group were lower than that in normal controls(P < 0.05); (2) The later the stage, the higher the mRNA expression of GATA-3 and Foxp3. The mRNA expression of GATA-3 in stage Binet B and stage Binet C of CLL patients were higher than that in stage Binet A (P < 0.05),and the mRNA expression of Foxp3 in stage Binet C was higher than that in stage of Binet A and Binet B (P < 0.05); the later the stage, the lower the ratio of T-bet/GATA-3 and RORγt/Foxp3. The ratio of T-bet/GATA-3 in stage of Binet A CLL patients was higher than that in stage Binet C (P < 0.05) and the ratio of RORγt/Foxp3 in stage of Binet A and stage of Binet B were higher than that in stage Binet C (P < 0.05).
This study found in the level of transcription factors in CLL patients that with the process of disease, the balance shifts from Th1/Th2 and Th17/Treg to Th17 and Treg, and Treg cell may play a critical immunosuppressive role in the development of CLL.
USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 in vitro, however the in vivo settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity in vivo, we characterized Usp21-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The Usp21-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged Usp21-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the Usp21-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The Usp21-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.
The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function.
We recently identified a novel susceptibility variant, rs865686, for estrogen-receptor positive breast cancer at 9q31.2. Here, we report a fine-mapping analysis of the 9q31.2 susceptibility locus using 43 160 cases and 42 600 controls of European ancestry ascertained from 52 studies and a further 5795 cases and 6624 controls of Asian ancestry from nine studies. Single nucleotide polymorphism (SNP) rs676256 was most strongly associated with risk in Europeans (odds ratios [OR] = 0.90 [0.88-0.92]; P-value = 1.58 × 10(-25)). This SNP is one of a cluster of highly correlated variants, including rs865686, that spans ∼14.5 kb. We identified two additional independent association signals demarcated by SNPs rs10816625 (OR = 1.12 [1.08-1.17]; P-value = 7.89 × 10(-09)) and rs13294895 (OR = 1.09 [1.06-1.12]; P-value = 2.97 × 10(-11)). SNP rs10816625, but not rs13294895, was also associated with risk of breast cancer in Asian individuals (OR = 1.12 [1.06-1.18]; P-value = 2.77 × 10(-05)). Functional genomic annotation using data derived from breast cancer cell-line models indicates that these SNPs localise to putative enhancer elements that bind known drivers of hormone-dependent breast cancer, including ER-α, FOXA1 and GATA-3. In vitro analyses indicate that rs10816625 and rs13294895 have allele-specific effects on enhancer activity and suggest chromatin interactions with the KLF4 gene locus. These results demonstrate the power of dense genotyping in large studies to identify independent susceptibility variants. Analysis of associations using subjects with different ancestry, combined with bioinformatic and genomic characterisation, can provide strong evidence for the likely causative alleles and their functional basis.
S100A7 is expressed in many squamous cell carcinomas (SCCs), such as SCC of the skin, and well-differentiated SCC always displays stronger staining of this protein. A431 cells, an epidermal cancer cell line, were selected as a cell model to investigate the roles and mechanism of S100A7 in SCC of the skin. In this study, we demonstrated that the overexpression of S100A7 in A431 cells significantly promoted cell proliferation in vitro and tumor growth in vivo, whereas it suppressed the expression of GATA-3, caspase-14 and three squamous differentiation markers, keratin-1, TG-1 and involucrin. Conversely, the overexpression of caspase-14 not only significantly decreased cell proliferation and delayed tumor growth but also markedly induced the expression of three squamous differentiation markers, whereas S100A7 and GATA-3 were not influenced. Further evidence showed that silencing GATA-3 greatly inhibited the expression of caspase-14 and three differentiation markers, while the expression of S100A7 was not changed; contrary results were obtained when overexpressing GATA-3. Importantly, restoring the expression of GATA-3 and caspase-14 in A431-S100A7 cells could bypass the ability of S100A7 to increase cell viability and repress squamous differentiation. These data suggested that S100A7 expression in SCC may play an important role in the maintenance of SCC cell dedifferentiation, at least in SCC of the skin.
Kruppel-like factor 4 (KLF4) induces tumorigenesis or suppresses tumor growth in a tissue-dependent manner. However, the roles of KLF4 in hematological malignancies and the mechanisms of action are not fully understood.
Inducible KLF4-overexpression Jurkat cell line combined with mouse models bearing cell-derived xenografts and primary T-cell acute lymphoblastic leukemia (T-ALL) cells from four patients were used to assess the functional role of KLF4 in T-ALL cells in vitro and in vivo. A genome-wide RNA-seq analysis was conducted to identify genes regulated by KLF4 in T-ALL cells. Chromatin immunoprecipitation (ChIP) PCR was used to determine direct binding sites of KLF4 in T-ALL cells.
Here we reveal that KLF4 induced apoptosis through the BCL2/BCLXL pathway in human T-ALL cell lines and primary T-ALL specimens. In consistence, mice engrafted with KLF4-overexpressing T-ALL cells exhibited prolonged survival. Interestingly, the KLF4-induced apoptosis in T-ALL cells was compromised in xenografts but the invasion capacity of KLF4-expressing T-ALL cells to hosts was dramatically dampened. We found that KLF4 overexpression inhibited T cell-associated genes including NOTCH1, BCL11B, GATA3, and TCF7. Further mechanistic studies revealed that KLF4 directly bound to the promoters of NOTCH1, BCL2, and CXCR4 and suppressed their expression. Additionally, KLF4 induced SUMOylation and degradation of BCL11B.
These results suggest that KLF4 as a major transcription factor that suppresses the expression of T-cell associated genes, thus inhibiting T-ALL progression.
Despite several common phenotypic features, chronic obstructive pulmonary disease (COPD) and severe asthma differ with regard to their causative factors and pathophysiology. Both diseases may be exacerbated by environmental factors, however, the molecular profiles of disease episodes have not been comprehensively studied. We identified differences in gene and protein expression profiles expressed by peripheral blood mononuclear cells (PBMC) of COPD patients, patients with atopic asthma and healthy subjects when challenged with exacerbating factors in vitro: lipopolysaccharide (LPS), house dust mite (HDM) and cat allergen.
PBMC isolated from patients with severe atopic asthma and COPD, as well as healthy subjects were stimulated with rDer p 1 DG, rFel d 1 DG and LPS. The changes in the expression of 47 genes belonging to five groups (phospholipase A2, eicosanoids, transcription factors, cytokines and airway remodeling) were studied using TaqMan low density array cards. Immunoblotting was used to study relative protein expression.
rDer p 1 significantly up-regulated the expression of PLA2G4A, PLA2G6, PLA2G15, CYSLTR1, LB4R2, PTGS1, PTGS2, FOXP1, GATA3, HDAC2, IREB2, PPARG, STAT4, TSLP and CHI3L1 genes in asthmatics in comparison to healthy subjects. LPS induced significant expression of ANXA1 and LTA4H in asthmatics when compared to COPD patients and healthy subjects. SOX6,STAT4 and IL1RL1 were induced in COPD after LPS stimulation. Analysis of protein expression revealed a pattern similar to mRNA expression.
LPS-induced exacerbation of asthma and COPD is characterized by differential expression of selected genes in PBMC. HDM allergen changed the expression profile of inflammatory genes between patients with asthma of atopic origin and healthy controls.
Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral infections in mammals and also in fish. It has been seen by others that ribavirin by itself is able to stimulate the immune system of mammals, causing a differentiation of T-cells to T helper 1 cells (Th)-1. In this work, we evaluated the immune effect of ribavirin in vitro on kidney cells from Atlantic salmon and in vivo by oral administration of ribavirin to Atlantic salmon. For this purpose, the transcripts of immune molecules Tbet, GATA3, CD8, CD4, IFNα, IFNγ, IL-4/13, IL-10, IL-12, IL-15 and TGF-B were quantified. The results show that ribavirin administered orally in food to Atlantic salmon increased IFNγ and CD4 transcripts in the in vivo assays and, in addition, increased IL-12, IL-15 and CD8 in the in vitro analyses, indicating that the treatment stimulates a Th1 type response in salmon.
The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.
Infants that develop severe bronchiolitis due to respiratory syncytial virus (RSV) are at increased risk of developing asthma later in life. We investigated a potential immunological mechanism for the association between RSV and the development of allergic inflammation. The enzyme indoleamine 2,3-dioxygenase (IDO) has been reported to induce selective apoptosis of T helper 1 (Th1) cells and contributed to Th2-biased immune responses.
To determine whether RSV infection in vitro could induce IDO expression and bioactivity in human dendritic cells, leading to a Th2-biased immune response.
Human peripheral blood monocytes from healthy adult donors were isolated, differentiated to dendritic cells (moDC), in vitro. We studied RSV infection and mechanisms of IDO activation in moDC with subsequent effect on T-bet expression.
We found that moDC were infected by RSV and that this induced IDO activation. RSV-induced IDO activity was inhibited by palivizumab, UV inactivation, TL4R inhibition, and ribavirin. However, blocking endosomal TLR function with chloroquine did not inhibit IDO activity. Selective inhibitors suggested that RSV-induced IDO activity was dependent on the retinoic acid-inducible gene-I (RIG-I) related pathway via NF-κB and p38 MAPK. Coculture of RSV-infected moDC with activated T cells, in a transwell system, suppressed expression of T-bet (a Th1-associated factor) but not GATA3 (a Th2 regulator). Inhibition of IDO activity with the competitive inhibitor, 1-methyl tryptophan, blocked the effect on T-bet expression.
Our data show for the first time that RSV can induce the expression and bioactivity of IDO in human moDC, in a virus replication-dependant fashion. We suggest that RSV activation of IDO could be a potential mechanism for the development of allergic diseases.
Distinguishing between invasive urothelial carcinoma from other genitourinary lesions such as prostatic and renal carcinomas can be difficult, and may require highly sensitive immunohistochemical markers. GATA-binding protein 3 (GATA3) has been reported in a high percentage of urothelial and breast carcinomas. Mouse monoclonal uroplakin II (UPII) and p40 antibodies have recently been developed and demonstrated high specificity in urothelial carcinoma. This study evaluated the immunohistochemical staining sensitivities of UPII, GATA3, p40, and p63 in the detection of invasive urothelial carcinoma. UPII, GATA3, and p40 were further tested for specificity in lung, breast, colon, kidney, and prostate cancers. In all invasive urothelial carcinoma cases, UPII, GATA3, p40, and p63 exhibited sensitivities of 77.7%, 83.5%, 85.4%, and 80.6%, respectively. The combination of UPII, GATA3, and p40 antibodies stained 94.2% (97/103) of all invasive urothelial carcinoma cases, including 92.2% (71/77) of grade 2-3 urothelial carcinomas. In addition, GATA3 and UPII showed negative staining in lung squamous cell carcinomas and p40 showed negative staining in breast infiltrating ductal carcinomas. The combination of UPII, GATA3, and p40 showed negative staining in lung adenocarcinoma, colon adenocarcinoma, and renal carcinomas. In conclusion, UPII, GATA3, and p40, when used in combination, are highly sensitive in the differential diagnosis of invasive urothelial carcinoma.
To investigate whether cellular immunity and humoral immunity are involved in trichlorethylene (TCE)-induced mixed allergy, then provide the scientific basis for the mechanism of this disease.
Guinea pigs and rats were tested for this study by application of guinea pig maximization test (GPMT), the animals were randomly divided into negative control, positive control and TCE treatment groups. Animals of these groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. After TCE administration, rat peripheral blood samples were collected by flow cytometry to detect lymphocytes CD3⁺, CD4⁺, CD8⁺. Guinea pig peripheral blood samples were collected to detect the levels of IgG, IgA, IgM, C3, C4, and the spleens were taken out from guinea pigs after various treatment, mRNA expression of GATA3, T-bet, CTLA4 and Foxp3 in lymphocytes of guinea pig spleen was detected by real-time fluorescent PCR assay. Additionally, TCE allergic dermatitis patients were selected for the study, the peripheral blood samples were collected from the TCE patients group and control group, quantitative PCR was applied to detect mRNA expression of immune-related genes Foxp3, GATA3, CTLA4, T-bet.
TCE induced obvious skin allergic reaction in guinea pigs, the sensitization rate was 83.3%, IgG levels in TCE group and positive control increased significantly. Additionally, mRNA expression levels of GATA3, T-bet, CTLA4 significantly elevated in TCE group and positive control, but Foxp3 mRNA levels decreased. The lymphocytes CD3⁺ ratio in TCE group and positive control of rats was higher than that in negative control, we found that there was no statistical difference of CD4⁺, CD8⁺, CD4⁺/CD8⁺ between TCE group and negative control of rats. The mRNA expression levels of Foxp3, GATA3, CTLA4 in TCE patients increased by 115%, 97%, 241%, respectively as compared with the control, T-bet levels decreased by 47%when compared with the control.
TCE could induce obvious changes of cellular immunity and humoral immunity in guinea pigs, rats, and TCE patients, these findings indicated that TCE-induced immunological disorder belongs to the mixed allergy with involvment of cellular immunity and humoral immunity, the mixed allergy might be type IV and type II allergy.
The association of ovarian Brenner tumors and adjacent mucinous tumors is well known but not completely understood. In this study, we analyzed immunohistochemical markers on Brenner tumors and their associated mucinous tumor to explore Mullerian as well as Wolffian and germ cell derivation and determine if the mucinous component is independent or related to the Brenner tumor. Of 32 consecutive cases of Brenner tumors, 8 were identified with significant mucinous component, and 7 additional cases included foci of mucinous epithelium within the Brenner transitional nests. All Brenner tumors were diffusely positive for GATA3 and negative for Paired box gene 8, PAX2, and Sal-like protein 4. Interestingly, the areas of mucinous epithelium as well as mucinous tumors, intermixed and adjacent to the Brenner tumor, were negative for all 4 markers; however, occasional basal-like cells retained expression of GATA3. The immunoprofile of mucinous tumors associated with Brenner tumors shares the lack of Mullerian markers PAX2 and Paired box gene 8 with the Brenner tumor but differs in the expression of GATA3 only in the Brenner tumor component.
The study was designed to test DNA Aβ42 immunization in mice as alternative approach for possible active immunotherapy in Alzheimer patients. As results, we found polarized Th2 immune responses, efficient Aβ42 antibody levels, and disappearance of antigen specific T cells. In-vivo TNFRSF4/25 antibody co-stimulation enhanced Aβ42 specific T cell responses with initial Th2 expansion and subsequent development of Aβ42 specific CD4+CD25+Foxp3+ cells. It showed that Th2 biased responses due to gene gun immunizations propagate the development of regulatory T cells. In conclusion, full-length DNA Aβ42 immunization into skin results in a regulatory response with minimal risk of inflammation and autoimmunity.
Notch is a single-pass transmembrane receptor protein expressed by T cells, which contributes to the pathogenesis of asthma through regulation of the development and differentiation of T cells. γ-Secretase inhibitor (GSI) acts as an effective blocker of Notch signalling. The present study aimed to investigate the role of GSI MW167 in T cell differentiation and antigen-induced airway inflammation. An OVA-induced airway inflammation mouse model was established. Blockade of Notch signalling was achieved using MW167. The expression of IL-4, IL-5, IFN-γ, Notch1 signalling and pro-inflammatory transcription factors in activated lung T cells was evaluated. Finally, the therapeutic effect of MW167 was investigated by haematoxylin and eosin staining, real-time PCR and ELISA. The expression of IL-4 and IL-5 decreased and that of IFN-γ increased significantly, and the protein expression levels of pro-inflammatory transcription factors reduced in active lung T cells after administration of MW167, compared to the control group. MW167 treatment prevented OVA-induced airway inflammation and histological changes. The serum and bronchoalveolar lavage fluid (BALF) levels of IL-4 and IL-5 in MW167-treated mice decreased significantly, whereas those of IFN-γ increased, relative to the levels in OVA-challenged animals treated with PBS. Our findings indicate that Notch signalling plays an important role in the pathogenesis of asthma and that MW167 may be a potential therapeutic target for allergen-induced airway inflammation.
Allergy to cow's milk increases the risk of sensitization to other foods in young children.
We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk.
BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT-treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays.
In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced TH2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region.
Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell-dependent mechanism.
Primary mediastinal seminomas are unusual tumors that can present in a pure form or as part of a mixed germ cell tumor. Contrary to testicular seminomas, little is known about the expression of novel immunohistochemical markers in mediastinal seminomas. This study investigates the immunohistochemical features of these tumors with a focus on novel markers. Thirty-two cases of primary mediastinal seminomas were reviewed; and representative whole-tissue sections were selected for immunohistochemical studies using antibodies directed against high molecular weight cytokeratin 5/6 (CK5/6), low molecular weight cytokeratin (CAM5.2), octamer-binding transcription factor 3/4 (OCT3/4), spalt-like transcription factor 4 (SALL4), GATA binding protein 3 (GATA-3), sry-related HMG box 2 (SOX2), SOX17, human T cell leukemia/lymphoma 1 (TCL1), glypican 3, melanoma associated antigen C2 (MAGEC2), and paired box gene 8 (Pax8). The percentage of positive tumor cells as well as the intensity of staining was evaluated and scored. Thirty-one cases (97%) expressed SOX17, whereas 29 cases (91%) were positive for OCT3/4 and SALL4, respectively. Twenty-eight cases (88%) expressed MAGEC2 and CAM5.2, respectively. Two cases (6%) were positive for Pax8, and a single case (3%) was positive for TCL1. None of the cases stained with CK5/6, GATA-3, SOX2, or glypican 3. Similar to testicular seminomas, mediastinal seminomas show consistent expression of OCT3/4, SALL4, SOX17, and MAGEC2 and are negative for SOX2, glypican 3, GATA-3, and CK5/6. Pax8 positivity is only inconsistently identified in mediastinal seminomas. Contrary to their testicular counterparts, mediastinal tumors show diffuse expression of low-molecular-weight cytokeratin in up to 90% of cases and are commonly negative for TCL1. Although there is some immunohistochemical overlap between testicular and mediastinal seminomas, considerable differences also exist and should be acknowledged when dealing with these tumors.
To investigate Th2 differentiation features of Mycobacterium tuberculosis heat resistant antigens (MTB-HAg)-activated human γδT cells and the regulation of transcription factor T-box expression in T cells (T-bet) and GATA-binding protein 3 (GATA-3) on differentiation.
Peripheral blood mononuclear cells (PBMCs) were stimulated with MTB-HAg to generate MTB-HAg-activated T cells (MTBAT) and expanded in the neutral condition or Th2 polarizing condition. After restimulation for 6 hours with phorbol myristate acetate (PMA, 10 ng/mL), ionomycin (500 ng/mL) and monensin (2.5 μmol/L), intracellular cytokines (IFN-γ, IL-4) of γδT cells and αβT cells among MTBAT were detected by four-color fluorescence mAb staining combined with flow cytometry. The highly purified γδT cells and CD4⁺ T cells were sorted by flow cytometer from MTBAT that were cultured in neutral and Th2 polarizing conditions for 28 days. The expressions of T-bet and GATA-3 mRNA in purified γδT cells and CD4⁺ T cells were determined by reverse transcription PCR (RT-PCR) technique.
γδT cells among MTBAT cultured in the neutral or Th2 polarizing condition predominantly produced IFN-γ, whereas the percentage of IFN-γ⁺ αβT cells significantly decreased in the Th2 polarizing condition as the culture time went by. Compared with the neutral condition, Th0 type (IFN-γ⁺ IL-4⁺) γδT cells significantly increased, and Th2 type (IFN-γ⁻ IL-4⁺) αβT cells also significantly increased in the Th2 polarizing condition. RT-PCR showed that mRNA expression of T-bet was still at a high level in γδT cells that were expanded in the Th2 polarizing condition, but at a low level in Th2 polarized CD4⁺ T cells. Moreover, the mRNA expressions of GATA-3 in both Th2 polarized γδT cells and CD4⁺T cells were up-regulated.
In the Th2 polarizing condition, the majority of γδT cells in MTBAT still remained Th1 profile, whereas the portion of γδT cells differentiated into Th0 type cells. Both transcription factor T-bet and GATA-3 failed to display a fully cross-regulation function in Th2 polarized γδT cells.
Group 2 innate lymphoid cells (ILC2s) play critical roles in anti-helminth immunity, airway epithelial repair, and metabolic homeostasis. Recently, these cells have also emerged as key players in the development of allergic inflammation at multiple barrier surfaces. ILC2s arise from common lymphoid progenitors in the bone marrow, are dependent on the transcription factors RORα, GATA3, and TCF-1, and produce the type 2 cytokines interleukin (IL)-4, IL-5, IL-9, and/or IL-13. The epithelial cell-derived cytokines IL-25, IL-33, and TSLP regulate the activation and effector functions of ILC2s, and recent studies suggest that their responsiveness to these cytokines and other factors may depend on their tissue environment. In this review, we focus on recent advances in our understanding of the various factors that regulate ILC2 function in the context of immunity, inflammation, and tissue repair across multiple organ systems.
To determine T box expressed in T cells (T bet), GATA binding protein-3 (GATA 3), and retinoid-related orphan nuclear receptor gammat (RORgammat) in rats with chronic obstructive pulmonary disease (COPD).
Thirty rats were randomly divided into a control and a COPD group. The COPD model was established through smoking and lipopolysaccharide (LPS) tracheal instillation. Pulmonary function of the rats was measured 28 d after the establishment of the COPD model by a spirometer. Enzyme linked immunosorbent assay was performed to detect serum γ interferon (IFN-γ), interleukin (IL)-4, and IL-17. The expressions of T-bet, GATA-3, and RORgammat protein in lung tissues were determined by Western blot.
Compared with the controls, the COPD rats had decreased pulmonary function and expression of serum IL-4, and increased INF-γ, IL- 17, Th1/Th2, T-bet, T bet /GATA-3, and RORgammat protein (P<0. 05). Forced expiratory volume in 0. 3 seconds (FEV 0.3) was negatively correlated with INF γ and T-bet/GATA-3. Forced vital capacity (FVC) was positively correlated with IL 4. FEV0.3/FVC was negatively correlated with Thl/Th2, T-bet and T-bet/GATA-3. Peak expiratory flow (PEF) was negatively correlated with IL-17, T bet, and RORgammat (P<0. 05). Thl/Th2 was positively correlated with T bet/GATA-3. IL-17 was positively correlated with RORgammat. T bet/GATA-3 was positively correlated with RORgammat (P<0. 05).
Imbalanced regulation of T bet / GATA 3 and RORgammat on Th1/Th2 and Th17 cells is associated with the occurrence of COPD.
It is suggested that regulatory immune cells play a critical role in cancer cell growth by facilitating cancer cells to escape from the immune surveillance. The generation of the immune regulatory cells in cancer has not been fully understood yet. This study aims to investigate the role of the hepatoma-derived growth factor (HDGF) in the generation of regulatory T cells (Treg).
CCL-9.1 cells (A mouse hepatoma cell line), were cultured. The expression of HDGF in CCL-9.1 cells was assessed by quantitative RT-PCR and Western blotting. The generation of Foxp3(+) T cells was assessed by cell culture and flow cytometry. The immune suppressor function of the Foxp3(+) T cells on CD8(+) T cell activities was assessed by the carboxyfluorescein succinimidyl ester (CFSE)-dilution assay and enzyme-linked immunosorbent assay.
The results showed that exposure to PolyIC markedly increased the expression of HDGF in CCL-9.1 cells. Coculture of CCL-9.1 cells and CD4(+) CD25(-) T cells in the presence of PolyIC generated the Forkhead box protein (Foxp)3(+) T cells. The exposure to HDGF increased the expression of Foxp3 and decreased the expression of GATA3 in CD4(+) T cells. After activation, the Foxp3(+) T cells suppressed the CD8(+) T cell proliferation and the release of the cytotoxic cytokines.
Liver cancer cell-derived HDGF can induce Foxp3(+) T cells; the latter has the immune suppressor functions on CD8(+) T cell activities.
Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4+ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4+CD25hiFoxp3+ regulatory T cells. These profiles included increased IL-17A+, IL-4+, RORC2+ and GATA3+CD4+ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1β and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.
We have demonstrated previously that gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin A (MAM) are of limited utility in triple-negative breast cancer (TNBC). GATA-binding protein 3 (GATA-3) is an emerging breast-associated immunohistochemical (IHC) marker with limited data in TNBC. Here, we examined GATA-3 expression in TNBC in comparison with GCDFP-15 and MAM.
We studied GATA-3, GCDFP-15 and MAM IHC expression in 62 primary and 68 metastatic TNBCs. In primary TNBCs, GATA-3 staining was observed in 25 cases (40%), including 16 cases that were negative for GCDFP-15 and MAM. In metastatic TNBCs, GATA-3 staining was observed in 30 cases (44%), including 16 cases that were negative for GCDFP-15 and MAM. The expression frequency of any of the markers was 56% in primary and 62% in metastatic TNBCs. However, when focal staining was excluded, the expression frequency of any marker dropped to 31% and 44%, respectively.
GATA-3 is expressed at a higher frequency by IHC in TNBC compared to GCDFP-15 and MAM, although the tissue specificity of the latter markers may be superior. When evaluating a triple-negative tumour, including GATA-3 in a panel of markers may increase the diagnostic accuracy for tissue origin in the appropriate clinical setting.
Dendritic cells play an important role in determining whether naïve T cells mature into either Th1 or Th2 cells. We determined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)-β production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demonstrated by an increase in the number of eosinophils in bronchoalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-responsiveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma.
Sepsis is characterised by the frequent presence of organ failure and marked immunologic alterations. We studied the association between the extent of organ failure and the transcriptomic response of septic patients.
Gene expression profiles in the blood of 74 surgical patients with sepsis were compared with those of 30 surgical patients with no sepsis. Differentially expressed genes were assessed for their correlation with the sequential organ failure (SOFA) score.
The expression levels of a group of genes participating in the cell cycle (HIST1H1C, CKS2, CCNA2, CDK1, CCNB2, CIT, CCNB1, AURKA, RAD51), neutrophil protease activity (ELANE, ADORA3, MPO, MMP8, CTSG), IL-1R and IL-18R response correlated directly with SOFA and mortality. Genes involved in T cell (LCK, CD3G, CD3D, ZAP70, ICOS, CD3E, CD28, IL2RB, CD8B, CD8A, CD40LG, IL23A, CCL5, SH2D1A, ITK, CD247, TBX21, GATA3, CCR7, LEF1, STAT4) and NK cell immunity (CD244, KLRK1, KLRD1) were inversely associated with SOFA and mortality.
The extent of organ failure in sepsis correlates directly with the existence of imbalanced innate and adaptive responses at the transcriptomic level. Quantification of the expression levels of the genes identified here could contribute to the simultaneous assessment of disease severity and immunological alterations in sepsis.
While 30%-70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention.
To investigate the expression of transcriptional factors (TFs) T-bet, GATA-3, RORγt and FOXP in peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) and to evaluate the correlation between the imbalances of Th1/Th2, Th17/Treg at the expression levels and liver cancer Methods: The peripheral venous blood was drawn from 20 HCC-patients (HCC-group) and 20 health participants (C-group). The expression levels of Th1, Th2 and Th17 and the major Treg-specific TFs T-bet, GATA-3, RORγt and FOXP3 in the PBMC were measured with quantitative real-time PCR(RT-qPCR).
The mRNA level of Th1-specific TF T-bet in HCC-group was significantly lower than that of C-group (52.34±34.07 VS 104.01±56.00, P<0.01); the mRNA level of Th2-specifc TF, GATA-3, in HCC group was significantly higher than that in C-group (1.38±1.15 VS 0.58±0.65, P<0.05) and T-bet mRNA/GATA-3 mRNA ratio was significantly lower in HCC-group than in C-group (86.01±116.71 VS 461.88±708.81, P<0.05). The mRNA level of Th17-specific TF RORγt in HCC-group was significantly higher than that of C-group (72.32±32.82 VS 33.07±22.86, P<0.01). Treg-specific TF FOXP3 mRNA level was significant higher in HCC-group than in C-group (3.17±1.59 VS 1.39±1.13, P<0.01) CONCLUSION: T-bet mRNA level was reduced whereas GATA-3 mRNA level was increased and T-bet/GATA-3 ratio was significantly reduced in PBMC, indicating that Th1/Th2 ratio was of imbalance at TF levels in PBMC of HCC, displaying Th2 thrift phenomena. The mRNA levels of RORγt and FOXP3 in PBMC of HCC were significantly increased, indicating the existence of a predominant phenomenon of Th17- and Treg-expressing PBMC in HCC.
GATA3 is a transcription factor, which is involved in the growth and differentiation of several human tissues. Immunohistochemical staining for this marker has proven to be useful in recognizing a number of tumors, most notably those in the urinary tract and breasts. To date, no study has specifically assessed the distribution of GATA3 among different histomorphologic subtypes of breast carcinoma. The surgical pathology archive at our institution was searched, to retrieve cases of breast carcinomas of the following microscopic types-ductal, lobular, mucinous, metaplastic, medullary, apocrine, signet-ring cell, and micropapillary. Tissue microarrays were created, with four 0.6-mm punch specimens from each case. The tissue microarrays were cut at a 5-μm thickness and stained with monoclonal antibodies to GATA3 (Biocare Medical Inc, Concord, CA), mammaglobin (Dako, Carpinteria, CA), and gross cystic disease fluid protein 15 (Dako). Tumors were considered to be positive for those markers if more than 5% of the cells were labeled. Of 55 ductal adenocarcinomas, 51 (92.7%) expressed GATA3. All 4 GATA3-negative tumors were Nottingham grade III lesions that were also nonreactive for estrogen receptor protein. GATA3 was present in 28 (96.6%) of 29 lobular adenocarcinomas, 10 (90.9%) of 11 apocrine adenocarcinomas, 10 (83.3%) of 12 medullary carcinomas, 5 (55.5%) of 9 metaplastic carcinomas, and 1 of 2 signet-ring cell carcinomas. Mucinous carcinomas (23 cases) and micropapillary carcinomas (12 cases) uniformly and strongly labeled for GATA3. GATA3 equaled or surpassed the sensitivity of mammaglobin and gross cystic disease fluid protein 15 in all histologic subgroups of breast cancer in the study. Although most ductal adenocarcinomas were labeled for GATA3, it was absent in high-grade tumors that also lacked estrogen receptor protein. Favorable prognosis types of breast carcinoma (eg, mucinous carcinoma) and aggressive variants such as micropapillary carcinoma were equally reactive for this marker. A proportion of medullary and metaplastic carcinomas was GATA3 negative (17% and 44%, respectively). Thus, those pathologic entities cannot be excluded diagnostically by an absence of GATA3 immunoreactivity.
Genome-wide association studies (GWASs) have revealed SNP rs889312 on 5q11.2 to be associated with breast cancer risk in women of European ancestry. In an attempt to identify the biologically relevant variants, we analyzed 909 genetic variants across 5q11.2 in 103,991 breast cancer individuals and control individuals from 52 studies in the Breast Cancer Association Consortium. Multiple logistic regression analyses identified three independent risk signals: the strongest associations were with 15 correlated variants (iCHAV1), where the minor allele of the best candidate, rs62355902, associated with significantly increased risks of both estrogen-receptor-positive (ER(+): odds ratio [OR] = 1.24, 95% confidence interval [CI] = 1.21-1.27, ptrend = 5.7 × 10(-44)) and estrogen-receptor-negative (ER(-): OR = 1.10, 95% CI = 1.05-1.15, ptrend = 3.0 × 10(-4)) tumors. After adjustment for rs62355902, we found evidence of association of a further 173 variants (iCHAV2) containing three subsets with a range of effects (the strongest was rs113317823 [pcond = 1.61 × 10(-5)]) and five variants composing iCHAV3 (lead rs11949391; ER(+): OR = 0.90, 95% CI = 0.87-0.93, pcond = 1.4 × 10(-4)). Twenty-six percent of the prioritized candidate variants coincided with four putative regulatory elements that interact with the MAP3K1 promoter through chromatin looping and affect MAP3K1 promoter activity. Functional analysis indicated that the cancer risk alleles of four candidates (rs74345699 and rs62355900 [iCHAV1], rs16886397 [iCHAV2a], and rs17432750 [iCHAV3]) increased MAP3K1 transcriptional activity. Chromatin immunoprecipitation analysis revealed diminished GATA3 binding to the minor (cancer-protective) allele of rs17432750, indicating a mechanism for its action. We propose that the cancer risk alleles act to increase MAP3K1 expression in vivo and might promote breast cancer cell survival.
Prenatal dexamethasone exposure has been reported to increase allergy potential in childhood possibly by interference with normal immunological development in utero. This study investigated the effects of prenatal dexamethasone on T helper cell immune responses in a rat model.
Pregnant rats received either dexamethasone 0.1 mg/kg/day or normal saline from gestational day 14-21. Off-springs were cared for by their biological mother, or cross-fostered by the opposing group. Spleen and blood samples were collected at post-natal day 7 and 120 and tested for mRNA expression and plasma cytokine levels of Th1/Th2/Th17 immune response.
Both Th1 (T-bet) and Th2 (GATA-3) mRNA expression were shown to have a significant increase in the prenatal dexamethasone exposure group at day 120 (p<0.05). The plasma levels for Th1 (IFNγ and IL-2) and Th2 (IL-4, IL-5, IL-13) were found to have no significant differences between the two group (p>0.05). The mRNA expression of Th17 (RORγt) showed a significant decrease at post-natal day 120 as well as the plasma level of IL-17A at day 7 (11.21±1.67 vs. 6.23±1.06 pg/ml, p = 0.02). Cross-fostering by a dexamethasone exposed mother resulted in a significant increase in Th1/Th2 mRNA expression (p<0.05) and decrease of Th17.
Prenatal dexamethasone exposure increased Th1, Th2 and decreased Th17 expression. Cross-fostering by a dexamethasone exposed mother results in more prominent increase of Th1 and Th2 expression.
The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority. In this study, we aimed to seek DNA methylation changes in peripheral blood and tissue biopsies from patients with inflammatory bowel disease. The promoter methylation status of genes involved in inflammation and autoimmunity was profiled using the Human Inflammatory Response and Autoimmunity EpiTect Methyl II Signature PCR Array profiles. Methylation was considered to be hypermethylated if >20% according to the instructions of the manufacturer. The microarrays were validated with Quantitative Real-time PCR. Regarding Crohn disease (CD) no gene appeared hypermethylated compared to healthy controls. In ulcerative colitis (UC) 5 genes (CXCL14, CXCL5, GATA3, IL17C, and IL4R) were hypermethylated compared to healthy controls. Some of the examined genes show different methylation patterns between CD and UC. Concerning tissue samples we found that all hypermethylated genes appear the same methylation pattern and confirmed a moderate-strong correlation between methylation levels in colon biopsies and peripheral blood (Pearson coefficients r=0.089-0.779, and r=0.023-0.353, respectively). The epigenetic changes observed in this study indicate that CD and UC exhibit specific DNA methylation signatures with potential clinical applications in IBD non-invasive diagnosis and prognosis.
Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis.
Type-2 innate lymphoid cells (ILC2) belong to an expanding family of innate lymphocytes that provide a potent source of immune effector cytokines at the initiation of immune responses. ILC2 arise, under the control of the transcription factors RORα and GATA3, from lymphoid progenitors in the bone marrow, to secrete type-2 cytokines including IL-5 and IL-13. Using experimental models, ILC2 have been implicated in allergic diseases, such as asthma and atopic dermatitis, but also in metabolic homeostasis. Furthermore, recent reports have indicated that ILC2 not only play roles at the initiation of type-2 immunity but can also contribute to chronic pathology, such as fibrosis, and can impact on the priming of the adaptive T-cell response. The identification of ILC2 in patients with allergic dermatitis and allergic rhinitis indicates that these cells may represent new therapeutic targets.
During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood.
A novel Gata3-enhancer-Gfp-based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path.
FGF signaling acts as a "motor" that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157-167, 2015. © 2014 Wiley Periodicals, Inc.
To generate high-titer monoclonal antibodies, strong immuno-stimulation must be used for eliciting an intense cellular immune response. Here, we report that antigen-specific antibody production was potentiated by Peptide-25 derived from Ag85B of Mycobacterium tuberculosis, and that the production of antigen-specific IgG1 in particular was markedly potentiated; specifically, this occurred because the use of Peptide-25 resulted in an increase in the number of antigen-specific antibody-producing cells. We studied the activation of T cells by the peptide by examining gene expression. The observed expression pattern of GATA-3 and T-bet suggests that the peptide modulates the Th1/Th2 balance during immunization. This potentiation, which was remarkably high in BALB/c mice, could be applied in the immunization performed for monoclonal antibody production in vivo and in vitro.
Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is a rare condition inherited as autosomal dominant trait and characterized by hypoparathyroidism, sensorineural deafness, and renal dysplasia. HDR syndrome is caused by haploinsufficiency of the GATA3 gene located on chromosome 10p15. Here, we report the case of a 32-day-old Korean male with HDR syndrome. He was presented due to repeated seizures over previous 3 days. The patient was born after 40 weeks of gestation with birth weight of 2930 g, and was the first-born baby of healthy Korean parents. Hypoparathyroidism was first noticed due to seizure. A multicystic left dysplastic kidney and vesicoureteral reflux were detected by ultrasound after birth. Auditory brainstem response (ABR) testing revealed that the patient had moderate sensorineural deafness, with hearing losses of 80 dB at the mid and higher frequencies for both ears. Echocardiography finding revealed secundum atrial septal deftect. Based on biochemical results and clinical findings, a presumptive diagnosis of HDR syndrome was made. GATA3 mutation analysis identified a heterozygous deletion, c.153del (p.Phe51Leufs*144) in exon 1 causing a frameshift mutation, which is a novel de novo mutation. Therefore, we suggest that HDR syndrome should be considered in the differential diagnosis in symptomatic or asymptomatic patients with hypoparathyroidism, and that renal ultrasound or ABR testing be performed to prevent a missed diagnosis. This is the first report on Korean patient with confirmed HDR syndrome with novel mutation.
Now a number of CD4+ T-lymphocytes, known as Th1, Th2, Treg and Th17, is currently identified and well- studied. The methods basing on the targeted regulation of differentiation process of the Th-lymphocytes that carry out the immune response polarization attract an attention of scientists dealing with a correction of immune-mediated. In the present study, endogenous beta-galactoside-binding protein of the lectin family, galectin-3, was investigated as a regulator of T-cell homeostasis. A galectin-3 is known to be actively produced by tumor cells in malignant transformation and able to influence the processes of signal transduction, cell-cell cooperation and the implementation of programmed death. As cell differentiation processes are directly connected with the regulation of gene expression, we investigated the effect of recombinant galectin-3 on expression of mRNA of transcription.factors, which guide the differentiation of CD4+ lymphocytes. The study was performed on peripheral blood mononuclear cells of healthy individuals. The gene expression levels were evaluated by a real-time PCR. In the experiments in vitro, it has been first found the recombinant galectin-3 (0.5 mg/mL) up-regulating the expression of transcription factors Gata-3 and Rorc mRNAs and down-regulating the mRNA expression of transcription factors T-bet and FoxP3. Up to a concentration of 1 mg/mL recombinant galectin-3 stimulates Th-cells by dose-dependent manner, whereas at higher concentrations stimulating effect weakens, and inhibiting action starts prevailing. Thus, one can suppose that galectin-3 through regulation of lymphocytes differentiation promote development of allergic, autoimmune and neoplastic diseases that allows us to consider the galectin-3 as a.potential target for therapy of these diseases.
Regulatory T cells (Treg cells) can express the transcription factors T-bet and GATA-3, but the function of this expression and whether such cells represent stable subsets is still unknown. By using various reporter tools, we found that the expression of T-bet and GATA-3 in Treg cells was dynamically influenced by the cytokine environment. Treg cell-specific deletion of the gene encoding either T-bet (Tbx21) or GATA-3 (Gata3) alone did not result in loss of Treg cell function; however, mice with combined deficiency in both genes in Treg cells developed severe autoimmune-like diseases. Loss of Treg cell function correlated with upregulation of expression of the transcription factor RORγt and reduced expression of the transcription factor Foxp3. Thus, in the steady state, activated Treg cells transiently upregulated either T-bet or GATA-3 to maintain T cell homeostasis.
Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.
In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.
These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.
We previously showed that Trypanosomacruzi infection in C57BL/6 mice results in a lethal infection linked to unbalanced pro- and anti-inflammatory mediators production. Here, we examined the dynamics of CD4(+)Foxp3(+) regulatory T (Treg) cells within this inflammatory and highly Th1-polarized environment. Treg cells showed a reduced proliferation rate and their frequency is progressively reduced along infection compared to effector T (Teff) cells. Also, a higher fraction of Treg cells showed a naïve phenotype, meanwhile Teff cells were mostly of the effector memory type. T. cruzi infection was associated with the production of pro- and anti-inflammatory cytokines, notably IL-27p28, and with the induction of T-bet and IFN-γ expression in Treg cells. Furthermore, endogenous glucocorticoids released in response to T. cruzi-driven immune activation were crucial to sustain the Treg/Teff cell balance. Notably, IL-2 plus dexamethasone combined treatment before infection was associated with increased Treg cell proliferation and expression of GATA-3, IL-4 and IL-10, and increased mice survival time. Overall, our results indicate that therapies aimed at specifically boosting Treg cells, which during T. cruzi infection are overwhelmed by the effector immune response, represent new opportunities for the treatment of Chagas disease, which is actually only based on parasite-targeted chemotherapy.
The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.
Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.
In this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.
In the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.
Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P ≤ 5 × 10(-8) in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P = 2.8 × 10(-10)) and rs3781093, OR, 1.73 (P = 3.2 × 10(-9)). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non-Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P = 6.29 × 10(-11)). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.
Pregnancy suppressive effect on autoimmune diseases including Multiple Sclerosis and Rheumatoid Arthritis may result from high levels of sex steroids such as estrogen and estriol. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of estrogen on MS alleviation. Female C57BL/6 mice were immunized with MOG35-55. Clinical scores and other relevant parameters were monitored daily. Brain and spinal cord histology was performed to measure lymphocyte infiltration and central nervous system demyelination. Th1/Th2/Th17 and Treg cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed by real-time PCR and T cell differentiation was explored through flow cytometry examination. Pregnancy and pregnancy level of estrogen alleviated clinical manifestations in EAE induced mice, reduced CNS demyelination and cell infiltration, suppressed spleen T cell proliferation, enhanced production of anti-inflammatory cytokines in splenocytes and increased the percentage of Th2 and Treg cells. Furthermore, the results of real-time PCR for transcription factors and related cytokines of Th1/Th2/Th17 and Treg cells in CNS showed reduced expression levels of Th1 and Th17 transcription factors, including T-bet and ROR-γt, and decreased Th1 and Th17 cytokines including IFN-γ, TNF-α, IL-17 and IL-23. These results are the first to indicate that pregnancy and pregnancy level of estrogen ameliorate the EAE condition by favoring Treg and Th2 differentiation through induced expression of Foxp3 and GATA3 in the CNS. Moreover, pregnancy and pregnancy level of estrogen decreased mRNA levels of T-bet and ROR-γt in the CNS.
Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3-4 month-old female tree shrews were orally administrated 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.
By binding to T cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 co-stimulation may rescue IR-stressed orthotopic liver transplants from innate immunity-driven inflammation.
We used wild type (WT) and TIM-3 transgenic (Tg) mice (C57BL/6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20 h at 4°C in UW solution) and syngeneic orthotopic liver transplantation (OLT).
Orthotopic liver transplants in WT or TIM-3Tg→TIM-3Tg groups were resistant against IR-stress, evidenced by preserved hepatocellular function (serum ALT levels) and liver architecture (Suzuki's score). In contrast, orthotopic liver transplants in WT or TIM-3Tg→WT groups were susceptible to IRI. TIM-3 induction in circulating CD4+ T cells of the recipient: (1) depressed T-bet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in orthotopic liver transplants; (2) promoted T cell exhaustion (PD-1, LAG-3) phenotype; and (3) depressed neutrophil and macrophage infiltration/function in orthotopic liver transplants. In parallel studies, we documented for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in vivo and in vitro. Moreover, exogenous recombinant Gal-9 (rGal-9) potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells.
Harnessing TIM-3/Gal-9 signalling at the T cell-hepatocyte interface facilitates homeostasis in IR-stressed orthotopic liver transplants. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients.
Thrombopoietin (TPO) is the principal regulator of thrombopoiesis and promotes the proliferation, differentiation and maturation of megakaryocytic progenitor cells in mammals. In this study we report on the molecular and functional characterization of goldfish TPO. Quantitative expression analysis of goldfish tpo revealed the highest mRNA levels in heart, followed by spleen, liver, brain, intestine and kidney tissues. Significant decrease of tpo and c-mpl expressions in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, indicates that TPO is not involved in monopoiesis. Recombinant goldfish TPO (rgTPO) alone did not induce significant proliferation of progenitor cells, but TPO in cooperation with recombinant goldfish kit ligand A (rgKITLA) supported proliferation of progenitor cells in a dose-dependent manner. In response to rgTPO or a combination of rgTPO and rgKITLA, the mRNA levels of thrombopoietic markers cd41 and c-mpl as well as thrombo/erythropoietic transcription factors gata1 and lmo2 in sorted progenitor cells were up-regulated, while the mRNA levels of granulopoietic markers (cebpα and gcsfr) and the lymphoid transcription factor gata3 were down-regulated. Furthermore, rgTPO and rgKITLA synergistically stimulated thrombocytic colony-formation. Our results demonstrate that goldfish TPO has similar functions to mammalian TPO as a regulator of thrombopoiesis, and suggests a highly conserved molecular mechanism of thrombocyte development throughout evolution of vertebrates.
Uroplakins are markers of terminally differentiated urothelium. Uroplakin II (UPII) is a newly described sensitive marker for urothelial carcinoma (UC). The expression profile of UPII in different types of UC and its utility in the diagnostic setting are needed. We evaluated UPII expression in bladder tissue microarrays, including urothelial neoplasm of low malignant potential (n = 8), low-grade papillary UC (n = 72), noninvasive high-grade papillary UC (n = 77), UC in situ (n = 27), and invasive high-grade UC (INVUC) (n = 122). UPII expression in 52 breast carcinomas and 38 high-grade prostate adenocarcinomas was also assessed. UPII expression was compared with GATA binding protein 3 (GATA3) and estrogen receptor for its role in facilitating the differential diagnosis of the above 3 types of malignancy. UPII labeling was seen in 83.0% of UC overall, including 95.7% of noninvasive UC and 65.6% of INVUC. UPII labeling was not found in any breast and prostate carcinomas. In comparison, GATA3 labeling was seen in 91.6% of all UCs, including 96.4% of noninvasive UCs and 85.1% of INVUC, with stronger intensity and extent compared with UPII (P < .005). GATA3 labeled 2 (5%) of 38 high-grade prostate adenocarcinoma. Estrogen receptor nuclear labeling was seen in 13.0% of UCs and 12.5% of prostate carcinomas. UPII was highly specific (100%) but only moderately sensitive for UC and can therefore be a potentially useful marker to identify urothelial lineage and help distinguish UC from prostate cancer or, in conjunction with GATA3, from metastatic breast cancer.
We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases.
We sought to investigate the role of DNA-PK in asthma.
Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study.
DNA-PK was essential for monocyte adhesion to TNF-α-treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs(+/-)) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of TH2-skewed OT-II wild-type CD4(+) T cells reversed IgE and TH2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs(+/-) mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4(+) T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4(+) T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4(+) T cells and prevented differentiation of TH1 and TH2 cells under respective TH1- and TH2-skewing conditions.
Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.
Mycosis fungoides (MF) is a neoplasm of skin-homing CD4(+) helper T (TH) lymphocytes with dysregulation of TH1 and TH2 immunity. Diagnosis of MF is challenging, as there is significant morphologic overlap with other dermatologic entities.
We investigated diagnostic utility of TH1- and TH2-specific markers, T-bet, and GATA-3, respectively, in MF and its reactive and neoplastic mimics.
Immunohistochemical staining for CD3/T-bet and CD3/GATA-3 was performed on inflammatory dermatoses (n = 56), MF (n = 37), Sezary syndrome (SS; n = 8), and cutaneous anaplastic large cell lymphoma (C-ALCL; n = 14).
Inflammatory dermatoses showed epidermal T cells predominantly expressing GATA-3, except psoriasis, which exhibited a mixed GATA-3/T-bet staining. In contrast, neoplastic T cells in patch stage MF showed markedly increased T-bet positivity with minimal GATA-3 expression. Plaque stage MF had a mixed T-bet/GATA-3 phenotype, whereas tumor stage MF and SS exhibited diffuse GATA-3 expression. C-ALCL lacked significant staining for both markers.
Sample size was relatively small.
A predominance of T-bet(+) T cells in the epidermis support patch stage MF over dermatitis. A predominance of GATA-3(+) T cells in the dermis support CD30(+) MF with large cell transformation over C-ALCL. These stains do not allow distinction between dermatitis and cutaneous infiltrates of SS.
Conventional karyotyping (550 bands resolution) is able to identify chromosomal aberrations >5-10 Mb, which represent a known cause of intellectual disability/developmental delay (ID/DD) and/or multiple congenital anomalies (MCA). Array-Comparative Genomic Hybridization (array-CGH) has increased the diagnostic yield of 15-20%.
In a cohort of 700 ID/DD cases with or without MCA, including 15 prenatal diagnoses, we identified a subgroup of seven patients with a normal karyotype and a large complex rearrangement detected by array-CGH (at least 6, and up to 18 Mb). FISH analysis could be performed on six cases and showed that rearrangements were translocation derivatives, indistinguishable from a normal karyotype as they involved a similar band pattern and size. Five were inherited from a parent with a balanced translocation, whereas two were apparently de novo. Genes spanning the rearrangements could be associated with some phenotypic features in three cases (case 3: DOCK8; case 4: GATA3, AKR1C4; case 6: AS/PWS deletion, CHRNA7), and in two, likely disease genes were present (case 5: NR2F2, TP63, IGF1R; case 7: CDON). Three of our cases were prenatal diagnoses with an apparently normal karyotype.
Large complex rearrangements of up to 18 Mb, involving chromosomal regions with similar size and band appearance may be overlooked by conventional karyotyping. Array-CGH allows a precise chromosomal diagnosis and recurrence risk definition, further confirming this analysis as a first tier approach to clarify molecular bases of ID/DD and/or MCA. In prenatal tests, array-CGH is confirmed as an important tool to avoid false negative results due to karyotype intrinsic limit of detection.
Currently, in the era of post-genomics, immunology is facing a challenging problem to translate mutant phenotypes into gene functions based on high-throughput data, while taking into account the classifications and functions of immune cells, which requires new methods.
Here we propose a novel application of a multidimensional analysis, Canonical Correspondence Analysis (CCA), to reveal the molecular characteristics of undefined cells in terms of cellular differentiation programmes by analysing two transcriptomic datasets. Using two independent datasets, whether RNA-seq or microarray data, CCA successfully visualised the cross-level relationships between genes, cells, and differentiation programmes, and thereby identified the immunological features of mutant cells (Gata3-KO T cells and Stat3-KO T cells) in a data-oriented manner. With a new concept, differentiation variable, CCA provides an automatic classification of cell samples, which had a high sensitivity and a comparable performance to other classification methods. In addition, we elaborate how CCA results can be interpreted, and reveal the features of CCA in comparison with other visualisation techniques.
CCA is a visualisation tool with a classification ability to reveal the cross-level relationships of genes, cells and differentiation programmes. This can be used for characterising the functional defect of cells of interest (e.g. mutant cells) in the context of cellular differentiation. The proposed approach fits with common hypothesis-oriented studies in immunology, and can be used for a wide range of molecular and genomic studies on cellular differentiation mechanisms.
To evaluate the prognostic significance of GATA-binding protein 3 (GATA-3), gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin (MGB) in invasive breast carcinomas (IBCs).
GATA-3, GCDFP-15 and MGB were expressed in 37.9% (370/976), 26.0% (254/978) and 35.3% (348/986) of this cohort of 1017 IBCs, respectively. GCDFP-15 was an independent favourable prognostic factor in all cases [disease-free survival (DFS), hazard ratio (HR) 0.587, P = 0.049; overall survival (OS), HR 0.512, P = 0.049], as well as in oestrogen receptor (ER)-negative (DFS, HR 0.353, P = 0.012; OS, HR 0.310, P = 0.017) and HER2-positive (DFS, HR 0.279, P = 0.036; OS, HR 0.235, P = 0.050) cases; it also refined the prognostication of molecular apocrine cancers. GATA-3 and MGB did not show any prognostic significance.
The commonly used breast carcinoma biomarkers vary in their prognostic implications. GCDFP-15 independently indicated a favourable prognosis, especially in ER-negative, HER2-positive and molecular apocrine cancers. GATA-3 and MGB were not associated with outcome.
Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.
GATA binding protein 3 (GATA3) is a regulator of mammary luminal cell differentiation, and an estrogen receptor (ER) associated marker in breast cancer. Tumor suppressor functions of GATA3 have been demonstrated primarily in basal-like breast cancers. Here, we focused on its function in luminal breast cancer, where GATA3 is frequently mutated, and its levels are significantly elevated.
GATA3 target genes were identified in normal- and luminal cancer- mammary cells by ChIP-seq, followed by examination of the effects of GATA3 expressions and mutations on tumorigenesis-associated genes and processes. Additionally, mutations and expression data of luminal breast cancer patients from The Cancer Genome Atlas were analyzed to characterize genetic signatures associated with GATA3 mutations.
We show that some GATA3 effects shift from tumor suppressing to tumor promoting during tumorigenesis, with deregulation of three genes, BCL2, DACH1, THSD4, representing major GATA3-controlled processes in cancer progression. In addition, we identify an altered activity of mutant GATA3, and distinct associated genetic signatures. These signatures depend on the functional domain mutated; and, for a specific subgroup, are shared with basal-like breast cancer patients, who are a clinical group with regard to considerations of mode of treatment.
The GATA3 dependent mechanisms may call for special considerations for proper prognosis and treatment of patients.
Circulating lymphocyte number was significantly decreased in patients with sepsis. However, it remains unknown which severity phase (sepsis, severe sepsis, and septic shock) does it develop and what happen on each subpopulation. Eight patients with differing severities of sepsis (31 sepses, 33 severe sepses, and 16 septic shocks) were enrolled. Quantitative real-time polymerase chain reaction (RT-PCR) of Th1, Th2, and Th17; regulatory T (Treg) cell-specific transcription factor T-bet; GATA-3; RORgammat (RORγt); forkhead box P3 (FOXP3); and IL-17 mRNA were performed, and the enzyme-linked immunosorbent assay (ELISA) was used to detect serum interferon (IFN)-γ, IL-4, and IL-10. In this study, the Th1, Th2, Treg transcription factors, and related cytokines IFN-γ, IL-4, and IL-10 levels of sepsis and severe sepsis patients in peripheral blood were significantly higher than those of the normal controls. Except for IL-17, the T-bet, GATA-3, and IFN-γ levels of septic shock patients were lower than those of sepsis patients. We also observed that the proportions of Th17/Treg in the sepsis and septic shock groups were inversed. From the above, the inflammatory response especially the adaptive immune response is still activated in sepsis and severe sepsis, but significant immunosuppression was developed in septic shock. In addition, the proportion of Th17/Treg inversed may be associated with the illness aggravation of patients with sepsis.
Hyperplastic polyps of the stomach are regarded as benign. However, in rare cases they may contain incipient primary carcinomas. To our knowledge, breast carcinoma metastatic to a gastric hyperplastic polyp has not yet been reported. We describe the case of a 69-year-old woman to whom a gastric polyp was endoscopically excised. The patient had previously undergone a right mastectomy for mixed, invasive ductal and lobular carcinoma 5 years earlier. Histological sections from the gastric lesion showed typical features of hyperplastic polyp with foci of poorly differentiated adenocarcinoma including signet ring cells infiltrating the lamina propria. The histologic findings were consistent with a primary gastric cancer. However, the carcinoma cells were immunopositive for estrogen and progesterone receptors and GATA3 and negative for CDX2, Hep Par 1, and MUC5AC. E-cadherin showed membranous reactivity in some of the carcinoma cells while in others it was negative. Accordingly, metastatic mixed, lobular and ductal breast carcinoma was diagnosed. We conclude that metastatic adenocarcinoma mimicking primary gastric cancer can be rarely encountered in hyperplastic gastric polyps.
Cytokine receptors can be markers defining different T-cell subsets and considered as therapeutic targets. The association of IL-6 and IL-6 receptor α (IL-6Rα) with asthma was reported, suggesting their involvement in asthma.
To determine whether and how IL-6Rα defines a distinct effector memory (EM) CD8+ T-cell population in health and disease.
EM CD8+ T cells expressing IL-6Rα (IL-6Rα(high)) were identified in human peripheral blood and analyzed for function, gene, and transcription factor expression. The relationship of these cells with asthma was determined using blood and sputum.
A unique population of IL-6Rα(high) EM CD8+ T cells was found in peripheral blood. These cells that potently proliferated, survived, and produced high levels of the Th2-type cytokines IL-5 and IL-13 had increased levels of GATA3 and decreased levels of T-bet and Blimp-1 in comparison with other EM CD8+ T cells. In fact, GATA3 was required for IL-6Rα expression. Patients with asthma had an increased frequency of IL-6Rα(high) EM CD8+ T cells in peripheral blood compared with healthy control subjects. Also, IL-6Rα(high) EM CD8+ T cells exclusively produced IL-5 and IL-13 in response to asthma-associated respiratory syncytial virus and bacterial superantigens.
Human IL-6Rα(high) EM CD8+ T cells is a unique cell subset that may serve as a reservoir for effector CD8+ T cells, particularly the ones producing Th2-type cytokines, and expand in asthma.
We evaluated the effects of T helper cell differentiation in a mite-allergic animal model treated with inhaled heparins of different molecular weight.
BALB/c mice were divided into four groups: 1. Control, 2. Mite intratracheal (mIT), 3. Inhaled heparin (hIN), 4. Inhaled low-molecular-weight heparin (lmwhIN). Groups 2, 3, and 4 were sensitized twice with Der p allergen subcutaneously on day 1 and day 8. Der p allergen was administered intratracheally on day 15. Groups 3 and 4 were treated with heparin or low-molecular-weight (lmw) heparin intranasally from day 1 to 22. Splenocytes from sacrificed mice stimulated with 16 µg/ml of Der p were cultured for 72 hours. Supernatants of splenocyte were collected to analyze the effect of Interleukin (IL)17-A/F, Interferon(IFN)-γ, IL-4, IL-13, and IL-10. Serum was also collected for Der P-specific IgE level on day 23. Total RNA was extracted from spleen tissue for mRNA expression. Gene expression of Foxp3, IL-10 IFN-γ, GATA3, IL-5, and RORγt were analyzed.
Both hIN and lmwhIN groups had lower serum IgE level than that of the mIT group (both p<0.0001). Both hIN and lmwhIN groups showed significantly decreased transcripts of GATA-3, IFN-γ, IL-5, and RORγt mRNA in their spleen. Regarding the supernatant of splenocyte culture stimulated with Der p, compared with the mIT group, there were significant decreases in IL-17A/F, IFN-γ, IL-4, IL-13, and IL-10 secretion in inhaled hIN and lmwhIN groups.
From this balb/c mice study, the analyses of mRNA and cytokines revealed that both intranasal heparin and lmw heparin treatment decreased the expression of Th1, Th2, and Th17 in spleen. The underlying mechanism(s) warrant further studies.
T helper type 2 (Th2) cells, which produce interleukin-4 (IL-4), IL-5 and IL-13, control immunity to all forms of allergic inflammatory responses. Interleukin-21 (IL-21) reduces allergic symptoms in murine models and inhibits IL-4-induced IgE secretion by B cells. However, whether or not IL-21 directly affects Th2 cells, which leads to reduced allergic symptoms, is unclear. In this study, we investigated the effects of IL-21 on the differentiation and effector functions of Th2 cells. We found that IL-21 reduced the number of differentiated Th2 cells and these Th2 cells showed a diminished Th2 cytokine production. Interleukin-21 suppressed Th2 cytokine production of already polarized Th2 cells by down-regulation of transcription factor GATA-3. It also induced apoptosis of Th2 cells with decreased anti-apoptotic factor Bcl-2. Intranasal administration of IL-21 at the beginning of ovalbumin (OVA) sensitization or before OVA challenge decreased Th2 cytokines in the bronchoalveolar lavage fluid of OVA/alum-immunized allergic mice. In addition, the inhibitory effects of IL-21 on Th2 effector functions can also be found in allergic patients. Our results demonstrate that IL-21 suppresses the development of Th2 cells and functions of polarized Th2 cells. Hence, the administration of IL-21 may be considered for use as a preventive and therapeutic approach when dealing with Th2-mediated allergic diseases.
Neuroblastoma is a common solid malignant tumor of the sympathetic nervous system, which contributes to 15% of cancer‑related mortality in children. The differentiation status of neuroblastoma is correlated with clinical outcome, and the induction of differentiation thus constitutes a therapeutic approach in this disease. However, the molecular mechanisms that control the differentiation of neuroblastoma remain poorly understood. The present study aimed to define whether GATA3 is involved in the differentiation of neuroblastoma cells. The results demonstrated that GATA3 is a prognostic marker for survival in patients with neuroblastoma, and that high‑level GATA3 expression is associated with increased self‑renewal and proliferation of neuroblastoma cells. Retinoic acid treatment led to GATA3 downregulation together with neuronal differentiation, suppression of cell proliferation and inhibition of tumorigenecity in neuroblastoma cells. These findings suggest that GATA3 is a key regulator of neuroblastoma differentiation, and provide a novel potential therapeutic strategy for the induction of neuroblastoma differentiation.
We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4(+) T-helper (Th) and CD8(+) cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO(+)CD62L(+)) and effector memory (CD45RO(+)CD62L(-)) subsets. We evaluated the phenotype of CD4(+) cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet(+)CD119(+)) and Th17 (RORγt(+)CD161(+)) populations, while having no effect on the Th2 population (GATA3(+)CD193(+)/GATA3(+)CD294(+)cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1β), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4(+)FoxP3(+) cells, and an increase of CD25(+)FoxP3(+) and CD39(+)FoxP3(+) Treg in the CD4(+) population. Induction of Treg cells was corroborated by the increased secretion of TGF-β. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.
Patient-derived human-in-mouse xenograft models of breast cancer (PDX models) that exhibit spontaneous lung metastases offer a potentially powerful model of cancer metastasis. In this study, we evaluated the malignant character of lung micrometastases that emerge in such models after orthotopic implantation of human breast tumor cells into the mouse mammary fat pad. Interestingly, relative to the parental primary breast tumors, the lung metastasis (met)-derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status. Epigenetic correlates were determined by gene array analyses. Lung met-derived tumors displayed differential expression of negative regulators of cell proliferation and metabolism and positive regulators of mammary epithelial differentiation. Clinically, this signature correlated with breast tumor subtypes. We identified hsa-miR-138 (miR-138) as a novel regulator of invasion and epithelial-mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Mechanistic investigations showed that GATA3 transcriptionally controlled miR-138 levels in lung metastases. Notably, the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes, or a proliferation gene signature. Our results highlight the loss of malignant character in some lung micrometastatic lesions and the epigenetic regulation of this phenotype.
Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely related stem/progenitor cells within the context of their overall niche.
The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1) alginate-encapsulated IPNV antigens (ENCAP); 2) soluble antigens (UNENCAP) or 3) untreated feed (control). This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1st oral boost and the 2nd, at 4 weeks after the 2nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-β and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p<0.04) in serum antibodies following the 1st boost but not after the 2nd boost. This coincided with significant up-regulation of CD4 and GATA3 genes. In contrast, serum antibodies in the UNENCAP group decreased both after the 1st and 2nd oral boosts. This was associated with significant up-regulation of FOXP3, TGF-β and IL-10 genes. The expression of IgT was not induced in the hindgut after the 1st oral boost but was significantly up-regulated following the 2nd one. CD4 and GATA3 mRNA expressions exhibited a similar pattern to IgT in the hindgut. IgM mRNA expression on the other hand was not differentially regulated at any of the times examined. Our findings suggest that 1) Parenteral prime with oil-adjuvanted vaccines followed by oral boost with ENCAP results in augmentation of the systemic immune response; 2) Symmetrical prime and boost (mucosal) with ENCAP results in augmentation of mucosal immune response and 3) Symmetrical priming and boosting (mucosal) with soluble antigens results in the induction of systemic immune tolerance.
The aim of this study is to investigate the expression pattern of histone deacetylase 9 in peripheral blood of patients with allergic asthma and its regulatory effect on the balance of Th17/Treg cells involved in the pathogenesis of asthma.
flap-Ub promoter-GFP-WRE vector was used to construct the Jurkat-HA-FOXP3 cell line. After histone deacetylase inhibitor-trichostatin A (TSA) treatment, FOXP3 and RORγt expression were detected by real-time-polymerase chain reaction (RT-PCR). BALB/c mice were randomly assigned to control group, TSA treatment and the asthma group. Serum Immunoglobulin E (IgE) was detected with enzyme-linked immunosorbent assay (ELISA), airway inflammation in lung tissue evaluated by haematoxylin/eosin staining, bronchoalveolar lavage fluid (BALF) cell number and differential counted, interleukin (IL)-17A and TGF-β concentrations in BALF measured with ELISA, and expression of RORγt and FOXP3 messenger RNA (mRNA)measured by RT-PCR. Forty-seven patients with asthma were recruited and assigned to intermittent, mild and moderate-severe group. GATA3, IL-4, histone deacetylases (HDAC) 9 mRNA expression level were measured by RT-PCR.
After TSA treatment, FOXP3 mRNA level was upregulated, while RORγt mRNA level was downregulated. FOXP3 protein level was also upregulated by TSA. In vivo, TSA treatment can inhibit IL-17 but promote transforming growth factor-beta production in the BALF of asthma mice, and inhibited the expression of Th17 cells and RORγt mRNA in lung; also can promote Foxp3 mRNA expression. GATA3, IL-4 mRNA expression levels were upregulated in patients with asthma than the healthy control. HDAC9 mRNA expression level was associated with the severity of disease.
The histone deacetylase inhibitor TSA can regulate the balance of Th17/Treg in asthma by regulating the activity of histone deacetylase.
Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.
The Sonic hedgehog (Shh) signaling pathway is crucial for pattern formation in early central nervous system development. By systematically analyzing high-throughput in situ hybridization data of E11.5 mouse brain, we found that Shh and its receptor Ptch1 define two adjacent mutually exclusive gene expression domains: Shh+Ptch1- and Shh-Ptch1+. These two domains are associated respectively with Foxa2 and Gata3, two transcription factors that play key roles in specifying them. Gata3 ChIP-seq experiments and RNA-seq assays on Gata3-knockdown cells revealed that Gata3 up-regulates the genes that are enriched in the Shh-Ptch1+ domain. Important Gata3 targets include Slit2 and Slit3, which are involved in the process of axon guidance, as well as Slc18a1, Th and Qdpr, which are associated with neurotransmitter synthesis and release. By contrast, Foxa2 both up-regulates the genes expressed in the Shh+Ptch1- domain and down-regulates the genes characteristic of the Shh-Ptch1+ domain. From these and other data, we were able to reconstruct a gene regulatory network governing both domains. Our work provides the first genome-wide characterization of the gene regulatory network involved in the Shh pathway that underlies pattern formation in the early mouse brain.
CD4(+) T cells orchestrate the immune response by differentiating into T helper (Th) or regulatory (Treg) cell subsets that secrete distinct sets of cytokines. They also play a critical role in the pathogenesis of autoimmunity, asthma, allergy and, likely, cancer. The mechanisms involved in the regulation of CD4(+) T cell homeostasis by galectin-1 remain poorly characterized. To investigate whether galectin-1 modulates the differentiation of CD4(+) T cells, the effects of galectin-1 on the mRNA expression levels of TBX21, GATA-3, FOXP3 and RORC in activated peripheral blood mononuclear cells were examined. The expression levels of GATA-3 and FOXP3 mRNA were up-regulated after treatment with 1.0 μg/ml galectin-1 and were unchanged (for GATA-3) or slightly elevated (for FOXP3) compared with untreated cells when 2.0 μg/ml galectin-1 was added. At the same time, at both concentrations of galectin-1, we observed reduced TBX21 and RORC mRNA expression levels. These findings support the concept that galectin-1 skews the differentiation of CD4(+) T cells towards Th2 and Treg cells.
Brenner tumors are composed of urothelial/transitional-type epithelium and, hence, are morphologically similar to Walthard nests and tubal/mesothelial transitional metaplasia. In this study, we analyzed immunohistochemical markers on Brenner tumors to explore Müllerian as well as Wolffian and germ cell derivation. We also attempted to explore their possible association with tubal/paratubal Walthard nests/transitional metaplasia, using the same immunostains. Thirty-two consecutive cases of Brenner tumors were identified. Thirteen (43%) of the patients had Walthard nests in the tubal/periovarian soft tissue. All Brenner tumors were diffusely positive for GATA3 (strongly positive in 30/32 and weakly positive in the remaining 2) and negative for PAX8, PAX2, and SALL4. Similarly, all Walthard nests were positive for GATA3, whereas only 3 (23%) of 13 showed occasional PAX8 expression; all were negative for PAX2 and SALL4. In our study, more than 40% of Brenner tumors had associated Walthard nests. The similar morphology and immunoprofile of Brenner tumors and Walthard nests suggest a probable link between Brenner tumors and Walthard nests. Two additional cases presented highlight small transitional lesions involving the ovary: a possible precursor lesion or the initial steps of Brenner tumor formation. Brenner tumors and most Walthard nests lacked staining for Müllerian (PAX8 and PAX2) and germ cell tumor markers (SALL4).
Mesonephric carcinomas are rare tumors predominantly arising in the uterine cervix from mesonephric remnants. Although the tumor has classic morphologic features, some cases can mimic Müllerian adenocarcinoma and be misdiagnosed, especially those with significant ductal pattern. Moreover, there is an overlap in immunohistochemical results with endometrial and endocervical carcinomas. In this study, we report 2 cases of mesonephric carcinosarcoma, originally diagnosed as Müllerian carcinomas, 1 presenting in the vagina; review immunohistochemical results including positivity for GATA-3, not previously reported and comment on the proposed panel of PAX8, p16, and estrogen receptors as discriminators of Müllerian adenocarcinoma (endocervical or endometrial) versus mesonephric carcinoma.
Astragalus membranaceus, a traditional Chinese herb, has been used to improve airway inflammation and asthma. The present study investigated whether A. membranaceus has immunotherapeutic effects on asthma, a chronic inflammatory mucosal disease that is associated with excess production of IgE, eosinophilia, T helper 2 (Th2) cytokines, and bronchial hyperresponsiveness. An ovalbumin (OVA)-induced, chronic inflammatory airway murine asthma model was used to examine the status of pulmonary inflammation after the administration of A. membranaceus. The IgE levels in serum and bronchoalveolar lavage fluid showed a tendency to decrease after the administration of A. membranaceus. The number of eosinophils decreased and infiltration of inflammatory cells and collagen deposition declined in lung sections after A. membranaceus administration. The RNA and protein levels of Th2 cytokines and the ratio of the GATA3/T-bet mRNA levels decreased after A. membranaceus treatment. Furthermore, the mRNA level of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor, increased in the lung tissues of A. membranaceus-treated mice. Finally, an A. membranaceus water extract activated PPARγ activity in either human embryonic kidney 293 (HEK293) or A549 cells in a PPARγ-responsive element-containing luciferase reporter assay. These results indicate that A. membranaceus has an inhibitory effect on airway inflammation in a murine model of asthma through modulating the imbalanced relationship between Th1 and Th2 cytokines.
Foxp3(+) regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6(-/-) ) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6(-/-) mice, increased numbers of Foxp3-negative 'exTreg' cells were found, particularly in the CD25(+) population. ExTreg cells from Bcl6(-/-) mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6(-/-) mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6(Foxp3-/-) ) mice were analysed. Bcl6(Foxp3-/-) mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6(-/-) mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6(Foxp3-/-) mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6(+) Treg cells. Bcl6(Foxp3-/-) mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses.
Naïve CD4(+) T cells are activated and differentiate to distinct lineages of T helper (Th) cells, which are involved in physiological and pathological processes by obtaining the potential to produce different lineage-specific cytokines that mediate adaptive immunity. In the past decade, our knowledge of Th cells has been significantly expanded with the findings of new lineages. Interleukin (IL)-9 producing T cells are recently identified. In consideration of the ability to preferentially secret IL-9, these cells are termed Th9 cells. Given the multiple function of IL-9, Th9 cells participate in the lesion of many diseases, such as allergic inflammation, tumor, and parasitosis. In this chapter, we will focus on the cytokines, co-stimulatory factors, and transcriptional signaling pathways, which regulate Th9 cells development as well as stability, plasticity, and the multiple roles of Th9 cells in vivo.
CD4+ T helper cells regulate appropriate cellular and humoral immune responses to a wide range of pathogens and get involved in many diseases progress. The balance of the earliest determined CD4+ T helper cell subsets, Th1 and Th2, play an important role in allergy and autoimmune diseases. During the research, Animal models in immunology research are necessary and always the powerful tools for the basic scientific research. With the new sequence technologies, the finding of key gene mutation in Th1/Th2 cells has been proved to be related to human diseases. Here, we review four animal models about four key genes in Th1/Th2 cells to introduce the balance between Th1/Th2 cells. Furthermore, the related genetic mutations in human diseases and the new therapies are reviewed in this chapter, which show the importance of Th1/Th2 cells in human diseases further.
The distinctive differentiated states of the CD4+ T helper cells are determined by the set of transcription factors and the genes transcribed by the transcription factors. In vitro induction models, the major determinants of the cytokines present during the T-cell receptor (TCR)-mediated activation process. IL-12 and IFN-γ make Naive CD4+ T cells highly express T-bet and STAT4 and differentiate to TH1 cells, while IL-4 make Naive CD4+ T cells highly express STAT6 and GATA3 and differentiated to TH2 cells. Even through T-bet and GATA3 are master regulators for TH1/TH2 cells differentiation. There are many other transcription factors, such as RUNX family proteins, IRF4, Dec2, Gfi1, Hlx, and JunB that can impair TH1/TH2 cells differentiation. In recent years, noncoding RNAs (microRNA and long noncoding RNA) join in the crowd. The leukocytes should migrate to the right place to show their impact. There are some successful strategies, which are revealed to targeting chemokines and their receptors, that have been developed to treat human immune-related diseases.
ASH2L is a component of MLL complexes that confer H3K4 trimethylation. The ASH2L gene is located at 8q11-12, which is often amplified in breast cancers. We found that increased ASH2L expression, which can result from gene amplification, is often correlated with increased ERα expression in both breast cancer cell lines and primary breast cancers. Forced expression of ASH2L induced ERα expression in mammary epithelial cells, whereas depletion of ASH2L suppressed ERα expression in breast cancer cells. To understand the mechanism by which ASH2L regulates ERα expression, we identified GATA3 as the binding protein of ASH2L. ASH2L was shown to potentiate the transcriptional activity of GATA3. ASH2L was recruited to the enhancer of the ERα gene through GATA3 to promote ERα transcription. This study established that ASH2L enhances ERα expression as a coactivator of GATA3 in breast cancers.
Caspase 14 is reduced in adenocarcinomas of the stomach and colon. In contrast, breast and lung adenocarcinomas frequently show an overexpression of caspase 14. Salivary gland adenocarcinomas have not been evaluated for potential aberrant caspase 14 expression.
Samples from salivary gland carcinomas (n = 43) were analysed by immunohistochemistry (caspase 14, filaggrin, GATA3 and Ki67) and fluorescence in situ hybridization.
Caspase 14 is not expressed in normal salivary glands, while in a subfraction of carcinomas (32%) an aberrant expression was found. Filaggrin could not be detected. Caspase 14 staining was not associated with tumour dedifferentiation, GATA3 expression or amplification of gene locus 19p13.
In summary, aberrant expression of caspase 14 can be found in a subfraction of salivary gland carcinomas but could not be used as a biomarker for a specific carcinoma subtype of the salivary gland.
Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the world's population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-γt, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1β, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3+ T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.
The prevalence of asthma in girls increases after puberty. Previous studies have detected associations between sex hormones and asthma, as well as between sex hormones and T helper 2 (Th2) asthma-typical immune responses. Therefore, we hypothesized that exogenous or endogenous sex hormone exposure (represented by oral contraceptive pill (OCP) use and early menarche, respectively) are associated with DNA methylation (DNA-M) of the Th2 transcription factor gene, GATA3, in turn affecting the risk of asthma in girls, possibly in interaction with genetic variants. Blood samples were collected from 245 female participants aged 18 years randomly selected for methylation analysis from the Isle of Wight birth cohort, UK. Information on use of OCPs, age at menarche, and concurrent asthma were assessed by questionnaire. Genome-wide DNA-M was determined using the Illumina Infinium HumanMethylation450 beadchip. In a first stage, we tested the interaction between sex hormone exposure and genetic variants on DNA-M of specific cytosine-phosphate-guanine (CpG) sites. In a second stage, we determined whether these CpG sites interact with genetic variants in GATA3 to explain the risk of asthma.
Interactions between OCP use and seven single nucleotide polymorphisms (SNPs) of GATA3 were analyzed for 14 CpG sites (stage 1). The interaction between OCP use and SNP rs1269486 was found to be associated with the methylation level of cg17124583 (P = 0.002, false discovery rate (FDR) adjusted P = 0.04). DNA-M of this same CpG site was also influenced by the interaction between age at menarche and rs1269486 (P = 0.0017). In stage 2, we found that cg17124583 modified the association of SNP rs422628 with asthma risk at the age of 18 years (P = 0.006, FDR adjusted P = 0.04). Subjects with genotype AG showed an increase in average risk ratio (RR) from 0.31 (95% CI: 0.10 to 0.8) to 11.65 (95% CI: 1.71 to 79.5) when methylation level increased from 0.02 to 0.12, relative to genotype AA.
A two-stage model consisting of genetic variants in the GATA3 gene, OCP use, age at menarche, and DNA-M may explain how sex hormones in women can increase the asthma prevalence after puberty.
The establishment and maintenance of mammary epithelial cell identity depends on the activity of a group of proteins, collectively called maintenance proteins, that act as epigenetic regulators of gene transcription through DNA methylation, histone modification, and chromatin remodeling. Increasing evidence indicates that dysregulation of these crucial proteins may disrupt epithelial cell integrity and trigger breast tumor initiation. Therefore, we explored in silico the expression pattern of a panel of 369 genes known to be involved in the establishment and maintenance of epithelial cell identity and mammary gland remodeling in cell subpopulations isolated from normal human mammary tissue and selectively enriched in their content of bipotent progenitors, committed luminal progenitors, and differentiated myoepithelial or differentiated luminal cells. The results indicated that, compared to bipotent cells, differentiated myoepithelial and luminal subpopulations were both characterized by the differential expression of 4 genes involved in cell identity maintenance: CBX6 and PCGF2, encoding proteins belonging to the Polycomb group, and SMARCD3 and SMARCE1, encoding proteins belonging to the Trithorax group. In addition to these common genes, the myoepithelial phenotype was associated with the differential expression of HDAC1, which encodes histone deacetylase 1, whereas the luminal phenotype was associated with the differential expression of SMARCA4 and HAT1, which encode a Trithorax protein and histone acetylase 1, respectively. The luminal compartment was further characterized by the overexpression of ALDH1A3 and GATA3, and the down-regulation of NOTCH4 and CCNB1, with the latter suggesting a block in cell cycle progression at the G2 phase. In contrast, myoepithelial differentiation was associated with the overexpression of MYC and the down-regulation of CCNE1, with the latter suggesting a block in cell cycle progression at the G1 phase.
Acute rejection is still a major cause of graft dysfunction and would jeopardize recipients' post-transplantation survival. Current studies demonstrate that Galectin-9 (Gal-9) is associated with down-regulation of pro-inflammatory cytokines, thus, possesses a negative immune regulatory role. However, the specific role of Gal-9 in liver transplant remains unknown.
To establish acute rejection models of rat liver transplantation (Lewis-BN, n = 45), recipients were randomly divided into following three groups: the transfected group (n = 15); the blank plasmid group (n = 15); and the control group (n = 15). The transfected group was perfused with Ad-galectin-9 through the portal vein during the cold ischemia period. The blank plasmid group was perfused with non-target vector, and the control group was perfused with saline. The acute rejection was assayed by pathological examination; Gal-9, T-bet, RORγt, GATA3 and Foxp3 mRNA expression was detected by real-time PCR; Western blots and enzyme-linked immunosorbent assays were performed to measure IFN-γ, IL-10 and IL-17 expression.
The pathological change of the transfected group was ameliorated than that of the other two groups. The Gal-9 mRNA level in the transfected group was much higher than that in the other two groups (*P < 0.05); T-bet and RORγt mRNA levels were significantly lower in the transfected group than in the other two groups while GATA3 and Foxp3 were not shown statistics significances (*P < 0.05). The IFN-γ and IL-17 levels in the transfected group were significantly lower than in the other two groups (*P < 0.05 for both protein and serum levels).
Up-regulation of Gal-9 in vivo turns immune system toward immnuosuppression and prolongs rats liver allograft survival by the diminishment of Th1/Th17.
Immunohistochemical expression of GATA-3 is seen predominantly in non-neoplastic bladder and breast epithelium and their respective carcinomas; however, data on expression in normal and lesional trophoblastic tissues are limited. Immunohistochemical staining for GATA-3 was assessed in a range of normal/lesional trophoblastic tissues and tumors in the differential diagnosis (n=445), including nonmolar products of conceptions/second and third trimester placentas/ectopic pregnancies, hydatidiform moles, placental site nodules, normal/exaggerated implantation sites, choriocarcinomas, epithelioid trophoblastic tumors, placental site trophoblastic tumors, atypical smooth muscle tumors (including leiomyosarcoma), and cervical and pulmonary squamous cell carcinomas. The extent of expression (0 to 4+) and intensity (weak to strong) were recorded. All cases with developing trophoblast/non-neoplastic trophoblastic proliferation and 81% of trophoblastic neoplasms were positive. Of all non-neoplastic trophoblast cell types, expression was observed in cytotrophoblast in 89% of cases, syncytiotrophoblast in 50%, intermediate trophoblast in 100%, and villous trophoblastic columns in 100%. Increasing gestational age was associated with a decrease in extent/intensity of expression in non-neoplastic cytotrophoblast and syncytiotrophoblast, whereas intermediate trophoblast maintained diffuse and strong expression from early to late gestation (P<0.0001). Eighty-nine percent of normal/exaggerated implantation sites showed 3+ or 4+ expression, whereas staining in 55% of placental site nodules was 1+ or 2+. Staining for GATA-3 was present in 78% of choriocarcinomas, 95% of epithelioid trophoblastic tumors, and 71% of placental site trophoblastic tumors. Although the number of choriocarcinomas and placental site trophoblastic tumors that showed a spectrum of expression ranging from negative to diffuse was relatively evenly distributed, 81% of epithelioid trophoblastic tumors had 3+ or 4+ staining. None of the atypical smooth muscle tumors and 3% of squamous cell carcinomas were positive, all of which exhibited weak staining. We conclude that GATA-3 is frequently expressed in normal and lesional trophoblastic tissues. It is also differentially expressed in intermediate trophoblast and cytotrophoblast/syncytiotrophoblast, which varies according to time during pregnancy. This study expands the spectrum of neoplasms known to express GATA-3. Thus, recognition of expression in trophoblastic tumors is important, because it can present a diagnostic pitfall in the assessment of suspected metastatic bladder or breast carcinomas involving the gynecologic tract. In the evaluation of diagnostically problematic tumors for which trophoblastic neoplasms are in the differential diagnosis, such as leiomyosarcoma and squamous cell carcinoma, GATA-3 can be included as part of an immunohistochemical panel particularly when other trophoblastic markers are either not available or yield ambiguous results.
The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4(+) FoxP3(+) Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild-type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4(+) FoxP3(+) Treg cells by application of diphtheria toxin. In Treg cell-depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA-3(+) T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)-4, IL-5, and IL-13. In contrast, only a mild increase in the Th1-associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.
GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.
The prevalence of black gram (Vigna mungo) induced allergic reactions are reported from several parts of the world including Asia and Australia. But, a thorough exploration of the allergic reactions induced by black gram proteins is still lacking. Therefore, efforts have been made to explore black gram allergy using in vivo and in vitro approaches. In this study, Simulated Gastric Fluid (SGF) assay and IgE immunoblotting were carried out to identify clinically relevant allergens of black gram. BALB/c mice and RBL-2H3 cells were used for elucidation of allergenic reactions of black gram proteins. Further, this study was extended to screen black gram sensitive patients among nasobronchial allergic patients on the basis of clinical history, skin prick test (SPT), specific IgE levels and IgE immunoblotting. Enhanced levels of specific IgE, IgG1/IgG2a (p < 0.05), histamine (p < 0.05), clinical symptoms, pathological indications in the lungs, intestine and spleen were evident in black gram sensitized BALB/c mice. Moreover, the expression of Th2 cytokine transcripts and GATA-3/T-bet ratio was found enhanced in the treated group. In vitro studies on RBL-2H3 cells,showed increased release of β-hexosaminidase (p < 0.05), histamine (p < 0.05), cysteinyl leukotriene (p<0.05) and prostaglandin D2 (p < 0.05). Further, 8.5% of screened patients were found allergic to black gram and concomitant sensitization with other allergens has shown the possibility of further enhancement in allergenic problem. Conclusively, the present study suggested that black gram consumption may be responsible for inducing immediate type of allergic sensitization in susceptible subjects.
Bronchial asthma is characterized by chronic lung inflammation, airway hyperresponsiveness, and airway remodelling. Astragaloside IV (3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosyl-cycloastragenol, AST), the primary pure saponin isolated from the root of Astragalus membranaceus, is an effective compound with distinct pharmacological effects including anti-inflammation, immunoregulation, and antifibrosis. However, the effect of AST on asthma remains unclear. In the present study, in the murine model of asthma, the airway hyperresponsiveness was relieved after treatment with AST, accompanied by a reduction of inflammatory cells. In addition, the levels of IL-4 and IL-5 decreased, while the IFN-γ level increased, in bronchoalveolar lavage fluid. The compound also significantly inhibited the synthesis of GATA-3-encoding mRNA and protein in addition to increasing the synthesis of T-bet-encoding mRNA and protein in both lung tissues and CD4+ T cells. Our findings indicate that AST treatment inhibits ovalbumin-induced airway inflammation by modulating the key master switches GATA-3 and T-bet, which results in committing T helper cells to a Th1 phenotype.
Ventricular septal defect (VSD), one of the most common types of congenital heart disease (CHD), results from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in development of CHD. This study was to characterize the expression of miRNAs that might be involved in the development or reflect the consequences of VSD.
MiRNA microarray analysis and reverse transcription-polymerase chain reaction (RT-PCR) were employed to determine the miRNA expression profile from 3 patients with VSD and 3 VSD-free controls. 3 target gene databases were employed to predict the target genes of differentially expressed miRNAs. miRNAs that were generally consensus across the three databases were selected and then independently validated using real time PCR in plasma samples from 20 VSD patients and 15 VSD-free controls. Target genes of validated 8 miRNAs were predicted using bioinformatic methods.
36 differentially expressed miRNAs were found in the patients with VSD and the VSD-free controls. Compared with VSD-free controls, expression of 15 miRNAs were up-regulated and 21 miRNAs were downregulated in the VSD group. 15 miRNAs were selected based on database analysis results and expression levels of 8 miRNAs were validated. The results of the real time PCR were consistent with those of the microarray analysis. Gene ontology analysis indicated that the top target genes were mainly related to cardiac right ventricle morphogenesis. NOTCH1, HAND1, ZFPM2, and GATA3 were predicted as targets of hsa-let-7e-5p, hsa-miR-222-3p and hsa-miR-433.
We report for the first time the circulating miRNA profile for patients with VSD and showed that 7 miRNAs were downregulated and 1 upregulated when matched to VSD-free controls. Analysis revealed target genes involved in cardiac development were probably regulated by these miRNAs.
Extracellular calcium participates in several key physiological functions, such as control of blood coagulation, bone calcification or muscle contraction. Calcium homeostasis in humans is regulated in part by genetic factors, as illustrated by rare monogenic diseases characterized by hypo or hypercalcaemia. Both serum calcium and urinary calcium excretion are heritable continuous traits in humans. Serum calcium levels are tightly regulated by two main hormonal systems, i.e. parathyroid hormone and vitamin D, which are themselves also influenced by genetic factors. Recent technological advances in molecular biology allow for the screening of the human genome at an unprecedented level of detail and using hypothesis-free approaches, such as genome-wide association studies (GWAS). GWAS identified novel loci for calcium-related phenotypes (i.e. serum calcium and 25-OH vitamin D) that shed new light on the biology of calcium in humans. The substantial overlap (i.e. CYP24A1, CASR, GATA3; CYP2R1) between genes involved in rare monogenic diseases and genes located within loci identified in GWAS suggests a genetic and phenotypic continuum between monogenic diseases of calcium homeostasis and slight disturbances of calcium homeostasis in the general population. Future studies using whole-exome and whole-genome sequencing will further advance our understanding of the genetic architecture of calcium homeostasis in humans. These findings will likely provide new insight into the complex mechanisms involved in calcium homeostasis and hopefully lead to novel preventive and therapeutic approaches. Keyword: calcium, monogenic, genome-wide association studies, genetics.
Breast cancer expression profiling has been used for determining biomarkers. Using gene expression profiles of 2,400 patients we identified keratin 19 (KRT19) as a highly deregulated gene in breast cancer. KRT19 expression is independent of patient race but correlates with disease grade, and ER, PR or HER2 expression. Expression of TPD52, GATA3 and KRT18 was increased in KRT19 expressing patients. Furthermore, KRT19 expression was associated with ER up-regulation and Luminal B gene signatures, as well as a constitutive RAF1 signaling pathway. Finally, KRT19 expression correlated with poor overall survival. Taken together, our results suggest that KRT19 expression can be used as a prognostic marker.
T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.
Chronic Chagas disease cardiomyopathy (CCC), a late consequence of Trypanosoma cruzi infection, is an inflammatory cardiomyopathy with prognosis worse than those of noninflammatory etiology (NIC). Although the T cell-rich myocarditis is known to play a pathogenetic role, the relative contribution of each of the functional T cell subsets has never been thoroughly investigated. We therefore assessed gene expression of cytokines and transcription factors involved in differentiation and effector function of each functional T cell subset (TH1/TH2/TH17/Treg) in CCC, NIC, and heart donor myocardial samples.
Quantitative PCR showed markedly upregulated expression of IFN-γ and transcription factor T-bet, and minor increases of GATA-3; FoxP3 and CTLA-4; IL-17 and IL-18 in CCC as compared with NIC samples. Conversely, cytokines expressed by TH2 cells (IL-4, IL-5, and IL-13) or associated with Treg (TGF-β and IL-10) were not upregulated in CCC myocardium. Expression of TH1-related genes such as T-bet, IFN-γ, and IL-18 correlated with ventricular dilation, FoxP3, and CTLA-4.
Results are consistent with a strong local TH1-mediated response in most samples, possibly associated with pathological myocardial remodeling, and a proportionally smaller FoxP3(+)CTLA4(+) Treg cell population, which is unable to completely curb IFN-γ production in CCC myocardium, therefore fueling inflammation.
GATA3 is implicated in mammary epithelial development and breast cancer progression and is an evolving immunohistochemical marker in breast cancer. Often associated with estrogen receptor (ER) signaling, GATA3 expression has been reported in ER-negative breast cancers, but systematic evaluation of GATA3 expression in a large set of triple-negative breast cancers (TNBC) is lacking. Given low sensitivities of mammaglobin (MGB) and GCDFP15 in metastatic TNBC, additional markers for site of origin identification would be useful in this context. We examined immunohistochemical expression of GATA3 in a large group of treatment-naive TNBC (n = 111) and ER-positive (n = 39) and HER2-positive (n = 31) breast cancers with commonly used antibody clones, HG3-31 (GATA3-H) and L50-823 (GATA3-L), and compared GATA3, MGB, and GCDFP15. Respectively, GATA3-L and GATA3-H were positive in 66% and 44% of TNBC (P = .002), 93% and 79% of ER-/HER2+ tumors (P = .596), and 100% of ER+/HER2- and ER+/HER2+ tumors (P = 1.00 each). GATA3-L was technically and diagnostically more sensitive than GATA3-H in TNBC and was technically more sensitive in other subtypes. MGB (26%) and GCDFP15 (16%) were less sensitive for TNBC than other subtypes (P < .001). Notably, 56% and 36% of MGB-/GCDFP15- TNBC were positive with GATA3-L and GATA3-H, respectively (P = .027). Seventy percent of TNBC were positive for GATA3-L, MGB, or GCDFP15 compared with 49% using GATA3-H in the panel. GATA3 is a diagnostically useful marker for TNBC and is more sensitive than MGB and GCDFP15 combined. GATA3-L is more sensitive for TNBC than GATA3-H, and an immunopanel of GATA3-L, MGB, and GCDFP15 provides optimal diagnostic sensitivity for TNBC.
The present study was designed to evaluate the efficacy of wild yam root extract (WYRE) as a potential demethylating agent using two breast cancer cell lines, MCF-7 (estrogen receptor positive; ER(+)) and MDA-MB-231 (Estrogen receptor negative; ER(-)), and a methylated gene, GATA3, as a potential marker of breast cancer development. The cells were treated with WYRE (0-50 μg/mL) for 72 h and used for viability, mRNA, and methylation analyses. WYRE significantly reduced viability of both cell lines and enhanced mRNA content of GATA3 in a concentration-dependent manner; however, DNMT mRNAs (DNMT1, 3A, 3B) were found to increase significantly only in MDA-MB-231 cells. Global DNA methylation, analyzed as 5'-methyl-2'-deoxycytidine (5-mC) and 5-hydroxymethylcytosine (5-hmC), showed a concentration-dependent enhancement of 5-mC with no alteration in 5-hmC level in MCF-7 cells; however, in MDA-MB-231 cells, in contrast to MCF-7 cells, 5-mC remained unaltered but 5-hmC reduced significantly in all WYRE concentrations (10-50 μg/mL) used in this study. Since 5-hmC is generated from 5-mC by ten-eleven-translocation (TET) enzymes, analysis of TET mRNAs (TET1, TET2, and TET3) in MDA-MB-231 cells indicated a concentration-dependent reduction in TET1 and induction of TET3; however, TET2 remained unaltered. No alterations in any of the TET mRNAs were found in MCF-7 cells. Methylation analysis of GATA3 promoter at specific locus indicates probable demethylating activity of WYRE in MDA-MB-231 cells. We conclude that activation of GATA3 gene in ER(-) MDA-MB-231 cells may occur by altering DNA methylation pattern on the promoter region which may be different from the mechanisms operated in ER(+) MCF-7 cells.
RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor α (ERα) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERα, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERα-complex formation, ERα-mediated gene expression, and ERα-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERα-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment.
T helper (Th)-2 cells are the major players in allergic asthma; however, the mechanisms that control Th2-mediated inflammation are poorly understood. Here we find that enhanced expression of Grail, an E3 ubiquitin ligase, in Th2 cells depends on interleukin (IL)-4-signalling components, signal transducer and activator of transcription 6 (Stat6) and Gata3, that bind to and transactivate the Grail promoter. Grail deficiency in T cells leads to increased expression of Th2 effector cytokines in vitro and in vivo and Grail-deficient mice are more susceptible to allergic asthma. Mechanistically, the enhanced effector function of Grail-deficient Th2 cells is mediated by increased expression of Stat6 and IL-4 receptor α-chain. Grail interacts with Stat6 and targets it for ubiquitination and degradation. Thus, our results indicate that Grail plays a critical role in controlling Th2 development through a negative feedback loop.
Myeloid derived suppressor cells (MDSCs) expand in cancer bearing hosts and contribute to tumor immune evasion. M2 macrophages constitute a major cellular component of cancer-related inflammation. However, the correlation between circulating MDSCs and infiltrating M2 macrophages in tumor tissues from patients with esophageal cancer (ECA), and its potential relationship with the polarization of Th2 cells remain unclear. In the present study, we showed the level of MDSCs in PBMC and Arg1 in plasma were significantly elevated in ECA patients, and the increased ratio of MDSC in PBMC was closely related to the expression of CD163 in cancer tissues. In addition, the ECA patients exhibited remarkable increases in the mRNA levels of IL-4 and GATA3, as well as the protein levels of IL-13 and IL-6, but IFN-γ and IL-12 in peripheral blood were decreased. Our data indicate that the increased Th2 cytokines are associated with MDSCs and M2 macrophages polarization, and foster the infiltration of CD163+M2 macrophages in cancer tissues, which promote the formation of immunosuppressive microenvironment in ECA patients.
Neuropathic pain is one of the most devastating kinds of chronic pain. Neuroinflammation has been shown to contribute to the development of neuropathic pain. We have previously demonstrated that lumbar spinal cord-infiltrating CD4(+) T lymphocytes contribute to the maintenance of mechanical hypersensitivity in spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we further examined the phenotype of the CD4(+) T lymphocytes involved in the maintenance of neuropathic pain-like behavior via intracellular flow cytometric analysis and explored potential interactions between infiltrating CD4(+) T lymphocytes and spinal cord glial cells.
We consistently observed significantly higher numbers of T-Bet(+), IFN-γ(+), TNF-α(+), and GM-CSF(+), but not GATA3(+) or IL-4(+), lumbar spinal cord-infiltrating CD4(+) T lymphocytes in the L5Tx group compared to the sham group at day 7 post-L5Tx. This suggests that the infiltrating CD4(+) T lymphocytes expressed a pro-inflammatory type 1 phenotype (Th1). Despite the observation of CD4(+) CD40 ligand (CD154)(+) T lymphocytes in the lumbar spinal cord post-L5Tx, CD154 knockout (KO) mice did not display significant changes in L5Tx-induced mechanical hypersensitivity, indicating that T lymphocyte-microglial interaction through the CD154-CD40 pathway is not necessary for L5Tx-induced hypersensitivity. In addition, spinal cord astrocytic activation, represented by glial fibillary acidic protein (GFAP) expression, was significantly lower in CD4 KO mice compared to wild type (WT) mice at day 14 post-L5Tx, suggesting the involvement of astrocytes in the pronociceptive effects mediated by infiltrating CD4(+) T lymphocytes.
In all, these data indicate that the maintenance of L5Tx-induced neuropathic pain is mostly mediated by Th1 cells in a CD154-independent manner via a mechanism that could involve multiple Th1 cytokines and astrocytic activation.
The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.
A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota.
We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice.
pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate.
These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.
In most patients with hypoparathyroidism (HP), the etiology is not defined clinically. Eight genes (AIRE, CASR, CLDN16, GATA3, GCM2, PTH, TBCE, and TRPM6) are known to be responsible genes associated with HP; however, no previous study has screened the eight responsible genes comprehensively in HP patients.
This study was conducted to determine the genetic defect in HP patients. We also described clinical and molecular findings of two HP patients with novel GCM2 mutations.
We enrolled 20 nonconsanguineous Japanese patients with child-onset permanent HP without 22q11 deletion. Mutations and genomic rearrangements involving the eight genes were screened by targeted next-generation sequencing (NGS). We also screened genetic rearrangements by array comparative genomic hybridization (aCGH) in the mutation-negative patients. A putative deletion, which was suspected by NGS, was additionally analyzed by droplet digital PCR (ddPCR) and junction PCR. Identified novel nucleotide-level GCM2 mutants were characterized in vitro.
We identified seven patients with a single gene disorder, including a CASR mutation, GATA3 mutations, and novel GCM2 mutations (R367Tfs*15, T370M, and the deletion encompassing exon 1). This submicroscopic deletion, which had been suspected by NGS, could not be detected by aCGH and was confirmed by ddPCR and junction PCR. Functional studies of R367Tfs*- and T370M-GCM2 demonstrated a reduction of target gene transactivation in both.
Using comprehensive NGS analyses, we identified the genetic defect in 35% of HP patients in our cohort and discovered novel GCM2 mutations including submicroscopic deletion that was undetectable by aCGH.
The pseudoangiosarcomatous pattern has been described mostly in cutaneous and some visceral squamous cell carcinomas and is unique for its striking morphologic resemblance to angiosarcoma. Herein, we describe the clinicopathologic features of 7 pseudoangiosarcomatous urothelial carcinomas that occurred in the urinary bladder. The patients included 6 men and 1 woman ranging in age from 47 to 87 years (median 70 y). The pseudoangiosarcomatous morphology was observed in 7 urothelial carcinomas including 3 with squamous differentiation and comprised 35% to 85% of the invasive tumor. Histologically, the pseudoangiosarcomatous carcinomas were characterized by tumor cell discohesion and lysis that created pseudolumina formations surrounded by attached residual tumor cells. Detached degenerating tumor cells variably admixed with inflammatory cells were common in the false lumina. Partly intact urothelial carcinoma nests contained irregular or cleft-like spaces and disintegrating tumor cells with stretched intercellular bridges. The tumor was commonly associated with a dense collagenous matrix, often surrounding the lytic nests. Similar tumor cell discohesion and breakdown were observed in 3 tumors with foci of squamous cell differentiation, distinguished by the presence of dyskeratosis and keratin formation. All 7 tumors contained other nonpseudoangiosarcomatous carcinoma components such as conventional urothelial carcinoma (5), squamous differentiation (4), sarcomatoid spindle cell carcinoma (2), small cell carcinoma (1), micropapillary carcinoma (1), and glandular differentiation (1). The pseudoangiosarcomatous urothelial carcinomas were all (7/7) diffusely CK7 positive, most (6/7) were GATA3 positive, and none (0/7) expressed vascular-associated markers. There was no evidence to suggest that apoptosis (by TUNEL assay and cleaved caspase-3 immunostaining) or loss of the adhesion molecules CD138 and e-cadherin were possible causes for the tumor cell discohesion and breakdown. All 7 tumors were high stage at cystectomy and included 1 pT3a, 2 pT3b, and 4 pT4a tumors, and 3 had pelvic lymph node involvement. Follow-up data available in 6 cases revealed a poor outcome with an overall median survival of 8.5 months. In conclusion, we present an unusual morphology of bladder carcinoma that has a striking resemblance to a malignant vasoformative tumor. Our series showed that bladder pseudoangiosarcomatous carcinoma morphology is associated with a higher tumor stage at cystectomy, commonly admixed with other aggressive carcinoma variant morphologies, and portend a poorer outcome. Knowledge of this pattern is also important to avoid misdiagnosis, particularly in limited tissue samples.
Gut microbiota mediated low-grade inflammation is involved in the onset of type 2 diabetes (T2DM). In this study, we used a high fat sucrose (HFS) diet-induced pre-insulin resistance and a low dose-STZ HFS rat models to study the effect and mechanism of Lactobacillus casei Zhang in protecting against T2DM onset. Hyperglycemia was favorably suppressed by L. casei Zhang treatment. Moreover, the hyperglycemia was connected with type 1 immune response, high plasma bile acids and urine chloride ion loss. This chloride ion loss was significantly prevented by L. casei via upregulating of chloride ion-dependent genes (ClC1-7, GlyRα1, SLC26A3, SLC26A6, GABAAα1, Bestrophin-3 and CFTR). A shift in the caecal microflora, particularly the reduction of bile acid 7α-dehydroxylating bacteria, and fecal bile acid profiles also occurred. These change coincided with organ chloride influx. Thus, we postulate that the prevention of T2DM onset by L. casei Zhang may be via a microbiota-based bile acid-chloride exchange mechanism.
This study was aimed to compare the expressions of specific transcription factors of CD4(+) T cell subset ( T-bet, GATA-3, RORγt and FoxP3 mRNA) in peripheral blood of patients with aplastic anemia(AA), myelodysplastic syndrome(MDS), and acute myeloid leukemia(AML), and investigate their immune status and pathogenesis, so as to provide experimental basis for the choice of clinical treatment. The expression of T-box (T-bet), GATA-3, ROR-γt and Foxp3 mRNA in PBMNC were examined by RT-PCR in 42 cases of MDS, including 22 refractory anemia(MDS-RA) and 20 refractory anemia with excess blasts (MDS-RAEB), in 23 cases of AA, 17 cases of AML patients and 16 healthy volunteers respectively. The results indicated that, compared with normal control group, expressions of T-bet and RORγt mRNA in AA patient group were significantly higher (P < 0.01), expression levels of GATA3 Foxp3 mRNA were lower (both P < 0.01). There was no significant difference in expression of T-bet and GATA3 mRNA between MDS group and normal control group, but the expression levels of Foxp3 and RORγt mRNA were higher than those in normal controls (P < 0.05); T-bet and RORγt in MDS-RA group were higher than those in the normal controls(P < 0.01), and GATA3 expression significantly reduced (P < 0.05), however, there was no significant difference in expression of Foxp3 between MDS-RA and the controls. Expression levels of T-bet and RORγt mRNA in patients with MDS-RAEB and AML were lower than those in normal controls (P < 0.05), but the expression levels of GATA3 and Foxp3 mRNA were significantly higher than those in normal controls (P < 0.01). It is concluded that the transcription factor expressions are different in PBMNC of patients among these three diseases. Immune-mediated excessive apoptosis may play an important role in pathogenesis, bone marrow failure in patients with AA and MDS-RA, and abnormal clones of immature cells may be one of main reasons for bone marrow failure in AML and late stage of MDS.
Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.
Cranial placodes contribute to many sensory organs and ganglia of the vertebrate head. The olfactory, otic, and lateral line placodes form the sensory receptor cells and neurons of the nose, ear, and lateral line system; the lens placode develops into the lens of the eye; epibranchial, profundal, and trigeminal placodes contribute sensory neurons to cranial nerve ganglia; and the adenohypophyseal placode gives rise to the anterior pituitary, a major endocrine control organ. Despite these differences in fate, all placodes are now known to originate from a common precursor, the preplacodal ectoderm (PPE). The latter is a horseshoe-shaped domain of ectoderm surrounding the anterior neural plate and neural crest and is defined by expression of transcription factor Six1, its cofactor Eya1, and other members of the Six and Eya families. Studies in zebrafish, Xenopus, and chick reveal that the PPE is specified together with other ectodermal territories (epidermis, neural crest, and neural plate) during early embryogenesis. During gastrulation, domains of ventrally (e.g., Dlx3/Dlx5, GATA2/GATA3, AP2, Msx1, FoxI1, and Vent1/Vent2) and dorsally (e.g., Zic1, Sox3, and Geminin) restricted transcription factors are established in response to a gradient of BMP and help to define non-neural and neural competence territories, respectively. At neural plate stages, the PPE is then induced in the non-neural competence territory by signals from the adjacent neural plate and mesoderm including FGF, BMP inhibitors, and Wnt inhibitors. Subsequently, signals from more localized signaling centers induce restricted expression domains of various transcription factors within the PPE, which specify multiplacodal areas and ultimately individual placodes. For further resources related to this article, please visit the WIREs website.
The author has declared no conflicts of interest for this article.
In the respiratory mucosa, interleukin (IL)-33, has been shown to enhance T helper 2 (TH2)-type responses through the master regulatory gene GATA-3. IL-33 is upregulated in ulcerative colitis (UC), and the aim was to assess if IL-33 holds a similar key position in the shaping of the immune response in experimental colitis (piroxicam-accelerated colitis (PAC) in IL-10 (-/-) mice, dextran sodium sulfate (DSS) model) and UC.
Colonic IL-33 expression was determined in UC (8 active UC, 8 quiescent UC, and 7 controls) and experimental colitis. Mesenteric lymph node (MesLN) T cells were isolated from PAC IL-10 (-/-) mice and stimulated with IL-33.
The colonic IL-33 expression was significantly upregulated all forms of colitis (P < 0.01) and correlated with disease severity score and inflammation (P < 0.001), and with GATA-3 expression levels (P < 0.01); no correlation with the TH1-specific T-bet expression was observed. MesLN T cells stimulated with IL-33 had increased GATA-3 expression, and showed an IL-33 dose-dependent increase in secreted TH2-type cytokines, whereas this effect was abolished by blocking IL-33 signaling. The non-TH2-type cytokine IL-17 was upregulated by IL-33 but in a T cell receptor dependent manner, as opposed to TH2-type cytokines, which required only IL-33 stimulation.
The study demonstrates that intestinal IL-33 is capable of inducing GATA-3 in mucosal T cells, and suggests that IL-33 is a key mediator of pathological TH2 and non-TH2-type responses in intestinal inflammation. Blocking IL-33 signaling could be a feasible option in the treatment of UC.
The identification of DC-derived signals orchestrating activation of Th1 and Th17 immune responses has advanced our understanding on how these inflammatory responses develop. However, whether specific signals delivered by DCs also participate in the regulation of Th2 immune responses remains largely unknown. In this study, we show that administration of antigen-loaded, IL-6-deficient DCs to naïve mice induced an exacerbated Th2 response, characterized by the differentiation of GATA-3-expressing T lymphocytes secreting high levels of IL-4, IL-5, and IL-13. Coinjection of wild type and IL-6-deficient bone marrow-derived dendritic cells (BMDCs) confirmed that IL-6 exerted a dominant, negative influence on Th2-cell development. This finding was confirmed in vitro, where exogenously added IL-6 was found to limit IL-4-induced Th2-cell differentiation. iNKT cells were required for optimal Th2-cell differentiation in vivo although their activation occurred independently of IL-6 secretion by the BMDCs. Collectively, these observations identify IL-6 secretion as a major, unsuspected, mechanism whereby DCs control the magnitude of Th2 immunity.
GATA3 as a diagnostic marker of metastatic breast carcinoma in cytology specimens has not been fully established.
Metastatic breast carcinoma was assessed for GATA3, mammaglobin, and GCDFP-15 immunohistochemistry on cell blocks. GATA3 was scored by intensity (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive), and area (0-100%). Mammaglobin (MMG) and GCDFP-15 staining was scored qualitatively (positive vs. negative). Results were correlated with specimen type (fine-needle aspiration vs. body fluid), breast prognostic markers estrogen receptor (ER), progesterone receptor (PR), Her-2/Neu (Her2), and Ki67, and with each other. Statistical significance was determined by chi-squared test and ANOVA for numerical variables. Alpha was set as 0.05. A total of 40 CB specimens containing metastatic breast carcinoma were studied.
GATA3 was positive in 32 (80%) cases. All ER-positive cases (n = 25) were positive for GATA3. Conversely, all GATA3-negative cases (n = 8) were triple-negative breast cancers. On qualitative univariate analysis, GATA3 was statistically associated with ER (P = 0.0001), and PR (P = 0.0468). GATA3 intensity was statistically associated with ER (P ≤ 0.0001), PR (P = 0.0157), Her2 (P = 0.0256), and cancer category (P = 0.0127). GATA3 staining was statistically associated with ER (P ≤ 0.0001), PR (P = 0.0160), Her2 (P = 0.0451), and cancer category (P = 0.0002). MMG and GCDFP-15 were directly compared to GATA3 in 35 samples. The sensitivity was 86% for GATA3, 26% for MMG, and 14% for GCDFP-15.
GATA3 is a more sensitive diagnostic marker of metastatic breast carcinoma in CB samples than MMG and GCDFP-15.
To observe the effect of needle pricking therapy on the expression of thymic stromal lymphopoietin (TSLP) protein, T-box expressed in T cells(T-bet)mRNA and GATA-binding protein-3 (GATA 3) mRNA in the lung tissue of mice with asthma, so as to explore its mechanism underlying improvement of asthma.
Thirty female BALB/c mice were randomly divided into control, model and needle pricking groups (10 mice/group). The asthma model was established by i. p. of Ovalbumin (OVA) suspension fluid (containing Aluminium Hydroxide 400 microg and OVA 100 microg, 50 microg/L) and forced inhalation of atomized OVA. A sharp needle was held to prick "Dazhui" (GV 14),"Feishu" (BL 13), "Dingchuan" (EX-B 1),"Fengmen" (BL 12),"Shenshu" (BL 23) and "Pishu" (BL 20) acupoint regions to let a little bit of white fibrous tissue out, then to insert into the acupoints to about 1 mm in depth, once a day for seven times. The lung tissue was taken for detecting the expression of T-bet mRNA and GATA-3 mRNA by RT-PCR and for determining the immunoactivity of TSLP by immunofluorescence method. In addition, HE staining was used to examine the pathologic changes of the lung tissue.
Compared with the control group, the expression levels of GATA-3 mRNA and TSLP protein of the lung tissue in the model group were significantly increased (P < 0.01), whereas that of T-bet mRNA was remarkably decreased (P < 0.05). Following needle prick stimulation treatment, the expression levels of GATA-3 mRNA and TSLP protein were markedly down-regulated (P < 0.01) and that of T-bet mRNA was obviously upregulated (P < 0.05). After the treatment, pathological changes including hyperemia of the pulmonary alveoli, epithelial thickening, narrowing of the lumina, and infiltration of many inflammatory cells around the tracheal blood vessels were improved.
Needle pricking therapy can regulate the expression of pulmonary TSLP protein and GATA 3 and T-bet genes in mice with asthma, which may contribute to its effect in improving pulmonary pathological changes of asthma mice.
Breast cancer prognosis and treatment is highly dependent on the molecular features of the primary tumors. These tumors release specific molecules into the environment that trigger characteristic responses into the circulatory cells. In this study we investigated the expression pattern of 84 genes known to be involved in breast cancer signaling in the peripheral blood of breast cancer patients with ER-, PR- primary tumors. The patients were grouped according to Her2 expression on the primary tumors in Her2+ and Her2- cohorts. Transcriptional analysis revealed 15 genes to be differentially expressed between the two groups highlighting that Her2 signaling in primary tumors could be associated with specific blood gene expression. We found CCNA1 to be up-regulated, while ERBB2, RASSF1, CDH1, MKI67, GATA3, GLI1, SFN, PTGS2, JUN, NOTCH1, CTNNB1, KRT8, SRC, and HIC1 genes were down-regulated in the blood of triple negative breast cancer patients compared to Her2+ cohort. IPA network analysis predicts that the identified genes are interconnected and regulate each other. These genes code for cell cycle regulators, cell adhesion molecules, transcription factors or signal transducers that modulate immune signaling, several genes being also associated with cancer progression and treatment response. These results indicate an altered immune signaling in the peripheral blood of triple negative breast cancer patients. The involvement of the immune system is necessary in favorable treatment response, therefore these results could explain the low response rates observed for triple negative breast cancer patients.
This study was conducted to explore the anti-asthma effects of synthetic salidroside on cytokines and airway inflammation of asthma induced by diisocyanate (TDI) in mice. The experiment was carried out 60 female BALB/C mice which were randomly assigned to six experimental groups: control, vehicle, model, dexamethasone (2 mg/kg), and salidroside (24 and 48 mg/kg). After the experiment, histological studies were evaluated by the hematoxylin and eosin staining, the bronchoalveolar lavage fluid (BALF) and blood were collected from the animals, and the composition of the induced inflammatory cells, and the concentrations of certain cytokines (IL-4, INF-γ) were evaluated. GATA3 and T-bet mRNAs were evaluated by QPCR. Our study demonstrated that salidroside inhibited TDI-induced increases in eosinophil count; IL-4 and INF-γ were recovered. Histological studies demonstrated that salidroside substantially inhibited TDI-induced eosinophilia in lung tissue. Salidroside can improve T-bet mRNA and reduce GATA3 mRNA in lung. These findings suggest that salidroside may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma by regulating GATA3/T-bet balance.
GATA3 is a transcription factor that is involved in the embryonic development of the parathyroid glands and in adult parathyroid cell proliferation. The aim of this study is to investigate the expression of GATA3 in parathyroid tumors and to determine whether it could be used as an immunohistochemical marker for parathyroid differentiation in tumors. Immunoreactivity for GATA3 was nuclear and was demonstrated in all 10 hyperplastic parathyroid glands, 22 parathyroid adenomas, and 6 parathyroid carcinomas; whereas, all thyroid tumors, renal cell carcinomas, thymic epithelial tumors, and carcinoid tumors investigated for comparison purposes were negative for this marker. It is concluded that GATA3 is a very sensitive and relatively specific immunohistochemical marker for parathyroid differentiation that can assist in the differential diagnosis of parathyroid tumors.
The preplacodal region (PPR) is a region of specialized ectoderm at the border of neural and nonneural ectoderm (NNE). Coordinated Bmp, Fgf, and Wnt signals are known to drive PPR development; however, the underlying mechanism is unknown.
We identified key components involved in PPR differentiation. The mesoderm/marginal Wnts at the early gastrula stage trigger differentiation by allowing the adjacent NNE border cells to start adopting caudal PPR fates; otherwise, the development of caudal PPR identity is hindered due to the persistent presence of gata3 mRNA. The caudal PPR fate dominates when foxi1 expression is enhanced at the late gastrula stage, and depleting Foxi1 after 6 hours postfertilization (hpf) reduces the otic-epibranchial placodal domain. When the Gata3 level is manipulated at the fertilized egg stage or near 6 hpf, the lens is always affected. In establishing PPR polarity, both Gata3 and Foxi1 inhibit Bmp signaling, whereas Foxi1 inhibits, but Gata3 enhances, Fgf sensitivity of the PPR cells.
Our study reveals that in zebrafish, (1) the PPR at the shield stage may enter a developmental state when the PPR cells preferentially adopt a particular placodal fate and (2) a network of genetically linked factors, including Wnt/beta-catenin, Fgfr, Bmp, Gata3, and Foxi1, direct the process of PPR differentiation.
TH2 cells can further differentiate into dual-positive TH2/TH17 cells. The presence of dual-positive TH2/TH17 cells in the airways and their effect on asthma severity are unknown.
We sought to study dual-positive TH2/TH17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients, examine their response to glucocorticoids, and define their relevance for disease severity.
Bronchoscopy and lavage were performed in 52 asthmatic patients and 25 disease control subjects. TH2 and TH2/TH17 cells were analyzed by using multicolor flow cytometry and confocal immunofluorescence microscopy. Cytokines were assayed by means of ELISA.
Dual-positive TH2/TH17 cells were present at a higher frequency in BAL fluid from asthmatic patients compared with numbers seen in disease control subjects. High-level IL-4 production was typically accompanied by high-level IL-17 production and coexpression of GATA3 and retinoic acid receptor-related orphan receptor γt. Increased presence of TH2/TH17 cells was associated with increased IL-17 production in lavage fluid. TH2/TH17 cell counts and IL-17 production correlated with PC20 for methacholine, eosinophil counts, and FEV1. TH2/TH17 cells, unlike TH2 cells, were resistant to dexamethasone-induced cell death. They expressed higher levels of mitogen-activated protein-extracellular signal-regulated kinase kinase 1, a molecule that induces glucocorticoid resistance. On the basis of the dominance of BAL fluid TH2 or TH2/TH17 cells, we identified 3 subgroups of asthma: TH2(predominant), TH2/TH17(predominant), and TH2/TH17(low). The TH2/TH17(predominant) subgroup manifested the most severe form of asthma, whereas the TH2/TH17(low) subgroup had the mildest asthma.
Asthma is associated with a higher frequency of dual-positive TH2/TH17 cells in BAL fluid. The TH2/TH17(predominant) subgroup of asthmatic patients manifested glucocorticoid resistance in vitro. They also had the greatest airway obstruction and hyperreactivity compared with the TH2(predominant) and TH2/TH17(low) subgroups.
Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.
Allergic disease is dominated by Th2 immune responses. Interleukin (IL)-4 and IL-13, representative Th2 cytokines, play pivotal roles in the pathogenic activation of the Th2 immune response. In this study, we found that cyanidin-3-glucoside chloride (C3G), an anthocyanin suppressed IL-4 and IL-13 produced in activated EL-4 T cells but not Th1 cytokines including IL-2, interferon-γ, or IL-12. IL-4 and IL-13 mRNA levels and luciferase activation in cells transiently transfected with IL-4 and IL-13 promoter reporter plasmids were significantly inhibited by C3G, suggesting that suppression might be, at least in part, regulated at the transcriptional level. Data from western blot and reverse transcription-polymerase chain reaction analyses of transcription factors involved in cytokine expression suggested that expression of GATA-3, but not T-bet, was downregulated in the nucleus by C3G. Taken together, our data indicate that C3G may has potential as an anti-allergic agent suppressing Th2 activation by downregulating Th2 cytokines and the GATA3 transcription factor in allergies.
The following are the International Society of Urological Pathology (ISUP) recommendations for the use of immunohistochemistry (IHC) in prostate specimens. Either high-molecular weight cytokeratin (34βE12 or CK5/6 or others) or p63 or a combination of the 2 with AMACR either in a double or triple cocktail is recommended for the workup of small foci of atypical glands suspicious for adenocarcinoma of the prostate (PCa). ERG is optional as it is present in only 40% to 50% of prostate cancers and also positive in high-grade prostatic intraepithelial neoplasia. In the setting of obvious carcinoma or benign glands, there is no justification to do basal cell stains and AMACR. If there is a Gleason score of 3+4=7 or a higher-grade cancer on at least 1 part, the workup of other parts with an atypical focus suspicious for Gleason score 3+3=6 cancer is not recommended. In the setting of Gleason score 4+3 or 4+4=8 cancer on at least 1 part, the extent of high-grade cancer could affect clinical treatment such that workup of other atypical possible high-grade cancer foci is justified. In the setting of Gleason score 4+3 or higher-grade cancer on at least 1 part, given that intraductal carcinoma in the vast majority of cases is considered extension of high-grade cancer into prostatic ducts and acini, it is not recommended in the setting of definitive invasive high-grade cancer that workup of additional cribriform lesions be pursued. In the setting of Gleason score 3+3 on at least 1 part, the number of positive cores and/or their location could possibly affect subsequent therapy in terms of suitability for active surveillance or focal therapy, such that unless one knows with certainty that it would not affect therapy, it is justified to perform an IHC workup of additional atypical foci. In the differential diagnosis of high-grade PCa versus urothelial carcinoma (UC), the primary option is to use prostate-specific antigen (PSA) as a first test to identify PCa and GATA3 to identify UC. If GATA3 is not available, then HMWCK and p63 can be used. If the tumor is PSA positive with intense staining and HMWCK and p63 negative, the findings are diagnostic of PCa. If the tumor is equivocal/weak/negative for PSA and negative/focal for p63 and HMWCK, then one needs to perform staining for P501S, NKX3.1, and GATA3. Some experts also include PAP in this second round of staining. If the tumor is negative for PSA and diffusely strongly positive for p63 and HMWCK, the findings are diagnostic of UC. If the tumor is negative for PSA and moderately to strongly positive for GATA3, it is diagnostic of UC. Laboratories should be encouraged to use GATA3 for UC and add P501S and NKX3.1 as prostate markers in addition to PSA, p63, and HMWCK. If GATA3, p501S, and NKX3.1 are not available in equivocal cases, the case should be sent out for consultation to laboratories with these antibodies. The article also covers the use of IHC in: (1) high-grade PCa versus bladder adenocarcinoma; (2) prostatic small cell carcinoma versus high-grade PCa; (3) metastatic carcinoma of unknown primary: rule out PCa; (4) nonspecific granulomatous prostatitis/xanthoma versus high-grade PCa; (5) adult prostate sarcoma versus sarcomatoid PCa; (6) colorectal adenocarcinoma versus high-grade PCa; and (7) prognostic IHC markers.
The bladder working group of the 2013 International Society of Urologic Pathology (ISUP) Conference on Best Practices Recommendation in the Application of Immunohistochemistry (IHC) in Urologic Pathology discussed 5 settings in which IHC is commonly used in clinical practice. With regard to markers for urothelial differentiation, the committee found that there is no ideal marker or established panel to confirm urothelial differentiation. On the basis of the differential diagnostic consideration, positivity for GATA3, CK20, p63, and either high-molecular weight cytokeratin (HMWCK) or cytokeratin (CK)5/6 is of value in proving urothelial differentiation in the appropriate morphologic and clinical context. With regard to the role of IHC in the distinction of reactive atypia from urothelial carcinoma in situ, the committee recommended that morphology remains the gold standard in this differential diagnosis and that, at best, the IHC panel of CK20/p53/CD44(s) has potential utility but is variably used and has limitations. The immunostaining pattern must be interpreted with strict morphologic correlation, because overreliance on IHC may be misleading, particularly in the posttreatment setting. IHC has no role in the distinction of dysplasia versus carcinoma in situ and in the grading of papillary urothelial carcinoma. IHC may have a limited but distinct role in staging of bladder cancer. In a subset of cases, depending on the clinical and histologic context, broad-spectrum cytokeratins (to identify early or obscured invasion) and desmin (distinction of muscle from desmoplasia and to highlight muscle contours for subclassification) may be helpful. Limited experience and conflicting data preclude smoothelin or vimentin to be recommended routinely for subclassifying muscle type at this time. In the workup of a spindled cell proliferation of the bladder and in limited specimens, we recommend an immunohistochemical panel of 6 markers including ALK1, SMA, desmin, cytokeratin (AE1/AE3), and p63 with either of HMWCK or CK5/6. Currently, there are no prognostic immunohistochemical or molecular studies that are recommended to be routinely performed on biopsy or resection specimens.
The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIPS, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIPS and c-FLIPL, were detected at the protein level although c-FLIPR was expressed at the mRNA level. The knockdown of c-FLIPL led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIPS diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.
New immunohistochemical (IHC) markers of urothelial carcinoma (UCa) and prostatic adenocarcinoma (PCa) have emerged in recent years, yet comparative studies to establish markers remain lacking. We aimed to identify an effective but parsimonious approach for poorly differentiated bladder neck lesions, to establish a best practice panel approach in a setting simulating prospective use.
We tested the performance of a panel of IHC markers on whole sections of a consecutive cohort of transurethral resection specimens of poorly differentiated, challenging bladder neck resections (n=36).
In the setting of poorly differentiated bladder neck carcinomas, biomarker sensitivities for UCa were as follows: GATA3, 100%; S100P, 88%; p63, 75%; and cytokeratin (CK) 5/6, 56%; specificities of each were 100%. CK7 and CK20 showed sensitivities of 75% and 63%, though these were only 85% and 80% specific. For PCa markers, NKX3.1, p501S, prostate-specific membrane antigen, and androgen receptor (AR) each showed 100% sensitivity, outperforming ERG (35%) and prostate-specific antigen (PSA; 25%). All the prostate histogenesis markers were 100% specific, except for AR, which was positive in 13% of the UCa cases.
Novel IHC markers show improved diagnostic performance that enables positive and negative support for identifying histogenesis with the use of as few as two markers for this critical therapeutic distinction. PSA underperforms newer markers.
Hosts are likely to respond to parasitic infections by a combination of resistance (expulsion of pathogens) and tolerance (active mitigation of pathology). Of these strategies, the basis of tolerance in animal hosts is relatively poorly understood, with especially little known about how tolerance is manifested in natural populations. We monitored a natural population of field voles using longitudinal and cross-sectional sampling modes and taking measurements on body condition, infection, immune gene expression, and survival. Using analyses stratified by life history stage, we demonstrate a pattern of tolerance to macroparasites in mature compared to immature males. In comparison to immature males, mature males resisted infection less and instead increased investment in body condition in response to accumulating burdens, but at the expense of reduced reproductive effort. We identified expression of the transcription factor Gata3 (a mediator of Th2 immunity) as an immunological biomarker of this tolerance response. Time series data for individual animals suggested that macroparasite infections gave rise to increased expression of Gata3, which gave rise to improved body condition and enhanced survival as hosts aged. These findings provide a clear and unexpected insight into tolerance responses (and their life history sequelae) in a natural vertebrate population. The demonstration that such responses (potentially promoting parasite transmission) can move from resistance to tolerance through the course of an individual's lifetime emphasises the need to incorporate them into our understanding of the dynamics and risk of infection in the natural environment. Moreover, the identification of Gata3 as a marker of tolerance to macroparasites raises important new questions regarding the role of Th2 immunity and the mechanistic nature of the tolerance response itself. A more manipulative, experimental approach is likely to be valuable in elaborating this further.
To observe the change of regulatory T cells(Tregs), fork head-like transcription factor 3 (Foxp3), T box expressed in T cells (T-bet) and GATA binding protein 3 (GATA3) in rat models of chronic obstructive pulmonary disease (COPD).
Thirty rats were randomly divided into control group and model group (n=15 each). The rats of model group were developed by lipopolysaccharide (LPS) and smoking. Interleukin-4 (IL-4) and γ-interferon (IFN-γ) in serum and bronchoalveolar lavage fluid (BALF) were detected 28 days after modeling by ELISA. Peripheral Tregs were detected by flow cytometry. The expressions of Foxp3, T-bet, GATA gene and protein in lung tissue were observed by reverse transcription PCR and Western blotting, respectively.
Compared with the control group, the model group had more serious inflammation in lung tissues, expressed the higher levels of IFN-γ in serum and BALF (P<0.01), T-bet mRNA and protein in lung tissue, and the lower levels of IL-4, CD4⁺ CD25⁺ Treg, CD4⁺ CD25⁺ Foxp3⁺ Treg (P<0.05), Foxp3, GATA-3 mRNA and protein (P<0.01). Correlation analysis showed that there were correlations between T-bet, GATA-3, Foxp3 expressions and IL-4, IFN-γ levels (P<0.05).
There is a relationship between inflammation and imbalance of in the expression of T-bet, GATA-3, Foxp3 in COPD.
A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.
In a previous study, we reported that intrathecal injection of mesenchymal stem cells (MSCs) slowed disease progression in G93A mutant superoxide dismutase1 transgenic mice. In this study, we found that intrathecal MSC administration vastly increased the infiltration of peripheral immune cells into the spinal cord of Amyotrophic lateral sclerosis (ALS) mice (G93A mutant superoxide dismutase1 transgenic). Thus, we investigated the immunomodulatory effect of MSCs on peripheral blood mononuclear cells (PBMCs) in ALS patients, focusing on regulatory T lymphocytes (Treg ; CD4(+) /CD25(high) /FoxP3(+) ) and the mRNA expression of several cytokines (IFN-γ, TNF-α, IL-17, IL-4, IL-10, IL-13, and TGF-β). Peripheral blood samples were obtained from nine healthy controls (HC) and sixteen patients who were diagnosed with definite or probable ALS. Isolated PBMCs from the blood samples of all subjects were co-cultured with MSCs for 24 or 72 h. Based on a fluorescence-activated cell sorting analysis, we found that co-culture with MSCs increased the Treg /total T-lymphocyte ratio in the PBMCs from both groups according to the co-culture duration. Co-culture of PBMCs with MSCs for 24 h led to elevated mRNA levels of IFN-γ and IL-10 in the PBMCs from both groups. However, after co-culturing for 72 h, although the IFN-γ mRNA level had returned to the basal level in co-cultured HC PBMCs, the IFN-γ mRNA level in co-cultured ALS PBMCs remained elevated. Additionally, the levels of IL-4 and TGF-β were markedly elevated, along with Gata3 mRNA, a Th2 transcription factor mRNA, in both HC and ALS PBMCs co-cultured for 72 h. The elevated expression of these cytokines in the co-culture supernatant was confirmed via ELISA. Furthermore, we found that the increased mRNA level of indoleamine 2,3-dioxygenase (IDO) in the co-cultured MSCs was correlated with the increase in Treg induction. These findings of Treg induction and increased anti-inflammatory cytokine expression in co-cultured ALS PBMCs provide indirect evidence that MSCs may play a role in the immunomodulation of inflammatory responses when MSC therapy is targeted to ALS patients. We propose the following mechanism for the effect of mesenchymal stem cells (MSCs) administered intrathecally in amyotrophic lateral sclerosis (ALS): MSCs increase infiltration of peripheral immune cells into CNS and skew the infiltrated immune cells toward regulatory T lymphocytes (Treg ) and Th2 lymphocytes. Treg and Th2 secret anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β. A series of immunomodulatory mechanism provides a new strategy for ALS treatment.
Micropapillary urothelial carcinoma (MPUC) is an uncommon variant of urothelial carcinoma (UC) with an aggressive clinical course. There have been limited studies on the UC markers GATA-binding protein 3 (GATA3), p63, and p40 in MPUC. Our study investigated the immunoreactivity of these 3 markers in MPUC compared with conventional UC of different grades and stages. Immunohistochemistry was performed on 62 cases of high-grade urothelial carcinoma (HGUC), 16 low-grade urothelial carcinoma (LGUC), and 20 MPUC. p63 expression was strong and diffuse in all LGUC, significantly decreased in high stage and HGUC, and virtually absent in MPUC. p40 expression was decreased in HGUC and markedly decreased in MPUC relative to LGUC. These results suggest that loss of p63 expression in a UC appears to be associated with adverse features--including cases with micropapillary differentiation. Decreased GATA3 expression was seen frequently in high-grade and high-pathologic stage (≥pT2) tumors but was retained in MPUC cases. The findings of retained GATA3 expression in MPUC, which often shows a loss of expression of other urothelial markers such as p63, may be helpful for determining the origin of micropapillary carcinoma of unknown primary. Compared with the traditional markers p63 and p40, GATA3 is the most sensitive marker of conventional UC and MPUC.
Despite the importance of 5-HT1A as a major target for the action of several anxiolytics/antidepressant drugs, little is known about its regulation in central serotonin (5-hydroxytryptamine, 5-HT) neurons.
We report that expression of 5-HT1A and the transcription factor Pet1 was impaired in the rostral raphe nuclei of mice lacking tryptophan hydroxylase 2 (Tph2) after birth. The downregulation of Pet1 was recapitulated in 5-Ht1a-/- mice. Using an explant culture system, we show that reduction of Pet1 and 5-HT1A was rescued in Tph2-/- brainstem by exogenous 5-HT. In contrast, 5-HT failed to rescue reduced expression of Pet1 in 5-Ht1a-/- brainstem explant culture.
These results suggest a causal relationship between 5-HT1A and Pet1, and reveal a potential mechanism by which 5-HT1A-Pet1 autoregulatory loop is maintained by 5-HT in a spatiotemporal-specific manner during postnatal development. Our results are relevant to understanding the pathophysiology of certain psychiatric and developmental disorders.
TiO2 nanoparticles (NPs) are used in the food industry but have potential toxic effects in humans and animals. The resulting immune response is driven by the production of Th2 cytokines IL-4 and IL-5, which contribute to the development of hepatic inflammation. However, TiO2 NPs have been demonstrated to impair liver function and cause liver inflammation in animal models, which may be associated with activation of Th2 factor-mediated pathways. Mice were administered a gavage instillation of 2.5, 5, or 10 mg/kg body weight TiO2 NPs for six consecutive months. We investigated whether TiO2 NPs activate the Th2 factor-mediated signaling pathway under TiO2 NP-induced hepatic toxicity. The results showed that mice exhibited an accumulation of titanium in the liver, which in turn led to reductions in body weight, increases in liver indices, liver dysfunction, infiltration of inflammatory cells, and hepatocyte apoptosis or necrosis. Furthermore, hepatic inflammation was accompanied by increased (0.67 ± 0.09- to 2.14 ± 0.19-fold) IL-4 expression and up-regulation of its target genes including IL-5 (0.1 ± 0.06- to 0.69 ± 0.12-fold), IL-12 (0.08 ± 0.03- to 0.83 ± 0.21-fold), IFN-γ (0.17 ± 0.09- to 0.87 ± 0.15-fold), GATA3 (0.05 ± 0.02- to 1.29 ± 0.18-fold), GATA4 (0.04 ± 0.01- to 0.87 ± 0.13-fold), T-bet (0.3 ± 0.06- to 0.93 ± 0.15-fold), RORγt (0.32 ± 0.11- to 1.67 ± 0.17-fold), STAt3 (0.16 ± 0.06- to 2.14 ± 0.23-fold), STAT6 (0.2 ± 0.05- to 0.63 ± 0.12-fold), eotaxin (0.53 ± 0.13- to 1.49 ± 0.21-fold), MCP-1 (0.5 ± 0.11- to 0.74 ± 0.18-fold), and MIP-2 (0.27 ± 0.07- to 0.71 ± 0.18-fold) and significant down-regulation of its target gene STAT1 (-0.15 ± 0.05 to -0.81 ± 0.11-fold). Taken together, the alteration of Th2 factor expression may be involved in the control of hepatic inflammation induced by chronic TiO2 NP toxicity.
The gastric ligaments are superficial cord-like structures, located on the lesser curvature of the stomach, that extend from the pylorus to the esophagus. These ligaments have been documented in a wide variety of mammalian species, including humans, but their composition and ontogeny is unexplored. Here, we demonstrate that, during ontogeny, the gastric ligaments are first visible as extensions from a C-shaped domain of Gata3-expressing cells that surround the future pylorus; this domain will later give rise to the pyloric outer longitudinal muscle (OLM). The open ends of the C are located ventrally, and, beginning at embryonic day (E) 13.5, the ligaments grow anteriorly from these points. Whereas most other ligaments of the stomach are neurovascular in nature, the gastric ligaments are composed of smooth muscle cells that mature between E14.5 and E16.5. The gastric ligaments coexpress the transcription factors Gata3, Nkx2-5, and Sox9, and germline loss of Gata3 or conditional deletion of Nkx2-5 abrogates Sox9 expression and impairs gastric ligament smooth muscle development; similar phenotypes were previously seen in the OLM. In accord with this molecular contiguity between the OLM and gastric ligaments, three-dimensional image reconstruction highlights physical contiguity between these smooth muscle structures, suggesting that they may work together as a unit to control flexure of the pyloric region, a function similar to the ligament of Treitz at the duodenojejunal junction. These findings may have implications for our understanding of normal pyloric sphincter function, as well as the common human congenital pathology idiopathic hypertrophic pyloric stenosis.
Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.
Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.
To evaluate GATA-3 immunohistochemical expression semiquantitatively in breast, gynecologic, gastric, pancreatic-biliary tract, urothelial, and vulvar/cervical squamous cell carcinomas.
GATA-3 expression was evaluated by immunohistochemistry in 198 invasive breast carcinomas on tissue microarrays. Tissue microarrays of other tissues included 144 gynecologic tumors, 28 bladder carcinomas, 63 cholangiocarcinomas, 20 pancreatic carcinomas, and 62 gastric carcinomas. Full tissue sections of 10 invasive squamous cell carcinomas were also stained. GATA-3 expression was semiquantitatively scored using an H-score method. H-score greater than 10 was considered a positive result.
Of 186 breast carcinomas, 95% were positive (mean H-score of 217). GATA-3 expression was uncommon in 139 nonsquamous gynecologic tumors, with often weak reactivity (mean H-score <50) seen in 18% of endocervical, 7% of endometrial, and 10% of ovarian tumors. Six (60%) of 10 squamous cell carcinomas expressed GATA-3 (mean H-score of 102). Of 22 urothelial carcinomas, 95% expressed GATA-3 (mean H-score of 170). A few cholangiocarcinomas (3%), pancreatic adenocarcinomas (10%), and gastric carcinomas (2%) weakly expressed GATA-3 (mean H-score <50).
Strong GATA-3 expression is a reliable marker of primary breast carcinoma in the appropriate clinical context. GATA-3 reactivity in around 70% of triple-negative breast carcinomas is also clinically useful. Significant reactivity in gynecologic squamous cell carcinomas suggests that GATA-3 alone cannot reliably distinguish these tumors from urothelial carcinoma.
GATA3 is a sensitive marker for urothelial carcinoma. We here evaluate, for the first time, GATA3 expression in small cell carcinoma of bladder and prostate and assess its utility in the differential diagnosis with small cell carcinoma of lung primary. Archival tissues from 60 small cell carcinomas (12 bladder, 15 lung, and 33 prostate primary cases) were used to build 2 tissue microarrays. We also assessed whole slide sections from 10 additional primary small cell carcinomas of bladder. GATA3 nuclear expression was evaluated using standard immunohistochemistry. Intensity (weak, moderate, and strong) and extent of expression were assessed in each tissue microarray spot. Extent positivity was categorized as focal (1%-25%), multifocal (>25%), and diffuse (>75%). Nuclear GATA3 expression was encountered in 7 bladder (7/22, 32%) and 2 lung (2/15, 13%) small cell carcinomas. All 33 primary prostate small cell carcinomas were negative. Among bladder tumors, strong and diffuse (>75%) GATA3 labeling was seen in 3 cases (3/22, 14%); focal positivity was observed in the 4 remaining cases (4/22, 18%). Both positive lung cases had only focal positivity. Our study is the first to reveal GATA3 expression in the small subset of lung small cell carcinoma that should be taken into consideration in assigning site of origin in advanced small cell carcinoma cases. Our novel finding of GATA3 positivity in one-third of bladder small cell carcinoma is of potential value in differentiating small cell carcinomas of prostate origin from those of bladder origin.
Group 2 innate lymphoid cells (ILC2s) were shown to be involved in the initiation and coordination of Th2-type immune responses in allergic disease animal models. Recently, ILC2s enrichment was noted in chronic rhinosinusitis (CRS) patients; however, the role of ILC2s in coordinating the Th2 response in CRS remains to be elucidated. Here, we characterize the ILC2 compartment in CRS by investigating the correlations between ILC2s, Th2 cells and Th2 cytokines expression in CRS patients.
We used flow cytometric analysis of sinonasal mucosal tissues of 29 CRS patients and 5 controls to quantify ILC2s and Th2 cells. Messenger RNA expression levels of IL-5, IL-13, IL-25, IL-33, TSLP and GATA3 were determined using qRT-PCR.
ILC2s were significantly enriched in nasal polyps (CRSwNP) patients. Multivariate linear regression showed a significant positive association of ILC2 numbers with CRSwNP and allergic CRS and a negative association with the number of previous endoscopic sinus surgeries. Group 2 innate lymphoid cell numbers significantly correlated with Th2 cell frequencies. Messenger RNA expression levels of IL-5 and IL-13 were increased in CRSwNP compared with controls, while mRNA levels of IL-25 and GATA3 were significantly reduced.
Our results characterize the complex interactions between ILC2s and other Th2 response elements in the context of CRS and suggest that ILC2 enrichment occurs in CRSwNP and in allergic CRS patients.
Nephrogenic adenoma (NA) is a benign lesion of the urinary tract associated with injury to the urothelium. The varied morphologic patterns of NA make it a potential diagnostic pitfall, because it can mimic closely prostatic adenocarcinoma and urothelial carcinoma. In current practice, an antibody cocktail comprising p63, CK903, and AMACR (PIN-4 cocktail) is frequently utilized to evaluate foci suspicious for prostatic adenocarcinoma. Although the staining characteristics of the individual components of the PIN-4 cocktail have been reported for NA, no study has described the expression patterns for NA when the stains are applied as a cocktail. GATA-3 is an emerging marker of urothelial carcinoma; however, the GATA-3 staining characteristics of NA have yet to be described. Sixty-three NA specimens (M:F=36:27, average age=51.4 y) from various locations in the urinary tract including urinary bladder (n=40), ureter (n=3), and urethra (n=20) were collected from the archives of 2 institutions. Immunohistochemical analysis with the PIN-4 cocktail and GATA-3 antibodies was performed, and the distribution and intensity of staining was recorded for each antibody in each case. PIN-4 cocktail staining revealed AMACR expression in 56% of cases, CK903 expression in 97%, and rare p63 positivity (in only 2 cases). Only 2 NA cases displayed an overall PIN-4 staining pattern compatible with prostate cancer. GATA-3 expression was noted in 40% of NAs. No correlation between AMACR, CK903, or GATA-3 positivity and histologic pattern or anatomic location was identified. Although heterogenous staining patterns were seen within individual cases, use of the PIN-4 cocktail effectively discriminates NA from prostate cancer because of the high frequency of coexpression of AMACR and CK903 within NA. In addition, GATA-3 is not a useful marker in differentiating between NA and urothelial carcinoma.
Rhabdoid sarcomas are highly malignant tumors that usually occur in young children. A key to the genesis of this tumor is the mutational loss of the BAF47 gene as well as the widespread epigenetic suppression of other key anticancer genes. The BRM gene is one such epigenetically silenced gene in Rhabdoid tumors. This gene codes for an ATPase catalytic subunit that shifts histones and opens the chromatin. We show that BRM is an epigenetically silenced gene in 10/11 Rhabdoid cell lines and in 70% of Rhabdoid tumors. Moreover, BRM can be induced by BAF47 re-expression and by Flavopiridol. By selective shRNAi knockdown of BRM, we show that BRM re-expression is necessary for growth inhibition by BAF47 re-expression or Flavopiridol application. Similar to lung cancer cell lines, we found that HDAC3, HDAC9, MEF2D and GATA3 controlled BRM silencing and that HDAC9 was overexpressed in Rhabdoid cancer cell lines. In primary BRM-deficient Rhabdoid tumors, HDAC9 was also found to be highly overexpressed. Two insertional BRM promoter polymorphisms contribute to BRM silencing, but only the -1321 polymorphism correlated with BRM silencing in Rhabdoid cell lines. To determine how these polymorphisms were tied to BRM silencing, we conducted ChIP assays and found that both HDAC9 and MEF2D bound to the BRM promoter at or near these polymorphic sites. Using BRM promoter swap experiments, we indirectly showed that both HDAC9 and MEF2D bound to these polymorphic sites. Together, these data show that the mechanism of BRM silencing contributes to the pathogenesis of Rhabdoid tumors and appears to be conserved among tumor types.
Chronic immune thrombocytopenia (cITP) is an autoimmune disease with disturbed cytokine profile. Although plasma levels of IL-27 are shown to be associated with cITP, its association with T cell subsets has not been studied. The objective of this study was to study the association between IL-27 and different T cell subsets in patients with cITP. Heparinized blood was collected from 31 patients with cITP and 36 healthy controls (platelet count <100 × 10(9)/l and 103-280 × 10(9)/l, respectively). The percentage of Th1, Th2 and Th17 cells in peripheral blood mononuclear cells (PBMCs) were enumerated by flow cytometry, and the mRNA levels of IL-27, T-bet, GATA-3 and retinoid-related orphan receptor gamma (RORγt) by real-time reverse transcriptase polymerase chain (RT-PCR). Plasma cytokine levels of IL-27, interferon-gamma (IFN-γ), IL-4 and IL-17A were estimated by flow cytometrix. The effect of exogenous recombinant IL-27(rhIL-27) on the differentiation of T cells into Th1, Th2 and Th17 cells was investigated by cell culture. The percentage of Th1 and Th17 cells and the plasma concentration and mRNA levels of IL-27 were significantly higher in cITP patients compared with healthy controls. Plasma levels of IL-27 correlated positively with percentage of Th1 cells in patients with cITP. Exogenous (rhIL-27) could significantly up-regulate the percentage of Th1 cells and down-regulate Th2 cells in vitro. Th17 cells were reduced in the presence of (rhIL-27) in controls but had no effect in patients with cITP. The up-regulation of IL-27 might cause Th1 differentiation and might be involved in the pathophysiology of cITP.
Serine protease inhibitor Kazal type-5 (SPINK5) plays a crucial role in deciding the timing of desquamation of the skin. Its gene expression is limited at the very surface of the stratum granulosum (SG), whereas expression of kallikreins (KLKs) encoding proteases is usually found throughout the stratum spinosum and SG.
To explore the difference in expression regulation of these proteases/inhibitors, the function of SPINK5 promoter was examined using luciferase assay.
Luciferase assay targeting the SPINK5 promoters (nucleotide -676/-532 and -318/-146 from the major transcription start site) showed high intensity in NHEK human keratinocyte. These two sites had neither common cis-elements nor GATA3 element but electrophoretic mobility shift assay showed similar retardation bands. Moreover, DNA footprinting did not display specific protected bands. Thus, we could not identify cis-element(s) that controlled these elements. Differentiation induced by high Ca(2+) medium failed to alter their luciferase activities. Transfection of GATA3 expressing vector significantly but slightly increased them and that of vector expressing its dominant negative form decreased.
Although GATA3 is reportedly important for inhibition of proliferation and induction of differentiation of keratinocytes, its effect on SPINK5 expression was indirect and GATA3 alone was insufficient for final differentiation of keratinocytes where full SPINK5 expression was observed.
Vestigial-like 1 (VGLL1) is a poorly characterized gene encoding a transcriptional co-activator structurally homologous to TAZ and YAP that modulates the Hippo pathway in Drosophila. In this study, we examined the expression of VGLL1 and its intronic miRNA, miR-934, in breast cancer. VGLL1 and miR-934 expression miRNA profiling was carried out on frozen samples of grade 3 invasive ductal carcinomas. VGLL1 protein was also examined in 433 sporadic and BRCA1-associated breast carcinomas on tissue microarrays. RNA-seq data from The Cancer Genome Atlas (TCGA) was used to confirm differences in VGLL1 and miR-934 expression in different breast cancer subtypes, and to correlate their expression with that of other genes and miRNAs. Of 28 miRNAs differentially expressed in estrogen receptor (ER)-positive and ER-negative grade 3 breast carcinomas, miR-934 was most strongly upregulated in ER-negative carcinomas, and its expression was correlated with that of VGLL1. Nuclear VGLL1 expression was observed in 13% of sporadic breast carcinomas, and while VGLL1 was only occasionally found in luminal A (0.70%) and B (5.60%) carcinomas, it was often expressed in HER2-positive (17%), triple-negative (TN) breast carcinomas (>40%) and BRCA1-associated TN carcinomas (>50%). These findings were confirmed in the TCGA dataset, which revealed positive associations with luminal progenitor genes (GABRP, SLC6A14, FOXC1, PROM1, and BBOX1) and strong negative correlations with ER-associated genes (ESR1, C6ORF211, GATA3, and FOXA1). Moreover, VGLL1 expression was associated with reduced overall survival. In conclusion, VGLL1 and miR-934 are mainly expressed in sporadic and BRCA1-associated TN basal-like breast carcinomas, and their coordinated expression, at least partially mediated by the direct modulation of ESR1, might be involved in the maintenance of a luminal progenitor phenotype.
Type 1 myeloid dendritic cells (mDCs) contribute to inception of allergic asthma (AA) and are regulated by epithelial-derived cytokines.
To evaluate whether mDCs from AA patients are primed for thymic stromal lymphopoietin (TSLP)-driven responses.
mDCs from 18 AA patients and 15 controls were purified using immunomagnetic sorting. Cells were pulsed with TSLP or Dermatophagoides pteronyssinus (Der p) allergen, before FACS phenotyping and co-culture with allogeneic CD4+ T cells. Bronchial biopsies from 15 AA patients and four controls were immunostained for CD1c and TSLP receptor (TSLPR).
Allergic asthma patients had a higher proportion of TSLPR+ mDCs, in blood and bronchial mucosa. When compared to mDCs from controls, both TSLP- and Der p-pulsed blood mDCs from AA patients induced increased polarization of CD4+ T cells into Th2 cells (IL-5, IL-13, and GATA3+), while only TSLP-mDCs promoted Th9 cells (IL-9 and PU.1+ /IRF4+). In addition, OX40L was induced upon TSLP stimulation and was required for the induction of Th2, but not Th9, cells. In contrast, development of Th9 cells in this model depended on TGF-β1.
Our data indicate overlapping but partially distinct effects of TSLP and Der p allergen pathways, showing that DCs are primed in human asthma for TSLP-driven induction of both Th2 and Th9 cells. This novel TSLP/mDC/Th9 axis operates through a distinct, OX40L-independent pathway. These data further highlight the TSLP pathway as a relevant target in human asthma.
The earliest recognizable stages of breast neoplasia are lesions that represent a heterogeneous collection of epithelial proliferations currently classified based on morphology. Their role in the development of breast cancer is not well understood but insight into the critical events at this early stage will improve efforts in breast cancer detection and prevention. These microscopic lesions are technically difficult to study so very little is known about their molecular alterations.
To characterize the transcriptional changes of early breast neoplasia, we sequenced 3'- end enriched RNAseq libraries from formalin-fixed paraffin-embedded tissue of early neoplasia samples and matched normal breast and carcinoma samples from 25 patients. We find that gene expression patterns within early neoplasias are distinct from both normal and breast cancer patterns and identify a pattern of pro-oncogenic changes, including elevated transcription of ERBB2, FOXA1, and GATA3 at this early stage. We validate these findings on a second independent gene expression profile data set generated by whole transcriptome sequencing. Measurements of protein expression by immunohistochemistry on an independent set of early neoplasias confirms that ER pathway regulators FOXA1 and GATA3, as well as ER itself, are consistently upregulated at this early stage. The early neoplasia samples also demonstrate coordinated changes in long non-coding RNA expression and microenvironment stromal gene expression patterns.
This study is the first examination of global gene expression in early breast neoplasia, and the genes identified here represent candidate participants in the earliest molecular events in the development of breast cancer.
We present the clinicopathological features of 56 cases of the nested variant of urothelial bladder carcinoma. This is an uncommon variant of bladder cancer, recognized by the current WHO classification of urologic tumors. The nested component represented 100 % of the tumor in 24 cases. The architectural pattern of the tumor varied from solid expansile to infiltrative nests characterized by deceptively bland histologic features resembling von Brunn nests. Typical features of high-grade conventional urothelial carcinoma were present in 32 cases. Most neoplastic cells had nuclei of low to intermediate nuclear grade with occasional nuclear enlargement, most frequently seen in deep areas of tumor. The nested component expressed cytokeratins 7, 20, CAM5.2, and high molecular weight (34ßE12), p63, Ki67, p53, p27, and GATA3. Tumor extension was T1 (n = 9), minimally T2 (n = 10), T2a (n = 1), T2b (n = 4), T3a (n = 8), T3b (n = 13), and T4a (n = 11). On follow-up, 36 of patients died of or were alive with disease from 2 to 80 months (mean 21 months). Four patients died of other causes. Eleven other patients remained disease free. Univariate survival analysis showed no differences for nested carcinoma compared with conventional urothelial carcinoma. As in conventional urothelial carcinoma, in nested carcinoma of the bladder pT category defined different survival groups. In summary, nested variant of urothelial bladder carcinoma is typically associated with advanced stage. In samples of limited volume, it may be misdiagnosed as proliferation of von Brunn nests or other nested-like bladder lesions, delaying definitive therapy.
Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.
Hypoparathyroidism, sensorineural deafness and renal dysplasia syndrome (HDRS) is comprised of a triad of conditions. It is an autosomal dominant condition caused by mutations in the GATA3 gene, located at 10p15, a critical region in the development of the embryonic parathyroid glands, inner ear, and kidneys. Here we describe the case of a patient with all three components of HDR syndrome diagnosed in the neonatal period who presented with cerebral infarction, hypocalcemia, and renal anomalies. Upon chromosomal microarray he was found to have an interstitial deletion at 10p, which produced a partial deletion in the GATA3 gene.
Research in mouse and human clearly identified subsets of T helper (Th) cells based on nuclear expression of specific lineage transcription factors. In swine, however, transcription factor based detection of functional subpopulations of porcine Th cells by flow cytometry is so far limited to regulatory T cells via Foxp3. T-bet and GATA-3 are the transcription factors that regulate commitment to Th1 or Th2 cells, respectively. In this study we prove GATA-3 and T-bet expression in porcine CD4(+) cells polarized in vitro. Importantly, GATA-3 and T-bet expressing cells were detectable in pigs infected with pathogens associated with Th2 and Th1 immune responses. Increased frequencies of GATA-3 positive CD4(+) cells are found in vivo in pigs experimentally infected with the nematode Trichuris suis, whereas porcine reproductive and respiratory syndrome virus (PRRSV) infection elicited T-bet positive CD4(+) T cells. Analysing the immune status of pre-weaning piglets with intrauterine growth restriction (IUGR) we found an increased expression of Foxp3, T-bet and GATA-3 in CD4(+) and CD4(+)CD8(+) double-positive T cells in systemic and intestinal compartments of IUGR piglets. Hence, we established the detection of porcine Th1 and Th2 cells via T-bet and GATA-3 and show that the porcine lineage transcription factors are differentially regulated very early in life depending on the developmental status.
Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24-48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT(2) Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-γ/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise.
Fumaric acid esters (FAEs) are widely used in Europe for the treatment of psoriasis because of their clinical efficacy and favourable safety profile. However, the mechanisms of action by which FAEs improve psoriasis remain largely unknown.
To identify pathways and mechanisms affected by FAE treatment and to compare these with pathways affected by treatment with the antitumour necrosis factor (anti-TNF)-α biologic etanercept.
In a prospective cohort study, 50 patients with plaque psoriasis were treated with FAEs for 20 weeks. Nine patients were randomly selected for gene expression profiling of plaque biopsies from week 0 and week 12. The groups consisted of FAE responders [> Psoriasis Area and Severity Index (PASI)-75 improvement] and nonresponders (< PASI-50 improvement). Changes in gene expression profiles were analysed using Ingenuity Pathway Analysis (IPA) and the outcome was compared with gene expression affected by etanercept.
Response to FAE treatment was associated with a ≥ 2-fold change (P < 0.05) in the expression of 458 genes. In FAE responders the role of interleukin-17A in the psoriasis pathway was most significantly activated. Glutathione and Nrf2 pathway molecules were specifically induced by FAE treatment and not by etanercept treatment, representing an FAE-specific effect in psoriatic skin. In addition, FAE treatment specifically induced the transcription factors PTTG1, NR3C1, GATA3 and NFκBIZ in responding patients.
FAE treatment induces glutathione and Nrf2 pathway genes in lesional skin of patients with psoriasis. In responders, FAEs specifically regulate the transcription factors PTTG1, NR3C1, GATA3 and NFκBIZ, which are important in normal cutaneous development, and the T-helper (Th)2 and Th17 pathways, respectively.
MicroRNAs (miRNAs) play important roles in leukocyte differentiation, although those utilised for specific programs and key functions remain incompletely characterised. As a global approach to gain insights into the potential regulatory role of miRNA in mast cell differentiation we characterised expression in BM cultures from the initiation of differentiation. In cultures enriched in differentiating mast cells we characterised miRNA expression and identified miRNA targeting the mRNA of putative factors involved in differentiation pathways and cellular identity. Detailed pathway analysis identified a unique miRNA network that is intimately linked to the mast cell differentiation program.
We identified 86 unique miRNAs with expression patterns that were up- or down- regulated at 5-fold or more during bone marrow derived mast cells (BMMC) development. By employing TargetScan and MeSH databases, we identified 524 transcripts involved in 30 canonical pathways as potentially regulated by these specific 86 miRNAs. Furthermore, by applying miRanda and IPA analyses, we predict that 7 specific miRNAs of this group are directly associated with the expression of c-Kit and FcεRIα and likewise, that 18 miRNAs promote expression of Mitf, GATA1 and c/EBPα three core transcription factors that direct mast cell differentiation. Furthermore, we have identified 11 miRNAs that may regulate the expression of STATs-3, -5a/b, GATA2 and GATA3 during differentiation, along with 13 miRNAs that target transcripts encoding Ndst2, mMCP4 and mMCP6 and thus may regulate biosynthesis of mast cell secretory mediators.
This investigation characterises changes in miRNA expression in whole BM cultures during the differentiation of mast cells and predicts functional links between miRNAs and their target mRNAs for the regulation of development. This information provides an important resource for further investigations of the contributions of miRNAs to mast cell differentiation and function.
Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. Irrespective of much research in RA disease, no drugs with capable safety profiles are yet available. Poly(ADP-ribose) polymerase-1 (PARP-1) synthesizes and transfers ADP ribose polymers to target proteins, and regulates DNA repair and genomic integrity maintenance. PARP-1 also plays a crucial role in the progression of the inflammatory response, and its inhibition confers protection in several models of inflammatory disorders. We investigated the possible anti-arthritic effects of the PARP-1 inhibitor 5-aminoisoquinolinone (5-AIQ) in a mouse model of adjuvant induced arthritis (AIA). In this study, we examined the effects of 5-AIQ on the key mediators of arthritic inflammation, namely, edema and arthritic score, T cell subsets, regulatory T (Treg) cells, IL-17A, GITR expressing cells, NF-kB p65, IkB-α and pro and anti-inflammatory mediators mRNA expression levels. PARP-1 inhibition 5-AIQ treatment significantly attenuated the severity of AIA, reduced the arthritis scores, a substantial reduction in the levels of T cell subsets, IL-17A, NF-kB p65, GITR expressing cells, and as well as the pro-inflammatory mediators. However, 5-AIQ significantly up-regulated the number of Tregs cells, IkB-α levels and mRNA expression of anti-inflammatory mediators. Our results suggest that treatment with 5-AIQ attenuated AIA in mice might offer a promising alternative/adjunct treatment for RA.
Carbon monoxide (CO) is endogenously produced by haeme oxygenase-1 and has profound effects on intracellular signalling processes, generating anti-inflammatory, antiproliferative and antiapoptotic effects. A boron-containing compound CORM-A1 is capable of releasing CO in such a way to mimic physiological functions of haeme oxygenase-1. Considering the importance of Th1/Th17 versus Th2 balance in the final outcome of immune and inflammatory responses in this study we focused on immune-modulatory effects of CORM-A1 on murine lymph node-derived T cells in vitro and its influence on T-cell proliferation, activation and differentiation. Anti-CD3/CD28 antibody-triggered lymph node cells proliferation remained unaffected after 24-hour CORM-A1 treatment, as well as the expression of the early activation marker CD25. However, CORM-A1 successfully reduced the secretion of the two representative pro-inflammatory cytokines, IFN-γ and IL-17, while the secretion of anti-inflammatory cytokine IL-4 remained unchanged. Furthermore, CORM-A1 efficiently reduced the percentage of CD4(+) IFN-γ(+) and CD4(+) IL-17(+) cells, whereas CD4(+) IL-4(+) cell population increased after treatment. Also, CORM-A1 significantly reduced expression of transcription factor RORγT, necessary for Th17 development, but the expression of Th1-related and Th2-related transcription factors (T-bet and GATA-3, respectively) remained unchanged. In conclusion, our findings indicate that CO has anti-inflammatory role through the regulation of balance between pro-inflammatory Th1/Th17 and anti-inflammatory Th2 cells. Observed immunomodulatory effects of CORM-A1 could be useful for developing novel therapeutic approaches in managing Th1/Th17-mediated immune disorders.
In T-cell acute lymphoblastic leukemia (T-ALL), several members of the NK-like (NKL) homeobox genes are aberrantly expressed. Here, we have analyzed the activity of NKL homeobox gene MSX1 using pediatric T-ALL in silico data, detecting overexpression in 11% of patients. Quantification of MSX1 transcripts in a panel of 24 T-ALL cell lines demonstrated overexpression in two examples. Comparative expression profiling indicated inhibition of the bone morphogenetic protein (BMP) signaling pathway, which was shown to inhibit MSX1 transcription. In the LOUCY cell line we identified conspicuous expression of CHRDL1 encoding a BMP inhibitor which mediated activation of MSX1. Promoter analyses demonstrated activation of CHRDL1 by oncogenic PITX1. Furthermore, knockdown and overexpression studies of hematopoietic transcription factors demonstrated that GATA2 and FOXC1 mediate activation and GATA3, LEF1, TAL1 and TOX repression of MSX1 transcription. Collectively, our findings suggest that MSX1 is physiologically restricted to lymphoid progenitors. The identification of deregulated BMP signaling may provide novel therapeutic options for the treatment of T-ALL.
Estrogen has potent immunomodulatory effects on proinflammatory responses, which can be mediated by serine proteases. We now demonstrate that estrogen increased the extracellular expression and IL-12-induced activity of a critical member of serine protease family Granzyme A, which has been shown to possess a novel inflammatory persona. The inhibition of serine protease activity with inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride significantly diminished enhanced production of proinflammatory interferon-γ, IL-1β, IL-1α, and Granzyme A activity even in the presence of a Th1-inducing cytokine, IL-12 from splenocytes from in vivo estrogen-treated mice. Inhibition of serine protease activity selectively promoted secretion of Th2-specific IL-4, nuclear phosphorylated STAT6A, signal transducer and activator of transcription (STAT)6A translocation, and STAT6A DNA binding in IL-12-stimulated splenocytes from estrogen-treated mice. Inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride reversed the down-regulation of Th2 transcription factors, GATA3 and c-Maf in splenocytes from estrogen-exposed mice. Although serine protease inactivation enhanced the expression of Th2-polarizing factors, it did not reverse estrogen-modulated decrease of phosphorylated STAT5, a key factor in Th2 development. Collectively, data suggest that serine protease inactivity augments the skew toward a Th2-like profile while down-regulating IL-12-induced proinflammatory Th1 biomolecules upon in vivo estrogen exposure, which implies serine proteases as potential regulators of inflammation. Thus, these studies may provide a potential mechanism underlying the immunomodulatory effect of estrogen and insight into new therapeutic strategies for proinflammatory and female-predominant autoimmune diseases.
The integral membrane protein 2a (Itm2a) is one of the BRICHOS domain-containing proteins and is structurally related to Itm2b and Itm2c. It is expressed preferentially in the T lineage among hematopoietic cells and is induced by MHC-mediated positive selection. However, its transcriptional regulation and function are poorly understood. Here we showed Itm2a to be a target gene of GATA-3, a T cell-specific transcription factor. Deficiency of Itm2a had little impact on the development and function of polyclonal T cells but resulted in a partial defect in the development of thymocytes bearing a MHC class I-restricted TCR, OT-I. In addition, Itm2a-deficient mice displayed an attenuated T helper cell-dependent immune response in vivo. We further demonstrated that Itm2b but not Itm2c was also expressed in T cells, and was induced upon activation, albeit following a kinetic different from that of Itm2a. Thus, functional redundancy between Itm2a and Itm2b may explain the minimal phenotype of Itm2a deficiency.
The current available data on GATA-binding protein 3 (GATA3) expression in sarcomatoid urothelial carcinoma are limited, especially in the non-tissue microarray-based setting. In this study, we analyzed the expression of GATA3 in sarcomatoid urothelial carcinoma of the bladder in cystectomy/cystoprostatectomy specimens. A search was made through our surgical pathology and consultation files for cystectomy/cystoprostatectomy specimens with a diagnosis of sarcomatoid urothelial carcinoma. Only cases with available tissue blocks were selected. Immunohistochemical staining for GATA3 was performed, and staining in adjacent/overlying conventional urothelial carcinoma and/or benign urothelium was also documented. Twenty-two cases were obtained. Of 22 cases, 16 (73%) of sarcomatoid urothelial carcinoma were positive for GATA3. In the 7 (27%) of 22 cases that were negative for GATA3, it was observed that these cases were predominantly composed either of pleomorphic undifferentiated sarcomatoid areas or foci composed of extensive heterologous elements (chondroid, osteoid, or rhabdoid). GATA3 staining was positive in the adjacent/overlying conventional urothelial carcinoma and/or benign urothelium in all cases. This is one of the largest studies to date analyzing the expression of GATA3 in sarcomatoid urothelial carcinoma in cystectomy/cystoprostatectomy specimens. GATA3 is expressed in most cases of sarcomatoid urothelial carcinoma. Negative expression may, however, be observed in cases composed predominantly of pleomorphic undifferentiated sarcomatoid areas or extensive heterologous elements. We recommend including GATA3 in the panel of immunohistochemical stains for sarcomatoid carcinomas of unknown origin, especially if a bladder primary is being considered in the differential diagnosis.
Kruppel-like factor (KLF) 13 is a transcription factor that positively regulates expression of the chemokine RANTES 3-5 d after activation of T cells. In this study, we document a key role for KLF13 in the expression of IL-4 in CD4(+) T cells. Gene expression analysis in activated T cells from Klf13(-/-) mice showed that IL-4, along with other Th2 cytokine genes, was downregulated when compared with cells from wild-type mice. The decreased levels of IL-4 were not associated with changes in expression of the Th2-inducing transcription factors GATA3 or c-Maf. Additional analysis revealed that KLF13 directly binds to IL-4 promoter regions and synergizes with c-Maf to positively regulate IL-4 expression. These results indicate that KLF13 is a positive regulator for differentiation of Th2 cells, as part of the transcriptional machinery that regulates IL-4 production in Th2 cells.
Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4(+) and CD8(+) cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages.
CD70-expressing CD4 T cells are enriched in RA and promote autoimmunity via co-stimulatory CD70-CD27 interaction. This study aimed to explore the phenotype and cytokine production of CD70(+) CD4 T cells in RA.
Peripheral blood mononuclear cells from 32 RA patients were isolated and frequencies of CD70(+) cells within different CD4 T subsets were analysed using flow cytometry. IFN-γ and IL-17 production were compared between the CD70(+) and CD70(-) cells. Expression of master transcription factors T-bet, GATA3 and retinoic acid-related orphan receptor gamma t (RORγt) were examined by real-time PCR. Results are presented as mean (s.e.m.).
CD4 T cells of healthy controls rarely expressed CD70 as compared with CD4 T cells of RA patients [mean 0.9% (s.e.m. 0.3%) vs 7.6 (0.6), P < 0.001]. In RA, CD70(+) cells were present within all CD4 T cell subsets, i.e. CD45RA(+)CCR7(+) naive, CD45RA(-)CCR7(+) central memory, CD45RA(-)CCR7(-) effector memory and CD45RA(+)CCR7(-) terminally differentiated effector memory T cells with a mean frequency of 3.9% (s.e.m. 1.1%), 4.0 (0.5), 4.2 (0.7) and 9.4 (4.3), respectively. As compared to CD70(-) CD4 T cells, CD70(+) CD4 T cells produced significantly more IFN-γ and IL-17 after short activation. CD70(+) CD4 T cells preferentially expressed transcription factor RORγt.
CD70(+) CD4 T cells are enriched in RA and may directly contribute to RA pathogenesis by producing IFN-γ and IL-17. Targeting CD70(+) CD4 T cells might offer new therapeutic opportunities in RA.
Epithelioid mesotheliomas and breast carcinomas can present a variety of morphologic patterns. Because of this, breast carcinomas that metastasize to the pleura and lung may be confused with mesotheliomas. The aim of the present study is to compare the immunohistochemical markers currently available for the diagnosis of these 2 malignancies and to determine the best panel of markers that can be used to assist in discriminating between them. Sixty epithelioid mesotheliomas and 80 breast carcinomas (40 triple negative and 40 estrogen receptor positive) were investigated for expression of the positive mesothelioma markers calretinin, keratin 5/6, mesothelin, podoplanin, thrombomodulin, and WT1; the positive carcinoma marker claudin 4; and the breast-associated markers gross cystic disease fluid protein 15 (GCDFP-15), mammaglobin, and GATA3. All of the epithelioid mesotheliomas reacted for calretinin and keratin 5/6, 93% for WT1; 88% for podoplanin; 77% for thrombomodulin; 23% for GATA3; and 0% for claudin 4, GCDFP-15, and mammaglobin, respectively. Of the triple-negative breast carcinomas, 100% expressed claudin 4; 5%, keratin 5/6; 30%, GATA3; 18%, mammaglobin; 15%, GCDFP-15; 56%, mesothelin; 38%, calretinin; 18%, thrombomodulin; 5%, WT1; and 3%, podoplanin. Among the estrogen receptor-positive breast carcinomas, 100% were claudin 4 and GATA3 positive; 70% expressed GCDFP-15; 63%, mammaglobin; 13%, calretinin; 13%, thrombomodulin; 8%, WT1; 5%, keratin 5/6; 3%, mesothelin; and 0%, podoplanin. It is concluded that podoplanin and WT1 are the best positive mesothelioma markers for differentiating epithelioid mesotheliomas from breast carcinomas. An accurate differential diagnosis can be reached with the use of these two markers in combination with the breast-associated markers GCDFP-15, mammaglobin, and GATA3.
CD4(+) Th2 development is regulated by the zinc finger transcription factor GATA3. Once induced by acute priming signals, such as IL-4, GATA3 poises the Th2 cytokine locus for rapid activation and establishes a positive-feedback loop that maintains elevated GATA3 expression. Type I IFN (IFN-α/β) inhibits Th2 cells by blocking the expression of GATA3 during Th2 development and in fully committed Th2 cells. In this study, we uncovered a unique mechanism by which IFN-α/β signaling represses the GATA3 gene in human Th2 cells. IFN-α/β suppressed expression of GATA3 mRNA that was transcribed from an alternative distal upstream exon (1A). This suppression was not mediated through DNA methylation, but rather by histone modifications localized to a conserved noncoding sequence (CNS-1) upstream of exon 1A. IFN-α/β treatment led to a closed conformation of CNS-1, as assessed by DNase I hypersensitivity, along with enhanced accumulation of H3K27me3 mark at this CNS region, which correlated with increased density of total nucleosomes at this putative enhancer. Consequently, accessibility of CNS-1 to GATA3 DNA binding activity was reduced in response to IFN-α/β signaling, even in the presence of IL-4. Thus, IFN-α/β disrupts the GATA3-autoactivation loop and promotes epigenetic silencing of a Th2-specific regulatory region within the GATA3 gene.
Our analysis of the tumors of 57 women with metastatic breast cancer with next generation sequencing (NGS) demonstrates that each patient's tumor is unique in its molecular fingerprint. We observed 216 somatic aberrations in 70 different genes, including 131 distinct aberrations. The most common gene alterations (in order of decreasing frequency) included: TP53, PIK3CA, CCND1, MYC, HER2 (ERBB2), MCL1, PTEN, FGFR1, GATA3, NF1, PIK3R1, BRCA2, EGFR, IRS2, CDH1, CDKN2A, FGF19, FGF3 and FGF4. Aberrations included mutations (46%), amplifications (45%), deletions (5%), splices (2%), truncations (1%), fusions (0.5%) and rearrangements (0.5%), with multiple distinct variants within the same gene. Many of these aberrations represent druggable targets, either through direct pathway inhibition or through an associated pathway (via 'crosstalk'). The 'molecular individuality' of these tumors suggests that a customized strategy, using an "N-of-One" model of precision medicine, may represent an optimal approach for the treatment of patients with advanced tumors.
Cell-mediated immunity is indispensable for host protection against tuberculosis (TB). Growth factor receptor bound protein 2-associated binder (Gab) 2, a scaffolding adaptor protein, negatively regulates signaling pathways critical for T cell-mediated immunity. We sought to investigate the clinical significance and immunological role of Gab2 in Mycobacterium tuberculosis infection. We evaluated Gab2 protein and messenger RNA (mRNA) expression in human patients with pulmonary TB and determined the correlation of the mRNA expression pattern with antigen-specific IFN-γ secretion. Subsequently, we carried out M. tuberculosis infection in Gab2-deficient and wild-type control mice to explore the immunological role of Gab2 by examining bacterial load, histological changes, cytokine secretion, and gene expression of immune-associated transcription factors. mRNA levels of Gab2 and its correlated family member, Gab1, were markedly decreased in untreated patients with pulmonary TB compared with healthy control subjects. Importantly, this decreased Gab2 expression to normal levels after bacterial load in the patient's sputum became undetectable under the standard anti-TB treatment, which negatively correlated with the level of M. tuberculosis antigen-specific IFN-γ secretion. In the M. tuberculosis infection mouse model, infected Gab2-deficient mice exhibited decreased bacterial load and milder lung pathological damage compared with infected wild-type mice, accompanied by decreased production of IL-2, IL-6, and granulocyte/macrophage colony-stimulating factor proinflammatory cytokines, and an increased T-cell-specific T-box transcription factor/GATA binding protein 3 expression ratio. Overall, our study indicates that down-regulation of Gab2 relates to a protective function during M. tuberculosis infection, revealing a potential negative regulatory role for Gab2 in immunity to TB.
Recent studies have demonstrated that extracellular adenosine 5'-triphosphate (eATP) is involved in allergic airway inflammation by activating purinergic receptors. eATP can be hydrolyzed by ectonucleotidases, such as CD39. In this study, we investigated the expression and distribution of CD39 in the lungs of mice, as well as the effects of apyrase on airway inflammation and the chemotactic migration of dendritic cells (DCs). A mouse model of asthma was developed with chicken ovalbumin (OVA)/aluminum hydroxide using female C57BL/6 mice. Apyrase was administered to OVA-sensitized mice prior to each challenge by intraperitoneal injection. The distribution of CD39 was detected by immunofluorescence. The mRNA and protein expression of CD39 was determined by quantitative PCR and western blot analysis, respectively. The levels of Th2 cytokines in the bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The effect of apyrase on the chemotactic migration of DCs towards ATP was explored by migration assay in vitro. In the lungs, CD39 was primarily located in the cytoplasm and cytomembrane of bronchial epithelial cells and CD39 expression was reduced in mice with allergic asthma. Treatment with apyrase markedly attenuated OVA-induced airway inflammation, including peribronchial eosinophilic inflammation and reduced the number of inflammatory cells, as well as the levels of cytokines in BALF. Furthermore, apyrase also markedly reduced the expression of GATA binding protein 3 (GATA3) and decreased the chemotactic migration of DCs towards ATP.Our data demonstrate that a reduction in CD39 expression may be associated with the development of allergic airway inflammation and that apyrase alleviates airway inflammation by decreasing the chemotactic migration of DCs towards eATP. Therefore, targeting at eATP or ectonucleotidases may provide a novel therapeutic approach for allergic asthma.
The mechanistic target of rapamycin (mTOR) signaling integrates diverse environmental cues, including growth factors, nutrients and immunological signals. Activation of mTOR signaling stimulates protein synthesis and anabolic metabolism and coordinates cell growth, proliferation and fate decisions. In recent years, mTOR signaling has been linked to the entire spectrum of T cell biology, ranging from T cell development and activation to lineage specification and memory formation. Mechanistically, mTOR activation profoundly affects the expression and activity of many immunologically relevant transcription factors to propagate immune signaling and mediate effector functions. These transcription factors orchestrate cell metabolism (MYC, SREBPs and HIF1), lineage differentiation (T-bet, GATA3, RORγt, FOXP3 and Eomesodermin) and immune activation and functions (NF-κB, FOXOs, IRF4, STATs and GFI-1). This review discusses how mTOR signaling, through impinging upon transcriptional factors, regulates T cell development, activation, and effector and memory differentiation.
The JmjC domain-containing H3K4 histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) (also known as KDM5B and PLU1) is overexpressed in breast cancer and is a potential target for breast cancer treatment. To investigate the in vivo function of JARID1B, we developed Jarid1b(-/-) mice and characterized their phenotypes in detail. Unlike previously reported Jarid1b(-/-) strains, the majority of these Jarid1b(-/-) mice were viable beyond embryonic and neonatal stages. This allowed us to further examine phenotypes associated with the loss of JARID1B in pubertal development and pregnancy. These Jarid1b(-/-) mice exhibited decreased body weight, premature mortality, decreased female fertility, and delayed mammary gland development. Related to these phenotypes, JARID1B loss decreased serum estrogen level and reduced mammary epithelial cell proliferation in early puberty. In mammary epithelial cells, JARID1B loss diminished the expression of key regulators for mammary morphogenesis and luminal lineage specification, including FOXA1 and estrogen receptor α. Mechanistically, JARID1B was required for GATA3 recruitment to the Foxa1 promoter to activate Foxa1 expression. These results indicate that JARID1B positively regulates mammary ductal development through both extrinsic and cell-autonomous mechanisms.
Recently, we have reported that lymphocyte-derived endogenous catecholamines (CAs) facilitate a shift in the T helper (Th)1/Th2 balance towards Th2. The purpose of this study was to explore the involvement of adrenoreceptors (ARs) in Th differentiation and function modulation by lymphocyte-derived CAs.
Lymphocytes were separated from the mesenteric lymph nodes of mice, stimulated with concanavalin A (Con A) and treated with pargyline, an inhibitor of CA degradation.
Pargyline downregulated the expression of Th1-relative factors, T-bet, interferon (IFN)-γ and interleukin (IL)-2, but upregulated the expression of Th2-relative factors, GATA-3, IL-4 and IL-10. Pargyline reduced the percentage of IFN-γ-producing CD4+ cells and the CD4+IFN-γ+/CD4+IL-4+ cell ratio, although it did not alter the proportion of IL-4-producing CD4+ cells. In addition, the percentage of CD4+CD26+ T cells and the CD4+CD26+/CD4+CD30+ cell ratio were also reduced in the pargyline-treated group. Furthermore, Con A-activated T cells treated with pargyline produced a lower level of IFN-γ and a higher level of IL-4 than the control group. All these effects were blocked by the α1-AR antagonist corynanthine or the β2-AR antagonist ICI 118551, but not by the α2-AR antagonist yohimbine or β1-AR antagonist atenolol.
These results imply that lymphocyte-derived CAs promote polarization of differentiation and function towards Th2 cells and that this effect is mediated by α1-AR and β2-AR.
Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group), the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group), as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF- β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group) compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF- β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.
Estrogen receptor alpha (ERα) expression is critical for breast cancer classification, high ERα expression being associated with better prognosis. ERα levels strongly correlate with that of GATA binding protein 3 (GATA3), a major regulator of ERα expression. However, the mechanistic details of ERα-GATA3 regulation remain incompletely understood. Here we combine mathematical modeling with perturbation experiments to unravel the nature of regulatory connections in the ERα-GATA3 network. Through cell population-average, single-cell and single-nucleus measurements, we show that the cross-regulation between ERα and GATA3 amounts to overall negative feedback. Further, mathematical modeling reveals that GATA3 positively regulates its own expression and that ERα autoregulation is most likely absent. Lastly, we show that the two cross-regulatory connections in the ERα-GATA3 negative feedback network decrease the noise in ERα or GATA3 expression. This may ensure robust cell fate maintenance in the face of intracellular and environmental fluctuations, contributing to tissue homeostasis in normal conditions, but also to the maintenance of pathogenic states during cancer progression.
In this study, OVA-induced asthma mice was taken as the model, and orally administered with different concentration of ethanol extracts of crude and processed Stemona tuberosa, in order to determine the cytokine level released from Th1 and Th2 in splenocytes. RT-PCR was carried out to determine the genetic expression of T-bet/GATA-3 in lung, and compare the differentiation between ethanol extracts of crude and processed S. tuberosa in therapeutic effect on asthma in mice. According to the results, compared with the crude samples, processed samples significantly increased the levels of inflammatory factor INF-gamma (P < 0.05) and decreased IL-5 (P < 0.05) in splenocytes. According to the RT-PCR results, the administration of processed samples could increase the ratio of T-bet/GATA-3 (P < 0.05). The experiment showed that ethanol extracts of both crude and processed S. tuberosa could treat asthma by regulating Th1/Th2 ratio, but processed samples showed more notable effect. This indicated that crude and processed S. tuberosa had significant pharmacological difference. Therefore, it was more rational to apply processed S. tuberosa in clinical treatment of asthma and chronic cough, which layed a foundation for further revealing the processing mechanism of S. tuberosa.
GATA3 has conventionally been regarded as a transcription factor that drives the differentiation of T helper (Th) 2 cells. Increasing evidence points to a function for GATA3 beyond controlling Th2 differentiation. GATA3 regulates T cell development, proliferation, and maintenance. Furthermore, recent studies have demonstrated important roles for GATA3 in innate lymphoid cells. Thus, GATA3 emerges as a factor with diverse functions in immune regulation, which are in some cases cell-type specific and in others shared by multiple cell types. Here, I discuss recent discoveries and the current understanding of the functions of GATA3 in immune regulation.
Data on immunohistochemical expression of novel and traditional urothelial markers in the wide range of urothelial carcinoma variants have so far been very limited. In this study, whole tissue sections from 130 bladder urothelial carcinoma and variants were stained with a panel of novel and traditional immunomarkers supportive of urothelial lineage. The positivity rates were as follows: (a) urothelial carcinomas with or without divergent differentiation: GATA3 (50%), S-100P (86%), uroplakin III (20%), thrombomodulin (40%), cytokeratin 7 (CK7) (80%), CK20 (55%), p63 (87%), and high molecular weight cytokeratin (HMCK) (89%); (b) urothelial carcinoma variants (micropapillary, plasmacytoid, nested, clear cell, and microcystic): GATA3 (88%), S-100P (96%), uroplakin III (33%), thrombomodulin (49%), CK7 (95%), CK20 (61%), p63 (69%), and HMCK (96%); and (c) undifferentiated carcinomas (lymphoepithelioma-like carcinoma, small cell carcinoma, sarcomatoid carcinoma and carcinoma with rhabdoid and giant cells): GATA3 (28%), S-100P (31%), uroplakin III (0%), thrombomodulin (22%), CK7 (50%), CK20 (3%), p63 (50%), and HMCK (49%). In urothelial carcinoma with squamous differentiation, GATA3 expression was lower (20%) in contrast to p63 and S-100P. In urothelial carcinoma with glandular differentiation, GATA3 (50%) and p63 (60%) expression was lower than S-100P (100%). p63 expression was relatively lower in micropapillary (54%) and plasmacytoid (50%) variants compared with the other urothelial carcinoma variants. This study provides comprehensive data for novel and traditionally used markers to support urothelial lineage in urothelial carcinoma variants. Our findings show that GATA3, S-100P, CK7, CK20, HMCK, and p63, in the appropriate differential diagnostic setting, are useful to support urothelial lineage of variant morphologies.
Senescence or biological aging impacts a vast variety of molecular and cellular processes. To date, it is unknown whether CD4(+) Th cells display an age-dependent bias for development into specific subpopulations. In this study, we show the appearance of a distinct CD4(+) T cell subset expressing IL-4 at an early stage of development in infant adenoids and cord blood that is lost during aging. We identified by flow cytometric, fluorescent microscopic, immunoblot, and mass spectrometric analysis a population of CD4(+) T cells that expressed an unglycosylated isoform of IL-4. This T cell subpopulation was found in neonatal but not in adult CD4(+) T cells. Furthermore, we show that the mRNA of the Th2 master transcription factor GATA3 is preferentially expressed in neonatal CD4(+) T cells. The Th2 phenotype of the IL-4(+)CD4(+) T cells could be reinforced in the presence of TGF-β. Although the IL-4(+)CD4(+) T cells most likely originate from CD31(+)CD4(+) T recent thymic emigrants, CD31 was downregulated prior to secretion of IL-4. Notably, the secretion of IL-4 requires a so far unidentified trigger in neonatal T cells. This emphasizes that cytokine expression and secretion are differentially regulated processes. Our data support the hypothesis of an endogenously poised cytokine profile in neonates and suggest a link between cytokine production and the developmental stage of an organism. The determination of the IL-4 isoform-expressing cells in humans might allow the identification of Th2 precursor cells, which could provide novel intervention strategies directed against Th2-driven immunopathologies such as allergies.
Innate lymphoid cells (ILCs) are lymphoid cells that do not express rearranged receptors and have important effector and regulatory functions in innate immunity and tissue remodeling. ILCs are categorized into 3 groups based on their distinct patterns of cytokine production and the requirement of particular transcription factors for their development and function. Group 1 ILCs (ILC1s) produce interferon γ and depend on Tbet, group 2 ILCs (ILC2s) produce type 2 cytokines like interleukin-5 (IL-5) and IL-13 and require GATA3, and group 3 ILCs (ILC3s) include lymphoid tissue inducer cells, produce IL-17 and/or IL-22, and are dependent on RORγt. Whereas ILCs play essential roles in the innate immune system, uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. In this review, we provide an overview of the characteristics of ILCs in the context of health and disease. We will focus on human ILCs but refer to mouse studies if needed to clarify aspects of ILC biology.
Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis includes genetic, environmental, and immunological factors, such as T helper cells and their secreted cytokines. T helper cells are classified as Th1, Th2, and Th17 cells. However, it is unclear which T helper cells are important in UC. Dextran sulfate sodium (DSS)-induced colitis is a commonly used model of UC. In this study, we induced DSS colitis in Th1 dominant (T-bet transgenic (Tg)) mice, Th2 dominant (GATA-3 Tg) mice, and Th17 dominant (RORγt Tg) mice to elucidate the roles of T helper cell in DSS colitis. The results showed that GATA-3 Tg mice developed the most severe DSS colitis compared with the other groups. GATA-3 Tg mice showed a significant decreased in weight from day 1 to day 7, and an increased high score for the disease activity index compared with the other groups. Furthermore, GATA-3 Tg mice developed many ulcers in the colon, and many neutrophils and macrophages were detected on day 4 after DSS treatment. Measurement of GATA-3-induced cytokines demonstrated that IL-13 was highly expressed in the colon from DSS-induced GATA-3 Tg mice. In conclusion, GATA-3 overexpression in T-cells and IL-13 might play important roles in the development of DSS colitis.
Differentiation of CD4(+) T cells into type 1 or type 2 subsets is mediated by the expression of the opposing lineage defining transcription factors T-bet and GATA-3. However, the existence of GATA-3(+) T-bet(+) CD4(+) T cells in mice suggests functional plasticity of these subsets. Little is known about type 1 and type 2 plasticity of human T-cell subsets in vivo. Here, we show that in the xenogeneic environment of humanized mice, which lacks a functional immune-regulatory network, human CD4(+) and, notably, CD8(+) T cells preferentially differentiate into interleukin (IL)-4(+) GATA-3(+) and IL-4(+) interferon-γ(+) GATA-3(+) T-bet(+) subsets. Treatment with recombinant human IL-12 or expansion of IL-12-producing human dendritic cells in vivo reverted this phenotype and led to the down-regulation of GATA-3 expression. These changes also correlated with improved antiviral immune responses in humanized mice. In conclusion, our study shows the capacity of human CD4(+) and CD8(+) T cells for stable co-expression of GATA-3 and T-bet in humanized mice and reveals a critical role for IL-12 in regulating this phenotype.
Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase-targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.
The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.
Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).
GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.
We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.
The cholinergic anti-inflammatory pathway can inhibit the inflammation of collagen induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). However, the immunologic mechanisms that provide a therapeutic effect against the auto-inflammatory disease are not yet elucidated. The present study explores the effect of cholinergic anti-inflammatory pathway on CD4+ T cell responses in CIA. Forty DBA/1 mice were divided into 4 groups: a control group, a CIA group, a vagotomy group, and a nicotine group. The degree of arthritis was measured by arthritis score and hematoxylin and eosin. ELISA was used to detect the serum concentration of IFN-γ, IL-4 and IL-17A. Flow cytometry was used to detect the cytokines and transcription factors (TFs) (the TFs of Th1, Th2, and Th17 cells are T-bet, RORγτ and GATA3 respectively) in the spleen. Immunohistochemistry was used to analyze RORγτ expression in the joint synovium. Arthritis in the nicotine group was significantly lightened compared with that in the CIA group and in the vagotomy group. Nicotine attenuated Th17 lineage by reducing IL-17A production and RORγτ expression. The expressions of IL-4 and GATA3 were increased in the same setting. However, the expressions of IFN-γ and T-bet had no difference between the nicotine and the CIA group. Nicotine may induce a shift to the Th2 lineage and improve the Th1/Th2 imbalance. Activating the cholinergic anti-inflammatory pathway with nicotine can inhibit Th17 cell responses and may improve the Th1/Th2 imbalance in CIA, providing a new justification for its application in the treatment of rheumatoid arthritis.
Systemic corticosteroids are the most effective anti-inflammatory drugs used for controlling chronic rhinosinusitis (CRS) symptoms. The potential mechanisms for their beneficial effects include increasing the number and function of T regulatory cells (Tregs), as reported in the local tissue post-intranasal steroid treatment. We investigated the effect of systemic corticosteroids on peripheral blood (PB) Tregs in subjects with CRS.
Twenty CRS subjects and 19 controls were recruited. PB mononuclear cells (PBMCs) were isolated from CRS subjects before and after systemic corticosteroid administration in the course of clinical treatment. Control subjects received no treatment and were studied at one visit. Nasal symptoms were recorded. CD4(+) CD25(+) Foxp3(+) cells (Tregs) were analyzed by flow cytometry. Messenger RNA (mRNA) levels for interferon γ (IFN-γ), interleukin 4 (IL-4), IL-10, IL-13, IL-17A, transforming growth factor β1 (TGF-β1), forkhead box P3 (FoxP3), and GATA-binding factor 3 (GATA-3) were measured in PBMCs using real-time polymerase chain reaction (PCR).
CRS subjects reported improved nasal symptoms (p = 0.005) and significantly reduced PB Tregs after treatment with corticosteroids (p = 0.042). The transcript levels of IL-4 and GATA-3 were significantly higher in the CRS subjects at their first visit when compared to controls (p = 0.019 and p = 0.05, respectively). Corticosteroid treatment lowered the transcript levels of immunoregulatory transcription factors [FoxP3 (p = 0.048) and GATA-3 (p = 0.012)] and IFN-γ (p = 0.036) in PB.
In contrast to prior work in local nasal tissue, our study reports reduced PB Tregs and decreased T helper 1 (T(H)1) and T(H)2 function after treatment with systemic corticosteroids. These data indicate that corticosteroid effects on Tregs in CRS are complex involving local signals in the tissue that are distinct from those in circulating cells.
Hispanic children have a higher incidence of acute lymphoblastic leukemia (ALL) than non-Hispanic whites but tend to be diagnosed at older ages. In genome-wide association studies, Native American ancestry and polymorphisms in six genes have been associated with ALL risk. In multivariable regression models, we investigated whether genomic ancestry, inherited risk SNPs, or acquired somatic alterations were associated with differences in age at diagnosis in Hispanic children with B-cell ALL. Genome-wide array data were used to estimate each participant's percent membership in the three Hispanic ancestral populations: Native American, African, and European. Each 20% increase in European ancestry was associated with a six month younger age at diagnosis (95% CI = 0.36-11.6 months, P = 0.037). Correspondingly, each 20% increase in Native American ancestry was associated with a six-month older age at diagnosis (P = 0.037). Both the TEL-AML1 translocation and high-hyperdiploidy were associated with younger age at diagnosis (24.4 months, P = 2.0 x 10(-4) and 12.4 months, P = 0.011, respectively), while CDKN2A and IKZF1 deletions were associated with older age at diagnosis (19.7 months, P = 7.0 x 10(-4) and 18.1 months, P = 0.012, respectively). No associations with age at diagnosis were observed for RAS mutation, PAX5 deletion or for known heritable risk alleles in IKZF1, CDKN2A, PIP4K2A, GATA3, ARID5B, or CEBPE. Because younger age at diagnosis is associated with improved treatment outcomes for children with ALL, the effect of European ancestry on ALL survival may be mediated by its effect on age at diagnosis, or by proxy, its association with more treatable molecular subtypes of ALL.
The GATA binding protein 3 (GATA3) is a member of a family of 6 GATA dual zinc finger transcription factors (GATA1-6), which are required for the development and morphogenesis of the mammary gland. GATA3 is considered to play a dual role in oncogenesis and cancer development, whereas somatic GATA3 mutations have been reported in breast cancer. Variants of the GATA3 genetic 3' untranslated region (3'UTR) microRNA (miRNA) binding sites have been associated with breast cancer risk. However, the roles of genetic variants in the GATA3 gene 3'UTR and its post-transcriptional regulation have not been fully elucidated. We discovered that rs1058240 in the GATA3 3'UTR displayed potential miRNA binding sites and this variant was found to be significantly associated with GATA3 mRNA expression (P=2.36E-07), suggesting that rs1058240 may be a putative variant mediating the post-transcriptional regulation of the GATA3 target gene. Further studies investigating the regulatory mechanism of GATA3 transcriptional activity are required to design novel strategies against breast cancer cell growth and differentiation.
The histopathologic distinction between primary adnexal carcinomas and metastatic adenocarcinoma to the skin from sites such as the breast, lung, and others often presents a diagnostic dilemma. Current markers of diagnostic utility include p63 and cytokeratin 5/6; however, their expression has been demonstrated in 11% to 22% and 27% of cutaneous metastases, respectively. Furthermore, the immunoreactivity of p40 and GATA3 in various cutaneous adnexal carcinomas has not been previously reported. In the present study, we compared the expression of p40, p63, cytokeratin 5/6, and GATA3 in a total of 143 cases, including 67 primary adnexal carcinomas and 76 cutaneous metastases. p40, p63, cytokeratin 5/6, and GATA3 expression was observed in 80%, 84%, 86%, and 47% of primary adnexal carcinoma, respectively, and in 8%, 17%, 26%, and 40% of cutaneous metastases, respectively. χ(2) Analysis revealed statistically significant P values (<.0001) for p40, p63, and cytokeratin 5/6 in distinguishing primary adnexal carcinoma from cutaneous metastases. In summary, while p63 and cytokeratin 5/6 have similar sensitivity (84% and 86%, respectively) in detecting primary adnexal carcinomas, p40 appeared to be the most specific marker (92%) with the best positive predictive value (90%). Since breast and lung are the most common sites of origin for cutaneous metastases, p40 is the best distinguishing marker in these settings. None of the four studied markers (p40, p63, cytokeratin 5/6, and GATA3) are helpful in distinguishing between primary adnexal carcinomas from cutaneous metastases of salivary gland or bladder malignancies.
GATA binding protein 3 (GATA-3) is a novel immunohistochemical marker for urothelial carcinoma (UC); however, few studies have investigated GATA-3's role as a marker for UC variants. We used immunohistochemistry to assess GATA-3 expression in different UC variants, including micropapillary (n = 46), sarcomatoid (n = 43), small cell carcinoma (n = 22), and plasmacytoid (n = 16) variants, and we also compared GATA-3 expression in conventional bladder UC (n = 103) to that in squamous cell carcinoma (n = 14). GATA-3 expression was present in 70% (72/103) of conventional bladder UCs and highly concordant between matched primary and metastatic UCs. The GATA-3 expression levels of the micropapillary variants (57%; 26/46) and plasmacytoid variants (44%; 7/16) were not significantly different from that of conventional UC. However, the GATA-3 expression levels of the sarcomatoid variants (16%; 7/43) and small cell carcinoma variants (5%; 1/22), which only weakly expressed the protein, were significantly lower than that of conventional UC (P < .001). Only 7% of squamous cell carcinomas (1/14) expressed GATA-3, and it was also significantly lower than that of conventional UC (P < .001). GATA-3 expression was not significantly associated with tumor stage or patients' clinical outcomes. In conclusion, GATA-3 expression differed among UC variants. GATA-3 is a useful marker for confirming the urothelial origin of micropapillary and plasmacytoid UC variants but not that of sarcomatoid or small cell carcinoma variants. GATA-3 can also be used in differentiating UC from squamous cell carcinoma.
Dietary factors, including meat, fruits, vegetables and fiber, are associated with colorectal cancer; however, there is limited information as to whether these dietary factors interact with genetic variants to modify risk of colorectal cancer. We tested interactions between these dietary factors and approximately 2.7 million genetic variants for colorectal cancer risk among 9,287 cases and 9,117 controls from ten studies. We used logistic regression to investigate multiplicative gene-diet interactions, as well as our recently developed Cocktail method that involves a screening step based on marginal associations and gene-diet correlations and a testing step for multiplicative interactions, while correcting for multiple testing using weighted hypothesis testing. Per quartile increment in the intake of red and processed meat were associated with statistically significant increased risks of colorectal cancer and vegetable, fruit and fiber intake with lower risks. From the case-control analysis, we detected a significant interaction between rs4143094 (10p14/near GATA3) and processed meat consumption (OR = 1.17; p = 8.7E-09), which was consistently observed across studies (p heterogeneity = 0.78). The risk of colorectal cancer associated with processed meat was increased among individuals with the rs4143094-TG and -TT genotypes (OR = 1.20 and OR = 1.39, respectively) and null among those with the GG genotype (OR = 1.03). Our results identify a novel gene-diet interaction with processed meat for colorectal cancer, highlighting that diet may modify the effect of genetic variants on disease risk, which may have important implications for prevention.
Newly identified nuocytes or group 2 innate lymphoid cells (ILC2s) play an important role in Th2 cell mediated immunity such as protective immune responses to helminth parasites, allergic asthma, and chronic rhinosinusitis. However, the contributions of ILC2s in the occurrence and development of cancer remain unknown. Our previous study found that there was a predominant Th2 phenotype in patients with gastric cancer. In this study, the ILC2s related genes or molecules in PBMC from patients with gastric cancer were measured, and the potential correlation between them was analyzed. The expression levels of RORα, GATA3, T1/ST2, IL-17RB, CRTH2, IL-33, IL-5, and IL-4 mRNA were significantly increased in patients, but no significant changes were found in ICOS, CD45, and IL-13 expression, and there was a positive correlation between RORα or IL-13 and other related factors, such as ICOS and CD45. The increased frequency of ILC2s was also found in PBMC of patients by flow cytometry. In addition, the mRNA of Arg1 and iNOS were also significantly increased in patients. These results suggested that there are polarized ILC2s in gastric cancer patients which might contribute to immunosuppressive microenvironment and closely related to the upregulation of MDSCs and M2 macrophages.
Although the involvement of helper T (Th) and regulatory T (Treg) cell-related immune molecules in pathogenesis of inflammatory bowel disease (IBD) is widely accepted, no discriminatory mucosal expression profiles of these molecules between ulcerative colitis (UC) and Crohn's disease (CD) have been clarified.
Mucosal expression of 17 cytokines and transcription factors related to Th1, Th2, Th17, and Treg were measured by quantitative PCR in endoscopic biopsies from inflamed (40 from UC [UCI] and 20 from CD [CDI]) and noninflamed (47, 22, and 25 from UC, CD, and controls, respectively) colon or ileum. The discriminatory power of these markers to differentiate between the 2 diseases was evaluated by linear discriminant analysis and, unsupervised, principal component analysis.
By univariate analysis, many targets were markedly increased in inflamed versus noninflamed areas. However, marker expression was almost comparable between UCI and CDI, with the largest difference in UCI-predominant interleukin (IL) 21 and IL-13 with area under the receiver operating characteristic curve (AUC) values of 0.704 and 0.664, respectively. In contrast, combinations of 2 to 7 markers improved UCI versus CDI discrimination with AUC = 0.875 to 0.975. Among these, a 5-maker set (interferon-γ, IL-12 p35, T-bet, GATA3, and IL-21) demonstrated an AUC of 0.949 and a misclassification rate of 8.3%. Principal component analysis also markedly separated UCI and CDI.
Inflamed mucosae from UC and CD could be discriminated with high accuracy using combinations of Th cell-related markers. Multigene analysis, possibly reflecting the underlying pathogenesis, is expected to be useful for diagnosis, monitoring and further defining distinctive characteristics in inflammatory bowel disease.
Genetic effects on asthma of genes in the T-helper 2 (Th2) pathway may interact with epigenetic factors including DNA methylation. We hypothesized that interactions between genetic variants and methylation in genes in this pathway (IL4, IL4R, IL13, GATA3, and STAT6) influence asthma risk, that such influences are age-dependent, and that methylation of some CpG sites changes over time in accordance with asthma transition. We tested these hypotheses in subsamples of girls from a population-based birth cohort established on the Isle of Wight, UK, in 1989.
Logistic regression models were applied to test the interaction effect of DNA methylation and SNP on asthma within each of the five genes. Bootstrapping was used to assess the models identified. From 1,361 models fitted at each age of 10 and 18 years, 8 models, including 4 CpGs and 8 SNPs, showed potential associations with asthma risk. Of the 4 CpGs, methylation of cg26937798 (IL4R) and cg23943829 (IL4) changes between ages 10 and 18 (both higher at 10; P = 9.14 × 10(-6) and 1.07 × 10(-5), respectively). At age 10, the odds of asthma tended to decrease as cg12405139 (GATA3) methylation increased (log-OR = -12.15; P = 0.049); this effect disappeared by age 18. At age 18, methylation of cg09791102 (IL4R) was associated with higher risk of asthma among subjects with genotype GG compared to AG (P = 0.003), increased cg26937798 methylation among subjects with rs3024685 (IL4R) genotype AA (P = 0.003) or rs8832 (IL4R) genotype GG (P = 0.01) was associated with a lower asthma risk; these CpGs had no effect at age 10. Increasing cg26937798 methylation over time possibly reduced the risk of positive asthma transition (asthma-free at age 10 → asthma at age 18; log-OR = -3.11; P = 0.069) and increased the likelihood of negative transition (asthma at age 10 → asthma-free at age 18; log-OR = 3.97; P = 0.074).
The interaction of DNA methylation and SNPs in Th2 pathway genes is likely to contribute to asthma risk. This effect may vary with age. Methylation of some CpGs changed over time, which may influence asthma transition.
Nucleocytoplasmic shuttling of macromolecules is a well-controlled process involving importins and exportins. These karyopherins recognize and bind to receptor-mediated intracellular signals through specific signal sequences that are present on cargo proteins and transport into and out of the nucleus through nuclear pore complexes. Nuclear localization signals (NLS) present on cargo molecules to be imported while nuclear export signals (NES) on the molecules to be exported are recognized by importins and exportins, respectively. The classical NLS are found on many transcription factors and molecules that are involved in the pathogenesis of allergic diseases. In addition, several immune modulators, including corticosteroids and vitamin D, elicit their cellular responses by regulating the expression and activity of importin molecules. In this review article, we provide a comprehensive list of importin and exportin molecules and their specific cargo that shuttled between cytoplasm and the nucleus. We also critically review the role and regulation of specific importin and exportin involved in the transport of activated transcription factors in allergic diseases, the underlying molecular mechanisms, and the potential target sites for developing better therapeutic approaches.
Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.
Although mast cells play a critical role in allergic reactions, the cells are also involved in the protective immunity in the body. This study aims to investigate the role of mast cells in immune regulation during aberrant T helper (Th)2 responses. In this study, an adoptive antigen-specific Th2 response model was established with mast cell-deficient mice to test the role of mast cell in the immune regulation. Cell culture was employed to test the role of mast cells in the modulation of the expression of B cell lymphoma 6 protein (Bcl-6) in Th2 cells. The results showed that after adoptive transfer with immune cells, the mast cell-deficient mice showed stronger Th2 pattern responses in the intestine than that in the mast cell-sufficient mice. Mast cell-derived mouse mast cell protease-6 increased the expression of Bcl-6 in Th2 cells. Bcl-6 inhibited the expression of GATA-3 in Th2 cells, subsequently, forkhead box P3 was increased and the Th2 cytokines were reduced in the cells; the cells thus showed the immune regulatory properties similar to regulatory T cells. We conclude that bedsides initiating immune inflammation, mast cells also contribute to the immune regulation on Th2 polarization.
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor commonly inactivated in glioblastoma multiforme (GBM), but the prognostic significance of PTEN remains controversial. Here, we demon- strate significant prognostic value of combined PTEN mutation and expression for the survival of patients with GBM on the basis of analysis of large-scale cancer genomic data. PTEN nonsense mutations associated with sig- nificantly shorter disease-free survival and overexpression of PTEN protein linked to shorter disease-free and overall survival of patients with GBM. PTEN nonsense mutations correlated with decreased p53 and Gata3 protein levels and increased genomic instability in human GBM tissues. Expression of nonsense PTEN mutant decreased p53 and Gata3 levels, producing increased DNA damage both in vitro and in vivo. Mice carrying xenograft tumors with nonsense PTEN mutant displayed significantly shorter survival. Our data demonstrated the prognostic value of combined PTEN mutation and protein expression for patients with GBM and highlighted distinct biologic effects of nonsense and missense mutations of PTEN.
The CD4(+) T-cell subgroups play central pathophysiological roles in Crohn's disease (CD); however, their clinical relevance requires additional clarification and remains controversial. We investigated their balance in Chinese CD patients and explored their clinical significance.
Peripheral blood mononuclear cells and serum were collected from 46 Chinese CD patients and 23 healthy donors. Circulating Treg, Th1, Th2, and Th17 cells were flow cytometrically analyzed. Subgroup-restricted transcription factor expression was determined by real-time polymerase chain reaction. Serum concentrations of the main cytokines produced by each subgroup were measured by cytometric bead arrays or enzyme-linked immunosorbent assay.
Lower Treg proportion (6.0 ± 1.2% vs 7.8 ± 1.5%, P = 0.030), FOXP3 mRNA expression (0.58-fold, P = 0.030), and circulating soluble TGFβ-1 (19.1 ± 9.9 vs 32.7 ± 16.8 ng/mL, P = 0.038) were observed in CD patients versus controls. The Th1 and Th17 proportions were higher in CD patients (17.8 ± 6.6% vs 7.8 ± 1.5%, P < 0.001; and 3.7 ± 1.8% vs 1.8 ± 0.7%, P = 0.022, respectively), as were transcription factors T-bet (4.6-fold, P = 0.043) and RORγt (14-fold, P < 0.001) and related cytokines (P < 0.05). Th2 proportion, GATA3 mRNA expression, and serum interleukin-4 concentration in CD patients were similar to controls (P > 0.05). Treg/Th1 and Treg/Th17 ratios were higher in inactive versus active CD patients (0.6 ± 0.4 vs 0.3 ± 0.1, P = 0.022; and 3.7 ± 2.0 vs 1.7 ± 1.4, P = 0.013, respectively). During follow-up, patients with lower Treg/Th1 and Treg/Th17 ratios were at higher recurrence risk.
Imbalances among Treg, Th1, and Th17 subgroups were found in Chinese CD patients. Treg/Th1 and Treg/Th17 ratios are associated with disease activity and are potential prognostic indicators for predicting CD recurrence.
Estrogen receptor (ER), gross cystic disease fluid protein 15 (GCDFP-15), and mammaglobin (MGB) are commonly used breast-specific immunomarkers; however, about half of metastatic breast carcinomas are negative for all three. GATA-binding protein 3 (GATA-3) has emerged recently as a sensitive and relatively specific immunomarker for breast and urothelial carcinomas, but the data documenting its expression in ER-negative breast carcinomas are limited; this often poses a dilemma in the setting of metastases. The purpose of this study is to investigate expression of GATA-3 in ER-negative breast carcinomas.
Immunohistochemical evaluation of GATA-3, GCDFP-15, and MGB on 96 ER-negative breast carcinomas was performed.
Overall, 69% (66/96), 15% (14/96), and 35% (34/96) of ER-negative breast carcinomas expressed GATA-3, GCDFP-15, and MGB, respectively.
Our data suggest that GATA-3 is, so far, the best breast-specific immunomarker, especially when encountering ER-negative metastatic breast carcinomas. GATA-3 should be included in the panel of immunomarkers in the workup of tumors of unknown primary.
GATA3 (GATA-binding protein 3) expression in urothelial carcinoma (UC) and mammary carcinomas has been recently reported. However, to the authors' knowledge, studies examining GATA3 staining of metastatic UC (MUC) in cytology specimens are lacking. Delta Np63 (p40) has been shown to be expressed highly selectively in squamous cell carcinomas (SCCs) but the literature concerning the expression of p40 in UC is limited and controversial. In the current study, the authors evaluated the usefulness of GATA3 and p40 in the diagnosis of MUC in cytology specimens.
Thirty-two MUC cytology cases and 44 controls (22 UC cases and 22 SCC cases) were stained for GATA3, p40, and p63 and nuclear staining intensity and the percentage of positive cells were recorded and compared.
MUC cytology cases stained positive for GATA3, p40, and p63 in 78.13%, 80.65%, and 61.29% of cases, respectively, with moderate/strong staining intensity. MUC cases had a significantly higher percentage of GATA3 positivity compared with SCC controls (P<.001), but GATA3 positivity was not found to be significantly different from UC controls (90.91%) (P = .28). For p40 positivity, there was no significant difference observed between MUC cases, UC controls (95.45%), and SCC controls (90.91%) (P=.29). p63 positivity was found to be significantly lower in MUC cases compared with UC controls (95.45%) and SCC controls (95.45%) (P<.01).
The results of the current study demonstrate that GATA3 is useful in confirming the diagnosis of MUC in cytology specimens and in distinguishing between MUC and SCC. p40 is a valuable adjunct to GATA3 in the diagnosis of MUC in cytology specimens, especially when SCC is not part of the clinical differential diagnosis.
Curcumin, a natural product derived from the plant Curcuma longa, has been found to have anti-inflammatory, antineoplastic and antifibrosis effects. It has been reported that curcumin attenuates allergic airway inflammation in mice through inhibiting NF-κB and its downstream transcription factor GATA3. It also has been proved the antineoplastic effect of curcumin through down-regulating Notch1 receptor and its downstream nuclear transcription factor NF-κB levels. In this study, we aimed to investigate the anti-inflammatory effect of curcumin on acute allergic asthma and its underlying mechanisms. 36 male BALB/c mice were randomly divided into four groups (normal, asthma, asthma+budesonide and asthma+curcumin groups). BALF (bronchoalveolar lavage fluid) and lung tissues were analyzed for airway inflammation and the expression of Notch1, Notch2, Notch3, Notch4 and the downstream transcription factor GATA3. Our findings showed that the levels of Notch1 and Notch2 receptors were up-regulated in asthma group, accompanied by the increased expression of GATA3. But the expression of Notch2 receptor was lower than Notch1 receptor. Curcumin pretreatment improved the airway inflammatory cells infiltration and reversed the increasing levels of Notch1/2 receptors and GATA3. Notch3 receptor was not expressed in all of the four groups. Notch4 receptor protein and mRNA expression level in the four groups had no significant differences. The results of the present study suggested that Notch1 and Notch2 receptor, major Notch1 receptor, played an important role in the development of allergic airway inflammation and the inhibition of Notch1-GATA3 signaling pathway by curcumin can prevent the development and deterioration of the allergic airway inflammation. This may be a possible therapeutic option of allergic asthma.
The prevalence of allergic asthma has been increased rapidly in recent years. About 20% of all these sufferers have experienced asthma exacerbation. Although corticosteroids and β-agonists therapy improves serious asthma symptoms, they can׳t completely cure these allergic diseases. BuShenYiQi Formula (BSYQF) has been widely used to treat bronchial asthma and its exacerbation for decades in Huashan Hospital of Fudan University, China. Nevertheless, the mechanisms of BSYQF' anti-asthmatic effects haven׳t been fully elucidated. In this study, we evaluated the involvement of Th1, Th2 and Th17 cells in the anti-asthmatic effects of BSYQF in Respiratory Syncytial Virus (RSV)-induced asthma exacerbated mice.
BALB/c mice were challenged with ovalbumin (OVA), followed by RSV infections for establishment of asthma exacerbated model. Airway hyperresponsiveness (AHR) was examined by direct airway resistance analysis. Bronchoalveolar lavage fluid (BALF) was assessed for inflammatory cell counts and secreted levels of cytokines. Lung tissues were detected for inflammatory cell infiltration and mucus hypersecretion. Subsequently, CD4(+)T cells and alveolar macrophages were sorted and purified from mice lungs in different groups. CD4(+)T cell subpopulations including the expression levels of important transcription factors in T lymphocyte polarization were examined. In asthma exacerbation group, the purified CD4(+)T cells and macrophages were co-cultured, and the changes of co-cultured cells with BSYQF treatment were further analyzed in vitro.
BSYQF significantly attenuated airway hyperresponsiveness and inhibited inflammatory cell infiltration, especially for excessive infiltration of eosinophils and neutrophils. Histopathological analysis showed that BSYQF could suppress airway inflammation and RSV replication. The decreases of antigen-specific IgE, IL-4, IL-5, IL-6, IL-17a and increases of IFN-γ, IL-12 were observed in BALF, lung homogenate or serum after BSYQF treatment. We further confirmed that BSYQF could down-regulate Th2-Th17 cell proportions with lower expressions of GATA3, STAT6 and RORγT, and up-regulate Th1 cell proportion with higher expression of T-bet. And as a result of strengthened Th1-response, activated macrophages were also observed by remarkable enhancement of signature gene expressions and phagocytosis.
BSYQF can significantly attenuate RSV-induced asthma exacerbation. These effects may be mediated at least partially by regulating the balance between Th1 and Th2-Th17 responses.
The relationships between tonsillar immune responses, and viral infection and allergy are incompletely known.
To study intratonsillar/nasopharyngeal virus detections and in vivo expressions of T-cell- and innate immune response-specific cytokines, transcription factors, and type I/II/III interferons in human tonsils.
Palatine tonsil samples were obtained from 143 elective tonsillectomy patients. Adenovirus, bocavirus-1, coronavirus, enteroviruses, influenza virus, metapneumovirus, parainfluenza virus, rhinovirus, and respiratory syncytial virus were detected using PCR. The mRNA expression levels of IFN-α, IFN-β, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-β, FOXP3, GATA3, RORC2, and Tbet were directly analyzed by quantitative RT-PCR.
Fifty percentage of subjects reported allergy, 59% had ≥1 nasopharyngeal viruses, and 24% had ≥1 intratonsillar viruses. Tonsillar virus detection showed a strong negative association with age; especially rhinovirus or parainfluenza virus detection showed positive association with IFN-γ and Tbet expressions. IL-37 expression was positively associated with atopic dermatitis, whereas IFN-α, IL-13, IL-28, and Tbet expressions were negatively associated with allergic diseases. Network analyses demonstrated strongly polarized clusters of immune regulatory (IL-10, IL-17, TGF-β, FOXP3, GATA3, RORC2, Tbet) and antiviral (IFN-α, IFN-β, IL-28, IL-29) genes. These two clusters became more distinctive in the presence of viral infection or allergy. A negative correlation between antiviral cytokines and IL-10, IL-17, IL-37, FOXP3, and RORC2 was observed only in the presence of viruses, and interestingly, IL-13 strongly correlated with antiviral cytokines.
Tonsillar cytokine expression is closely related to existing viral infections, age, and allergic illnesses and shows distinct clusters between antiviral and immune regulatory genes.
Abnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development.
Examination of Isl1 expression in developing mouse stomach by immunohistochemistry, whole mount in situ hybridization and real-time quantitative PCR demonstrated that Isl1 is highly expressed in developing mouse stomach, principally in the smooth muscle layer of the pylorus. Isl1 expression was also examined by immunofluorescence in human hypertrophic pyloric stenosis where the majority of smooth muscle cells were found to express Isl1. Isl1 function in embryonic stomach development was investigated utilizing a tamoxifen-inducible Isl1 knockout mouse model. Isl1 deficiency led to nearly complete absence of the pyloric outer longitudinal muscle layer at embryonic day 18.5, which is consistent with Gata3 null mouse phenotype. Chromatin immunoprecipitation, luciferase assays, and electrophoretic mobility shift assays revealed that Isl1 ensures normal pyloric development by directly targeting Gata3.
This study demonstrates that the Isl1-Gata3 transcription regulatory axis is essential for normal pyloric development. These findings are highly clinically relevant and may help to better understand pathways leading to pyloric disease.
To explore the hypothesis that blood transfusion contributes to an immunosuppressed phenotype in severely injured patients.
Despite trauma patients using disproportionately large quantities of blood and blood products, the immunomodulatory effects of blood transfusion in this group are inadequately described.
A total of 112 ventilated polytrauma patients were recruited. Messenger RNA (mRNA) was extracted from PAXGene tubes collected within 2 hours of the trauma, at 24 hours, and at 72 hours. T-helper cell subtype specific cytokines and transcription factors were quantified using real-time polymerase chain reaction.
Median injury severity score was 29. Blood transfusion was administered to 27 (24%) patients before the 2-hour sampling point. Transfusion was associated with a greater immediate rise in IL-10 (P = 0.003) and IL-27 (P = 0.04) mRNA levels. Blood products were transfused in 72 (64%) patients within the first 24 hours. There was an association between transfusion at 24 hours and higher IL-10 (P < 0.0001), lower Foxp3 (P = 0.01), GATA3 (P = 0.006), and RORγt (P = 0.05) mRNA levels at 24 hours. There were greater reductions in T-bet (P = 0.03) mRNA levels and lesser increases in TNFα (P = 0.015) and IFNγ (P = 0.035) at 24 hours in those transfused. Multiple regression models confirmed that the transfusion of blood products was independently associated with altered patterns of gene expression. Blood stream infections occur in 15 (20.8%) of those transfused in the first 24 hours, compared with 1 patient (2.5%) not transfused (OR = 10.3 [1.3-81], P = 0.008).
The primarily immunosuppressive inflammatory response to polytrauma may be exacerbated by the transfusion of blood products. Furthermore, transfusion was associated with an increased susceptibility to nosocomial infections.
Aplastic anemia (AA) is a type of bone marrow hematopoietic system disease. The immune mediated hematopoietic inhibition is recognized as the most common pathogenesis of AA. However, the roles of the T-bet/GATA-3-mediated cell immune disorder in aplastic anemia (AA) is still unknown.
Experimental samples were obtained from 27 patients with AA, including 15 cases of severe AA (SAA) and 12 cases of immune mediated AA (MAA), and 25 healthy volunteers (control group). The secretory levels of IFN-gamma and IL-4 cytokines were determined by ELISA. The mRNA expression levels of transcription factors T-bet, GATA-3, and FoxP3 were measured in PBMCs by RT-PCR. Th1, Th2, and T lymphocyte subsets were detected in peripheral blood by flow cytometry.
Compared to the healthy control group, the expression of T-bet mRNA and the percentage of Th1-type cells in the AA group significantly increased (p < 0.01), while the expression of GATA-3 and FoxP3 mRNA and the percentage of Th2-type cells decreased sharply (p < 0.05, p < 0.01). Compared with MAA, the expression of T-bet mRNA and the percentage of Th1-type cells increased significantly in SAA (p < 0.01); meanwhile, the expression of GATA-3 mRNA and the proportion of Th2-type cells decreased noticeably (p < 0.05, p < 0.01). Particularly, the percentage of CD3+ and CD3+CD8+ T cells in the AA group increased (p < 0.05), while the percentage of CD3+CD4+, CD4+CD25+, and CD4+CD8+ cells decreased (p < 0.05, p < 0.01).
Abnormal expression of the transcription factors T-bet and GATA-3 contributes to the imbalance of Thl/Th2 lymphocytes associated with immune dysfunction, leading to the development and progression of AA.
Twelve cases of urothelial cell papillary carcinoma with a whorled pattern of growth are described. This variant is reported with clinicopathological correlations and immunohistochemical findings. All cases showed this peculiar and distinctive curlicue histological pattern, ranging from 50% to 100% of the neoplastic population. Despite the disordered/turbulent growth pattern, the cytological grade was uniformly low. All the lesions were Ta staged and no patient experienced progression after transurethral resection, while 2 showed clinical recurrences. The immunophenotype (low p53, high p27, low Ki67, and high GATA3) of the cases contributes to define this rare variant as a low-grade tumor.
The ectoenzyme CD39 hydrolyzes extracellular adenosine 5'-triphosphate (ATP), which possesses pro-inflammatory properties. However, the role of CD39 in allergic asthma has not been fully elucidated. A total of 18 patients with persistent asthma who were allergic to house dust mites and 19 healthy volunteers were enrolled in this study. The expression of CD39, GATA3, RAR-related orphan receptor γ (ROR-γt) and forkhead box P3 (FoxP3) mRNA in peripheral blood mononuclear cells (PBMCs) was determined by SYBR-Green I quantitative polymerase chain reaction (qPCR). The cytokines interleukin (IL)-4, IL-17A, transforming growth factor β (TGF-β) and DP.sIgE were detected by enzyme-linked immunosorbent assay. Our data demonstrated that the expression of CD39 mRNA in PBMCs from asthmatic patients was significantly lower compared to that in normal controls [(1.49±0.59)×10(-3) vs. (2.17±0.77)×10(-3), respectively; P<0.01]. CD39 mRNA was negatively correlated with serum IL-4, IL-17A and GATA3 expression (r=-0.468, P<0.05; r=-0.550, P<0.05; and r=-0.424, P<0.01, respectively) and positively correlated with FoxP3 and TGF-β expression (r=0.373, P<0.05; and r=0.425, P<0.05, respectively). There was no obvious correlation between CD39 and ROR-γt expression (r=-0.259, P=0.122). These data suggested that CD39 mRNA expression was downregulated in allergic asthma, which was positively correlated with serum IL-4, IL-17A and GATA3 expression and negatively correlated with serum TGF-β and FoxP3 expression, whereas there was no correlation with ROR-γt. Therefore, it was hypothesized that CD39 may participate in the occurrence and progression of allergic asthma.
The transcription factor GATA2 plays pivotal roles in early renal development, but its distribution and physiological functions in adult kidney are largely unknown. We examined the GATA2 expression pattern in the adult kidney by tracing green fluorescent protein (GFP) fluorescence in Gata2(GFP/+) mice that recapitulate endogenous GATA2 expression and found a robust GFP expression specifically in the renal medulla. Upon purification of the GFP-positive cells, we found that collecting duct (CD)-specific markers, including aquaporin 2 (Aqp2), an important channel for water reabsorption from urine, were abundantly expressed. To address the physiological function of GATA2 in the CD cells, we generated renal tubular cell-specific Gata2-deficient mice (Gata2-CKO) by crossing Gata2 floxed mice with inducible Pax8-Cre mice. We found that the Gata2-CKO mice showed a significant decrease in Aqp2 expression. The Gata2-CKO mice exhibited high 24-h urine volume and low urine osmolality, two important signs of diabetes insipidus. We introduced biotin-tagged GATA2 into a mouse CD-derived cell line and conducted chromatin pulldown assays, which revealed direct GATA2 binding to conserved GATA motifs in the Aqp2 promoter region. A luciferase reporter assay using an Aqp2 promoter-reporter showed that GATA2 trans activates Aqp2 through the GATA motifs. These results demonstrate that GATA2 regulates the Aqp2 gene expression in CD cells and contributes to the maintenance of the body water homeostasis.
The epidermis is maintained by epidermal stem cells (ESCs) that reside in distinct niches and contribute to homeostasis and wound closure. Keratinocytes at the nonhealing edges of venous ulcers (VUs) are healing-incompetent, hyperproliferative, and nonmigratory, suggesting deregulation of ESCs. To date, genes which regulate ESC niches have been studied in mice only. Utilizing microarray analysis of VU nonhealing edges, we identified changes in expression of genes harboring regulation of ESCs and their fate. In a prospective clinical study of 10 VUs, we confirmed suppression of the bone morphogenetic protein receptor (BMPR) and GATA binding protein 3 (GATA3) as well as inhibitors of DNA-binding proteins 2 and 4 (ID2 and ID4). We also found decreased levels of phosphorylated glycogen synthase kinase 3 (GSK3), nuclear presence of β-catenin, and overexpression of its transcriptional target, c-myc, indicating activation of the Wnt pathway. Additionally, we found down-regulation of leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1), a gene important for maintaining ESCs in a quiescent state, and absence of keratin 15 (K15), a marker of the basal stem cell compartment suggesting local depletion of ESCs. Our study shows that loss of genes important for regulation of ESCs and their fate along with activation of β-catenin and c-myc in the VU may contribute to ESC deprivation and a hyperproliferative, nonmigratory healing incapable wound edge.
Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).
In the examination of >3000 radical prostatectomy specimens and their seminal vesicles (SVs), we came across a unique case of an intraepithelial abnormality in preexisting ducts and acini in the left SV of a 73-yr-old patient with prostatic adenocarcinoma. At low magnification, the epithelial lining was thicker than the surrounding normal ducts with obliteration of the acinar lumen. At high magnification, there were varying degrees of cell stratification. Prostate-specific antigen, prostate-specific acid phosphatase, prostate-specific membrane antigen, prostein (P501S), alpha-methylacyl-CoA racemase, and GATA binding protein 3 (GATA3) were negative; p63, 34betaE12, cytokeratin 5/6, and p53 were positive in cells in basal and suprabasal positions, whereas CA-125 was expressed in the luminal cells. The case shows morphologic and immunohistochemical features similar to those of basal cell hyperplasia of the prostate and is different from the early neoplastic epithelial changes of the SV in the transgenic adenocarcinoma mouse prostate model (ie, SV intraepithelial neoplasia). To the best of our knowledge, there are no previous reports of such a change in a human SV. The clinical significance of this lesion is not known and is overshadowed by the presence of prostate cancer.
Innate lymphoid cells (ILCs) are critical in innate immune responses to pathogens and lymphoid organ development. Similar to CD4(+) T helper (Th) cell subsets, ILC subsets positive for interleukin-7 receptor α (IL-7Rα) produce distinct sets of effector cytokines. However, the molecular control of IL-7Rα(+) ILC development and maintenance is unclear. Here, we report that GATA3 was indispensable for the development of all IL-7Rα(+) ILC subsets and T cells but was not required for the development of classical natural killer cells. Conditionally Gata3-deficient mice had no lymph nodes and were susceptible to Citrobactor rodentium infection. After the ILCs had fully developed, GATA3 remained important for the maintenance and functions of ILC2s. Genome-wide gene expression analyses indicated that GATA3 regulated a similar set of cytokines and receptors in Th2 cells and ILC2s, but not in ILC3s. Thus, GATA3 plays parallel roles in regulating the development and functions of CD4(+) T cells and IL-7Rα(+) ILCs.
To report the auditory and vestibular phenotypes of patients with GATA3 mutation.
Case series of 6 patients.
Tertiary referral center.
All patients had the classic triad of GATA3 deficiency: hypoparathyroidism, hearing loss, and renal dysplasia. Patients (29-60 yr old; mean age, 42.5 yr; 3 male and 3 female subjects) were confirmed to have heterozygous mutations involving GATA3 by Sanger sequencing.
Behavioral audiometry, distortion product otoacoustic emissions (DPOAEs), and auditory brainstem responses (ABRs) were used to assess hearing. Rotational vestibular testing was used to assess vestibular function.
All patients with GATA3 mutation presented with hearing loss during childhood. The mean 3-frequency (0.5/1/2 kHz) pure tone average was 67 dB HL (range, 50-83 dB HL; SD, 9.3). The average speech discrimination score was 73% (range, 36%-100%; SD, 15.9). DPOAEs were absent in all patients. ABRs were remarkably robust and provided no evidence of retrocochlear dysfunction. Some patients complained of dizziness, but rotary chair testing was normal across participants for whom testing occurred.
Patients with GATA3 mutation present with early-onset sensorineural hearing loss (SNHL). DPOAEs were absent, supporting outer hair cell dysfunction, whereas ABRs were present and robust. Rotational vestibular testing revealed no evidence of abnormal horizontal semicircular canal function.
Gene variants known to contribute to Autoimmune Addison's disease (AAD) susceptibility include those at the MHC, MICA, CIITA, CTLA4, PTPN22, CYP27B1, NLRP-1 and CD274 loci. The majority of the genetic component to disease susceptibility has yet to be accounted for.
To investigate the role of 19 candidate genes in AAD susceptibility in six European case-control cohorts.
A sequential association study design was employed with genotyping using Sequenom iPlex technology. In phase one, 85 SNPs in 19 genes were genotyped in UK and Norwegian AAD cohorts (691 AAD, 715 controls). In phase two, 21 SNPs in 11 genes were genotyped in German, Swedish, Italian and Polish cohorts (1264 AAD, 1221 controls). In phase three, to explore association of GATA3 polymorphisms with AAD and to determine if this association extended to other autoimmune conditions, 15 SNPs in GATA3 were studied in UK and Norwegian AAD cohorts, 1195 type 1 diabetes patients from Norway, 650 rheumatoid arthritis patients from New Zealand and in 283 UK Graves' disease patients. Meta-analysis was used to compare genotype frequencies between the participating centres, allowing for heterogeneity.
We report significant association with alleles of two STAT4 markers in AAD cohorts (rs4274624: P = 0.00016; rs10931481: P = 0.0007). In addition, nominal association of AAD with alleles at GATA3 was found in 3 patient cohorts and supported by meta-analysis. Association of AAD with CYP27B1 alleles was also confirmed, which replicates previous published data. Finally, nominal association was found at SNPs in both the NF-κB1 and IL23A genes in the UK and Italian cohorts respectively.
Variants in the STAT4 gene, previously associated with other autoimmune conditions, confer susceptibility to AAD. Additionally, we report association of GATA3 variants with AAD: this adds to the recent report of association of GATA3 variants with rheumatoid arthritis.
The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-τ, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic research that is focused on peri-implantation and placenta development or as donor cells for transgenic animal production.
Adenocarcinoma of the paraurethral glands represents a very rare neoplasm of the urinary tract. Due to the rarity of this disease, there is no standard therapeutic approach. We report a case of adenocarcinoma of the paraurethral glands in a 56-year-old woman, presenting with abnormal serous vaginal discharges. The radiologic examination revealed a 5-cm mass around the urethra, which underwent surgical resection. After surgical resection, the histology revealed a moderately differentiated adenocarcinoma, probably arising from the paraurethral glands. One month later, a pelvic recurrent mass was radiologically diagnosed; consequently, an anterior pelvic exenteration with lymph node dissection was performed. Histological examination revealed a moderately differentiated adenocarcinoma, with glandular and micropapillary architecture, with multiple lymph node metastases. The absence of modifications such as urethritis cystic glandularis on the urethral mucosa, as well as the lack of a lesion in situ, associated with the immunohistochemical expression of PAX8 and negativity for GATA3 and S100p, suggested that the adenocarcinoma originated from the paraurethral glands rather than from the urethral mucosa. Post-surgery CT scans revealed no evidence of metastatic disease. The patient received 6 courses of adjuvant chemotherapy with carboplatin and paclitaxel. One year after the pelvic exenteration, because of inguinal lymph node progression, an inguinal lymphadenectomy was performed. Four months later, a TC-PET revealed a multidistrectual lymph node and a lung micronodule disease progression. Invasive micropapillary carcinomas have been characterized as a rare distinctive variant of carcinomas in several anatomic sites and are distinguished by a marked tendency to lymphovascular invasion, justifying the association with high-stage disease and poor prognosis. In the present case, both the poor prognosis connected with micropapillary structure and the lymph node involvement, encouraged adjuvant cisplatinum-based chemotherapy.
The hygiene hypothesis states that children exposed to higher loads of microbes such as farmers' children suffer less from allergies later in life. Several immunological mechanisms underpinning the hygiene hypothesis have been proposed such as a shift in T helper cell balance, T regulatory cell activity, or immune regulatory mechanisms induced by the innate immunity.
To investigate whether the proposed immunological mechanisms for the hygiene hypotheses are found in farmers' children.
We assessed gene expression levels of 64 essential markers of the innate and adaptive immunity by quantitative real-time PCR in white blood cells in 316 Swiss children of the PARSIFAL study to compare farmers' to non-farmers' expressions and to associate them to the prevalence of asthma and rhinoconjunctivitis, total and allergen-specific IgE in serum, and expression of Cε germ-line transcripts.
We found enhanced expression of genes of the innate immunity such as IRAK-4 and RIPK1 and enhanced expression of regulatory molecules such as IL-10, TGF-β, SOCS4, and IRAK-2 in farmers' children. Furthermore, farmers' children expressed less of the TH1 associated cytokine IFN-γ while TH2 associated transcription factor GATA3 was enhanced. No significant associations between the assessed immunological markers and allergic diseases or sensitization to allergens were observed.
Farmers' children express multiple increased innate immune response and immune regulatory molecules, which may contribute to the mechanisms of action of the hygiene hypothesis.
Mammary epithelial cell identity depends on a set of genes epigenetically-regulated by maintenance proteins, the best-characterized of which belong to the Trithorax and Polycomb groups. Perturbations in expression of these proteins may disrupt cell identity and trigger tumor initiation.
The pattern of expression of a panel of genes involved in control of cell identity and mammary gland remodeling was investigated in two precancerous lesions, atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) and compared to the corresponding histologically normal tissue.
ADH and DCIS showed a close association in overexpression of Polycomb complex components, silencing of Homeobox A (HOXA) cluster gene, and overexpression of the genes involved in estrogen signaling, specifically, forkhead box A1 (FOXA1) and GATA binding protein 3 (GATA3) pioneer factors, and estrogen receptor-1 (ESR1).
Our findings support the hypothesis that disruption of epigenetic control is associated with loss of cell identity and acquisition of a constitutive estrogen-dependent terminally-differentiated luminal phenotype.
Uromodulin is a kidney specific glycoprotein whose expression can modulate kidney homeostasis. However, the set of sequence specific transcription factors that regulate the uromodulin gene UMOD and their upstream binding locations are not well characterized. We built a high resolution map of its transcriptional regulation.
We applied in silico phylogenetic footprinting on the upstream regulatory regions of a diverse set of human UMOD orthologs to identify conserved binding motifs and corresponding position specific weight matrices. We further analyzed the predicted binding motifs by motif comparison, which identified transcription factors likely to bind these discovered motifs. Predicted transcription factors were then integrated with experimentally known protein-protein interactions available from public databases and tissue specific expression resources to delineate important regulators controlling UMOD expression.
Analysis allowed the identification of a reliable set of binding motifs in the upstream regulatory regions of UMOD to build a high confidence compendium of transcription factors that could bind these motifs, such as GATA3, HNF1B, SP1, SMAD3, RUNX2 and KLF4. ENCODE deoxyribonuclease I hypersensitivity sites in the UMOD upstream region of the mouse kidney confirmed that some of these binding motifs were open to binding by predicted transcription factors. The transcription factor-transcription factor network revealed several highly connected transcription factors, such as SP1, SP3, TP53, POU2F1, RARB, RARA and RXRA, as well as the likely protein complexes formed between them. Expression levels of these transcription factors in the kidney suggest their central role in controlling UMOD expression.
Our findings will form a map for understanding the regulation of uromodulin expression in health and disease.
GATA binding protein 3 is a zinc finger transcription factor with high affinity for urothelial tissue and is a promising immunohistochemical marker in detection of urothelial carcinomas (UC). We studied its usefulness in metastatic high-grade UC. This study was performed on cell blocks (CB) of fine needle aspirates from 25 cases of metastatic high-grade UC in patients with previously resected high-grade UC. Immunohistochemical staining for GATA3, thrombomodulin, uroplakin, cytokeratin 7, and cytokeratin 20 was performed. Twenty-three of 25 cases of metastatic UC expressed GATA3 (92%); positive staining for cytokeratin 7 was present in 23 of 25 cases (92%), 20 of 25 (80%) stained for thrombomodulin, and 13 of 25 (52%) stained for cytokeratin 20. No case expressed uroplakin. Five hundred forty-seven non-urothelial carcinomas, including breast ductal carcinoma (77), hepatocellular carcinoma (100), colonic adenocarcinoma (81), pancreatic adenocarcinoma (28), gastric adenocarcinoma (31), endometrial carcinoma (27), ovarian serous carcinoma (27), lung adenocarcinoma (27), lung squamous cell carcinoma (26), malignant melanoma (27), renal cell carcinoma (48), and prostatic adenocarcinoma (48) in tissue microarrays were also analyzed and were GATA3 negative except for 35 of 77 (45.5%) of GATA3 positive breast ductal carcinoma. GATA3 has high sensitivity and specificity for detection of metastatic UC and thus may play an important role in diagnosing metastatic UC in CB samples.
Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4(+) T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN-γ/IL-4 double-positive cells. These IFN-γ(+) IL-4(+) cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN-γ(+) IL-4(-) (Th1) and IFN-γ(-) IL-4(+) (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN-γ(+) IL-4(+) cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co-existence of Th1 and Th2 characteristics.
Differentiation of preadipocytes into adipocytes and the formation of the subsequent adipose tissue are critical for mammalian growth and development. The molecular mechanism relating to preadipocyte differentiation and adipogenesis from the perspective of miRNAs is not yet completely understood. Here we investigated whether miR-183 functioned in the differentiation process. Both gain-of-function and loss-of-function assays demonstrated that miR-183 positively regulated 3T3-L1 differentiation by enhancing the expression of adipogenic marker genes such as CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin and fatty acid synthase (FAS), as well as the triglyceride content and accumulation of lipid droplets. Meanwhile, low-density lipoprotein receptor-related protein 6 (LRP6) was known to impair the canonical Wnt/β-catenin signaling pathway and thereafter reduce c-myc and nuclear β-catenin protein. We showed that the inhibition of LRP6 by siRNA promoted 3T3-L1 adipogenic differentiation and adipogenesis. Further analysis showed that mouse miR-183 gene had its own transcription unit containing CpG islands, transcription start site (TSS), coding sequence (CDS) and polyA signal within the flanking sequences 2500 nt upstream and downstream of mouse miR-183 in genome. The core promoter of miR-183 gene was identified and transcription factor GATA3 (GATA binding protein 3) significantly inhibited the expression of mature miR-183 by binding to its core promoter in vivo, as indicated by the chromatin immunoprecipitation (ChIP) assay. These results suggest that miR-183, though negatively regulated by transcription factor GATA3, enhances 3T3-L1 preadipocyte differentiation and adipogenesis through the inhibition of the canonical Wnt/β-catenin signaling pathway by targeting LRP6.
Estrogen receptor (ER) is essential for estrogen-dependent growth, and its level of expression is considered a crucial determinant of response to endocrine therapy and prognosis in ER-positive breast cancer. On the other hand, the clinical role of progesterone receptor (PgR) in ER-positive breast cancer remains controversial, although testing of PgR by immunohistochemistry (IHC) has become routine. Recent studies indicated that plasma estradiol levels were related to the expression levels of estrogen-responsive genes in ER-positive breast cancer tissues in both pre- and postmenopausal women. In this study, we analyzed the expression levels of estrogen-responsive genes (PgR and TFF1), a progesterone-responsive gene (RANKL), ER-related genes (FOXA1 and GATA3), HER2, Ki67 and p53 in ER-positive, HER2-negative breast cancer tissues by IHC. Correlations between the expression levels of these molecular markers and clinicopathological factors, including prognosis, were compared between pre- and postmenopausal women. Serum levels of estrone, estradiol, progesterone, and testosterone were also measured. Expression levels of PgR, TFF1, RANKL, and GATA3 were significantly higher in premenopausal women than in postmenopausal women. Serum estradiol levels were positively correlated with Ki67 labeling index (LI) in premenopausal women, but not in postmenopausal women. High expression of FOXA1 and GATA3 was significantly associated with improved disease-free survival in premenopausal women, but not in postmenopausal women, whereas high expression of PgR and low expression of p53 were significantly correlated with the improved disease-free survival in postmenopausal women, but not in premenopausal women. Moreover, the best cutoff points of Ki67 LI for disease-free survival were 30 % for premenopausal women and 14 % for postmenopausal women. Expression levels of ER, TFF1, and RANKL were not associated with the disease-free survival in either pre- or postmenopausal women. Our results suggest that the mechanisms of development and estrogen-dependent growth of ER-positive breast cancer might differ according to menopausal status.
Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58-81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.
Persistent hepatitis B virus (HBV) infection is characterized by a weak adaptive immune response, which is considered to be due to an imbalance of T helper cell types 1 and 2 (Th1/Th2). Suppressors of cytokine signaling (SOCS) family members, particularly SOCS1 and SOCS3, have been demonstrated to be important in the regulation of T cell differentiation. Previous studies by our group showed that the expressed and purified fusion protein of cytoplasmic transduction peptide (CTP) and HBV core antigen 18‑27 (HBcAg18‑27)‑tapasin was able to enter the cytoplasm of bone marrow‑derived dendritic cells (BMDCs), promoting the maturation of BMDCs and efficiently enhancing T cell immune responses in vitro. In the present study, HBcAg‑specific immune responses induced by CTP‑HBcAg18‑27‑tapasin in HBV were assessed in transgenic mice, and SOCS1 and SOCS3 were identified as negative regulators of this response. The Th1/Th2 cytokine ratio was analyzed by ELISA. The expression of T cell‑specific T‑box transcription factor (T‑bet) and GATA‑binding protein 3 (GATA‑3), SOCS1 and SOCS3 were detected by real‑time quantitative polymerase chain reaction and western blot analysis. The results demonstrated that CTP‑HBcAg18‑27‑tapasin significantly increased the Th1/Th2 cytokine ratio in HBV transgenic mice. CTP‑HBcAg18‑27‑tapasin immunization more efficiently suppressed the expression of serum hepatitis B surface antigen (HBsAg), HBV DNA as well as liver HBsAg and HBcAg in HBV transgenic mice. Furthermore, CTP‑HBcAg18‑27‑tapasin promotes T‑bet but reduces GATA‑3 expression. In addition, the expression of SOCS1 and SOCS3 was significantly downregulated in the CTP‑HBcAg18‑27‑tapasin group compared with the control groups. In conclusion, the present study demonstrated that CTP‑HBcAg18‑27‑tapasin enhanced the Th1/Th2 cytokine ratio and antiviral immunity by suppressing SOCS1/3 in HBV transgenic mice.
Radiation-induced gastrointestinal toxicity, including its shift of the immune balance, remains a major limitation to delivering tumoricidal doses of abdominal radiation therapy. This study evaluates the effect on the colon's innate and adaptive immune responses to moderate irradiation doses and the therapeutic possibilities of maintaining immune homeostasis. We investigated whether administration of the TLR4 agonist LPS or of the TLR5 agonist flagellin, 3 days after a single 20-Gy colorectal irradiation, modified recruitment of neutrophils, NK cells, or CD4⁺ or CD8⁺ T cells, 7 days postirradiation. Flow cytometric analysis showed that LPS and flagellin reduced irradiation-induced neutrophil infiltration and normalized NK frequency. LPS normalized the CD4⁺ population and enhanced the CD8⁺ population, whereas flagellin maintained the radiation-induced elevation in the frequencies of both. Irradiation also modified TLR4 and TLR5 expression on the surface of both populations, but LPS and flagellin each subsequently normalized them. LPS and flagellin were strong inducers of Th1 cytokines (IL-12p35, IL-12p40, and IFN-γ) and thus, contributed to a shift from the Th2 polarization induced by irradiation toward a Th1 polarization, confirmed by an increase of the T-bet:GATA3 ratio, which assesses the Th1 or Th2 status in mixed cell populations. LPS and flagellin treatment resulted in overexpression of FoxP3, IL-2Rα (CD25), IL-2, and OX40, all expressed specifically and involved in high levels of Treg cell expansion. We observed no variation in Treg function-related expression of IL-10 or CTLA-4. These data suggest that the use of TLR ligands limits the effects of irradiation on innate and adaptive immunity.
Barakat syndrome (also known as HDR syndrome) is an autosomal dominant disorder characterized by hypoparathyroidism, sensorineural deafness, and renal disease caused by mutation of the GATA3 gene located at chromosome 10p15. The exact prevalence of this disorder is not known but is very rare, with only about a dozen cases reported in the literature. Here, we report a case of 58-year-old man from Ardabil who presented with seizure due to hypocalcemia. Further history revealed bilateral deafness. Audiogram confirmed sensorineural hearing loss of both sides. His laboratory data were consistent with hypoparathyroidism and renal failure. He was diagnosed to have Barakat syndrome based on his clinical and laboratory data. In conclusion, we need to be aware of rare inherited conditions in a patient with abnormal physical and laboratory findings even though their initial presentation was seizure and hypocalcemia.
Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development, by using lineage tracing and transfer studies. PLZF(high) cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1(+)DX5(-) 'NK-like' cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZF(high) precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.
Bavachinin, which can be isolated from the Chinese herb Fructus Psoraleae, has the potential as a potent anti-asthma drug. However, the extremely low water solubility of Bavachinin limits its application. In this study, two new derivatives of Bavachinin, i.e., compounds A and B, whose water solubility is better than that of Bavachinin, were synthesized via biotransformation. A comparative investigation was then performed on the effects of these two new derivatives, along with Bavachinin, on T cell differentiation. The results showed that they have different effects. Bavachinin and compound B inhibited green fluorescent protein (GFP) production from the T cells of IL-4-GFP-enhanced transcript (4GET) mice, whereas compound A did not. The effect was mainly attributed to the inhibition of GATA-3 protein production. Bavachinin and compound B can inhibit the production of GATA-3 mRNA, but they showed different effects on the production of T-bet mRNA. Compound B increased the production of T-bet mRNA, whereas Bavachinin did not. The results will be very useful for optimizing Bavachinin so that potent anti-allergic drugs can be developed. The structure-activity relationship of Th2 was revealed based on the difference between Bavachinin and compound B. This finding can enrich the database of preliminary drug screening from their chemical structures.
GATA binding protein 3 (GATA3) was recently proposed to function as a tumor suppressor gene in some types of human cancer. This study aims to investigate GATA3 expression and its prognostic significance in primary gastric adenocarcinoma.
Using real-time quantitative PCR (RT-qPCR) and immunohistochemical staining methods, GATA3 expression was analyzed in tissue samples from a consecutive series of 402 gastric adenocarcinoma patients who underwent resections between 2003 and 2006. The relationship between GATA3 expression, clinicopathological factors, and patient survival was investigated. The expression status of GATA3 was shown to be clearly reduced in the tumor tissue samples compared with that in the matched adjacent non-tumor tissue samples by RT-qPCR (P = 0.0014). Immunohistochemistry analysis indicated that GATA3 expression was significantly decreased in 225 of the 402 (56%) gastric adenocarcinoma cases. Reduced GATA3 expression was also observed in patients with large tumors (P = 0.017), signet ring cell carcinoma or mucinous carcinoma (P = 0.005) and tumors with lymphatic or venous invasion (P = 0.040). Additionally, reduced expression of GATA3 was more commonly observed in tumors that were staged as T4a/b (P<0.001), N3 (P<0.001), or M1 (P<0.001). Kaplan-Meier survival curves revealed that reduced expression of GATA3 was associated with poor prognosis in gastric adenocarcinoma patients (P<0.001). Multivariate Cox analysis identified GATA3 expression as an independent prognostic factor for overall survival (HR = 5.375, 95% CI = 3.647-7.921, P<0.001). To investigate the predictive ability of the models with and without containing GATA3 gene expression, Harrell's c-index was calculated as a measure of predictive accuracy of survival outcome. The c-index values revealed that model containing GATA3 expression (c-index = 0.897) had superior discrimination ability to the model without containg it (c-index = 0.811).
Our data suggest that GATA3 plays an important role in tumor progression and that reduced GATA3 expression independently predicts an unfavorable prognosis in primary gastric adenocarcinoma patients.
High-grade prostate adenocarcinoma can have overlapping morphologic features with high-grade urothelial carcinoma, a critical distinction as treatments differ substantially. Pseudopapillary features have not previously been described in high-grade acinar prostate adenocarcinoma. We reviewed our consult cases (2006 to 2013) for cases of high-grade prostate adenocarcinoma, in which the Gleason score was equal to 5+4=9 or 5+5=10, and the differential diagnosis included high-grade urothelial carcinoma. We identified 7 consult cases of high-grade prostate adenocarcinoma with pseudopapillary features, mimicking urothelial carcinoma. Three cases were originally misdiagnosed as urothelial carcinoma. In 3 cases, the outside diagnosis was urothelial carcinoma versus prostate adenocarcinoma, and 1 case had no submitting diagnosis. All cases were transurethral resections, with tumor involving the prostatic urethra in 5 cases and 6 with bladder involvement. Three patients had a known history of prostate adenocarcinoma. The tumors grew in nests and sheets, 1 with microacinar differentiation and another with focal, rare glands. In places, tumors formed papillary-appearing structures with central blood vessels. In most cases, the nuclei were uniform with prominent nucleoli. One case had pleomorphic giant cell features, and another had sarcomatoid features. Necrosis was present in 2 cases. One case had a separate focus of low-grade noninvasive urothelial carcinoma present in the bladder and a better-differentiated prostate adenocarcinoma (Gleason score 4+3=7) involving the prostate on needle biopsy. In all cases, the pseudopapillary areas showed negative immunohistochemical staining for bladder markers including GATA3 (5 cases), p63 (5 cases), CK903 (4 cases), and thrombomodulin (3 cases). All cases showed positivity for prostatic markers including PSA (5 cases), p501s (6 cases), PSMA (4 cases), and NKX3.1 (5 cases). One case showed focal, nonspecific staining for p63, and another showed focal staining for p63 and CK903 in an area with squamous differentiation, contributing to the diagnostic difficulty. In summary, high-grade prostate adenocarcinoma can present in the urinary bladder and prostatic urethra, clinically mimicking urothelial carcinoma. Although high-grade prostate adenocarcinoma typically has relatively uniform cytology, it can have pleomorphic giant cell features overlapping with urothelial carcinoma. The presence of pseudopapillary features in high-grade prostate adenocarcinoma is a newly recognized morphologic overlap that can lead to further diagnostic difficulty in distinguishing the 2 entities. For high-grade tumors involving the prostatic urethra without typical admixed lower-grade prostate adenocarcinoma, immunohistochemical studies for bladder and prostate markers should be carried out.
The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+) stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim) peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim) than CD56(bright) peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+)NKG2A(+) NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.
The cell of origin and the tumor microenvironment's role remain elusive for the most common peripheral T-cell lymphomas (PTCLs). As macrophages promote the growth and survival of malignant T cells and are abundant constituents of the tumor microenvironment, their functional polarization was examined in T-cell lymphoproliferative disorders. Cytokines that are abundant within the tumor microenvironment, particularly interleukin (IL)-10, were observed to promote alternative macrophage polarization. Macrophage polarization was signal transducer and activator of transcription 3 dependent and was impaired by the Janus kinase inhibitor ruxolitinib. In conventional T cells, the production of T helper (Th)2-associated cytokines and IL-10, both of which promote alternative macrophage polarization, is regulated by the T-cell transcription factor GATA-binding protein 3 (GATA-3). Therefore, its role in the T-cell lymphomas was examined. GATA-3 expression was observed in 45% of PTCLs, not otherwise specified (PTCL, NOS) and was associated with distinct molecular features, including the production of Th2-associated cytokines. In addition, GATA-3 expression identified a subset of PTCL, NOS with distinct clinical features, including inferior progression-free and overall survival. Collectively, these data suggest that further understanding the cell of origin and lymphocyte ontogeny among the T-cell lymphomas may improve our understanding of the tumor microenvironment's pathogenic role in these aggressive lymphomas.
The GATA3 gene (GATA-binding protein 3) is one of the most frequently mutated genes in breast cancer. The objective of the current study was to determine the clinicopathologic characteristics of patients with breast cancer harboring GATA3 mutations.
The authors examined the somatic mutation status of GATA3 and performed survival analysis in The Cancer Genome Atlas (TCGA) cohort (n=934) and the Fudan University Shanghai Cancer Center (FUSCC) cohort (n=308). Patient characteristics, including age; menopausal status; tumor laterality; tumor size; lymph node status; tumor grade; molecular subtypes; adjuvant radiotherapy, chemotherapy, and endocrine therapy; and prognosis, together with PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) and TP53 (tumor protein p53) mutation status, were collected.
GATA3 mutations were detected in 8.8% of patients (82 of 934 patients) in the TCGA cohort and 14.9% of patients (46 of 308 patients) in the FUSCC cohort. GATA3 mutations were found to be significantly associated with luminal-like breast cancer (P=.002 in the TCGA cohort and P<.001 in the FUSCC cohort), and were highly mutually exclusive to PIK3CA mutations (P=.001 in the TCGA cohort and P=.003 in the FUSCC cohort) and TP53 mutations (P<.001 in both cohorts). Furthermore, GATA3 mutations were correlated with improved overall survival in the entire population (P=.025 in the TCGA cohort and P = .043 in the FUSCC cohort) as well as in patients with luminal-like disease who received adjuvant endocrine therapy.
GATA3 mutations mainly occur in patients with luminal-like breast cancer and have identifiable clinicopathologic and genetic characteristics, highlighting a subgroup of patients with breast cancer in whom limited therapy may be appropriate.
The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuR(fl/+)) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuR(fl/fl)) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.
Destructive periodontitis is associated with a Th1-Th17 immune response and activation of RANKL-induced osteoclasts. In addition, Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17 response. This study aimed to investigate whether these P. gingivalis serotypes induce higher osteoclasts activation, by increased Th17-associated RANKL production, and an antigen-specific memory T-lymphocyte response.
The RANKL production and TRAP(+) osteoclast induction were quantified on naïve T lymphocytes stimulated with dendritic cells primed with the P. gingivalis serotypes. The T-bet, GATA-3, RORC2 and Foxp3 expression was correlated with RANKL production. The frequency of proliferating memory T lymphocytes in response to P. gingivalis serotypes was determined in both periodontitis and healthy subjects.
T lymphocytes stimulated by K1 or K2-primed dendritic cells elicited higher levels of RANKL and TRAP(+) osteoclasts than cells stimulated with the other serotypes. RANKL positively correlated with RORC2. Whereas periodontitis patients had a higher frequency of memory T lymphocytes responding to K1 or K2, healthy subjects had a higher frequency of memory T lymphocytes responding to K4 or K(-) .
P. gingivalis serotypes K1 and K2, but not others, are associated with an increased production of the osteoclastogenesis-related factor RANKL. This important information suggests that these serotypes could elicit a greater bone resorption in vivo and have a role in the periodontitis pathogenesis.
Vitamin D suppresses inflammation and vitamin D deficiency is linked to the severity of asthma symptoms. T-helper type 9 (TH9) cells are important in the pathogenesis, yet the effects of vitamin D on this subset of inflammatory T-helper cells from patients with chronic asthma is unknown.
To determine the effects of vitamin D and dexamethasone on TH9 memory cells from adults with chronic persistent asthma and on a recall response to dust mite allergen.
T-helper memory cells were cultured with cytokines that drive TH9 polarization with vitamin D and/or dexamethasone. Peripheral blood mononuclear cells (PBMCs) from patients with radioallergosorbent test results for house dust mite were stimulated with allergen in the presence or absence of vitamin D. Intracellular cytokines, transcription factors, and identification of cell surface phenotypic markers were determined by flow cytometry.
Vitamin D decreased interleukin (IL)-9, IL-5, and IL-8 but increased IL-13(+) cells in TH9 cultures. Transcription factors PU.1 and interferon regulatory factor 4 were downregulated by vitamin D but not GATA3 and c-MAF. When PBMCs from patients with positive radioallergosorbent test results were stimulated with dust mite allergen, vitamin D decreased IL-9, IL-5, and IL-13 in T-helper cells (CD4(+)). TH9 cells present in a recall response were classically TH2 (CD294(+)), and polarization by transforming growth factor-β and IL-4 altered that phenotype.
Vitamin D decreased inflammatory cytokine profiles in TH9 memory cells and CD4(+) cells stimulated with dust mite allergen. Vitamin D is additive with dexamethasone in decreasing inflammatory cytokine production from T-cell subsets implicated in asthma.
Allergic sensitization is initiated by allergen-specific Th2-cell responses. Data on early allergen-specific T-cell responses in allergic children are scarce. We hypothesized that allergen-specific Th2-cell responses can be detected preceding sensitization. Therefore, peripheral blood mononuclear cells (PBMC) of nonsensitized, 'not-yet' sensitized or sensitized children were cultured with highly purified allergens. Cytokine levels in supernatant were determined using multiplex assay and GATA3 expression by flow cytometry. PBMC of sensitized children aged 3 and 5 years showed higher production of IL4, IL5 and IL13 and higher expression of GATA3 in response to purified allergens compared to nonsensitized children. PBMC of children that were 'not-yet' sensitized already showed higher levels of IL5 and IL13 and higher GATA3 expression at age 3 years. This shows that allergen-specific in vitro Th2 responses precede the detection of allergen-specific IgE, which can provide a window of opportunity for novel therapeutic interventions.
T helper (Th)2 cells have been proposed to play a neuroprotective role in multiple sclerosis (MS). This is mainly based on "loss-of-function" studies in an animal model for MS, experimental autoimmune encephalomyelitis (EAE), using blocking antibodies against Th2 related cytokines, and knockout mice lacking Th2-related molecules. We tested whether an increase of Th2 responses ("gain-of-function" approach) could alter EAE, the approach of novel GATA binding protein 3 (GATA3)-transgenic (tg) mice that overexpress GATA3, a transcription factor required for Th2 differentiation. In EAE induced with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, GATA3-tg mice had a significantly delayed onset of disease and a less severe maximum clinical score, compared with wild-type C57BL/6 mice. Histologically, GATA3-tg mice had decreased levels of meningitis and demyelination in the spinal cord, and anti-inflammatory cytokine profiles immunologically, however both groups developed similar levels of MOG-specific lymphoproliferative responses. During the early stage, we detected higher levels of interleukin (IL)-4 and IL-10, with MOG and mitogen stimulation of regional lymph node cells in GATA3-tg mice. During the late stage, only mitogen stimulation induced higher IL-4 and lower interferon-γ and IL-17 production in GATA3-tg mice. These results suggest that a preexisting bias toward a Th2 immune response may reduce the severity of inflammatory demyelinating diseases, including MS.
Interleukin (IL)-17A contributes to the development of asthma, especially in severe asthma which has characteristic neutrophil infiltration in airways. However, IL-17A-blocking antibody could escalate T helper (Th) 2 cytokines, such as IL-13, IL-4 in murine models. We aimed at determining the effect of mycobacterial Ag85A and IL-17A fusion protein—Ag85A-IL-17A on airway inflammation in a murine model of asthma. IL-17A recombinant protein fused mycobacterial immunodominant antigen Ag85A was constructed, expressed and purified. The fusion protein was then administrated into BALB/c mice and its anti-inflammatory effects in the infiltration of inflammatory cells, Th2/Th17 cytokines in BALF, histopathological changes of lung tissues as well as chemokines in lung tissues were evaluated in the murine model of asthma. We found that administration of mycobacterial Ag85A and IL-17A fusion protein induced IL-17A specific immunoglobulin (Ig)G in sera and significantly decreased IL-17A and IL-6 levels in bronchoalveolar lavage fluid (BALF). Ag85A-IL-17A vaccinated mice also showed marked reduction in the infiltration of inflammatory cells in peribronchiolar region and significant decrease in total cells, eosinophil cells and neutrophil cells in BALF. The increased levels of IL-13 and IL-4 in BALF of ovalbumin-sensitized mice were significantly reduced by the administration of Ag85A-IL-17A. Furthermore, CD3+CD4+IL-13+ splenocytes stimulated with OVA and CXCL1 mRNA, CCL2 mRNA and GATA-3 mRNA expressed in lung tissues were decreased markedly in Ag85A-IL-17A vaccinated group. Our results demonstrate remarkable antiallergic effects of Ag85A-IL-17A in a murine model of asthma and it may have protective effects on allergic asthma.
The transcription factor GATA3 is known as a breast tumor suppressor as well as a urothelial marker, and its loss is often seen in high-grade invasive bladder cancer. Nonetheless, GATA3 functions in bladder cancer cells remain largely unknown. In this study, we assessed the effects of GATA3 silencing via RNA interference on cell migration, invasion, and proliferation of bladder cancer. GATA3 expression was downregulated in all four bladder cancer lines examined, compared with a non-neoplastic urothelial line SVHUC. Knockdown of GATA3 in the bladder cancer lines (5637, TCC-SUP, J82) resulted in promotion of cell migration and invasion as well as increases in the expression of their related molecules, such as vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9, and the activity of MMP-2 and MMP-9. GATA3 loss was also associated with an increasing level of a mesenchymal marker N-cadherin and a decreasing level of an epithelial marker β-catenin. Consistent with these findings, enforced expression of GATA3 in UMUC3 inhibited cell migration and invasion. However, GATA3 showed marginal effects on bladder cancer cell viability and the expression of cell cycle- or apoptosis-related molecules. Additionally, in contrast to bladder cancer lines, no significant effects of GATA3 silencing on cell migration were seen in SVHUC. These findings suggest that GATA3 plays an important role in the prevention of bladder cancer progression and metastasis by inhibiting cell migration and invasion as well as epithelial-to-mesenchymal transition.
We present the case of a Dutch family with a new mutation (c523_528dup) in GATA3 causing HDR syndrome. HDR syndrome is characterised by hypoparathyroidism, deafness and renal defects. In this study, we describe the audiometric characteristics of 5 patients from this family. Their hearing impairment was congenital, bilateral and symmetric. Audiograms showed mild-to-moderate hearing impairment with a flat audiogram configuration. Higher frequencies tended to be affected more strongly. Cross-sectional analyses showed no progression, and a mean audiogram was established. Psychophysical measurements in 3 HDR patients - including speech reception in noise, loudness scaling, gap detection and difference limen for frequency - were obtained to assess hearing function in greater detail. Overall, the results of the psychophysical measurements indicated characteristics of outer hair cell loss. CT scanning showed no anomalies in 3 of the HDR patients. Although 2 patients displayed vestibular symptoms, no anomalies in the vestibular system were found by vestibulo-ocular examination. Our results are in agreement with the theory that outer hair cell malfunctioning can play a major role in HDR syndrome.
Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease. CAKUT can be caused by monogenic mutations; however, data are lacking on their frequency. Genetic diagnosis has been hampered by genetic heterogeneity and lack of genotype-phenotype correlation. To determine the percentage of cases with CAKUT that can be explained by mutations in known CAKUT genes, we analyzed the coding exons of the 17 known dominant CAKUT-causing genes in a cohort of 749 individuals from 650 families with CAKUT. The most common phenotypes in this CAKUT cohort were vesicoureteral reflux in 288 patients, renal hypodysplasia in 120 patients, and unilateral renal agenesis in 90 patients. We identified 37 different heterozygous mutations (33 novel) in 12 of the 17 known genes in 47 patients from 41 of the 650 families (6.3%). These mutations include (number of families): BMP7 (1), CDC5L (1), CHD1L (5), EYA1 (3), GATA3 (2), HNF1B (6), PAX2 (5), RET (3), ROBO2 (4), SALL1 (9), SIX2 (1), and SIX5 (1). Furthermore, several mutations previously reported to be disease-causing are most likely benign variants. Thus, in a large cohort over 6% of families with isolated CAKUT are caused by a mutation in 12 of 17 dominant CAKUT genes. Our report represents one of the most in-depth diagnostic studies of monogenic causes of isolated CAKUT in children.
Except after neck surgery, hypoparathyroidism is a rare disease caused by defects in genes involved in parathyroid gland development (TBX1/22q11.2 del, GCMB, GATA3, TBCE) or function [calcium sensing receptor (CASR), GNA11, PTH], or the autoimmune polyglandular syndrome type 1 (AIRE). Approximately 90% of sporadic cases and 30% of familial cases of isolated hypoparathyroidism remain unexplained. Recurrent missense mutations in AP2S1, a calcium-sensing receptor regulator, have been recently identified in familial hyperparathyroidism.
The aim of the study was to investigate AP2S1 as a putative hypoparathyroidism-causing gene.
Sequencing analysis and quantitative genomic PCR of the AP2S1 gene in a large cohort of 10 index cases (from nine families) and 50 sporadic cases affected with isolated hypoparathyroidism were investigated.
None of the 60 patients presented with nucleotidic changes or copy number variation in the AP2S1 gene, thereby excluding AP2S1 defects as a frequent cause of isolated hypoparathyroidism.
The usefulness of GATA3 (GATA-binding protein 3 to DNA sequence [A/T]GATA[A/G]) as a marker for metastatic breast carcinoma in serous effusion specimens was investigated.
Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial, and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from the breast (30 specimens), female genital tract (13 specimens), gastrointestinal tract (7 specimens), lung adenocarcinoma (9 specimens), pancreas (1 specimen), kidney (1 specimen), and bladder (1 specimen). The breast carcinoma cases included 15 ductal carcinomas and 8 lobular carcinomas; the histology subtype was not available for 7 specimens. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and gross cystic disease fluid protein of 15 kilodaltons (GCDFP-15) to compare their sensitivity with GATA3.
Positive nuclear staining for GATA3 was found to be present in 90% of metastatic breast carcinoma specimens (27 of 30 specimens). All nonbreast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases demonstrated weak positivity of benign lymphoid cells. Staining results were unambiguous because all positive cases demonstrated intense nuclear staining in > 50% of tumor cells. Mammaglobin (57% staining; 17 of 30 cases) and GCDFP-15 (33% staining; 10 of 30 cases) were found to be less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only 1 additional case compared with those stained with GATA3.
GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration.
Group 3 innate lymphoid cells (ILC3) include IL-22-producing NKp46(+) cells and IL-17A/IL-22-producing CD4(+) lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and ILC2 development from hematopoietic stem cells (HSCs) and for IL-5 and IL-13 production by T cells and ILC2, the role for Gata3 in the generation or function of other ILC subsets is not known. We found that abundant GATA-3 protein is expressed in mucosa-associated ILC3 subsets with levels intermediate between mature B cells and ILC2. Chimeric mice generated with Gata3-deficient fetal liver hematopoietic precursors lack all intestinal RORγt(+) ILC3 subsets, and these mice show defective production of IL-22 early after infection with the intestinal pathogen Citrobacter rodentium, leading to impaired survival. Further analyses demonstrated that ILC3 development requires cell-intrinsic Gata3 expression in fetal liver hematopoietic precursors. Our results demonstrate that Gata3 plays a generalized role in ILC lineage determination and is critical for the development of gut RORγt(+) ILC3 subsets that maintain mucosal barrier homeostasis. These results further extend the paradigm of Gata3-dependent regulation of diversified innate ILC and adaptive T cell subsets.
High-fat diets (HFD) promote the development of both obesity and fatty liver disease through the up-regulation of hepatic lipogenesis. Insulin resistance, a hallmark of both conditions, causes dysfunctional fuel partitioning and increases in lipogenesis. Recent work has demonstrated that systemic insulin resistance occurs in as little as the first 72 hours of an HFD, suggesting the potential for hepatic disruption with HFD at this time point. The current study sought to determine differences in expression of lipogenic genes between sexes in 3-month-old male and female Long-Evans rats after 72 hours of a 40% HFD or a 17% fat (chow) diet. Owing to the response of estrogen on hepatic signaling, we hypothesized that a sexual dimorphic response would occur in the expression of lipogenic enzymes, inflammatory cytokines, apoptotic, and cell repair and remodeling genes. Both sexes consumed more energy when fed an HFD compared with their low fat-fed controls. However, only the males fed the HFD had a significant increase in body fat. Regardless of sex, HFD caused down-regulation of lipogenic and inflammatory genes. Interestingly, females fed an HFD had up-regulated expression of apoptotic and cell repair-related genes compared with the males. This may suggest that females are more responsive to the acute hepatic injury effects caused by HFDs. In summary, neither male nor female rats displayed disrupted hepatic metabolic pathways after 72 hours of the HFD treatment. In addition, female rats appear to have protection from increases in fat deposition, possibly due to increased caloric expenditure; male rats fed an HFD were less active, as demonstrated by distance traveled in their home cage.
Airway inflammation in allergic asthma reflects a threshold response of the innate immune system, including group 2 innate lymphoid cells (ILC2), followed by an adaptive Th2 cell-mediated response. Transcription factor Gata3 is essential for differentiation of both Th2 cells and ILC2. We investigated the effects of enforced Gata3 expression in T cells and ILC2 on the susceptibility of mice to allergic airway inflammation (AAI). We used CD2-Gata3 transgenic (Tg) mice with enforced Gata3 expression driven by the CD2 promoter, which is active both in T cells and during ILC2 development. CD2-Gata3 Tg mice and wild-type (WT) littermates were analyzed in mild models of AAI without adjuvants. Whereas OVA allergen exposure did not induce inflammation in WT controls, CD2-Gata3 Tg mice showed clear AAI and enhanced levels of IL-5 and IL-13 in bronchoalveolar lavage. Likewise, in house dust mite-driven asthma, CD2-Gata3 Tg mice were significantly more susceptible to AAI than WT littermates, whereby both ILC2 and Th2 cells were important cellular sources of IL-5 and IL-13 in bronchoalveolar lavage and lung tissue. Compared with WT littermates, CD2-Gata3 Tg mice contained increased numbers of ILC2, which expressed high levels of IL-33R and contributed significantly to early production of IL-4, IL-5, and IL-13. CD2-Gata3 Tg mice also had a unique population of IL-33-responsive non-B/non-T lymphoid cells expressing IFN-γ. Enforced Gata3 expression is therefore sufficient to enhance Th2 and ILC2 activity, and leads to increased susceptibility to AAI after mild exposure to inhaled harmless Ags that otherwise induce Ag tolerance.
Determining the indications for adjuvant chemotherapy (CT) in patients with hormone receptor (HR)-positive/HER2-negative breast cancer are difficult. The transcription factors GATA-binding protein 3 (GATA-3) and Forkhead-box protein A1 (FOXA1) are crucial for the hormone responsive phenotype of breast cancer. This study evaluated whether the expression of GATA-3 and FOXA1 is a prognostic and predictive marker of outcomes in patients with HR-positive/HER2-negative breast cancer.
The expression of GATA-3 and FOXA1 was analyzed immunohistochemically in 214 patients with invasive breast cancer to evaluate the association with the clinicopathological features and the prognosis.
GATA-3 expression was positively correlated with FOXA1 expression (P < 0.0001). Both GATA-3 and FOXA1 were positively correlated with ER (P < 0.0001 each) and PR expression (P = 0.0001 and P = 0.0009, respectively), and inversely correlated with nuclear grade (P = 0.0002 and P = 0.0018, respectively) and Ki67 index (P = 0.0052 and P = 0.0049, respectively). Expression of GATA-3 and FOXA1 was associated with better prognosis. FOXA1 was an independent favorable prognostic marker in HR-positive/HER2-negative breast cancer. Disease-free survival rates were similar in patients with HR-positive/HER2-negative breast cancer and high FOXA1 expression given adjuvant hormone therapy (HT) alone and those given CT plus HT.
GATA-3 and FOXA1 are associated with a less aggressive phenotype and a better prognosis in patients with HR-positive/HER2-negative breast cancer. FOXA1 may be useful in identifying those patients who may not require adjuvant CT.
The T-cell immunoglobulin and mucin domain-(Tim)-1 molecule and Tim-3 are mainly expressed on activated T helper (Th) 2 and Th1 cells, respectively, and have been implicated in the pathogenesis of some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between Tims and excessive immune responses in ITP remains unclear.
Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Tim-1, Tim-3, T-box transcription factor (T-bet) and GATA binding protein 3 (GATA-3) were measured in the peripheral blood mononuclear cells (PBMCs) of 45 newly diagnosed patients with active ITP, 34 ITP patients in remission and 31 healthy volunteers.
Tim-3 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Tim-1 mRNA was not significantly declined, which resulted in a decreased ratio of Tim-3 to Tim-1 in ITP patients with active disease. During the remission stages, the levels of these transcription factors were comparable with those observed in healthy controls.
The reduced levels of Tim-3/Tim-1 in PBMCs during active stages of the disease suggest a possible role in the pathogenesis and course of ITP. Regulating the balance of Tim-1 and Tim-3 in ITP patients could also be a therapeutic approach against ITP.
The ability to self-renew, be easily expanded in vitro and differentiate into different mesenchymal tissues, render mesenchymal stem cells (MSCs) an attractive therapeutic method for degenerative diseases. The subsequent discovery of their immunosuppressive ability encouraged clinical trials in graft-versus-host disease and auto-immune diseases. Despite sharing several immunophenotypic characteristics and functional capabilities, the differences between MSCs arising from different tissues are still unclear and the published data are conflicting.
Here, we evaluate the influence of human MSCs derived from umbilical cord matrix (UCM), bone marrow (BM) and adipose tissue (AT), co-cultured with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNC), on T, B and natural killer (NK) cell activation; T and B cells' ability to acquire lymphoblast characteristics; mRNA expression of interleukin-2 (IL-2), forkhead box P3 (FoxP3), T-bet and GATA binding protein 3 (GATA3), on purified T cells, and tumor necrosis factor-alpha (TNF-α), perforin and granzyme B on purified NK cells.
MSCs derived from all three tissues were able to prevent CD4+ and CD8+ T cell activation and acquisition of lymphoblast characteristics and CD56 dim NK cell activation, wherein AT-MSCs showed a stronger inhibitory effect. Moreover, AT-MSCs blocked the T cell activation process in an earlier phase than BM- or UCM-MSCs, yielding a greater proportion of T cells in the non-activated state. Concerning B cells and CD56 bright NK cells, UCM-MSCs did not influence either their activation kinetics or PHA-induced lymphoblast characteristics, conversely to BM- and AT-MSCs which displayed an inhibitory effect. Besides, when co-cultured with PHA-stimulated MNC, MSCs seem to promote Treg and Th1 polarization, estimated by the increased expression of FoxP3 and T-bet mRNA within purified activated T cells, and to reduce TNF-α and perforin production by activated NK cells.
Overall, UCM-, BM- and AT-derived MSCs hamper T cell, B cell and NK cell-mediated immune response by preventing their acquisition of lymphoblast characteristics, activation and changing the expression profile of proteins with an important role in immune function, except UCM-MSCs showed no inhibitory effect on B cells under these experimental conditions. Despite the similarities between the three types of MSCs evaluated, we detect important differences that should be taken into account when choosing the MSC source for research or therapeutic purposes.
Type 1 diabetes is due to destruction of pancreatic β-cells. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing β-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1β + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.
The natural product oleanolic acid is commonly found in a variety of medicinal plants. It is a triterpenoid compound known for its anti-inflammatory effects as well as various other pharmacological properties. The aim of the current study was to use a mouse model of allergic asthma to investigate whether oleanolic acid has anti-asthmatic effects, and if so, to determine the mechanism of these effects. Oleanolic acid suppressed eosinophil infiltration, allergic airway inflammation, and Penh, which occurred by suppressing the production of IL-5, IL-13, IL-17, and ovalbumin-specific IgE through the upregulation of T-bet and Foxp3, and the downregulation of GATA-3 and RORγt. The therapeutic effect of oleanolic acid was due to suppression of Th2 cytokines (IL-5 and IL-13), B cell-dependent production of OVA-specific IgE, and Gr-1 expression through the T-bet, GATA-3, retinoic acid-related orphan receptor γ t (RORγ t) and forkhead box p3 (Foxp3) transcription pathways. It is therefore reasonable to suggest that the anti-inflammatory and anti-asthmatic effects of oleanolic acid may be exerted through inhibition of the GATA-3 and RORγt pathways.
A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1-can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.
Moderate physical activity has been shown to promote immunity. Different moderate physical activities may have different effects on immunity. This study investigated the impacts of a 12-week regular music aerobic exercise (MAE) program on leukocyte distribution, lymphocyte subsets, and lymphocyte polarization.
The study used a case-control design with pretest and posttest. Forty-seven middle-age women were recruited for this study. Three participants dropped out, 22 completed the 12-week MAE program, and the other 22 participants who had heat-intolerance or limited schedule eligibility were enrolled as the control group without the MAE exercise.
Results showed that the MAE exercise for 12 weeks didn't change red blood cells or total leukocytes but increased lymphocyte counts. The women in MAE group revealed significant increases (P ≤ 0.01) of CD3CD4, CD3CD8, and CD4CD25 cells, associated with Treg polarization showing enhanced FoxP3 but not T-bet, Gata-3, or RORγT expression (P < .01). The control group without exercise revealed insignificant change of lymphocyte subsets or lymphocyte polarization.
This study shows that MAE increases specific lymphocyte subsets and enhances Treg cell differentiation. It is suggested to encourage moderate physical activity of music aerobic exercise to enhance lymphocyte function of middle-aged women.
The aim of the present study was to investigate the therapeutic effect of halofuginone (HF) in the treatment of idiopathic thrombocytopenic purpura (ITP) and explore the underlying mechanism. Sixty ITP mice were divided into four groups including control group, low dose group (25 mg/kg HF), medium dose group (50 mg/kg HF), and high dose group (100 mg/kg HF). Corresponding dose of HF was administrated by gavage daily in HF groups for 7 days, and the same volume of saline was given in control group. Platelet counts were 28.87 ± 3.91 × 10(9)/L, 57.13 ± 2.75 × 10(9)/L, 86.73 ± 3.06 × 10(9)/L and 89.73 ± 2.84 × 10(9)/L in control group, low dose group, medium dose group, and high dose group respectively, on day 7 after intragastrically administration of HF or saline. Compared with control group, three HF groups showed significantly increased levels of INF-γ and IL-2 (all P < 0.05), and significantly decreased concentrations of IL-4 and IL-10(all P < 0.05). The expression of T-bet mRNA increased and the expression of GATA-3 mRNA decreased (all P < 0.05) in ITP mice after intragastric administration with different dose of HF. HF significantly recovered peripheral platelet counts in ITP mice through promoting Th1 cell differentiation and attenuating Th2 differentiation in ITP mice.
Variants of the Bach2 gene are linked to vitiligo, celiac disease, and type 1 diabetes, but the underlying immunological mechanisms are unknown. In this study, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence and governing the differentiation, activation, and survival of Foxp3(+) regulatory T (Treg) cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of Th1/Th2-type cytokines. Without Bach2, Treg cells exhibit diminished Foxp3 expression, depleted numbers, hyperactivation, enhanced proliferation, and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim/Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios(-)Foxp3(+) Treg cells and a Treg cell transcriptome skewed toward the Th1/Th2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: 1) is indispensable for TCR/TGF-β-induced Foxp3 expression; and 2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3-driven Th2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal Th2-type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells, and protects against immune-mediated diseases.
Asthma is one of the serious global health problems and cause of huge mortality and morbidity. It is characterized by persistent airway inflammation, airway hyperresponsiveness, increased IgE levels and mucus hypersecretion. Asthma is mediated by dominant Th2 immune response, causing enhanced expression of Th2 cytokines. These cytokines are responsible for the various pathological changes associated with allergic asthma.
The role of Th2 cells in the pathogenesis of the asthma is primarily mediated through the cytokine IL-13, also produced by type 2 innate lymphoid cells, that comes under the transcriptional regulation of GATA3. In this review we will try to explore the link between IL-13 and GATA3 in the progression and regulation of asthma and its possible role as a therapeutic target.
Inhibition of GATA3 activity or blockade of GATA3 expression may attenuate the interleukin-13 mediated asthma phenotypes. So, GATA3 might be a potential therapeutic target for the treatment of allergic asthma.
Follicular helper T (Tfh) cells are dedicated to providing help to B cells and are strongly associated with antibody-mediated autoimmune disease. B cell lymphoma 6 (Bcl-6) is a key transcription factor of Tfh cells, and IL-21 is known to be a critical cytokine produced by Tfh cells. We silenced Bcl-6 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of Bcl-6 short hairpin RNAs (shRNAs) in experimental autoimmune myasthenia gravis (EAMG). Our data demonstrate that CD4(+)CXCR5(+)PD-1(+) Tfh cells, Bcl-6 and IL-21 were significantly increased in EAMG mice, compared with controls. In addition, we found that frequencies of Tfh cells were positively correlated with the levels of serum anti-AChR Ab. In-vivo transduction of lenti-siRNA-Bcl6 ameliorates the severity of ongoing EAMG with decreased Tfh cells, Bcl-6 and IL-21 expression, and leads to decreased anti-AChR antibody levels. Furthermore, we found that siRNA knockdown of Bcl-6 expression increases the expression of Th1(IFN-γ, T-bet) and Th2 markers (IL-4 and GATA3), but failed to alter the expression of Th17-related markers (RORγt, IL-17) and Treg markers (FoxP3). Our study suggests that Tfh cells contribute to the antibody production and could be one of the most important T cell subsets responsible for development and progression of EAMG or MG. Bcl-6 provides a promising therapeutic target for immunotherapy not only for MG, but also for other antibody-mediated autoimmune diseases.
GATA3 belongs to the GATA transcription factor family and is a crucial regulator of lymphocyte differentiation. More recently, GATA3 was shown to be involved in skin cell lineage determination, in morphogenesis and maintenance of hair follicle keratinocytes as well as in epidermal barrier formation in mouse. In human, the potential role of GATA3 in the regulation of interfollicular epidermal homeostasis was still poorly explored. We thus investigated whether GATA3 could play a role in the regulation of proliferation and/or differentiation processes in human primary keratinocytes. We silenced the expression of GATA3 by small interfering RNA in either proliferating or differentiated human primary keratinocytes and analyzed the effect on cell proliferation and differentiation. We showed that GATA3 inhibition increased cell number, BrdU incorporation and expression of the proliferation markers PCNA and Ki67, demonstrating that GATA3 can inhibit keratinocyte proliferation. Moreover, GATA3 seems to be able to induce keratinocyte differentiation since its silencing leads to a decrease of both early and late differentiation markers such as Keratins 1 and 10, Involucrin and Loricrin. Our results demonstrate that GATA3 transcription factor inhibits proliferation and induces differentiation of primary keratinocytes, which suggest that it may regulate human interfollicular epidermal renewal.
Allergies are mainly characterized as an unrestrained Th2-biased immune response. Epidemiological data associate protection from allergic diseases with the exposure to certain infectious agents during early stages of life. Modulation of the immune response by pathogens has been considered to be a major factor influencing this protection. Recent evidence indicates that immunoregulatory mechanisms induced upon infection ameliorate allergic disorders. A longitudinal study has demonstrated reduced frequency and incidence of asthma in children who reported a prior infection with Salmonella. Experimental studies involving Salmonella enterica serovar Typhimurium-infected murine models have confirmed protection from induced allergic airway inflammation; however, the underlying cause leading to this amelioration remains incompletely defined. In this study, we aimed to delineate the regulatory function of Salmonella Typhimurium infection in the amelioration of allergic airway inflammation in mice. We observed a significant increase in CD11b+ Gr1+ myeloid cell populations in mice after infection with S. Typhimurium. Using in vitro and in vivo studies, we confirmed that these myeloid cells reduce airway inflammation by influencing Th2 cells. Further characterization showed that the CD11b+ Gr1+ myeloid cells exhibited their inhibitory effect by altering GATA-3 expression and interleukin-4 (IL-4) production by Th2 cells. These results indicate that the expansion of myeloid cells upon S. Typhimurium infection could potentially play a significant role in curtailing allergic airway inflammation. These findings signify the contribution of myeloid cells in preventing Th2-mediated diseases and suggest their possible application as therapeutics.
Often the distinction of cutaneous apocrine carcinoma from metastatic mammary apocrine carcinoma to the skin can be a diagnostic dilemma because both tumors share similar histologic features and have overlapping immunohistochemical profile. We compared the expression of adipophilin, cytokeratin 5/6, p63, GATA3, mammaglobin, androgen receptor, estrogen receptor, progesterone receptor, and HER2 by immunohistochemistry in 14 cutaneous apocrine carcinomas (11 primary tumors, 3 metastases) and 26 primary apocrine carcinomas of the breast. Whereas focal adipophilin staining was seen in 36% (5/14) of cutaneous apocrine carcinoma, strong and diffuse adipophilin staining was seen in 88% (22/25) of mammary apocrine carcinoma (P = .0013). Differences in estrogen receptor and progesterone receptor expression were also statistically significant (P = .018 and .043). Androgen receptor was strongly positive in all cutaneous and mammary cases. Although there was no significant difference in the frequency of expression of cytokeratin 5/6, p63, HER2, GATA3, and mammaglobin in cutaneous apocrine carcinoma versus mammary apocrine carcinoma, strong and diffuse cytokeratin 5/6 and/or mammaglobin expression were seen only in cutaneous apocrine carcinoma. In conclusion, cutaneous apocrine carcinoma is likely adipophilin- ER+ PR+/- HER2- and can exhibit strong and diffuse cytokeratin 5/6 and/or mammaglobin expression. On the contrary, a mammary apocrine carcinoma is likely adipophilin+ ER- PR- and often exhibit 3+ HER2 with corresponding HER2 gene amplification. A panel of adipophilin, ER, PR, HER2, cytokeratin 5/6, and mammaglobin may be helpful in distinguishing cutaneous apocrine carcinoma from mammary apocrine carcinoma.
To investigate the role of polymorphic variants of immune-response modifying genes in predisposition to asthma.
The analysis of restriction fragments length polymorphism was used to investigate 10 single-nucleotide polymorphisms: IFNG rs2069705, IFNGR2 rs17880053, IL4 rs 2070874, IL4RA rs 1805010, GATA3 rs10905277, TBX21 rs11652969, PIASY rs3760903, PIAS3 rs12756687, STATS rs16967593, and SOCS5 rs6737848 in 106 asthma patients and 115 healthy people.
The rs6737848 SOCS5 polymorphism was significantly associated with asthma in additive model (p = 0.05, OR = 0.338, 95% CI 0.158-0.723) and in dominant model (p = 0.02, OR = 0.284, CI 0.126-0.638). None of the polymorphisms of the studied genes was associated with total IgE levels.
This is the first report on the association of rs6737848 SOCS5 with asthma.
Expression of the estrogen receptor-α (ERα) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the -150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.
Gastric cancer is a major health problem worldwide; it is the second most common cause of cancer death in the world. Recent studies indicate that the high-mobility group (HMG) of chromosomal proteins is associated with cancer progression. However, HMGB3 has been little studied. We analyzed the co-expression network between HMGB3 and differentially-expressed genes in the GSE17187 database, identifying the relevant transcription factors, and the conserved domain of HMGB3 to understand the underlying regulation mechanisms involved in gastric cancer. Thirty-one relationships between 11 differentially-expressed genes were included in a co-expression network; many of these genes have been identified as related to cancer, including TBX5 and TFR2. Further analysis identified nine transcription factors, these being GATA3, MZF1, GATA1, GATA2, SRY, REL, NFYB, NFYC, and NFYA, which could interact with HMGB3 to regulate target gene expression and consequently regulate gastric cancer cell proliferation, migration and invasion. The HMG-box domain was very similar in various species, with only a few amino acid changes, indicating conserved functions in HMG-box. This information helps to provide insight into the molecular mechanisms of HMGB3 in human gastric cancer.
Abnormal cellular immunity induced by deranged Th1/Th2 profile has been revealed to play a critical role in the pathogenesis of immune thrombocytopenic purpura (ITP). Correction of the shifted Th1/Th2 balance represents a potential therapeutic approach to treat ITP. Here, we investigated the effects of IL-11 on the restoration of Th1/Th2 balance in the peripheral blood mononuclear cells (PBMCs) isolated from adult ITP patients. As shown here, we observed a higher ratio of T-bet/GATA-3 gene expression by quantitative real-time PCR in the PBMCs from ITP patients, consistent with the presence of an abnormally high Th1/Th2 ratio. Remarkably, upon IL-11 treatment, a reversal of T-bet/GATA-3 ratio in ITP was achieved and was shown to be responsible for the restoration of Th1/Th2 balance, with IL-11 at 100 ng/ml demonstrating the highest efficiency. T-bet and GATA-3 are the two transcriptional factors that have been indicated to be the master regulators for Th1 and Th2 lineage commitment, respectively. In the presence of 100 ng/ml IL-11, GATA-3 transcript abundance rose up to ~85-fold of that measured in untreated cells, whereas T-bet transcripts were lowered merely to ~41%, suggesting that GATA-3 was the major contributor for the reversal of T-bet/GATA-3 ratio. Thus, our findings may very well encourage the development of novel medicines that specifically target and correct the T-bet/GATA-3 imbalance identified in ITP.
Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor γ (PPARγ). Previous two-dimensional proteomics studies using C3H10T1/2 murine mesenchymal pluripotent stem cells revealed that prolyl hydroxylase domain protein (PHD) levels significantly increased during rosiglitazone-induced adipocyte differentiation (RIAD). In this study, we investigated the functional role played by PHD during RIAD. Three PHD isoforms (PHD1, 2, and 3) were found to be up-regulated in C3H10T1/2 cells during RIAD, whereas PHD knockdown and treatment with PHD inhibitors (dimethyloxalyl glycine or ethyl-3,4-dihydroxybenzoate) blocked RIAD. PHD inhibition was found to be associated with increases in the levels of anti-adipogenic proteins such as GATA-3, KLF-2, and transcriptional coactivator with PDZ binding motif (TAZ), with their reduced ubiquitination, suggesting that PHDs evoke the ubiquitination/proteasomal degradation of anti-adipogenic proteins. On the other hand, MG-132 (a proteasomal inhibitor) prevented the degradation of anti-adipogenic proteins and retarded RIAD. PPARγ antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPARγ binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPARγ activation. Consistent with in vitro results, oral administration of rosiglitazone to ob/ob mice for 2 weeks increased adipose PHD levels and decreased anti-adipogenic protein levels by increasing their ubiquitination. These results suggest that rosiglitazone increases PHD expression in a PPARγ-dependent manner and that this leads to the commitment of anti-adipogenic proteins to the ubiquitination-proteasomal pathway and to the subsequent induction of adipocyte differentiation.
As the conduit for nutrients and growth signals, the placenta is critical to establishing an environment sufficient for fetal growth and development. To better understand the mechanisms regulating placental development and gene expression, we characterized the transcriptome of term placenta from 20 healthy women with uncomplicated pregnancies using RNA-seq. To identify genes that were highly expressed and unique to the placenta we compared placental RNA-seq data to data from 7 other tissues (adipose, breast, hear, kidney, liver, lung, and smooth muscle) and identified several genes novel to placental biology (QSOX1, DLG5, and SEMA7A). Semi-quantitative RT-PCR confirmed the RNA-seq results and immunohistochemistry indicated these proteins were highly expressed in the placental syncytium. Additionally, we mined our RNA-seq data to map the relative expression of key developmental gene families (Fox, Sox, Gata, Tead, and Wnt) within the placenta. We identified FOXO4, GATA3, and WNT7A to be amongst the highest expressed members of these families. Overall, these findings provide a new reference for understanding of placental transcriptome and can aid in the identification of novel pathways regulating placenta physiology that may be dysregulated in placental disease.
In vertebrates, six GATA transcription factors, GATA1 through GATA6, have been identified and GATA1-3 is known to be involved in hematopoietic developments, while GATA4-6 play roles in cardiac and endoderm developments. Recently, we and others have found that GATA2 and GATA3 found in the trophectoderm plays a role in gene expression specific to this cell type, but GATA4-6 have not been well characterized in early embryonic developments. Using quantitative polymerase chain reaction (qPCR) and in situ hybridization, we examined the expression of GATA4, 5 and 6 messenger RNAs (mRNAs) in ovine conceptuses and uteri during the peri-implantation period. In ovine conceptuses, GATA4, 5 and 6 transcripts were present on days 15, 17 and 21 (day 0 = day of mating), and high GATA5 and 6 mRNAs were found on day 21, most of which were localized in the trophectoderm and endoderm. Moreover, minute and substantial GATA4 and 5 mRNAs were found in days 15 and 21 uterine endometria, respectively. Increase in GATA4-6 transcripts in day 21 uteri indicates that in addition to GATA1-3, GATA4-6 may also play a potentially novel role in the development of ovine trophectoderm, endoderm and/or uterine endometria following conceptus attachment to the uterine epithelium.
Information flow through neural circuits is determined by the nature of the synapses linking the subtypes of neurons. How neurons acquire features distinct to each synapse remains unknown. We show that the transcription factor Mafb drives the formation of auditory ribbon synapses, which are specialized for rapid transmission from hair cells to spiral ganglion neurons (SGNs). Mafb acts in SGNs to drive differentiation of the large postsynaptic density (PSD) characteristic of the ribbon synapse. In Mafb mutant mice, SGNs fail to develop normal PSDs, leading to reduced synapse number and impaired auditory responses. Conversely, increased Mafb accelerates synaptogenesis. Moreover, Mafb is responsible for executing one branch of the SGN differentiation program orchestrated by the Gata3 transcriptional network. Remarkably, restoration of Mafb rescues the synapse defect in Gata3 mutants. Hence, Mafb is a powerful regulator of cell-type specific features of auditory synaptogenesis that offers a new entry point for treating hearing loss. DOI: http://dx.doi.org/10.7554/eLife.01341.001.
Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.
To report on the clinicopathological features of 20 cases of microcystic urothelial bladder carcinoma.
The extent of microcystic component varied from 50-100% of the specimens. The cysts were round-oval and of varying sizes; the periphery of large cysts was frequently punctuated by many smaller cysts. The cysts were lined by urothelial, low columnar cells or by a single layer of flattened epithelium of low-intermediate nuclear grade. Focal high-grade conventional urothelial carcinoma was present in eight cases. Immunohistochemistry demonstrated variable positivity for cytokeratins 7 and 20, MUC1, MUC5AC, p63 and GATA3. Extent of expression of Ki67, p53 and p27(kip1) ranged from 20-60%, 10-40% and 10-30% of cells, respectively. On follow-up, 11 patients died of disease at 11-56 months and three patients were alive with disease at 26-37 months. Univariate survival analysis showed no differences for microcystic carcinoma versus conventional urothelial carcinoma (P = 0.548).
Microcystic urothelial carcinoma may pose diagnostic difficulties, especially in limited biopsy samples, where it may be mistaken for cystitis glandularis or adenocarcinoma of the bladder. Histological features, clinical history and appropriate immunohistochemical studies should help to distinguish it from its mimics. Aggressiveness seems to be related to higher stage at diagnosis.
A novel polysaccharide (LCP50W) with a molecular weight of 4.72 × 10(4) Da was isolated from the pulp tissues of Litchi chinensis . The chemical structure of LCP50W was characterized using physicochemical and instrumental analyses. The results indicated that the main chain of LCP50W consisted of (1→3)-linked β-L-rhamnopyranosyl, (1→6)-linked α-D-glucopyranosyl, and (1→2,6)-linked α-D-glucopyranosyl residues, which branched at O-6. The three branches consisted of (1→2)-linked α-L-rhamnopyranosyl, (1→3)-linked α-D-galactopyranosyl, and (1→3)-linked α-L-mannopyranosyl residues, terminated with (1→)-linked α-L-arabinopyranosyl residues, respectively. The in vitro immunomodulatory assay revealed that LCP50W promoted the proliferation of mouse splenocytes and enhanced the cytotoxicity of NK cells. LCP50W boosted the secretion of Th1 cytokine IFN-γ while it inhibited the secretion of Th2 cytokine IL-4; it also enhanced the expression of T-bet while it inhibited the expression of GATA-3. Additionally, LCP50W promoted the development of cell cycle toward the S phase.
Naive T helper cells differentiate into functionally distinct effector subsets that drive specialized immune responses. Recent studies indicate that some of the effector subsets have plasticity. Here, we used an EAE model and found that Th17 cells deficient in the transcription factor BCL11B upregulated the Th2-associated proteins GATA3 and IL-4 without decreasing RAR-related orphan receptor γ (RORγt), IL-17, and GM-CSF levels. Surprisingly, abnormal IL-4 production affected Th17 cell trafficking, diverting migration from the draining lymph nodes/CNS route to the mesenteric lymph nodes/gut route, which ameliorated EAE without overt colitis. T helper cell rerouting in EAE was dependent on IL-4, which enhanced retinoic acid (RA) production by dendritic cells, which further induced expression of gut-homing receptors CCR9 and α4β7 on Bcl11b-deficient CD4+ T cells. Furthermore, IL-4 treatment or Th2 immunization of wild-type mice with EAE caused no alteration in Th17 cytokines or RORγt, but diverted T helper cell trafficking to the gut, which improved EAE outcome without overt colitis. Our data demonstrate that Th17 cells are permissive to Th2 gene expression without affecting Th17 gene expression. This Th17 plasticity has an impact on trafficking, which is a critical component of the immune response and may represent a possible avenue for treating multiple sclerosis.
Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naïve ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4(+) and CD8α(+) cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α(+) cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.
Transcription factor GATA binding protein 3 (GATA3) has been suggested as a marker of urothelial carcinoma of the bladder and upper urinary tract. Its expression in primary and metastatic renal tumors has not been fully determined. We evaluated GATA3 expression in 47 oncocytomas, 196 primary renal cell carcinomas (RCCs) (71 clear cell, 53 papillary, 21 Xp11.2, 33 chromophobe RCCs, and 18 collecting duct carcinomas [CDC]), and 43 unrelated metastatic RCCs (41 clear cell and 2 Xp11.2 RCC). GATA3 nuclear expression was evaluated in tissue microarrays built from archival tissues using immunohistochemistry. Intensity (0 to 3+) and extent (percentage) of expression were assessed. Several cutoff values (>0%, >5%, and >10%) were evaluated to indicate GATA3 positivity. Among oncocytomas, 9 (19%) of 47 had some degree of nuclear GATA3 expression with median extent of 0% (0%-100%). When using either 5% or 10% cutoff values, 5 (11%) of 47 oncocytomas were positive. In primary RCCs, 6 (3%) of 196 had some degree of nuclear expression with a median extent of 0% (0%-100%). When using either 5% or 10% cutoff values, 2 cases remained positive (1%) (Xp11.2 and CDC). All metastatic RCCs were negative. We found an overall lack of GATA3 expression in primary and metastatic RCCs. GATA3 is expressed in a minority of oncocytomas, Xp11.2-RCC, and CDC. Given GATA3 positivity in upper urinary tract urothelial carcinoma, our findings support a role for GATA3 in the differential diagnosis of primary renal masses and a utility in the interrogation of metastatic tumors of unknown primary in the presence of a renal mass.
We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.
Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.
The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.
We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.
The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-κB (NF-κB) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.
Id-1 is a member of the helix-loop-helix family of proteins that regulates the activity of transcription factors to suppress cellular differentiation and to promote cell growth. Overexpression of Id-1 in tumor cells correlates with increased malignancy and resistance to chemotherapy and radiotherapy. Id-1B is an isoform generated by alternative splicing that differs from the classical Id-1 in the 13-C-terminal amino acids, whose function is at present unknown. We have studied the role of Id-1B in cancer and its expression in healthy/malignant lung tissues. Overexpression of Id-1B in A549 lung and PC3 prostate cancer cells reduced anchorage-dependent and independent proliferation and clonogenic potential. Moreover, it increased the proportion of cells in the G0/G1 phase of the cell cycle and p27 levels, while reduced phospho-Erk and cyclin A levels. Through microarray analysis, we identified genes involved in cell growth and proliferation that are specifically deregulated as a consequence of Id-1B overexpression, including IGF2, BMP4, Id2, GATA3, EREG and AREG. Id-1B overexpressing cells that were treated with 4Gy irradiation dose were significantly less resistant to cell death. In vivo assays demonstrated that tumors with high Id-1B levels exhibited less growth (p<0.01), metabolic activity (glucose uptake) and angiogenesis (p<0.05) compared to tumors with low Id-1B expression; mice survival was significantly extended (p<0.05). Quantification by qRT-PCR revealed that expression of Id-1B was significantly lower (p<0.01) in human lung tumors compared to their matched nonmalignant counterparts. In conclusion, our results demonstrate that Id-1B decreases the malignancy of lung and prostate cancer cells, sensitizes them to radiotherapy-induced cell death, and counteracts the protumorigenic role of the classical form of Id-1.
Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.
To analyze the dysregulated genes among the differentially expressed genes in 41 nasopharyngeal biopsy samples and identify their protective transcriptional factors.
The differentially expressed gene profiles were obtained by analyzing both types I and II nasopharyngeal carcinoma (NPC_I and NPC_II, respectively) using EXCEL and Bioinformatics tools. The transcriptional factors were further studied only when (1) the difference in the binding sites of the differentially expressed genes between NPC_I and NPC_II groups was statistically significant, (2) the expressions of the transcription factors were correlated with the gene expressions in the samples, and (3) the transcription factors affected at least 40% of the expression of the related genes.
In NPC_I samples, 80 transcription factors were found to be up-regulated, in which RUNX3, GATA3, NR3C1, NRF1, RXRA, SMAD7, TBP, and ZBTB6 were positive factors and HLF and MTF1 were negative factors, involved in the regulation of the genes in T cell receptor signaling pathway. No eligible transcription factors were found in association with down-regulated genes in NPC_I compared to NPC_II gene expression profiles.
The over-expressed genes in NPC_I are mainly related to immune responses, and we found 8 positive factors and 2 negative factors that regulate the genes in T cell receptor signaling pathway. The 10 transcription factors may serve as potential therapeutic targets for NPC_I. We failed to identify any transcription factors associated with down-regulated genes in NPC_I relative to NPC_II possibly as a result of multiple factors that affect the differential gene expressions in NPC_II including the transcription factors, DNA phosphorylation and modification, chromosome variation and environmental factors.
National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 suggested the efficacy of adjuvant trastuzumab, even in HER2-negative breast cancer. This finding prompted us to develop a predictive model for degree of benefit from trastuzumab using archived tumor blocks from B-31.
Case subjects with tumor blocks were randomly divided into discovery (n = 588) and confirmation cohorts (n = 991). A predictive model was built from the discovery cohort through gene expression profiling of 462 genes with nCounter assay. A predefined cut point for the predictive model was tested in the confirmation cohort. Gene-by-treatment interaction was tested with Cox models, and correlations between variables were assessed with Spearman correlation. Principal component analysis was performed on the final set of selected genes. All statistical tests were two-sided.
Eight predictive genes associated with HER2 (ERBB2, c17orf37, GRB7) or ER (ESR1, NAT1, GATA3, CA12, IGF1R) were selected for model building. Three-dimensional subset treatment effect pattern plot using two principal components of these genes was used to identify a subset with no benefit from trastuzumab, characterized by intermediate-level ERBB2 and high-level ESR1 mRNA expression. In the confirmation set, the predefined cut points for this model classified patients into three subsets with differential benefit from trastuzumab with hazard ratios of 1.58 (95% confidence interval [CI] = 0.67 to 3.69; P = .29; n = 100), 0.60 (95% CI = 0.41 to 0.89; P = .01; n = 449), and 0.28 (95% CI = 0.20 to 0.41; P < .001; n = 442; P(interaction) between the model and trastuzumab < .001).
We developed a gene expression-based predictive model for degree of benefit from trastuzumab and demonstrated that HER2-negative tumors belong to the moderate benefit group, thus providing justification for testing trastuzumab in HER2-negative patients (NSABP B-47).
Contact hypersensitivity (CHS) mouse model induced by 2, 4-dinitrofluorobenzene (DNFB) is thought to be a T helper 1 (Th1)-dominant response and used for investigating anti-inflammatory and immunosuppressive agents. However, it is hardly used for screening large-scale drugs because of the large number of animals and complex mechanisms involved in-vivo. In this study, we focused on whether T lymphocytes from CHS mouse model could maintain the state of immune response in-vitro and explored a suitable time for drugs screening. The results showed that CD4(+) T cells of CHS mice were higher compared with those in normal group. The expression of T-bet and GATA3 showed a Th1 shift and the levels of interleukin (IL)-2 and IL-4 also showed similar trend. Furthermore, IL-6 produced by T lymphocytes from CHS mice had a high level too. Then, we detected the effects of dexamethasone (DEX), cyclosporine A (CsA) and mycophenolate mofetil (MMF) on T lymphocytes in-vitro, and the data displayed that these immunosuppressive drugs could all inhibit the proliferation of T lymphocytes significantly. These findings suggested that T lymphocytes from CHS mice could mimic a similar immune response in-vitro, and it's also a suitable method for screening anti-inflammatory and immunosuppressive agents.
After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.
Previous data showed that the predominance of intraglomerular T-bet or GATA3 is correlated with different mechanisms of rejection, suggesting that the ratio of T-bet/GATA3 might be used to distinguish antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR).
We compared the intraglomerular T-bet/GATA3 ratio in ABMR and TCMR. The intragraft expression of T-bet and GATA3 was studied via immunohistochemistry. The correlation of the diagnosis of AMR with the ratio of T-bet/GATA3 was examined.
Both intraglomerular T-bet- and GATA3-expressing cells were increased during acute rejection. T-bet/GATA3>1 was strongly correlated with ABMR (93.3% versus 18.2%). The incidence of positive HLA-I/II antibodies and glomerulitis is significantly higher in T-bet/GATA3>1 group (P < 0.001, 0.013, resp.). The scores of peritubular capillary inflammation and glomerulitis were also higher in T-bet/GATA3>1 group (P = 0.052, P < 0.001, resp.). Nevertheless, T-bet/GATA3>1 is also correlated with C4d-negative ABMR and resistance to steroid treatment. Compared with C4d deposition, T-bet/GATA3>1 had a slight lower (90% versus 100%) specificity but a much higher (87.5% versus 68.8%) sensitivity.
Our data suggested that intraglomerular predominance of T-bet over GATA3 might be used as diagnosis maker of ABMR in addition to C4d, especially in C4d-negative cases.
Transforming growth factor β (TGFβ) is a potent and context-dependent regulator of tumor progression. TGFβ promotes the lung metastasis of basal-like (but not the luminal-like) breast cancer. Here, we demonstrated that fascin, a pro-metastasis actin bundling protein, was a direct target of the canonical TGFβ-Smad4 signaling pathway in basal-like breast cancer cells. TGFβ and Smad4 induced fascin overexpression by directly binding to a Smad binding element on the fascin promoter. We identified GATA3, a transcription factor crucial for mammary gland morphogenesis and luminal differentiation, as a negative regulator of TGFβ- and Smad4-induced fascin overexpression. When ectopically expressed in basal-like breast cancer cells, GATA-3 abrogated TGFβ- and Smad4-mediated overexpression of fascin and other TGFβ response genes, invadopodium formation, cell migration, and invasion, suggesting suppression of the canonical TGFβ-Smad signaling axis. Mechanistically, GATA3 abrogated the canonical TGFβ-Smad signaling by abolishing interactions between Smad4 and its DNA binding elements, potentially through physical interactions between the N-terminal of GATA3 and Smad3/4 proteins. Our findings provide mechanistic insight into how TGFβ-mediated cell motility and invasiveness are differentially regulated in breast cancer.
Zinc oxide nanoparticles (ZNPs) have been used in dietary supplements and may cause an immunomodulatory effect. The present study investigated the effect of ZNPs on antigen-specific immune responses in mice sensitized with the T-cell-dependent antigen ovalbumin (OVA). BALB/c mice were intraperitoneally administered ZNPs (0.25, 0.5, 1 and 3mg) once, in combination with OVA, and the serum antibodies, splenocyte reactivity and activation of antigen-presenting cells were examined. The serum levels of OVA-specific IgG1 and IgE were found significantly enhanced by treatment with ZNPs over control. An increased level of IL-2, IL-4, IL-6, IL-17 and decreased level of IL-10 and TNF-α in splenocytes administered with ZNPs were observed in comparison with control. The ZNPs and OVA-stimulated T lymphocytes showed enhanced proliferation compared with control. Macrophages and B cells showed high expression of MHC class II, whereas higher expression of CD11b in macrophages of the ZNPs and ZNPs/OVA treated groups was observed. The lungs and spleen had increased eosinophils and mast cell numbers. Also, myeloperoxidase activity in lungs was found to be increased by 2.5-fold in the case of ZNPs and 3.75-fold increase in ZNPs/OVA, whereas in intestine, there was significant increase in both the groups. Increased expression of the genes for GATA-3, SOCS-3, TLR-4, IL-13 and IL-5 in the intestine was observed. Collectively, these data indicate that systemic exposure to a single administration of ZNPs could enhance subsequent antigen-specific immune reactions, including the serum production of antigen-specific antibodies, and the functionality of T cells.
T-box expressed in T cells (TBET) and guanine adenine thymine adenine sequence-binding protein 3 (GATA3) play important roles in the differentiation of Th1 and Th2 subsets, which contributes to the progression of acute coronary syndrome (ACS).
This study aimed to investigate the temporal change of TBET/GATA3 mRNA ratio in ACS.
Thirty-three patients suspected of ACS with symptom onset within 24 hours were recruited. Blood samples were taken after arrival at the emergency department and at hourly intervals until the 6th hour. The mRNA expressions of TBET and GATA3 were quantified by a real-time RT-qPCR.
The TBET/GATA3 mRNA ratio was elevated dramatically in patients with acute myocardial infarction (AMI) and exhibited biphasic M-shaped release kinetics with two distinct peaks. The ratio was elevated 2 hours after symptom onset, dropped to the lowest level at 10 hours, and rose to the second peak at 14 hours. A similar biphasic M-shaped curve was observed in AMI patients with blood samples taken prior to any intervention.
The TBET/GATA3 mRNA ratio was elevated in AMI patients throughout most of the first 20 hours after symptom onset. The biphasic M-shaped release kinetics was more likely to reflect pathophysiological changes rather than treatment effects.
Lymph node involvement is a major prognostic variable in breast cancer. Whether the molecular mechanisms that drive breast cancer cells to colonize lymph nodes are shared with their capacity to form distant metastases is yet to be established. In a transcriptomic survey aimed at identifying molecular factors associated with lymph node involvement of ductal breast cancer, we found that luminal differentiation, assessed by the expression of estrogen receptor (ER) and/or progesterone receptor (PR) and GATA3, was only infrequently lost in node-positive primary tumors and in matched lymph node metastases. The transcription factor GATA3 critically determines luminal lineage specification of mammary epithelium and is widely considered a tumor and metastasis suppressor in breast cancer. Strong expression of GATA3 and ER in a majority of primary node-positive ductal breast cancer was corroborated by quantitative RT-PCR and immunohistochemistry in the initial sample set, and by immunohistochemistry in an additional set from 167 patients diagnosed of node-negative and -positive primary infiltrating ductal breast cancer, including 102 samples from loco-regional lymph node metastases matched to their primary tumors, as well as 37 distant metastases. These observations suggest that loss of luminal differentiation is not a major factor driving the ability of breast cancer cells to colonize regional lymph nodes.
Intragastric immunization with recombinant chimeric immunogen, SBR-CTA2/B, constructed from the saliva-binding region (SBR) of Streptococcus mutans antigen AgI/II and the A2/B subunits of cholera toxin (CT) induces salivary and circulating antibodies against S. mutans that protect against dental caries. We previously found that SBR-CTA2/B activated dendritic cells (DC) in the Peyer's patches (PP) and mesenteric lymph nodes (MLN). To identify the cells involved in the intestinal uptake of SBR-CTA2/B and the initiation of immune responses, mice were immunized intragastrically with fluorescein-labeled SBR-CTA2/B or SBR, and intestinal cells were examined by imaging flow cytometry after fluorescent staining for cell surface markers. SBR-CTA2/B was preferentially taken up by CD103(+) DC in the PP and by both CD103(+) and CD11c(+) DC in intestinal lamina propria (LP), whereas SBR was taken up to a lesser extent by PP CD11c(+) DC, within 2 to 16 h. By 16 h, CD103(+) and CD11c(+) DC containing fluorescein-labeled SBR-CTA2/B were found in MLN and showed upregulation of the chemokine receptor CCR7. Large numbers of SBR-CTA2/B-containing DC were found interacting with CD4(+) (T helper) cells, which costained for nuclear transcription factors T-bet or RORγt, identifying them as Th1 or Th17 cells. In contrast, SBR-containing CD11c(+) DC interacted preferentially with GATA3(+) (Th2) cells. No SBR- or SBR-CTA2/B-containing DC were found interacting with Foxp3(+) (T regulatory) cells. We conclude that the coupling of SBR to CTA2/B enhances its immunogenicity by promoting uptake by DC in both PP and LP and that these antigen-containing DC migrated to MLN and interacted preferentially with Th1 and Th17 cells to induce active immune responses.
The rapidly growing collection of diverse genome-scale data from multiple tumor types sheds light on various aspects of the underlying tumor biology. With the objective to identify genes of importance for breast tumorigenesis in men and to enable comparisons with genes important for breast cancer development in women, we applied the computational framework COpy Number and EXpression In Cancer (CONEXIC) to detect candidate driver genes among all altered passenger genes. Unique to this approach is that each driver gene is associated with several gene modules that are believed to be altered by the driver. Thirty candidate drivers were found in the male breast cancers and 67 in the female breast cancers. We identified many known drivers of breast cancer and other types of cancer, in the female dataset (e.g. GATA3, CCNE1, GRB7, CDK4). In contrast, only three known cancer genes were found among male breast cancers; MAP2K4, LHP, and ZNF217. Many of the candidate drivers identified are known to be involved in processes associated with tumorigenesis, including proliferation, invasion and differentiation. One of the modules identified in male breast cancer was regulated by THY1, a gene involved in invasion and related to epithelial-mesenchymal transition. Furthermore, men with THY1 positive breast cancers had significantly inferior survival. THY1 may thus be a promising novel prognostic marker for male breast cancer. Another module identified among male breast cancers, regulated by SPAG5, was closely associated with proliferation. Our data indicate that male and female breast cancers display highly different landscapes of candidate driver genes, as only a few genes were found in common between the two. Consequently, the pathobiology of male breast cancer may differ from that of female breast cancer and can be associated with differences in prognosis; men diagnosed with breast cancer may consequently require different management and treatment strategies than women.
Our previous genome-wide linkage scan mapped five loci for caries experience. The purpose of this study was to fine map one of these loci, the locus 13q31.1, in order to identify genetic contributors to caries.
Seventy-two pedigrees from the Philippines were studied. Caries experience was recorded and DNA was extracted from blood samples obtained from all subjects. Sixty-one single nucleotide polymorphisms (SNPs) in 13q31.1 were genotyped. Association between caries experience and alleles was tested. We also studied 1,481 DNA samples obtained from saliva of subjects from the USA, 918 children from Brazil, and 275 children from Turkey, in order to follow up the results found in the Filipino families. We used the AliBaba2.1 software to determine if the nucleotide changes of the associated SNPs changed the prediction of the presence of transcription-binding site sequences and we also analyzed the gene expression of the genes selected based on binding predictions. Mutation analysis was also performed in 33 Filipino individuals of a segment of 13q31.1 that is highly conserved in mammals.
Statistically significant association with high caries experience was found for 11 markers in 13q31.1 in the Filipino families. Haplotype analysis also confirmed these results. In the populations used for follow-up purposes, associations were found between high caries experience and a subset of these markers. Regarding the prediction of the transcription-binding site, the base change of the SNP rs17074565 was found to change the predicted-binding of genes that could be involved in the pathogenesis of caries. When the sequence has the allele C of rs17074565, the potential transcription factors binding the sequence are GR and GATA1. When the subject carries the G allele of rs17074565, the potential transcription factor predicted to bind to the sequence is GATA3. The expression of GR in whole saliva was higher in individuals with low caries experience when compared to individuals with high caries experience (p = 0.046). No mutations were found in the highly conserved sequence.
Genetic factors contributing to caries experience may exist in 13q31.1. The rs17074565 is located in an intergenic region and is predicted to disrupt the binding sites of two different transcription factors that might be involved with caries experience. GR expression in saliva may be a biomarker for caries risk and should be further explored.
IL-6 plays a pivotal role in favoring T-cell commitment toward a Th17 cell rather than Treg-cell phenotype, as established through in vitro model systems. We predicted that in the absence of IL-6, mice infected with the gastrointestinal helminth Heligmosomoides polygyrus would show reduced Th17-cell responses, but also enhanced Treg-cell activity and consequently greater susceptibility. Surprisingly, worm expulsion was markedly potentiated in IL-6-deficient mice, with significantly stronger adaptive Th2 responses in both IL-6(-/-) mice and BALB/c recipients of neutralizing anti-IL-6 monoclonal Ab. Although IL-6-deficient mice showed lower steady-state Th17-cell levels, IL-6-independent Th17-cell responses occurred during in vivo infection. We excluded the Th17 response as a factor in protection, as Ab neutralization did not modify immunity to H. polygyrus infection in BALB/c mice. Resistance did correlate with significant changes to the associated Treg-cell phenotype however, as IL-6-deficient mice displayed reduced expression of Foxp3, Helios, and GATA-3, and enhanced production of cytokines within the Treg-cell population. Administration of an anti-IL-2:IL-2 complex boosted Treg-cell proportions in vivo, reduced adaptive Th2 responses to WT levels, and fully restored susceptibility to H. polygyrus in IL-6-deficient mice. Thus, in vivo, IL-6 limits the Th2 response, modifies the Treg-cell phenotype, and promotes host susceptibility following helminth infection.
Age-adjusted mortality rates for prostate cancer are higher for African-American men compared with those of European ancestry. Recent data suggest that West African men also have elevated risk for prostate cancer relative to European men. Genetic susceptibility to prostate cancer could account for part of this difference. We conducted a genome-wide association study (GWAS) of prostate cancer in West African men in the Ghana Prostate Study. Association testing was performed using multivariable logistic regression adjusted for age and genetic ancestry for 474 prostate cancer cases and 458 population-based controls on the Illumina HumanOmni-5 Quad BeadChip. The most promising association was at 10p14 within an intron of a long non-coding RNA (lncRNA RP11-543F8.2) 360 kb centromeric of GATA3 (p = 1.29E-7). In sub-analyses, SNPs at 5q31.3 were associated with high Gleason score (≥7) cancers, the strongest of which was a missense SNP in PCDHA1 (rs34575154, p = 3.66E-8), and SNPs at Xq28 (rs985081, p = 8.66E-9) and 6q21 (rs2185710, p = 5.95E-8) were associated with low Gleason score (<7) cancers. We sought to validate our findings in silico in the African Ancestry Prostate Cancer GWAS Consortium, but only one SNP, at 10p14, replicated at p < 0.05. Of the 90 prostate cancer loci reported from studies of men of European, Asian or African-American ancestry, we were able to test 81 in the Ghana Prostate Study, and 10 of these replicated at p < 0.05. Further genetic studies of prostate cancer in West African men are needed to confirm our promising susceptibility loci.
GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.
Chronic rhinosinusitis (CRS) is characterized by local inflammation of the sinonasal tissues. CRS patients with nasal polyps and asthma often develop acute exacerbation of sinonasal symptoms after upper respiratory tract infections. However, the influence of concomitant asthma on the nasal immune response to viral infection remains unclear.
Specimens of nasal polyp and mucosal tissues were obtained from 3 groups of CRS patients (n = 14 per group): 1) patients without asthma (CRS group), 2) patients with aspirin-tolerant asthma (ATA group), and 3) patients with aspirin-intolerant asthma (AIA group). Nasal fibroblasts isolated from the specimens were stimulated with poly I:C. CXCL10 expression was analyzed by the quantitative real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay. Biopsy specimens from CRS patients without asthma were subjected to immunohistochemistry for detection of T-bet and GATA-3 expression in CD3+ T cells by double labeling.
Nasal fibroblasts from the ATA and AIA groups showed significantly enhanced expression of CXCL10 mRNA and protein after poly I:C stimulation compared with cells from the CRS group and the control group (normal nasal mucosa). In addition to T helper (Th)2 cells, there was more abundant infiltration of Th1 cells into tissues from the AIA and ATA groups.
Our findings suggest that CRS associated with asthma may become intractable through the over-production of CXCL10 in response to viral infection.
CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events.
GATA3 is a transcription factor important in the differentiation of breast epithelia, urothelia, and subsets of T lymphocytes. It has been suggested to be useful in the evaluation of carcinomas of mammary or urothelial origin or metastatic carcinomas, but its distribution in normal and neoplastic tissues is incompletely mapped. In this study, we examined normal developing and adult tissues and 2040 epithelial and 460 mesenchymal or neuroectodermal neoplasms for GATA3 expression to explore its diagnostic value in surgical pathology, using monoclonal antibody (clone L50-823) and Leica Bond automated immunohistochemistry. GATA3 was expressed in trophoblast, fetal and adult epidermis, adult mammary and some salivary gland and sweat gland ductal epithelia, urothelia, distal nephron in developing and adult tissues, some prostatic basal cells, and subsets of T lymphocytes. It was expressed stronger in fetal than in adult mesothelia and was absent in respiratory and gastrointestinal epithelia. In epithelial neoplasms, GATA3 was expressed in >90% of primary and metastatic ductal and lobular carcinomas of the breast, urothelial, and cutaneous basal cell carcinomas and trophoblastic and endodermal sinus tumors. In metastatic breast carcinomas, it was more sensitive than GCDFP. Among squamous cell carcinomas, the expression was highest in the skin (81%) and lower in cervical (33%), laryngeal (16%), and pulmonary tumors (12%). Common positivity was found in skin adnexal tumors (100%), mesothelioma (58%), salivary gland (43%), and pancreatic (37%) ductal carcinomas, whereas frequency of expression in adenocarcinomas of lung, stomach, colon, endometrium, ovary, and prostate was <10%. Chromophobe renal cell carcinoma was a unique renal tumor with frequent positivity (51%), whereas oncocytomas were positive in 17% of cases but other types only rarely. Among mesenchymal and neuroectodermal tumors, paragangliomas were usually positive, which sets these tumors apart from epithelial neuroendocrine tumors. Mesenchymal tumors were only sporadically positive, except epithelia of biphasic synovial sarcomas. GATA3 is a useful marker in the characterization of not only mammary and urothelial but also renal and germ cell tumors, mesotheliomas, and paragangliomas. The multiple specificities of GATA3 should be taken into account when using this marker to detect metastatic mammary or urothelial carcinomas.
Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have been regarded as useful sources for cell-based transplantation therapy. However, immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hESC lines (NTU1 and H9), hiPSC lines, and their derivatives (including stem cell-derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune-related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC-I) molecules, β2-microglobulin, and HLA-E in undifferentiated stem cells. The levels were increased after cotreatment with interferon-γ and/or in vitro differentiation. Antigen-presenting cell markers (CD11c, CD80, and CD86) and MHC-II (HLA-DP, -DQ, and -DR) remained low throughout the treatments. Recognition of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hESCs induced a cell number-dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly, activation of lymphocytes by differentiated hiPSCs or H9 cells became blunted at higher cell numbers. Real-time reverse transcriptase PCR (RT-PCR) showed significant differential expression of immune privilege genes (TGF-β2, Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, IL-1RN, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59, and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It was concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidence suggests some level of potential immune privilege. In addition, differential immunogenicity may exist between different pluripotent stem cell lines and their derivatives.
Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (T(H)2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen-induced lung inflammation. Gfi1 'preferentially' regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.
Recent genomic profiling of childhood acute lymphoblastic leukemia (ALL) identified a high-risk subtype with an expression signature resembling that of Philadelphia chromosome-positive ALL and poor prognosis (Ph-like ALL). However, the role of inherited genetic variation in Ph-like ALL pathogenesis remains unknown. In a genome-wide association study (GWAS) of 511 ALL cases and 6,661 non-ALL controls, we identified a susceptibility locus for Ph-like ALL (GATA3, rs3824662; P = 2.17 × 10(-14), odds ratio (OR) = 3.85 for Ph-like ALL versus non-ALL; P = 1.05 × 10(-8), OR = 3.25 for Ph-like ALL versus non-Ph-like ALL), with independent validation. The rs3824662 risk allele was associated with somatic lesions underlying Ph-like ALL (CRLF2 rearrangement, JAK gene mutation and IKZF1 deletion) and with variation in GATA3 expression. Finally, genotype at the GATA3 SNP was also associated with early treatment response and risk of ALL relapse. Our results provide insights into interactions between inherited and somatic variants and their role in ALL pathogenesis and prognosis.
Excessive uterine bleeding is the most common and problematic side effect of RU486 medical abortion. Shenghua Decoction (SHD) is a well-known traditional Chinese herbal prescription for reducing uterine bleeding induced by RU486 medical abortion. However, its therapeutic mechanism still remains unclear. The Th1/Th2/Th17/Treg paradigm plays an important role in achieving maternal-fetal immunotolerance and its bias participates in RU486-induced abortion. Our previous research on mice demonstrated that the uterine bleeding volume is negatively related to the proportions of Th1 and Th17 cells whereas positively related to the proportions of Th2 and Treg cells. Additionally, Th1-type cytokine inducing effect was identified in our previous study. Therefore, it was hypothesized that SHD reduced the uterine bleeding in RU486 medical abortion by inducing Th1/Th2/Th17/Treg paradigm bias. The purpose of this study was to determine the regulatory effect and the mechanism of SHD on human decidual Th1/Th2/Th17/Treg paradigm for alleviating uterine bleeding in RU486 medical abortion.
90 women within seven weeks of a normal intrauterine pregnancy, who elected for termination of pregnancy, were divided into three groups; vacuum aspiration group, RU486 group, and SHD-RU486 group. Duration of uterine bleeding was recorded and volume of uterine bleeding was measured by the method of alkaline hematin photometric. To determine the regulatory effect of SHD on Th1/Th2/Th17/Treg paradigm, the proportions of Th1/Th2/Th17/Treg cells in the decidua of different groups were analyzed using a FACS calibur. Correlation was analyzed in order to demonstrate the relationship between the Th1/Th2/Th17/Treg paradigm and the uterine bleeding in RU486 medical abortion. Moreover, to elucidate the mechanism underlying the T-cell paradigm regulating of SHD, the mRNA and protein expressions of subset-specific transcription factors (T-bet, GATA-3, RORγt, and Foxp3) for the differentiation of Th1/Th2/Th17/Treg paradigm in human decidual CD4(+) T cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) assay and western blot analysis respectively. Moreover, the mRNA expression of the characteristic cytokines of Th1/Th2/Th17/Treg paradigm (IFNγ, IL-4, IL-17A, TGF-β) were analyzed by RT-PCR assay.
Compared with RU486 group, both the uterine bleeding volume and duration reduced significantly in SHD-RU486 group. Both the duration and the volume of the uterine bleeding demonstrated negative correlation with the proportions of Th1 and Th17 cells, whereas showed positive correlation with Th2 and Treg cells. SHD increased the proportions of Th1 and Th17 cells whereas decreased those of Th2 and Treg cells. Thus, the ratios of Th1/Th2 and Th17/Treg cells elevated markedly after SHD treatment. SHD promoted the mRNA as well as the protein expressions of subset-specific transcription factors for the differentiation of Th1 and Th17 subsets (T-bet and RORγt) while inhibited those of Th2 and Treg cells (GATA-3 and Foxp3). Moreover, the mRNA expression of Th1- and Th17- type cytokines (IFNγ and IL-17A) was up-regulated while that of Th2-type and Treg-produced cytokines (IL-4 and TGF-β) was down-regulated significantly after SHD administration.
Th1/Th2/Th17/Treg paradigm bias was involved in RU486 medical abortion. SHD reduced the uterine bleeding efficiently by inducing Th1 and Th17 skews in the maternal-fetal of RU486 medical abortion patients. The regulatory effect of SHD on Th1/Th2/Th17/Treg paradigm in RU486 medical abortion is attributed to the modulation of transcription and protein expression of subset-specific transcription factors for T-cell subsets differentiation and their characteristic cytokines.
The penile urethra has a distinctive morphology not yet fully characterized by immunohistochemistry. In addition, both urothelial and squamous cell carcinomas have been reported in the penile urethra, and the distinction between these 2 tumors might be difficult. The purposes of this study are to assess the histology and immunohistochemical profile (CK20, CK7, p63, and GATA3) of the penile urethra and to assess the usefulness of Trans-acting T-cell-specific transcription factor (GATA3) and human papillomavirus detection in distinguishing urothelial versus squamous cell carcinomas. Normal penile urethra was evaluated in 11 total penectomies. The penile urethra was lined by 2 cell layers: a superficial single layer of CK7+, CK20-, and p63- columnar cells and a deep stratified layer of CK7-, CK20-, and p63+ cubical cells. Both layers were GATA3+, supporting urothelial differentiation. In addition, 2 tissue microarrays and 6 surgical specimens of primary tumors of the penile urethra (3 urothelial and 3 squamous cell carcinomas) were evaluated for GATA3 expression. In the tissue microarrays, 22 of 25 upper tract urothelial carcinomas and 0 of 38 penile squamous cell carcinomas were GATA3+. In the surgical specimens, GATA3 was positive in all urothelial carcinomas and negative in all squamous cell carcinomas. Human papillomavirus was detected in 2 of 3 squamous cell carcinomas and in 0 of 3 of the urothelial carcinomas. In conclusion, the penile urethra is covered by epithelial cells that are unique in morphology and immunohistochemical profile. In addition, our study suggests that GATA3 and human papillomavirus detection are useful markers for distinguishing urothelial carcinomas from squamous cell carcinomas of the penile urethra.
Biodegradable poly(lactic-co-glycolic acid) (PLGA) porous films are developed to support mammary cell growth and function. Such porous polymer matrices of PLGA are generated using the easily implemented water-templating "breath-figure" technique that allows water droplets to penetrate the nascent polymer films to create a rough porous polymer film. Such breath figure-based micropatterned porous films show higher epithelial differentiation and growth than the corresponding flat 2D films, and represent the first instance of using them for tissue culture. Specifically, the breath figure morphology supports robust acinar growth with almost double the number of lobular-alveolar units compared to the 2D cultures. Gene profile analysis indicates that the cells grown on porous polymer films show enhanced expressions of mammary differentiation genes (GATA3, EMA, and INTEGB4) but lower the expression of mesenchymal gene (CALLA). Hormonal stimulation of these cultures dramatically increases expression of progenitor marker gene Notch1. Importantly, cells grown on porous PLGA films exhibit an enhanced resistance to doxorubicin treatment in comparison to 2D cultures. Breath-figure PLGA films show promise in mimicking in vivo mammary functions and can potentially be used to screen chemotherapeutic drugs. The simplicity and ease of fabrication of these polymer films is especially appealing to the development of effective biomaterials to support cell culture and differentiation.
To explore the relationship between the dendritic cell (DC) subsets and abnormal expression of transcription factors Gata-3 and T-bet in patients with immune thrombocytopenia (ITP).
The plasmacytoid DC (pDC) and myeloid DC (mDC) of 33 ITP (16 untreated, 17 remitted) patients and 12 healthy controls were analyzed by flow cytometry (FCM) . The expressions of Gata-3 mRNA and T-bet mRNA in peripheral blood mononuclear cell (PBMNC) were detected by reverse transcription-polymerase chain reaction (RT-PCR) .The levels of interleukin-4 (IL-4) and interferon-gamma (IFN-γ) were measured by FCM in 33 ITP patients and 12 healthy controls.
The percentage of pDC in PBMNC was 0.49% ± 0.18% in untreated and it was higher than that in remitted ITP patients (0.27% ± 0.17%) and in controls (0.32% ± 0.13%) (both P < 0.05). The percentage of mDC in PBMNC was 0.23% ± 0.17% in untreated, which was lower than that in remitted ITP patients (0.33% ± 0.18)% and in controls (0.31% ± 0.11%), but no statistic difference in mDC expression existed among 3 groups (P > 0.05). pDC/mDC ratios was (3.15 ± 2.01) in untreated ITP patients and it was higher than that in remitted ITP patients (0.81 ± 0.32) and in controls (1.07 ± 0.44) (both P < 0.05). The relative mRNA expression levels of Gata-3 were 2775 ± 489, 1357 ± 307 and 652 ± 165 respectively. And the expression of Gata-3mRNA in untreated group was higher than that in remission group or healthy controls (both P < 0.05). The relative mRNA expression levels of T-bet were 782 ± 394, 583 ± 176 and 576 ± 120. No statistic difference in T-bet expression existed among 3 groups (P > 0.05). Gata-3mRNA/T-bet mRNA ratio was (4.13 ± 1.69 ) in untreated group and it was higher than that of remission group (2.45 ± 0.69) or controls (1.15 ± 0.27) (both P < 0.05). The level of IL-4 in the untreated group was 9.14% ± 4.34% and it was higher than that of remission group (4.78% ± 1.69%) or controls (4.86% ± 1.41%). The level of IFN-γ in the untreated group was lower than that of controls (P < 0.05). Significant positive correlations existed between Gata-3 and pDC/mDC ratio (r = 0.585, P < 0.01). Significant positive correlations existed between Gata-3 and IL-4 ( r = 0.463, P < 0.05).
The mechanism of ITP may be due to a disorder of DC subsets and a high expression of Gata-3.
Current research on antigen specific CD4+ T cells indicates that there is functional and phenotypic heterogeneity within these populations, but the extent of this heterogeneity is poorly described. The CD134/CD25 assay allows live isolation of antigen specific cells in vitro for down-stream molecular analysis. Antigen specific CD4+ T cells were examined at the molecular level by lineage specific transcription factor profiling using qualitative multiplex single cell RT-PCR and Lock Nucleic Acid (LNA) probes allowed unbiased amplification and delineation of expression of Tbx21, Gata3, Rorc, Foxp3 and Bcl-6. It overcomes the limitations of previous assays by allowing identification of transcription factor mRNA in single antigen specific cells with high sensitivity (down to 10 femtograms) and specificity. Patterns of responses can be robustly characterized using <200 cells based on exact binomial calculations. These results are reproducible with a CV of ≈6%. The patterns of heterogeneity are stable within an individual antigen specific response but vary between responses to different antigens. Responses to CMV have a Th1 predominant profile (35.6% of responding cells expressing tbx21) whereas responses to Tetanus Toxoid have a Th2 biased profile (22% of responding cells expressing gata3), with unexpectedly high levels of Treg cells found in both populations. Here we describe a methodology that allows live isolation of Ag specific cells and transcription factor profiling at a single cell level to robustly delineate the different CD4+ T cell subsets within this population. This novel method is a powerful tool that can be used to study CD4+ T cell heterogeneity within extremely small populations of cells and where cell numbers are limited.
Bladder urothelial carcinoma (BUC) accounts for ∼90% of all cases of bladder cancer. Reduced expression of TGFBR3 has been frequently observed in several types of human cancers. However, little is known about whether expression of TGFBR3 reduced in BUC and the underlying mechanisms. In the present study, we performed quantitative real-time PCR to examine the mRNA expression of TGFBR3 and GATA3, and bisulfite genomic sequencing to evaluate the methylation status in TGFBR3 and GATA3 promoter regions in fresh tumor and the corresponding paracarcinoma tissues from 29 patients with BUC. As a result, the expression of TGFBR3 and GATA3, a transcriptional factor of the TGFBR3 gene, were found to be co-downregulated in BUC. Moreover, our findings indicated that GATA3 promoter methylation was one of the reasons for silencing of GATA3 and TGFBR3 in BUC, albeit TGFBR3 methylation and mutation were not associated with reduced expression of TGFBR3 mRNA in BUC. In summary, our findings suggest that methylation in the GATA3 promoter region may inhibit the expression of GATA3 mRNA, which leads to the reduced expression of TGFBR3 mRNA in BUC.
GATA3 has been recognized as a promising marker for primary urothelial carcinoma (UC), consistently showing higher expression levels than urothelial markers thrombomodulin and uroplakin III. However, expression of GATA3 in comparison with UC-associated markers CK7 and p63 has not been systematically studied. Moreover, no studies have been conducted to establish GATA3 sensitivity in regional metastases. In this study, high-density tissue microarrays were constructed from 69 matched paired primary and metastatic bladder tumors including pure urothelial UCs with papillary (n=48) or flat phenotype (n=9), mixed tumors with micropapillary, glandular, small cell, squamous, giant cell, and plasmacytoid features (n=9), and 3 adenocarcinomas. GATA3 was expressed in 62/69 (90%) primary UC and 64/69 (93%) metastases, with significantly higher staining intensity in nodal metastases (P=0.03). In primary tumors, GATA3 was positive in 44/48 (92%) papillary UCs, 9/9 (100%) flat UCs, 8/9 (89%) mixed UCs, and 1/3 (33%) adenocarcinomas, whereas in metastases these numbers were 45/48 (94%), 9/9 (100%), 8/9 (89%), and 2/3 (67%), respectively. The majority of positive cases showed strong diffuse nuclear reactivity: 75% of primary UCs and 79% of metastases. GATA3 sensitivity in primary and metastatic UCs was comparable to that of CK7 and superior to that of p63 (P<0.05). GATA3 specificity was computed in comparison with its morphologic mimics expressing CK7 and p63, including 208 primary and 24 metastatic tumors from the lung, cervix, and head and neck regions. Strong GATA3 expression was present in 2/51 (4%) cervical carcinomas, whereas weak GATA3 expression was present in 7/51 (14%) cervical, 6/74 (8%) head and neck cancers, and 2/83 (3%) lung carcinomas. Remaining 191 primary and 24 metastatic tumors were GATA3 negative. Therefore, specificity of GATA3 calculated on the basis of morphologic and immunophenotypic UC mimics from lung, cervix, head and neck was 92%. Our findings demonstrate high sensitivity and specificity of the GATA3 diagnostic marker, with not only maintained but increased expression in regional metastases.
Complement is undeniably quintessential for innate immunity by detecting and eliminating infectious microorganisms. Recent work, however, highlights an equally profound impact of complement on the induction and regulation of a wide range of immune cells. In particular, the complement regulator CD46 emerges as a key sensor of immune activation and a vital modulator of adaptive immunity. In this review, we summarize the current knowledge of CD46-mediated signalling events and their functional consequences on immune-competent cells with a specific focus on those in CD4(+) T cells. We will also discuss the promises and challenges that potential therapeutic modulation of CD46 may hold and pose.
Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3.
We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively.
Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development.
Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.
Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes.
Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified.
Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others.
These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response.
Allergy is a complex disease that is likely to involve dysregulated CD4+ T cell activation. Here we propose a novel methodology to gain insight into how coordinated behaviour emerges between disease-dysregulated pathways in response to pathophysiological stimuli. Using peripheral blood mononuclear cells of allergic rhinitis patients and controls cultured with and without pollen allergens, we integrate CD4+ T cell gene expression from microarray data and genetic markers of allergic sensitisation from GWAS data at the pathway level using enrichment analysis; implicating the complement system in both cellular and systemic response to pollen allergens. We delineate a novel disease network linking T cell activation to the complement system that is significantly enriched for genes exhibiting correlated gene expression and protein-protein interactions, suggesting a tight biological coordination that is dysregulated in the disease state in response to pollen allergen but not to diluent. This novel disease network has high predictive power for the gene and protein expression of the Th2 cytokine profile (IL-4, IL-5, IL-10, IL-13) and of the Th2 master regulator (GATA3), suggesting its involvement in the early stages of CD4+ T cell differentiation. Dissection of the complement system gene expression identifies 7 genes specifically associated with atopic response to pollen, including C1QR1, CFD, CFP, ITGB2, ITGAX and confirms the role of C3AR1 and C5AR1. Two of these genes (ITGB2 and C3AR1) are also implicated in the network linking complement system to T cell activation, which comprises 6 differentially expressed genes. C3AR1 is also significantly associated with allergic sensitisation in GWAS data.
Animal development progresses through the stepwise deployment of gene regulatory networks (GRN) encoded in the genome. Comparative analyses in different species and organ systems have revealed that GRN blueprints are composed of subcircuits with stereotypical architectures that are often reused as modular units. In this review, we report the evidence for the GRN underlying renal primordium development. In vertebrates, renal development is initiated by the induction of a field of intermediate mesoderm cells competent to undergo lineage specification and nephric (Wolffian) duct formation. Definition of the renal field leads to the activation of a core regulatory subcircuit composed of the transcription factors Pax2/8, Gata3 and Lim1. These transcription factors turn on a second layer of transcriptional regulators while also activating effectors of tissue morphogenesis and cellular specialization. Elongation and connection of the nephric duct to the cloaca (bladder/urethra primordium) is followed by metanephric kidney induction through signals emanating from the metanephric mesenchyme. Central to this process is the activation and positioning of the glial cell line-derived neurotrophic factor (Gdnf)-Ret signaling pathway by network subcircuits located in the mesenchyme and epithelial tissues of the caudal trunk. Evidence shows that each step of the renal primordium developmental program is regulated by structured GRN subunits organized in a hierarchical manner. Understanding the structure and dynamics of the renal GRN will help us understand the intrinsic phenotypical variability of congenital anomalies of the kidney and urinary tract and guide our approaches to regenerative medicine.
The inositol-requiring enzyme 1α (IRE1α) is a serine-threonine kinase that plays crucial roles in activating the unfolded protein response. Studies suggest that IRE1α is activated during thymic T cell development and in effector CD8(+) T cells. However, its role in regulating T helper cell differentiation remains unknown. We find that IRE1α is up-regulated and activated upon CD4(+) T cell activation and plays an important role in promoting cytokine IL-4 production. CD4(+) T cells from IRE1α KO mice have reduced IL-4 protein expression, and this impaired IL-4 production is not due to the altered expression of Th2 lineage-specific transcription factors, such as GATA3. Instead, IL-4 mRNA stability is reduced in IRE1α KO T cells. Furthermore, treatment of T cells with an IRE1α-specific inhibitor, 4μ8C, leads to a block in IL-4, IL-5, and IL-13 production, confirming the role of IRE1α in the regulation of IL-4. This study identifies a regulatory function for IRE1α in the promotion of IL-4 in T cells.
The essential effect of vitamin A on immune function occurs through various mechanisms including direct effect on Th1-Th2 balance modulation. However, it is unclear whether or not vitamin A can regulate Th1-Th2 balance under a strong Th1-polarizing condition. Therefore, the purpose of our study was to examine the effect of vitamin A metabolite all-trans retinoic acid (ATRA) on Th1-Th2 differentiation in CD4+ T cells under GATA-3 deficiency, which can induce Th1-polarizing condition. In the present study, GATA-3 deficiency T cells were induced by siRNA and checked by real-time quantitative PCR and western blot. GATA-3 deficiency CD4+ T cells and normal CD4+ T were treated for 48 h with or without ATRA. The expression of Th1 and Th2 cytokines were detected by qPCR and ELISA. The results would contribute to clarify the knowledge of the role of vitamin A in regulating Th1-Th2 balance under some special conditions, and help to explain the mechanism of immune regulatory function of vitamin A.
T cell commitment and αβ/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αβ/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αβ/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αβ-committed cells do not express the pre-TCR and revealed a major stochastic component in αβ-lineage specification.
Invariant natural killer T cells (iNKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of iNKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8(+) T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, iNKT cell-derived IL-4 altered immunological properties under normal steady-state conditions.
Breast cancer is a collection of diseases with distinct molecular traits, prognosis, and therapeutic options. Luminal A breast cancer is the most heterogeneous, both molecularly and clinically. Using genomic data from over 1,000 Luminal A tumors from multiple studies, we analyzed the copy number and mutational landscape of this tumor subtype. This integrated analysis revealed four major subtypes defined by distinct copy-number and mutation profiles. We identified an atypical Luminal A subtype characterized by high genomic instability, TP53 mutations, and increased Aurora kinase signaling; these genomic alterations lead to a worse clinical prognosis. Aberrations of chromosomes 1, 8, and 16, together with PIK3CA, GATA3, AKT1, and MAP3K1 mutations drive the other subtypes. Finally, an unbiased pathway analysis revealed multiple rare, but mutually exclusive, alterations linked to loss of activity of co-repressor complexes N-Cor and SMRT. These rare alterations were the most prevalent in Luminal A tumors and may predict resistance to endocrine therapy. Our work provides for a further molecular stratification of Luminal A breast tumors, with potential direct clinical implications.
The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4(+) T cells. However, the role of the Th1 CD4(+) T cell differentiation program on the ability to control parasitemia and susceptibility to ECM disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background, we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM. T-bet-deficient (Tbx21(-/-)) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 ± 3.1% versus 10.9 ± 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only 30% (3/10) of Tbx21(-/-) mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx21(-/-) mice was associated with diminished numbers of IFN-γ-producing CD4(+) T cells in the spleen and a lower accumulation of CD4(+) and CD8(+) T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3(+) CD4(+) T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.
Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+) (Th1) activated CD69(+)CD4(+) T cells (p<0.001) and reduced percentage of Gata-3(+) (Th2) activated CD69(+)CD4(+)T cells (p<0.001) was detected in the mesenteric lymph nodes (MLN) of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+)Foxp3(+)) could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+) /T-bet(+) (Th1-Tregs) was significantly reduced (p<0.05) in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.
These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.
Differential diagnosis of collecting duct carcinoma (CDC) from invasive upper tract urothelial carcinoma (UTUC) can be challenging. PAX8 and p63 are 2 markers often used in this setting. GATA binding protein 3 (GATA3) is a marker of urothelial differentiation. We investigated GATA3 expression in CDC and UTUC and its use in this differential. Eighteen CDC and 25 UTUC cases were used to build 2 tissue microarrays. GATA3, p63, and PAX8 nuclear expression was evaluated using standard immunohistochemistry. Staining intensity and percentage of positive cells were assessed. Sensitivity, specificity, and positive and negative predictive values of the markers and their combination were also evaluated. We found GATA3 positivity in 22 (88%) of 25 UTUCs and 1 (6%) of 18 CDCs. The median GATA3 extent of expression was higher in UTUC than in CDC (74% versus 0%, P = .00). We found p63 positivity in 23 (92%) of 25 UTUCs and 2 (11%) of 18 CDCs. PAX8 was positive in 3 (12%) of 25 UTUCs and all (100%) CDCs. GATA3 sensitivity and specificity for UTUC were 88% and 94%, respectively. p63 sensitivity and specificity for UTUC were 92% and 89%, respectively. The p63+/PAX8- profile showed higher sensitivity for UTUC than did the GATA3+/PAX8- profile (80% versus 76%). Both showed a specificity of 100% for UTUC. GATA3+ or p63+/PAX8- sensitivity and specificity for UTUC were 84% and 100%, respectively. Immunohistochemical expression of GATA3 was higher in UTUC, suggesting a potential role for distinguishing UTUC from CDC. Adding this marker to the combination panel of p63 and PAX8 might improve its performance in the diagnosis of epithelial neoplasms involving the renal sinus.
Bone morphogenetic proteins (BMPs) are known to induce diverse differentiation fates in human embryonic stem cells (hESCs). In the present study, we compared the potency at which BMP5, BMP10 and BMP13, which are members of distinct BMP subgroups due to differences in sequential and structural homology, induce differentiation in hESCs and human induced pluripotent stem cells (hiPSCs). We observed, in agreement with previous BMP4 model studies, that all ligands induced differentiation to the trophoblast lineage in the absence of bFGF. However, distinct BMPs exerted differences in the kinetics of induced differentiation, with BMP10 being the most potent. hiPSCs and hESCs shared comparable expression patterns of BMP type-I and -II receptor subtypes, which might explain conserved properties with respect to ligand potency and activation of SMAD-dependent (via SMAD1/5/8) and -independent (via MAPK p38) signal transduction pathways. The tested BMPs had distinct and also conserved target genes such as CDX2, DLX3, DLX5, GATA2, GATA3, HAND1, ID2, MSX2 and TFAP2A, known to be associated with the emergence of trophoblast cells. hESCs induced expression of the BMP antagonist NOGGIN as a protection mechanism to constrict extensive BMP action. Unlike BMP4, BMP10 has been shown to be resistant to NOGGIN-induced inhibition which in part might explain its potency. BMPs, in particular BMP4, are commonly used cytokines in differentiation protocols to generate diverse mesoderm- and endoderm-derivates from human pluripotent stem cells. Our study has identified BMP10, a cardiac-specific protein, as a superior alternative to BMP4 for inducing trophoblast differentiation in human pluripotent stem cells.
Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.
Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.
GATA-3 is a newly described marker that labels urothelial and breast carcinoma. However, no prior study has evaluated the expression of GATA-3 in primary bladder adenocarcinoma. Tissue microarrays (TMAs) containing 46 primary bladder adenocarcinomas were constructed. They contained 19 signet ring cell (SRC) and 27 conventional adenocarcinomas. Three additional cases of SRC using routine sections were included resulting in a total of 22 SRCs. In addition, TMAs containing 32 primary gastric signet ring adenocarcinomas and 36 primary lobular breast carcinomas were evaluated. The TMAs were subjected to immunohistochemical analysis for GATA-3, with nuclear labeling scored by intensity and percentage labeling. Breast and urothelial TMAs were also labeled for estrogen receptor, progesterone receptor, and gross cystic duct fluid protein. Diffuse nuclear GATA-3 labeling was seen in 9/22 (41.0%) SRCs and in 2/27 (7.0%) conventional adenocarcinomas (P=0.01). Extracellular mucin production was seen in 12 SRCs. One of 12 (8.0%) SRCs with extracellular mucin was GATA-3 positive, and 8/10 SRCs without extracellular mucin was GATA-3 positive (P=0.005). No nuclear GATA-3 labeling was seen in any gastric signet ring carcinoma. Diffuse, moderate to strong nuclear GATA-3 labeling was seen in 36/36 (100%) primary lobular breast carcinomas. Nuclear GATA-3 labeling is a useful marker for primary adenocarcinomas of the urinary bladder with signet ring features and can be helpful in distinguishing primary signet ring carcinomas of the urinary bladder from gastric signet ring carcinomas. GATA-3 is rarely positive in bladder adenocarcinomas that lack signet ring features and in SRCs displaying extracellular mucin production.
Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.
Although intergenic long noncoding RNAs (lincRNAs) have been linked to gene regulation in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identified 1,524 lincRNA clusters in 42 T cell samples, from early T cell progenitors to terminally differentiated helper T cell subsets. Our analysis revealed highly dynamic and cell-specific expression patterns for lincRNAs during T cell differentiation. These lincRNAs were located in genomic regions enriched for genes that encode proteins with immunoregulatory functions. Many were bound and regulated by the key transcription factors T-bet, GATA-3, STAT4 and STAT6. We found that the lincRNA LincR-Ccr2-5'AS, together with GATA-3, was an essential component of a regulatory circuit in gene expression specific to the TH2 subset of helper T cells and was important for the migration of TH2 cells.
Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy.
Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3(+) (Th2-polarized) lymphocytes; and the number of tumor-infiltrating T-bet(+) (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy.
The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02 ± 0.17 vs. 0.5 ± 0.12 (P = 0.01) and 1.1 ± 0.15 vs. 0.56 ± 0.15 (P = 0.04), respectively. Ratio of GATA-3(+) (Th2-polarized) lymphocytes to T-bet(+) (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85 ± 0.94 vs. 0.98 ± 0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure.
The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.
The fibroblast growth factor receptor 2 (FGFR2) locus has been consistently identified as a breast cancer risk locus in independent genome-wide association studies. However, the molecular mechanisms underlying FGFR2-mediated risk are still unknown. Using model systems we show that FGFR2-regulated genes are preferentially linked to breast cancer risk loci in expression quantitative trait loci analysis, supporting the concept that risk genes cluster in pathways. Using a network derived from 2,000 transcriptional profiles we identify SPDEF, ERα, FOXA1, GATA3 and PTTG1 as master regulators of fibroblast growth factor receptor 2 signalling, and show that ERα occupancy responds to fibroblast growth factor receptor 2 signalling. Our results indicate that ERα, FOXA1 and GATA3 contribute to the regulation of breast cancer susceptibility genes, which is consistent with the effects of anti-oestrogen treatment in breast cancer prevention, and suggest that fibroblast growth factor receptor 2 signalling has an important role in mediating breast cancer risk.
Innate lymphocyte populations play a central role in conferring protective immunity at the mucosal frontier. In this study, we demonstrate that T cell factor 1 (TCF-1; encoded by Tcf7), a transcription factor also important for NK and T cell differentiation, is expressed by multiple innate lymphoid cell (ILC) subsets, including GATA3(+) nuocytes (ILC2) and NKp46(+) ILCs (ILC3), which confer protection against lung and intestinal inflammation. TCF-1 was intrinsically required for the differentiation of both ILC2 and NKp46(+) ILC3. Loss of TCF-1 expression impaired the capacity of these ILC subsets to produce IL-5, IL-13, and IL-22 and resulted in crippled responses to intestinal infection with Citrobacter rodentium. Furthermore, a reduction in T-bet expression required for Notch-2-dependent development of NKp46(+) ILC3 showed a dose-dependent reduction in TCF-1 expression. Collectively, our findings demonstrate an essential requirement for TCF-1 in ILC2 differentiation and reveal a link among Tcf7, Notch, and Tbx21 in NKp46(+) ILC3 development.
Dendritic cells (DCs) are the central architects of the immune response, inducing inflammatory or tolerogenic immunity, dependent on their activation status. As such, DCs are highly attractive therapeutic targets and may hold the potential to control detrimental immune responses. TIM-4, expressed on APCs, has complex functions in vivo, acting both as a costimulatory molecule and a phosphatidylserine receptor. The effect of TIM-4 costimulation on T cell activation remains unclear. In this study, we demonstrate that Ab blockade of DC-expressed TIM-4 leads to increased induction of induced regulatory T cells (iTregs) from naive CD4(+) T cells, both in vitro and in vivo. iTreg induction occurs through suppression of IL-4/STAT6/Gata3-induced Th2 differentiation. In addition, blockade of TIM-4 on previously activated DCs still leads to increased iTreg induction. iTregs induced under TIM-4 blockade have equivalent potency to control and, upon adoptive transfer, significantly prolong skin allograft survival in vivo. In RAG(-/-) recipients of skin allografts adoptively transferred with CD4(+) T cells, we show that TIM-4 blockade in vivo is associated with a 3-fold prolongation in allograft survival. Furthermore, in this mouse model of skin transplantation, increased induction of allospecific iTregs and a reduction in T effector responses were observed, with decreased Th1 and Th2 responses. This enhanced allograft survival and protolerogenic skewing of the alloresponse is critically dependent on conversion of naive CD4(+) to Tregs in vivo. Collectively, these studies identify blockade of DC-expressed TIM-4 as a novel strategy that holds the capacity to induce regulatory immunity in vivo.
Probiotics are believed to have interaction with immune cells through sustained effects on gene expression of different cytokines and transcription factors. The present randomized doubled-blind controlled clinical trial was performed recruiting 75 individuals with BMI 25-35, who were randomly assigned to the following three groups: Group 1 (n = 25) who consumed regular yogurt as part of a low calorie diet [RLCD], group 2 (n = 25) who received probiotic yogurt with a LCD [PLCD] and group 3 (n = 25) who consumed probiotic yogurt without LCD [PWLCD] for 8 week. Participants in PLCD and PWLCD groups received 200 g/day yogurt containing Lactobacillus acidophilus La5, Bifidobacterium Bb12, and lactobacillus casei DN001 10(8) cfu/gr. The expression of the FOXP3, T-bet, GATA3, TNF-α, IFN-γ, TGF-β, and ROR-γt in PBMCs genes were assessed, before and after intervention. In three groups, ROR-γt expression was reduced (P = 0.007) and FOXP3 was increased (P < 0.001). The expression of TNFα, TGFβ, and GATA3 genes did not change among all groups after intervention. Interestingly, the expression of T-bet gene, which was significantly decreased in PLCD and PWLCD groups (P < 0.001), whereas gene expression of IFN-γ decreased in all three groups. Our results suggest that weight loss diet and probiotic yogurt had synergistic effects on T-cell subset specific gene expression in peripheral blood mononuclear cells among overweight and obese individuals.
Asthmatic inflammation is mediated by a type 2 helper T cell (Th2) cytokine response, and blocking Th2 cytokine production is proven to have a potent therapeutic effect against asthmatic inflammation. Using IL-4-green fluorescent protein (GFP) reporter mice, we demonstrated that Bavachinin, a single compound isolated from a Chinese herb, significantly inhibited Th2 cytokine production, including IL-4, IL-5 and IL-13. Notably, this compound almost completely blocked inflammation in the ovalbumin (OVA)-sensitized animal asthma model. Furthermore, we demonstrated that this chemical selectively affects the level of GATA-3, most likely by affecting the stability of GATA-3 mRNA. Our results demonstrate, for the first time, the potential therapeutic value of this single compound derived from Chinese herbs.
While most factors used as reprogramming transgenes can be replaced by other means, Oct4 has remained essential until now. Three recent papers have now broken this barrier through the use of opposing lineage specifying transgenes and chemical modulation, thus signifying a milestone in advancing our understanding of pluripotency induction.
Endothelial microparticles (EMPs) can be involved in inflammatory process, blood coagulation, and regulation of vascular function. However, it remains unclear whether EMPs participate in the pathogenesis of ACS. The purpose of this study is to investigate the impact of EMPs on Th1/Th2 development and functions in vitro.
Eight-five patients were allocated into SAP group (n=27), UAP group (n=28), and AMI group (n=30). Twenty hospitalized patients with normal coronary angiography were recruited as controls. The frequency of EMPs, IFN-γ, and IL-4 levels were measured, and the correlation between EMPs and Th1/Th2 cytokine was analyzed. PBMCs isolated from patients with ACS were treated in vitro with EMPs. This was followed by flow cytometry for Th1/Th2 counts, real-time PCR and western blotting for T-bet and GATA mRNA and protein expression, and ELISA for IFN-γ, TNF-α, IL-4, and IL-10.
This study proved that the frequency of EMPs was significantly increased in ACS patients. There was a significant positive correlation between EMPs and IFN-γ. EMPs could significantly upregulate the differentiation and function of Th1 through increasing the expression of T-bet mRNA and protein. Furthermore, this study also indicated that EMP treatment in vitro could promote the expression of TNF-α, which exerts adverse effects on the pathogenesis and progression of atherosclerosis.
EMPs may be involved in the immune and inflammatory processes that take part in artery atherosclerosis and that they do so by regulating Th1/Th2 differentiation and function. They may play an important role in the pathogenesis of coronary atherosclerosis and plaque instability.
To detect the expression levels of Th1/Th2 cytokines in the acute injury of brain and spinal cord in rats, and explore their possible roles in sustained secondary injury.
The acute brain and spinal cord injury models in SD rats were established and randomly divided into injury group and lipopolysaccharide (LPS) treatment group, and each group was experimentally intervened in the brain and spinal cord, respectively. The mRNA of Th1/Th2 cytokines were determined by quantitative real-time PCR (qRT-PCR) and analyzed in correlation.
Compared with the control group, in injury and LPS treatment groups, the level of IFN-γ (as a Th1 associated cytokine) in brain tissue significantly increased (P<0.05), while the expression of T-bet did not significantly change; in spinal cord, the levels of IFN-γ and transcription factors T-bet, HLX rose significantly (P<0.05). The cytokine IL-4 and transcription factor GATA3 in both injury and LPS treatment groups were as low as those in the control group.
The expressions of Th1 associated cytokines, especially soluble cytokine IFN-γ, increased in brain and spinal cord injury. The up-regulated T-bet and HLX expressions were only observed in spinal injury.
Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreases the risk of breast cancer and its recurrence. However, the biology of COX-2 in the multicellular tumor microenvironment is poorly defined.
Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice that were deficient in mammary epithelial cell COX-2 (COX-2(MEC)KO) compared to wild type (WT) mice. Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, for proliferation, apoptosis, angiogenesis and immune microenvironment. Lentiviral shRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breast cancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenic recipient mice, with or without depletion of CD8+ immune cells.
Mammary tumor onset was delayed, and multiplicity halved, in COX-2(MEC)KO mice compared to WT. COX-2(MEC)KO tumors showed decreased expression of Ki67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2, endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumor vascularization. COX-2(MEC)KO tumors contained more CD4+ T helper (Th) cells and CD8+ cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of Th markers Tbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla, a M2 macrophage marker, lower, in COX-2(MEC)KO tumor infiltrating leukocytes compared to WT, suggesting a prevalence of pro-immune Th1 over immune suppressive Th2 lymphocytes, and reduced macrophage polarization to the immune suppressive M2 phenotype. Enhanced immune surveillance in COX-2(MEC)KO tumors was coincident with increased intratumoral CXCL9, a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitory receptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was also decreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumors in immune competent mice was markedly suppressed. However, robust growth of COX-2KD tumor cells was evident when recipients were depleted of CD8+ cells.
The data strongly support that, in addition to its angiogenic function, tumor cell COX-2 suppresses intratumoral cytotoxic CD8+ immune cell function, possibly through upregulation of immune checkpoints, thereby contributing to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.
CD4(+) T-helper cells (THs) dominate the classical Hodgkin lymphoma (CHL) microenvironment, but their role is poorly understood. Advances in flow cytometry and immunohistochemistry permit more detailed investigation of this aspect of CHL pathophysiology. To address the hypothesis that the TH-infiltrate, rather than being TH2-enriched, senescent and hypofunctional, is TH1 and activation marker-rich, cytokine-secretory and proliferative, we applied comprehensive flow cytometric immunophenotyping and functional assays of cytokine secretion/proliferation to TH cells from 18 CHL-derived single-cell suspensions (SCSs) compared to reactive lymph nodes (RLNs). CHL-derived TH cells express TH1-associated CXCR3/CCR5 and TNFα/IFNγ/interleukin-2 (IL-2) and less TH2-associated CCR3/CCR4, with no IL-4/IL-13. They lack exhaustion-/suppression-associated PD1, CD57 and terminally differentiated effector memory cells, with more central memory cells, activation-associated partners of Hodgkin Reed Sternberg (HRS) cell-expressed CD30/OX40-L/ICOS-L, and other activation markers. TH cell lines established from CHL and RLN-derived SCSs remain cytokine-secretory. We confirmed and extended these studies using tissue microarray immunohistochemistry (TMA-IHC) from a large CHL tissue bank (n = 122) and demonstrate TH1-associated TBET is abundant in CHL, and TH2-associated CMAF/GATA3 and exhaustion-associated PD1 expressed at significantly lower levels. These molecular insights into the CHL-associated TH offer potential diagnostic, prognostic and pharmacologically modifiable therapeutic targets and do not support the established view of a TH2-enriched, senescent/exhausted, hypofunctional, hypoproliferative infiltrate.
Group 2 innate lymphoid cells (ILC2s) play critical roles in anti-helminth immunity and airway epithelial repair. Recently, these cells have also emerged as key players in the development of allergic inflammation at multiple barrier surfaces. ILC2s arise from common lymphoid progenitors in the bone marrow, are dependent on the transcription factors RORα, GATA3, and TCF-1 and produce the type 2 cytokines IL-4, IL-5, IL-9, and/or IL-13. The epithelial cell-derived cytokines IL-25, IL-33, and TSLP regulate the activation and effector functions of ILC2s, and recent studies suggest that their responsiveness to these cytokines and other factors may depend on their tissue environment. In this review, we focus on recent advances in our understanding of how ILC2s are differentially regulated in the context of allergic inflammation and discuss the therapeutic potential of targeting ILC2s in the treatment of allergic diseases.
Renal calyx carcinoma (RCXC) may mimic collecting duct carcinoma (CDC) or urothelial carcinoma (UC) of the renal pelvis. RCXC is distinguished from CDC and UC of the renal pelvis as having the tumor epicenter in the renal calyx, with limited involvement of the surrounding renal pelvis surface urothelium. In this study, we summarize our experience with this entity. Ten cases of RCXC, including 9 cases with urothelial differentiation (RCXC-UC) and 1 case with salivary gland-type differentiation (RCXC-SC), were identified. Ten consecutive cases of UC were selected for comparison, with extensive renal pelvis involvement and with secondary renal parenchymal invasion. Two cases of collecting duct carcinoma (CDC) were also examined. Immunohistochemistry (IHC) was performed on representative tissue blocks for PAX8, PAX2, CK5, CK7, CK20, p63, GATA3, AMACR, RCC, CD10, vimentin, S100, and MSA. The 10 cases of RCXC (M:F=4:6, ages: 62-91 years, mean: 76) presented with renal masses of 3-6cm. Ureteroscopic studies and renal pelvic washings showed atypical/malignant cells in three cases. Seven patients were treated with nephrectomy followed by radiation±chemotherapy, and all cases developed metastases to lymph nodes or liver/lung/bone. In all 7 cases with nephrectomy, there was extensive renal parenchymal involvement with infiltrating borders and diffuse spread along collecting ducts. Six RCXC-UC contained focal squamous differentiation. The RCXC-SC displayed features of adenoid cystic and basaloid features. In situ UC, with or without papillary components, was identified in the calyces in all 7 nephrectomy cases with remaining renal pelvis harboring small tumor burden in 5 cases, and no tumor in another 2 cases. Of the three cases without nephrectomy, no tumor in the renal pelvis could be visualized with endoscopy, however one case was associated with UC of the urinary bladder. Of 10 control UC cases, tumor was limited to the tip of renal papilla in 7 cases, extensive in 3 cases, and with no extension into the collecting ducts. RCXC-UC were all positive for p63, CK5, CK7, and PAX2, with all negative for RCC. PAX8 (70% positive) and GATA3 (50% positive) reactivity was variable. The 10 UC cases shared IHC properties with RCXC-UC but frequent negativity for PAX8 and positivity for GATA3. RCXC is an aggressive neoplasm with high risk of metastases. Similar to CDC, it is located in the renal papilla and rarely with clinically visible renal pelvis tumor or ureteral urine positive for tumor cells. Unlike CDC and non-calyceal UC, RCXC shows predominantly urothelial and squamous differentiation and is associated with an in situ component of adjacent renal calyces. By IHC, RCXC exhibited features intermediate between UC and CDC with decreased or negative immunoreactivity for PAX8 and GATA.
GATA-3 and estrogen receptor (ER) are involved in a positive cross-regulatory loop and are frequently coexpressed in breast cancers. GATA-3 expression was shown to be an independent predictor of overall and disease-free survival in some studies, whereas others showed no difference. However, the studies used different cutoff values for determining GATA-3 positivity and analyzed outcomes in patients who received systemic therapy together with those who did not. We investigated GATA-3 expression and correlated clinicopathologic findings and outcomes in 516 women who received systemic chemotherapy and/or hormonal therapy. Nuclear staining of 1% or greater was considered positive for GATA-3, ER and progesterone receptor (PR). Of 516 cases, 436 (84.5%) were GATA-3+. GATA-3+ tumors were more likely to be grade 1 or 2, ER+, PR+, non-triple-negative phenotypes (all P < .0001), and higher stage (P = .01). ER-/GATA-3+ tumors, compared with ER-/GATA-3- tumors, had worse breast cancer survival (BCS) (P = .02) and a trend for worse overall survival (OS) (P = .05) in univariate analysis. However, there was no difference in OS and BCS between patients who received chemotherapy and/or hormonal therapy among GATA-3-positive and GATA-3-negative groups. GATA-3+ tumors are correlated with lower grade, ER+, PR+, and non-triple-negative phenotypes. Although there was no difference in OS and BCS between GATA-3-positive and GATA-3-negative groups, there was an adverse effect of GATA-3 expression in the ER-negative subgroup of patients who received systemic therapy.
Acute lymphoblastic leukemia (ALL) is the major pediatric cancer diagnosed in economically developed countries with B-cell precursor (BCP)-ALL, accounting for approximately 70% of ALL. Recent genome-wide association studies (GWAS) have provided the first unambiguous evidence for common inherited susceptibility to BCP-ALL, identifying susceptibility loci at 7p12.2, 9p21.3, 10q21.2, and 14q11.2. To identify additional BCP-ALL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1658 cases and 4723 controls, with validation in 1449 cases and 1488 controls. Combined analysis identified novel loci mapping to 10p12.2 (rs10828317, odds ratio [OR] = 1.23; P = 2.30 × 10(-9)) and 10p14 marked by rs3824662 (OR = 1.31; P = 8.62 × 10(-12)). The single nucleotide polymorphism rs10828317 is responsible for the N215S polymorphism in exon 7 of PIP4K2A, and rs3824662 localizes to intron 3 of the transcription factor and putative tumor suppressor gene GATA3. The rs10828317 association was shown to be specifically associated with hyperdiploid ALL, whereas the rs3824662-associated risk was confined to nonhyperdiploid non-TEL-AML1 + ALL. The risk allele of rs3824662 was correlated with older age at diagnosis (P < .001) and significantly worse event-free survivorship (P < .0001). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to BCP-ALL and the influence of constitutional genotype on disease development.
Idiopathic thrombocytopenic purpura (ITP) is an acquired autoimmune disorder. Both impaired platelet production and T cell-mediated effects play a role in ITP thrombocytopenia. A Th1 polarization of the immune response, up-regulation of Th17 cells and decreased number of Treg cells have been demonstrated in ITP patients. High-dose dexamethasone was administered as first-line therapy in adult patients with ITP. However, the mechanism of effects of dexamethasone on ITP is still unclear. In this study, we tested the effectiveness of high-dose dexamethasone as initial treatment in adults with immune thrombocytopenic purpura. PBMCs were isolated from Donors, ITP and Treatment groups. T cell subsets were analyzed by FCM and transcriptional factors were checked by Real-time PCR. We found that dexamethasone returned the ratio of Th1/Th2 and the number of Th17 and Treg cells to the normal levels. Furthermore, we identified that dexamethasone corrected the T cell subset levels through inhibiting GATA3 and FOXp3 expression and promoting RORγt expression. Taken together, we reported a previously unrecognized mechanism on dexamethasone in the ITP treatment.
CD226 costimulatory signals strongly promote Th1 differentiation, enhancing IFN-γ production by naive T cells. We recently reported that knockdown of CD226 on human T cells resulted in a decrease in T-bet and IFN-γ expression. However, the role of CD226 on Th2 and Th17 cells remains unknown. In this study, we found that CD226 and its ligand CD155 were decreased on Th2-polarized naive T cells, whereas both were highly expressed under Th17 conditions. Most IFN-γ- and IL-17-producing cells expressed high levels of CD226, but production of IL-13 did not correlate with CD226 expression. CD226 knockdown by lentiviral transduction resulted in increased STAT-6 phosphorylation, enhanced GATA3 expression, and consequently higher production of IL-4 and IL-13. Under Th17 conditions, CD226-depleted cells showed slightly impaired IL-17 secretion, suggesting that CD226 contributes, in part, to IL-17 production but is dispensable for Th17 cell generation. In line with these results, CD226 blockade with neutralizing Abs efficiently inhibited T cell activation and proliferation and production of IFN-γ and IL-17, whereas IL-13 secretion remained functional. Taken together, our results establish an important role for CD226 in differentially regulating the proinflammatory (Th1/Th17)/anti-inflammatory (Th2) balance, suggesting that the CD226/CD155 interaction could potentially be targeted in therapeutic approaches to human autoimmune diseases.
Differentiated T helper (Th) cell lineages are thought to emerge from alternative cell fate decisions. However, recent studies indicated that differentiated Th cells can adopt mixed phenotypes during secondary immunological challenges. Here we show that natural primary immune responses against parasites generate bifunctional Th1 and Th2 hybrid cells that co-express the lineage-specifying transcription factors T-bet and GATA-3 and co-produce Th1 and Th2 cytokines. The integration of Th1-promoting interferon (IFN)-γ and interleukin (IL)-12 signals together with Th2-favoring IL-4 signals commits naive Th cells directly and homogeneously to the hybrid Th1/2 phenotype. Specifically, IFN-γ signals are essential for T-bet(+)GATA-3(+) cells to develop in vitro and in vivo by breaking the dominance of IL-4 over IL-12 signals. The hybrid Th1/2 phenotype is stably maintained in memory cells in vivo for months. It resists reprogramming into classic Th1 or Th2 cells by Th1- or Th2-promoting stimuli, which rather induce quantitative modulations of the combined Th1 and Th2 programs without abolishing either. The hybrid phenotype is associated with intermediate manifestations of both Th1 and Th2 cell properties. Consistently, hybrid Th1/2 cells support inflammatory type-1 and type-2 immune responses but cause less immunopathology than Th1 and Th2 cells, respectively. Thus, we propose the self-limitation of effector T cells based on the stable cell-intrinsic balance of two opposing differentiation programs as a novel concept of how the immune system can prevent excessive inflammation.
Polarization of cell phenotypes, a common strategy to achieve cell type diversity in metazoa, results from binary cell-fate decisions in the branching pedigree of development. Such "either-or" fate decisions are controlled by two opposing cell fate-determining transcription factors. Each of the two distinct "master regulators" promotes differentiation of its respective sister lineage. But they also suppress one other, leading to their mutually exclusive expression in the two ensuing lineages. Thus, promiscuous coexistence of the antagonist regulators in the same cell, the hallmark of the common "undecided" progenitor of two sister lineages, is considered unstable. This antagonism ensures robust polarization into two discretely distinct cell types. But now the immune system's T-helper (Th) cells and their two canonical subtypes, Th1 and Th2 cells, tell a different story, as revealed in three papers recently published in PLOS Biology. The intermediate state that co-expresses the two opposing master regulators of the Th1 and Th2 subtypes, T-bet and Gata3, is highly stable and is not necessarily an undecided precursor. Instead, the Th1/Th2 hybrid cell is a robust new type with properties of both Th1 and Th2 cells. These hybrid cells are functionally active and possess the benefit of moderation: self-limitation of effector T cell function to prevent excessive inflammation, a permanent risk in host defense that can cause tissue damage or autoimmunity. Gene regulatory network analysis suggests that stabilization of the intermediate center in a polarizing system can be achieved by minor tweaking of the architecture of the mutual suppression gene circuit, and thus is a design option readily available to evolution.
The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.
Distinguishing metastatic carcinoma of breast origin (MCBO) to lung from primary lung carcinomas (PLC) is a diagnostic quandary with clinical ramifications. Immunostains CK7, CK20, ER, PR, and Mammaglobin as well as pertinent negative stains are utilized but prove insufficient. We set out to identify stains either alone or as a group that would better discern between these 2 entities.
Tissue microarrays of 109 MCBO to lung and 102 PLC were stained with CK7, CK20, ER, PR, AR, Mammaglobin, Napsin A, GATA-3, and TTF-1. An H-score was calculated for each case and stain.
The highest area under the receiver-operating characteristic curves for each stain was seen with GATA-3 (0.817), Napsin A (0.817), and TTF-1 (0.854). When all possible combinations were analyzed, GATA-3 and TTF-1 proved to correctly classify with the highest accuracy (0.934). Combinations of GATA-3 and Napsin A (0.920) and GATA-3, Napsin A, and TTF-1 (0.933) were not significantly different from GATA-3 and TTF-1. The odds ratios for each stain and combination of stains showed that those for GATA-3 and TTF-1 were divergent, signifying that cases with higher H-scores for GATA-3 and TTF-1 were more likely to be classified as MCBO and PLC, respectively.
GATA-3 and TTF-1 can correctly classify a case as either MCBO or PLC in 93.4% of cases. Although highly specific and sensitive for PLC, Napsin A in lieu of TTF-1 or as an additional stain did not improve classification accuracy.
Th2 cells are crucially important in allergic disease and the possible involvement of Treg and Th17 cells has not been clearly identified.
To identify the mRNA expression of T cell transcription factors in nasal mucosa in patients with allergic rhinitis (AR) and to reveal their correlations with clinical features.
Eighteen patients with AR and 12 controls with turbinate hypertrophy were included. mRNA expression of the following transcriptional factors in nasal mucosa were measured by quantitative polymerase chain reaction; T-bet (Th1), GATA3 (Th2), retinoic acid-related orphan receptor C (RORC; Th17), and forkhead box P3 (Foxp3; Treg). mRNA expression was compared among groups and correlation between mRNA expression level and clinical features (rhinitis symptoms, eosinophil count, and IgE) were also investigated.
GATA3 and RORC were significantly increased and Foxp3 was significantly decreased in the AR group. Moderate-to-severe AR group also had increased expression of GATA3 and RORC than mild AR group, suggesting severity of AR influence expression of transcription factors. Correlation analysis showed that none of these transcription factors were associated with severity of clinical symptoms, eosinophil counts and skin prick test severity and that IgE level was significantly correlated with expression level of GATA3 and RORC, suggesting an association of IgE production with Th2 and Th17 cells.
Increased mRNA expression of GATA3 (Th2), increased expression of RORC and decreased expression of Foxp3 may be important in pathogenesis of AR. GATA3 and RORC may be closed related with IgE level.
Infections with the parasitic flagellate Ichthyobodo necator (Henneguy, 1883) cause severe skin and gill disease in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) juveniles. The epidermal disturbances including hyperplasia and mucous cell exhaustion caused by parasitization are known, but no details on specific cellular and humoral reactions have been presented. By applying gene expression methods and immunohistochemical techniques, further details of immune processes in the affected skin can be presented. A population of I. necator was established in the laboratory and used to induce an experimental infection of juvenile rainbow trout. The course of infection was followed by sampling for parasite enumeration, immunohistochemistry (IHC) and quantitative PCR (qPCR) on days 0, 5, 9 and 14 post-infection. IHC showed a significant increase in the occurrence of IgM-positive cells in the skin of the infected fish, whereas IgT-positive cells were eliminated and the number of CD8-positive cells declined. qPCR studies supported the IHC findings showing a significant increase in IgM and a decrease in the CD8 gene expression. In addition, genes encoding innate immune genes such as lysozyme, SAA and cathelicidin 2 were up-regulated. Expression of cytokines (IL-1β, IL-4/13A, IL-6, IL-8, IL-10), the cell marker CD4 and the transcription factor GATA3 showed a significant increase after infection. Cytokine profiling including up-regulation of IL-4/13A and IL-10 genes and transcription factor GATA3 connected to the proliferation of IgM producing lymphocytes suggests a partial shift towards a Th2 response associated with the I. necator infection.
Interleukin-19 (IL-19) is a putative Th2, anti-inflammatory interleukin. Its expression and potential role in atherogenesis are unknown. IL-19 is not detected in normal artery and is expressed to a greater degree in plaque from symptomatic versus asymptomatic patients, suggesting a compensatory counter-regulatory function. We tested whether IL-19 could reduce atherosclerosis in susceptible mice and identified plausible mechanisms.
LDLR(-/-) mice fed an atherogenic diet and injected with either 1.0 or 10.0 ng/g per day recombinant mouse IL-19 had significantly less plaque area in the aortic arch compared with controls (P<0.0001). Weight gain, cholesterol, and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19-treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, interferon-γ, interleukin-1β, and interleukin-12β and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19-treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated that IL-19-treated mice have significantly less macrophage infiltrate compared with controls (P<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells, vascular smooth muscle cells, and bone marrow-derived macrophages with IL-19 resulted in a significant decrease in chemokine mRNA and mRNA stability protein human antigen R.
These data suggest that IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.
Individual genetic variations may have a significant influence on the survival of metastatic prostate cancer (PCa) patients. We aimed to identify target genes and their variations involved in the survival of PCa patients using a single nucleotide polymorphism (SNP) panel. A total of 185 PCa patients with bone metastasis at the initial diagnosis were analyzed. Germline DNA in each patient was genotyped using a cancer SNP panel that contained 1,421 SNPs in 408 cancer-related genes. SNPs associated with survival were screened by a log-rank test. Fourteen SNPs in 6 genes, XRCC4, PMS1, GATA3, IL13, CASP8, and IGF1, were identified to have a statistically significant association with cancer-specific survival. The cancer-specific survival times of patients grouped according to the number of risk genotypes of 6 SNPs selected from the 14 SNPs differed significantly (0-1 v. 2-3 v. 4-6 risk genotypes; P = 7.20 × 10(-8)). The high-risk group was independently associated with survival in a multivariate analysis that included conventional clinicopathological variables (P = 0.0060). We identified 14 candidate SNPs in 6 cancer-related genes, which were associated with poor survival in patients with metastatic PCa. A panel of SNPs may help predict the survival of those patients.
GATA3 is a member of a group of zinc-finger transcription factors that is involved in cell development and differentiation. Recent studies have shown that, among tumors, GATA3 is commonly expressed in both urothelial tumors and breast epithelial neoplasms. With the exception of salivary gland and parathyroid tumors, GATA3 has been reported to be either absent or only rarely expressed in other epithelial tumors. Owing to its restricted expression in urothelial and breast carcinomas, GATA3 has proved to be a useful immunohistochemical marker for assisting in distinguishing these 2 groups of neoplasms from other malignancies with which they may be confused.
Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision.
During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3(DTR) mice (which allow specific conditional depletion of Foxp3(+) T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4(+) and CD8(+) T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4(+) T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.
We encountered an undescribed histologic feature of papillary urothelial neoplasms: "urothelial eddy", which was histologically reminiscent of squamous eddy of irritated follicular keratosis of the skin. A review of 756 patients with transurethral resection of bladder tumor revealed 10 patients (1.3%) of papillary urothelial neoplasms with urothelial eddies. All cases were male with a median age of 65 years. Urothelial eddies were characterized by small ovoid nests of ovoid to spindle cells arranged in an onion-skin pattern with fine cytoplasmic processes within wide intercellular space. The cytoplasmic processes mimicked intercellular bridges but ultrastructurally were cytoplasmic microvillous projections. They were of papillary urothelial neoplasm of low malignant potential in seven patients and low-grade urothelial carcinoma in three patients. Nine patients presented as non-invasive tumor and one patient showed microinvasion within papillary stalks. Six patients showed an inverted growth pattern. Their immunoprofile was more similar to that of conventional urothelial carcinoma rather than squamous cell carcinoma: high expressions of GATA3, S100P, uroplakin III, and cytokeratin 7; and low expressions of high molecular weight cytokeratin and p53. The Ki-67 labeling index was low (mean and median values, 2% each). During the follow-up period (mean, 88.7 months), four patients, including the microinvasive patient, showed recurrence with the same grade and stage but neither progressed into muscle-invasive tumor nor caused death. Our results suggest that urothelial eddy is a rare aberrant histology of papillary urothelial neoplasms with indolent behavior and should be discriminated from squamous differentiation of urothelial carcinoma, which has a poor prognosis.
To investigate the potential therapeutic effects of mesenchymal stem cells (MSCs) in inflammatory bowel disease (IBD), we transplanted MSCs into an experimental model of IBD.
A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg body weight) was administered to female BALB/c mice. Bone marrow mesenchymal stem cells (BMSCs) were derived from male green fluorescent protein (GFP) transgenic mice and were transplanted intravenously into the experimental animals after disease onset. Clinical activity scores and histological changes were evaluated. GFP and Sex determining region Y gene (SRY) expression were used for cell tracking. Ki67 positive cells and Lgr5-expressing cells were determined to measure proliferative activity. Inflammatory response was determined by measuring the levels of different inflammatory mediators in the colon and serum. The inflammatory cytokines included tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-17, IL-4, IL-10, and transforming growth factor (TGF-β). Master regulators of Th1 cells (T-box expressed in T cells, T-bet), Th17 cells (retinoid related orphan receptor gamma(t), RORγt), Th2 cells (GATA family of transcription factors 3, GATA3) and regulatory T cells (forkhead box P3, Foxp3) were also determined.
Systemic infusion of GFP-BMSCs ameliorated the clinical and histopathologic severity of colitis, including body weight loss, diarrhea and inflammation, and increased survival (P < 0.05). The cell tracking study showed that MSCs homed to the injured colon. MSCs promoted proliferation of intestinal epithelial cells and differentiation of intestinal stem cells (P < 0.01). This therapeutic effect was mainly mediated by down-regulation of both Th1-Th17-driven autoimmune and inflammatory responses (IL-2, TNF-α, IFN-γ, T-bet; IL-6, IL-17, RORγt), and by up-regulation of Th2 activities (IL-4, IL-10, GATA-3) (P < 0.05). MSCs also induced activated CD4(+)CD25(+)Foxp3(+) regulatory T cells (TGF-β, IL-10, Foxp3) with a suppressive capacity on Th1-Th17 effecter responses and promoted Th2 differentiation in vivo (P < 0.05).
MSCs are key regulators of immune and inflammatory responses and may be an attractive candidate for cell-based therapy of IBD.
We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells.
Translating the developmental program encoded in the genome into cellular and morphogenetic functions requires the deployment of elaborate gene regulatory networks (GRNs). GRNs are especially crucial at the onset of organ development where a few regulatory signals establish the different programs required for tissue organization. In the renal system primordium (the pro/mesonephros), important regulators have been identified but their hierarchical and regulatory organization is still elusive. Here, we have performed a detailed analysis of the GRN underlying mouse pro/mesonephros development. We find that a core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a deeper layer of transcriptional regulators while activating effector genes responsible for cell signaling and tissue organization. Among the genes directly affected by the core components are the key developmental molecules Nephronectin (Npnt) and Plac8. Hence, the pro/mesonephros GRN links together several essential genes regulating tissue morphogenesis. This renal GRN sheds new light on the disease group Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) in that gene mutations are expected to generate different phenotypic outcomes as a consequence of regulatory network deficiencies rather than threshold effects from single genes.
22q11 deletion syndrome is one of the most common chromosomal deletion syndromes and is usually caused by a 1.5-3.0 Mb deletion at chromosome 22q11.2. It is characterized by hypocalcemia resulting from hypoplasia of the parathyroid glands, hypoplasia of the thymus, and defects of the cardiac outflow tract. We encountered a Japanese boy presenting with an unusually severe phenotype of 22q11 deletion syndrome, including progressive renal failure and severe intellectual disabilities. Diagnostic testing using fluorescent in situ hybridization revealed deletion of the 22q11 region, but this did not explain the additional complications. Copy number analysis was therefore performed using whole genome single nucleotide polymorphism (SNP) assay, which identified an additional de novo deletion at 10p14. This region is the locus for hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome caused by haploinsufficiency of GATA3. Together, these two syndromes sufficiently explain the patient's phenotype. This is the first known case report of the co-occurrence of 22q11 deletion syndrome and HDR syndrome. As the two syndromes overlap clinically, this study indicates the importance of carrying out careful clinical and genetic assessment of patients with atypical clinical phenotypes or unique complications. Unbiased genetic analysis using whole genome copy number SNP arrays is especially useful for detecting such rare double mutations.
Red kidney bean (Phaseolus vulgaris L.) is one the most commonly consumed legumes that requires an in depth understanding of its allergenicity. Therefore, the aim of this study was to explore the allergenicity of red kidney bean proteins following oral exposure in BALB/c mice and elucidate the levels of Th1/Th2 transcription factors induced by red kidney bean proteins in rat basophilic leukemia cells (RBL-2H3 cells) passively sensitized with the sera of red kidney bean sensitized mice. Red kidney bean proteins showed enhanced levels of total and specific IgE, anaphylactic symptoms, thymic stromal lymphopoietin (TSLP) and peritoneal albumin over control. Enhanced release of β-hexosaminidase along with up regulated expressions of GATA-3, STAT-6, T-bet, c-MAF and NFAT were observed in the RBL-2H3 cells exposed with red kidney bean proteins when compared to that of the controls. Taken together, exposure of red kidney bean proteins may cause allergic symptoms in mice and the ambivalent effect on Th2/Th1 transcription factors in RBL-2H3 cells.
GATA-3 is a transcription factor involved in human growth and development. Recent studies found its association with breast cancer, however, its expression profile in renal cell carcinoma (RCC) has not been investigated.
The study included 35 patients submitted to radical nephrectomy with confirmed pathological diagnosis of RCC. Normal control kidney tissues were obtained from 25 living kidney donors and tissues were biopsied before implantation. The majority of RCC samples were diagnosed as clear cell renal cell carcinoma (94.3%) except for 1 case of papillary RCC and 1 case of collecting duct carcinoma. GATA-3 expression was evaluated by quantitative PCR and Western blotting (WB) in RCC samples and normal kidneys respectively, immunohistochemical staining was performed as well. Meanwhile, the GATA-3 expression in two cancer cell lines (786-O, ACHN) and normal kidney epithelial cells (HK-2) was detected by PCR and WB. In addition, renal cancer cells and HK-2 cells were cultivated and detected by confocal microscopy for the exact intra-cellular localization of GATA-3.
Data showed a significant down-regulation of GATA-3 expression present in neoplastic tissues compared with normal tissues; similarly, GATA-3 was significantly attenuated in all renal cancer cell lines compared with normal HK-2 cells. Confocal displayed a strong cytoplasmic immno-fluorescence activity of GATA-3 with peri-nuclear zone in HK-2, whereas the intensity in cancer cells was markedly weaker than that of HK-2.
In summary, our present study clarifies that the aberrant expression profile of GATA-3 in human RCC is possibly involved with tumorigenesis, and the complicated mechanism is worthy of further investigation.
To explore the expression and clinical significance of T cell immunoglobulin mucin (TIM)-1, TIM-3 and T cell-specific transcription factors T-bet and GATA-3 in spleen mononuclear cells in patients with primary immune thrombocytopenia (ITP).
The spleen samples were obtained from 17 active ITP patients and 10 controls with spleen traumatic rupture. By using real-time quantitative polymerase chain reaction, the mRNA expressions of TIM-3, TIM1, T-bet and GATA-3 were studied in all subjects.
TIM-3 mRNA levels of active ITP patients were significantly decreased to (29 ± 16)% of that of control, TIM-1 mRNA levels of active ITP patients increased to (3.20 ± 2.18) folds of that of control, but the difference was not significant. The ratio of TIM-1/ TIM-3 was elevated in active ITP patients. T-bet mRNA levels were up-regulated in ITP patients by (2.82 ± 1.57) folds (P<0.05) and the expression of GATA3 was decreased by 14% folds (P<0.05) compared to controls. The ratio of T-bet/GATA3 were significantly elevated in ITP patients.
The imbalance between TIM-3 and TIM-1 expression might play an important role in pathogenesis of ITP.
Phyllodes tumors are uncommon breast tumors that account for less than 0.5% of all breast malignancies. After metastases develop, the prognosis is poor, with very few patients living more than 1 year. The biology of this unusual cancer is not understood and, consequently, no potential targets for treatments are currently available. There has been an exponential increase in the number of commercially available tumor profiling services. Herein, we report a case of metastatic malignant phyllodes tumor for which a comprehensive molecular analysis was performed by using Clinical Laboratory Improvement Amendments (CLIA)-certified labs, providing new insights into the potential opportunities for molecularly targeted therapies for this extremely rare disease.
Next-generation sequencing was performed by using the FoundationOne™ platform (Foundation Medicine, Cambridge, MA). Whole-genome array-based comparative genomic hybridization (array CGH) was performed by using the DNAarray™ (CombiMatrix Diagnostics, Irvine, CA). Immunohistochemical and morphoproteomics analysis were performed at Consultative proteomics®, The University of Texas, UT Health Medical School, Houston,TX (Robert E Brown Lab); Clarient Diagnostics, Aliso Viejo, CA; and Caris Life Sciences Target one, Irving, TX, USA.
Next-generation sequencing showed 3 aberrant genes: activating mutation Q61L on NRAS; inactivating mutations Q504* and K740* on RB1; and TP53 loss. Whole-genome array-based comparative genomic hybridization (array CGH) revealed amplifications of chromosome (chr.) 1 (CKS1B gene), chr. 8 (MYC gene), and chr. 9 (CDKN2A gene) Deletions of chr. 17 (TP53), chr. 10 (GATA3), chr. 11 (FGF4 and CCND1 genes), and chr.22 (PDGFβ). Immunohistochemical analysis for relevant markers showed a positive staining for transducing-like enhancer of split (TLE) 3; secreted protein acidic and rich in cysteine (SPARC) was expressed at 2-3+ in the cytoplasm of the tumors cells, whereas mammalian target of rapamycin (mTOR) was expressed up to 2+ in the nuclei of the tumor cells.
We describe for the first time an NRAS mutation with concomitant activation of PI3K/Akt/mTOR in phyllodes tumor. We also found markers for sensitivity to taxane-based therapies, especially albumin-bound paclitaxel. Exploring the biology of rare malignancies by CLIA certified labs may be reasonable strategy for the development of targeted treatments.
The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.
The accumulated uremic toxins inhibit the expression of various renal transporters and this inhibition may further reduce renal function and subsequently cause the accumulation of uremic toxins. However, the precise mechanism of the nephrotoxicity of uremic toxins on renal transport has been poorly understood. Here we report that indoxyl sulfate, one of the potent uremic toxins, directly suppresses the renal-specific organic anion transporter SLCO4C1 expression through a transcription factor GATA3. The promoter region of SLCO4C1 gene has several GATA motifs, and indoxyl sulfate up-regulated GATA3 mRNA and subsequently down-regulated SLCO4C1 mRNA. Overexpression of GATA3 significantly reduced SLCO4C1 expression, and silencing of GATA3 increased SLCO4C1 expression vice versa. Administration of indoxyl sulfate in rats reduced renal expression of slco4c1 and under this condition, plasma level of guanidinosuccinate, one of the preferable substrates of slco4c1, was significantly increased without changing plasma creatinine. Furthermore, in 5/6 nephrectomized rats, treatment with oral adsorbent AST-120 significantly decreased plasma indoxyl sulfate level and conversely increased the expression of slco4c1, following the reduction of plasma level of guanidinosuccinate. These data suggest that the removal of indoxyl sulfate and blocking its signal pathway may help to restore the SLCO4C1-mediated renal excretion of uremic toxins in CKD.
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.
Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells.
We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and TH cell functional differentiation.
A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T(H)1, T(H)2, and T(H)17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA.
Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1, IL13, and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T(H)2 and T(H)17 CD4 T cells, respectively, without affecting GATA3, retinoic acid-related orphan receptor γt (RORγt), or T(H)1 cells and IFNG and T-bet expression. Short hairpin RNA-mediated silencing of Cyp11a1 in polarized T(H)2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels.
Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.
Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c(+) MHCII(+) DCs in the immune-sampling Peyer's Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine.
Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in ∼62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD.
The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor.
Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.
The transcription factor GATA3 is a key regulator of mammary gland development and a definitive marker of luminal breast cancer. However, the molecular mechanisms underlying the role of GATA3 in breast carcinogenesis is still not fully understood. We report here that GATA3 promotes cell proliferation and tumorigenesis by facilitating the G1/S transition through its transcription regulation of the CCND1 gene in breast cancer cells. We found that GATA3 is physically associated with poly-ADP ribose polymerase-1 (PARP1), an enzyme modifying nuclear proteins by poly(ADP-ribosyl)ation. We showed that PARP1 acts as a transcription coactivator for GATA3 in breast cancer cells and demonstrated that GATA3 cooperates with PARP1 in transactivation of the CCND1 gene. We demonstrated that PARP1 competes with linker histone H1 to maintain a transcriptional competent chromatin environment for CCND1 gene. Our results unveiled a molecular basis for the coordinated regulation between GATA3 and PARP1 in transcription activation, providing a mechanism for GATA3 in breast carcinogenesis.
The key molecular events required for the formation of ductal carcinoma in situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here, we show that the nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal three-dimensional mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both before DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ breast cancer-initiating cell (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. The loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, human epidermal growth factor receptor 2 (HER2) and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 has a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through the maintenance of BCIC, facilitates formation of DCIS, a necessary step before development of invasive disease.
Role of inflammation-induced oxidative stress in the pathogenesis and progression of chronic inflammatory airways diseases has received increasing attention in recent years. Nuclear factor erythroid 2-related factor 2 is the primary transcription factor that regulates the expression of antioxidant and detoxifying enzymes. Graptopetalum paraguayense E. Walther, a vegetable consumed in Taiwan, has been used in folk medicine for protection against liver injury through elevating antioxidation. Recently, we found that gallic acid is an active compound of Graptopetalum paraguayense E. Walther, which has been reported to inhibit T-helper 2 cytokines. Currently, we assumed that Graptopetalum paraguayense E. Walther may potentially protect against ovalbumin-induced allergy and airway inflammation. Results demonstrated that Graptopetalum paraguayense E. Walther ethanolic extracts (GPE) clearly inhibited airway inflammation, mucus cell hyperplasia, and eosinophilia in OVA-challenged mice. Additionally, GPE also prevented T-cell infiltration and Th2 cytokines, including interleukin- (IL-)4, IL-5, and IL-13 generations in bronchial alveolar lavage fluid. The adhesion molecules ICAM-1 and VCAM-1 were substantially reduced by GPE treatment mediated by Nrf2 activation. Moreover, GPE attenuated GATA3 expression and inhibited Th2 signals of the T cells. These findings suggested that GPE ameliorated the development of airway inflammation through immune regulation.
Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors with a high rate of local recurrence and a significant tendency to metastasize. Its dismal outcome points to the urgent need to establish better therapeutic strategies for patients harboring MPNSTs. The investigations of genomic and molecular aberrations in MPNSTs which detect many chromosomal aberrations, pathway abnormalities, and specific molecular aberrant events would supply multiple potential therapy targets and contribute to achievement of personalized medicine. The involved genes in the significant gains aberrations include BIRC5, CCNE2, DAB2, DDX15, EGFR, DAB2, MSH2, CDK6, HGF, ITGB4, KCNK12, LAMA3, LOXL2, MET, and PDGFRA. The involved genes in the significant deletion aberrations include CDH1, GLTSCR2, EGR1, CTSB, GATA3, SULT2A1, GLTSCR2, HMMR/RHAMM, LICAM2, MMP13, p16/INK4a, RASSF2, NM-23H1, and TP53. These genetic aberrations involve in several important signaling pathways such as TFF, EGFR, ARF, IGF1R signaling pathways. The genomic and molecular aberrations of EGFR, IGF1R, SOX9, EYA4, TOP2A, ETV4, and BIRC5 exhibit great promise as personalized therapeutic targets for MPNST patients.
Many lines of evidence propose that psoriasis is a T cell-mediated disease where T cell activation is followed by secretion of inflammatory cytokines. To elucidate the functional state of T cells in guttate psoriasis, we analysed mRNA expression levels of T-bet and GATA-3 for Th1 and Th2 differentiation, respectively together with Th1 (IFN-γ) and Th2 (IL-4) cytokine mRNA expression. Relative quantification of T-bet, GATA-3, IFN-γ and Th2, and IL-4 transcripts in peripheral blood leukocytes (PBL) was conducted by real-time reverse transcriptase PCR (RT-PCR). Serum levels of IFN-γ and Th2 and IL-4 were also determined by ELISA. GATA-3 and IL-4 mRNA expression levels were lower in psoriatic patients as compared to normal healthy controls. The expression levels of T-bet and IFN-γ and Th2 genes were relatively similar in the patients and controls. In addition, a marked decrease in plasma IL-4 levels was observed in the psoriasis group, while no differences were observed with regard to levels of IFN-γ and Th2 between patients and normal subjects. Furthermore, a clear correlation between decreased IL-4 mRNA expression and IL-4 (P < 0.05) was revealed. These results suggested that altered balance between Th1 and Th2 cells transcription factor genes and they products may be implicated in the pathogenesis of psoriasis.
Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.
The DNAzyme hgd40 was shown to effectively reduce expression of the transcription factor GATA-3 RNA which plays an important role in the regulation of Th2-mediated immune mechanisms such as in allergic bronchial asthma. However, uptake, biodistribution and pharmacokinetics of hgd40 have not been investigated yet. We examined local and systemic distribution of hgd40 in naive mice and mice suffering from experimental asthma. Furthermore, we evaluated the pharmacokinetics as a function of dose following single and repeated administration in rats and dogs. Using intranasal administration of fluorescently labeled hgd40 we demonstrated that the DNAzyme was evenly distributed in inflamed asthmatic mouse lungs within minutes after single dose application. Systemic distribution was investigated in mice using radioactive labeled hgd40. After intratracheal application, highest amounts of hgd40 were detected in the lungs. High amounts were also detected in the bladder indicating urinary excretion as a major elimination pathway. In serum, low systemic hgd40 levels were detected already at 5 min post application (p.a.), subsequently decreasing over time to non-detectable levels at 2h p.a. As revealed by Single Photon Emission Computed Tomography, trace amounts of hgd40 were detectable in lungs up to 7 days p.a. Also in the toxicologically relevant rats and dogs, hgd40 was detectable in blood only shortly after inhalative application. The plasma pharmacokinetic profile was dose and time dependent. Repeated administration did not lead to drug accumulation in plasma of dogs and rats. These pharmacokinetic of hgd40 provide guidance for clinical development, and support an infrequent and convenient dose administration regimen.
The embryonic spinal cord in mice is organized into eleven progenitor domains. Cells in each domain first produce neurons and then switch to specifying glia. Five of these domains known as p3, pMN, p2, p1 and p0 are located in the ventral spinal cord and each expresses a unique code of transcription factors (TFs) that define the molecular profile of progenitor cells. This code is largely responsible for determining the subtype specification of neurons generated from each domain. Pax6 codes for a homedomain-containing TF that plays a central role in defining the molecular boundaries between the two ventral-most domains, p3 and pMN. Using fate mapping and gene expression studies we show that PAX6, in addition to each patterning function, is expressed in a group of late born interneurons that derive from the p2 and p0 domains. The p2-derived neurons represent a subset of late born V2b interneurons and their specification depends on Notch signaling. The V0 neurons represent V0v ventral neurons expressing Pax2. Our data demonstrate that interneuron diversity in the ventral spinal cord is more complex than originally appreciated and point to the existence of additional mechanisms that determine interneuron diversity, particularly in the p2 domain.
IgG4-related sclerosing cholangitis and type 1 autoimmune pancreatitis (IgG4-SC/AIP) are characterized by massive lymphoplasmacytic infiltration including Th2 and regulatory T cells (Tregs). This study was conducted to address which chemotactic factors are involved in this condition.
Chemokine expression profiles in tissue were examined in IgG4-SC/AIP (n=17), classical primary sclerosing cholangitis (IgG4(low) PSC, n=17), PSC with elevated serum/tissue IgG4 levels (IgG4(high) PSC, n=5), and primary biliary cirrhosis (n=7). We focused on five chemotactic factors/receptors (CCL1-CCR8, CCL17/CCL22-CCR4), given that CCR4 and CCR8 are predominantly expressed in both Th2 and Tregs.
In conjunction with higher expression levels of IL-4 and IL-10, expression values of CCL1 and CCR8 transcripts were significantly higher in IgG4-SC/AIP than in IgG4(low) PSC (p=0.002) and IgG4(high) PSC (p=0.023). CCL1 and CCR8 were also overexpressed in IgG4(high) PSC than in IgG4(low) PSC (p=0.023). No difference was seen for CCL17, CCL22, and CCR4. In situ hybridization revealed CCL1 to be predominantly expressed in the pancreatic duct epithelium, peribiliary glands, and vascular endothelial cells including the ones involved in obliterative phlebitis in IgG4-SC/AIP, in contrast to IgG4(high) PSC where this chemotactic factor was positive in several infiltrating lymphocytes. These CCL1-expressing sites were infiltrated by CCR8(+) lymphocytes. On immunohistochemistry, GATA3(+) Th2 lymphocytes and FOXP3(+) Tregs were significantly larger in number in IgG4-SC/AIP, with the GATA3(+)/T-bet(+) cell ratio to be shifted in favour of Th2 in periductal and perivascular areas.
CCL1-CCR8 interaction may play a critical role in lymphocytic recruitment in IgG4-SC/AIP, leading to duct-centred inflammation and obliterative phlebitis.
Polysaccharopeptide (PSP) is well known for its immunoregulatory effects. In the present study, the effect of PSP on white blood cell (WBC) count, T lymphocyte subsets, B lymphocytes, Th1/Th2 balance and negative immune regulators was investigated using an immunosuppressed mouse model. The results demonstrated that the WBC count and the absolute number of CD3+CD4+ T cells, CD3+CD8+ T cells and CD3‑CD19+ B cells in the peripheral blood were increased in PSP‑treated groups as compared with the cyclophosphamide (Cy) group. In addition, PSP reduced interleukin (IL)‑4 and GATA binding protein 3 (GATA‑3) mRNA relative expression levels and elevated the ratios of IL‑2/IL‑4 and the transcription factors, T‑box‑containing protein/GATA‑3. The relative mRNA expression levels of the forkhead/winged‑helix transcription factor box protein 3 (Foxp3), programmed death‑1 (PD‑1) and IL‑10 were also downregulated by PSP. These observations indicate that the immunoregulatory effects of PSP are associated with restoration of WBC number, improving the absolute number of T lymphocyte subsets and B lymphocytes, inducing the Th1/Th2 response and downregulating the negative immune regulators, Foxp3, PD‑1 and IL‑10.
T-cell acute lymphoblastic leukemia (T-ALL) is caused by a defect in T-cell maturation to the mature T cell. T-ALL is a poor prognostic hematopoietic malignancy. In order to establish a successful therapeutic treatment plan, it is essential to understand the biology of T-cell development and molecules that contribute to this process. This study uses Jurkat T cells, as a well-established model for in vitro study of T-ALL to investigate the role of the microRNA (miRNA), miR-146a, on gene expressions involved in T-cell differentiation.
The permanent over-expression of miR-146a was established using a lentivector that expressed GFP hsa-mir-146a miRNA. We used quantitative real-time polymerase chain reaction and flow cytometry for T-cell differentiation to monitor induction of the differentiation process by assessing changes in expression of some distinct transcription factors and cell surface markers.
Ectopic expression of miR-146a resulted in significant up-regulation of PU.1, c-Fos, CCAAT/enhancer-binding protein alpha (C/EBPα) and GATA3, and slight up-regulation of Foxp3 and Runx1. There was a significant, moderate down-regulation in the expressions of Notch1, LIM-domain only (Lmo2), son of sevenless 1 (SOS1), Ikaros, and signal transducer and activator of transcription 3 (STAT3).
Our results indicated that ectopic expression of miR-146a could not independently induce differentiation in lymphoblastic cells. However, the expression of multiple genes involved in T-cell differentiation and T-cell CD markers were found to be affected. These results have suggested a potential tumor suppressive, immunomodulatory and cell activator role for miR146-a.
Triptolide, a type of diterpenoid, is the active compound of Tripterygium wilfordii; it plays roles in anti-inflammatory and immune response regulation. Our objective was to investigate the mechanism of the inhibitory effect of triptolide on interleukin-13 (IL-13) gene expression in activated T lymphocytes. Understanding the molecular mechanism by which triptolide exerts a therapeutic function may be useful in developing a pharmaceutical treatment for asthma.
Peripheral blood mononuclear cells (PBMC) and Hut-78 cells were stimulated with anti-CD3/CD28 with or without co-incubation with triptolide. The alteration of IL-13 messenger RNA (mRNA), expression and protein level were analysed using real-time reverse transcription polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay, respectively. The intracellular distribution profile of transcription factor GATA3 and nuclear factor of activated T cells (NFAT1) were analysed by Western blotting. The binding rates of GATA3 and NFAT1 to the promoter sequence of IL-13 were analysed by chromatin immunoprecipitation (ChIP) PCR.
In PBMC, the release of IL-13 was dependent on anti-CD3/CD28 stimulation. Its release could be inhibited by triptolide at the concentration of 500 nmol. In Hut-78 cells, IL-13 mRNA and protein expression were increased with anti-CD3/CD28 stimulation and significantly inhibited by incubation with 28 nmol triptolide. This concentration of triptolide also significantly inhibited the nuclear translocation of GATA3 and NFAT1 reducing the binding rate to the IL-13 gene promoter.
Triptolide inhibits IL-13 gene transcription and protein expression by inhibiting GATA3 and NFAT1 nuclear translocation and their binding rates to the IL-13 gene promoter region.
The aim of this study was to evaluate the effects of yerba mate extract and its principal bioactive compounds on adipogenesis. The anti-adipogenic effects of yerba mate, chlorogenic acid, quercetin and rutin were evaluated in 3T3-L1 cells using a PCR array. The results obtained in vitro were validated in vivo in a high-fat diet-induced model of obesity. The in vitro and in vivo results demonstrated that yerba mate extract down-regulated the expression of genes that regulate adipogenesis, such as Creb-1and C/EBPα, and the extract up-regulated the expression of genes related to the inhibition of adipogenesis, including Dlk1, Gata2, Gata3, Klf2, Lrp5, Pparγ2, Sfrp1, Tcf7l2, Wnt10b, and Wnt3a. In summary, it was demonstrated that yerba mate and its bioactive compounds regulate the expression of genes related to in vitro adipogenesis. Furthermore, yerba mate might regulate adipogenesis through the Wnt pathway.
Recent studies have shown that T helper type-2 (Th2) cells can induce the apoptosis of CD4+CD25+ Treg cells or resist the immunosuppressive effect of Treg cells. We hypothesize that an imbalance of Th2/Treg is present in patients with allergic asthma.
Twenty-two patients with mild asthma, 17 patients with moderate to severe asthma, and 20 healthy donors were enrolled. All patients were allergic to house dust mites. The proportion of peripheral blood CD4+CD25+ Treg cells and Th2 cells were determined by flow cytometry. The concentration of interleukin (IL)-10, transforming growth factor (TGF)-β and IL-4 in plasma was determined by enzyme linked immunosorbent assay. In these subjects, peripheral blood mononuclear cells from 17 mild asthmatic patients, 13 moderate to severe asthmatic patients and 14 healthy donors were acquired and expression of forkhead box P3 (Foxp3) and GATA-3 mRNA was detected by reverse-transcriptase polymerase chain reaction.
Compared with healthy donors and patients with mild asthma, the percent of CD4+CD25+ Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. There were no significant differences in Foxp3 mRNA expression among three groups, but a downward trend seen among patients with asthma. However, the percent of Th2 cells, IL-4 levels and expression of GATA-3 mRNA was markedly higher in patients with mild and moderate to severe asthma than in the control group. The ratio of Th2/Treg and their cytokines was increased in allergic asthma, especially for moderate to severe asthma. The ratio of GATA-3/Foxp3 mRNA was also increased in allergic asthma. In patients with moderate to severe asthma, the percentage of peripheral blood Treg cells was negatively correlated to the percentage of Th2 cells and IL-4 levels.
The decline of CD4+CD25+ Treg cells in patients with moderate to severe asthma may play an important role in progress of the disease. Furthermore, the deficiency of CD4+CD25+ Treg cells was associated with the over-expression of Th2 response.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in Helicobacter pylori-induced gastric carcinogenesis. While the mechanism is not well understood, BM-MSCs have been shown to contribute to the immunosuppressive response found in a number of diseases. Here, BM-MSCs were transplanted into the stomach of mice with a 44-week mouse-adapted H. pylori infection. At day 28 post-transplantation, BM-MSCs migrated from the subserosal to the mucosal layer of the stomach. The grafted BM-MSCs significantly stimulated systemic and local interleukin-10 (IL-10)-secreting T cell and regulatory T cell (Treg) functions. This observation was correlated with an increased percentage of CD4⁺IL-10⁺ cells and CD4⁺CD25⁺FoxP3⁺ cells in splenic mononuclear cells compared with H. pylori-infected mice not receiving BM-MSCs. Moreover, inhibitory cytokines IL-10 and transforming growth factor-β1 increased in the gastric tissue, while there was a decrease in inflammatory interferon-γ (IFN-γ). BM-MSC-transplanted mice also developed elevated IL-10/IFN-γ secreting and Treg/Th17 ratios. A coculture system in the presence or absence of BM-MSCs was also established to evaluate the immune responses in vitro. An increase in IL-10-secreting T cells and Tregs, associated with increased expression of Gata-3 and FoxP3, generation of IL-10 in the supernatant, and proliferation of gastric epithelial cells (GECs) was observed. These findings demonstrate that transplantation of BM-MSCs into a chronic H. pylori-infected mouse model results in the generation of an immunosuppressive environment. The local and systemic immunosuppression mediated by BM-MSCs likely contributed to an environment that is compatible with the development of H. pylori-induced gastric cancer.
Breast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate-derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival and is required in models of endocrine resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for patients with breast cancer treated with endocrine therapy.
Fibrocytes are hematopoietic stem cell-derived fibroblast precursors that are implicated in chronic inflammation, fibrosis, and wound healing. Myeloid-derived suppressor cells (MDSCs) expand in cancer-bearing hosts and contribute to tumor immune evasion. They are typically described as CD11b⁺HLA-DR⁻ in humans. We report abnormal expansions of CD11b⁺HLA-DR⁺ myeloid cells in peripheral blood mononuclear fractions of subjects with metastatic pediatric sarcomas. Like classical fibrocytes, they display cell surface α smooth muscle actin, collagen I/V, and mediate angiogenesis. However, classical fibrocytes serve as antigen presenters and augment immune reactivity, whereas fibrocytes from cancer subjects suppressed anti-CD3-mediated T-cell proliferation, primarily via indoleamine oxidase (IDO). The degree of fibrocyte expansion observed in individual subjects directly correlated with the frequency of circulating GATA3⁺CD4⁺ cells (R = 0.80) and monocytes from healthy donors cultured with IL-4 differentiated into fibrocytes with the same phenotypic profile and immunosuppressive properties as those observed in patients with cancer. We thus describe a novel subset of cancer-induced myeloid-derived suppressor cells, which bear the phenotypic and functional hallmarks of fibrocytes but mediate immune suppression. These cells are likely expanded in response to Th2 immune deviation and may contribute to tumor progression via both immune evasion and angiogenesis.
We review the syndrome of hypoparathyroidism, deafness, and renal anomalies (HDR syndrome).
The current understanding and relevant literature pertaining to the background, genetic considerations, clinical features, prognosis, and treatment of HDR syndrome are reviewed.
The combination of hypoparathyroidism, deafness, and renal anomalies constitutes an unusual syndrome associated most commonly with haploinsufficiency in GATA3, which encodes a transcription factor that binds to the (A/T) GATA (A/G) consensus DNA sequence. Sensorineural hearing loss is the most consistently expressed clinical feature, being present in almost all affected individuals, and the combination of hypoparathyroidism and hearing impairment occurs in well over 90% of those affected, with various renal anomalies being the most heterogeneous feature of the classic triad. We characterize, in tabular form, the individual cases described in the literature and propose a classification scheme based on the presence or absence of renal anomalies. We also include the specific genetic abnormality and renal anomaly associated with each individual case.
HDR syndrome is a heterogeneous syndrome most commonly associated with GATA3 haploinsufficiency.
The development of macrophages is a highly regulated process requiring coordination amongst transcription factors. The presence/absence, relative levels, antagonism, or synergy of all transcription factors involved is critical to directing lineage cell fate and differentiation. While relative levels of many key myeloid transcription factors have been determined in mammalian macrophage differentiation, a similar set of studies have yet to be conducted in a teleost system. In this study, we report on the mRNA levels of transcription factors (cebpa, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1) in sorted goldfish progenitor cells, monocytes, and macrophages from primary kidney macrophage cultures. The mRNA levels of runx1 and pu.1 were significantly higher, gata3 and pax5 mRNA levels were lower, in monocytes compared to progenitors, and the mRNA levels of cjun, egr1, gata2, gata3, mafb and pax5 were significantly decreased in macrophages compared to progenitor cells. The relative mRNA levels of the interferon regulatory factor family of transcription factors, irf1, irf2, irf5, irf7, irf8 and irf9 in sorted progenitors, monocytes and macrophages were also measured. In contrast to other irf family transcription factors examined, irf8 mRNA levels were increased in monocytes compared to progenitors by greater than three-fold, suggesting that irf8 is important for monopoiesis. Lastly, we show the differential regulation of myeloid transcription factor mRNA levels in sorted progenitor cells from 1, 2, or 3-day old cultures in response to the recombinant goldfish growth factors, rgCSF-1 and rgKITLA.
Group 2 innate lymphoid cells (ILC2s; also called nuocytes, innate helper cells, or natural helper cells) provide protective immunity during helminth infection and play an important role in influenza-induced and allergic airway hyperreactivity. Whereas the transcription factor GATA binding protein 3 (Gata3) is important for the production of IL-5 and -13 by ILC2s in response to IL-33 or -25 stimulation, it is not known whether Gata3 is required for ILC2 development from hematopoietic stem cells. Here, we show that chimeric mice generated with Gata3-deficient fetal liver hematopoietic stem cells fail to develop systemically dispersed ILC2s. In these chimeric mice, in vivo administration of IL-33 or -25 fails to expand ILC2 numbers or to induce characteristic ILC2-dependent IL-5 or -13 production. Moreover, cell-intrinsic Gata3 expression is required for ILC2 development in vitro and in vivo. Using mutant and transgenic mice in which Gata3 gene copy number is altered, we show that ILC2 generation from common lymphoid progenitors, as well as ILC2 homeostasis and cytokine production, is regulated by Gata3 expression levels in a dose-dependent fashion. Collectively, these results identify Gata3 as a critical early regulator of ILC2 development, thereby extending the paradigm of Gata3-dependent control of type 2 immunity to include both innate and adaptive lymphocytes.
MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translational repression or RNA degradation. The aberrant expression of miRNAs might be involved in human diseases, including cancer. The expression of miR-206 in estrogen receptor alpha (ER-α)-positive human breast cancer tissues is well known. However, the expression and regulation of miR-206 in the developing mammary gland has not yet been studied. To understand the effects of miR-206 on mammary gland development, we have profiled gene expression in scramble-transfected and miR-206-overexpressing developing mammary buds. The genes that are potentially regulated by miR-206 in the mammary epithelium and/or mesenchyme, such as Tachykinin1 and Gata3, are known to be breast cancer markers. The expression of Wnt, which is involved in gland positioning, and of the transcription factors Tbx3 and Lef1, which are essential for mammary gland development, changes after miR-206 overexpression. Using a mammary bud in vitro culture system, we have demonstrated that miR-206 acts downstream of ER-α during mammary gland growth. Thus, miR-206 might be a novel candidate for morphogenesis during the initiation of mammary gland formation and the regulation of genes related to mammary gland development and breast cancer.
Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.
The T helper 17 (Th17) cells have been suggested to have osteoclast activating effects while T helper 2 (Th2) cells are considered to have an osteoprotective role in periodontitis. This study was to compare the markers of Th17 cells (RORC2 and IL-17 genes) with that of Th2 cells (IL-4 and GATA-3 genes) between healthy and periodontitis tissues.
The samples were obtained from patients with periodontitis and healthy tissues. The mRNA expression levels of IL-17, RORC2, IL-4 and GATA-3 were measured in both groups using quantitative RT-PCR. The results were compared using SPSS 11.0. Correlation coefficient was analyzed by Spearman's rho test. Mann-Whitney was used to measure the difference between IL-17 and IL-4 as well as RORC2 and GATA-3.
In periodontal lesions, the expression levels of all markers were significantly higher than the healthy tissue (p≤ 0.001). The results showed a significant increase in the number of markers of Th17 (RORC2 and IL-17 genes) compared to markers of Th2 (GATA-3, IL-4) in patients with periodontitis compared to controls (p≤0.002). A positive correlation between IL-17 and RORC2 (p≤0.05) and between IL-4 and GATA-3 (p≤0.001) was found.
The results show that expression levels of IL-4, GATA-3, IL-17 and RORC2 increase significantly in periodontal lesions compared with the controls. In periodontal lesions, IL-17 levels are significantly greater than IL-4, which plays a protective role against alveolar bone loss.
The importance of the estrogen receptor (ER) in breast cancer (BCa) development makes it a prominent target for therapy. Current treatments, however, have limited effectiveness, and hence the definition of new therapeutic targets is vital. The ER is a member of the nuclear hormone receptor superfamily of transcription factors that requires co-regulator proteins for complete regulation. Emerging evidence has implicated a small number of histone methyltransferase (HMT) and histone demethylase (HDM) enzymes as regulators of ER signalling, including the histone H3 lysine 9 tri-/di-methyl HDM enzyme KDM4B. Two recent independent reports have demonstrated that KDM4B is required for ER-mediated transcription and depletion of the enzyme attenuates BCa growth in vitro and in vivo. Here we show that KDM4B has an overarching regulatory role in the ER signalling cascade by controlling expression of the ER and FOXA1 genes, two critical components for maintenance of the estrogen-dependent phenotype. KDM4B interacts with the transcription factor GATA-3 in BCa cell lines and directly co-activates GATA-3 activity in reporter-based experiments. Moreover, we reveal that KDM4B recruitment and demethylation of repressive H3K9me3 marks within upstream regulatory regions of the ER gene permits binding of GATA-3 to drive receptor expression. Ultimately, our findings confirm the importance of KDM4B within the ER signalling cascade and as a potential therapeutic target for BCa treatment.
The severity of most human birth defects is highly variable. Our ability to diagnose, treat and prevent defects relies on our understanding of this variability. Mutation of the transcription factor GATA3 in humans causes the highly variable hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. Although named for a triad of defects, individuals with HDR can also exhibit craniofacial defects. Through a forward genetic screen for craniofacial mutants, we isolated a zebrafish mutant in which the first cysteine of the second zinc finger of Gata3 is mutated. Because mutation of the homologous cysteine causes HDR in humans, these zebrafish mutants could be a quick and effective animal model for understanding the role of gata3 in the HDR disease spectrum. We demonstrate that, unexpectedly, the chaperone proteins Ahsa1 and Hsp90 promote severe craniofacial phenotypes in our zebrafish model of HDR syndrome. The strengths of the zebrafish system, including rapid development, genetic tractability and live imaging, make this an important model for variability.
Autonomic neurons and chromaffin cells, which constitute the autonomic nervous system, are derived from a common progenitor from the neural crest, and its development is controlled by a network of transcription factors, including the master regulator, Phox2b, and its downstream, Gata3. Anti-Phox2b and anti-Gata3 antibodies were applied to a total of 77 autonomic nervous system tumors, including 35 paragangliomas, 21 pheochromocytomas, 9 neuroblastomas, 4 ganglioneuroblastomas, and 8 ganglioneuromas, as well as their potential morphologic mimics, including tumors of the small round cell tumor group, neuroendocrine carcinomas of lung and gastrointestinal tract (carcinoid tumors/neuroendocrine tumors, large cell neuroendocrine carcinomas, and small cell carcinomas), Merkel cell carcinomas, benign and malignant tumors of thyroid, parathyroid, and adrenal cortex, and malignant melanomas. A variety of nonendocrine/neuroendocrine carcinomas were also studied. Gata3 expression was seen in 89% of paragangliomas, 95% of pheochromocytomas, and all neuroblastomas, ganglioneuroblastomas, and ganglioneuromas, as well as in all parathyroid tumors, a majority of urothelial and mammary carcinomas, and a subset of squamous cell carcinomas, but all other tumors were negative. Phox2b expression was seen in all neuroblastomas, ganglioneuroblastomas, and ganglioneuromas and in 40% of paragangliomas, but pheochromocytomas and all other tumors were negative. Gata3 is a highly reliable marker for paragangliomas, pheochromocytomas, and neuroblastic tumors to distinguish from their simulators. This is an additional utility for this marker, which is used for the diagnosis of urothelial and mammary carcinomas. Phox2b is also highly specific, but its low sensitivity to paragangliomas and pheochromocytomas would limit the utility only to neuroblastic tumors.
GATA-3 controls T helper type 2 (TH2) differentiation. However, whether GATA-3 regulates the function of mature T cells beyond TH2 determination remains poorly understood. We found that signaling via the T cell antigen receptor (TCR) and cytokine stimulation promoted GATA-3 expression in CD8(+) T cells, which controlled cell proliferation. Although GATA-3-deficient CD8(+) T cells were generated, their peripheral maintenance was impaired, with lower expression of the receptor for interleukin 7 (IL-7R). GATA-3-deficient T cells had defective responses to viral infection and alloantigen. The proto-oncoprotein c-Myc was a critical target of GATA-3 in promoting T cell proliferation. Our study thus demonstrates an essential role for GATA-3 in controlling the maintenance and proliferation of T cells and provides insight into immunoregulation.
Andrographolide, an active component in traditional anti-diabetic herbal plants, is a diterpenoid lactone isolated from Andrographis paniculata because of its potent anti-inflammatory and hypoglycemic effects. However, the effect of andrographolide on the development of diabetes in autoimmune non-obese diabetic (NOD) mice remains unknown. This study aimed to investigate the protective effects of andrographolide on the development of autoimmune diabetes and clarify the underlying mechanism. NOD mice were randomly divided into four groups and administered with water and andrographolide at 50, 100, and 150mg/kg body weight for four weeks. ICR mice were also selected as the control group. Oral glucose tolerance and histopathological insulitis were examined. Th1/Th2/Th17 cytokine secretion was determined by ELISA. The transcriptional profiles of T-bet, GATA3, and RORγt in the pancreatic lymphatic node samples derived from the NOD mice were detected by RT-PCR. After four weeks of oral supplementation, andrographolide significantly inhibited insulitis, delayed the onset, and suppressed the development of diabetes in 30-week-old NOD mice in a dose dependent manner. This protective status was correlated with a substantially decreased production of interferon (IFN)-γ and interleukin (IL)-2, increased IL-10 and transforming growth factor (TGF)-β, and a reduced IL-17. Andrographolide also increased GATA3 mRNA expression but decreased T-bet and RORγt mRNA expressions. Our results suggested that andrographolide prevented type 1 diabetes by maintaining Th1/Th2/Th17 homeostasis.
We performed a whole-transcriptome correlation analysis, followed by the pathway enrichment and testing of innate immune response pathway analyses to evaluate the hypothesis that transcriptional activity can predict cortical gray matter thickness (GMT) variability during normal cerebral aging.
Transcriptome and GMT data were available for 379 individuals (age range=28-85) community-dwelling members of large extended Mexican American families. Collection of transcriptome data preceded that of neuroimaging data by 17 years. Genome-wide gene transcriptome data consisted of 20,413 heritable lymphocytes-based transcripts. GMT measurements were performed from high-resolution (isotropic 800 μm) T1-weighted MRI. Transcriptome-wide and pathway enrichment analysis was used to classify genes correlated with GMT. Transcripts for sixty genes from seven innate immune pathways were tested as specific predictors of GMT variability.
Transcripts for eight genes (IGFBP3, LRRN3, CRIP2, SCD, IDS, TCF4, GATA3, and HN1) passed the transcriptome-wide significance threshold. Four orthogonal factors extracted from this set predicted 31.9% of the variability in the whole-brain and between 23.4 and 35% of regional GMT measurements. Pathway enrichment analysis identified six functional categories including cellular proliferation, aggregation, differentiation, viral infection, and metabolism. The integrin signaling pathway was significantly (p<10(-6)) enriched with GMT. Finally, three innate immune pathways (complement signaling, toll-receptors and scavenger and immunoglobulins) were significantly associated with GMT.
Expression activity for the genes that regulate cellular proliferation, adhesion, differentiation and inflammation can explain a significant proportion of individual variability in cortical GMT. Our findings suggest that normal cerebral aging is the product of a progressive decline in regenerative capacity and increased neuroinflammation.
The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.
Airway hyperreactivity and inflammation are important factors in the aggravation of lung function. Suplatast tosilate (IPD) is a novel and unique anti‑asthma clinical compound. However, the mechanisms of IPD action in the inhibition of asthma remain to be elucidated. The present study aimed to investigate the role of the GATA binding protein 3 (GATA‑3)/interleukin (IL)‑5 signaling pathway in IPD‑induced inhibition of asthma. Sprague‑Dawley rats were sensitized by intraperitoneal injection with ovalbumin (OVA) to establish an animal model of asthma. IPD was administered continuously (C‑IPD) or at a later stage (L‑IPD). Budesonide (BUD) was used as a positive control. Airway resistance and the expression of genes at the mRNA and protein levels were measured. Morphological changes in lung tissue and the percentage of eosinophils (EOS) in peripheral blood were observed and correlation analysis was performed. The results revealed that sensitization by OVA significantly increased airway resistance and the percentage of EOS in peripheral blood and induced significant inflammatory changes in lung tissue, as demonstrated by thick epithelium, goblet cell hyperplasia and submucosal cell infiltration. In addition, sensitization by OVA was found to markedly upregulate IL‑5 mRNA and protein expression. Airway resistance was found to positively correlate with the expression of IL‑5 in the rat lung tissues. Sensitization by OVA was also observed to markedly enhance GATA‑3 protein expression and GATA‑3 levels were found to positively correlate with airway resistance and IL‑5 levels. Similar to the effect of BUD, treatment with C‑IPD or L‑IPD was found to significantly attenuate OVA‑induced increases in airway resistance and the percentage of EOS in peripheral blood. Notably, treatment with C‑IPD or L‑IPD markedly reduced the OVA-induced expression of IL‑5 and GATA‑3. In the present study, IPD intervention was demonstrated to ameliorate airway hyperreactivity and inflammation and the mechanisms may involve inhibition of the GATA‑3/IL‑5 signaling pathway.
The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on Chenopodium album allergy, using a BALB/c mouse model.
The BALB/c mice had already been sensitized to C. album via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR.
Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or C. album pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased.
Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response.
The mechanisms underlying the silencing of alternative fate potentials in very early B cell precursors remain unclear. Using gain- and loss-of-function approaches together with a synthetic Zinc-finger polypeptide (6ZFP) engineered to prevent transcription factor binding to a defined cis element, we show that the transcription factor EBF1 promotes B cell lineage commitment by directly repressing expression of the T-cell-lineage-requisite Gata3 gene. Ebf1-deficient lymphoid progenitors exhibited increased T cell lineage potential and elevated Gata3 transcript expression, whereas enforced EBF1 expression inhibited T cell differentiation and caused rapid loss of Gata3 mRNA. Notably, 6ZFP-mediated perturbation of EBF1 binding to a Gata3 regulatory region restored Gata3 expression, abrogated EBF1-driven suppression of T cell differentiation, and prevented B cell differentiation via a GATA3-dependent mechanism. Furthermore, EBF1 binding to Gata3 regulatory sites induced repressive histone modifications across this region. These data identify a transcriptional circuit critical for B cell lineage commitment.
The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.
The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin-22-pSTAT3-RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.
T helper type 2 (Th2) cells are critical to host defence against helminth infection and the pathogenesis of allergic diseases. The differentiation of Th2 cells from naive CD4 T cells is controlled by intricate transcriptional mechanisms. At the precursor stage of naive CD4 T cells, transcriptional mechanisms maintain the potential and in the meantime prevent spontaneous differentiation to Th2 fate. In addition, intrachromosomal interactions important for co-ordinated expression of Th2 cytokines pre-exist in naive CD4 T cells. Upon T-cell receptor (TCR) engagement, naive CD4 T cells are induced by polarizing signals of the interleukin-4/Stat6 and Jagged/Notch pathways to up-regulate the expression of GATA-3. Once up-regulated, GATA-3 drives Th2 and suppresses Th1 differentiation in a cell autonomous fashion. In this stage of differentiation, the Th2 cytokine locus, as well as the interferon-γ locus, undergoes chromatin remodelling and epigenetic modifications that contribute to the somatic memory of Th2 cytokine gene expression pattern. Once differentiated, Th2 effector cells promptly produce Th2 cytokines upon TCR stimulation, which is regulated by concerted actions of GATA-3, TCR signalling, enhancers and the Th2 locus control region. This review provides a detailed account of the transcriptional regulatory events at these different stages of Th2 differentiation.
HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.
Tubular adenomas in the urinary tract with the same appearance as those in the gastrointestinal tract have not yet been described in the literature. We herein report 4 cases of tubular adenomas in the urinary tract encountered within our consult practice. This lesion was defined by the presence of a collection of small round tubular glands with intestinal-type epithelium showing moderate dysplasia, identical to the histology of tubular adenomas in the intestinal tract. Patients ranged in age from 37 to 63 years (mean, 45 years), with 3 of the 4 being male (male-to-female ratio, 3:1). The locations were urinary bladder, prostatic urethra and ureter with hematuria, polyps, and obstructive mass as their presentations, respectively. One lesion was large measuring 1.4 cm associated with pseudoinvasion as well as invasive adenocarcinoma. Immunohistochemically, the tubular adenomas stained positive for CDX2 and CK20, while negative for GATA3 and CK7. One case showed positive nuclear β-catenin staining. Tubular adenoma of the urinary tract is a rare lesion, and recognition of this entity will encourage further reports and help to better understand the relation of tubular adenoma to concurrent and subsequent urinary tract malignancies.
Etiologic diagnosis of seizure requires proper consideration of apparently unrelated clinical features of the patient. Here, we report the case of a patient of status epilepticus with moderate-to-severe bilateral sensorineural deafness. Investigations showed extensive intracranial calcification, hypoparathyroidism and unilateral renal agenesis. The features were consistent with Barakat syndrome, a rare developmental disorder associated with mutations in the GATA3 gene. To the best of our knowledge, this is the first reported case of Barakat syndrome from India.
Mice homozygous for the juvenile alopecia mutation (jal) display patches of hair loss that appear as soon as hair develops in the neonatal period and persist throughout life. Although a report initially describing this mouse variant suggested that jal maps to mouse Chromosome 13, our preliminary mapping analysis did not support that claim.
To map jal to a particular mouse chromosome, we produced a 103-member intraspecific backcross panel that segregated for jal, and typed it for 93 PCR-scorable, microsatellite markers that are located throughout the mouse genome. Only markers from the centromeric tip of Chromosome 2 failed to segregate independently from jal, suggesting that jal resides in that region. To more precisely define jal's location, we characterized a second, 374-member backcross panel for the inheritance of five microsatellite markers from proximal Chromosome 2. This analysis restricted jal's position between D2Mit359 and D2Mit80, an interval that includes Il2ra (for interleukin 2 receptor, alpha chain), a gene that is known to be associated with alopecia areata in humans. Complementation testing with an engineered null allele of Il2ra, however, showed that jal is a mutation in a distinct gene. To further refine the location of jal, the 374-member panel was typed for a set of four single-nucleotide markers located between D2Mit359 and D2Mit80, identifying a 0.55 Mb interval where jal must lie. This span includes ten genes-only one of which, Gata3 (for GATA binding protein 3)-is known to be expressed in skin. Complementation testing between jal and a Gata3 null allele produced doubly heterozygous, phenotypically mutant offspring.
The results presented indicate that the jal mutation is a mutant allele of the Gata3 gene on mouse Chromosome 2. We therefore recommend that the jal designation be changed to Gata3jal, and suggest that this mouse variant may provide an animal model for at least some forms of focal alopecia that have their primary defect in the hair follicle and lack an inflammatory component.
To explore the role of sinomenine in treatment of allergic rhinitis mice model and its possible mechanism.
We used ovalbumin (OVA) to make allergic rhinitis model of BALB/c mice. Saline was used in the control group. When we challenged the mice with OVA intranasally, the mice in sinomenine treatment group were feed by the food containing sinomenine. Mice were then killed 24 h after the last OVA challenge. The noses of mice from each group were removed en bloc and fixed, then each section was stained with hematoxylin and eosin. ELISA assay was used to measure the concentration of anti-OVA IgE, IL-4 and IFN-gamma. The proteins expressive level of T-bet and GATA3 were examined.
Nasal mucosa of the mice in sinomenine treatment group were not hyperplasia and without obvious infiltration of eosinophils. The concentration of anti-OVA IgE, IL-4 and IFN-gamma in the serum and T-bet and GATA3 expression levels of sinomenine treatment group were lower than those of allergic rhinitis group.
The sinomenine can be used to treat allergic rhinitis mice, and the mechanism may rely on the improvements of the Th1/Th2 imbalance.
Estrogen receptor alpha (ERα) is a cellular receptor for the female sex hormone estrogen and other natural and synthetic ligands and play critical roles in normal development and physiology and in the etiology and treatment of endocrine-related diseases. ERα is a member of the nuclear receptor superfamily of transcription factors and regulates target gene expression in a ligand-dependent manner. It has also been shown to interact with G-protein coupled receptors and associated signaling molecules in the cytoplasm. Transcriptionally, ERα either binds DNA directly through conserved estrogen response element sequence motifs or indirectly by tethering to other interacting transcription factors and nucleate transcriptional regulatory complexes which include an array of co-regulator proteins. Genome-scale studies of ERα transcriptional activity and localization have revealed mechanistic complexity and insights including novel interactions with several transcription factors, including FOXA1, AP-2g, GATA3, and RUNX1, which function as pioneering, collaborative, or tethering factors. The major challenge and exciting prospect moving forward is the comprehensive definition and integration of ERα complexes and mechanisms and their tissue-specific roles in normal physiology and in human diseases.
Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2ß were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.
Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.
Macelignan isolated from Myristica fragrans Houtt. is widely used for spice and flavoring for foods, and has been reported to have anti-inflammatory activity. The aim of this study was to investigate the effects of macelignan on allergic lung inflammation with a murine model of experimental asthma.
Fungal protease mixed with chicken egg ovalbumin allergen was used as a challenge to induce murine experimental asthma. To determine its effects on allergy and inflammation, macelignan was administered orally during allergen challenge, and the symptoms of allergic asthma and its underlined mechanisms were examined.
Treatment with macelignan attenuated eosinophilic airway inflammation and airway hyper-responsiveness. With the administration of macelignan, interleukin-4 (IL-4) producing cells, but not interferon-γ (IFN-γ) or IL-17 producing cells, were diminished in the lungs. Additionally, activation of the T helper type 2 (Th2) cell-specific master transcription factor, GATA3 was decreased with macelignan treatment. Finally, production of IL-4 but not IFN-γ or IL-17, by CD4(+) T cells was reduced with stimulation when combined with the administration of macelignan.
Our data show that macelignan has anti-inflammatory effects on Th2 cell-mediated allergic lung inflammation and could potentially provide a novel preventative and/or therapy for the treatment of allergic diseases.
Development of a novel and effective vaccine against Mycobacterium tuberculosis (M.tb) is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e) and fms-like tyrosine kinase 3 ligand (FL) genes (termed Esat-6/3e-FL), and was enveloped with chitosan (CS) nanoparticles (nano-chitosan). The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL) was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against M.tb H37Rv challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing M.tb infection in mice.
Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness. Numbers of CD8(+)IL-13(+) T cells are increased in asthmatics and during the development of experimental asthma in mice. In an atopic environment rich in IL-4, these CD8(+) T cells mediate asthmatic responses, but the mechanisms regulating the conversion of CD8(+) effector T cells from IFN-γ- to pathogenic IL-13-producing effector cells that contribute to an asthma phenotype have not been defined. Here, we show that cholesterol side-chain cleavage P450 enzyme, Cyp11a1, is a key regulator of CD8(+) T-cell conversion. Expression of the gene, protein, and enzymatic activity of Cyp11a1 were markedly increased in CD8(+) T cells differentiated in the presence of IL-2 plus IL-4 compared with cells differentiated in IL-2 alone. Inhibition of Cyp11a1 enzymatic activity with aminoglutethimide or reduction in the expression of Cyp11a1 using short hairpin RNA prevented the IL-4-induced conversion of IFN-γ- to IL-13-producing cells without affecting expression of the lineage-specific transcription factors T-box expressed in T cells (T-bet) or GATA binding protein 3 (GATA3). Adoptive transfer of aminoglutethimide-treated CD8(+) T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation. We demonstrate that Cyp11a1 controls the phenotypic conversion of CD8(+) T cells from IFN-γ to IL-13 production, linking steroidogenesis in CD8(+) T cells, a nonclassical steroidogenic tissue, to a proallergic differentiation pathway.
Hair cells of the developing mammalian inner ear are progressively defined through cell fate restriction. This process culminates in the expression of the bHLH transcription factor Atoh1, which is necessary for differentiation of hair cells, but not for their specification. Loss of several genes will disrupt ear morphogenesis or arrest of neurosensory epithelia development. We previously showed in null mutants that the loss of the transcription factor, Gata3, results specifically in the loss of all cochlear neurosensory development. Temporal expression of Gata3 is broad from the otic placode stage through the postnatal ear. It therefore remains unclear at which stage in development Gata3 exerts its effect. To better understand the stage specific effects of Gata3, we investigated the role of Gata3 in cochlear neurosensory specification and differentiation utilizing a LoxP targeted Gata3 line and two Cre lines. Foxg1(Cre)∶Gata3(f/f) mice show recombination of Gata3 around E8.5 but continue to develop a cochlear duct without differentiated hair cells and spiral ganglion neurons. qRT-PCR data show that Atoh1 was down-regulated but not absent in the duct whereas other hair cell specific genes such as Pou4f3 were completely absent. In addition, while Sox2 levels were lower in the Foxg1(Cre):Gata3(f/f) cochlea, Eya1 levels remained normal. We conclude that Eya1 is unable to fully upregulate Atoh1 or Pou4f3, and drive differentiation of hair cells without Gata3. Pax2-Cre∶Gata3(f/f) mice show a delayed recombination of Gata3 in the ear relative to Foxg1(Cre):Gata3(f/f) . These mice exhibited a cochlear duct containing patches of partially differentiated hair cells and developed only few and incorrectly projecting spiral ganglion neurons. Our conditional deletion studies reveal a major role of Gata3 in the signaling of prosensory genes and in the differentiation of cochlear neurosenory cells. We suggest that Gata3 may act in combination with Eya1, Six1, and Sox2 in cochlear prosensory gene signaling.
Development of asthma in childhood is linked to viral infections of the lower respiratory tract in early life, with subsequent chronic exposure to allergens. Progression to persistent asthma is associated with a Th2-biased immunological response and structural remodelling of the airways. The underlying mechanisms are unclear, but could involve epigenetic changes. To investigate this, we employed a recently developed mouse model in which self-limited neonatal infection with a pneumovirus, followed by sensitisation to ovalbumin via the respiratory tract and low-level chronic challenge with aerosolised antigen, leads to development of an asthmatic phenotype. We assessed expression of microRNA by cells in the proximal airways, comparing changes over the period of disease progression, and used target prediction databases to identify genes likely to be up- or downregulated as a consequence of altered regulation of microRNA. In parallel, we assessed DNA methylation in pulmonary CD4(+) T cells. We found that a limited number of microRNAs exhibited marked up- or downregulation following early-life infection and sensitisation, for many of which the levels of expression were further changed following chronic challenge with the sensitizing antigen. Targets of these microRNAs included genes involved in immune or inflammatory responses (e.g. Gata3, Kitl) and in tissue remodelling (e.g. Igf1, Tgfbr1), as well as genes for various transcription factors and signalling proteins. In pulmonary CD4(+) T cells, there was significant demethylation at promoter sites for interleukin-4 and interferon-γ, the latter increasing following chronic challenge. We conclude that, in this model, progression to an asthmatic phenotype is linked to epigenetic regulation of genes associated with inflammation and structural remodelling, and with T-cell commitment to a Th2 immunological response. Epigenetic changes associated with this pattern of gene activation might play a role in the development of childhood asthma.
GATA3 is a zinc finger transcription factor that regulates the normal development of many tissues and cell types. Recent studies have shown that immunohistochemical nuclear staining for GATA3 among tumors is highly restricted to carcinomas of breast and urothelial origin; however salivary gland tumors have not been tested. Given that breast and salivary gland tissues are very similar with respect to embryologic development and structure, we performed GATA3 staining on a spectrum of salivary gland neoplasms. GATA3 immunohistochemistry was performed on a diverse collection of 180 benign and malignant salivary gland neoplasms including 10 acinic cell carcinomas, 2 adenocarcinomas not otherwise specified, 41 adenoid cystic carcinomas, 2 epithelial-myoepithelial carcinomas, 1 low grade cribriform cystadenocarcinoma, 15 mammary analogue secretory carcinomas, 7 metastatic squamous cell carcinomas, 27 mucoepidermoid carcinomas, 2 oncocytic carcinomas, 5 oncocytomas, 34 pleomorphic adenomas, 4 polymorphous low grade adenocarcinomas, 25 salivary duct carcinomas, and 5 Warthin tumors. Staining for GATA3 was observed in 92/180 (51 %) of salivary gland tumors. GATA3 staining was observed in most of the tumor types, but diffuse immunolabeling was consistently seen in salivary duct carcinoma (25 of 25) and mammary analogue secretory carcinoma (15 of 15)-the two tumor types that most closely resemble breast neoplasia. Background benign salivary gland tissue was also usually weakly positive in both acini and ducts. GATA3 immunostaining is not restricted to tumors of breast and urothelial origin. Rather, it is expressed across many different types of salivary gland neoplasms. As a result, salivary gland origin should be considered in the differential diagnosis of a GATA3-positive carcinoma, particularly in the head and neck. Although GATA3 immunohistochemistry is not helpful in resolving the differential diagnosis between a primary salivary gland neoplasm and metastatic breast cancer, it may have some utility in subtyping salivary gland tumors, particularly salivary duct carcinoma and mammary analogue secretory carcinoma.
GATA3 is a zinc-finger transcription factor, which is expressed in various normal and neoplastic tissues. Amongst tumors, it labels urothelial carcinoma, collecting duct carcinoma of the kidney, breast carcinoma, lymphoma and, uncommonly, endometrial carcinoma. Few studies have investigated its positivity in various neoplasms that may mimic urothelial neoplasms. In this study, we evaluated GATA3 expression in urinary bladder paragangliomas, which may closely mimic urothelial carcinomas. We retrieved 12 cases of paragangliomas from the urinary bladder and 20 cases of paragangliomas from non-urologic sites using the Hopkins Pathology Data Base system. GATA3 was positive in 10 of the 12 (83%) urinary bladder paragangliomas studied on routine slide sections. Most (6/12) of the staining was diffusely strong (3+) staining, whereas the rest (4/12) that were positive showed mixed intensities (strong 3+ to moderate 2+). The 20 paragangliomas from other sites were constructed into tissue microarrays, wherein three cores from each tumor were taken. Fifteen out of 20 (75%) paragangliomas outside of the bladder were positive for GATA3 staining. Moderate (2+) or strong (3+) staining was seen in 13/20 (65%) of extravesical paragangliomas, ranging from 5 to 100% of the cell labeling (mean 59%, median 60%). In the remaining 7/20 (35%) cases, only weak (2/7) or negative (5/7) immunoreactivity for GATA3 was seen. An additional 15 cases of metastatic paraganglioma from various primary sites were retrieved with 12 of 15 (80%) metastatic paragangliomas staining positively for GATA3. Overall, for paragangliomas, regardless of site, 78.7% were positive for GATA3. Recognition of this finding will aid pathologists in preventing a misdiagnosis of a urothelial tumor based on GATA3 expression, which is critical given the differences in treatment, follow-up and prognosis between bladder paragangliomas and urothelial carcinoma.
To observe the influence of Sijunzi decoction and Yupingfeng powder on the expression of the relevant DNAs of janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway of the brain in spleen-deficiency model rats.
Eighty male Wistar rats of sanitary degree were divided randomly into four groups: normal group, model group, treatment group 1, treatment group 2. Besides the rats in the normal group, all the rats in other 3 groups were prepared as spleen deficiency model. The treatment group 1 were treated with Sijunzi decoction and the treatment group 2 were treated with Yupingfeng powder. After treatment for 6 weeks, perfusion was given and the brain was taken for detection of the expression of the relevant DNAs of JAK-STAT signal pathway of the brain in SD rats bygene chip method.
Spleen deficiency could lead to increase expression of JAK1, STAT1 and Interleukin 4 (IL-4) in the brain, but the decrease expression of Suppressor of cytokine signaling 1 (SOCS1), prolactin receptor (PRLR) and binding protein 3 (GATA 3). Sijunzi decoction could increase expression of STAT3, Prolactin (PRL) and GATA3, but decrease expression of JAK1, STAT, STAT4, Interleukin 10 receptor, alpha (IL10RA), Coagulation factor II (F2), PRLR, MAD homolog 3 (SMAD3) and IL-4. Yupingfeng powder could decrease expression of JAK1, STAT1, STAT4, SOCS4_ predicted, Epidermal growth factor receptor (EGFR), PRLR, High mobility group AT-hook 1 (HMGA10), IL-4.
Sijunzi decoction and Yupingfeng powder can improve immune function of the rat through influencing the genetic expression of JAK-STAT signal pathway.
Transforming growth factor (ß1TGFß1) can promote proliferation in late stage cancers but acts as a tumor suppressor in normal epithelial cells and in early stage cancers. Although, the TGFß pathway has been shown to play a key role in tumorigenesis and metastasis, only a limited number of models have been developed to understand this process. Here, we present a novel model system to discern this paradoxical role of TGFß1 using the MDA-MB-231 (MB-231) cell line. The MB-231 triple-negative breast cancer cell line has been extensively characterized and has been shown to continue to proliferate and undergo epithelial-to-mesenchymal transition (EMT) upon TGFß1 stimulation. We have previously shown by microarray analysis that expression of GATA3 in MB-231 cells results in reprogramming of these cells from a basal to a luminal subtype associated with a reduction of metastasis and tumorigenesis when implanted as xenografts. We now demonstrate that GATA3 overexpression in these cells results in a reduction of TGFß1 response, reversal of EMT, and most importantly, restoration of sensitivity to the inhibitory effects on proliferation of TGFß1. Microarray analysis revealed that TGFß1 treatment resulted in reduction of several cell cycle effectors in 231-GATA3 cells but not in control cells. Furthermore, our microarray analysis revealed a significant increase of BMP5 in 231-GATA3 cells. We demonstrate that combined treatment of MB-231 control cells with TGFß1 and BMP5 results in a significant reduction of cellular proliferation. Thus, this model offers a means to further investigate potentially novel mechanisms involved in the switch in response to TGFß1 from tumor promoter to tumor suppressor through the reprogramming of a triple-negative breast cancer cell line by the GATA3 transcription factor.
We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes.
The innate lymphoid cell (ILC) family has recently expanded with the discovery of type-2 innate lymphoid cells (ILC2). These cells arise from lymphoid progenitors in the bone marrow and, under the control of the transcriptional regulators RORα and Gata3, they mature to give rise to IL-5, IL-9 and IL-13 producing ILC2. These cells are critical components of the innate immune response to parasitic worm infections and have also been implicated in the pathogenesis of asthma and allergy. Recent advances in our understanding of the molecular regulation of ILC2 development and function now present the opportunity to develop new genetic models to assess ILC2 immune function and to investigate possible therapeutic interventions.
The role of Th17 cells and associated cytokines was investigated in oral lichen planus.
14 consecutive patients with oral lichen planus were investigated. For biological studies, tissues were taken from reticular or erosive lesions and from normal oral mucosa (controls) of the same patient. mRNA expression for IL-17F, IL-17A, MCP-1, IL-13, IL-2, IL-10, IL-1β, RANTES, IL-4, IL-12B, IL-8, IFN-γ, TNF-α, IL-1α, IL-18, TGF-β1, IL-23R, IL-7, IL-15, IL-6, MIG, IP-10, LTB, VEGF, IL-5, IL-27, IL-23A, GAPDH, PPIB, Foxp3, GATA3, and RORC was measured using the QuantiGene 2.0.
Results showed that Th17-type and Th0-type molecules' mRNAs, when compared with results obtained from tissue controls, were increased in biopsies of erosive lesions, whereas Th2-type molecules' mRNAs were increased in reticular lesions. When the CD4+ T-cell clones, derived from oral lichen planus tissues and tissue controls, were analyzed, a higher prevalence of Th17 (confirmed by an increased CD161 expression) and Th0 CD4+ T clones was found in erosive lesions, whereas a prevalence of Th2 clones was observed in reticular lesions.
Our data suggest that Th17, Th0, and Th2 cells, respectively, may have a role in the pathogenesis of erosive and reticular oral lichen planus.
Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis.
Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets.
We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27-17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52-4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of "luminal-like" genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers.
We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR activation or cancer stem cells may need to be developed.
Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.
This study investigates the effects and possible molecular mechanisms of corilagin extraction on prevention of Schistosoma japonicum ova-induced granulomas and liver fibrosis. As a result, under a light microscope, when compared to a model group, the corilagin group showed smaller granulomas, less liver cell denaturation and less inflammatory cell infiltration, and the connective tissues were significantly decreased. By Masson staining, the liver sections from the corilagin group showed less collagen distributed around granulomas, decreased liver fibrosis in the portal tracts and less formed interlobular tissue. The expression of hydroxyproline, IL-13 in liver and GATA3 in spleen in the model group was significantly higher than that in the normal group (P less than 0.05 or 0.01), while the level of hydroxyproline, IL-13 and GATA3 in the corilagin group were significantly lower than that in the model group (P less than 0.05). In conclusion, corilagin extraction can decrease the level of Th2-associated profibrotic cytokine IL-13, and down-regulate the transcription of GATA3 mRNA in spleen cells, which alleviate the hepatic fibrosis caused by egg granuloma in Schistosoma japonicum infection.
Brahma (BRM) is a novel anticancer gene, which is frequently inactivated in a variety of tumor types. Unlike many anticancer genes, BRM is not mutated, but rather epigenetically silenced. In addition, histone deacetylase complex (HDAC) inhibitors are known to reverse BRM silencing, but they also inactivate it via acetylation of its C-terminus. High-throughput screening has uncovered many compounds that are effective at pharmacologically restoring BRM and thereby inhibit cancer cell growth. As we do not know which specific proteins, if any, regulate BRM, we sought to identify the proteins, which underlie the epigenetic suppression of BRM. By selectively knocking down each HDAC, we found that HDAC3 and HDAC9 regulate BRM expression, whereas HDAC2 controls its acetylation. Similarly, we ectopically overexpressed 21 different histone acetyltransferases and found that KAT6A, KAT6B and KAT7 induce BRM expression, whereas KAT2B and KAT8 induce its acetylation. We also investigated the role of two transcription factors (TFs) linked to either BRM (GATA3) or HDAC9 (MEF2D) expression. Knockdown of either GATA3 and/or MEF2D downregulated HDAC9 and induced BRM. As targets for molecular biotherapy are typically uniquely, or simply differentially expressed in cancer cells, we also determined if any of these proteins are dysregulated. However, by sequencing, no mutations were found in any of these BRM-regulating HDACs, HATs or TFs. We selectively knocked down GATA3, MEF2D, HDAC3 and HDAC9, and found that each gene-specific knockdown induced growth inhibition. We observed that both GATA3 and HDAC9 were greatly overexpressed only in BRM-negative cell lines indicating that HDAC9 may be a good target for therapy. We also found that the mitogen-activated protein (MAP) kinase pathway regulates both BRM acetylation and BRM silencing as MAP kinase pathway inhibitors both induced BRM as well as caused BRM deacetylation. Together, these data identify a cadre of key proteins, which underlie the epigenetic regulation of BRM.
The functional plasticity of CD8(+) T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness and inflammation, and yet remains mechanistically undefined. We demonstrate that CD8(+) T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8(+) T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the stepwise molecular conversion of CD8(+) T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA polymerase II (Pol II) recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage after TCR stimulation, indicating that IL-4-induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro, an atopic (IL-4) environment poises CD8(+) T cells via stepwise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.
Innate lymphoid cells (ILCs) are an emerging group of innate lymphocytes that share functional and transcriptional attributes with the various T helper cell effector fates (e.g. Th1, Th2, Th17). ILCs are substantially represented in the intestinal mucosa but are rare in secondary lymphoid organs. They play important roles in epithelial homeostasis, tissue repair and in immunity to intestinal infections. They are also involved in immune-mediated pathology. Here, we will review the emerging roles of the transcription factors T-bet and Gata3 in the development, lineage specification and function of distinct ILC lineages. We will also highlight the requirement of these transcriptional programs for the control of infections and the pathogenesis of inflammatory diseases.
Activated TCD4(+) cells are detected in human atherosclerotic plaques which indicate their participation in disease progression and destabilization. Among these cells, IFN-γ-producing T cells (TH1) are recognized as having a pro-atherogenic role. Recently, the IL-17-producing T helper lineage of cells (TH17) has been identified in atherosclerotic lesions. They have been linked to atheroma development through the production of pro-inflammatory mediators present in these lesions. Furthermore, IL-22 producing TCD4(+) cells (TH22) have been identified in the atheromatous environment, but their presence and function has not been investigated. The aim of this study was to analyze the immune response mediated by pro-inflammatory subtypes of TCD4(+) cells in atheromatous lesions. Atherosclerotic plaques of 57 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Three carotid fragments from organ donors were used as control. mRNA analysis showed expression of TH1 (IFN-γ, T-bet, IL-2, IL-12p35, TNF-α and IL-18); TH2 (GATA-3); TH17 (IL-17A, IL-17RA, Rorγt, TGF-β, IL-6, IL-1β, IL-23p19, CCL20, CCR4 and CCR6) and TH22 (IL-22 and Ahr) related markers. Asymptomatic patients showed higher expression of mRNA of IL-10, TGF-β, CCR4 and GATA-3 when compared to symptomatic ones. Immunohistochemistry analysis showed higher levels of IL-23, TGF-β, IL-1β and IL-18 in macrophages and foam cells in unstable lesions compared to stable and control ones. In vitro stimulation of atheroma cells induced IL-17 and IFN-γ production. Finally we were able to detect, the following subpopulations of TCD3(+) cells: TCD4(+) IFN-γ(+), TCD4(+)IL-17(+), TCD4(+)IL-4(+), TCD4(+)IL-22(+) and double positive cells (IFN-γ/IL-17(+), IFN-γ/IL-22(+) or IL-17/IL-22(+)). Our results showed the presence of distinct TCD4(+) cells subsets in human carotid lesions and suggest that interactions among them may contribute to the atheroma progression and destabilization.
GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4-nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.
In this issue of Blood, García-Ojeda et al demonstrate a key mechanism that drives T and B lymphocytes to divergent fates from early in their development.
GATA transcription factor family members have been found to involve in the growth and differentiation of mammary gland. Among them GATA-3 is regarded as the most critical regulator involving the tumorigenesis of breast cancer (BC). Recently, trichorhinophalangeal syndrome-1 gene (TRPS-1), a new GATA family member, has been identified to be highly prevalent in breast cancer. Compared with ER-negative breast cancer, the expression of TRPS-1 is higher in ER-positive breast cancer and was significantly correlates with estrogen receptor, progesterone receptor, and GATA-3, indicating it may serve as a ductal epithelial cell-specific regulator in the differentiation of breast ductal epithelial cells. Studies have shown that miR221/222 is able to downregulate the expression of an epithelial cell marker E-cadherin by targeting TRPS-1, resulting in mammary epithelial cells transition to mesenchymal cell (EMT). In addition, it has been well accepted that, and the Science and Technology Bureau of Jiaxing (2012AY1071-2)TRPS-1 plays a role in the differentiation of several other cell types including kidney nephric mesenchymal cells, columnar chondrocytes, and osteoclasts, indicating that TRPS-1 involves in mesenchymal-to-epithelial cell transition (MET). In this article, we summarize the roles of GATA transcription factor TRPS-1 in ductal epithelial cells and the roles of its gene and protein expressions in predicting the prognosis of breast cancer.
Autoinflammatory attacks in familial Mediterranean fever (FMF) are accompanied by elevated levels of interleukin-6 (IL-6), and are controllable by IL-1-targeting drugs. In combination, IL-6 and IL-1 are known to be potent inducers of T helper (Th) 17 cells development. Therefore, we studied the Th17 population size, and activation potential, of FMF patients. Based on the relative mRNA expression of the Th1, Th2, Treg and Th17 transcription factors T-bet, GATA3, FOXP3 and retinoic acid-related orphan receptor γT (RORγT), respectively, the Th17 population in peripheral blood mononuclear cells (PBMCs) of healthy subjects was estimated at 2.5% of the entire Th population and 4.4% in FMF patients in remission (n=6 for each group, P=0.03). IL-17 secretion after universal stimulation of the T-cell receptor in PBMCs culture was twice higher in cultures of patients with frequent attacks (n=18) than in those of patients with infrequent attacks (n=10, 1124±266 vs 615±196 pg ml(-1), P=0.009). IL-17 secretion correlated well with IL17A mRNA level. Part of the increased secretion was related to the deleterious, MEFV p.M694V homozygous genotype (n=19, 1.5-fold, P=0.03). Almost all IL-17 producer cells were CD4-positive (CD4(+)IL-17(+)). In conclusion, frequent attacks and the deleterious FMF genotype appear to drive FMF patients to a heightened Th17 response.
Immunohistochemistry for PAX8 and GATA3 are sensitive markers for renal cell carcinoma and urothelial carcinoma, respectively. However, there are limited data on these markers in sarcomatoid renal cell carcinoma (SARCRCC) and sarcomatoid urothelial carcinoma (SARCUC). Tissue microarrays (TMAs) were constructed from 45 cases of SARCRCC and 45 cases of SARCUC of the lower urinary tract, with an additional 11 SARCUCs of the upper tract. PAX8 and GATA3 were also evaluated in TMAs from 161 sarcomas from other sites, 14 atypical epithelioid angiomyolipomas (AMLs) of the kidney, 23 bladder inflammatory myofibroblastic tumors (IMTs), and 2 bladder and 4 renal leiomyosarcomas. In the SARCRCC, PAX8 and GATA3 were positive in the sarcomatoid areas in 31 (69%) and 0 (0%) of cases, respectively. In the bladder SARCUC, GATA3 and PAX8 were positive in 14 (31%) and 2 (4%) of cases, respectively. Of the 11 SARCUCs of the upper urinary tract, 2 (18%) cases were PAX8 positive and 2 (18%) separate cases were GATA3 positive. Only 1 tumor present on the sarcoma TMAs, a Ewing sarcoma/primitive neuroectodermal tumor, was PAX8 positive, and all sarcomas were GATA3 negative. Of the AMLs, IMTs, and leiomyosarcoma, only 1 case of IMT showed moderate GATA3 positivity, and all were negative for PAX8. PAX8 can be used to distinguish SARCCRCC from atypical epithelioid AMLs and primary renal or retroperitoneal sarcomas. However, in a kidney/renal pelvic tumor, PAX8 shows overlap in staining between SARCUC and SARCRCC. GATA3 lacks sensitivity but is more specific for SARCUC.
Patients with ulcerative colitis (UC) who are in clinical remission may still have underlying endoscopic inflammation, which is associated with inferior clinical outcomes. The goal of this study was to determine the prevalence of active endoscopic disease, and factors associated with it, in patients with UC who are in clinical remission.
Prospective observational study in a single center. Patients with UC in clinical remission (by Simple Clinical Colitis Activity Index) were enrolled prospectively at the time of surveillance colonoscopy. Disease phenotype, endoscopic activity (Mayo subscore), and histologic score (Geboes) were recorded, and blood was drawn for peripheral blood biomarkers.
Overall, 149 patients in clinical remission were prospectively enrolled in this cohort; 81% had been in clinical remission for >6 months, and 86% were currently prescribed maintenance medications. At endoscopy, 45% of patients in clinical remission had any endoscopic inflammation (Mayo endoscopy subscore >0), and 13% had scores >1. In a multivariate model, variables independently associated with a Mayo endoscopic score >1 were remission for <6 months (P = 0.001), white blood count (P = 0.01), and C-reactive protein level (P = 0.009). A model combining these 3 variables had a sensitivity of 94% and a specificity of 73% for predicting moderate-to-severe endoscopic activity in patients in clinical remission (area under the curve, 0.86). In an unselected subgroup of patients who had peripheral blood mononuclear cell messenger RNA profiling, GATA3 messenger RNA levels were significantly higher in patients with endoscopic activity.
Duration of clinical remission, white blood count, and C-reactive protein level can predict the probability of ongoing endoscopic activity, despite clinical remission in patients with UC. These parameters could be used to identify patients who require intensification of treatment to achieve mucosal healing.
Hematopoiesis is a classic system with which to study developmental potentials and to investigate gene regulatory networks that control choices among alternate lineages. T-cell progenitors seeding the thymus retain several lineage potentials. The transcription factor PU.1 is involved in the decision to become a T cell or a myeloid cell, and the developmental outcome of expressing PU.1 is dependent on exposure to Notch signaling. PU.1-expressing T-cell progenitors without Notch signaling often adopt a myeloid program, whereas those exposed to Notch signals remain in a T-lineage pathway. Here, we show that Notch signaling does not alter PU.1 transcriptional activity by degradation/alteration of PU.1 protein. Instead, Notch signaling protects against the downregulation of T-cell factors so that a T-cell transcriptional network is maintained. Using an early T-cell line, we describe two branches of this network. The first involves inhibition of E-proteins by PU.1 and the resulting inhibition of Notch signaling target genes. Effects of E-protein inhibition can be reversed by exposure to Notch signaling. The second network is dependent on the ability of PU.1 to inhibit important T-cell transcription factor genes such as Myb, Tcf7 and Gata3 in the absence of Notch signaling. We show that maintenance of Gata3 protein levels by Myb and Notch signaling is linked to the ability to retain T-cell identity in response to PU.1.
Somatic and germline mutations in the dual zinc-finger transcription factor GATA3 are associated with breast cancers expressing the estrogen receptor (ER) and the autosomal dominant hypoparathyroidism-deafness-renal dysplasia syndrome, respectively. To elucidate the role of GATA3 in breast tumorigenesis, we investigated 40 breast cancers that expressed ER, for GATA3 mutations. Six different heterozygous GATA3 somatic mutations were identified in eight tumors, and these consisted of: a frameshifting deletion/insertion (944_945delGGinsAGC), an in-frame deletion of a key arginine residue (991_993delAGG), a seven-nucleotide frameshifting insertion (991_992insTGGAGGA), a frameshifting deletion (1196_1197delGA), and two frameshifting single nucleotide insertions (1224_1225insG found in three tumors and 1224_1225insA). Five of the eight mutations occurred in tumors that retained GATA3 immunostaining, indicating that absence of GATA3 immunostaining is an unreliable predictor of the presence of GATA3 mutations. Luciferase reporter assays, electrophoretic mobility shift assays, immunofluorescence, invasion and proliferation assays demonstrated that the GATA3 mutations resulted in loss (or reduction) of DNA binding, decrease in transactivational activity, and alterations in invasiveness but not proliferation. The 991_992insTGGAGGA (Arg330 frameshift) mutation led to a loss of nuclear localization, yet the 991_993delAGG (Arg330deletion) retained nuclear localization. Investigation of the putative nuclear localization signal (NLS) sites showed that the NLS of GATA3 does not conform to either a classical mono- or bi-partite signal, but contains multiple cooperative NLS elements residing around the N-terminal zinc-finger which comprises residues 264-288. Thus, approximately 20 % ER-positive breast cancers have somatic GATA3 mutations that lead to a loss of GATA3 transactivation activity and altered cell invasiveness.
GATA3 is an enriched transcription factor in mammary epithelium. To date, there has been no study on the relationship between microsatellites in the GATA3 gene and breast cancer risk. In this study, we investigated the existence of polymorphisms in the cytosine-thymine (CT) dinucleotide repeat in intron 3 of the GATA3 gene and its association with breast cancer risk. A case-control study of 206 breast cancer patients and 262 controls was conducted in Iranian women. Several different CT repeat alleles of GATA3 were detected in both the patients and controls. The frequencies of 17 and 18 alleles in patients were significantly lower than controls. Our findings demonstrate that women who carry 17-CT (OR = 0.5; p = 0.003) or 18-CT (OR = 0.41, p = 0.02) alleles of GATA3 gene are at lower risk of developing breast cancer. The highest protection against breast cancer was observed with heterozygotes of 16/17 repeats (OR = 0.12, p = 0.02). Also, the presence of the 17-CT allele has a positive relation with estrogen receptor expression. However, we found that the allelic length of GATA3 polymorphisms had no significant effect on the age onset or grade of the disease, as well as the expression of progesterone receptors and HER2.
Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression.
Spiral ganglion neurons (SGNs) play a key role in hearing by rapidly and faithfully transmitting signals from the cochlea to the brain. Identification of the transcriptional networks that ensure the proper specification and wiring of SGNs during development will lay the foundation for efforts to rewire a damaged cochlea. Here, we show that the transcription factor Gata3, which is expressed in SGNs throughout their development, is essential for formation of the intricately patterned connections in the cochlea. We generated conditional knock-out mice in which Gata3 is deleted after SGNs are specified. Cochlear wiring is severely disrupted in these animals, with premature extension of neurites that follow highly abnormal trajectories toward their targets, as shown using in vitro neurite outgrowth assays together with time-lapse imaging of whole embryonic cochleae. Expression profiling of mutant neurons revealed a broad shift in gene expression toward a more differentiated state, concomitant with minor changes in SGN identity. Thus, Gata3 appears to serve as an "intermediate regulator" that guides SGNs through differentiation and preserves the auditory fate. As the first auditory-specific regulator of SGN development, Gata3 provides a useful molecular entry point for efforts to engineer SGNs for the restoration of hearing.
Infectious pancreatic necrosis virus (IPNV) is a highly contagious disease causing high mortalities in juvenile salmonids. Lack of correlation between neutralizing antibodies and infecting virus suggests a likelihood of involvement of the cellular mediated immune response in vaccine protection. To elucidate the kinetics of CD4 and CD8 T-cells responses in vaccine protection, Atlantic salmon (Salmo salar L) were vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine and challenged by cohabitation using a highly virulent Norwegian Sp strain. Analysis of T-cell gene expression in lymphoid organs (headkidney and spleen) showed that GATA-3 was positively correlated with increase in antibody levels when T-bet was low. Conversely, T-bet and FoxP3 were positively correlated with viral infection and negatively correlated with increase in antibody levels. Among the CD8+ T cell genes, expression of eomes and CD8α were positively correlated with increase in viral copy numbers and negatively correlated with increase in antibody levels. Up-regulation of granzyme A was highly correlated with increase in viral copy numbers in the LoAg and control groups indicating that this gene could save as a diagnostic marker of acute infection for IPNV during acute infection. In contrast, its down regulation in the HiAg which had low viral copy numbers corresponded with high antibody levels. Overall, these data show that the kinetics of CD4 and CD8 T-cell genes expression follow the same pattern as that observed in higher vertebrates. These findings suggest that functional signatures of the cellular mediated immune response could be evolutionary conserved across the vertebrate taxa and that they can effectively be used to monitor vaccine protection and infection progression of IPNV in Atlantic salmon.
Evidence suggests that prenatal exposure to air pollution affects the ontogeny and development of the fetal immune system. The aim of this study was to investigate the effect of maternal exposure to airborne particulate matter (PM) on immune function in postnatal offspring. Pregnant female ICR mice were intralaryngopharyngeally administered with 30 μl of phosphate buffered solution (the control group) or resuspended PM of Standard Reference Material 1649a at 0.09 (low), 0.28 (medium), 1.85 (high) or 6.92 (overdose) μg/μl once every three days from day 0 to 18 of pregnancy (n=8-10). Offspring were sacrificed on postnatal day 30. Interleukin-4 and interferon-γ levels in plasma and splenocytes, splenic lymphocyte proliferation, and expressions of GATA-3 and T-bet mRNA in the spleen were tested. The spleen and thymus were histopathologically examined. The offspring of the medium, high and overdose PM-exposed dams showed significantly suppressed splenocyte proliferation. Decreased interferon-γ and increased interleukin-4 levels in the blood and splenocytes, and lowered T-bet and elevated GATA-3 mRNA expressions were found in the spleen in the medium, high and overdose groups when compared with the control or low dose group (P<0.05). Histopathology revealed prominent tissue damage in the spleen and thymus in the overdose group. These results suggest that exposure of pregnant mice to PM modulates the fetal immune system, resulting in postnatal immune dysfunction by exacerbation of Thl/Th2 deviation. This deviation is associated with altered T-bet and GATA-3 gene expressions.
The transcriptional repressor Bcl6 is a critical regulator of T helper cell fate, and inhibits Th2-type inflammation. We have found that microRNA-21 (miR-21) is a novel target gene for Bcl6 in Treg cells. Bcl6 represses and Stat3 activates miR-21 transcription through a Stat3 binding element in the promoter, indicating opposing regulation of miR-21 by the two transcription factors via the same DNA site. Ectopic expression of miR-21 promoted Th2 differentiation in non-polarized T cells. The pro-Th2 activity of miR-21 was associated with increased Gata3 expression and decreased expression of the miR-21 target gene Sprouty1. Increased miR-21 promoted Th2 and Treg gene expression in wild-type Tregs. MiR-21 could thus help promote the Th2 bias of Bcl6-deficient conventional T cells and Treg cells. MiR21 expression is increased in Th2-type inflammation, and our results define miR-21 as a critical target of Bcl6, thus providing a new link between Bcl6 and Th2 inflammation. Finally, our results reveal a novel T cell autonomous role for miR-21 in promoting Th2 differentiation.
The aim of this study was to explore the role of immune modulation therapy in regulating the imbalance of Th1/Th2, serum IFN-γ, IL-4 and the T-cell-specific transcription factors T-bet/GATA-3 in peripheral blood in aging male patients with chronic cardiac insufficiency (CCI).
In total, 156 participants were divided into three groups: the CCI intervention group, which received regular therapy and thymopetidum (20 mg intramuscular injection, once every other day for 3 months; n = 70), the CCI control group, which received regular therapy (n = 56) and 50 healthy individuals older than 57 years of age, who served as normal controls.
Before therapy, in comparison with the control group, levels of left ventricular end diastolic diameter, NT-proBNP, C-reactive protein (CRP), Th1, Th1/Th2, IFN-γ, and T-bet mRNA and T-bet/GATA-3 mRNA all increased, and the level of left ventricular ejection fraction (LVEF), 6MWT, Th2, IL-4, and GATA-3 mRNA also decreased in both the CCI intervention and control groups. Linear correlation analysis indicated that LVEF was inversely correlated with serum NT-proBNP, CRP, Th1/Th2, IFN-γ and T-bet mRNA/GATA-3 mRNA, and was positively correlated with plasma IL-4. After 3 months of therapy, levels of left ventricular end diastolic diameter, NT-proBNP, CRP, Th1, Th1/Th2, IFN-γ, T-bet mRNA and T-bet/GATA-3 mRNA decreased in the two CCI subgroups, but levels in the CCI intervention group were lower in comparison to the control group. Levels of LVEF, 6MWT, Th2 and GATA-3 mRNA increased in the two CCI subgroups, while levels in the CCI intervention group were higher in comparison with the control group. Plasma levels of IL-4 showed no change after treatment.
Immune modulation improved cardiac function of CCI patients and was associated with amelioration of T-helper superficial transcription factor polarization and its related cytokine imbalance. Immune modulation might be a new treatment strategy for aging CCI patients.
In malaria-endemic areas, a proportion of individuals becomes chronic carriers of parasites with few or no clinical signs. There is little information on cellular immune responses in asymptomatic parasite carriers.
In 80 schoolchildren residing in a malaria-endemic area of Flores Island, Indonesia, T-helper subsets, regulatory T-cell (Treg) frequencies, tumor necrosis factor receptor type II (TNFRII) expression on Tregs, and cytokine responses induced by Plasmodium falciparum-infected red blood cells (RBCs) were measured, and values for asymptomatic infected subjects were compared to those for uninfected controls. To ascertain that alterations found were due to the presence of malaria parasites, the immune responses were analyzed in 16 children before and 1 month after antimalarial treatment.
TNFRII expression, a marker of activation on Tregs, was higher during infection but decreased upon treatment. GATA3-positive cells and the level of interleukin 13 secretion in response to P. falciparum-infected RBCs appeared to be suppressed by plasmodial infection, as both increased after antimalarial treatment. TNFRII expression on Tregs correlated positively with TNF in response to P. falciparum-infected RBCs, but this association disappeared following treatment.
Malaria parasites associated with asymptomatic infections seem to result in increased TNFRII expression on Tregs, as well as suppressed Th2 cytokine responses, features that might be important for survival of the parasites in asymptomatic carriers.
The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

Ki67 is widely used in order to distinguish the "A" and "B" subtypes of luminal-type breast cancer. This study aimed to validate the prognostic value of adding p53 to Ki67 for characterizing luminal-type breast cancer.
Immunostaining for Ki67, p53, and the molecular markers HER2, CK5/6, CK14, EGFR, FOXA1, GATA3, and P-cadherin was examined hormone receptor (HR)-positive cancer tissues from 150 patients. The prognostic value of an immunohistochemical panel comprising Ki67 and p53 was compared with that of the single Ki67 labeling index (LI), and uni- and multivariate analyses were performed.
Division of the patients based on the immunohistochemistry results into favorable- (low Ki67 LI, p53-negative) and unfavorable- (high Ki67 LI and/or p53-positive) phenotype groups yielded distinctly different Kaplan-Meier's curves of both disease-free (P<0.0001) and overall survival (P=0.0007). These differences were much more distinct than those between the corresponding low Ki67 LI vs. high Ki67LI curves. While the prognostic values of the other molecular markers were not significant, combined Ki67-p53 status was an independent prognostic factor by multivariate analysis.
These data indicate that an immunohistochemical panel comprising Ki67 and p53 is a practical tool for management of patients with HR-positive breast cancer.
Tumour cells are influenced by their microenvironment, which can promote uncontrolled growth, invasion and metastasis. The GATA3 transcription factor is now shown to regulate the tumour microenvironment by inducing the expression of miR-29b in cancer cells. This microRNA in turn inhibits the expression of genes involved in angiogenesis and extracellular matrix signalling and remodelling to suppress metastasis.
GATA3 plays an integral role in breast luminal cell differentiation and is implicated in breast cancer progression. GATA3 immunohistochemistry is a useful marker of breast cancer; however, its use in specific subtypes is unclear. Here, we evaluate GATA3 expression in 86 invasive ductal carcinomas including triple-negative, Her-2, and luminal subtypes, in addition to 13 metaplastic carcinomas and in 34 fibroepithelial neoplasms. In addition, we report GATA3 expression in matched primary and metastatic breast carcinomas in 30 patients with known estrogen receptor (ER), progesterone receptor (PR), and Her-2 status, including 5 with ER and/or PR loss from primary to metastasis. Tissue microarrays containing 5 to 10 cores per tumor were stained for GATA3, scored as follows: 0 (0-5%), 1+ (6%-25%), 2+ (26%-50%), 3+ (51%-75%), and 4+ (>75%). GATA3 labeling was seen in 67% (66/99) of primary ductal carcinomas including 43% of triple-negative and 54% of metaplastic carcinomas. In contrast, stromal GATA3 labeling was seen in only 1 fibroepithelial neoplasm. GATA3 labeling was seen in 90% (27/30) of primary breast carcinomas in the paired cohort, including 67% of triple-negative carcinomas. GATA3 labeling was overwhelmingly maintained in paired metastases. Notably, GATA3 was maintained in all "luminal loss" metastases, which showed ER and/or PR loss. In conclusion, GATA3 expression is maintained between matched primary and metastatic carcinomas including ER-negative cases. GATA3 can be particularly useful as a marker for metastatic breast carcinoma, especially triple-negative and metaplastic carcinomas, which lack specific markers of mammary origin. Finally, GATA3 labeling may help distinguish metaplastic carcinoma from malignant phyllodes tumors.
Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they reportedly promote JAK2-oligomerization and autonomous signalling, Affected entities are promising candidates for therapy with JAK2 signalling inhibitors. While JAK2-translocations occur in myeloid, B-cell and T-cell lymphoid neoplasms, our findings suggest their incidence among the last group is low. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in a trio of cell lines established at indolent (MAC-1) and aggressive (MAC-2A/2B) phases of a cutaneous T-cell lymphoma (CTCL). To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 top upregulated genes in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2, highlighting its role as a central signalling target of JAK2 translocation neoplasia. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive lymphoma cells, was partially restored by PCM1-JAK2 knockdown. Treatment with a selective JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive confirmed knockdown results and highlighted JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5, supporting a general paradigm of STAT5 activation by JAK2 alterations in lymphoid malignancies. MAC-1/2A/2B--the first JAK2-translocation leukemia/lymphoma cell lines described--display conspicuous JAK/STAT signalling accompanied by T-cell developmental and autoimmune disease gene expression signatures, confirming their fitness as CTCL disease models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements.
Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.
Despite advances in our understanding of breast cancer, patients with metastatic disease have poor prognoses. GATA3 is a transcription factor that specifies and maintains mammary luminal epithelial cell fate, and its expression is lost in breast cancer, correlating with a worse prognosis in human patients. Here, we show that GATA3 promotes differentiation, suppresses metastasis and alters the tumour microenvironment in breast cancer by inducing microRNA-29b (miR-29b) expression. Accordingly, miR-29b is enriched in luminal breast cancers and loss of miR-29b, even in GATA3-expressing cells, increases metastasis and promotes a mesenchymal phenotype. Mechanistically, miR-29b inhibits metastasis by targeting a network of pro-metastatic regulators involved in angiogenesis, collagen remodelling and proteolysis, including VEGFA, ANGPTL4, PDGF, LOX and MMP9, and targeting ITGA6, ITGB1 and TGFB, thereby indirectly affecting differentiation and epithelial plasticity. The discovery that a GATA3-miR-29b axis regulates the tumour microenvironment and inhibits metastasis opens up possibilities for therapeutic intervention in breast cancer.
Henoch-Schönlein purpura (HSP), the most common type of leukocytoclastic vasculitis, is caused by T cell-mediated autoimmune reactions. In this study, we analyze histone modification patterns in peripheral blood mononuclear cells (PBMCs) of HSP patients, and investigate the expression levels of inflammatory cytokines (IFN-γ, IL-2, IL-4, IL-6 and IL-13), transcription factors (T-bet, GATA-3 and TIM-1) and chemokines (CXCL4 and CXCL10) in HSP patients. Our results show that histone H3 acetylation and methylation are significantly enhanced in PBMCs from HSP patients. We also demonstrate specifically that marked increases in histone H3 acetylation and H3 lysine 4 trimethylation occur at the IL-4 loci in these patients. In addition, the expression levels of IL-4, IL-6, IL-13, GATA-3, TIM-1 and CXCL4 are also increased. These findings suggest that abnormal histone modifications are present in the PBMCs of patients with HSP, possibly contributing to the activation of pathological immune responses associated with HSP.
Genotoxic stress and RAS induce the expression of CD155, a ligand for the immune receptors DNAM-1, CD96 and TIGIT. Here we show that antigen-presenting cells upregulate CD155 expression in response to Toll-like receptor activation. Induction of CD155 by Toll-like receptors depended on MYD88, TRIF and NF-κB. In addition, IRF3, but not IRF7, modulated CD155 upregulation in response to TLR3 signals. Immunization of CD155-deficient mice with OVA and the TLR9 agonist CpG resulted in increased OVA-specific IgG2a/c titers when compared to wild type mice. Splenocytes of immunized CD155-deficient mice secreted lower levels of IL-4 and fewer IL-4 and GATA-3 expressing CD4(+) T cells were present in the spleen of Cd155(-/-) mice. Our data suggest that CD155 regulates T(h)2 differentiation. Targeting of CD155 in immunization protocols using peptides may represent a promising new approach to boost protective humoral immunity in viral vaccines.
Glandular neoplasms involving the urinary bladder carry a challenging differential diagnosis including primary and secondary processes. We investigated the potential diagnostic utility of cadherin-17 and GATA3 in 25 primary adenocarcinomas of the urinary bladder, as compared with other commonly used markers including β-catenin and p63. Urothelial carcinoma with glandular differentiation (11), colorectal adenocarcinoma secondarily involving the bladder (25), and primary colorectal adenocarcinoma (22) were also analyzed and the results were compared using a Fisher exact test. Cadherin-17 was expressed in 23/25 primary bladder adenocarcinomas (92%), 23/25 colorectal adenocarcinomas involving the bladder (92%), 21/22 primary colorectal adenocarcinomas (95%) and entirely negative (0/11) in both components of urothelial carcinoma with glandular differentiation (P<0.001). In urothelial carcinoma with glandular differentiation, positive nuclear staining for GATA3 was evident in the urothelial component for 18% (2/11) and the glandular component for 9% (1/11) with additional tumors showing only cytoplasmic staining. Nuclear reactivity for GATA3 was not present in primary bladder adenocarcinoma and primary/secondary colorectal adenocarcinoma (P<0.05). Positive nuclear and cytoplasmic immunostaining for β-catenin was evident in 21/22 primary colorectal adenocarcinomas (95%) and 23/25 cases of secondary involvement by colorectal adenocarcinoma (92%). In contrast, positive membranous and cytoplasmic staining for β-catenin was observed in 23/25 primary bladder adenocarcinomas (92%) and 11/11 urothelial carcinomas with glandular differentiation (100%, P<0.001). p63 was expressed only in the urothelial component of urothelial carcinoma with glandular differentiation and not in the glandular component (P<0.001). In summary, cadherin-17 is a relatively specific and sensitive marker for primary adenocarcinoma of the urinary bladder, distinguishing it from urothelial carcinoma with glandular differentiation. However, it does not distinguish primary bladder adenocarcinoma from secondary involvement by colorectal adenocarcinoma. The pattern of reactivity for β-catenin remains the most useful marker for distinguishing these two tumors.
To observe the effect of autoblood acupoint-injection (ABAI) on expression levels of pulmonary transacting T-cell-specific transcription factor GATA 3 (involving Th 2 cytokine expression), Th 1-specific T-box transcription factor T-box expressed in T-cells (T-bet) proteins and genes in asthmatic rats so as to explore its mechanisms underlying asthma relief.
Forty-eight male SD rats were randomized into normal control (n = 8), model (n = 10), saline acupoint-injection (SAI, n = 10), ABAI (n = 10), and Dexamethasone (DXM, n = 10) groups. Asthma model was established during 28 days by 10% Ovalbumin + 10% aluminium hydroxide solution injection (i. p.) and vapourized 2% Ovalbumin inhaling for 14 days. For rats of the ABAI group, 0.4 mL autoblood was injected into the bilateral "Feishu" (BL 13) or "Shenshu" (BL 23) alternately, once every other day for six times. For rats of the DXM group, 50% DXM solution (0.5 mg/kg, i. p.) was given from the 17th day on after starting the modeling, once every other day for 11 days. Pulmonary GATA 3 and T-bet protein expression was detected by immunohistochemistry, and GATA 3 mRNA and T-bet mRNA expression detected by real time-PCR.
In comparison with the normal group, pulmonary GATA 3 protein and mRNA expression levels in the model group were up-regulated significantly (P < 0.01), while T-bet mRNA expression in the model group was down-regulated obviously (P < 0.01). Compared to the model group, GATA 3 protein and mRNA expression levels were down-regulated significantly in both ABAI and DXM groups (P < 0.01), while T-bet protein expression in ABAI group and T-bet mRNA expression in both ABAI and DXM groups were up-regulated significantly (P < 0.01, P < 0.05). No significant differences were found between model and SAI groups, and between ABAI and DXM groups in GATA 3 protein expression levels; and between ABAI and DXM groups in GATA 3 mRNA expression levels; between normal and model groups, and between SAI and ABAI groups in T-bet protein expression levels; between model and SAI groups and between ABAI and DXM groups in T-bet mRNA expression levels (P > 0.05). The ratio of GATA 3 mRNA/T-bet mRNA expression was significantly higher in the model group than in the normal group (P < 0.01), while obviously lower in the SAI, ABAI and DXM groups than in the model group (P < 0.05, P < 0.01). Additionally, the ratios of GATA 3 mRNA/T-bet mRNA in ABAI and DXM groups were comparable (P > 0.05).
Autoblood acupoint injection is comparable to DXM intraperitoneal injection in down-regulating asthma-induced increase of pulmonary GATA 3 protein and mRNA expression as well as ratio of GATA 3 mRNA/T-bet mRNA, and in up-regulating asthma-induced decrease of T-bet mRNA expression in asthma rats, which may contribute to their effects in relieving asthma.
Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. Epithelial-to-mesenchymal transition has been shown to correlate with therapy resistance, but the functional link and signalling pathways remain to be elucidated. Here we report that microRNA-30c, a human breast tumour prognostic marker, has a pivotal role in chemoresistance by a direct targeting of the actin-binding protein twinfilin 1, which promotes epithelial-to-mesenchymal transition. An interleukin-6 family member, interleukin-11 is identified as a secondary target of twinfilin 1 in the microRNA-30c signalling pathway. Expression of microRNA-30c inversely correlates with interleukin-11 expression in primary breast tumours and low interleukin-11 correlates with relapse-free survival in breast cancer patients. Our study demonstrates that microRNA-30c is transcriptionally regulated by GATA3 in breast tumours. Identification of a novel microRNA-mediated pathway that regulates chemoresistance in breast cancer will facilitate the development of novel therapeutic strategies.
Plant inflorescence meristems and floral meristems possess specific boundary domains that result in proper floral organ separation and specification. HANABA TARANU (HAN) encodes a boundary-expressed GATA3-type transcription factor that regulates shoot meristem organization and flower development in Arabidopsis thaliana, but the underlying mechanism remains unclear. Through time-course microarray analyses following transient overexpression of HAN, we found that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ specification. Transient overexpression of HAN also represses the expression of HAN and three other GATA3 family genes, HANL2 (HAN-LIKE 2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED), and GNL (GNC-LIKE), forming a negative regulatory feedback loop. Genetic analysis indicates that HAN and the three GATA3 family genes coordinately regulate floral development, and their expression patterns are partially overlapping. HAN can homodimerize and heterodimerize with the three proteins encoded by these genes, and HAN directly binds to its own promoter and the GNC promoter in vivo. These findings, along with the fact that constitutive overexpression of HAN produces an even stronger phenotype than the loss-of-function mutation, support the hypothesis that HAN functions as a key repressor that regulates floral development via regulatory networks involving genes in the GATA3 family, along with genes involved in hormone action and floral organ specification.
To explore the effects of FLT3 signaling on the accumulation and maturation of pulmonary conventional dendritic cells (cDCs) and determine whether or not the inhibition of FLT3 signaling may attenuate acute lung inflammation/injury (ALI).
C57BL/6 mice were pretreated separately with FLT3-ligand (FLT3L) and lestaurtinib for 5 days. Murine model of ALI was subsequently induced by an intra-tracheal instillation of lipopolysaccharide (LPS) and lung specimens were harvested 24 hours later. The accumulation and maturation status of pulmonary cDCs were assessed by flow cytometry. Lung myeloperoxidase (MPO) activity was measured to evaluate the infiltration of neutrophils. The ratio between transcription factors T-bet and GATA-3 mRNA was determined to estimate the balance of Th1/Th2 response. Lung injury was estimated by lung wet weight/body weight ratio (LWW/BW) and histopathological assessment.
LPS challenge resulted in rapid accumulation and maturation of pulmonary cDCs. FLT3L pretreatment further stimulated the accumulation and maturation of pulmonary cDCs, leading to a marked deterioration of LWW/BW and lung histopathological changes. Meanwhile, the lung MPO activity and T-bet/GATA-3 mRNA ratio were boosted by the administration of FLT3L. In contrast, the lestaurtinib pretreatment inhibited the accumulation and maturation of pulmonary cDCs, leading to a significant improvement of LWW/BW and lung histopathological changes. The administration of lestaurtinib also suppressed the lung MPO activity and T-bet/GATA-3 mRNA ratio in the lung.
FLT3 signaling attenuates ALI by regulating the accumulation and maturation of pulmonary cDCs, indicating a potential pharmacotherapy for ALI.
In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8(+) T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8(+) T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8(+) T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3(+) and CCR5(+) T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8(+) T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44(hi)CD62L(hi) central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8(+) T cells into established tumors to operate significant tumor cell lysis.
Submicroscopic duplications in the Miller-Dieker critical region have been recently described as new genomic disorders. To date, only a few cases have been reported with overlapping 17p13.3 duplications in this region. Also, small deletions that affect chromosome region 10p14→pter are rarely described in the literature. In this study, we describe, to our knowledge for the first time, a 5-year-old female patient with intellectual disability who has an unbalanced 10;17 translocation inherited from the father. The girl was diagnosed by subtelomeric FISH and array-CGH, showing a 4.43-Mb heterozygous deletion on chromosome 10p that involved 14 genes and a 3.22-Mb single-copy gain on chromosome 17p, which includes the critical region of the Miller-Dieker syndrome and 61 genes. The patient's karyotype was established as 46,XX.arr 10p15.3p15.1(138,206-4,574,436)x1,17p13.3(87,009-3,312,600)x3. Because our patient exhibits a combination of 2 imbalances, she has phenotypic features of both chromosome abnormalities, which have been reported separately. Interestingly, the majority of patients who carry the deletion 10p have visual and auditory deficiencies that are attributed to loss of the GATA3 gene. However, our patient also presents severe hearing and visual problems even though GATA3 is present, suggesting the involvement of different genes that affect the development of the visual and auditory systems.
Human embryonic stem cells (hESCs) can be used to model the cellular and molecular mechanisms that underlie embryonic development. Understanding the cellular mechanisms and pathways involved in extraembryonic (ExE) differentiation is of great interest because of the important role of this process in maternal health and fertility. Fibroblast growth factor 2 (FGF-2) is widely used to maintain the self-renewal of hESCs and induced pluripotent stem cells, while all trans retinoic acid (RA) is used to facilitate the directed differentiation of hESCs. Here, we monitored the RA induced differentiation of hESCs to the ExE lineage with and without FGF-2 over a 7-day period via global transcriptional profiling. The stemness markers POU5F1, NANOG and TDGF1 were markedly downregulated, whereas an upregulation of the ExE markers KRT7, CGA, DDAH2 and IGFBP3 was observed. Many of the differentially expressed genes were involved in WNT and TGF-β signaling. RA inactivated WNT signaling even in the presence of exogenous FGF-2, which that promotes the maintenance of the pluripotent state. We also show that BMP4 was upregulated and that RA was able to modulate the TGF-β signaling pathway and direct hESCs toward the ExE lineage. In addition, an epigenetic study revealed hypermethylation of the DDAH2, TDGF1 and GATA3 gene promoters, suggesting a role for epigenetic regulation during ExE differentiation. These data reveals that the effect of RA prevails in the presence of exogenous FGF-2 thus resulting in the direction of hESCs toward the ExE lineage.
This study aimed to determine if pneumococcal polysaccharide vaccine (PPV) could suppress allergic inflammation in an allergic rhinitis mouse model and to explore whether differences exist regarding the effect of PPV according to timing of administration.
In vivo study using an animal model.
Catholic Research Institutes of Medical Science.
BALB/c mice were divided into control, Der f, Pre-S, and Post-S groups. The allergen was Dermatophagoides farinae (Der f). Pneumococcal polysaccharide vaccine was administered before (Pre-S) or after (Post-S) sensitization. Allergic symptoms and eosinophils in nasal mucosa, interferon-γ, interleukin (IL)-13, and IL-10 in nasal lavage fluid and serum Der f-specific IgE were measured. T-bet, GATA-3, and Foxp3 mRNA in spleen were determined by real-time polymerase chain reaction. Flow cytometry of CD4(+)CD25(+)Foxp3(+) T cells in spleen was analyzed.
In the Pre-S group, symptom score, serum Der f-specific IgE, eosinophils, IL-13, and GATA-3 mRNA were decreased (P < .05), and IL-10, Foxp3 mRNA, and CD4(+)CD25(+)Foxp3(+) T cells were increased compared with those in Der f group (P < .05). In the Post-S group, symptom score, serum Der f-specific IgE, and GATA-3 mRNA were decreased (P < .05), and Foxp3 mRNA and CD4(+)CD25(+)Foxp3(+) T cells were increased compared with those in the Der f group (P < .05).
These results suggest that PPV administered before or after sensitization suppresses Th2 response and enhanced induction of regulatory T cells in an allergic rhinitis model. In addition, there was no significant difference between the degrees of effects in these 2 conditions. In the future, we can consider PPV to be a preventative agent for allergic rhinitis.
CD4(+) helper T cells are essential for immune responses and differentiate in the thymus from CD4(+) CD8(+) "double-positive" (DP) thymocytes. The transcription factor Runx3 inhibits CD4(+) T-cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4(+) T cells. The transcription factor Thpok is upregulated in CD4-differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4(+) T-cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4(+) T-cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high-level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok-deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4-lineage gene-expression, Gata3 represses CD8-lineage gene expression. These findings identify Gata3 as a critical pivot of CD4-CD8 lineage differentiation.
T-bet and GATA-3 are known to regulate cytokine expression in T lymphocytes, and cytokines have been implicated in endometrial regulation and implantation. Previous work showed that female steroid hormones modulate the expression of T-bet in endometrial epithelial cells, suggesting a mechanism for local immune regulation in the human endometrium. We hypothesized that stromal cells are involved in immune regulation, as they have been shown to exert paracrine effects on other endometrial cells and compartments and also secrete cytokines. The objective of this study was to examine the modulation of the gene expression of T-bet and GATA-3, and of the cytokines interferon γ (IFN-γ) and interleukin 4 (IL-4), by female steroid hormones, in human endometrial stromal cells (HESC) in long-term cultures (30 days) mimicking the normal menstrual cycle. T-bet and GATA-3 messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction, and intracellular protein production was demonstrated by immunoblotting. In addition, secretion of IL-4 and IL-15 was measured by enzyme-linked immunosorbent assay. T-bet and IL-4 mRNA expression increased and GATA-3 decreased under decidualization conditions; IFN-γ was not detected. Secretion of IL-15 increased during decidualization, and IL-15 upregulated T-bet gene expression. In conclusion, gene expression of T-bet and GATA-3 by endometrial stromal cells is under hormonal conditions mimicking decidualization, and the results are consistent with an autocrine regulatory mechanism of IL-15 secretion and T-bet expression.
LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.
Chronic allergic asthma is characterized by Th2-typed inflammation, and contributes to airway remodeling and the deterioration of lung function. However, the initiating factor that links airway inflammation to remodeling is unknown. Thymic stromal lymphopoietin (TSLP), an epithelium-derived cytokine, can strongly activate lung dendritic cells (DCs) through the TSLP-TSLPR and OX40L-OX40 signaling pathways to promote Th2 differentiation. To determine whether TSLP is the underlying trigger of airway remodeling in chronic allergen-induced asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extracts for up to 5 consecutive weeks. We showed that repeated respiratory exposure to HDM caused significant airway eosinophilic inflammation, peribronchial collagen deposition, goblet cell hyperplasia, and airway hyperreactivity (AHR) to methacholine. These effects were accompanied with a salient Th2 response that was characterized by the upregulation of Th2-typed cytokines, such as IL-4 and IL-13, as well as the transcription factor GATA-3. Moreover, the levels of TSLP and transforming growth factor beta 1 (TGF-β1) were also increased in the airway. We further demonstrated, using the chronic HDM-induced asthma model, that the inhibition of Th2 responses via neutralization of TSLP with an anti-TSLP mAb reversed airway inflammation, prevented structural alterations, and decreased AHR to methacholine and TGF-β1 level. These results suggest that TSLP plays a pivotal role in the initiation and persistence of airway inflammation and remodeling in the context of chronic allergic asthma.
Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) "master genes". EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.
Using a Slug-lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10-20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.
We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways.
The E2A transcription factors promote the development of thymus-seeding cells, but it remains unknown whether these proteins play a role in T lymphocyte lineage specification or commitment. Here, we showed that E2A proteins were required to promote T-lymphocyte commitment from DN2 thymocytes and to extinguish their potential for alternative fates. E2A proteins functioned in DN2 cells to limit expression of Gata3, which encodes an essential T-lymphocyte transcription factor whose ectopic expression can arrest T-cell differentiation. Genetic, or small interfering RNA-mediated, reduction of Gata3 rescued T-cell differentiation in the absence of E2A and restricted the development of alternative lineages by limiting the expanded self-renewal potential in E2A−/− DN2 cells. Our data support a novel paradigm in lymphocyte lineage commitment in which the E2A proteins are necessary to limit the expression of an essential lineage specification and commitment factor to restrain self-renewal and to prevent an arrest in differentiation.
In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3- and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3-overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.
Transcription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. While GATA-3 is required for T-cell specification, its mechanism of action is poorly understood. We show that GATA-3 works in concert with Notch1 to commit thymic progenitors to the T-cell lineage via 2 distinct pathways. First, GATA-3 orchestrates a transcriptional “repertoire” that is required for thymocyte maturation up to and beyond the pro-T-cell stage. Second, GATA-3 critically suppresses a latent B-cell potential in pro–T cells. As such, GATA-3 is essential to sealing in Notch-induced T-cell fate in early thymocyte precursors by promoting T-cell identity through the repression of alternative developmental options.
Bladder cancer is the 4(th) most common cancer among men in the U.S. We analyzed variant genotypes hypothesized to modify major biological processes involved in bladder carcinogenesis, including hormone regulation, apoptosis, DNA repair, immune surveillance, metabolism, proliferation, and telomere maintenance. Logistic regression was used to assess the relationship between genetic variation affecting these processes and susceptibility in 563 genotyped urothelial cell carcinoma cases and 863 controls enrolled in a case-control study of incident bladder cancer conducted in New Hampshire, U.S. We evaluated gene-gene interactions using Multifactor Dimensionality Reduction (MDR) and Statistical Epistasis Network analysis. The 3'UTR flanking variant form of the hormone regulation gene HSD3B2 was associated with increased bladder cancer risk in the New Hampshire population (adjusted OR 1.85 95%CI 1.31-2.62). This finding was successfully replicated in the Texas Bladder Cancer Study with 957 controls, 497 cases (adjusted OR 3.66 95%CI 1.06-12.63). The effect of this prevalent SNP was stronger among males (OR 2.13 95%CI 1.40-3.25) than females (OR 1.56 95%CI 0.83-2.95), (SNP-gender interaction P = 0.048). We also identified a SNP-SNP interaction between T-cell activation related genes GATA3 and CD81 (interaction P = 0.0003). The fact that bladder cancer incidence is 3-4 times higher in males suggests the involvement of hormone levels. This biologic process-based analysis suggests candidate susceptibility markers and supports the theory that disrupted hormone regulation plays a role in bladder carcinogenesis.
Ustekinumab is a fully human anti-p40 monoclonal antibody which neutralizes interleukin (IL)-12 and IL-23, thereby interfering with T-helper (Th)1/Th17 pathways and keratinocyte activation, and is highly effective in the treatment of psoriasis. During ustekinumab treatment, some of our patients noticed reduced koebnerization of noninvolved skin and less new plaque formation.
To determine whether ustekinumab improves psoriasis-related gene expression and tape-strip responses in noninvolved skin.
Before and 4 weeks after ustekinumab treatment, noninvolved skin was tape-stripped. After 5 h, biopsies were taken from untouched and tape-stripped skin. The mRNA expression of psoriasis-related markers such as NGF, GATA3 and IL-22RA1, and several antimicrobial peptides (AMP) was quantified. Leucocyte counts and a broad range of inflammatory serum proteins were analysed to gain insight into the systemic alterations.
Four weeks following a single ustekinumab injection, NGF showed a significant decrease, whereas GATA3 and IL-22RA1 expression increased, indicative of reduced responsiveness to epidermal triggering. This was accompanied by an increase of the inflammation-related serum proteins GPNMB, MST1 and TRADD. The baseline and tape-strip-induced mRNA expression of the AMP human β-defensin-2 (hBD-2), S100A7 and LL-37 remained unaltered. Clinically, after 4 weeks, eight out of 11 patients showed a 50% psoriasis area and severity index (PASI) improvement, which was accompanied by a significant reduction in serum hBD-2 levels. No changes were noted in total leucocytes, C-reactive protein and erythrocyte sedimentation rate.
These findings indicate that ustekinumab reduces psoriasis-related gene expression in noninvolved psoriatic skin, making it more resistant to exogenous triggering, without disturbing its antimicrobial response. In parallel, ustekinumab modulates important circulating inflammation-related proteins.
There are few studies on the natural history of milk allergy. Most are single-site and not longitudinal, and these have not identified a means for early prediction of outcomes.
Children aged 3 to 15 months were enrolled in an observational study with either (1) a convincing history of egg allergy, milk allergy, or both with a positive skin prick test (SPT) response to the trigger food and/or (2) moderate-to-severe atopic dermatitis (AD) and a positive SPT response to milk or egg. Children enrolled with a clinical history of milk allergy were followed longitudinally, and resolution was established by means of successful ingestion.
The cohort consists of 293 children, of whom 244 were given a diagnosis of milk allergy at baseline. Milk allergy has resolved in 154 (52.6%) subjects at a median age of 63 months and a median age at last follow-up of 66 months. Baseline characteristics that were most predictive of resolution included milk-specific IgE level, milk SPT wheal size, and AD severity (all P < .001). Baseline milk-specific IgG4 level and milk IgE/IgG4 ratio were not predictive of resolution and neither was expression of cytokine-inducible SH2-containing protein, forkhead box protein 3, GATA3, IL-10, IL-4, IFN-γ, or T-bet by using real-time PCR in CD25-selected, casein-stimulated mononuclear cells. A calculator to estimate resolution probabilities using baseline milk IgE level, SPT response, and AD severity was devised for use in the clinical setting.
In this cohort of infants with milk allergy, approximately one half had resolved over 66 months of follow-up. Baseline milk-specific IgE level, SPT wheal size, and AD severity were all important predictors of the likelihood of resolution.
Expression of GATA-3 in female breast cancers has been linked to estrogen receptor (ER) expression and, in turn, to improved outcomes. However, GATA-3 has not been studied in male breast cancers. Nineteen male breast carcinomas (average age: 63 years) and 164 female breast carcinomas (average age: 57 years) were immunostained for GATA-3. Results were compared to age, tumor size, tumor grade, lymph node status, distant metastases, survival, and positivity for ER, progesterone receptor (PR), and HER2/neu. Six of 19 (31.6%) male and 135 of 164 (82.3%) female breast carcinomas were GATA-3 positive (P < .001). In women, 82.1% of GATA-3-positive cancers were grade 1 or 2, whereas 75.9% of GATA-3-negative cancers were grade 3 (P < .001); no such significant correlation was seen in men. Unlike female cancers, male cancers showed no correlation between GATA-3 positivity and ER positivity, PR positivity, or distant metastases. Nodal metastasis and HER2 status were not linked to GATA-3 in either sex. Seventeen (89.5%) men were alive at follow-up (average: 61 months); only 1 died of disease. Most women (159/164, 97.0%) were also alive at follow-up (average: 41 months), with a higher proportion of GATA-3-negative women dead than GATA-3-positive women (3/29 [10.3%] vs. 2/135 [1.5%], P = .039). GATA-3 is expressed less often in male than female breast cancers. Male cancers show no correlation between GATA-3 positivity and ER/PR positivity or distant metastases, unlike female cancers. There appears to be no link between GATA-3 positivity and survival in men, whereas in women, GATA-3-positive tumors are typically lower grade with a better prognosis.
DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.
In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.
The Th2 locus control region (LCR) has been shown to be important in efficient and coordinated cytokine gene regulation during Th2 cell differentiation. However, the molecular mechanism for this is poorly understood. To study the molecular mechanism of the Th2 LCR, we searched for proteins binding to it. We discovered that transcription factor YY1 bound to the LCR and the entire Th2 cytokine locus in a Th2-specific manner. Retroviral overexpression of YY1 induced Th2 cytokine expression. CD4-specific knockdown of YY1 in mice caused marked reduction in Th2 cytokine expression, repressed chromatin remodeling, decreased intrachromosomal interactions, and resistance in an animal model of asthma. YY1 physically associated with GATA-binding protein-3 (GATA3) and is required for GATA3 binding to the locus. YY1 bound to the regulatory elements in the locus before GATA3 binding. Thus, YY1 cooperates with GATA3 and is required for regulation of the Th2 cytokine locus and Th2 cell differentiation.
T-bet plays an important role in immunoregulation; it induces the differentiation of Th1 together with the homeobox transcription factor gene Hlx. Recent studies show that T-bet and Th1-associated factors are critical in regulating tumor development. However, the contributions of Hlx in the occurrence and development of cancer remain unknown. In this study, the Hlx, T-bet, Runx3, and IFN-γ were measured in PBMC from patients with gastric cancer and the correlation between Hlx and T-bet or IFN-γ was assessed. The expression levels of Hlx, T-bet, and IFN-γ were significantly decreased, and there was a positive correlation between Hlx and T-bet or IFN-γ. In addition, the Runx3 expression was also downregulated with the lower T-bet mRNA level. These results suggested that the decreased Hlx expression was closely associated with T-bet and Runx3 downregulations and may contribute to the development of gastric cancer.
Reporting in this issue of Developmental Cell, Kizil et al. (2012) identify the transcription factor Gata3 as an important player in regeneration of the zebrafish adult telencephalon. Other injured tissues like the heart and the fin also upregulate Gata3 in response to injury, suggesting that Gata3 is a general response to tissue damage.
T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions.
GATA-3, a C2C2-type zinc finger transcription factor, regulates many steps of T cell development and differentiation. It is also required for optimal production of type 2 cytokines by CD8(+) T cells. However, its role in the development and function of this subset of T cells is still poorly characterized. In this paper, we report that GATA-3 is required for MHC-mediated positive selection and final maturation of CD8 single-positive thymocytes. Deficiency of GATA-3 mediated by a CD4cre transgene led to age-dependent lymphadenopathy partly because of abnormal expansion of CD8(+) T cells driven by a cell-extrinsic mechanism. Paradoxically, GATA-3-deficient CD8(+) T cells were hyporesponsive to Ag stimulation due to a defect in the maintenance/progression, but not initiation, of activation signals. More importantly, GATA-3-deficient CD8(+) T cells were less efficient in killing Ag-bearing tumor cells in vivo. Taken together, our data further expand the role of GATA-3 in T cells.
The analysis of Fgf10 mouse mutants has demonstrated a critical role for this ligand in neurosensory development of the vertebrate inner ear, and we have been looking to define the direct upstream regulators of Fgf10 in this sensory organ, as part of constructing the programme of early inner ear development. Through the analysis of reporter constructs in transgenic mouse embryos and neonatal mice, in this report we define a minimal 1400 bp enhancer from the 5' flanking region of Fgf10. This enhancer drives reporter transgene expression in a manner that recapitulates endogenous expression of Fgf10, from its initial onset in the invaginating otic placode and onwards throughout gestation, controlling Fgf10 expression in all developing sensory patches and in the developing VIIIth ganglion. This regulatory region includes three putative Gata3 binding sites that we demonstrate directly interacts with Gata3 protein through the DNA binding domain with differing affinities. Site directed mutagenesis of all three sites and functional testing in transgenic embryos using reporter transgenes reveals an absolute requirement for Gata3 in controlling Fgf10 expression. Transgenic analysis of individual Gata3 binding site mutations illustrates that only one of these binding sites is necessary for reporter expression. Together these data demonstrate that Gata3 directly activates Fgf10 in the early inner ear, and does so through a single binding site.
Hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome (MIM 146255) is a rare autosomal dominant disorder caused by mutations in the gene encoding GATA3, a dual zinc-finger transcription factor involved in vertebrate embryonic development. In this clinical case study we report on a follow-up of a phenotype associated with a GATA3 mutation. HDR syndrome was clinically diagnosed at age of 1.5 years in a boy with a de novo heterozygous missense (c.815C→T) mutation, Thr272Ile, in exon 4 of the GATA3 gene. Both parents were negative for Thr272Ile.At age of 17 months, the patient had a weight of 10.7, a body length of 78 cm, and a head circumference of 47.5 cm. By the age of 7 years, growth is age-appropriate, severe bilateral hearing loss (dB 60) was corrected by hearing aids. However, cognitive development (auditory sensory me-mory and language abilities) is at the lower ends of the test scores.In conclusion, a mildly impaired clinical course was achieved by the age of 7 years in a patient with HDR syndrome; this report adds to the body of data on genotype-phenotype analysis in HDR syndrome. ·
Allergic asthma is a chronic airway disorder characterized by airway hyperresponsiveness to allergens, chronic airway inflammation, airway edema, increased mucus secretion, excess production of Th2 cytokines, and eosinophil accumulation in the lungs. Ursolic acid is known for its pharmacological effects, such as its anti-tumor, anti-inflammatory and antimicrobial activities. To investigate the anti-asthmatic effects and mechanism of ursolic acid, we studied the development of pulmonary eosinophilic inflammation and enhanced pause (Penh) in a mouse model of allergic asthma. In this study, BALB/c mice were systemically sensitized to ovalbumin followed by intratracheal, intraperitoneal, and aerosol allergen challenges. We investigated the effect of ursolic acid and Cyclosporin A (CsA) on Penh, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokines, IL-17 production, and ovalbumin specific IgE production in a mouse model of asthma. In BALB/c mice, ursolic acid had suppressed eosinophil infiltration, allergic airway inflammation, and Penh, which occurred by suppressing the production of IL-5, IL-13, IL-17, and ovalbumin-specific IgE by blocking the GATA-3 and STAT6 pathways. Our data suggest the therapeutic mechanism of ursolic acid in asthma is based on reductions of Th2 cytokines (IL-5 and IL-13), ovalbumin-specific IgE production, and eosinophil infiltration via the Th2-GATA-3, STAT6, and IL-17-NF-κB pathways.
This study investigated whether an imbalance in Th1/Th2 cells is involved in the post-resuscitation myocardial immune dysfunction.
26 Wuzhishan miniature pigs were randomly divided into return of spontaneous circulation (ROSC) group (n=20) and sham-operated group (n=6), 20 pigs were subjected to 8min of electrically induced cardiac arrest, After successful ROSC, the 16 surviving pigs were randomly assigned to be sacrificed (n=8 per group) at 12 and 24h after ROSC, respectively. CD4(+) and CD8(+) lymphocyte subsets were determined by flow cytometry, interleukin (IL)-4 and interferon (IFN)-γ in the myocardium were measured by ELISA, and protein and mRNA levels of GATA-3 and T-bet were detected in the myocardium by Western blotting and quantitative real-time PCR in the post-ROSC group (n=8 per group) at 12 and 24h after ROSC and sham-operated group (n=6) at 24h after ROSC, respectively.
CD4(+) lymphocyte subsets were significantly lower in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC. The levels of myocardium IFN-γ were markedly increased, while IL-4 was significantly decreased in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC. Protein expression and mRNA levels of T-bet were markedly increased in the myocardium of pigs in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC, while GATA-3 was significantly reduced (P<0.05).
The myocardial immune dysfunction induced by the change in expression levels of the transcription factors GATA-3 and T-bet may be involved in the process of post-resuscitation myocardial injury in a porcine model of cardiac arrest.
Even though the systemic level of SCCA1, a serine protease inhibitor, was shown to be elevated in asthma, its physiological role is unknown.
We sought to determine the effect of SCCA1 on apoptosis, cytokine expression and mucus production by A549 cells and define the effect of promoter variants on gene expression and association with asthma.
SCCA levels were measured by ELISA. Promoter variants were determined by direct sequencing. 442 asthmatic children and 191 controls were genotyped by RFLP. The functional effect of the polymorphisms was assessed in transient transfection experiments using reporter constructs. A transcription factor ELISA was used for differential binding of GATA proteins to the variant region. The effects of SCCA1 on cytokine synthesis, mucus production and apoptosis were determined in A549 cells transfected with SCCA1 pcDNA vector. MUC5AC expression in A549 cells was determined with RT-PCR.
SCCA1 protein level was significantly higher in asthmatic children compared to healthy controls. Four polymorphisms SCCA1 promoter that were in linkage disequilibrium were associated with skin test positivity in asthmatic children and showed higher promoter activity and higher binding of GATA-2 and GATA-3 after IL-4 + IL-13 stimulation. IL-6, IL-8 levels were significantly higher in cells transfected with SCCA1 whereas RANTES increased only after IL-4 stimulation. Transfection of A549 cells with SCCA1 resulted in decreased MUC5AC expression and conferred protection against apoptosis.
Our results showed that SCCA1 has diverse effects on many of the cellular events that characterize asthma and its role extends beyond protease inhibition.
Abstract Background: To detect genes associated with the expression of ESR1 and PGR - as well as of their protein products, estrogen receptor (ER) and progesterone receptor (PR) - 221 de-identified invasive ductal carcinomas of the breast were investigated. Our long-term goal is to decipher relationships between the expression of ER- and PR-associated genes and breast cancer behavior to improve diagnostics and identify new molecular targets for drug design.
Frozen tissue sections were evaluated for structural integrity and pathology after hematoxylin and eosin staining. ER and PR protein levels were quantified by either enzyme immunoassay or radio-ligand binding assay. Total RNA preparations were reverse transcribed for qPCR measurements of ESR1, PGR and 31 gene candidates.
Both ESR1 and PGR expression levels were correlated with their cognate receptor protein expression (Pearson correlations of 0.82 and 0.68, p<0.001, respectively), to assess molecular relationships between clinically relevant biomarkers in tissue specimens. Coordinate expression of EVL, NAT1, TBC1D9, SCUBE2, RABEP1, SLC39A6, TCEAL1, FUT8, XBP1, PTP4A2 or GATA3 with either ESR1 or PGR was detected.
Examination of relationships between ESR1 and PGR gene expression and that of other genes of interest indicated: a high degree of correlation between ESR1 levels and expression of NAT1, SCUBE2, XBP1 and GATA3; and a high degree of correlation between PGR expression and that of NAT1, ESR1, SCUBE2 and RABEP1. These results suggest that direct relationships of these genes exist with estrogen and progestin receptor mediated pathways. Pathway analysis software provided additional evidence of gene interactions.
Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by a GATA3 gene mutation. Here we report a novel mutation of GATA3 in a patient diagnosed with HDR syndrome at the age of 58 with extensive intracranial calcification.
A 58-year-old Japanese man showed severe hypocalcemia and marked calcification in the basal ganglia, cerebellum, deep white matter, and gray-white junction on computed tomography (CT). The serum intact parathyroid hormone level was relatively low against low serum calcium concentration. The patient had been diagnosed with bilateral sensorineural deafness in childhood and had a family history of hearing disorders. Imaging studies revealed no renal anomalies. The patient was diagnosed with HDR syndrome, and genetic testing was performed.
Genetic analysis of GATA3 showed a novel nonsense mutation at codon 198 (S198X) in exon 3. The S198X mutation leads to a loss of two zinc finger deoxyribonucleic acid (DNA) binding domains and is considered to be responsible for HDR syndrome.
We identified a novel nonsense mutation of GATA3 in an adult patient with HDR syndrome who showed extensive intracranial calcification.
Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.
Carp kidney leukocytes co-cultured with a supporting cell layer resulted in proliferation of polyclonal CD4(+) αβT cells as described previously. These bulk-cultured T cells expressed transcripts for both T helper 1 cells (Th1) master regulator (T-bet) and T helper 2 cells (Th2) master regulator (GATA-3). To identify the Th subsets in bulk-cultured T cells, single cells were picked up from the bulk culture, proliferated, and characterized. The majority of the clones displayed characteristics consistent with CD4(+) αβT cell identity. These clones expressed both TCRα and TCRβ, but could not produce a TCRγδ heterodimer since they typically only expressed either TCRγ or TCRδ. These clones also expressed the TCR co-receptor genes CD4-1 or CD4-2, whereas they did not express CD8α or CD8β. In addition, GATA-3 was expressed whereas T-bet was not. Among these clones, one clone (KoThL5) continued to proliferate on the supporting cells and was successively transferred for more than 10 months and 90-100 passages. To characterize the KoThL5 cells by their cytokine production profile, they were stimulated with PHA and investigated by real-time RT-PCR. mRNA expression of Th2-related cytokine (IL-4/13B) was only enhanced in KoThL5 cells whereas both Th1-related cytokine (IFNγ) and Th2-related cytokines (IL-4/13A and IL-4/13B) were significantly enhanced in bulk-cultured T cells. Taken together, KoThL5 cells share some features with mammalian Th2 cells. This is the first study to describe in vitro cultures of teleost cell with Th2-like features. The KoThL5 cell line has considerable potential for addressing questions concerning the properties of teleost Th2 cells.
Atopic dermatitis (AD) is a chronic disease with Th2-type-cytokine dominant profile. Several cytokines and related peptides had been tried for the treatment of AD but with unsuccessful results; a part of the reason is the limitation of their biological half time. We have recently developed a highly efficient mouse dominant negative IL-4/IL-13 DNA vaccine, which blocks both IL-4 and IL-13 signal transductions, resulting in the amelioration of atopic reaction. At the next step, the further consistent protein supplementation system is required for stable suppression of allergic reaction. To examine the effects of mutant IL-4/13 protein supplementation from skin, a keratinocyte-specific dominant negative IL-4-transgenic mouse line (IL-4DM) was established. The anti-inflammatory function was evaluated measuring ear thickness and analyzing histological change in mice AD model induced by repeated elicitation of oxazolone. In IL-4DM, ear thickness was suppressed significantly in the early phase of the elicitation schedule of contact hypersensitivity response. We next transplanted IL-4DM skin to normal control mice, and investigated effects in inflammatory reactions. In IL-4DM-skin-grafted mice, the inflammatory response was suppressed significantly similarly in the early phase accompanied with lesional suppressed Th2-type-cytokine signaling transduction. IL-4DM skin has the anti-inflammatory function especially in the acute phase of AD. Although there are several issues to be addressed for human application, the present results implicated that the gene manipulated skin transplantation is a potent therapeutic strategy to control allergic reactions.
A 49-year-old female patient presented with anemia of unclear origin (hemoglobin level 6 µg/dl). During coloscopy a circular wall edema in the right flexure of the colon was observed and gastroduodenoscopy gave rise to the suspicion of fundic gland polyps (Elster's cysts) of the mucosa. In the lamina propria of the gastric mucosa, infiltration of a poorly differentiated carcinoma with signet ring cell morphology could be histologically identified resulting in the suspected diagnosis of a primary gastric signet ring carcinoma. Using immunohistochemistry it could be shown that the tumor cells expressed cytokeratin 7 (CK7), estrogen receptors and Gata 3 but not CK20 or Cdx2. The diagnosis of metastases of lobular breast cancer in the gastric and colon mucosa could be made.
Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells.
Glucocorticoid receptor (GR) variants have been found to be associated with stress-related disorders. Our previous in vivo study revealed that the CC allele of GR BclI single-nucleotide polymorphism (SNP) was more common in the high-stress group, which had lower levels of both regulatory T cells (Treg) and Th1 cytokine. The current study was to investigate the associations between GR BclI polymorphism and immunomodulatory response to stress hormone in vitro in human peripheral blood mononuclear cells (PBMC). Blood samples were collected from 18 normal volunteers including 9 subjects with BclI polymorphism GG allele and 9 with wild-type (WT) CC allele. PBMC were cultured with 10(-8) m dexamethasone (DEX), which mimics the plasma cortisol level observed during periods of psychological stress for 24 h and 11 days. Gene expressions of transcription factors, stress hormone and cytokine receptors were analysed by real-time RT-PCR. FoxP3 mRNA was significantly altered in the BclI WT (decreased at 24 h and increased at 11 days) but not in the GG allele. GR mRNA was up-regulated at 24 h and down-regulated at 11 days in CC alleles (P < 0.01 and P < 0.05), rather than in GG alleles. The expression of β-2 adrenergic receptor (β2AR) was increased at 24 h in both CC and GG alleles (P < 0.01 and P < 0.001), but decreased significantly at 11 days in only GG alleles. Expression of T-bet and GATA-3 was altered simultaneously in 24-h culture with DEX from both groups. The BclI polymorphism of GR identifies different immunomodulatory responses to corticosteroids, which may explain, at least in part, the variability in individual sensitivity to stressors.
Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.
The adult zebrafish brain, unlike mammalian counterparts, can regenerate after injury owing to the neurogenic capacity of stem cells with radial glial character. We hypothesized that injury-induced regenerative programs might be turned on after injury in zebrafish brain and enable regenerative neurogenesis. Here we identify one such gene-the transcription factor gata3-which is expressed only after injury in different zebrafish organs. Gata3 is required for reactive proliferation of radial glia cells, subsequent regenerative neurogenesis, and migration of the newborn neurons. We found that these regeneration-specific roles of Gata3 are dependent on the injury because Gata3 overexpression in the unlesioned adult zebrafish brain is not sufficient to induce neurogenesis. Thus, gata3 acts as a specific injury-induced proregenerative factor that is essential for the regenerative capacity in vertebrates.
Allergic asthma is associated with Th2-mediated inflammation. Several flavonoids were isolated from Glycyrrhiza uralensis, one of the herbs in the anti-asthma herbal medicine intervention. The aim of this investigation was to determine whether Glycyrrhiza uralensis flavonoids have inhibitory effects on memory Th2 responses in vitro and antigen-induced Th2 inflammation in vivo. The effects of three Glycyrrhiza uralensis flavonoids on effector memory Th2 cells, D10.G4.1 (D10 cells), were determined by measuring Th2 cytokine production. Isoliquiritigenin, 7, 4'-dihydroxyflavone (7, 4'-DHF) and liquiritigenin significantly suppressed IL-4 and IL-5 production in a dose-dependent manner, 7, 4'-DHF being most potent. It was also evaluated for effects on D10 cell proliferation, GATA-3 expression and IL-4 mRNA expression, which were suppressed, with no loss of cell viability. Chronic treatment with 7, 4'-DHF in a murine model of allergic asthma not only significantly reduced eosinophilic pulmonary inflammation, serum IgE levels, IL-4 and IL-13 levels, but also increased IFN-γ production in lung cell cultures in response to antigen stimulation.
Mouse mesenchymal stem cells (MSCs) can generate sensory neurons and produce inner ear hair cell-like cells. An equivalent source from humans is highly desirable, given their potential application in patient-specific regenerative therapies for deafness. In this study, we explored the ability of human MSCs (hMSCs) to differentiate into otic lineages.
hMSCs were exposed to culture media conditioned by human fetal auditory stem cells.
Conditioned media induced the expression of otic progenitor markers PAX8, PAX2, GATA3 and SOX2. After 4 weeks, cells coexpressed ATOH1, MYO7A and POU4F3 (indicators of hair cell lineage) or neuronal markers NEUROG1, POU4F1 and NEFH. Inhibition of WNT signaling prevented differentiation into otic progenitors, while WNT activation partially phenocopied results seen with the conditioned media.
This study demonstrates that hMSCs can be driven to express key genes found in the otic lineages and thereby promotes their status as candidates for regenerative therapies for deafness.
Loricrin is a major component of the epidermal cornified cell envelope and is expressed only in terminally differentiated keratinocytes. This cell differentiation-specific expression pattern suggests specific regulatory mechanisms for activation of loricrin gene transcription in differentiated keratinocytes. Here, we identified a positive regulatory element in the proximal promoter region of the loricrin gene involved in activation of its expression in differentiated keratinocytes. A database search indicated that this sequence contained a GATA-3 binding motif. Constructs with point mutations in the GATA-3 binding motif showed decreased reporter activity, indicating that GATA-3 positively regulates loricrin gene transcription. Western blotting analysis indicated that GATA-3 is more abundant in differentiated than in undifferentiated keratinocytes. Cotransfection experiments indicated that GATA-3 activates transcription of the loricrin gene in a cooperative manner with c-Fos and Sp1. These findings indicate that GATA-3 contributes to keratinocyte differentiation-specific activation of loricrin gene transcription via interaction with c-Fos and Sp1.
The co-infection of HIV and helminth parasites, such as Schistosoma spp, has increased in sub-Saharan Africa. Many HIV vaccine candidate studies have been completed or are in ongoing clinical trials, but it is not clear how HIV vaccines might affect the course of schistosome infections. In this study, we immunized S. mansoni-infected mice with an efficient DNA vaccine that included HIV gag. Using this model, we found that Th2 cytokines, such as IL-4 and IL-13, were highly induced after schistosome infection. Treatment of infected mice with the HIV DNA vaccine resulted in a significant attenuation of this rise in IL-13 expression and an increase in expression of the Th1 cytokine, TNF-α. However, vaccine administration did not significantly influence the expression of IL-4, or IFN-γ, and did not affect T cell proliferative capacity. Interestingly, the IL-4 (+) IFN-γ (+) phenotype appears in schistosome-infected mice that received HIV vaccination, and is associated with the expression of transcription factors GATA3 (+) T-bet (+) in these mice. These studies indicate that DNA vaccination can have an impact on ongoing chronic infection.
In amyotrophic lateral sclerosis (ALS) mice, regulatory T-lymphocytes (Tregs) are neuroprotective, slowing disease progression. To address whether Tregs and FoxP3, a transcription factor required for Treg function, similarly influence progression rates of ALS patients, T-lymphocytes from patients were assessed by flow cytometry. Both numbers of Tregs and their FoxP3 protein expressions were reduced in rapidly progressing ALS patients and inversely correlated with progression rates. The mRNA levels of FoxP3, TGF-β, IL4 and Gata3, a Th2 transcription factor, were reduced in rapidly progressing patients and inversely correlated with progression rates. Both FoxP3 and Gata3 were accurate indicators of progression rates. No differences in IL10, Tbx21, a Th1 transcription factor or IFN-γ expression were found between slow and rapidly progressing patients. A 3.5-year prospective study with a second larger cohort revealed that early reduced FoxP3 levels were indicative of progression rates at collection and predictive of future rapid progression and attenuated survival. Collectively, these data suggest that Tregs and Th2 lymphocytes influence disease progression rates. Importantly, early reduced FoxP3 levels could be used to identify rapidly progressing patients.
GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.
The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.
A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
Kinases have been implicated in the immunopathological mechanisms of Systemic Lupus Erythematosus (SLE). v-akt murine-thymoma viral-oncogene-homolog 1 (AKT1) and mitogen-activated-protein-kinase 1 (MAPK1) gene expressions in peripheral mononuclear cells from thirteen SLE patients with inactive or mild disease were evaluated using quantitative real-time reverse-transcription polymerase-chain-reaction and analyzed whether there was any correlation with T-helper (Th) transcription factors (TF) gene expression, cytokines, and S100A8/S100A9-(Calprotectin). Age- and gender-matched thirteen healthy controls were examined. AKT1 and MAPK1 expressions were upregulated in SLE patients and correlated with Th17-(Retinoic acid-related orphan receptor (ROR)-C), T-regulatory-(Treg)-(Transforming Growth Factor Beta (TGFB)-2), and Th2-(interleukin (IL)-5)-related genes. MAPK1 expression correlated with Th1-(IL-12A, T-box TF-(T-bet)), Th2-(GATA binding protein-(GATA)-3), and IL-10 expressions. IL-10 expression was increased and correlated with plasma Tumor Necrosis Factor (TNF)-α and Th0-(IL-2), Th1-(IL-12A, T-bet), GATA3, Treg-(Forkhead/winged-helix transcription factor- (FOXP)-3), and IL-6 expressions. FOXP3 expression, FOXP3/RORC, and FOXP3/GATA3 expression ratios were increased. Plasma IL-1β, IL-12(p70), Interferon-(IFN)-γ, and IL-6 cytokines were augmented. Plasma IL-1β, IL-6, IL-2, IFN-γ, TNF-α, IL-10, and IL-13 correlated with C-reactive protein, respectively. Increased Calprotectin correlated with neutrophils. Conclusion, SLE patients presented a systemic immunoinflammatory activity, augmented AKT1 and MAPK1 expressions, proinflammatory cytokines, and Calprotectin, together with increased expression of Treg-related genes, suggesting a regulatory feedback opposing the inflammatory activity.
Epidemiologic studies have reported the association between fine particles (aerodynamic diameter ≤ 2.5 μm; PM2.5) and health effects, but the immunological mechanisms are not clear. To investigate the dose and time-dependent role of toll-like receptor (TLR) and Th1/Th2 shift in local and systemic inflammation induced by PM2.5, mice were subjected to intratracheal instillation of 2.5, 5, or 10 mg/kg PM2.5 in this study. After 24 h, 72 h, 7 days, and 14 days, mice were sacrificed to measure TLR2 and TLR4 expressions and Th1/Th2 related cytokines in bronchoalveolar lavage fluid (BALF) and peripheral blood. Histopathological changes in lung were also examined. Inflammatory infiltration and macrophages with engulfed particles were found by lung histopathology after PM2.5 exposure. TLR4 positive cells decreased in BALF but increased in blood at 24 h after the exposure. The low percentage of TLR4 positive cells continued to day 14 in BALF, but recovered at day 7 and decreased further to lower than the control value at day 14 in blood. TLR2 positive cell changed similar to TLR4 in BALF on the dose effects. In BALF at 24 h after the exposure, the Th2 related cytokines IL-5 and IL-10 increased dose-dependently; and in blood, the Th2 related cytokines IL-4, IL-5, and IL-10 also increased. These results suggest that acute exposure of PM2.5 leads to acute inflammatory responses locally and systemically in mice. TLR2 and TLR4 are involved in this process and PM2.5 can drive a Th2-biased immune response.
Both T-bet and GATA-3, Th1/Th2 lineage-specific transcription factors, play important roles in the development of T cells and Th1/Th2 differentiation. In this study, T-bet and GATA-3 genes were cloned from grass carp (Ctenopharyngodon idellus). The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp T-bet contained 608 aa, which possessed a T-box DNA binding domain. The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp GATA-3 contained 396 aa, which possessed two consensus zinc finger domains (C-X(2)-C-X(17)-C-X(2)-C). The YxKxHxxxRP motif, KRRLSA and LMEKs/n sequences were also conserved in this GATA-3. Phylogenetic analysis indicated that grass carp T-bet and GATA-3 group with their known counterparts with zebrafish T-bet and GATA-3 as the closest neighbor, respectively. RT-qPCR results showed that grass carp T-bet gene was highly expressed in head kidney, followed by spleen, and low expressed in gill, liver, kidney, and intestine, while GATA-3 gene was highly expressed in intestine, followed by spleen, and low expressed in gill, liver, kidney, and head kidney. Grass carp is one of the "four important domestic fish" in China and often infected by grass carp reovirus (GCRV). As yet, there is no evidence that T-bet and GATA-3 (Th1/Th2 subsets) are involved in anti-virus immune of teleost fish. In this study, by RT-qPCR, we analyzed the expression dynamics of grass carp T-bet and GATA-3 genes with IFN-γ gene in response to GCRV infection for the first time. The expression dynamics showed that three genes might be crucially modulated by in vivo GCRV infection: (1) GCRV mainly induced a T-bet expression profile comparing to the GATA-3 expression, while the higher expression profiles of IFN-γ correlated with the up-regulation of T-bet; (2) T-bet/IFN-γ and GATA-3 expression changes suggest that in GCRV-infected grass carp, the common immune state of head kidney further heightens, whereas the common physiological state of intestine transforms to an anti-virus immune state. From this finding, we realize that GCRV mainly induces a Th1 response, and Th1 cell-mediated recognition mechanisms play very important roles in anti-virus cellular immune of grass carp.
Distinction between primary lung carcinomas and metastases from other sites, especially the urinary tract, is a common diagnostic dilemma. As urothelial carcinomas can demonstrate a broad range of morphology and frequently demonstrate squamous differentiation, discerning metastatic urothelial carcinoma to the lung from primary pulmonary squamous cell carcinoma can be challenging.
To investigate immunostains that may aid in the distinction of urothelial carcinoma metastatic to the lung.
Staining patterns of 14 markers in primary urothelial carcinoma of the bladder and primary squamous cell carcinoma of the lung were examined to establish a diagnostic panel. These antibodies were subsequently tested on tumors taken from 30 patients with a paired urinary tract and metastatic lung lesion.
The best markers to distinguish poorly differentiated metastatic urothelial carcinoma from primary pulmonary squamous cell carcinoma were CK7, CK20, GATA-3, CK14, desmoglein-3, and uroplakin III, with the utility of the latter dependent upon the quantity of tissue available for analysis. The observed percentage positive staining in nonmetastatic urothelial carcinoma versus primary pulmonary squamous cell carcinoma with these antibodies was as follows: CK7 (100% versus 33%), CK20 (54% versus 7%), GATA-3 (78% versus 23%), CK14 (32% versus 77%), desmoglein-3 (11% versus 87%), and uroplakin III (14% versus 0%). Similar expression patterns were observed among the paired cases.
When interpreted in correlation with clinical history and histomorphology, a panel of immunostains including CK7, CK20, GATA-3, CK14, desmoglein-3, and uroplakin III may be a useful adjunct in the distinction of metastatic urothelial carcinoma to the lung.
High-throughput chromatin immunoprecipitation has become the method of choice for identifying genomic regions bound by a protein. Such regions are then investigated for overrepresented sequence motifs, the assumption being that they must correspond to the binding specificity of the profiled protein. However this approach often fails: many bound regions do not contain the 'expected' motif. This is because binding DNA directly at its recognition site is not the only way the protein can cause the region to immunoprecipitate. Its binding specificity can change through association with different co-factors, it can bind DNA indirectly, through intermediaries, or even enforce its function through long-range chromosomal interactions. Conventional motif discovery methods, though largely capable of identifying overrepresented motifs from bound regions, lack the ability to characterize such diverse modes of protein-DNA binding and binding specificities. We present a novel Bayesian method that identifies distinct protein-DNA binding mechanisms without relying on any motif database. The method successfully identifies co-factors of proteins that do not bind DNA directly, such as mediator and p300. It also predicts literature-supported enhancer-promoter interactions. Even for well-studied direct-binding proteins, this method provides compelling evidence for previously uncharacterized dependencies within positions of binding sites, long-range chromosomal interactions and dimerization.
MicroRNA (miR)-155 is a critical player in both innate and adaptive immune responses. It can influence CD4(+) T cell lineage choice. To clarify the role of miR-155 in CD4(+) CD25(+) regulatory T (Treg)/T helper (Th)17 cell differentiation and function, as well as the mechanism involved, we performed gain-and loss-of-function analysis by transfection pre-miR-155 and anti-miR-155 into purified CD4(+) T cells. The results showed that miR-155 positively regulated both Treg and Th17 cell differentiation. It also induced the release of interleukin (IL)-17A by Th17 cells, but not the release of IL-10 and transforming growth factor (TGF)-β1 by Treg cells. Furthermore, we found that miR-155 reacted through regulating Janus kinase/signal transducer and activator of transcription (JAK/STAT) rather than TGF-β/mothers against decapentaplegic homolog (SMAD) signaling pathway in the process of Treg and Th17 cells differentiation. This may because suppressors of cytokine signaling (SOCS)1, the important negative regulator of JAK/STAT signaling pathway, was the direct target of miR-155 in this process, but SMAD2 and SMAD5 were not. Therefore, we demonstrated that miR-155 enhanced Treg and Th17 cells differentiation and IL-17A production by targeting SOCS1.
Preplacodal ectoderm (PPE) and neural crest (NC) are specified at the interface of neural and nonneural ectoderm and together contribute to the peripheral nervous system in all vertebrates. Bmp activates early steps for both fates during late blastula stage. Low Bmp activates expression of transcription factors Tfap2a and Tfap2c in the lateral neural plate, thereby specifying neural crest fate. Elevated Bmp establishes preplacodal competence throughout the ventral ectoderm by coinducing Tfap2a, Tfap2c, Foxi1 and Gata3. PPE specification occurs later at the end of gastrulation and requires complete attenuation of Bmp, yet expression of PPE competence factors continues well past gastrulation. Here we show that competence factors positively regulate each other's expression during gastrulation, forming a self-sustaining network that operates independently of Bmp. Misexpression of Tfap2a in embryos blocked for Bmp from late blastula stage can restore development of both PPE and NC. However, Tfap2a alone is not sufficient to activate any other competence factors nor does it rescue individual placodes. On the other hand, misexpression of any two competence factors in Bmp-blocked embryos can activate the entire transcription factor network and support the development of NC, PPE and some individual placodes. We also show that while these factors are partially redundant with respect to PPE specification, they later provide non-redundant functions needed for development of specific placodes. Thus, we have identified a gene regulatory network that coordinates development of NC, PPE and individual placodes in zebrafish.
The Delta/Notch signal transduction pathway is central to T cell differentiation from haemopoietic stem cells (HSCs). Although T cell development is well characterized using expression of cell surface markers, the detailed mechanisms driving differentiation have not been established. This issue becomes central with observations that adult HSCs exhibit poor differentiation towards the T cell lineage relative to neonatal or embryonic precursors. This study investigates the contribution of Notch signalling and stromal support cells to differentiation of adult and Cord Blood (CB) human HSCs, using the Notch signalling OP9Delta co-culture system. Co-cultured cells were assayed at weekly intervals during development for phenotype markers using flow cytometry. Cells were also assayed for mRNA expression at critical developmental stages. Expression of the central thymocyte marker CD4 was initiated independently of Notch signalling, while cells grown with Notch signalling had reduced expression of CD4 mRNA and protein. Interruption of Notch signalling in partially differentiated cells increased CD4 mRNA and protein expression, and promoted differentiation to CD4(+) CD8(+) T cells. We identified a set of genes related to T cell development that were initiated by Notch signalling, and also a set of genes subsequently altered by Notch signal interruption. These results demonstrate that while Notch signalling is essential for establishment of the T cell lineage, at later stages of differentiation, its removal late in differentiation promotes more efficient DP cell generation. Notch signalling adds to signals provided by stromal cells to allow HSCs to differentiate to T cells via initiation of transcription factors such as HES1, GATA3 and TCF7. We also identify gene expression profile differences that may account for low generation of T cells from adult HSCs.
The precise mechanism of characteristic Th2 predominance at maternal-fetal interface remains unresolved. In the present study, we investigated roles of the decidua-derived CCL2 in Th2 predominance at maternal-fetal interface. FCM shows that 55% CD56(+)CD16(-)CD3(-) decidual NK, 52% CD4(+) T cells and 75% CD14(+) monocytes express CCR2. Recombinant human CCL2 (rhCCL2) and the decidual stromal cells (DSCs)-derived supernatant can enhance proliferation and inhibit apoptosis of these decidual leukocytes (DLCs), and promote Th2 cytokines production, IL-4 and IL-10, with an increase in GATA-3 transcription. They also inhibit the secretion of Th1 cytokines, TNF-α and IFN-γ, with a decrease in T-bet transcription It is concluded that the secreted CCL2 by decidual stromal cells increases GATA-3 transcription and decreases T-bet transcription in the decidual leukocytes, which contributes to Th2 polarization at maternal-fetal interface. Furthermore, the Th2 cytokines, IL-4 and IL-10, rather than Th1 cytokines, was shown to increase CCL2 secretion of DSC.
Innate lymphoid cells (ILCs) reside at mucosal surfaces and control immunity to intestinal infections. Type 2 innate lymphoid cells (ILC2s) produce cytokines such as IL-5 and IL-13, are required for immune defense against helminth infections, and are involved in the pathogenesis of airway hyperreactivity. Here, we have investigated the role of the transcription factor GATA-3 for ILC2 differentiation and maintenance. We showed that ILC2s and their lineage-specified bone marrow precursors (ILC2Ps), as identified here, were characterized by continuous high expression of GATA-3. Analysis of mice with temporary deletion of GATA-3 in all ILCs showed that GATA-3 was required for the differentiation and maintenance of ILC2s but not for RORγt(+) ILCs. Thus, our data demonstrate that GATA-3 is essential for ILC2 fate decisions and reveal similarities between the transcriptional programs controlling ILC and T helper cell fates.
Type 2 innate lymphoid cells (ILC2s) are part of a large family of ILCs that are important effectors in innate immunity, lymphoid organogenesis, and tissue remodeling. ILC2s mediate parasite expulsion but also contribute to airway inflammation, emphasizing the functional similarity between these cells and Th2 cells. Consistent with this, we report that the transcription factor GATA3 was highly expressed by human ILC2s. CRTH2(+) ILC2s were enriched in nasal polyps of patients with chronic rhinosinusitis, a typical type 2-mediated disease. Nasal polyp epithelial cells expressed TSLP, which enhanced STAT5 activation, GATA3 expression, and type 2 cytokine production in ILC2s. Ectopic expression of GATA3 in Lin(-)CD127(+)CRTH2(-) cells resulted in induction of CRTH2 and the capacity to produce high amounts of type 2 cytokines in response to TSLP plus IL-33. Hence, we identify GATA3, potently regulated by TSLP, as an essential transcription factor for the function of human ILC2s.
There is an emerging body of research demonstrating that the co-expression of key lineage-specifying transcription factors, commonly referred to as 'master regulators', affects the functional capabilities and flexibility of CD4(+) T cell subsets. Here, we discuss how the natural co-expression of these lineage-specifying transcription factors has challenged the concept that the expression of a single 'master regulator' strictly establishes an absolute CD4(+) T cell phenotype. Instead, it is becoming clear that the interplay between the lineage-specifying (or lineage-defining) transcription factors, including T-bet, GATA3, RORγt, BCL-6 and FOXP3, contributes to the fate and flexibility of CD4(+) T cell subtypes. This in turn has led to the realization that CD4(+) T cell phenotypes are more diverse than previously recognized.
To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.
Lymphocytes isolated from peripheral blood of leukemic children were induced with γ- interferon (IFN-γ), CD3McAb and interluki-2 (IL-2), and co-cultured with dendritic cells (DCs) to generate DC-CIK cells. The morphology and immunophenotype of these cells were determined by a light microscopy and flow cytometry, respectively. IL-2 and IFN-γ levels released by DC-CIK cells were quantified by ELISA. Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by MTT assay. FCM was used to detect CD4(+)CD25(+)Treg cells, while RT-PCR and Western blot were used to determine mRNA and protein expressions of Foxp3 and GATA3 in DC-CIK cells treated with T-bet monoclonal antibody.
Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. The main effector cells in this population were CD3(+)CD8(+) cells and CD3(+)CD56(+) cells. We demonstrated a time dependent increase in IL-2 and IFN-γ levels after induction. DC-CIK cells were cytotoxic to B95 cells, Jhhan cells and M07e cells, with the highest cytotoxicity towards B95 cells. Treatment with mouse anti-human T-bet monoclonal antibody resulted in an increase in the proportion of CD4(+)CD25(+)Treg cells and elevation of Foxp3 and GATA3 mRNA and protein levels.
DC-CIK cells induced with cytokines were strongly cytotoxic towards a number of cancer cell lines. Foxp3 and GATA3 were implicated in the T-bet mediated anti-neoplastic effects of DC-CIK cells via activation of the Th1 pathway and suppression of the Th2 and Treg pathways.
The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.
HDR syndrome (hypoparathyroidism, sensorineural deafness, renal abnormalities) (OMIM #146265) is a rare autosomal dominant disorder caused by mutations in the GATA-3 gene (OMIM 13120), a transcription factor coding for a protein involved in vertebrate embryonic development. More than a hundred cases with variable renal features have been described so far. Here, we report on a patient suffering from HDR syndrome with glomerular nephropathy. Hypoparathyroidism appeared early in childhood but the subsequent features of HDR occurred later in the form of bilateral sensorineural deafness and renal insufficiency associated with nephrocalcinosis. HDR was not initially diagnosed due to the appearance of a transitory cardiac involvement and atypical renal symptoms (diffuse proliferative glomerulonephritis characterized by a self-limiting nephrotic syndrome).
HDR syndrome with glomerular nephropathy has not yet been reported to our knowledge. Further studies of GATA-3 are needed to explore the involvement of this transcription factor in the development of HDR in humans, particularly in the kidneys.
Immunosuppressive CD11b(+)Gr1(+) myeloid-derived suppressor cells and TGF-β have been shown to negatively regulate host immunity against allografts. Our results demonstrated that Smad3-deficient mice or mice reconstituted with Smad3-deficient hematopoietic cells rejected allogeneic skin or heart grafts in a significantly slower manner compared with littermates or wild-type (WT) control mice. Transplanted Smad3(-/-) recipients produced markedly less anti-donor IgG Abs, especially IgG1 and IgG2b subclasses. T cells in alloskin-grafted Smad3-deficient mice were more likely to participate in a Th2-type immune response, as evidenced by more Th2-specific transcription factor, GATA3 expression, and increased IL-4 and IL-10 production, as well as less Th1-specific transcription factor, T-bet expression, and decreased IL-2 and IFN-γ production. More CD11b(+)Gr1(+) neutrophil infiltration and less monocyte/macrophage and T cell infiltration in allografts were observed in Smad3(-/-) recipients compared with WT recipients. Increased CXCL1 and CXCL2 as well as decreased CCL3, MCP-1, and RANTES chemokines in allografts of Smad3(-/-) recipients were consistently detected by real-time PCR. Further studies indicated that the increased CD11b(+)Gr1(+) myeloid cells in Smad3-deficient mice were immunosuppressive and responsible for the delayed allograft rejection mainly via an NO-dependent pathway. Thus, this study identifies Smad3 as an intrinsic negative regulator that critically inhibits the differentiation and function of immunosuppressive CD11b(+)Gr1(+) myeloid-derived suppressor cells.
T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.
The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the aorta-gonads-mesonephros (AGM) region of the embryo. We have recently identified the transcription factor Gata3 as being upregulated in this tissue specifically at the time of HSC emergence. We now demonstrate that the production of functional and phenotypic HSCs in the AGM is impaired in the absence of Gata3. Furthermore, we show that this effect on HSC generation is secondary to the role of Gata3 in the production of catecholamines, the mediators of the sympathetic nervous system (SNS), thus making these molecules key components of the AGM HSC niche. These findings demonstrate that the recently described functional interplay between the hematopoietic system and the SNS extends to the earliest stages of their codevelopment and highlight the fact that HSC development needs to be viewed in the context of the development of other organs.
The purpose of the present study was to investigate the effect of nucleotide-binding oligomerization domain 1 (NOD1), an innate immune sensor, on allergic inflammation and induction of regulatory T cells in a mouse model of allergic rhinitis. We also aimed to explore whether there were differences in the effect of NOD1 ligand according to the timing of administration. Study Design An in vivo study using an animal model.
Catholic Research Institutes of Medical Science.
Forty BALB/c mice were divided into 4 groups: control, OVA, pre-NOD1, and post-NOD1. Ovalbumin (OVA) was used for sensitization and challenge. The pre-NOD1 group received NOD1 ligand intranasally before sensitization, whereas the post-NOD1 group received it after sensitization. The effects of allergic inflammation and regulatory T cells were compared among the groups.
In the post-NOD1 group, serum OVA-specific IgE, eosinophil counts, interleukin (IL)-13 levels, and GATA-3 mRNA expression were significantly increased and Foxp3(+) mRNA expression and CD4(+) Foxp3(+) T cells were decreased compared with the OVA group. In the pre-NOD1 group, Foxp3 mRNA expression and CD4(+) Foxp3(+) T cells were significantly decreased compared with the OVA group. Although not significant, the pre-NOD1 group showed increases in serum OVA-specific IgE, eosinophil counts, IL-13 levels, and GATA-3 mRNA expression compared with the OVA group.
The innate immune response through NOD1 enhances allergen-specific Th2 response and suppresses induction of regulatory T cells in a mouse model of allergic rhinitis, and the effects are different depending on the timing of exposure to NOD1 ligand.
Previously we have screened out Insulin-like Growth Factor Binding Protein 7 (IGFBP7) as a differentially expressed gene in post-implantation uterus versus pre-implantation uterus by suppressive subtractive hybridation. However its function in uterus was not clearly identified. In this research, the expression and function of IGFBP7 during post-implantation were studied. We found that IGFBP7 was mainly located in the glandular epithelium and the stroma, and was upregulated after embryo implantation. The vector pCR3.1-IGFBP7-t expressing partial IGFBP7 was constructed. Inhibition of IGFBP7 by specific DNA immunization induced significant reduction of implanted embryos and pregnancy rate. The number of implanted embryos (5.68 ± 0.46) was significantly reduced after immunization with pCR3.1-IGFBP7-t, as compared with that of the mice immunized with the control vector (12.29 ± 0.36) or saline (14.58 ± 0.40) (p<0.01). After specific inhibition of IGFBP7, the T helper type 1 (Th1) cytokine IFNγ, was significantly elevated (p<0.05) and the Th2 cytokines IL-4 and IL-10, were reduced in uteri (p<0.05). The increase of Tbet and the decrease of Gata3 were found in mice peripheral lymphocytes by flow cytometry. The expression of decidualization marker IGFBP1 and angiogenesis regulator VEGF were declined in uteri (p<0.05). The expression of apoptosis-associated proteins, caspase3 and Bcl-2, were also declined (p<0.05). These results showed that inhibition of IGFBP7 induced pregnancy failure by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization.
We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.
Urothelial neoplasms with squamous morphology raise the differential diagnosis between pure primary squamous cell carcinoma, urothelial carcinoma with squamous differentiation and secondary involvement by squamous cell carcinoma, for example, from uterine cervix. Accurate identification between these entities is critical due to differing prognosis and therapeutic strategies. We evaluated the utility of an immunohistochemical panel of 3 urothelial-associated antibodies (uroplakin III, S100P, and GATA3) and two squamous-associated antibodies (CK14 and desmoglein-3) in 50 primary urothelial neoplasms: 15 pure urothelial carcinomas, 12 pure squamous cell carcinomas and 23 urothelial carcinomas with squamous differentiation. Squamous differentiation was defined by intercellular bridges or evidence of keratinization. Pure squamous cell carcinomas were positive for CK14 (100%) and desmoglein-3 (75%), negative for GATA3 and uroplakin III; one case was S100P positive (9%). Pure urothelial carcinomas had an opposite pattern and were positive for S100P (93%), GATA3 (93%), and uroplakin III (67%) and were negative for desmoglein-3; CK 14 was positive in 27% of cases; 74% of urothelial carcinomas with squamous differentiation had expression of urothelial and squamous associated markers (S100P, 83%; GATA3, 35%; uroplakin III, 13%; CK14, 87%; and desmoglein-3, 70%), although reactivity for individual markers within some tumors did not always correspond with morphologic differentiation. Of the remaining 26%, 4 showed an overall "squamous" immunoprofile, whereas 2 cases showed a "urothelial" immunoprofile. Our study showed that a panel of five antibodies identifies squamous and urothelial differentiation in most instances suggesting potential diagnostic utility.
This study is to elucidate the immunoregulation mechanisms of artesunate (AST) on allergic contact dermatitis (ACD). Pharmacodynamics analyses, HE staining, semi-quantitative RT-PCR and Western blotting were used to explore the effects of AST on the related cytokines, transcription factor and signaling molecule of ACD respectively. The results indicated that topical administration of AST not only reduced the increase of ear swelling, spleen index and inflammatory cells infiltration in ACD mice, but also inhibited remarkably the expression of IFN-gamma, T-bet and NF-kappaB p65. It's suggested that AST could exhibit suppressive effects on inflammatory response and immune function of ACD, which indicates the possibility of developing AST as a novel immunoregulatory agent in the treatment of ACD and other immune-related diseases.
Diverse mechanisms regulate development of GABAergic neurons in different regions of the central nervous system. We have addressed the roles of a proneural gene, Ascl1, and a postmitotic selector gene, Gata2, in the differentiation of GABAergic neuron subpopulations in three diencephalic prosomeres: prethalamus (P3), thalamus (P2) and pretectum (P1). Although the different proliferative progenitor populations of GABAergic neurons commonly express Ascl1, they have distinct requirements for it in promotion of cell-cycle exit and GABAergic neuron identity. Subsequently, Gata2 is activated as postmitotic GABAergic precursors are born. In P1, Gata2 regulates the neurotransmitter identity by promoting GABAergic and inhibiting glutamatergic neuron differentiation. Interestingly, Gata2 defines instead the subtype of GABAergic neurons in the rostral thalamus (pTh-R), which is a subpopulation of P2. Without Gata2, the GABAergic precursors born in the pTh-R fail to activate subtype-specific markers, but start to express genes typical of GABAergic precursors in the neighbouring P3 domain. Thus, our results demonstrate diverse mechanisms regulating differentiation of GABAergic neuron subpopulations and suggest a role for Gata2 as a selector gene of both GABAergic neuron neurotransmitter and prosomere subtype identities in the developing diencephalon. Our results demonstrate for the first time that neuronal identities between distinct prosomeres can still be transformed in postmitotic neuronal precursors.
The mechanisms regulating breast cancer differentiation state are poorly understood. Of particular interest are molecular regulators controlling the highly aggressive and poorly differentiated traits of basal-like breast carcinomas. Here we show that the Polycomb factor EZH2 maintains the differentiation state of basal-like breast cancer cells, and promotes the expression of progenitor associated and basal-lineage genes. Specifically, EZH2 regulates the composition of basal-like breast cancer cell populations by promoting a ‘bi-lineage’ differentiation state, in which cells co-express basal- and luminal-lineage markers. We show that human basal-like breast cancers contain a subpopulation of bi-lineage cells, and that EZH2-deficient cells give rise to tumors with a decreased proportion of such cells. Bi-lineage cells express genes that are active in normal luminal progenitors, and possess increased colony-formation capacity, consistent with a primitive differentiation state. We found that GATA3, a driver of luminal differentiation, performs a function opposite to EZH2, acting to suppress bi-lineage identity and luminal-progenitor gene expression. GATA3 levels increase upon EZH2 silencing, mediating a decrease in bi-lineage cell numbers. Our findings reveal a novel role for EZH2 in controlling basal-like breast cancer differentiation state and intra-tumoral cell composition.
Transcription factors (TFs) can regulate different sets of genes to determine specific cell types by means of combinatorial codes. We previously identified closely-spaced TF binding motifs located 8.2-8.5 kb 5' to the ATG of the murine Pou4f3 gene, a gene required for late hair cell (HC) differentiation and survival. These motifs, 100% conserved among four mammalian species, include a cluster of E-boxes preferred by TCF3/ATOH1 heterodimers as well as motifs for GATA factors and SP1. We hypothesized that these factors might interact to regulate the Pou4f3 gene and possibly induce a HC phenotype in non-sensory cells of the cochlea. Cochlear sensory epithelium explants were prepared from postnatal day 1.5 transgenic mice in which expression of GFP is driven by 8.5 kb of Pou4f3 5' genomic DNA (Pou4f3/GFP). Electroporation was used to transfect cells of the greater epithelial ridge with multiple plasmids encoding human ATOH1 (hATOH1), hTCF3 (also known as E2A or TEF2), hGATA3, and hSP1. hATOH1 or hTCF3 alone induced Pou4f3/GFP cells but hGATA3 and hSP1 did not. hATOH1 but not hTCF3 induced conversion of greater epithelial ridge cells into Pou4f3/GFP and myosin VIIa double-positive cells. Transfection of hATOH1 in combination with hTCF3 or hGATA3 induced 2-3X more Pou4f3/GFP cells, and similarly enhanced Pou4f3/GFP and myosin VIIa double-positive cells, when compared to hATOH1 alone. Triple or quadruple TF combinations were generally not more effective than double TF combinations except in the middle turn, where co-transfection of hATOH1, hE2A, and hGATA3 was more effective than hATOH1 plus either hTCF3 or hGATA3. The results demonstrate that TFs can cooperate in regulation of the Pou4f3 gene and in the induction of at least one other element of a HC phenotype. Our data further indicate that combinations of TFs can be more effective than individual TFs in the inner ear.
Distinguishing invasive high-grade urothelial carcinoma (UC) from other carcinomas occurring in the genitourinary tract may be difficult. The differential diagnosis includes high-grade prostatic adenocarcinoma, spread from an anal squamous cell carcinoma (SCC), or spread from a uterine cervical SCC. In terms of metastatic UC, the most common problem is differentiating spread of UC to the lung from a primary pulmonary SCC. Immunohistochemical analysis (IHC) for GATA binding protein 3 (GATA3), thrombomodulin (THROMBO), and uroplakin III was performed on a tissue microarray (TMA) containing 35 cases of invasive high-grade UC. GATA3 IHC was also performed on TMAs containing 38 high-grade (Gleason score ≥8) prostatic adenocarcinomas, representative tissue sections from 15 invasive anal SCCs, representative tissue sections from 19 invasive cervical SCCs, and TMAs with 12 invasive cervical carcinomas of the cervix [SCC (n=10), SCC with neuroendocrine features (n=1), and adenosquamous carcinoma (n=1)]. In addition, GATA3 IHC was performed on representative tissue sections from 15 pulmonary UC metastases and a TMA with 25 SCCs of the lung and 5 pulmonary non-small cell carcinomas with squamous features. GATA3, THROMBO, and uroplakin III were positive in 28 (80%), 22 (63%), and 21 (60%) cases of high-grade UC, respectively. All cases of GATA3-positive staining were nonfocal; 25 (89%) cases demonstrated moderate to strong staining, and 3 (11%) demonstrated weak staining. Of the 7 cases that failed to express GATA3, 5 were positive for THROMBO and/or uroplakin III, whereas 2 were negative for all 3 markers. None of the 38 high-grade prostatic adenocarcinomas was positive for GATA3. Weak GATA3 staining was present in occasional basal cells of benign prostate glands, in a few benign atrophic glands, and in urothelial metaplasia. Of the 15 cases of anal SCCs, 2 (7%) cases showed focal weak staining, and 1 (3%) showed focal moderate staining. Weak staining was also rarely observed in the benign anal squamous epithelium. Of the 31 uterine cervical carcinomas, 6 (19%) showed weak GATA3 staining (3 nonfocal and 3 focal), and 2 (6%) demonstrated focal moderate staining. Twelve (80%) of the metastatic UCs to the lung were positive for GATA3, with 11 cases showing diffuse moderate or strong staining and 1 case showing focal moderate staining. None of the pulmonary SCCs or non-small cell carcinomas with squamous features was GATA3 positive. GATA3 IHC is a sensitive marker for UC, and positive staining in UC is typically nonfocal and moderate or strong in intensity. GATA3 is also highly specific in excluding high-grade prostate adenocarcinoma. Although some cervical and anal SCCs can be GATA3 positive, unlike in UC, staining is more commonly focal and weak. GATA3 is also a useful maker when diagnosing metastatic UC to the lung.
Resveratrol is a bioactive polyphenol enriched in red wine that exhibits many beneficial health effects via multiple mechanisms. However, it is unclear whether resveratrol is beneficial for the prevention of food allergy. This study investigated whether resveratrol inhibited the development of food allergy by using a mouse model of the disease.
Mice fed standard diet or standard diet plus resveratrol were sensitized by intragastric administration of ovalbumin (OVA) and mucosal adjuvant cholera toxin (CT). Several manifestations of food allergy were then compared between the mice. The effects of resveratrol on T cells or dendritic cells were also examined by using splenocytes from OVA-specific T cell-receptor (TCR) transgenic DO11.10 mice or mouse bone marrow-derived dendritic cells (BMDCs) in vitro. We found that mice fed resveratrol showed reduced OVA-specific serum IgE production, anaphylactic reaction, and OVA-induced IL-13 and IFN-ã production from the mesenteric lymph nodes (MLNs) and spleens in comparison to the control mice, following oral sensitization with OVA plus CT. In addition, resveratrol inhibited OVA plus CT-induced IL-4, IL-13, and IFN-ã production in splenocytes from DO11.10 mice associated with inhibition of GATA-3 and T-bet expression. Furthermore, resveratrol suppressed the OVA plus CT-induced CD25 expression and IL-2 production in DO11.10 mice-splenocytes in association with decreases in CD80 and CD86 expression levels. Finally, resveratrol suppressed CT-induced cAMP elevation in association with decreases in CD80 and CD86 expression levels in BMDCs.
Ingestion of resveratrol prevented the development of a food allergy model in mice. Given the in vitro findings, resveratrol might do so by inhibiting DC maturation and subsequent early T cell activation and differentiation via downregulation of CT-induced cAMP activation in mice. These results suggest that resveratrol may have potential for prophylaxis against food allergy.
Immune tolerance is instituted early in life, during which time regulatory T (T(reg)) cells have an important role. Recurrent infections with respiratory syncytial virus (RSV) in early life increase the risk for asthma in adult life. Repeated infection of infant mice tolerized to ovalbumin (OVA) through their mother's milk with RSV induced allergic airway disease in response to OVA sensitization and challenge, including airway inflammation, hyper-reactivity and higher OVA-specific IgE, as compared to uninfected tolerized control mice. Virus infection induced GATA-3 expression and T helper type 2 (T(H)2) cytokine production in forkhead box P3 (FOXP3)(+) T(reg) cells and compromised the suppressive function of pulmonary T(reg) cells in a manner that was dependent on interleukin-4 receptor α (IL-4Rα) expression in the host. Thus, by promoting a T(H)2-type inflammatory response in the lung, RSV induced a T(H)2-like effector phenotype in T(reg) cells and attenuated tolerance to an unrelated antigen (allergen). Our findings highlight a mechanism by which viral infection targets a host-protective mechanism in early life and increases susceptibility to allergic disease.
Conventional dendritic cells (cDCs) have been reported to participate in the pathophysiology of acute lung injury (ALI). Fms-like tyrosine kinase 3 (FLT3) signaling represents a highly specific pathway for the manipulation of cDCs in vivo. The purpose of this study was to clarify the effect of FLT3 signaling on the accumulation and maturation of pulmonary cDCs, and whether inhibition of FLT3 signaling may attenuate acute lung inflammation and lung injury. C57BL/6 mice were pretreated with FLT3-ligand (FLT3L) and lestaurtinib separately for five consecutive days. A murine model of ALI was subsequently generated by intra-tracheal instillation of lipopolysaccharide (LPS) and lung specimens were harvested 24 h later. Flow cytometry was conducted to measure the accumulation and maturation of pulmonary cDCs. IL-6, IFN-γ, IL-4, MPO activity and transcription factor T-bet/GATA-3 mRNA ratio were quantified to evaluate lung inflammation. Lung injury was estimated by lung wet weight/body weight ratio (LWW/BW) and histopathological analysis. LPS challenge resulted in rapid accumulation and maturation of pulmonary cDCs. FLT3L pretreatment further stimulated the accumulation and maturation of pulmonary cDCs, leading to a markedly increased LWW/BW and aggravated lung histopathology. Meanwhile, lung MPO activity, T-bet/GATA-3 mRNA ratio and concentrations of IL-6 and IFN-γ were elevated by FLT3L administration. In contrast, lestaurtinib pretreatment inhibited the accumulation and maturation of pulmonary cDCs, leading to a significantly decreased LWW/BW and improved lung histopathology. Lestaurtinib administration also suppressed lung MPO activity, T-bet/GATA-3 mRNA ratio and production of IL-6 and IFN-γ. Our findings show that FLT3 signaling ameliorates ALI by regulating the accumulation and maturation of pulmonary cDCs, suggesting an innovative pharmacotherapy for ALI.
The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.
Highly regulated expression of the negative costimulatory molecule cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells modulates T-cell activation and proliferation. CTLA-4 is preferentially expressed in Th2 T cells, whose differentiation depends on the transcriptional regulator GATA3. Sézary syndrome (SS) is a T-cell malignancy characterized by Th2 cytokine skewing, impaired T-cell responses, and overexpression of GATA3 and CTLA-4. GATA3 is regulated by phosphorylation and ubiquitination. In SS cells, we detected increased polyubiquitinated proteins and activated GATA3. We hypothesized that proteasome dysfunction in SS T cells may lead to GATA3 and CTLA-4 overexpression. To test this hypothesis, we blocked proteasome function with bortezomib in normal T cells, and observed sustained GATA3 and CTLA-4 upregulation. The increased CTLA-4 was functionally inhibitory in a mixed lymphocyte reaction (MLR). GATA3 directly transactivated the CTLA-4 promoter, and knockdown of GATA3 messenger RNA and protein inhibited CTLA-4 induction mediated by bortezomib. Finally, knockdown of GATA3 in patient's malignant T cells suppressed CTLA-4 expression. Here we demonstrate a new T-cell regulatory pathway that directly links decreased proteasome degradation of GATA3, CTLA-4 upregulation, and inhibition of T-cell responses. We also demonstrate the requirement of the GATA3/CTLA-4 regulatory pathway in fresh neoplastic CD4+ T cells. Targeting of this pathway may be beneficial in SS and other CTLA-4-overexpressing T-cell neoplasms.
The TCF7L2 transcription factor is linked to a variety of human diseases, including type 2 diabetes and cancer. One mechanism by which TCF7L2 could influence expression of genes involved in diverse diseases is by binding to distinct regulatory regions in different tissues. To test this hypothesis, we performed ChIP-seq for TCF7L2 in six human cell lines.
We identified 116,000 non-redundant TCF7L2 binding sites, with only 1,864 sites common to the six cell lines. Using ChIP-seq, we showed that many genomic regions that are marked by both H3K4me1 and H3K27Ac are also bound by TCF7L2, suggesting that TCF7L2 plays a critical role in enhancer activity. Bioinformatic analysis of the cell type-specific TCF7L2 binding sites revealed enrichment for multiple transcription factors, including HNF4alpha and FOXA2 motifs in HepG2 cells and the GATA3 motif in MCF7 cells. ChIP-seq analysis revealed that TCF7L2 co-localizes with HNF4alpha and FOXA2 in HepG2 cells and with GATA3 in MCF7 cells. Interestingly, in MCF7 cells the TCF7L2 motif is enriched in most TCF7L2 sites but is not enriched in the sites bound by both GATA3 and TCF7L2. This analysis suggested that GATA3 might tether TCF7L2 to the genome at these sites. To test this hypothesis, we depleted GATA3 in MCF7 cells and showed that TCF7L2 binding was lost at a subset of sites. RNA-seq analysis suggested that TCF7L2 represses transcription when tethered to the genome via GATA3.
Our studies demonstrate a novel relationship between GATA3 and TCF7L2, and reveal important insights into TCF7L2-mediated gene regulation.
Allergic skin inflammation such as atopic dermatitis (AD) is characterized by edema and infiltration with various inflammatory cells such as mast cells, basophils, eosinophils and T cells. Thymic stromal lymphopoietin (TSLP) is produced mainly by epidermal keratinocytes, as well as dermal fibroblasts and mast cells in the skin lesions of AD. Omega-3 polyunsaturated fatty acids in fish oil can reduce inflammation in allergic patients. Fermentation has a tremendous capacity to transform chemical structures. The antiinflammatory effects of fish oil have been described in many diseases, but the beneficial effects by which fermented olive flounder oil (FOF) modulates the allergic response is poorly understood. In this study, we produced FOF and tested its ability to suppress the various allergic inflammatory responses. The ability of FOF to modulate the immune system was investigated using a mouse model of AD. The FOF-treated group showed significantly decreased immunoglobulin E (IgE) and histamine in serum. Also, the increased TSLP expression was significantly inhibited in the FOF group; the FOF-treated group was not appreciably different from the hydrocort cream treatment group. In addition, FOF treatment resulted in a smaller spleen size with reduced the thickness and length compared to the induction group. Splenocytes from mice treated with FOF produced significantly less IFN-γ, IL-4, T-box transcription factor (T-bet) and GATA binding protein 3 (GATA3) expression compared with the induction group. These results suggest that FOF may be effective in treating the allergic symptoms of AD. 5.

Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries). Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i) trophoblast proliferation and differentiation or (ii) embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i) stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii) mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this "mining", we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n = 64) that we used as biological "gold standard". When successful, the "mining" approach would identify them all, as well as novel ones.
To gain knowledge on molecular-genetic regulations in a non model organism, we offer an approach based on literature-mining and score arrangement of data from model organisms. This approach was applied to identify novel transcription factors during bovine blastocyst elongation, a process that is not observed in rodents and primates. As a result, searching through human and mouse corpuses, we identified numerous bovine homologs, among which 11 to 14% of transcription factors including the gold standard TF as well as novel TF potentially important to gene regulation in ruminant embryo development. The scripts of the workflow are written in Perl and available on demand. They require data input coming from all various databases for any kind of biological issue once the data has been prepared according to keywords for the studied topic and species; we can provide data sample to illustrate the use and functionality of the workflow.
To do so, we created a workflow that allowed the pipeline processing of literature data and biological data, extracted from Web of Science (WoS) or PubMed but also from Gene Expression Omnibus (GEO), Gene Ontology (GO), Uniprot, HomoloGene, TcoF-DB and TFe (TF encyclopedia). First, the human and mouse homologs of the bovine proteins were selected, filtered by text corpora and arranged by score functions. The score functions were based on the gene name frequencies in corpora. Then, transcription factors were identified using TcoF-DB and double-checked using TFe to characterise TF groups and families. Thus, among a search space of 18,670 bovine homologs, 489 were identified as transcription factors. Among them, 243 were absent from the high-throughput data available at the time of the study. They thus stand so far for putative TF acting during bovine embryo elongation, but might be retrieved from a recent RNA sequencing dataset (Mamo et al. , 2012). Beyond the 246 TF that appeared expressed in bovine elongating tissues, we restricted our interpretation to those occurring within a list of 50 top-ranked genes. Among the transcription factors identified therein, half belonged to the gold standard (ASCL2, c-FOS, ETS2, GATA3, HAND1) and half did not (ESR1, HES1, ID2, NANOG, PHB2, TP53, STAT3).
A workflow providing search for transcription factors acting in bovine elongation was developed. The model assumed that proteins sharing the same protein domains in closely related species had the same protein functionalities, even if they were differently regulated among species or involved in somewhat different pathways. Under this assumption, we merged the information on different mammalian species from different databases (literature and biology) and proposed 489 TF as potential participants of embryo proliferation and differentiation, with (i) a recall of 95% with regard to a biological gold standard defined in 2011 and (ii) an extension of more than 3 times the gold standard of TF detected so far in elongating tissues. The working capacity of the workflow was supported by the manual expertise of the biologists on the results. The workflow can serve as a new kind of bioinformatics tool to work on fused data sources and can thus be useful in studies of a wide range of biological processes.
Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contrast to this innate immune deficit, CGD patients and animal models display a predisposition toward autoimmune disease and enhanced response to Helicobacter pylori and influenza virus infection. These data imply an altered, perhaps augmented, adaptive immune response in CGD. As previous data demonstrated functional NOX2 expression in T cells, our goal here was to determine if NOX2-deficient T cells are inherently altered in their responses. Activation of purified naive CD4(+) T cells from NOX2-deficient mice led to augmented IFN-γ and diminished IL-4 production and an increased ratio of expression of the T(H)1-specific transcription factor T-bet versus the T(H)2-specfic transcription factor GATA-3, consistent with a T(H)1 skewing of naïve T cells. Selective inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Exposure to antioxidants inhibited, while pro-oxidants augmented T(H)2 cytokine secretion and STAT5 phosphorylation, supporting the redox dependence of these signaling changes. These data suggest that TCR-induced ROS generation from NOX2 activation can regulate the adaptive immune response in a T-cell-inherent fashion, and propose a possible role for redox signaling in T helper differentiation.
The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.
The identifying of binding sites for transcription factors is a key component of gene regulatory network analysis. This is often done using position-weight matrices (PWMs). Because of the importance of in silico mapping of tentative binding sites, we previously developed an approach for PWM optimization that substantially improves the accuracy of such mapping.
The present work implements the optimization algorithm applied to the existing PWM for GATA-3 transcription factor and builds a new di-nucleotide PWM. The existing available PWM is based on experimental data adopted from Jaspar. The optimized PWM substantially improves the sensitivity and specificity of the TF mapping compared to the conventional applications. The refined PWM also facilitates in silico identification of novel binding sites that are supported by experimental data. We also describe uncommon positioning of binding motifs for several T-cell lineage specific factors in human promoters.
Our proposed di-nucleotide PWM approach outperforms the conventional mono-nucleotide PWM approach with respect to GATA-3. Therefore our new di-nucleotide PWM provides new insight into plausible transcriptional regulatory interactions in human promoters.
T-bet and GATA-3 are transcriptional factors involved in Th1 and Th2 cell differentiation, although their concomitant roles at protein levels in target organs during human allergic disease have not been assessed.
We investigated the expression of T-bet and GATA-3 in nasal and cutaneous models of Th2 (grass-pollen allergen) and a cutaneous model of Th1 (PPD) responses in man.
Nasal biopsies were obtained at 8 h and skin biopsies at 8 and 48 h after allergen and PPD challenges, respectively, from 10 allergic rhinitics and 6 non-atopic controls. T cells were assessed using immunofluorescence microscopy.
There were increases in CD3(+)STAT6(+)cells (P = 0.01 for nose and skin) and CD3(+)GATA3(+)cells (P = 0.03 for skin) in response to allergen compared with diluent in allergics. When compared with non-atopics after allergen challenge the difference between the two groups was also significant for CD3(+)STAT6(+) (P = 0.001 and 0.03) and for CD3(+)GATA3(+)cells (P = 0.04 and 0.001) for nose and skin respectively. Following PPD challenge CD3(+)STAT4(+)cells and CD3(+)T-bet(+)cells increased in both groups compared with diluent (P = 0.02 and 0.03 for both TFs), whereas only CD3(+)T-bet(+) cells were significantly greater in non-atopics compared with allergics (P = 0.04). The ratio of GATA3(+):T-bet(+) T cells in allergen-induced responses was significantly greater in the allergics (P = 0.008 and 0.01 nose and skin respectively), whereas the ratio of T-bet:GATA3(+)T cells was significantly higher in the non-atopics during PPD-induced responses (P = 0.003).
Dysregulation of Th1 transcription may contribute to heightened expression of STAT6 and GATA3 leading to exaggerated Th2-driven manifestations of allergic disease.
A characteristic feature of anaplastic large cell lymphoma is the significant repression of the T-cell expression program despite its T-cell origin. The reasons for this down-regulation of T-cell phenotype are still unknown. To elucidate whether epigenetic mechanisms are responsible for the loss of the T-cell phenotype, we treated anaplastic large cell lymphoma and T-cell lymphoma/leukemia cell lines (n=4, each) with epigenetic modifiers to evoke DNA demethylation and histone acetylation. Global gene expression data from treated and untreated cell lines were generated and selected, and differentially expressed genes were evaluated by real-time reverse transcriptase polymerase chain reaction and western blot analysis. Additionally, histone H3 lysine 27 trimethylation was analyzed by chromatin immunoprecipitation. Combined DNA demethylation and histone acetylation of anaplastic large cell lymphoma cells was not able to reconstitute their T-cell phenotype. Instead, the same treatment induced in T cells: (i) an up-regulation of anaplastic large cell lymphoma-characteristic genes (e.g. ID2, LGALS1, c-JUN), and (ii) an almost complete extinction of their T-cell phenotype including CD3, LCK and ZAP70. In addition, suppressive trimethylation of histone H3 lysine 27 of important T-cell transcription factor genes (GATA3, LEF1, TCF1) was present in anaplastic large cell lymphoma cells, which is in line with their absence in primary tumor specimens as demonstrated by immunohistochemistry. Our data suggest that epigenetically activated suppressors (e.g. ID2) contribute to the down-regulation of the T-cell expression program in anaplastic large cell lymphoma, which is maintained by trimethylation of histone H3 lysine 27.
The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.
The present study evaluated the expression profile of 19 genes previously reported in microarray studies and associated with resistance or sensitivity to vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), prednisolone (F8A, CDK2AP1, BLVRB, CD69), daunorubicin (MAP3K12, SHOC2, PCDH9, EGR1, KCNN4) and l-asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). We studied 140 bone marrow samples at diagnosis from children with acute lymphoblastic leukemia (ALL) treated according to the Brazilian Childhood Leukemia Treatment Group (GBTLI) ALL-99 protocol. The expression profiles of the genes listed above were analyzed by real-time quantitative polymerase chain reaction (PCR) and then related to the clinical and biological prognostic factors. The results showed significant associations (p ≤ 0.05) between the expression levels of genes GPR56, BLVRB, IGFBP7 and white blood cell (WBC) count at diagnosis; GATA3, MAN1A1, CD44, MAP3K12, CLEC11A, SHOC2 and CD10 B-lineage ALL; TCFL5 and bone marrow status at day 14; MAP3K12 and TRIM24 and bone marrow status at day 28; and CD69, TCFL5 and TRIM24 genes and ETV6/RUNX1 positive ALL. The up-regulation of SHOC2 was also associated with better 5-year event-free survival (EFS) in univariate and multivariate analysis (p = 0.02 and p = 0.03, respectively). These findings highlight genes that could be associated with clinical and biological prognostic factors in childhood ALL, suggesting that these genes may characterize and play a role in the treatment outcome of some ALL subsets.
To investigate the expression pattern of histone deacetylase 9 (HDAC9) in peripheral blood of asthmatics and its effect on immune cells (Th2, Th17, Tregs) involved in the pathogenesis of asthma.
Forty-seven asthmatics from Ruijin Hospital were recruited and assigned to intermittent, mild and moderate-severe groups. Lung function test and Asthma Control Questionnaire were performed to evaluate asthma control and severity. Twenty healthy donors were enrolled as controls. GATA3, IL-4, and HDAC9 mRNA expression levels were measured by SYRB Green Real-time PCR. The cytokine IL-17-mainly produced by Th17 cells and TGF-β-mainly produced by Treg cells, were measured by ELISA.
The GATA3 and IL-4 mRNA expression levels (28.12 ± 7.57 and 743.6 ± 312.8) were up-regulated in asthmatics as compared to the healthy controls [0.56 ± 0.22, 0.7 ± 0.8 (U = 16.00, 37.00, P < 0.01)]. The HDAC9 mRNA expression levels of intermittent, mild and moderate-severe groups were 3.20 ± 0.50, 89.6 ± 18.0, 323.0 ± 65.3, respectively, which were associated with the severity of disease (H = 11.32, P < 0.05). The level of IL-17 in asthmatic group was (83 ± 55) ng/L, which was up-regulated as compared to the healthy control [(34 ± 22) ng/L (U = 153.50, P < 0.01)]. The level of TGF-β was decreased in the asthmatic groups as compared to the healthy control, but the difference did not reach significance. HDAC9 mRNA expression level was positively correlated with GATA3 mRNA expression level (r = 0.482, P < 0.05), and also with IL-4 mRNA expression (r = 0.432, P < 0.05) and IL-17 (r = 0.538, P < 0.05), but negatively correlated with TGF-β (r = -0.417, P < 0.05). In patients with moderate-severe asthma, HDAC9 mRNA expression level was negatively correlated with FEV(1)% (r = -0.657, P < 0.05).
HDAC9 mRNA expression was up-regulated in peripheral blood of asthmatics, which was not only associate with the Th2 master transcriptional factors GATA3, cytokine IL-4 mRNA, Th17 and Treg cell-related cytokines, but also with FEV(1)% in moderate-severe asthma.
Blockage of nerve growth factor (NGF) by anti-NGF antibodies can inhibit allergic airway hyper-responsiveness in mice. This study was aimed at determining the mechanisms underlying the action of anti-NGF in vivo.
BALB/c mice were sensitized with ovalbumin (OVA) and treated with anti-NGF. At 1 day after the last challenge, their airway responsiveness and inflammation were examined and the levels of cytokine and transcription factor mRNA transcripts in the lungs and cytokines in the bronchoalveolar lavage fluid were determined. The frequency of different functional T cells and the levels of serum OVA-specific antibodies were measured.
OVA challenge induced severe airway resistance, inflammation, higher levels of IL-4, TNFα, IL-17A, TGFβ, GATA-3 and RORγT expression and increased Th2 and Th17 cells and IgE responses, but decreased IFNγ and IL-10 responses, T-bet and Foxp3 expression and Th1 and Tregs. Treatment with anti-NGF significantly reduced allergic airway resistance and inflammation, up-regulated IFNγ, IL-10, TGFβ, T-bet, and Foxp3 expression, increased Th1 and Tregs, but down-regulated IL-4, TNFα, IL-17A, RORγT and GATA-3 expression and reduced Th2 and Th17 cells, accompanied by increased serum IgG2a.
Anti-NGF inhibits allergic airway inflammation by modulating the balance of pro- and anti-asthmatic T cell responses in the lungs of mice.
Cells that belong to the family of innate lymphoid cells (ILCs) not only form a first line of defense against invading microbes, but also play essential roles in tissue remodeling and immune pathology. Rorγt(+) ILCs, producing the cytokines IL-22 and IL-17, include lymphoid tissue inducer (LTi) cells which are critical for the formation of lymphoid structures. Recently another ILC subset has been identified, which is dependent on RORα for its development and is dedicated to the production of the Th2 cytokines IL-5 and IL-13. These ILCs have been termed type 2 ILCs. All ILC subets are considered to belong to the same family that also includes natural killer cells because they all rely on the common γ-chain (γc) of the IL-2 receptor for their development and function, share a lymphoid morphology and depend on the transcriptional repressor Id2 for their development. Other transcription factors, including Notch, and the aryl hydrocarbon receptor (AhR) in RORγt(+) ILCs and GATA3 in type 2 ILCs, also play roles in the development, survival, and function of these ILC subpopulations. Here we review the current knowledge with regard to the transcription factors involved in the development and functions of ILCs.
Interleukin 6 (IL-6) is a protein secreted by T cells and macrophages and plays an important role in immune response. IL-6 regulates the proliferation and differentiation of T cells, and elicits immunoglobulin production in B cells. In this study, the cDNA il-6 (gil-6) sequence of the orange spotted grouper (Epinephelus coioides) was obtained. The deduced IL-6 (gIL-6) protein comprised 223 amino acids, the sequence shared approximately 30% similarity with mammalian IL-6, and between 47% and 69% similarity with other available teleost IL-6. The protein comprises the signal peptide, the IL-6 family signature, and conserved amino acid residues found in IL-6 sequences of other teleost. In order to understand the bioactivity and influence of gIL-6 on humoral immune response, recombinant gIL-6 (rgIL-6) synthesized by prokaryotes was injected into orange spotted groupers, and the immune-related gene expression at various times in various organs was observed. Our results revealed that the Th1 specific transcription factor t-bet was down-regulated and Th2 specific transcription factors gata3, and c-maf were up-regulated in immune organs, following IL-6 stimulation. Additionally, higher levels of igm mRNA and translated protein were detected in rgIL-6 stimulated fish. These results indicate that IL-6 in groupers regulates the differentiation of naїve T helper cells into Th2 cells and elicits the production of antibodies.
CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.
Immune balances are important for many diseases including ulcerative colitis (UC). This study aimed to explore the role of the balance between CD8+ CD28+ and CD8+ CD28- T lymphocytes for the immunological pathogenesis of UC.
Sixteen patients with UC, 16 patients with irritable bowel syndrome (IBS) and 15 healthy volunteers were enrolled. The frequencies of CD8+ CD28+ and CD8+CD28- T lymphocytes in peripheral blood and colon tissue were tested using flow cytometry and immunofluorescent, respectively. The cytokines of the two lymphocytes were detected by protein chips and ELISA. The expression of the signal transducers, the JAK3 and STAT6, as well the transcription factors, the NFATc2 and GATA3, was all detected by both western blot and immunohistochemistry.
For UC patients, the frequencies of CD8+ CD28+ T lymphocytes, together with the ratios of CD8+ CD28+ / CD8+ CD28- T lymphocytes in blood and colon tissue, were significantly lower than those in both IBS patients and healthy volunteers. But the frequencies of CD8+ CD28- T lymphocytes in blood and colon tissue of the UC patients were significantly higher than the other two groups. The concentration of IL-7 and -13, and the expression of JAK3 and STAT6 in UC patients, were significantly lower when compared with the other two groups. Conversely, the concentration of IL-12p40 and -15, and the expression of GATA3 and NFATc2 in UC patients, were significantly higher than both IBS and control group.
The balance of CD8+ CD28+ / CD8+ CD28- T lymphocytes plays a vital role in UC, while the balance tilt towards CD8+ CD28+ T lymphocytes is beneficial for patients with UC.
Homeobox genes encode transcription factors impacting key developmental processes including embryogenesis, organogenesis, and cell differentiation. Reflecting their tight transcriptional control, homeobox genes are often embedded in large non-coding, cis-regulatory regions, containing tissue specific elements. In T-cell acute lymphoblastic leukemia (T-ALL) homeobox genes are frequently deregulated by chromosomal aberrations, notably translocations adding T-cell specific activatory elements. NKX3-1 is a prostate specific homeobox gene activated in T-ALL patients expressing oncogenic TAL1 or displaying immature T-cell characteristics. After investigating regulation of NKX3-1 in primary cells and cell lines, we report its ectopic expression in T-ALL cells independent of chromosomal rearrangements. Using siRNAs and expression profiling, we exploited NKX3-1 positive T-ALL cell lines as tools to investigate aberrant activatory mechanisms. Our data confirmed NKX3-1 activation by TAL1/GATA3/LMO and identified LYL1 as an alternative activator in immature T-ALL cells devoid of GATA3. Moreover, we showed that NKX3-1 is directly activated by early T-cell homeodomain factor MSX2. These activators were regulated by MLL and/or by IL7-, BMP4- and IGF2-signalling. Finally, we demonstrated homeobox gene SIX6 as a direct leukemic target of NKX3-1 in T-ALL. In conclusion, we identified three major mechanisms of NKX3-1 regulation in T-ALL cell lines which are represented by activators TAL1, LYL1 and MSX2, corresponding to particular T-ALL subtypes described in patients. These results may contribute to the understanding of leukemic transcriptional networks underlying disturbed T-cell differentiation in T-ALL.
Evidence suggests that nebulized lidocaine is beneficial in asthma therapy, but to what extent and the mechanisms underlying this effect remain poorly understood. The aim of this study was to assess the impact of lidocaine treatment using a murine model of allergic asthma characterized by expression of pivotal features of the disease: inflammation, mucus production, and lung remodeling.
A/J mice sensitized with ovalbumin were treated with inhaled lidocaine or vehicle immediately after ovalbumin intranasal challenges. Lung function, total and differential leukocytes in bronchoalveolar lavage fluid, peribronchial eosinophil density, interleukin (IL)-4, IL-5 and eotaxin-1 levels, epithelial mucus, collagen, extracellular-matrix deposition, matrix metalloproteinase-9 activity, and GATA-3 expression were evaluated. Between five and eight animals per group were used.
Inhaled lidocaine inhibited ovalbumin-induced airway hyperreactivity to methacholine, and accumulation of lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid 24 h after the last allergen provocation. Lidocaine administration also prevented other pathophysiological changes triggered by ovalbumin in lung tissue, including peribronchial eosinophil and neutrophil infiltration, subepithelial fibrosis, increased content of collagen and mucus, matrix metalloproteinase-9 activity, and increased levels of IL-4, IL-5, IL-13, and eotaxin-1. Furthermore, inhaled lidocaine inhibited lung tissue GATA-3 expression in ovalbumin-challenged mice. We also demonstrated that lidocaine inhibited the expression of GATA-3 in ovalbumin-stimulated T cells in vitro.
Inhaled lidocaine prevents eosinophilic inflammation, overproduction of mucus, and peribronchial fibrosis in a murine model of asthma, and impaired airway hyperreactivity, possibly by inhibiting allergen-evoked GATA-3 expression and the subsequent up-regulation of proinflammatory cytokines and chemokines.
Natural killer (NK) cells are a subset of lymphocytes crucial for innate and adaptive immune responses. Here we show a stimulatory effect of cryptotanshinone (CTS) and tanshinone IIA (TS), isolated from Salvia miltiorrhiza Bunge, on the differentiation of NK cells. In the presence of IL-15, tanshinones increased NK cell maturation, NK cell differentiation and the expression of several transcription factors, including Id2, GATA3, T-bet, and Ets-1. Additionally, tanshinones increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, the p38 inhibitor SB203580 blocked the developmental effects of the tanshinones and suppressed Id2, T-bet, and Ets-1 expression during NK cell differentiation. These results suggest that tanshinones significantly increased IL-15-induced NK cell differentiation via enhancing the p38 phosphorylation and the expression of transcription factors.
IL-4 and signal transducer and activator of transcription 6 (STAT6) play an important role in the progression of allergic airway disease (AAD) or asthma. IL-4 and STAT6 mediate T(H)2 responses in T cells and immunoglobulin class-switching to IgE in B cells. Both T(H)2 responses and IgE promote the asthmatic condition. We have previously demonstrated that poly (ADP-ribose) polymerase (PARP) 14, a member of the PARP family of proteins, regulates the transcription function of STAT6. However, the role of PARP-14 in AAD is not known.
Here we investigate the role of PARP-14 and the enzyme activity associated with it in a model of AAD dependent on airway hyperresponsiveness and lung inflammation. We also elucidate the mechanism by which PARP-14 regulates AAD.
The role of PARP-14 and its enzyme activity in AAD and T(H)2 differentiation were examined by using a murine model of AAD and in vitro T(H) cell differentiation.
PARP-14-deficient animals show reduced lung pathology and IgE levels when compared with control animals. Treating mice with a pharmacologic inhibitor for PARP activity reduced the severity of airway hyperresponsiveness and lung inflammation. Mechanistically, our data indicate that PARP-14 and its enzyme activity aid in the differentiation of T cells toward a T(H)2 phenotype by regulating the binding of STAT6 to the Gata3 promoter.
PARP-14 and the catalytic activity associated with it promote T(H)2 differentiation and AAD in a murine model, and targeting PARP-14 might be a potential new therapy for allergic asthma.
IL-4 produced by Th2 cells can block cytokine production by Th1 cells, and Th1 IFN-γ is known to counterregulate Th2 immune response, inhibiting allergic eosinophilia. As intrauterine undernutrition can attenuate lung inflammation, we investigated the influence of intrauterine undernourishment on the Th1/Th2 cytokine balance and allergic lung inflammation. Intrauterine undernourished offspring were obtained from dams fed 50% of the nourished diet of their counterparts and were immunized at 9 weeks of age. We evaluated the cell counts and cytokine protein expression in the bronchoalveolar lavage, mucus production and collagen deposition, and cytokine gene expression and transcription factors in lung tissue 21 days after ovalbumin immunization. Intrauterine undernourishment significantly reduced inflammatory cell airway infiltration, mucus secretion and collagen deposition, in rats immunized and challenged. Intrauterine undernourished rats also exhibited an altered cytokine expression profile, including higher TNF-α and IL-1β expression and lower IL-6 expression than well-nourished rats following immunization and challenge. Furthermore, the intrauterine undernourished group showed reduced ratios of the IL-4/IFN-γ and the transcription factors GATA-3/T-Bet after immunization and challenge. We suggest that the attenuated allergic lung inflammation observed in intrauterine undernourished rats is related to an altered Th1/Th2 cytokine balance resulting from a reduced GATA-3/T-bet ratio.
To investigate the role of immunological parameters in tumorigenesis of cervical cancer in women infected with high risk human papillomavirus (hr-HPV), and determine whether key findings with human material can be recapitulated in the mouse TC1 carcinoma model which expresses hr-HPV epitopes.
Epithelial and lymphoid cells in cervical tissues were analyzed by immunohistochemistry and serum IL10 levels were determined by ELISA. Tumor draining lymph nodes were analyzed in the mouse TC1 model by flow cytometry.
The mucosa was infiltrated by CD20+ and CD138+ cells already at cervical intraepithelial neoplasia 1 (CIN1) and infiltration increased in cervical intraepithelial neoplasia 3 (CIN3)/carcinoma in situ (CIS) and invasive cervical cancer (ICC), where it strongly correlated with infiltration by CD32B+ and FoxP3+ lymphocytes. GATA3+ and T-bet+ lymphoid cells were increased in ICC compared to normal, and expression in epithelial cells of the Th2 inflammation-promoting cytokine TSLP and of IDO1 was higher in CIN3/CIS and ICC. As a corollary, serum levels of IL10 were higher in women with CIN3/CIS or ICC than in normals. Finally we demonstrated in the mouse TC1 carcinoma, which expresses hr-HPV epitopes, an increase of cells expressing B cell or plasma cell markers or Fc receptors in tumor-draining than distal lymph nodes or spleen.
hr-HPV initiates a local Th2 inflammation at an early stage, involving antibody forming cells, and fosters an immunosuppressive microenvironment that aids tumor progression.
Several studies provided evidence of innate interferons (IFNs) regulating T(H)2 cytokine production using purified CD4(+) memory cells and T(H)2 polarisation via interleukin-4 (IL-4). Vitally, none of these previous studies examined IFN attenuation of T(H)2 responses to allergen or antigen. This study therefore sought to investigate the abrogation of specific allergen- and antigen-stimulated T(H)2 response in peripheral blood mononuclear cells (PBMC) derived from 12 sensitised individuals by IFN-β and IFN-λ. PBMC were cultured in the presence of house dust mite (HDM) allergen, rhinovirus (RV), influenza vaccine and tetanus toxoid (TT)±either IFN-β or IFN-λ for 3 and 5 days. IFN-γ, IL-5 and IL-13 protein levels were measured by ELISA. Quantitative PCR (qPCR) was used to investigate induction of genes involved in control of T(H)2 cytokines. No alteration in T(H)1 IFN-γ allergen/antigen response was observed with addition of IFN-β or IFN-λ. Consistent abrogation of T(H)2 response to HDM and influenza was observed with IFN-β at both time points; attenuation was observed by day 5 with RV and TT. IFN-λ had no consistent effect on T(H)2 production except in the presence of RV (multiplicity of infection=5); a decrease in IL-5 alone was observed in the presence of trivalent inactivated influenza vaccine. GATA binding protein 3 (GATA3) and suppressors of cytokine signalling3 mRNA were differentially regulated in HDM and influenza-stimulated cultures±IFN-β. We concluded that IFN-β produced a strong and consistent abrogation of T(H)2 cytokine production in the presence of a range of allergen and antigen stimulants.
Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a new disease entity characterized by high serum IgG4 levels, IgG4-positive plasmacytic infiltration, and fibrosis in various organs. The purpose of this study was to determine the mechanism of upregulation of IgG4 class switch recombination in IgG4-RD.
We extracted RNA from peripheral blood mononuclear cells (PBMCs) of patients with IgG4-RD (n = 6), Sjögren syndrome (SS) (n = 6), and healthy controls (n = 8), from CD3-positive T cells and CD20-positive B cells sorted from PBMCs of patients with IgG4-RD (n = 3), SS (n = 4), and healthy controls (n = 4), as well as from labial salivary glands (LSGs) of patients with IgG4-RD (n = 11), SS (n = 13), and healthy controls (n = 3). The mRNA expression levels of IgG4-specific class switch-related molecules, such as Th2 cytokines (IL-4 and IL-13), Treg cytokines (IL-10 and TGF-β), and transcriptional factors (GATA3 and Foxp3) were examined with quantitative polymerase chain reaction (PCR). IgG4-nonspecific class switch-related molecules, such as CD40, CD154, BAFF, APRIL, IRF4, and AID, were also examined.
The expression levels of Treg cytokines (IL-10 and TGF-β) and AID were significantly higher in LSGs of IgG4-RD than in SS and the controls (P < 0.05, each). In contrast, those of CD40 and CD154 were significantly lower in PBMCs of IgG4-RD than in SS (P < 0.05, each), whereas CD40 in CD20-positive B cells and CD154 in CD3-positive T cells were comparable in the three groups.
Overexpression of IL-10, TGF-β, and AID in LSGs might play important roles in the pathogenesis of IgG4-RD, such as IgG4-specific class-switch recombination and fibrosis. IgG4 class-switch recombination seems to be mainly upregulated in affected organs.
Elevated expression of FoxM1 in breast cancer correlates with an undifferentiated tumor phenotype and a negative clinical outcome. However, a role for FoxM1 in regulating mammary differentiation was not known. Here, we identify another function of FoxM1, the ability to act as a transcriptional repressor, which plays an important role in regulating the differentiation of luminal epithelial progenitors. Regeneration of mammary glands with elevated levels of FoxM1 leads to aberrant ductal morphology and expansion of the luminal progenitor pool. Conversely, knockdown of FoxM1 results in a shift toward the differentiated state. FoxM1 mediates these effects by repressing the key regulator of luminal differentiation, GATA-3. Through association with DNMT3b, FoxM1 promotes methylation of the GATA-3 promoter in an Rb-dependent manner. This study identifies FoxM1 as a critical regulator of mammary differentiation with significant implications for the development of aggressive breast cancers.
To find additional susceptibility loci for lung cancer, we tested promising associations from our previous genome-wide association study (GWAS) of lung cancer in the Chinese population in an extended validation sample size of 7,436 individuals with lung cancer (cases) and 7,483 controls. We found genome-wide significant (P < 5.0 × 10(-8)) evidence for three additional lung cancer susceptibility loci at 10p14 (rs1663689, close to GATA3, P = 2.84 × 10(-10)), 5q32 (rs2895680 in PPP2R2B-STK32A-DPYSL3, P = 6.60 × 10(-9)) and 20q13.2 (rs4809957 in CYP24A1, P = 1.20 × 10(-8)). We also found consistent associations for rs247008 at 5q31.1 (IL3-CSF2-P4HA2, P = 7.68 × 10(-8)) and rs9439519 at 1p36.32 (AJAP1-NPHP4, P = 3.65 × 10(-6)). Four of these loci showed evidence for interactions with smoking dose (P = 1.72 × 10(-10), P = 5.07 × 10(-3), P = 6.77 × 10(-3) and P = 4.49 × 10(-2) for rs2895680, rs4809957, rs247008 and rs9439519, respectively). These results advance our understanding of lung cancer susceptibility and highlight potential pathways that integrate genetic variants and smoking in the development of lung cancer.
Ipilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4(+) and CD8(+) T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.
Microarray analysis of flow-cytometry purified CD4(+) and CD8(+) T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.
Ipilimumab up-regulated Ki67 and ICOS on CD4(+) and CD8(+) cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8(+) at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4(+) and CD8(+) cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES(+)CD8(+), GranzymeB(+)EOMES(+)CD8(+) and decreased Ki67(+)EOMES(+)CD4(+) T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67(+)CD8(+) T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67(+)EOMES(+)CD8(+) T cells were associated with relapse (p ≤ 0.001), and low Ki67(+)EOMES(+)CD4(+) T cells were associated with irAE (p ≤ 0.008).
Up-regulation of proliferation and activation signals in CD4(+) and CD8(+) T cells were pharmacodynamic markers for ipilimumab. Ki67(+)EOMES(+)CD8(+) and Ki67(+)EOMES(+)CD4(+)T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.
Cooperation between the innate and adaptive immune responses is critical for enabling protective immunity against various invading microbes. Distinct types of effector T cells have different functions in adaptive immune responses. Th1 cells play important roles in the control of intracellular bacteria by producing IFN-γ to activate macrophages and in anti-viral immunity by producing IFN-γ and activating cytotoxic T lymphocytes. Th2 cell-derived cytokines are important in activating mast cells, eosinophils, and goblet cells in anti-helminth immunity. Th17 cells are pivotal for the inflammatory response mediated by neutrophils, which resists extracellular bacterial infection. In all cases, it is critical that the innate immune responses limit the growth and expansion of invading microbes until antigen-specific adaptive immune responses are established. Recent studies have identified multiple subsets in innate lymphocytes corresponding to previously defined Th subsets. Classical natural killer cells, RORγ(+) lymphoid tissue inducer-related cells, and Th2-type innate lymphocytes play distinct roles in innate immune responses by producing Th1, Th17, and Th2 cytokines, respectively. Cooperation between innate lymphocytes and antigen-specific T and B cells are likely important in protective immunity against distinct types of microbes. The most recently identified subset is the RORγ-independent Lin(-)Thy-1(+)IL-7R(+)GATA3(+) innate lymphocyte subset such as natural helper (NH) cell, which is Id2- and IL-7-dependent. This population produces Th2 cytokines, most notably IL-5 and IL-13, and plays a major role in innate immune responses during anti-helminth immunity. In addition, these cells are likely involved in the pathophysiology of some types of allergic diseases. We summarize here current knowledge regarding various innate lymphocyte subsets. In particular, we focus on the Th2-type innate lymphocyte subset.
Churg-Strauss syndrome (CSS) is a rare systemic vasculitis associated with eosinophilia and asthma. We assessed the local immune response in airways of CSS patients with different activity of the disease.
Concentration of IL-5, CCL17, CCL22 and CCL26 (ELISA) together with cell expression of T-helper-related genes (real-time PCR array) were measured in bronchoalveolar lavage fluid (BALF) sampled from 11 patients with active CSS, 11 patients with CSS in remission and 9 control subjects with bronchial asthma.
In active CSS, both BALF and blood eosinophil counts were increased (P<0.01). BALF cells in active disease were characterized by an increased expression of Th2 and regulatory-type transcripts: STAT6, STAT3, GATA3, IL4, IL5 and IL10 as compared with asthmatics, and STAT5A, CCR4, FOXP3, IL4, IL5 and IL10 when compared with inactive CSS. There was significant increase in BALF concentration of IL-5 and CCL26 in exacerbation of CSS. CCR4-active chemokines were detected more frequently in active disease. We found a strong positive correlation between clinical parameters of disease activity (BVAS, eosinophilia) and expression of IL4, IL5, IL10 and STAT5A.
These results indicate that as compared with asthma, active-CSS patients have much stronger local Th2 response in the airways. Airway cells may contribute to lung eosinophilia in CSS by producing IL-5 and eosinophil active chemokines.
The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program.
Vaccination remains one of the most effective approaches to prevent the spread of infectious diseases. Immune responses to vaccination can be enhanced by inclusion of adjuvant in a vaccine. Paclitaxel extracted from the bark of the Pacific yew tree Taxus brevifola was previously demonstrated to have adjuvant property. Compared to paclitaxel, docetaxel is another member of taxane family, and is more soluble in water and easier to manipulate in medication. To investigate the adjuvant effect of this compound, we measured the immune responses induced by co-administration of a split inactivated influenza H1N1 vaccine antigen with docetaxel.
When co-administered with docetaxel, lower dose antigen (equivalent to 10 ng HA) induced similar levels of IgG and IgG isotypes as well as HI titers to those induced by higher dose antigen (equivalent to 100 ng HA). Docetaxel promoted splenocyte responses to H1N1 antigen, ConA and LPS, mRNA expressions of cytokines (IFN-gamma, IL-12, IL-4 and IL-10) and T-bet/GATA-3 by splenocytes. The enhanced immunity was associated with up-expressed microRNAs (miR-155, miR-150 and miR-146a) in docetaxel-stimulated RAW264.7 cells. Docetaxel promoted similar IgE level to but alum promoted significantly higher IgE level than the control.
Docetaxel has adjuvant effect on the influenza H1N1 vaccine by up-regulation of Th1/Th2 immune responses. Considering its unique vaccine adjuvant property as well as the safe record as an anti-neoplastic agent clinically used in humans during a long period, docetaxel should be further studied for its use in influenza vaccine production.
The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3 (GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy.
Tumor specimens obtained before neoadjuvant chemotherapy [paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic subtyping.
Seventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were significantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR) was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. multivariate analysis showed that tumor size, human epidermal growth factor receptor 2 (her2), and gata3 were independent predictors of pcr.
GATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and were mostly classified as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor response to chemotherapy.
To observe the effect of moxibustion pretreatment on the expression of leukocytic T-box transcription factor expressed in T cells (T-bet) mRNA and trans-acting T-cell-specific transcription factor GATA-3 mRNA in athletes undergoing heavy load training, so as to study its mechanism underlying prevention of imbalance of immune function.
Twelve male middle-long distance running athletes were equally randomized into control group and treatment group. The althelets in the treatment group accepted alternate moxibustion pretreatment of Guanyuan (CV 12) + bilateral Zusanli (ST 36) or Guanyuan (CV 12) + bilateral Sanyinjiao (SP 6) for 30 min, once a day for 4 weeks, and beginning from the 1st day of the heavy load training on till the end of the modulatory training. The althletes of the control group were asked to conduct simple heavy load running training. Leukocytes in the collected venous blood samples were acquired following erythrocyte lysis, centrifugation and removal of the supernatant. The expression levels of leukocytic T-bet mRNA and GATA-3 mRNA were detected before, 3 weeks after the heavy load running training and 1 week after the adjustment training.
In comparison with pre-training, the expression levels of leukocytic T-bet mRNA were increased slightly after heavy load running training (7.24%, 16.84%) and after adjustment training (4.52%, 14.8%) in both control and treatment groups (P > 0.05). After heavy load running training, the expression levels of leukocytic GATA-3 mRNA were dow-regulated mildly (13.14%, 34.04%) in both control and treatment groups (P > 0.05), but upregulated considerably in the control group (59.12%, P < 0.05) rather than in the treatment group (-17.02%, P > 0.05). The GATA-3 mRNA expression level of the treatment group was significantly lower than that of the control group (P < 0.05). The ratios of leukocytic T-bet mRNA/GATA-3 mRNA were increased by 13.58% and 75.16% after heavy load running training and decreased by 29.63% and increased by 35.4%, respectively after adjustment training in the control and treatment groups in comparison with pre-training. No significant differences were found between two groups in the expression levels of T-bet mRNA and the ratios of T-bet mRNA/GATA-3 mRNA at the 3 time-points, and in the expression levels of GATA-3 mRNA before and after heavy load running training (P > 0.05).
Moxibustion pretreatment can inhibit the expression of leukocytic GATA-3 mRNA after adjustment training in the middle-long distance heavy load running athletes, which may contribute to its effect in regulating imbalance of Th 1/Th 2 after heavy load training.
In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.
Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-β and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T(H)2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T(H)2 cytokine, and prevented binding of GATA-3 to this promoter. T(H)2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-β to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.
The diagnosis of diffuse pontine tumors has largely been made on the basis of MRI since the early 1990 s. In cases of tumors considered "typical," as a rule, no biopsy specimen has been obtained, and the tumors have been considered diffuse intrinsic pontine gliomas (DIPGs). There have been sporadic reports that primitive neuroectodermal tumors (PNETs) of the pons may not be distinguishable from the DIPGs by radiological imaging. This study presents 2 cases of diffuse pontine PNETs with molecular evidence that these are indeed PNETs, distinct from DIPGs, thus supporting biopsy of diffuse pontine tumors as a standard of care.
Biopsy specimens were obtained from 7 diffuse pontine tumors and snap frozen. Two of these 7 tumors were identified on the basis of pathological examination as PNETs. All 7 of the diffuse pontine tumors were analyzed for gene expression using the Affymetrix HG-U133 Plus 2.0 GeneChip microarray. Gene expression was compared with that of supratentorial PNETs, medulloblastomas, and low- and high-grade gliomas outside the brainstem.
Unsupervised hierarchical clustering analysis of gene expression demonstrated that pontine PNETs are most closely related to PNETs of the supratentorial region and not with gliomas. They do not cluster with the 5 DIPGs in the study. Thirty-eight genes, including GATA3, are uniquely differentially expressed in pontine PNETs compared with other types of pediatric brain tumors, including DIPGs and other PNETs at a false discovery rate statistical significance of less than 0.05.
The cluster and individual gene expression analyses indicate that pontine PNETs are intrinsically different from DIPGs. The 2 pontine PNET cases cluster with supratentorial PNETs, rather than with DIPGs, suggesting that these tumors should be treated with a PNET regimen, not with DIPG therapy. Since diagnosis by imaging is not reliable and the biology of the tumors is disparate, a biopsy should be performed to enable accurate diagnosis and direct potentially more effective treatments.
The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs).
To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used.
Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay.
We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.
To investigate the association of the expressions of Th1 cytokines in gastric cancer tissue and the prognosis of patients with gastric cancer after radical resection.
Fifty-eight patients with gastric cancer treated at the Zhongshan Hospital of Fudan University were retrospectively analyzed. The expressions of Th1 cytokines mRNA were detected in tumor tissues by real-time polymerase chain reaction(RT-PCR) method in Th1 cells including TRAV10, IRF1, TBX21, CD3Z, GZMB, GATA3, and IFNG. Association of Th1 cytokines mRNA expressions and prognosis was evaluated.
The median follow up was 42.5(1-64) months. The 1-year survival rate was 84.5% and the 3-year survival rate was 72.4%. Univariate Cox regression analysis showed that lymph node metastasis, distant metastasis, TNM staging, lymphovascular invasion, GNLY mRNA expression, and the overall expression level of the 8 types of Th1 cells were associated with the prognosis of patients with gastric cancer(all P<0.05). The 3-year survival was 86.2% in patients with increased expression of mRNA and 58.6% in those with decreased expression. The 3-year survival was 79.6% in patients with any increase in the 8 Th1 cytokines and 33.3% in those with consistent downregulation of the 8 cytokines. Multivariate Cox analysis showed that lymph node metastasis and the overall expression level of 8 Th1 cytokines were independent risk factors associated with the prognosis of patients with gastric cancer in this cohort(both P<0.05).
The mRNA expression of GNLY is associated with the prognosis of patients with gastric cancer but is not an independent risk factor. The combination of mRNA expressions of the eight cytokines is an independent prognostic factor.
Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.
To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.
GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.
T-cell development and differentiation is coordinated by a multitude of signaling molecules and transcription factors that impart distinct functional properties to progenitors. In this review, we focus on the role of the T lineage-enriched chromatin organizer and regulator SATB1 in T-cell differentiation. SATB1 mediates Wnt signaling by recruiting β-catenin to its genomic targets and coordinates T helper type 2 (T(H)2) differentiation by positively regulating GATA-3. In contrast, maintenance of regulatory T cell (Treg) functions are dependent on inhibition of SATB1-mediated modulation of global chromatin organization. We discuss how regulation of the activity of SATB1 has a critical role in driving these two important differentiation pathways in T cells.
GATA3 expression has been reported in urothelial and breast carcinomas; however, the published data on GATA3 expression in tumors from other organs are limited. Immunohistochemical evaluation of GATA3 expression in 1,110 carcinomas and 310 cases of normal tissue using tissue microarray sections, 48 breast and bladder biopsy specimens, and 53 breast fine-needle aspiration biopsy specimens was performed. Sixty-two of 72 urothelial carcinomas (86%) and 138 of 147 breast carcinomas (94%) tested positive for GATA3. All other cases, except for 2 of 96 endometrial carcinomas, tested negative for GATA3. On fine-needle aspiration biopsy samples, 88% of primary breast carcinomas and 82% of metastatic breast carcinomas tested positive for GATA3. Our study revealed that GATA3 is a sensitive and specific marker for the diagnosis of breast and urothelial carcinomas. When working on a tumor of unknown origin, GATA3 should be routinely included in the initial screening panel if either a breast or urothelial primary tumor is suspected.
Hair follicles (HFs) are equipped with stem cell niches that allow regeneration. Tumor necrosis factor-α converting enzyme (TACE), also known as A disintegrin and metalloproteinase 17, is a proteolytic enzyme that regulates a variety of cell surface molecules including TNF-α, via ectodomain shedding. We found TACE expression on mouse HFs and conditionally depleted it in cells that expressed sex-determining region Y-related high-mobility-group box 9 (SOX9) transcription factor, an HF stem cell transcription factor (Tace(flox/flox) -Sox9-Cre, hereafter, "Tace/Sox9"). Tace/Sox9 mice were born with brittle hair with prolonged anagen phase. They underwent diffuse, progressive, and ultimately whole-body hair loss by 20 weeks old. Tace/Sox9 HFs lacked CD34(+) bulge cells as demonstrated via immunofluorescence microscopy and flow cytometry. Real-time PCR revealed downregulation of transcription factors Sox9, Lhx2, and Gata3 and upregulation of Lef1. In vitro colony-forming capacity was abolished in Tace/Sox9 keratinocytes, and HFs exhibited increased proliferation in situ, collectively demonstrating that Tace/Sox9 mice failed to establish the bulge niche and to maintain "stemness" of HF stem cells. Epidermal growth factor receptor (EGFR) signaling was impaired in Tace/Sox9 keratinocytes, and mice depleted of Egfr in SOX9-expressing tissues exhibited hair phenotype nearly identical to Tace/Sox9 mice, demonstrating EGFR signaling as a pathway downstream of TACE in HF homeostasis. This study provides mechanistic implication for human TACE-deficiency and for hair abnormality caused by EGFR inhibitors.
To study the differentiation of Th1/Th2 on the levels of transcription factors and cytokines production in patients with laryngeal squamous cell carcinoma in peripheral blood mononuclear cells (PBMC). In addition, the relation of p53 and T-bet, GATA3 expression was discussed for understanding the role of T-bet and GATA3 in metastasis.
The gene expression of Th1/Th2 type transcription factors T-bet, GATA3 and cytokines IFN-gamma, IL-4 were determined by RT-PCR and realtime RT-PCR from 49 patients with laryngeal squamous cell carcinoma and 30 normal controls. The expression of p53 was analyzed by immunohistochemistry.
Predominant expression of Th2 type cytokines was detected in 49 laryngeal squamous cell carcinoma patients. The expression rates of T-bet, GATA3, IFN-gamma and IL-4 was 42.86% (21/49), 71.43% (35/49), 26.53% (13/49), 63.27% (33/49) respectively. The expression rates of T-bet and IFN-gamma in patients were lower than in control group (P < 0.05), but the results of GATA3 and IL-4 were opposite (P < 0.05). The similar results were obtained by realtime PCR. The expression of p53 in patients was accompanied with lower expression of T-betmRNA and higher expression of GATA3mRNA.
There is predominant expression of Th2 type transcription factors and cytokines in PBMC of laryngeal squamous cell carcinoma patients. T-bet and GATA3 can be used as reference indicators for the metastasis of laryngeal squamous cell carcinoma.
Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-β, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem-spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Bötzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.
We have investigated the inflammatory infiltrate in post-transplant dn-AIH, a form of late insidious graft rejection, focusing on transcription factors defining effector and T-regs, using an antigen retrieval immunohistochemical method on archived liver tissue, and compared it with ACR and classical AIH. Paraffin-embedded liver biopsies from pediatric patients with dn-AIH (n = 10), ACR (n = 10), and AIH (n = 13) were selected randomly and stained using antibodies directed to CD4, CD8, T-bet (marker of Th1 polarization), GATA-3 (marker of Th2 polarization), FOXP3 (marker for T regulatory cells), IL-17, CD56 (NK cells), and perforin. Portal and lobular lymphocytic infiltrate was assessed semi-quantitatively. Prominent CD4, CD8, and T-bet positivity were present in both the lobular and portal infiltrate of all three conditions. Overall T-bet score of lobular inflammation in the dn-AIH group was lower than in the ACR and AIH groups (p = 0.02). In contrast, most samples showed absent or minimal GATA-3 positivity. FOXP3, CD56, IL-17, and perforin staining of mild to moderate severity were present in all three groups in both the portal and lobular infiltrate. A Th1 polarization of the inflammatory infiltrate characterizes dn-AIH, but also ACR and AIH.
CD4⁺ T helper cells are playing critical roles in host defense to pathogens and in the maintenance of immune homeostasis. Naïve CD4⁺T cells, upon antigen-specific recognition, receive signals to differentiate into distinct effector T helper cell subsets characterized by their pattern of cytokine production and specific immune functions. A tight balance between these different subsets ensures proper control of the immune response. There is increasing evidence revealing an important role for Notch signaling in the regulation of CD4⁺T helper cell differentiation or function in the periphery. However, the exact mechanisms involved remain unclear and appear contradictory. In this review, we summarize current knowledge and discuss recent advances in the field to reconcile different views on the role of Notch signaling in the differentiation of functional T helper subsets.
To observe the therapeutic effect and mechanism of new percutaneous absorption herbal patch for asthma of paracmasis, and to optimize the form of the patch.
One hundred and twenty cases of paracmasis asthma were randomly divided into medicine patch group, medicinal vesiculation group and western medication group with 40 cases for each. The new percutaneous absorption herbal patch was applied on medicine patch group. Traditional medicinal herbs cake of the hospital were applied on medicinal vesiculation group. Feishu (BL 13), Fengmen (BL 12) and Dazhui (GV 14) were adopted for both groups. Each patch was applied for 6 hours and once every other day. Accuhaler was applied on the western medicine group with 2 inhalations a time and twice a day. Clinical symptom scores, number of attacks and asymtomatic days were observed right before, after and half a year after the treatment. Meanwhile, the expression level of IgE, IL-4, GATA-3 mRNA and T-bet mRNA were observed and compared before and after the treatment.
Clinical symptom scores of all the 3 groups were improved (all P < 0.01). The differences of the total effective rate, number of attacks and asymtomatic days of all the 3 groups are without statistical significance (all P > 0.05). However, the long-term therapeutic effect in half a year after the treatment showed that the total effective rate of the medicine patch group was 80.0% (32/40), and the medicinal vesiculation group was 70.0% (28/40). Both of the them were obviously higher than 47.5% (19/40) of the western medicine group (P < 0.01, P < 0.05). And the medicine patch group surpassed the other 2 groups in controlling the number of attacks and increasing the asymtomatic days (P < 0.05, P < 0.01). The level of IgE and IL-4 of all the 3 groups decreased sharply after the treatment (P < 0.05, P < 0.01). And there was no statistic significance in differences among groups (all P > 0.05). The level of GATA-3 mRNA was obviously decreased, while the level of T-bet mRNA was obviouly increased in the medicine patch and medicinal vesiculation groups (P < 0.05, P < 0.01). And the medicine patch group had obvious superiority on increasing the level of T-bet mRNA when compared with the medicinal vesiculation and western medicine groups (P < 0.01, P < 0.05).
It is concluded that the new percutaneous absorption herbal patch has exact effect on asthma. The treatment may reverse the imbalance condition of Th1/Th2 through regulation on cell factor and its specific transcription factors.
GABAergic neurons in the ventral mesodiencephalic region are highly important for the function of dopaminergic pathways that regulate multiple aspects of behavior. However, development of these neurons is poorly understood. We recently showed that molecular regulation of differentiation of the GABAergic neurons associated with the dopaminergic nuclei in the ventral midbrain (VTA and SNpr) is distinct from the rest of midbrain, but the reason for this difference remained elusive. Here, we have analyzed the developmental origin of the VTA and SNpr GABAergic neurons by genetic fate mapping. We demonstrate that the majority of these GABAergic neurons originate outside the midbrain, from rhombomere 1, and move into the ventral midbrain only as postmitotic neuronal precursors. We further show that Gata2, Gata3 and Tal1 define a subpopulation of GABAergic precursors in ventral rhombomere 1. A failure in GABAergic neuron differentiation in this region correlates with loss of VTA and SNpr GABAergic neurons in Tal1 mutant mice. In contrast to midbrain, GABAergic neurons of the anterior SNpr in the diencephalon are not derived from the rhombomere 1. These results suggest unique migratory pathways for the precursors of important GABAergic neuron subpopulations, and provide the basis for understanding diversity within midbrain GABAergic neurons.
Although GATA binding protein 3, a zinc finger transcription factor and an estrogen receptor-regulated gene, has recently been suggested as a marker for urothelium, prognostic significance of GATA binding protein 3 expression in bladder tumor remains unclear. We immunohistochemically stained for GATA binding protein 3 in urothelial neoplasm and matched nonneoplastic bladder tissue specimens. GATA binding protein 3 was positive in 125 (86%; 13 [9%] weak, 44 [30%] moderate, and 68 [47%] strong) of 145 bladder tumors, which was significantly lower than in benign urothelium (104/106 [98%]; 3 [3%] weak, 30 [28%] moderate, and 71 [67%] strong) (P=.001). Fifty (98%) of 51 low-grade tumors were GATA binding protein 3 positive, whereas 75 (80%) of 94 high-grade carcinomas were GATA binding protein 3 positive (P=.002). Similarly, 78 (98%) of 80 non-muscle-invasive tumors expressed the GATA binding protein 3, compared with 47 (72%) of 65 muscle-invasive tumors (P<.001). Conversely, among 68 cases treated with cystectomy, significantly lower expression of GATA binding protein 3 was found in pN0 tumors (32/47 [68%]) than in node-positive tumors (20/21 [95%]) (P=.027). Kaplan-Meier and log-rank tests further revealed that overall positivity (P=.048) or strong positivity (P=.025) of GATA binding protein 3 correlated with progression of muscle-invasive tumors. Multivariate analysis identified high GATA binding protein 3 expression as a strong prognosticator for progression (P=.052) and cancer-specific survival (P=.040) of muscle-invasive tumors. Moreover, there were significant correlations between GATA binding protein 3 expression vs androgen receptor overexpression, estrogen receptor α overexpression, or loss of estrogen receptor β expression. Thus, compared with benign urothelium, a significant decrease in the expression of GATA binding protein 3 in urothelial neoplasms was seen. Loss of GATA binding protein 3 was associated with high-grade and/or muscle-invasive tumors, whereas strong expression was an independent predictor of poor prognosis.
Lineage commitment studies in mammary glands have focused on identifying cell populations that display stem or progenitor properties. However, the mechanisms that control cell fate have been incompletely explored. Herein we show that zinc finger protein 157 (Zfp157) is required to establish the balance between luminal alveolar pStat5- and Gata-3-expressing cells in the murine mammary gland. Using mice in which the zfp157 gene was disrupted, we found that alveologenesis was accelerated concomitantly with a dramatic skewing of the proportion of pStat5-expressing cells relative to Gata-3⁺ cells. This suppression of the Gata-3⁺ lineage was associated with increased expression of the inhibitor of helix-loop-helix protein Id2. Surprisingly, Gata-3 becomes dispensable in the absence of Zfp157, as mice deficient for both Zfp157 and Gata-3 lactate normally, although the glands display a mild epithelial dysplasia. These data suggest that the luminal alveolar compartment of the mammary gland is comprised of a number of distinct cell populations that, although interdependant, exhibit considerable cell fate plasticity.
Using homozygosity mapping and locus resequencing, we found that alterations in the homeodomain of the IRX5 transcription factor cause a recessive congenital disorder affecting face, brain, blood, heart, bone and gonad development. We found through in vivo modeling in Xenopus laevis embryos that Irx5 modulates the migration of progenitor cell populations in branchial arches and gonads by repressing Sdf1. We further found that transcriptional control by Irx5 is modulated by direct protein-protein interaction with two GATA zinc-finger proteins, GATA3 and TRPS1; disruptions of these proteins also cause craniofacial dysmorphisms. Our findings suggest that IRX proteins integrate combinatorial transcriptional inputs to regulate key signaling molecules involved in the ontogeny of multiple organs during embryogenesis and homeostasis.
T cell development constitutes a multistage process allowing the dissection of events resulting in cellular commitment and functional specification in a specialized microenvironment. This process is guided by the appropriate expression of regulatory genetic factors like transcriptional activators or repressors which are, in part, dependent on instructive signals of the microenvironment. To date, it remains unclear whether exactly the same genetic mechanism acts in adult compared to fetal T cell development. In order to directly compare T cell commitment during adult and fetal differentiation, we isolated subsequent stages of intrathymic subpopulations starting with early canonical T cell progenitors up to irreversibly committed T cell precursors. The genome-wide analysis revealed several distinct gene clusters with a specific pattern of gene regulation for each subset. The largest cluster contained genes upregulated after transition through the most primitive pool into the next transitory population with a consistently elevated expression of elements associated with ongoing T cell fate specification, like Gata3 and Tcf7, in fetal progenitors. Furthermore, adult and fetal T cell progenitors occupied distinct "transcriptional territories" revealing a precise land map of the progression to final T cell commitment operating in different developmental windows. The presence and/or elevated expression of elements associated with an ongoing establishment of a T cell signature in the most primitive fetal subset is highly suggestive for an extrathymic initiation of T cell specification and underlines the fundamental differences in fetal versus adult lymphopoiesis.
Chronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor for T helper (Th) cell differentiation and function. The aim of this study was to investigate the effect of T-bet overexpression on Th cell differentiation and the possible mechanism in chronic hepatitis B (CHB) patients. CD4+ T cells from the peripheral blood of 23 CHB patients, 8 acute hepatitis B (AHB) patients and 10 healthy controls were isolated. T-bet mRNA expression of CD4+ T cells was detected by quantitative real-time polymerase chain reaction (PCR). The T-bet DNA fragment was subcloned into the pGC-FU vector containing GFP to generate a recombinant lentiviral vector, pGC-FU-T-bet, while a no-load pGC-FU vector was used as the negative control. After transduction into CD4+ T cells from another 22 CHB patients, the induction of Th1- and Th2-type cytokines was assayed by an enzyme-linked immunosorbent assay (ELISA), and RT-PCR and western blot analysis were used to measure the mRNA and transcription levels of H2.0-like homeobox (HLX1), GATA-3 and STAT-6. T-bet mRNA expression in CD4+ T cells from AHB patients was enhanced compared with CHB patients and healthy controls. Th1-type cytokines and HLX1 expression was upregulated, while Th2-type cytokines and GATA-3 and STAT-6 expression was repressed after lentiviral introduction of T-bet. In conclusion, lentivirally overexpressed T-bet regulates Th cell lineage commitment in CHB patients, which may be mediated by regulating HLX1, GATA-3 and STAT-6 expression.
The development of T cells from multipotent progenitors in the thymus occurs by cascades of interactions between signaling molecules and transcription factors, resulting in the loss of alternative lineage potential and the acquisition of the T-cell functional identity. These processes require Notch signaling and the activity of GATA3, TCF1, Bcl11b, and the E-proteins HEB and E2A. We have shown that HEB factors are required to inhibit the thymic NK cell fate and that HEBAlt allows the passage of T-cell precursors from the DN to DP stage but is insufficient for suppression of the NK cell lineage choice. HEB factors are also required to enforce the death of cells that have not rearranged their TCR genes. The synergistic interactions between Notch1, HEBAlt, HEBCan, GATA3, and TCF1 are presented in a gene network model, and the influence of thymic stromal architecture on lineage choice in the thymus is discussed.
Most studies about functions of aryl hydrocarbon receptor (AhR) in the pathogenesis of asthma have been carried out with non-physiological industrial by-products such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene. In the present study, effects of 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of AhR, on the pathogenesis of asthma were examined and then underlying mechanisms of its immumodulatory effects were investigated. FICZ significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs. Flow cytometric analysis of mediastinal lymph nodes showed that IL-4 producing cells decreased in FICZ-treated mice compared with PBS control. Next, effects of FICZ on in vitro Th2 differentiation and expression of the Th2 transcription factor GATA-3 were examined. CD4+ T cells were isolated from the spleen and incubated under the Th2 differentiation conditions. FICZ inhibited both Th2 differentiation and the expression of GATA-3. Finally, activation of STAT6, which is necessary for Th2 differentiation, was inhibited by FICZ.
Aberrant transcriptional activities have been documented in breast cancers. Studies often find some transcription factors to be inappropriately regulated and enriched in certain pathological states. The promoter regions of most target genes have binding sites for their transcription factors. An ample of evidence supports their combinatorial effect on their shared target gene expressions. Here, we used a new statistic method, bivariate CID, to predict combinatorial interaction activity between ERα and a transcription factor (E2F1or GATA3 or ERRα) in regulating target gene expression via four regulatory mechanisms. We identified gene sets in three signal transduction pathways perturbed in breast tumors: cell cycle, VEGF, and PDGFRB. Bivariate network analysis revealed several target genes previously implicated in tumor angiogenesis are among the predicted shared targets, including VEGFA, PDGFRB. In summary, our analysis suggests the importance for the multivariate space of an inferred ERα transcriptional regulatory network in breast cancer diagnostic and therapeutic development.
Chrysin, a flavonoid obtained from various natural sources, has been reported to possess anti-inflammatory, antitumor, antioxidant and anti-allergic activities. However, its anti-inflammatory and immunoregulatory activities in asthma animal models are poorly understood. In the present study, we examined the effects of chrysin on airway inflammation and the possible mechanisms through which it acts in a murine model of allergic asthma. BALB/c mice sensitized and challenged to ovalbumin (OVA) were administered intragastrically with chrysin at a dose of 50 mg/kg daily. Chrysin significantly suppressed OVA-induced airway hyperresponsiveness (AHR) to acetylcholine chloride (Ach). Chrysin administration significantly inhibited the total inflammatory cell and eosinophil counts in bronchoalveolar lavage fluid (BALF) and total immunoglobulin E (IgE) levels in serum. Histological examination of lung tissue demonstrated that chrysin significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. In addition, chrysin triggered a switch of the immune response to allergens towards a T-helper type 1 (Th1) profile by modulating the transcription factors T-bet and GATA-3 in allergic mice. These data suggest that chrysin exhibits anti-inflammatory and immunoregulatory properties and provides new insights into the immunopharmacological role of chrysin in terms of its effects in a murine model of asthma.
Epidermal Langerhans cells (LCs) are dendritic APCs that play an important role in cutaneous immune responses. LCs are associated with epidermal nerves and the neuropeptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) inhibit LC Ag presentation for Th1-type immune responses. Here, we examined whether PACAP or VIP modulates LC Ag presentation for induction of IL-17A-producing CD4(+) T cells. Treatment with VIP or PACAP prior to in vitro LC Ag presentation to CD4(+) T cells enhanced IL-17A, IL-6, and IL-4 production, decreased interferon (IFN)-γ and interleukin (IL)-22 release, and increased RORγt and Gata3 mRNA expression while decreasing T-bet expression. The CD4(+) T-cell population was increased in IL-17A- and IL-4-expressing cells and decreased in IFN-γ-expressing cells. Addition of anti-IL-6 mAb blocked the enhanced IL-17A production seen with LC preexposure to VIP or PACAP. Intradermal administration of VIP or PACAP prior to application of a contact sensitizer at the injection site, followed by harvesting of draining lymph node CD4(+) T cells and stimulation with anti-CD3/anti-CD28 mAbs, enhanced IL-17A and IL-4 production but reduced production of IL-22 and IFN-γ. PACAP and VIP are endogenous mediators that likely regulate immunity and immune-mediated diseases within the skin.
In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.
Puumala hantavirus (PUUV) infection, also known as nephropathia epidemica, is the most common cause of hemorrhagic fever with renal syndrome (HFRS) in Europe. The pathogenesis of PUUV nephropathia epidemica is complex and multifactorial, and the risk factors for severe acute kidney injury (AKI) during acute PUUV infection are not well defined. We conducted a prospective study of hospitalized patients with PUUV infection in Tampere, Finland to identify acute illness risk factors for HFRS severity. Serial daily blood and urine samples were collected throughout acute illness and at 2 week and 6 month convalescent visits. By univariate analyses, the maximum white blood cell count during acute illness was a risk factor for severe AKI. There were no significant associations between PUUV-induced AKI severity and platelet counts, C-reactive protein, or alanine aminotransferase levels. Maximum plasma interleukin (IL)-6, urine IL-6, and urine IL-8 concentrations were positively associated with PUUV-induced AKI. Finally, the maximum urinary sediment GATA-3 mRNA level was positively correlated with the peak fold-change in serum creatinine, regardless of AKI severity classification. By multivariate analyses, we found that the maximum levels of leukocytes and urinary sediment GATA-3 mRNA during acute illness were independent risk factors for severe PUUV-induced AKI. We have identified novel acute illness risk factors for severe PUUV-induced AKI.
Allergic asthma is a common inflammatory disease regulated by the T helper (Th) cells. Interferon regulatory factor 4 (IRF4) plays an important role in the differentiation of Th cells. This study investigated whether IRF4 is involved in the systemic immune responses in allergic asthma patients.
Peripheral blood mononuclear cells (PBMCs) were isolated from allergic asthmatics (n = 29) and healthy controls (n = 12). The mRNA and protein levels of IRF4 in PBMCs were measured by quantitative RT-PCR and western blotting. The frequencies of Th1, Th2, and Th17 cells were analyzed by flow cytometry. The related cytokine (interferon-γ, interleukin-4 (IL-4), and IL-17) concentrations in plasma and culture supernatants were measured by ELISA. The levels of GATA binding protein 3 (GATA3), retinoic acid-related orphan receptor γt (RORγt), and forkhead box P3 (FOXP3), the master transcription factors controlling Th2, Th17, and T regulatory cells differentiation, respectively, were examined by quantitative RT-PCR.
The levels of IRF4 were elevated in allergic asthmatics compared with those in healthy controls. The frequencies of Th2 and Th17 cells as well as the concentrations of Th2- and Th17-related cytokines were higher in plasma from asthma patients than those from healthy controls; similar results were observed in culture supernatants. IRF4 mRNA levels were positively correlated with GATA3, RORγt, and FOXP3 mRNA levels in allergic asthmatics but not in healthy controls.
Our results suggested that IRF4 may play a role in the systemic inflammation of allergic asthma patients by facilitating the differentiation of Th2 and Th17 cells at least at the transcriptional level.
The downstream of kinase (DOK)-1 is involved in the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in the pathogenesis of asthma has not been defined. In this study, we have demonstrated a novel regulatory role of DOK-1 in airway inflammation and physiologic responses in a murine model of asthma using lentiviral vector containing DOK-1 cDNA or DOK-1-specific ShRNA. The OVA-induced inflammatory cells, airway hyperresponsiveness, Th2 cytokine expression, and mucus response were significantly reduced in DOK-1 overexpressing mice compared to OVA-challenged control mice. The transgenic introduction of DOK-1 significantly stimulated the activation and expression of STAT-4 and T-bet, while impressively inhibiting the activation and expression of STAT-6 and GATA-3 in airway epithelial cells. On the other hand, DOK-1 knockdown mice enhanced STAT-6 expression and its nuclear translocation compared to OVA-challenged control mice. When viewed in combination, our studies demonstrate DOK-1 regulates allergen-induced Th2 immune responses by selective stimulation and inhibition of STAT-4 and STAT-6 signaling pathways, respectively. These studies provide a novel insight on the regulatory role of DOK-1 in allergen-induced Th2 inflammation and airway responses, which has therapeutic potential for asthma and other allergic diseases.
Transplant glomerulopathy is an important cause of late graft loss. Inflammatory lesions including glomerulitis and peritubular capillaritis, suggestive of endothelial injury, are prominent in this condition but the mechanism underlying this inflammation remains unclear. Here we measured the expression of T-bet (a member of the T-box family of transcription factors regulating Th1 lineage commitment) and its relationship with inflammation in 70 patients with transplant glomerulopathy. Within this cohort, 32 patients were diagnosed with transplant glomerulopathy, 23 with interstitial fibrosis/tubular atrophy, and 15 with stable grafts. There was a significant increase in T-bet expression in both glomerular and peritubular capillaries of the transplant glomerulopathy group. This expression was strongly correlated with CD4(+), CD8(+), and CD68(+) cell infiltration within glomerular and peritubular capillaries. The expression of GATA3, a Th2 regulator, was rarely found in the transplant glomerulopathy group. Transplant glomerulopathy was associated with diffuse peritubular capillary dilation without reduced capillary density. Moreover, the degree of capillary dilation was significantly correlated with the number of infiltrating CD68(+) cells. Since endothelial injury is a typical lesion that follows alloantibody reactivity, our results suggest that T-bet is involved in the pathogenesis of this glomerulopathy.
Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.
Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the -237 polymorphic site in the 5' promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis.
Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. Previous studies have shown that genistein decreased airway inflammation in allergic asthma. However, studies on how genistein affects immunoreactions in asthma are very limited.
It was the aim of this study to investigate the effect of genistein on T helper 1 (Th1)/Th2 cytokines in a murine asthma model and to explore its underlying mechanisms.
The asthma model was set up both in vivo and in vitro: the mice were divided into four groups in vivo, i.e. control group, ovalbumin-sensitized (OVA) group, Gen20 group (20 mg/kg genistein) and Gen40 group (40 mg/kg genistein), and into three groups in vitro, i.e. control group, OVA group, genistein group. Changes in lung histology were observed and concentrations of interleukin-4, interleukin-5 and interferon-γ in bronchoalveolar lavage fluid and serum were measured by enzyme-linked immunosorbent assay. The mRNA expression of GATA binding protein 3 (GATA-3), signal transducer and activator of transcription 6 (STAT-6) and T-box transcription factor (T-bet) in the lungs and CD4+ T cells of each group were detected by real-time PCR and the corresponding proteins were detected by Western blot.
The results showed that genistein attenuated OVA-induced airway inflammation, decreased Th2-type cytokines and increased Th1-type cytokines. Additionally, our data suggested that genistein may modulate the Th1/Th2 reaction by inhibiting GATA-3 and STAT-6 production while increasing T-bet production.
Genistein may modulate the immunomodulatory actions caused by Th1/Th2 cytokines in asthma, at least partially, by the down-regulation of GATA-3 and STAT-6 and the up-regulation of T-bet.
T cell development comprises a stepwise process of commitment from a multipotent precursor. To define molecular mechanisms controlling this progression, we probed five stages spanning the commitment process using RNA-seq and ChIP-seq to track genome-wide shifts in transcription, cohorts of active transcription factor genes, histone modifications at diverse classes of cis-regulatory elements, and binding repertoire of GATA-3 and PU.1, transcription factors with complementary roles in T cell development. The results highlight potential promoter-distal cis-regulatory elements in play and reveal both activation sites and diverse mechanisms of repression that silence genes used in alternative lineages. Histone marking is dynamic and reversible, and though permissive marks anticipate, repressive marks often lag behind changes in transcription. In vivo binding of PU.1 and GATA-3 relative to epigenetic marking reveals distinctive factor-specific rules for recruitment of these crucial transcription factors to different subsets of their potential sites, dependent on dose and developmental context.
Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively.
Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively.
Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.
Erythropoietin acts by binding to its cell surface receptor on erythroid progenitor cells to stimulate erythrocyte production. Erythropoietin receptor expression in nonhematopoietic tissue, including skeletal muscle progenitor cells, raises the possibility of a role for erythropoietin beyond erythropoiesis. Mice with erythropoietin receptor restricted to hematopoietic tissue were used to assess contributions of endogenous erythropoietin to promote skeletal myoblast proliferation and survival and wound healing in a mouse model of cardiotoxin induced muscle injury. Compared with wild-type controls, these mice had fewer skeletal muscle Pax-7(+) satellite cells and myoblasts that do not proliferate in culture, were more susceptible to skeletal muscle injury and reduced maximum load tolerated by isolated muscle. In contrast, mice with chronic elevated circulating erythropoietin had more Pax-7(+) satellite cells and myoblasts with increased proliferation and survival in culture, decreased muscle injury, and accelerated recovery of maximum load tolerated by isolated muscle. Skeletal muscle myoblasts also produced endogenous erythropoietin that increased at low O(2). Erythropoietin promoted proliferation, survival, and wound recovery in myoblasts via the phosphoinositide 3-kinase/AKT pathway. Therefore, endogenous and exogenous erythropoietin contribute to increasing satellite cell number following muscle injury, improve myoblast proliferation and survival, and promote repair and regeneration in this mouse induced muscle injury model independent of its effect on erythrocyte production.
Mucosal cytokine profile determines T cell differentiation and may play an important role in the clinical course of inflammatory bowel disease (IBD). Cytokines from different T helper (Th) cell subsets are elevated in inflamed mucosa of patients with ulcerative colitis (UC), contributing to the inflammation. The aim of this study was to determine the predictive value of pre-treatment mucosal cytokine profile in response to therapy with the anti-TNF agent infliximab (IFX).
The expression of Th1, Th17, Th2 and T-regulatory (Treg)-related cytokines was quantified by real-time PCR in mucosal biopsies from 74 UC patients before initiation of IFX induction therapy. Clinical and endoscopic effects were assessed after three infusions. Remission was defined as ulcerative colitis disease activity index (UCDAI) below 3.
Higher gene expression levels of IL-17A and IFN-γ were significantly associated with remission after three IFX infusions (OR = 5.4, p = 0.013 and OR = 5.5, p = 0.011, respectively). IL-17A and IFN-γ mRNA expression showed positive correlation. Th2 and Treg-related mediators were not significantly associated with clinical outcome, but were expressed at higher levels in UC patients compared with the controls. Immunohistochemistry (IHC) confirmed the presence of cells expressing both IL-17A and IFN-γ.
High expression of Th1- and Th17-related cytokines in the mucosa of UC patients can potentially predict a favorable outcome of IFX induction therapy. Th2 and Treg-related mediators do not appear useful as predictive markers.
Naïve CD4(+) T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4(+) T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4(+) T cell plasticity and long-term CD4(+) T cell memory.
Cytokine signaling pathways play a central role in the pathogenesis of inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease (CD) have unique as well as overlapping phenotypes, susceptibility genes, and gene expression profiles. This study aimed to delineate patterns within cytokine signaling pathways in colonic mucosa of UC and CD patients, explore molecular diagnostic markers, and identify novel immune mediators in IBD pathogenesis.
We quantified 70 selected immune genes that are important in IBD signaling from formalin-fixed, paraffin-embedded (FFPE) colon biopsy samples from normal control subjects and UC and CD patients having either severe colitis or quiescent disease (n = 98 subjects). We utilized and validated a new modified real-time reverse-transcription polymerase chain reaction (RT-PCR) technique for gene quantification.
Expression levels of signaling molecules including IL-6/10/12/13/17/23/33, STAT1/3/6, T-bet, GATA3, Foxp3, SOCS1/3, and downstream inflammatory mediators such as chemokines CCL-2/11/17/20, oxidative stress inducers, proteases, and mucosal genes were differentially regulated between UC and CD and between active and quiescent disease. We also document the possible role of novel genes in IBD, including SHP-1, IRF-1,TARC, Eotaxin, NOX2, arginase I, and ADAM 8.
This comprehensive approach to quantifying gene expression provides insights into the pathogenesis of IBD by elucidating distinct immune signaling networks in CD and UC. Furthermore, this is the first study demonstrating that gene expression profiling in FFPE colon biopsies might be a practical and effective tool in the diagnosis and prognosis of IBD and may help identify molecular markers that can predict and monitor response to individualized therapeutic treatments.
Nasal polyposis (NP) is a Th2-skewed inflammatory disorder, but it is unclear what role regulatory T cells (T-reg) play in disease pathology. We investigated the expression profiles of T-reg and T-helper-cell-associated genes and their response to glucocorticosteroid (GC) treatment in Chinese patients with NP.
Biopsies were obtained from 29 non-treated NP patients for comparison with inferior turbinates collected from healthy controls. In 13 patients, NP samples were collected both before and after short-term oral GC treatment. Levels of mRNA for T-cell markers were determined by microarray and quantitative PCR. Cellular infiltrates were assessed by histo- and immunohistochemistry.
FOXP3(+) T-reg were increased in GC-naïve NP, and numbers were negatively correlated with eosinophil infiltration. Helios staining was not detected, suggesting that FOXP3(+) cells in NP are not thymus-derived T-reg. Compared with controls, mRNA levels corresponding to T-reg genes were significantly increased in NP (FOXP3, TGFB1, IL10, SMAD3, IL2RA, and JAK3), but transcription factors associated with Th2 (GATA3) or Th17 responses (RORc) were significantly reduced. FOXP3 mRNA levels positively correlated with other T-reg cell markers. Microarray analysis showed that most Th2-related markers (e.g., Eotaxin-1, CCL13, and CCL18) were upregulated in GC-naïve NP vs controls. GC therapy significantly suppressed eosinophilic inflammation in NP, but did not significantly alter the expression levels of T-reg/Th2-associated genes.
Upregulation of FOXP3(+) -inducible T-reg cells and downregulation of Th2 and Th17 markers in NP indicate a regulatory response occurring at a site of persistent mucosal inflammation. However, immune regulation fails to control the underlying tissue pathology. Expression of T-reg/Th2 markers after GC treatment was unaltered, suggesting that T-cell-driving NP inflammatory mediators are GC resistant.
Cell surface 6-sulfated glycans play important roles in various immunological events through cell-to-cell interactions. The 6-sulfation process is mediated by 6-sulfotransferase family isoenzymes. We previously demonstrated that GlcNAc6ST-1, one of the isoenzyme genes, is induced by GATA-3 and NF-κB in human helper T (Th) cells. However, transcriptional regulation of HEC-GlcNAc6ST, another isoenzyme important in Th cells, remains unclear.
5'-RACE analysis, chromatin immunoprecipitation, and reporter assays were performed to reveal transcriptional regulation of HEC-GlcNAc6ST. RNA-knockdown and forced expression experiments were performed to demonstrate the contribution of HEC-GlcNAc6ST to the 6-sulfated glycan expression.
We identified potential binding sites of Sp1, T-bet, and GATA-3 in the HEC-GlcNAc6ST promoter. Reporter assays indicated that transfection of Sp1 enhanced the activity, whereas mithramycin A, an Sp1-specific inhibitor, repressed it. Transfection of T-bet increased the activity, which was inhibited by introducing a mutation into the potential T-bet binding site. GATA-3 alone could not elevate the activity, although co-transfection of protein kinase A, which is known to enhance IL-5 transcription in Th2 cells through phosphorylation of GATA-3, caused elevation. RNA-knockdown and forced expression of HEC-GlcNAc6ST in Jurkat cells down- and up-regulated α2,6-sialylated 6-sulfo N-acetyllactosamine, a preferential ligand for B-cell-specific CD22 antigen, respectively. From these results, we concluded that T-bet and GATA-3 as well as Sp1 control the expression of glycan with cell-adhesion activity by regulating HEC-GlcNAc6ST transcription in Th cells.
These results may provide a clue to biological regulation of Th-cell interaction with selectins and other carbohydrate-recognition molecules by T-bet and GATA-3.
Allergic diseases are characterized by an overreaction characterized by Th2-type cell response, and as a consequence, an IgE-switched B cell immunity. Obviously, type-2 cytokines (IL-4, IL-5, IL-9, IL-13) and particularly IL-4 have been identified as potential targets for allergy treatment. While initial experiences using anti-IL-4 principles in asthma were rather ambiguous, more recent studies using an IL-4 mute in blocking the IL-4 and IL-13 receptor have shown promising results. Furthermore, our understanding of IL-4 biology is more specific and may promote more targeted interventions. A key function of IL-4 is the induction of 'master switch' transcription factor GATA3 that drives Th2 differentiation and also effectively inhibits the induction of regulatory T cells. Consequences for treatment of allergic diseases are also discussed.
DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases.
T cell Ig and ITIM domain (TIGIT) is a newly identified receptor expressed on T cells that binds to CD155 on the dendritic cell surface, driving them to a more tolerogenic phenotype. Given that TIGIT contains an ITIM motif in its intracellular domain and considering the potential importance of the TIGIT/CD226 pathway in human autoimmune disease, we investigated the specific role of TIGIT in human CD4(+) T cells. Using an agonistic anti-TIGIT mAb, we demonstrate a direct inhibitory effect on T cell proliferation with a decrease in expression of T-bet, GATA3, IFN regulatory factor 4, and retinoic acid-related orphan receptor c with inhibition of cytokine production, predominantly IFN-γ. Knockdown of TIGIT expression by short hairpin RNA resulted in an increase of both T-bet and IFN-γ mRNA and protein expression with concomitant decrease in IL-10 expression. Increases in IFN-γ with TIGIT knockdown could be overcome by blocking CD226 signaling, indicating that TIGIT exerts immunosuppressive effects by competing with CD226 for the same CD155 ligand. These data demonstrate that TIGIT can inhibit T cell functions by competing with CD226 and can also directly inhibit T cells in a T cell-intrinsic manner. Our results provide evidence for a novel role of this alternative costimulatory pathway in regulating human T cell responses associated with autoimmune disease.
Our previous investigation demonstrated that ginsenoside Rg3 was active in promotion of the immune response. In this study, two epimers, 20(R)-Rg3 and 20(S)-Rg3, were compared for their adjuvant effects on the immune response against ovalbumin (OVA). BALB/c mice were immunized subcutaneously with 10 μg of OVA alone or with 10 μg of OVA mixed in 20(R)-Rg3 (50 μg) or 20(S)-Rg3 (50 μg) on days 1 and 15. Two weeks after the last immunization, blood was sampled to test IgG and the IgG subclasses as well as IFN-γ and IL-5; splenocytes were prepared to measure proliferative responses to stimulations with Con A, LPS and OVA and mRNA expressions of cytokines and transcription factors by reverse transcription-PCR. Results indicated that both 20(R)-Rg3 and 20(S)-Rg3 exhibited the adjuvant effect on OVA-induced immune responses. 20(R)-Rg3 promoted significantly higher serum-specific IgG and the IgG isotype responses in association with highly up-regulated serum IFN-γ and IL-5 than 20(S)-Rg3. In addition, 20(R)-Rg3 significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as mRNA expression of IFN-γ, IL-12, IL-4 and IL-10 and transcription factors T-bet and GATA-3 by splenocytes when compared with the 20(S)-Rg3. Therefore, ginsenoside Rg3 is stereospecific in stimulation of the immune response, and 20(R)-Rg3 has more potent adjuvant activity than 20(S)-Rg3.
The double-positive (DP) to single-positive (SP) transition during T cell development is initiated by downregulation of the E protein transcription factors HEB and E2A. Here, we have demonstrated that in addition to regulating the onset of this transition, HEB and E2A also play a separate role in CD4(+) lineage choice. Deletion of HEB and E2A in DP thymocytes specifically blocked the development of CD4(+) lineage T cells. Furthermore, deletion of the E protein inhibitors Id2 and Id3 allowed CD4(+) T cell development but blocked CD8(+) lineage development. Analysis of the CD4(+) lineage transcriptional regulators ThPOK and Gata3 placed HEB and E2A upstream of CD4(+) lineage specification. These studies identify an important role for E proteins in the activation of CD4(+) lineage differentiation as thymocytes undergo the DP to SP transition.
Depending on polarizing cytokine signals during activation by antigen, naïve CD4+ T cells can be stimulated and differentiated into distinct functional CD4+ T cell subsets such as Th1, Th2 and Tr1 cells. Among them, Th2 cells are pathogenic in allergic diseases such as asthma, which are characterized by transcription factor GATA3 expression and IL-4, IL-5, IL-6, and IL-10 cytokine secretion. The overlapping expression of some signature cytokines by Th2 and other subsets of CD4+ T cells may not only indicate the plasticity of CD4+ T cells, but could also suggest the possibility of the deletion of a single signature cytokine gene leading to the functional differentiation of naïve CD4+ T cells into effector Th1 or Tr1 cells under Th2 differentiation conditions. In this work, we stimulated naïve CD4+ T cells derived from OT II mice or OT II mice that were deficient in individual cytokines (IL-4, IL-5, IL-6 and IL-10) with OVA-pulsed dendritic cells (DC(OVA)) in the presence of IL-4 and anti-IFN-γ, to generate OVA-specific wild-type (WT) Th2, and Th2(IL-4 KO), or Th2(IL-5 KO), or Th2(IL-6 KO), or Th2(IL-10 KO) cells, and to assess their capacity in modulating DC(OVA)-induced CD8+ cytotoxic T lymphocyte (CTL) responses, and antitumor immunity in WT C57BL/6 mice. We conclusively demonstrate that GATA-3-expressing Th2 cells enhance DC(OVA)-induced CTL responses via IL-6 secretion. We also show that IL-6 and IL-10 gene deficient Th2(IL-6 KO) and Th2(IL-10 KO) cells, but not IL-4 and IL-5 gene deficient Th2(IL-4 KO) and Th2(IL-5 KO) cells, behave like functional Tr1 and Th1 cells by inhibiting and enhancing DC(OVA)-induced OVA-specific CD8+ CTL responses and antitumor immunity, respectively. We further elucidate that inhibition and enhancement of DC(OVA)-induced OVA-specific CTL responses by Th2(IL-6 KO) and Th2(IL-10 KO) cells are mediated by their immune suppressive IL-10 and pro-inflammatory IL-6 secretion, respectively. Taken together, our study suggests that deletion of a single cytokine gene IL-6 and IL-10 makes CD4+ Th2 cells become effector CD4+ Tr1- and Th1-like cells, respectively. Our data thus not only provide new evidence for another type of CD4+ T cell plasticity, but also have a potential to impact the development of a new direction in immunotherapy of allergic diseases.
T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.
Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.
This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.
Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical practice. The pathogenesis of DILI usually involves the participation of the parent drug or metabolites that either affect cellular function or elicit an immune response. However, the mechanisms leading to DILI are unknown in most cases. Methimazole (MTZ) is used as an antithyroid drug and is well known to have induced liver injuries such as cholestatic hepatitis in a small number of human cases. Immune-mediated reactions were also suggested to play a role in MTZ-induced acute liver injury, but the mechanism underlying this process has not been elucidated. To address this issue, we measured plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, hepatic glutathione levels, hepatic expression of CD4⁺ Th cell-related transcriptional factors, cytokines and chemokines, plasma interleukin (IL)-4 levels and histopathological changes in the liver following MTZ (450 mg kg⁻¹ , p.o.) administration in mice. The hepatic expression of mRNA for Th2 cell-related factors, such as GATA-binding protein, macrophage inflammatory protein-2 (MIP-2) and plasma IL-4 levels, as well as plasma AST and ALT levels, was significantly increased in mice treated with MTZ. These changes were markedly enhanced by pre-treatment with L-buthionine sulfoximine (3 mmol kg⁻¹, i.p.) and MTZ (15 mg kg⁻¹, p.o.). Neutralization of IL-4 using a monoclonal anti-mouse IL-4 antibody (100 µg/mouse, single i.p.) suppressed the hepatotoxic effect of MTZ. In conclusion, this report is the first to demonstrate that Th2 cytokine-mediated immune responses are involved in MTZ-induced acute liver injury in mice.
Haploinsufficiency of a region located distal to 10p14 designated HDR1, is responsible for hypoparathyroidism, sensorineural deafness, and renal anomalies (HDR syndrome). Haploinsufficiency of a more proximal region, located on 10p13-10p14, designated as DGCR2 is associated with congenital heart defects and thymus hypoplasia/aplasia or T cell defect. We describe a patient showing facial dysmorphisms, delayed psychomotor development and bilateral sensorineural hearing loss and carrying a 10p14 deletion, the smallest deletion found in the literature so far. Our patient, carrying a partial deletion of the DGCR2 region and of the HDR1 region, including the GATA3 gene, showed, unexpectedly, only few of the clinical features of DiGeorge 2 syndrome (psychomotor retardation, palpebral ptosis, epicanthic folds, anteverted nares, cryptorchidism, hand/foot abnormalities) and did not show other typical signs, such as cardiac defect, cleft palate, and abnormal T cell levels. Of the three characteristic features of the HDR syndrome, our patient had only sensorineural deafness. On the basis of the revision of the other cases reported in the literature with a deletion including the 10p14 region, we suggest that GATA3 haploinsufficiency, although not recorded for each patient, is responsible for deafness. The present case shows that even this small 10p deletion is responsible for a specific phenotype. We also underline the importance of CGH-array, in order to obtain a more precise physical mapping of the 10p deletions and an accurate genotype-phenotype correlation.
We review the molecular basis of auditory development and evolution. We propose that the auditory periphery (basilar papilla, organ of Corti) evolved by transforming a newly created and redundant vestibular (gravistatic) endorgan into a sensory epithelium that could respond to sound instead of gravity. Evolution altered this new epithelia's mechanoreceptive properties through changes of hair cells, positioned the epithelium in a unique position near perilymphatic space to extract sound moving between the round and the oval window, and transformed its otolith covering into a tympanic membrane. Another important step in the evolution of an auditory system was the evolution of a unique set of "auditory neurons" that apparently evolved from vestibular neurons. Evolution of mammalian auditory (spiral ganglion) neurons coincides with GATA3 being a transcription factor found selectively in the auditory afferents. For the auditory information to be processed, the CNS required a dedicated center for auditory processing, the auditory nuclei. It is not known whether the auditory nucleus is ontogenetically related to the vestibular or electroreceptive nuclei, two sensory systems found in aquatic but not in amniotic vertebrates, or a de-novo formation of the rhombic lip in line with other novel hindbrain structures such as pontine nuclei. Like other novel hindbrain structures, the auditory nuclei express exclusively the bHLH gene Atoh1, and loss of Atoh1 results in loss of most of this nucleus in mice. Only after the basilar papilla, organ of Corti evolved could efferent neurons begin to modulate their activity. These auditory efferents most likely evolved from vestibular efferent neurons already present. The most simplistic interpretation of available data suggest that the ear, sensory neurons, auditory nucleus, and efferent neurons have been transformed by altering the developmental genetic modules necessary for their development into a novel direction conducive for sound extraction, conduction, and processing.
The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits β1, β2, and/or β5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the 'immunoproteasome'. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes.
A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1-/- mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified.
The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rβ, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1.
Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.
The aim of this study was to investigate the effects of transcription factors T-bet, GATA-3 in the pathogenesis of Hench-Schonlein purpura (HSP) in children, the relationship between CD4(+)CD25(+)regulatory T cells, transcription factor FoxP3 and the development of child HSP, and the molecular mechanisms of Th1/Th2 imbalance of child HSP at acute phase, so as to may provide a new approach and strategy for the treatment of HSP at the molecular levels. The expression of T-bet, GATA-3 and FoxP3 mRNA were detected by real time PCR using SYBR Green I in 46 patients with HSP at acute phase and 30 healthy children as controls. The expression of T lymphocyte subsets CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative level of GATA-3 mRNA in peripheral blood mononuclear cells of patients with HSP was significantly higher than those of the control group (964.30 ± 655.18 vs 78.09 ± 57.20, P < 0.01). The relative level of T-bet mRNA in peripheral blood mononuclear cells of patients with HSP was lower than those of the control group (53.98 ± 35.79 vs 181.56 ± 96.90, P < 0.01). The expression level of FoxP3 mRNA with HSP was lower than that of the control group (32.17 ± 23.04 vs 147.91 ± 99.15, P < 0.01). The result of CD4(+)CD25(+) Treg with HSP was lower than those of the control group [(5.34 ± 2.51)% vs (7.85 ± 1.97)%, P < 0.01)]. It is concluded that Th1/Th2 imbalance exists in acute phase of child HSP, especially predominant activation of Th2, which correlates with the abnormal expression of transcription factor T-bet and GATA-3 mRNA. At acute phase of child HSP, the expression of CD4(+)CD25(+)Treg and its special transcription factor FoxP3 mRNA are down-regulated. Treg cells decreases, which indicates that insufficient immunosuppressive effects resulting from the reduction of Treg cells may be one of the important reason in the immune imbalance of HSP acute phase. This study provides experimental evidence for illustrating the pathogenesis of HSP from the molecular mechanism of Treg cells and its regulation, and also provides a new thinking and new strategies for the treatment of HSP at molecular levels.
Invariant NKT (iNKT) cells are a conserved αβTCR(+) T cell population that can swiftly produce large amounts of cytokines, thereby activating other leukocytes, including neutrophilic granulocytes (neutrophils). In this study, we investigated the reverse relationship, showing that high neutrophil concentrations suppress the iNKT cell response in mice and humans. Peripheral Vα14 iNKT cells from spontaneously neutrophilic mice produced reduced cytokines in response to the model iNKT cell Ag α-galactosyl ceramide and expressed lower amounts of the T-box transcription factor 21 and GATA3 transcription factor than did wild-type controls. This influence was extrinsic, as iNKT cell transcription factor expression in mixed chimeric mice depended on neutrophil count, not iNKT cell genotype. Transcription factor expression was also decreased in primary iNKT cells from the neutrophil-rich bone marrow compared with spleen in wild-type mice. In vitro, the function of both mouse and human iNKT cells was inhibited by coincubation with neutrophils. This required cell-cell contact with live neutrophils. Neutrophilic inflammation in experimental peritonitis in mice decreased iNKT cell T-box transcription factor 21 and GATA3 expression and α-galactosyl ceramide-induced cytokine production in vivo. This was reverted by blockade of neutrophil mobilization. Similarly, iNKT cells from the human peritoneal cavity expressed lower transcription factor levels during neutrophilic peritonitis. Our data reveal a novel regulatory axis whereby neutrophils reduce iNKT cell responses, which may be important in shaping the extent of inflammation.
During pregnancy the maternal immune system has to coordinate uterine spiral-artery remodelling, trophoblast invasion, and acceptance of the semi-allogenic fetus simultaneously. As dysregulation of the immune system is associated with adverse pregnancy outcomes, we analysed first-trimester deciduas of pregnancies for immune parameters in later complicated pregnancies. Higher IL6 and macrophage mRNA expression, and lower ratios of regulatory macrophages were found in first-trimester deciduas of pregnancies later complicated with pregnancy-induced hypertension. Lower Gata3 (Th2) mRNA expression was found in deciduas of pregnancies with later foetal growth restriction. Our results suggest that adverse pregnancy outcomes are associated with immunological disturbances in first-trimester deciduas.
To explore the expression of T-bet/GATA-3 in nasal mucosa tissue of allergic rhinitis rat and to investigate the association between the expression of T-bet/GATA-3 and the eosinophil count.
Twenty SD rats were randomly divided into a control group and an allergic rhinitis group. The allergic rhinitis rat model was induced with ovalbumin. The total eosinophils were counted in the nasal mucosa. The concentrations of IL-4, IL-5 and IFN-gamma in nasal lavage fluid were measured by ELISA. The mRNA and protein expressions of IL-4, IL-5, IFN-gamma, T-bet and GATA-3 in the nasal mucosa were detected by RT-PCR and Western blot respectively.
The main inflammatory cells were eosinophils in the nasal mucosa of allergic rhinitis rats. The level of IL-4, IL-5 and IFN-gamma in control group was significantly higher than that in allergic rhinitis group (P < 0.01). The mRNA and protein expression of IFN-gamma and T-bet in allergic rhinitis group was significantly higher than that in control group (P < 0.01). While the mRNA and protein expression of IL-4, IL-5 and GATA-3 in control group was significantly higher than that in allergic rhinitis group (P < 0.01). The ratio of protein expression of T-bet and GATA-3 was negatively correlated with the eosinophil count, IL-4 and IL-5, but positively with the concentrations of IFN-gamma.
The imbalance of transcription factor GATA-3 and T-bet has a close correlation with the eosinophil count, and may play a key role in the formation of allergic rhinitis.
The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4(+) splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.
In asthma, T helper 2 (T(H)2)-type cytokines such as interleukin (IL)-4, IL-5, and IL-13 are produced by activated CD4(+) T cells. Dendritic cells played an important role in determining the fate of naive T cells into either T(H)1 or T(H)2 cells. We determined whether RG-II regulates the T(H)1/T(H)2 immune response by using an ovalbumin-induced murine model of asthma. RG-II reduced IL-4 production but increased interferon- gamma production, and inhibited GATA-3 gene expression. RG-II also inhibited asthmatic reactions including an increase in the number of eosinophils in bronchoalveolar lavage fluid, an increase in inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyperresponsiveness. This study provides evidence that RG-II plays a critical role in ameliorating the pathogenic process of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of RG-II in terms of its effects in a murine model of asthma.
To study mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4 and T-bet) in peripheral blood of the patients with allergic dermatitis induced by trichloroethylene (TCE).
The peripheral blood samples were collected from 8 healthy workers (control group) and 8 patients with allergic dermatitis induced by TCE (case group). Real-time quantitative PCR was applied to detect mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4, T-bet).
The mRNA expression levels of Foxp3, GATA3 and CTLA4 genes increased by 115%, 97% and 241% in case group, as compared with control group (P < 0.01). The mRNA expression level of T-bet gene decreased by 47% in case group, as compared with control group (P < 0.01).
The mRNA expression levels of some immune-related genes changed in patients with allergic dermatitis induced by TCE, those genes may play an important role in TCE-induced allergy.
The objective of the present study was to evaluate the expression of T-box expressed in T cells (T-bet) and GATA binding protein 3 (GATA-3) in the eutopic endometrium from women with endometriosis. Endometrial tissues were collected from 20 women with laparoscopically confirmed endometriosis and 20 women without endometriosis. T-bet and GATA-3 expression was measured by quantitative real-time PCR (qPCR), immunohistochemistry and Western blot analysis. Eutopic endometrial tissues from patients with endometriosis expressed lower levels of T-bet mRNA and high levels of GATA-3 mRNA, leading to a significant lower T-bet/GATA-3 mRNA ratio (P<0.05). Western blot analysis showed that the T-bet/GATA-3 protein ratio in endometriosis group was also statistically lower than that in the control group (P<0.05). These results suggested that T-bet and GATA-3 may act as cytokine regulatory genes, and the Th2-specific transcription factor, GATA-3, probably plays an essential role in the immune response and the development of endometriosis.
As a member of the T cell immunoglobulin domain and mucin domain (TIM) gene family, TIMD4 plays an important role in the immune response. To understand its function more precisely, we isolated it and analyzed its subcellular localization, expression pattern, and associations. The porcine TIMD4 gene included nine exons and eight introns with an open reading frame of 1086 bp encoding 361 amino acids. It had relatively high levels in liver, lymph, and spleen. The fusion protein was localized mainly in the cytoplasm of pig kidney cells (PK15). The promoter region contained a TATA box and GATA3 consensus sites. A single nucleotide polymorphism was identified in intron 3 of the porcine TIMD4 gene, and analysis indicated that it had significant associations with the 17-day red blood cell count (p = 0.0106), hemoglobin (p = 0.0149), and hematocrit (p = 0.0063) and with 32-day hemoglobin (p = 0.0140).
Changes in the Th1/Th2 immune balance may play a role in increasing the incidence of radiation-induced toxicity. This study evaluates the consequences of Th1 deficiency on intestinal response (fibrosis and T cell trafficking) to abdominal irradiation and examines in mucosa and mesenteric lymph nodes (MLN) the differential involvement of the two Th1 pathways, T-bet/STAT1 and IL-12/STAT4, in controlling this balance in mice.
Using T-bet-deficient mice (T-bet-/-), we evaluated the mRNA and protein expression of the Th1 pathways (IFN-γ, T-bet/STAT1, and IL-12/STAT4) and the CD4+ and CD8+ populations in ileal mucosa and MLN during the first 3 months after 10 Gy abdominal irradiation.
The T-bet-deficient mice showed an increased fibrotic response to radiation, characterized by higher TGF-β1, col3a1 expression, and collagen deposition in mucosa compared with wild-type mice. This response was associated with drastically lower expression of IFN-γ, the hallmark Th1 cytokine. Analysis of the Th1 expression pathways, T-bet/STAT1 and IL-12/STAT4, showed their equal involvement in the failure of Th1 polarization. A minimal IFN-γ level depended on the IL-23-p19/STAT4 level. In addition, the radiation-induced deficiency in the priming of Th1 by IFN-γ was related to the defective homing capacity of CD8+ cells in the mucosa.
Irradiation induces Th2 polarization, and the Th2 immune response may play a role in potentiating irradiation-induced intestinal collagen deposition.
Recent studies in rodents indicate that the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome and a proinflammatory shift in the T cell population in adipose tissue (AT) contribute to AT inflammation and insulin resistance. We investigated: (1) the interplay between the NLRP3 inflammasome and T cell populations in abdominal subcutaneous AT in obese and lean humans in relation to AT inflammatory processes, and (2) involvement of the NLRP3 inflammasome and T cell populations in insulin resistance. Abdominal subcutaneous AT biopsies were collected in 10 obese men with impaired glucose tolerance and 9 lean normal glucose tolerant age-matched controls. AT gene expression of NLRP3 inflammasome-related genes and markers of T cell populations, chemoattraction, macrophage infiltration and other aspects of inflammation were examined. Furthermore, we examined systemic adaptive immune activation and insulin sensitivity (hyperinsulinemic-euglycemic clamp). CASPASE-1 mRNA and the proportion of T(h)1 transcripts (TBX21/CD3ɛ) were significantly higher in AT from obese compared with lean subjects. CASPASE-1 expression and a relative increase in T(h)1 transcripts in AT were strongly associated with insulin resistance and impairments in glucose homeostasis. Gene expression of NLRP3, CASPASE-1, CD3ɛ (pan T cells), TBX21 (T(h)1 cells) and RORC (T(h)17 cells) was positively, whereas GATA3 (T(h)2 cells) was inversely correlated with AT inflammation. Our data suggest that NLRP3 inflammasome activation and a T(h)1 shift in the T cell population in AT of obese subjects is related to insulin resistance and impaired glucose metabolism, which may be explained by AT inflammatory processes.
The diagnosis of prostatic adenocarcinoma relies on a constellation of architectural, cytological, and immunohistochemical features. Although the diagnosis of prostatic adenocarcinoma is straightforward in most cases, due to earlier detection of the disease in the modern era, pathologists have become increasingly challenged in diagnosing small foci of cancer when only a few atypical glands are present in needle biopsies. Immunohistochemistry has therefore become an essential tool in the evaluation of such foci to confirm the absence of basal cells. In this context, the 2 most commonly used basal cell markers are anti-keratin 34BE12 and p63. Furthermore, α-methylacyl-CoA racemase, a marker found to be overexpressed in the cytoplasm of prostatic adenocarcinoma glands, is also commonly used in routine practice. Another diagnostic role of immunohistochemistry is to confirm the prostatic origin of the tumor in the primary or metastatic setting of high-grade prostatic adenocarcinoma, which may be confused with nonprostatic carcinomas. We herein review the utility as well as the limitations of immunohistochemistry in the diagnosis of prostatic adenocarcinoma, and we describe the most important pitfalls in the interpretation of various immunostains that pathologists should be aware of to minimize misdiagnoses.
To explore the regulatory mechanism of Xiaoyin Recipe () on the T helper 1/T helper 2 (Th1/Th2) immune balance.
Thirty-six experimental animals were divided into three groups, 12 rats in each group: blank control group (B group), negative control group (N group), and Xiaoyin Recipe treatment group (T group). The latter two groups received immunization of experimental autoimmune thyroiditis (EAT), and T group were treated with Xiaoyin Recipe for a month. Then, the expression of Th1-Th2-related genes in peripheral blood mononuclear cells (PBMCs) were screened with Oligo GEArray Rat Th1-Th2-Th3 Microarray. The expressions of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), T-box expressed in T-cells (T-bet), and GATA-binding protein-3 (GATA-3) were detected by real-time polymerase chain reaction (RT-PCR).
Gene array screening showed that compared to N group, in T group after Xiaoyin Recipe treatment, 3 genes were upregulated in EAT rats, including interleukin-27 receptor alpha (IL-27rα), glomulin (Glmn), and GATA-3, while 38 genes were downregulated, such as CD28, IL-18, signal transducer, and activator of transcription 1 (STAT1), T-bet, TNF receptor superfamily member 4 (TNFRSF4), TNF ligand superfamily member 5 (TNFSF5), and TNF receptor superfamily member 5 (TNFRSF5). While RT-PCR showed that there was an increased level of TNF-α mRNA (P<0.01), an elevated ratio of T-bet/GATA-3, and a decreased level of IL-10 mRNA in PBMC of N and T group compared to B group (P <0.01); and after treatment with Xiaoyin Recipe, IL-10 mRNA level increased (P <0.01), while TNF-α mRNA level and T-bet/GATA-3 ratio decreased in T group compared to N group (P <0.01).
Xiaoyin Recipe for psoriasis could induce a Th1/Th2 balance drift toward Th2 in PBMC of EAT rats and thus improve the conditions.
Interleukin 1 receptor antagonist (IL-1Ra)-deficient BALB/c mice develop spontaneous arthritis resembling human rheumatoid arthritis. We herein report that infection with Toxoplasma gondii, an intracellular protozoan, is capable of ameliorating the spontaneous development of arthritis in IL-1Ra-deficient mice. The onset of arthritis development was delayed and the severity score of arthritis was significantly suppressed in T. gondii-infected mice. Expression of IL-12p40 mRNA from CD11c(+) cells of mesenteric lymph nodes (mLN) and spleen markedly increased at 1 week after peroral infection. While CD11c(+) cells also produced IL-10, IL-1β, and IL-6, CD4(+) T cells from T. gondii-infected mice expressed significantly high levels of T-bet and gamma interferon (IFN-γ) mRNA in both mLN and spleen. Levels of GATA-3/IL-4 mRNA or RORγt/IL-17 mRNA decreased in the infected mice, indicating Th1 cell polarization and the reduction of Th2 and Th17 cell polarization. The severity of arthritis was related to Th1 cell polarization accompanied by Th17 cell reduction, demonstrating the protective role of the T. gondii-derived Th1 response against Th17 cell-mediated arthritis in IL-1Ra-deficient mice.
To determine the effect of regulatory T cells on the ratio of Th1/Th2 differentiation in peripheral blood mononuclear cells of chronic hepatitis B patients, FoxP3, the essential transcription factor for Tcells differentiation and function, was knocked down by FoxP3 siRNA, which was affirmed by real-time polymerase chain reaction for decreased expression of FoxP3. Then, at different time points, comparing with control groups, we detected enhanced lymphocyte proliferation and up-regulated Th1-type cytokines, but down-regulated Th2-type cytokines by ELISA. Finally, to research the involved mechanisms, an increased ratio of T-bet/GATA-3 mRNA expression was found by real-time polymerase chain reaction. Our results suggest that siRNA-mediated knockdown of FoxP3 could regulate the immune balance of Th1/Th2 in chronic hepatitis B patients, which may be mediated partly by regulating transcription factors T-bet and GATA-3.
The 40S ribosomal S6 kinase 1 (S6K1) is an important regulator of cell growth. Expression of S6K1 is often elevated in breast cancer cells. However, the transcriptional mechanism of S6K1 overexpression is not understood. In this report, we demonstrate that estrogen activates expression of S6K1 via estrogen receptor (ER)α in ER-positive breast cancer cells. We also show that estrogen acts on the proximal promoter of the S6K1 gene in a mechanism involving the transcriptional factor GATA-3. Finally, we provide data that support the importance of estrogenic regulation of S6K1 expression in breast cancer cell proliferation. S6K1 directly phosphorylates and regulates ligand-independent activity of ERα, while ERα upregulates S6K1 expression. This S6K1-ERα relationship creates a positive feed-forward loop in control of breast cancer cell proliferation. Furthermore, the co-dependent association between S6K1 and ERα may be exploited in the development of targeted breast cancer therapies.
Antipsychotic drugs (APDs) are widely used to alleviate a number of psychic disorders and may have immunomodulatory effects. However, the previous studies of cytokine and immune regulation in APDs are quite inconsistent. The aim of this study was to examine the in vitro effects of different ADPs on cytokine production by peripheral blood mononuclear cells (PBMCs). We examined the effects of risperidone, clozapine, and haloperidol on the production of phorbol myristate acetate and ionomycin-induced interferon-γ (IFN-γ)/interleukin (IL)-4 in PBMCs by using intracellular staining. Real-time quantitative PCR and Western blot were used to further examine the expression changes of some critical transcription factors related to T-cell differentiation in antipsychotic-treated PBMCs. Our results indicated that clozapine can suppress the stimulated production of IFN-γ by 30.62%, whereas haloperidol weakly enhances the expression of IFN-γ. Differences in IL-4 production or in the number of CD4+ T cells were not observed in cells treated with different APDs. Furthermore, clozapine and risperidone inhibited the T-bet mRNA and protein expression, which are critical to Th1 differentiation. Also, clozapine can enhance the expression of Signal Transducer and Activator of Transcription 6 and GATA3, which are critical for the differentiation of Th2 cells. The results suggested that clozapine and haloperidol may induce different immunomodulatory effects on the immune system.
Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.
The pathogenesis of focal segmental glomerulosclerosis (FSGS) appears to be associated with type-2 cytokines and podocyte dysfunction. In this study, we tested the hypothesis that immunization with the polysaccharide fraction of Propionibacterium acnes (PS), a pro-Th1 agonist, may subvert the type-2 profile and protect podocytes from adriamycin-induced glomerulosclerosis. Adriamycin injection resulted in albuminuria and increased serum creatinine in association with loss of glomerular podocin and podoplanin expression, which is consistent with podocyte dysfunction. Renal tissue analysis revealed the expression of transcripts for GATA3 and fibrogenic-related proteins, such as TGF-β, tissue inhibitor of metalloproteinase-1 (TIMP-1) and metalloproteinase 9 (MMP9). In association with the expression of fibrogenic transcripts, we observed peri-glomerular expression of α-smooth muscle actin (α-SMA), indicating epithelial-to-mesenchymal transition, and increased expression of proliferating cell nuclear antigen (PCNA) in tubular cells, suggesting intense proliferative activity. Previous immunization with PS inhibited albuminuria and serum creatinine in association with the preservation of podocyte proteins and inhibition of fibrogenic transcripts and the expression of α-SMA and PCNA proteins. Tissue analysis also revealed that PS treatment induced expression of mRNA for GD3 synthase, which is a glycosiltransferase related to the synthesis of GD3, a ganglioside associated with podocyte physiology. In addition, PS treatment inhibited the influx of inflammatory CD8(pos) and CD11b(pos) cells to kidney tissue. Finally, PS treatment on day 4 post-ADM, a period when proteinuria was already established, was able to improve renal function. Thus, we demonstrate that the PS fraction of P. acnes can inhibit FSGS pathogenesis, suggesting that immunomodulation can represent an alternative approach for disease management.
NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that mediates the upregulation of a battery of cytoprotective genes in response to cell stress. Recent studies showed that Nrf2 also modulates immune responses and exhibits anti-inflammatory activity. In this article, we demonstrate that a common food preservative, tert-butylhydroquinone, can activate Nrf2 in T cells, as evidenced by Nrf2 binding to the antioxidant response element and the subsequent upregulation of Nrf2 target genes. The activation of Nrf2 suppresses IFN-γ production, while inducing the production of the Th2 cytokines IL-4, IL-5, and IL-13. Nrf2 activation also suppresses T-bet DNA binding and promotes GATA-binding protein 3 DNA binding. Collectively, the present studies suggested that Nrf2 activation skews CD4(+) T cells toward Th2 differentiation and, thus, represents a novel regulatory mechanism in CD4(+) T cells. Further studies are needed to determine whether the commercial use of Nrf2 activators as food preservatives promotes food allergies in humans.
Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.
Asthma is a chronic allergic disorder characterised by chronic inflammation. The balance of type I and type II (CD4+) T helper cells is of critical importance. In asthma there is an overexpression of T(H)2 cytokines, such as IL-4, IL-5 and IL-13. The genes encoding these cytokines are located together the same chromosomal region, 5q31.1 in humans. Here we confirm a central role for the transcription factors NFAT and GATA3 in the regulation of human IL-4 and IL-13. Chromatin Conformation Capture (3C) demonstrated the formation of specific ligation products containing spliced IL-4 and IL-13 DNA sequences in human T(H)2 polarised HuT-78 cells. This suggests that co-ordinate expression of T(H)2 cytokines, under the control of GATA3 and NFAT1 is due to the formation of a chromatin hub by DNA looping.
Polarized T helper type 2 (Th2) response is linked with fibrosis. Here, we evaluated the effect of the anti-fibrotic agent pirfenidone on Th type 1 (Th1) and Th2 responses. For in vivo testing; Wistar rats were made cirrhotic by intraperitoneal administration of thioacetamide. Once hepatic damage was established, pirfenidone was administered intragastrically on a daily basis during three weeks. Gene expression of Th marks was evaluated by RT-PCR and Western blot assays from liver homogenates. Pirfenidone therapy induced down-regulation of Th2 transcripts and proteins (GATA3 and IL-4), without affecting significantly Th1 genes expression (T-bet and IFN-γ). We found that the activated form of p38 MAPK (identified by Western blot) was reduced by pirfenidone treatment, which is consistent with the anti-Th2 activity observed. Pirfenidone reduced GATA3 nuclear localization without modifying its DNA binding activity (evaluated by electrophoretic mobility shift assay). For in vitro testing; human naive CD4+ T cells were cultured in either Th1 or Th2 polarizing conditions in the presence of pirfenidone and flow cytometric analysis of intracellular synthesis of IFN-γ and IL-4 was conducted. Pirfenidone impaired development of Th2 subpopulation. In conclusion, pirfenidone is capable of impairing Th2 differentiation and limits Th2 profibrogenic response. The mechanism involves p38 inhibition and regulation of GATA3 expression and translocation.
We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45γ and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.
To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast cancer.
Tissue microarray and individual paraffin tissue slides from 131 patients were used for the study. The association of FOXO1, GATA3 and Annexin-1 expression with clinicopathological features of breast cancer and disease outcome was examined in retrospective samples. Kaplan-Meier survival curves and Cox regression with multivariate analysis were used for assessing the relative risk (RR) and disease-free survival (DFS). The expression of FOXO1, GATA3 and Annexin-1 were determined by immunohistochemistry and the association among the three proteins was analyzed by Logistic regression analysis.
The nuclear expression of FOXO1 was observed in most of the normal breast tissues and 51.3% of the malignant breast tissues. GATA3 and Annexin-1 were expressed at 73% and 24.6% respectively in breast cancer tissues. The expression of FOXO1, GATA3 and Annexin-1 were all inversely correlated with lymph node-positive tumors. Both FOXO1 and Annexin-1 expression were also inversely associated with HER2-overexpressing tumors. FOXO1 expression was significantly associated with both GATA3 and Annexin-1 expression. In addition, Multivariate analyses confirm that only FOXO1 levels independently predict DFS.
FOXO1 expression in breast cancer is regulated by the PI3K/Akt pathway. The expression of FOXO1 is also associated with GATA3 and/or Annexin-1. Restoring or targeting FOXO1 to the cell nucleus in breast cancer tissues may improve response to therapy and disease outcome. Further clinical studies are warranted to test this hypothesis.
The aim of this study was to identify candidate causal single nucleotide polymorphisms (SNPs) and candidate causal mechanisms of psoriasis and Behcets's disease (BD) and to generate an SNP → gene → pathway hypothesis. A psoriasis genome-wide association study (GWAS) dataset that included 436,192 SNPs in 1,409 psoriasis cases and 1,436 controls of European descent and a BD GWAS dataset that contained 310,324 SNPs in 1,215 BD cases and 1,278 controls were used in this study. Identify candidate causal SNPs and pathways (ICSNPathway) analysis was applied to the GWAS datasets. ICSNPathway analysis identified 15 candidate causal SNPs and 28 candidate causal pathways. The top five candidate causal SNPs were rs1063478 (P = 1.45E-10), rs8084 (P = 2.20E-08), rs7192 (P = 5.18E-08), rs20541 (P = 5.30E-06), and rs1130838 (P = 5.65E-06), which with the exception of rs20541 [interleukin (IL)-13] are at human leukocyte antigen (HLA) loci. These candidate causal SNPs and pathways provided ten hypothetical biological mechanisms. The most strongly associated pathway concerned HLA. When HLA loci were excluded, ICSNPathway analysis provided one hypothetical biological mechanism. rs20541 (non_synonymous_coding) → IL-13 → dendritic cell involvement in the regulation of Th1 and Th2 development, and the GATA3 pathway. ICSNPathway analysis identified four candidate causal SNPs, eleven candidate causal pathways, and three hypothetical biological mechanisms. One of them was as follows: rs2072895 (non_synonymous_coding & splice-site) and rs2735059 (non_synonymous_coding) → HLA-F → type I diabetes mellitus, antigen processing and presentation, and autoimmune thyroid disease. The application of ICSNPathway analysis to GWAS dataset of psoriasis and BD resulted in the identification of candidate causal SNPs and candidate pathways that might contribute to psoriasis susceptibility.
Regulatory T cells (Treg) play some important roles in allergic rhinitis. The most specific marker for Treg is FOXP3, a recently identified transcription factor that is essential for Treg development. In order to clarify the levels of Treg in allergic nasal mucosa, we studied the relationship between FOXP3-expressing cells and Th1-Th2 balance in nasal mucosa by means of immunohistochemistry. Human turbinates were obtained after turbinectomy from 26 patients (14 patients with perennial allergic rhinitis and 12 patients with nonallergic rhinitis). To identify the cells expressing the FOXP3 protein, double immunostaining was performed by using anti-FOXP3 antibody and anti-CD3 antibody. There was no significant difference in the percentage of FOXP3+CD3+ cells among CD3+ cells in the nasal mucosa of two groups. The proportion of FOXP3+CD3+ cells tend to be correlated positively with GATA3+CD3+ cells/T-bet+CD3+ cells ratio (R = 0.56, P = 0.04). A positive correlation with GATA3+CD3+/T-bet+CD3+ ratio and FOXP3+CD3+/CD3+ ratio suggests the role of local regulatory T cells as a minimal control of the chronic allergen exposure in nasal mucosa.
This study was conducted to investigate the effect of morphine on CD4-positive T cells differentiation and the transcriptional factors induced by phorbol myristate acetate (PMA) and ionomycin. CD4-positive lymphocytes separated from healthy volunteers were incubated by PMA (25 ng/ml) + ionomycin (1 μg/ml) with or without the presence of morphine, ketamine, or naloxone. Th subsets, supernatant cytokines, and transcriptional factors were detected 4 h later. Th1 and Th2 cells, levels of INF-γ, IL-2, IL-4 and the activities of T-bet and GATA3 were significantly increased after incubation with PMA and ionomycin. However, the number of Th1 cells, Th1/ Th2, the levels of INF-γ and INF-γ/IL-4, and the activities and protein levels of T-bet and GATA3 were decreased after incubation with PMA and ionomycin in the presence of morphine. Naloxone can abolish morphine's suppressive effect on Th cell differentiation. Morphine has a negative effect on Th cell balance induced by PMA and ionomycin, the mechanism is related to T-bet and GATA3.
The purpose of this study was to investigate the effects of chronically inhaled particulate matter <2.5 μm (PM(2.5)) on inflammatory cell populations in the lung and systemic circulation. A prominent component of air pollution exposure is a systemic inflammatory response that may exaggerate chronic diseases such as atherosclerosis and insulin resistance. T cell response was measured in wild-type C57B/L6, Foxp3-green fluorescent protein (GFP) "knockin," and chemokine receptor 3 knockout (CXCR3(-/-)) mice following 24-28 wk of PM(2.5) or filtered air. Chronic PM(2.5) exposure resulted in increased CXCR3-expressing CD4(+) and CD8(+) T cells in the lungs, spleen, and blood with elevation in CD11c(+) macrophages in the lung and oxidized derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine in wild-type mice. CXCR3 deficiency decreased T cells in the lung. GFP(+) regulatory T cells increased with PM(2.5) exposure in the spleen and blood of Foxp3-GFP mice but were present at very low levels in the lung irrespective of PM(2.5) exposure. Mixed lymphocyte cultures using primary, PM(2.5)-treated macrophages demonstrated enhanced T cell proliferation. Our experiments indicate that PM(2.5) potentiates a proinflammatory Th1 response involving increased homing of CXCR3(+) T effector cells to the lung and modulation of systemic T cell populations.
Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.
The study was to explore the potential immunoregulatory mechanisms linking fine particles and cardiac injury. Wistar kyoto (WKY) rats were exposed by intratracheal instillation to fine particles with the doses of 0.0, 1.6, 8.0 and 40.0mg/kg b.w., respectively. The exposure was conducted once a day, for three consecutive days. Twenty-four hours after the last exposure, the rats were sacrificed. Th1- and Th2-related transcription factors and cytokines were assessed in left ventricle of rats. The mRNA expressions of Th1- and Th2-related transcription factors signal transducer and activator of transcriptionl 1 (STAT1), signal transducer and activator of transcriptional 6 (STAT6), GATA-3 and T-bet were assessed in left ventricle of rats using real-time PCR. Meanwhile, the levels of Th1- and Th2-related cytokines IL-4, IL-13 and interferon gamma (IFN-γ) were determined by ELISA kits in cardiac homogenate supernatant of rats. Furthermore, the protein expression of IL-4 and IFN-γ were detected in myocardium by Western blot. The results of cardiac histology demonstrated exacerbated cardiac lesions and histological characterization of inflammation and degeneration in rats after exposure to fine particles. Moreover, fine particles induced significant increase of IL-4 and IL-13 and decrease of IFN-γ in myocardium of rats. The mRNA expression of STAT1, STAT6 and GATA-3 were up-regulated in left ventricle of rats in a dose-dependent manner, whereas T-bet was significantly down-regulated. The variations of these cytokines demonstrated the imbalance of Th1 and Th2 cytokines existed in cardiac injuries induced by fine particle. The imbalance of Th1/Th2 cytokines might be one of the mechanisms of immunotoxicity of cardiovascular system induced by ambient fine particles.
Allergic rhinitis (AR) is a disease in which T-helper (Th)2 response is predominant and its pathogenic mechanism is still poorly understood.
To evaluate the possible role of Th1, Th2 and regulatory-T (Treg) cells in the pathogenesis of AR.
This case-control study enrolled 41 patients with seasonal AR (10-62 years old), sensitive to olive pollens, and 15 healthy controls (18-60 years old). Nasal biopsy was performed and specimens of nasal lavage fluid were obtained from all participants. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and transforming growth factor-β (TGF-β) were measured in nasal lavage fluid specimens. The expression of FOXP3, GATA-3 and T-bet was measured by immunohistochemical methods in the nasal biopsy specimens.
The levels of IFN-γ in the group with AR were significantly lower than those in the control group (p = 0.008). The levels of IL-4, IL-10 and TGF-β did not differ between the two groups. The expression of FOXP3 and T-bet in patients with AR was significantly lower than that in the control group (both p = 0.001). Expression of GATA-3 in the nasal mucosa was similar between the groups (p = 0.2). The ratios of T-bet/GATA-3 and FOXP3/GATA-3 in the AR group were significantly lower than those in the control group (p = 0.001).
Insufficient Treg and Th1 cells may be associated with the allergic inflammation that may be attributed to the Th2 immune response in patients suffering from AR who are sensitive to olive pollen.
Systemic lupus erythematosus (SLE) is characterized by disease flares and remission. We hypothesize that in clinically quiescent SLE patients, the mRNA level of target genes in the urinary sediment is an early indicator of disease flare.
From a cohort of 134 adult SLE patients prospectively followed for 56 weeks, we identified 19 patients with a single disease flare. The mRNA level of eight pre-defined target genes in their urinary sediment before disease flare was compared to 19 matched controls with no disease flare during the same period.
Urinary mRNA level remained static in the control group during the study period. Before disease flare, there was a significant increase in the mRNA level of monocyte chemotactic protein (MCP)-1 and forkhead box P3 (FOXP3), and decrease in interleukin (IL)-17 and GATA-3, in the urinary sediment. The mRNA level of FOXP3 in urinary sediment increases 8 weeks prior to a flare, which precedes the corresponding change in serum complement and anti-DNA antibody titer, while that of MCP-1, IL-17, and GATA3 began to change 4 weeks prior to a flare. The same pattern of change in urinary mRNA level was observed in patients with mild-to-moderate or severe flare, and those with renal or non-renal flare. The SLE Disease Activity Index (SLEDAI) score at the time of flare significantly correlated with the change in urinary level of IL-17 (r=-0.462, p=0.046) and GATA-3 (r=-0.455, p=0.05), but not MCP-1 or FOXP3, prior to the flare.
Monitoring of MCP-1, IL-17, GATA-3 and FOXP3 mRNA level in urinary sediment may provide an early clue for detecting disease flare in SLE patients.
In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs.
The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.
Contact hypersensitivity (CHS) is thought to be associated mainly with the activation of T helper (Th) type 1 cells. However, evidence also suggests that Th type 2 cells (Th2) and cytokines play roles in the development of CHS in humans. The present study examines the Th2 response during the development of CHS in response to 2,4,6-trinitrochlorobenzene (TNCB) in GATA-3-transgenic (GATA-3 Tg) mice. GATA-3 Tg mice were immunized with 7% TNCB applied to abdominal shaved skin. Seven days later, the mice were challenged with 1% TNCB applied to the left ear. Ear swelling, cytokine production in the skin of the ear, and the levels of IgE, IgG1 and IgG2a were measured. Furthermore, we examined the effects of medical treatment on TNCB-induced contact dermatitis using this model. The ear-swelling responses of TNCB-sensitized/challenged GATA-3 Tg mice were significantly greater than those of similarly treated wild-type (WT) mice. The expression of both IL-5 and IL-13 in TNCB sensitized/challenged skin tissues and the IgE response after challenge were obviously increased in the GATA-3 Tg mice, whereas the expression of IFN-γ was identical in the challenged skin tissues of GATA-3-Tg and WT mice. When TNCB-sensitized GATA-3 Tg mice were treated with a high dose of tacrolimus, ear swelling was not significantly decreased, compared with the results in WT mice. These results suggest that GATA-3-induced Th2-dominant responses play a critical role in the pathogenesis of allergic types of dermatitis, such as atopic dermatitis, and may lead to useful new drug development in the future.
Autonomic neuron development is controlled by a network of transcription factors, which is induced by bone morphogenetic protein signalling in neural crest progenitor cells. This network intersects with a transcriptional program in migratory neural crest cells that pre-specifies autonomic neuron precursor cells. Recent findings demonstrate that the transcription factors acting in the initial specification and differentiation of sympathetic neurons are also important for the proliferation of progenitors and immature neurons during neurogenesis. Elimination of Phox2b, Hand2 and Gata3 in differentiated neurons affects the expression of subtype-specific and/or generic neuronal properties or neuron survival. Taken together, transcription factors previously shown to act in initial neuron specification and differentiation display a much broader spectrum of functions, including control of neurogenesis and the maintenance of subtype characteristics and survival of mature neurons.
The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes.
In the thymus, developing T cells receive signals that determine lineage choice, specificity, MHC restriction and tolerance to self-antigen. One way in which thymocytes receive instruction is by secretion of Sonic hedgehog (Shh) from thymic epithelial cells. We have previously shown that Hedgehog (Hh) signalling in the thymus decreases the CD4:CD8 single-positive (SP) thymocyte ratio. Here, we present data indicating that double-positive (DP) thymocytes are Hh-responsive and that thymocyte-intrinsic Hh signalling plays a role in modulating the production of CD4(+) (SP4), CD8(+) (SP8) and unconventional T-cell subsets. Repression of physiological Hh signalling in thymocytes altered the proportions of DP and SP4 cells. Thymocyte-intrinsic Hh-dependent transcription also attenuated both the production of mature SP4 and SP8 cells, and the establishment of peripheral T-cell compartments in TCR-transgenic mice. Additionally, stimulation or withdrawal of Hh signals in the WT foetal thymus impaired or enhanced upregulation of the CD4 lineage-specific transcription factor Gata3 respectively. These data together suggest that Hh signalling may play a role in influencing the later stages of thymocyte development.
The development of the inner ear sensory epithelia involves a complex network of transcription factors and signaling pathways and the whole process is not yet entirely understood. GATA3 is a DNA-binding factor that is necessary for otic morphogenesis and without GATA3 variable defects have been observed already at early stages in mouse embryos. In the less severe phenotypes, one small oval shaped vesicle is formed whereas in the more severe cases, the otic epithelium becomes disrupted and the endolymphatic domain becomes separated from the rest of the otic epithelium. Despite these defects, the early sensory fate specification occurs in Gata3-/- otic epithelium. However, due to the early lethality of Gata3-deficient embryos, the later morphogenesis and sensory development have remained unclear. To gain information of these later processes we produced drug-rescued Gata3-/- embryos that survived up to late gestation. In these older Gata3-/- embryos, a similar variability was observed as earlier. In the more severely affected ears, the development of the separate endolymphatic domain arrested completely whereas the remaining vesicle formed an empty cavity with variable forms, but without any distinguishable otic compartments or morphologically distinct sensory organs. However, the dorsal part of this vesicle was able to adopt a sensory fate and to produce some hair cells. In the less severe cases of Gata3-/- ears, distinct utricular, saccular and cochlear compartments were present and hair cells could be detected in the vestibular sensory epithelia. Although clear cristae and maculae formed, the morphology and size of these sensory areas were abnormal and they remained often un-separated. In contrast to the vestibule, the cochlear sensory compartment remained more immature and no hair or supporting cells could be detected. Our results suggest that GATA3 is critical for normal vestibular and cochlear morphogenesis and that it is especially important for cochlear sensory differentiation.
Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.
Thymic epithelial cells (TECs) are necessary for normal T cell development. Currently, one transcription factor, Foxn1 is known to be necessary for the progression of fetal TEC differentiation. However, some aspects of fetal TEC differentiation occur in Foxn1 mutants, suggesting the existence of additional transcriptional regulators of TEC differentiation. The goal of this study was to identify some of the additional candidate transcription factors that may be involved in the specification and/or differentiation of TECs during fetal development.
We identified candidate fetal TEC transcriptional regulators via data and text mining. From our data mining we selected the transcription factors Foxg1, Isl1, Gata3, Nkx2-5, Nkx2-6 and Sox2 for further studies. Whole mount in situ hybridizations confirmed the expression of these transcription factors within subdomains of the third pharyngeal pouch from E9.5-E10.5. By E11.5 days Foxg1 and Isl1 transcripts were the only mRNAs from this group of genes detected exclusively within the thymus domain of the third pouch. Based on this initial in situ hybridization analysis, we focused on defining the expression of Foxg1 and Isl1 during multiple stages of thymus development and TEC differentiation. We found that Foxg1 and Isl1 are specifically expressed in differentiating TECs during fetal and postnatal stages of thymus development. In addition, we found differential expression of Islet1 and Foxn1 within the fetal and postnatal TEC population.
Our studies have identified two developmental transcription factors that are excellent candidate regulators of thymic epithelial cell specification and differentiation during fetal development. Our results suggest that Foxg1 and Isl1 may play a role in the regulation of TEC differentiation during fetal and postnatal stages. Our results also demonstrate heterogeneity of TECs marked by the differential expression of transcription factors, potentially providing new insights into the regulation of TEC differentiation.
Parathyroid developmental anomalies, which result in hypoparathyroidism, are common and may occur in one in 4,000 live births. Parathyroids, in man, develop from the endodermal cells of the third and fourth pharyngeal pouches, whereas, in the mouse they develop solely from the endoderm of the third pharyngeal pouches. In addition, neural crest cells that arise from the embryonic mid- and hindbrain also contribute to parathyroid gland development. The molecular signaling pathways that are involved in determining the differentiation of the pharyngeal pouch endoderm into parathyroid cells are being elucidated by studies of patients with hypoparathyroidism and appropriate mouse models. These studies have revealed important roles for a number of transcription factors, which include Tbx1, Gata3, Gcm2, Sox3, Aire1 and members of the homeobox (Hox) and paired box (Pax) families.
To investigate kinetic expression of genes T-bet and GATA-3 in blood mononuclear cells (BMCs) in allergic rhinitis rats model at different stages of development of allergic rhinitis.
Twenty SD rats (including 10 male and 10 female) were divided into 2 groups, experimental group and control group, randomly, 10 rats for each group. Ten rats in experimental group were sensitized and intranasally challenged by OVA, aluminium hydroxide hydrate gel and Bordelella pertussis inactive microorganism suspension (B. pertussis) adjuvants, as allergic rhinitis models, Ten rats in control group were investigated using physiological saline only. BMCs were separated from 2 ml blood which was extracted from rat heart at the end of sensitization, 10- hour after the first challenge and 10-hour after the final challenge, respectively. RT-PCR was utilized to detect the expression of T-bet and GATA-3.
At the end of sensitization, 10-hour after the first challenge and 10-hour after the final challenge, in experimental group, Relative quantitation of expression of T-bet was 0.404 +/- 0.187, 1.676 +/- 0.708, 0.503 +/- 0.514 and that of GATA-3 was 0.434 +/- 0.147, 0.600 +/- 0.480, 1.029 +/- 0.690, respectively. While, In control group, Relative quantitation of expression of T-bet was 0.487 +/- 0.212, 0.486 +/- 0.148, 0.495 +/- 0.103 and and its of GATA-3 was 0.596 +/- 0.249, 0.474 +/- 0.101, 0.550 +/- 0.119, respectively. At 10-hour after the first challenge, relative quantitation of expression of T-bet in experimental group was increased markedly and there was significant difference compared with contol group (t=4.18, P<0.01). In experimental group, The amount of expression of T-bet at 10-hour after first challenge was higher than it at 10-hour after the final challenge and there was markedly difference (t=5.14, P<0.01). The amount of expression of T-bet at 10-hour after first challenge was increased significantly compared with it at the end of sensitization (t= 5.27, P<0.01). while, the expression of GATA-3 at 10-hour after final challenge was markedly increased compared with it at the end of sensitization (t= 3.51, P<0.05) and was higher than it at 10-hour after first challenge (t=2.53, P<0.05). At 10-hour after final challenge, The amount of expression of GATA-3 in experimental group was significantly higher than it in control group (t=2.71, P<0.05). However, both the expression of GATA-3 and T-bet had not markedly changed in control group.
It is thought that the development of allergic rhinitis is a successive and sequencing kinetic course, and imbalance of expression of GATA-3 and T-bet may be genetic base on allergic rhinitis, both GATA-3 and T-bet were involved in allergic rhinitis only in different phase of development of allergic rhinitis.
Glial cells missing B (GCMB) is a transcription factor that is expressed in the parathyroid hormone (PTH)-secreting cells of the parathyroid glands. Several mutations in GCMB have been reported to cause hypoparathyroidism (HP). We identified a family with two individuals in two generations (mother and son), who are affected by autosomal-dominant hypoparathyroidism (AD-HP). A novel heterozygous mutation in exon 2 of GCMB was identified in both affected individuals that changes cysteine at position 106 of the putative DNA-binding domain of GCMB to arginine (C106R).
We performed mutational analysis of the genes encoding GCMB, pre-pro PTH, GATA3 and CaSR using polymerase chain reaction (PCR)-amplified genomic DNA. The identified GCMB mutant was characterized by functional studies including nuclear localization, electrophoretic mobility shift assays (EMSA) and luciferase reporter assays, and homology modelling was performed to generate a three-dimensional structural model for the DNA-binding domain of GCMB to predict the structural consequences of the identified mutation.
The C106R mutant of GCMB failed to interact with the DNA consensus recognition motif, as determined by EMSA. Furthermore, in comparison with wild-type GCMB, the C106R mutant demonstrated reduced transactivation in luciferase reporter assays; however, the mutant GCMB failed to reduce the activity of the wild-type protein. Consistent with the EMSA findings, homology modelling analysis suggested that replacement of cysteine 106 with arginine would interfere with DNA binding.
We have identified a novel GCMB mutation that may explain AD-HP in our family. However, the exact mechanism by which this heterozygous mutation leads to the disease in the described family remains to be elucidated.
Many transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-driven oncogenicity and empowers IL-17-producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b-mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this "Th17 mimic" pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.
Effects of icariin on airway inflammation in asthmatic rats and the intervention of LPS induced inflammation are interfered with the machanism of icariin. Our study aimed to observe the effect of icariin on ovalbumin-induced imbalance of Th1/Th2 cytokine expression and its mechanism.
Sixty male SD rats were randomly divided into control group (PBS), asthma group (ovalbumin (OVA)-induced), dexamethasone group, and OVA+icariin low, medium and high dose groups (5, 10, 20 mg/kg, respectively). Each group had ten rats. The model of OVA sensitization was a rat asthma model. Enzyme-linked immunosorbent assay (ELISA) method was used to observe the effects of icariin on interleukin-4 (IL-4) and inerferon γ (IFN-γ) in rats' lung tissue. Immunohistochemical staining was applied to detect the intervention effects of icariin on T cells (T-bet) and gatabinding protein 3 (GATA-3) in rat pulmonary tissue. Realtime RT-PCR was used to observe the intervention effects of icariin on T-bet and GATA-3 mRNA expression in rat pulmonary tissue and spleen lymphocytes. Western blotting was used to observe the icariin intervention effects on T-bet, GATA-3 and nuclear factor-Kappa B (NF-κB) p65 protein expressions in rat pulmonary tissue.
The ELISA results from pulmonary tissue showed that IL-4 expression was significantly reduced (P < 0.05), while the IFN-γ expression increased but not significantly when we compared OVA+icariin medium and high dose groups with the asthma group. Immunohistochemical staining of pulmonary tissue showed that the GATA-3 decreased significantly while the T-bet staining did not change in the OVA+icariin high dose group. In pulmonary tissue and spleen lymphocytes T-bet and GATA-3 mRNA expressions were significantly reduced (P < 0.05) in icariin treatment groups compared with the asthma model group. GATA-3 and T-bet mRNA in rat spleen lymphocytes in the asthma group were higher than in the control group. GATA-3 mRNA expression in pulmonary tissue significantly decreased (P < 0.05) while T-bet mRNA expression decreased but not significantly in the icariin treatment group compared with the asthma group. T-bet and GATA-3 protein expressions in pulmonary tissue increased significantly compared with the asthma group, which meant that icariin could inhibit the increase of GATA-3 protein, but not of T-bet. The bronchus, blood vessels and periphery pulmonary tissue had infiltration of inflammatory cells in the OVA+icariin high dose group while NF-κB p65 cells were reduced, and expression of NF-κB p65 in this group was less than in the asthma group. The expression of total p65 protein decreased with icariin treatment while the expression of cytoplasmic p65 protein increased.
Icariin could regulate the imbalance of Th1/Th2 cytokines in asthmatic rat pulmonary tissue. Icariin could regulate the imbalance of Th1/Th2 associated transcription factors T-bet and GATA-3 in asthmatic rat pulmonary tissue and spleen lymphocytes. Icariin could inhibit the activation of NF-κB p65 protein in asthmatic rat pulmonary tissue.
The c-Myb and GATA-3 transcription factors play important roles in T cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. In this article, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE-IL-13 promoter by ∼160-fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin, and mixed lineage leukemia (MLL) bound to CGRE in human primary CD4(+) effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4(+) effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.
Recent reports have revealed that CD4(+) T(h) cell subsets have the ability to alter their gene expression pattern in response to extracellular stimuli. We previously highlighted the plasticity of T(h)1 cells by demonstrating that T(h)1 cells gain the capacity to produce IL-3, IL-9, IL-13 and granulocyte macrophage colony-stimulating factor in response to antigen, IL-2 and IL-18, and based on their unique function, we designated these activated T(h)1 cells as 'super T(h)1 cells'. However, the precise molecular mechanism underlying IL-13 production by super T(h)1 cells has not been elucidated. Here, we show that the GATA-binding protein 3 (Gata3) is essentially required for II13 gene expression in super T(h)1 cells. Gata3 is synergistically induced in T-box expressed in T-cells (T-bet)-expressing T(h)1 cells when co-stimulated with anti-CD3, IL-18 and IL-4 through the activation of nuclear factor of activated T cells, nuclear factor kappa-light-chain-enhancer of activated B cells and signal transducer and activator of transcription 6, respectively. However, Gata3 induction is not satisfactory, and additional TCR or anti-CD3 signaling is prerequisite for triggering IL-13 production by Gata3 plus T-bet-expressing T(h)1 cells. These findings suggest that Gata3, which is not originally expressed in T(h)1 cells, alters the cytokine production profile by T(h)1 cells.
Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent.
We studied the immunological response to GA and its relationship with disease activity.
Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing-remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI.
The expression of T helper type 1 (Th1) and Th17 cytokines and transcription factors was reduced during long-term treatment, but there was no relationship between the expression of cytokines and transcription factors and anti-GA antibodies. High expression of mRNA encoding GATA3 and lymphotoxin-β (LT-β) was associated with low disease activity in Gd-enhanced MRI studies. None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment.
The observed relationship between the expression of mRNA encoding GATA3 and LT-β expression and MRI disease activity deserves further analysis in future studies. The development of anti-GA antibodies was observed in all patients treated with GA, but this was not related with measures of cellular immunity, clinical or MRI disease activity.
Some organ-transplanted patients achieve a state of "operational tolerance" (OT) in which graft function is maintained after the complete withdrawal of immunosuppressive drugs. We used a gene panel of regulatory/inflammatory molecules (FOXP3, GATA3, IL10, TGFB1, TGFBR1/ TBX21, TNF and IFNG) to investigate the gene expression profile in peripheral blood mononuclear cells of renal-transplanted individuals experiencing OT compared to transplanted individuals not displaying OT and healthy individuals (HI). OT subjects showed a predominant regulatory (REG) profile with higher gene expression of GATA3, FOXP3, TGFB1 and TGFB receptor 1 compared to the other groups. This predominant REG gene expression profile displayed stability over time. The significant GATA3 gene and protein expressions in OT individuals suggest that a Th2 deviation may be a relevant pathway to OT. Moreover, the capacity of the REG/INFLAMMA gene panel to discriminate OT by peripheral blood analysis indicates that this state has systemic repercussions.
Follicular T helper (Tfh) cells provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated protein (SAP) prevent GC formation because of defective T cell-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally competent "Tfh-like" cells that expressed interleukin-21, Tfh cell markers, and Bcl6 and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wild-type, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3, and Rorc in Tfh-like and ex vivo Tfh cells and on Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.
It has been indicated that T-box 21 (TBX21) and H 2.0-like homeobox (HLX) are transcription factors related to the differentiation of T helper 1 cells, whereas GATA-binding protein 3 is the master transcription factor of T helper 2 cells.
We genotyped -1514T/C (rs17250932) and -1993T/C (rs4794067) polymorphisms of TBX21, - 742C/G polymorphism (rs2184658) of HLX and -1420G/A polymorphism (rs1269486) of GATA3 in genomic DNA samples from Japanese patients; 51 patients with severe Hashimoto's disease (HD), 39 with mild HD, 66 with intractable Graves' disease (GD), in whom remission was difficult to induce, 47 with GD in remission and 79 healthy volunteers.
The T alleles of the TBX21-1514T/C and -1993T/C polymorphisms were more frequent in patients with intractable GD than in those with GD in remission. Among individuals with the TBX21-1993TT genotype, the G allele of HLX-742C/G polymorphism, which correlates with low HLX expression, was more frequent in patients with intractable GD than in those with GD in remission.
Functional polymorphisms in TBX21 are associated with the development of autoimmune thyroid diseases and prognosis of GD, and a functional polymorphism in HLX in combination with the TBX21 polymorphism is also associated with the prognosis of GD.
Metabolic syndrome (MetS) and benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS) are often associated. One of their common denominators is hypogonadism. However, testosterone supplementation is limited by concerns for potential prostatic side effects. The objective was to determine whether MetS-associated prostate alterations are prevented by testosterone supplementation. We used a previously described animal model of MetS, obtained by feeding male rabbits a high-fat diet (HFD) for 12 weeks. Subsets of HFD rabbits were treated with testosterone or with the farnesoid X receptor agonist INT-747. Rabbits fed a standard diet were used as controls. HFD-animals develop hypogonadism and all the MetS features: hyperglycemia, glucose intolerance, dyslipidemia, hypertension, and visceral obesity. In addition, HFD-animals show a prostate inflammation. Immunohistochemical analysis demonstrated that HFD-induced prostate fibrosis, hypoxia, and inflammation. The mRNA expression of several proinflammatory (IL8, IL6, IL1β, and TNFα), T lymphocyte (CD4, CD8, Tbet, Gata3, and ROR γt), macrophage (TLR2, TLR4, and STAMP2), neutrophil (lactoferrin), inflammation (COX2 and RAGE), and fibrosis/myofibroblast activation (TGFβ, SM22α, αSMA, RhoA, and ROCK1/ROCK2) markers was significantly increased in HFD prostate. Testosterone, as well as INT-747, treatment prevented some MetS features, although only testosterone normalized all the HFD-induced prostate alterations. Interestingly, the ratio between testosterone and estradiol plasma level retains a significant, negative, association with all the fibrosis and the majority of inflammatory markers analyzed. These data highlight that testosterone protects rabbit prostate from MetS-induced prostatic hypoxia, fibrosis, and inflammation, which can play a role toward the development/progression of BPH/LUTS.
GATA-3 is a master transcription factor of the Th2 cells. We have identified GATA-3 cDNA and its splice variant in Atlantic cod. Cod GATA-3 (GmGATA-3) has a 1320 b p open reading frame encoding a polypeptide of 440 amino acids with two zinc finger domains that are well conserved within teleosts and higher vertebrates. The GATA-3 cDNA splice variant without zinc finger domains was shown to contain an 828 b p open reading frame encoding a polypeptide of 276 amino acids. Both GATA-3 proteins fused with RFP-tag were identified in or close to the nuclei 48 h after the plasmids were transfected in CHSE-214 cells. The full length GATA-3 with two zinc finger domains has a transcriptional function confirmed by transfection with GATA-3 reporter vector along with expression constructs of GATA-3 plasmids in CHSE-214 cells, whereas the GATA-3 splice variant without zinc finger domain did not enhance the activity of the GATA-3 reporter vector, and no interference was found between these two GATA-3 variants. RT-PCR analysis revealed that the two Atlantic cod GATA-3 variants were strongly expressed in the gills and infection with live Vibrio anguillarum induced the spleen expression of both GmGATA-3L and GmGATA-3S. Unexpectedly, PMA increased the expression of the GATA-3 splice variant in vivo and especially in vitro, with an increase of more than 100,000-fold in head kidney leukocytes at 24 and 48 h. On the other hand, there were no significant increases at the transcript level of full length GATA-3 between Poly I:C and β-glucan treatment groups compared to controls.
Tregs not only keep immune responses to autoantigens in check, but also restrain those directed toward pathogens and the commensal microbiota. Control of peripheral immune homeostasis by Tregs relies on their capacity to accumulate at inflamed sites and appropriately adapt to their local environment. To date, the factors involved in the control of these aspects of Treg physiology remain poorly understood. Here, we show that the canonical Th2 transcription factor GATA3 is selectively expressed in Tregs residing in barrier sites including the gastrointestinal tract and the skin. GATA3 expression in both murine and human Tregs was induced upon TCR and IL-2 stimulation. Although GATA3 was not required to sustain Treg homeostasis and function at steady state, GATA3 played a cardinal role in Treg physiology during inflammation. Indeed, the intrinsic expression of GATA3 by Tregs was required for their ability to accumulate at inflamed sites and to maintain high levels of Foxp3 expression in various polarized or inflammatory settings. Furthermore, our data indicate that GATA3 limits Treg polarization toward an effector T cell phenotype and acquisition of effector cytokines in inflamed tissues. Overall, our work reveals what we believe to be a new facet in the complex role of GATA3 in T cells and highlights what may be a fundamental role in controlling Treg physiology during inflammation.
Th17 cells are highly pathogenic in a variety of immune-mediated diseases, and a thorough understanding of the mechanisms of cytokine-mediated suppression of Th17 cells has great therapeutic potential. In this article, we characterize the regulation of both in vitro- and in vivo-derived Th17 cells by IL-4. We demonstrate that IL-4 suppresses reactivation of committed Th17 cells, even in the presence of TGF-β, IL-6, and IL-23. Downregulation of IL-17 by IL-4 is dependent on STAT6 and mediated by inhibition of STAT3 binding at the Il17a promoter. Although Th1 cytokines were shown to induce IFN-γ expression by Th17 cells, IL-4 does not induce a Th2 phenotype in Th17 cells. Suppression by IL-4 is stable and long-lived when applied to immature Th17 cells, but cells that have undergone multiple rounds of stimulation, either in vivo during a Th17-mediated inflammatory disease, or in vitro, become resistant to suppression by IL-4 and lose the ability to signal through IL-4R. Thus, although IL-4 is a potent suppressor of the Th17 genetic program at early stages after differentiation, prolonged stimulation renders Th17 cells impervious to regulatory cytokines.
T lymphocytes, which are central players in orchestrating immune responses, consist of several subtypes with distinct functions. The thymus is an organ where hematopoietic progenitors undergo sequential developmental processes to give rise to this variety of T-cell subsets with diverse antigen specificity. In the periphery, naive T cells further differentiate into effector cells upon encountering antigens. There are several developmental checkpoints during T-cell development, where regulation by a combination of transcription factors imprints specific functional properties on precursors. The transcription factors E2A, GATA-binding protein 3 (Gata3) and RUNT-related transcription factor (Runx) are involved at various stages in the differentiation of double-negative thymocytes and in β-selection, as are transcription factors from the Notch signaling pathway; other transcription factors such as B-cell lymphoma/leukemia 11b (Bcl11b), myeloblastosis viral oncogene homolog (Myb) and inhibitor of DNA binding 3 (Id3) are involved at specific stages. Differentiation of T cells into helper versus cytotoxic cells involves not only antagonistic interplay between Runx and T(h) inducing POZ-Kruppel factor (ThPOK) but also complex interactions between MAZR, Gata3 and Myb in the activation and silencing of genes such as Cd4 and Cd8 as well as the gene that encodes ThPOK itself. A wide range of well-defined transcription factors, including signal transducer and activator of transcriptions (STATs), T-bet, Gata3, nuclear factor of activated T cell (NFAT), adaptor-related protein complex 1 (AP-1) and nuclear factor κB (NF-κB), are known to shape T(h)1/T(h)2 differentiation. Runx and Gata3 also operate in this process, as do c-Maf and recombining binding protein for immunoglobulin Jκ region (RBP-J) and the chromatin-reorganizing protein special AT-rich sequence-binding protein 1 (SATB1). In this review, we briefly discuss how T-cell characteristics are acquired and become divergent from the point of view of transcriptional regulation.
We have previously reported that transgenic overexpression of CD200 in either mouse skin graft donors or recipients significantly enhances skin allograft survival. By focused microarray analysis we showed this enhanced graft survival is associated with increased expression of Foxp3, GITR, CTLA-4 and CCR4 mRNA, all genes related to T(reg) cell induction/function, and of Gata3, IL-4, IL-5, IL-13, and somewhat surprisingly, of T-bet, INF-γ and granzyme b. Gene-specific real-time PCR and immunohistochemistry analysis confirmed an increase in Foxp3(+) T(reg) cells in both the skin grafts and draining lymph nodes (DLNs) of CD200(tg) recipient mice at both 7/14 days post engraftment, as well as providing evidence for increased expression of the ligands for CCR4, CCL17 and CCL22 in both locations. Following lentivirus-mediated shRNA treatment of Dox-treated CD200(tg) mice to attenuate expression of CCR4 mRNA, the increased localization of T(reg) cells in skin/DLN of CD200(tg) recipients was abolished, and the enhanced graft survival similarly reversed. We conclude that enhanced CCR4 dependent migration of Foxp3(+) T(reg) to grafted tissue and DLNs is an essential step in the graft prolongation afforded by overexpression of CD200.
Serum levels of a secreted glycoprotein YKL-40 are elevated in patients with a wide range of cancers including breast, colorectal, and ovarian cancers. Furthermore, these increased levels correlate with poorer survival of cancer patients, suggesting that serum levels of YKL-40 might be a prognostic biomarker. However, the tissue expression of YKL-40 and its relationship with clinical outcomes and other potential markers are poorly understood.
Tissue samples from invasive breast cancers, breast ductal carcinoma in situ (DCIS), and cancer-free reduction mammoplasty were enrolled. YKL-40 expression was measured using immunohistochemistry and evaluated by a semi-quantification assay. Statistical analyses explored the relationship of YKL-40 with clinical outcome and other breast cancer biomarkers.
Breast ductal carcinoma in situ expressed low and moderate levels of YKL-40. In the subset of 203 patients with invasive cancer, YKL-40 levels were positively correlated with tumour grade (P<0.0001) and Her2/neu (P<0.01), but negatively correlated with oestrogen (P<0.0001) and progesterone receptor (P<0.0001). YKL-40 levels were inversely correlated with expressions of GATA3 (P=0.0137) and E-cadherin (P=0.0417).
These data demonstrate that expression levels of YKL-40 are associated with tumour grade, poor differentiation, and other breast cancer markers, highlighting that tissue levels of YKL-40 serve as a valuable biomarker for breast cancer diagnosis and prognosis.
Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.
The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of "luminal-like" cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.
Forkhead Box P3 (Foxp3)-expressing regulatory T (Treg) cells are central to maintaining self-tolerance and immune homeostasis. How Treg cell function and Foxp3 expression are regulated is an important question under intensive investigation. Here, we have demonstrated an essential role for the transcription factor GATA-3, a previously recognized Th2 cell master regulator, in controlling Treg cell function. Treg cell-specific GATA-3 deletion led to a spontaneous inflammatory disorder in mice. GATA-3-null Treg cells were defective in peripheral homeostasis and suppressive function, gained Th17 cell phenotypes, and expressed reduced amounts of Foxp3. In addition, GATA-3 controlled Foxp3 expression by binding to and promoting the activity of cis-acting elements of Foxp3. Furthermore, the combined function of GATA-3 and Foxp3 was essential for Foxp3 expression. These findings provide insights into immune regulatory mechanisms and uncover a critical function of GATA-3 in Treg cells and immune tolerance.
Although recent reports suggest a possible role for an imbalance in Th1 and Th2 proinflammatory cytokines in the development and progression of glomerulonephritis, there is little information on Th1 or Th2 predominance in Henoch-Schönlein purpura nephritis (HSPN). Since T-bet and GATA-3 are transcriptional factors that regulate the differentiation of helper T lymphocytes into Th1 and Th2, we examined the relative mRNA expression of T-bet and GATA-3 in the urinary sediment of children with proteinuric HSPN by real-time quantitative PCR. Eight consecutive patients with proteinuric HSPN (4 with grade IIIa and 4 with grade IIIb according to the International Study of Kidney Disease in Children criteria) and 20 healthy subjects were enrolled in this study. The relative expression level of T-bet was significantly higher in the urinary sediment of patients with HSPN at presentation than in that of the healthy controls (p = 0.021), while the relative expression of GATA-3 was significantly lower in the urinary sediment of patients than in that of controls (p = 0.002). Urinary mRNA expression of T-bet correlated with the urinary protein/creatinine ratio (r = 0.608, p = 0.013), and correlated inversely with serum level of total protein (r = -0.574, p = 0.020). Moreover, a significantly increased intensity of T-bet immunostaining was observed in the glomeruli in the biopsy specimen of all study patients. All patients received immunosuppressive therapy. Repeat measurements of urinary mRNA expression of T-bet and GATA-3 after treatment revealed that the expression of T-bet in patients had significantly decreased relative to the baseline (p = 0.003), while the expression of GATA-3 remained static. In a patient subjected to a post-treatment renal biopsy, the increased intensity of immunostaining of T-bet had clearly diminished following immunosuppressive treatment, in accordance with a significant decrease in urinary mRNA expression of T-bet. These observations suggest that patients with proteinuric HSPN demonstrate increased T-bet and depressed GATA-3 expression in the urinary sediment, indicating a possible shift in Th1/Th2 balance towards Th1 predominance.
Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative-type breast cancers (BTNBCs) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of lysyl oxidase (LOX), a metastasis-promoting, matrix-remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by small interfering RNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between LOX and GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBCs to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBCs.
Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.
Beta-arrestin 2 has been shown to participate in the pathogenesis of asthma by inducing Th2 cell migration to the lungs. Whether beta-arrestin 2 regulates cytokine production of CD4+ T cells is still unknown. The aim of the present study was to investigate the effect of beta-arrestin 2 on the cytokine production of CD4+ T lymphocytes and the mechanism involved in a mouse model for asthma. After silencing beta-arrestin 2 expression in CD4+ T lymphocytes from asthmatic mice by RNA interference (RNAi), the interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) levels in CD4+ T lymphocyte culture supernatants with or without terbutaline stimulation were determined. Cell-surface beta2 adrenergic receptor (beta2AR) as well as GATA3 expression of CD4+ T lymphocytes were also measured. CD4+ T lymphocytes of mice with allergic asthma expressed higher levels of beta-arrestin 2 on both mRNA and protein levels. beta-arrestin 2 RNAi decreased IL-4 (43.16%) and GATA3 (protein 77.21%, mRNA 62.98%) expression after terbutaline stimulation. Cell-surface beta2AR of CD4+ T lymphocytes decreased (15.27%) after terbutaline treatment, but recovered after beta-arrestin 2 RNAi down-modulation. These findings demonstrate that beta-arrestin 2 regulates IL-4 production and GATA3 expression of CD4+ T lymphocytes partly through the beta2AR signaling pathway in an allergic asthma model.
The transcription factor GATA3 plays an essential role during T cell development and T helper 2 (Th2) cell differentiation. To understand GATA3-mediated gene regulation, we identified genome-wide GATA3 binding sites in ten well-defined developmental and effector T lymphocyte lineages. In the thymus, GATA3 directly regulated many critical factors, including Th-POK, Notch1, and T cell receptor subunits. In the periphery, GATA3 induced a large number of Th2 cell-specific as well as Th2 cell-nonspecific genes, including several transcription factors. Our data also indicate that GATA3 regulates both active and repressive histone modifications of many target genes at their regulatory elements near GATA3 binding sites. Overall, although GATA3 binding exhibited both shared and cell-specific patterns among various T cell lineages, many genes were either positively or negatively regulated by GATA3 in a cell type-specific manner, suggesting that GATA3-mediated gene regulation depends strongly on cofactors existing in different T cells.
HIV coinfection modifies the clinical course of leishmaniasis by promoting a Th2 pattern of cytokine production. However, little information is available regarding the lymphocytic response in untreated coinfected patients. This work presents the immunophenotyping of Leishmania-stimulated T cells from a treatment-naÏve HIV+ patient with ML. Leishmania braziliensis antigens induced CD69 expression on CD3+CD4+ and CD3+CD8+ cells. It also increased IL-4 intracellular staining on CD3+CD4+GATA3- population and decreased the percentage of CD3+CD4+IL-17+ cells. This suggests that modulations in the IL-4R/STAT6 pathway and the Th17 population may serve as parasitic evasion mechanisms in HIV/ML. Further studies are required to confirm these results.
The effects of T helper (Th) cells on alcoholic cardiomyopathy have not been extensively investigated. Strain of mice with Th1 or Th2 immunological background were utilized in this study in order to explore the role of Th1/Th2 in chronic alcohol-induced cardiac fibrosis.
C57BL/6 WT or Balb/c mice were treated with alcohol for 90 days. Then cardiac structure and function were analyzed via echocardiography. Spleen CD4+CD25+Foxp3+ Tregs were determined by flow cytometry. The hearts were stained using haematoxylin and eosin (HE) and Masson's trichome. Myocardial ultrastructure was observed by electron microscopy. Expression of T-bet, GATA-3, IL-4 and IFN-gamma were determined by real-time RT-PCR. The heart was dilated significantly in the C57BL/6 WT+alcohol group and Balb/c+alcohol group compared with the controls. CD4+CD25+Foxp3+ Tregs were not statistically different. Masson's trichome staining revealed that fibrosis was more pronounced in the alcohol treated groups than the controls. Fibrosis was more evident in the Balb/c+alcohol group compared to the C57BL/6 WT+alcohol group. Alcohol consumption caused a decrease in the Th1 polarization and an increase in the Th2 response.
Chronic alcohol consumption induced a Th2 response within the Th1/Th2 balance. Th2 response is one of the underlying mechanism involved in alcohol-induced cardiac fibrosis.
Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.
The aims were to clarify the effect of placental trophoblasts on T lymphocytes by assessing production of cytokines and expression of transcription factors regulating Th1, Th2, and Th17 immunity in T lymphocytes. Placental trophoblasts were isolated and conditioned medium was made after trophoblast cultivation for 72 h. T lymphocytes were cultured in presence or absence of conditioned medium. ELISA was used to detect concentration of IL-2, TNF-α, IFN-γ, IL-4, IL-10, and IL-17 in supernatants of T cell and real-time PCR was used to detect the status of Th1 (T-bet, STAT-4), Th2 (GATA-3, STAT-6), and Th17 (RORC) immunity in T lymphocyte. We found that the level of IL-2, IFN-γ, TNF-α, and IL-17 was significantly decreased when T lymphocytes were cultured in conditioned medium compared with control, while IL-10 and IL-4 level were not significantly changed. The presence of conditioned medium significantly decreased the ratio of Th1/Th2. The expression of GATA-3 and STAT-6 were significantly increased and STAT-4 was reduced when T lymphocyte was cultured in conditioned medium, while the expression of T-bet and RORC was not significantly different. We concluded that placental trophoblast-induced shift of Th1/Th2 balance toward Th2 and inhibition of Th17 might be among the mechanisms involved in maternal tolerance to fetus.
We evaluated the use of immunological biomarkers in transconjunctival immunotherapy by using cholera toxin B for treating experimental allergic conjunctivitis in a mouse model.
Balb/c mice were sensitized using intraperitoneal injections of ovalbumin (OVA) and were then divided into two groups. The first group was treated by topical instillation of OVA after the instillation of combined OVA and cholera toxin B (CTB) solution B group). The second group was treated by topical instillation of OVA alone (allergy group). The control group consisted of nonsensitized mice undergoing topical OVA instillation only. The numbers of eosinophils and CD4-positive lymphocytes in the conjunctiva were determined histologically, and the observation of immunoglobulin A (IgA)-positive cells in the conjunctiva was performed by immunohistochemistry. Cytokine concentration in the conjunctiva was determined by the protein-array methods. Messenger RNA expression of T-cell-specific markers, such as T-bet, GATA-3, and Foxp3, in the conjunctiva was detected by reversed transcriptase polymerase chain reaction.
The number of eosinophils and CD4-positive lymphocytes increased significantly in the allergy group compared with the control group (P < 0.001) but showed no difference between the CTB group and control group. Concentrations of interleukin 4 (IL-4) (P < 0.05), B-lymphocyte chemoattractant (P < 0.01), and thymus-expressed chemokine (P < 0.05) in the conjunctiva were significantly higher in the CTB group than in the other two groups. GATA-3 messenger RNA (mRNA) in the conjunctiva was expressed in the three groups, but T-bet and Foxp3 were not detected.
Transconjunctival immunotherapy using CTB can be evaluated by histological examination of eosinophils and CD4-positive T cells, and a mucosal immunity-associated chemokine and a helper T-cell-17-associated chemokine as biomarkers.
The vertebrate thymus provides an inductive environment for T-cell development. Within the mouse thymus, Notch signals are indispensable for imposing the T-cell fate on multipotential haematopoietic progenitors, but the downstream effectors that impart T-lineage specification and commitment are not well understood. Here we show that a transcription factor, T-cell factor 1 (TCF-1; also known as transcription factor 7, T-cell specific, TCF7), is a critical regulator in T-cell specification. TCF-1 is highly expressed in the earliest thymic progenitors, and its expression is upregulated by Notch signals. Most importantly, when TCF-1 is forcibly expressed in bone marrow (BM) progenitors, it drives the development of T-lineage cells in the absence of T-inductive Notch1 signals. Further characterization of these TCF-1-induced cells revealed expression of many T-lineage genes, including T-cell-specific transcription factors Gata3 and Bcl11b, and components of the T-cell receptor. Our data suggest a model where Notch signals induce TCF-1, and TCF-1 in turn imprints the T-cell fate by upregulating expression of T-cell essential genes.
To investigate the effects of shikonin on phorbol myristate acetate (PMA) plus cyclic adenosine monophosphate (cAMP)-induced T helper (T(H)) 2 cell cytokine production, and the underlying mechanism.
We used activated EL-4 murine T-lymphoma cells, which produce interleukin (IL)-4 and IL-5, but not interferon (IFN)-γ, as T(H)2 cell-like cells and treated them with PMA+cAMP to investigate the effects of shikonin on T(H)2 cytokines, transcriptional factors, and the related mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway.
The data show that shikonin inhibited the PMA+cAMP-induced mRNA and protein expression of IL-4 and IL-5 via the downregulation of GATA-binding protein-3 (GATA-3) and c-musculoaponeurotic fibrosarcoma (Maf) but not T-box expressed in T cells (T-bet). Moreover, shikonin suppressed the phosphorylation of p38, inhibitor of κB (IκB) kinase (IKK)-β and IκB-α, and the subsequent IκB-α degradation induced by PMA+cAMP; however, the PMA+cAMP-induced phosphorylation of extracellular signal-related kinase (ERK), which resulted in minor inhibition and phosphorylation of c-Jun N-terminal kinase (JNK), seemed to be unaffected by shikonin treatment.
This study suggests that downregulation of GATA-3 and c-Maf via the suppression of p38, IKK-β and IκB-α phosphorylation might contribute to the inhibitory effect of shikonin on mitogen-induced IL-4 and IL-5 production in EL-4T cells. Furthermore, shikonin is a potential drug for treating allergic diseases.
To study the Uygur medicine Hyssopus officinalis L on T-bet, GATA-3, STAT-3 mRNA levels of asthma rats in order to explore the mechanism of its treatment of asthma.
Rats were randomly divided into normal control group, asthma model group and dexamethasone group and water extract of Hyssopus officinalis L high and low dose group. The rats were sensitized with OVA, Al(OH)(3); and DPT vaccine and then challenged with inhalation of aerosolized OVA solution for Preparation of asthma model and the level of T-bet, GATA-3, STAT-3 mRNA were detected with RT-PCR.
The normal control group and model group, model group and treatment group, the expression of T-bet, GATA-3 and STAT-3 mRNA in the lung tissue was statistically significant differences(P<0.05). Compared with model group, after treatment of Hyssopus officinalis L the expression of GATA-3 and STAT-3 mRNA of asthma rats significantly reduced (P<0.05), but the expression of T-bet mRNA was significantly higher (P<0.05).The expression of GATA-3 and STAT-3 mRNA of Hyssopus officinalis L high-dose treatment group was lower than the low-dose treatment group (P<0.05), but T-bet mRNA that was higher(P<0.05). The expression of T-bet mRNA has negative correlation with GATA-3 mRNA (r=-0.696), the expression of STAT-3 mRNA has correlation with T-bet mRNA and GATA-3 mRNA(r=-0.767, 0.772), P<0.05.
Hyssopus officinalis L probably regulates the differentiation of Th1, Th2 and Th17 on transcription level to play the role of anti-inflammatory.
The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons from embryonic day 12, when SG neurons first extend projections, up until postnatal day 15, after the onset of hearing. For comparison, we also analyzed the closely related vestibular ganglion (VG). Gene ontology analysis confirmed enriched expression of genes associated with gene regulation and neurite outgrowth at early stages, with the SG and VG often expressing different members of the same gene family. At later stages, the neurons transcribe more genes related to mature function, and exhibit a dramatic increase in immune gene expression. Comparisons of the two populations revealed enhanced expression of TGFβ pathway components in SG neurons and established new markers that consistently distinguish auditory and vestibular neurons. Unexpectedly, we found that Gata3, a transcription factor commonly associated with auditory development, is also expressed in VG neurons at early stages. We therefore defined new cohorts of transcription factors and axon guidance molecules that are uniquely expressed in SG neurons and may drive auditory-specific aspects of their differentiation and wiring. We show that one of these molecules, the receptor guanylyl cyclase Npr2, is required for bifurcation of the SG central axon. Hence, our dataset provides a useful resource for uncovering the molecular basis of specific auditory circuit assembly events.
To investigate the effect of N-methyl-D-aspartate (NMDA) receptor antagonist on T helper (TH) cell differentiation and intracellular transcriptional factors in vitro.
Ten male healthy volunteers (aged 20-45 y, BMI 18-25) were enrolled in this study. Twenty milliliters peripheral blood was collected in the morning from fasted volunteers and peripheral blood mononuclear cells (PBMC) were isolated. PBMC were incubated with phorbol-myristate-acetate (25 ng/ml) (PMA) plus ionomycin (1 μg/ml) in the presence of ketamine or MK-801 at 37 °C. TH subsets, supernatant interferon γ (IFN-γ), interleukin 4 (IL-4), and intracellular transcriptional factors T-bet and GATA3 were analyzed 4 h later.
The number of TH0 cells was kept constant and at baseline before PMA and ionomycin stimulation in each group. TH1 cells, TH2 cells, IFN-γ and IL-4 levels were significantly increased after PMA and ionomycin stimulation. Ketamine and MK-801 decreased TH1 cells, TH2 cells, IFN-γ and IL-4 levels but increased the ratio of TH1/TH2 and IFN-γ/IL-4 in the presence of PMA and ionomycin. Ketamine or MK-801 alone had no effect on either of them. T-bet and GATA3 activities in PBMC were significantly increased after PMA and ionomycin stimulation. Ketamine and MK-801 decreased T-bet and GATA3 activities but increased the ratio of T-bet/GATA3 following PMA and ionomycin stimulation. Ketamine or MK-801 alone had no effect on the activity of T-bet, GATA3 or T-bet/GATA3.
NMDA antagonist can suppress TH cell differentiation and subsequent cytokines production but increase TH1/TH2 ratio following PMA and ionomycin stimulation, it may be related to its regulation on T-bet and GATA3 activities.
It has been almost a quarter century since it was first appreciated that a class of oncogenes contained in rapidly transforming avian retroviruses encoded DNA-binding transcription factors. As with other oncogenes, genetic recombination with the viral genome led to their overexpression or functional alteration. In the years that followed, alterations of numerous transcription factors were shown to be causatively involved in various cancers in human patients and model organisms. Depending on their normal cellular functions, these factors were subsequently categorized as proto-oncogenes or tumor suppressor genes. This review focuses on the role of GATA transcription factors in carcinogenesis. GATA factors are zinc finger DNA binding proteins that control the development of diverse tissues by activating or repressing transcription. GATA factors thus coordinate cellular maturation with proliferation arrest and cell survival. Therefore, a role of this family of genes in human cancers is not surprising. Prominent examples include structural mutations in GATA1 that are found in almost all megakaryoblastic leukemias in patients with Down syndrome; loss of GATA3 expression in aggressive, dedifferentiated breast cancers; and silencing of GATA4 and GATA5 expression in colorectal and lung cancers. Here, we discuss possible mechanisms of carcinogenesis vis-à-vis the normal functions of GATA factors as they pertain to human patients and mouse models of cancer.
Nonanastomotic biliary strictures represent a serious complication after orthotopic liver transplantation (OLT). This study investigates the potential role of mucins in bile duct injury after OLT.
Sprague-Dawley rats were divided into four groups: normal group (Normal, n=5), sham-operated group (Sham, n=20), OLT group with 1 hr donor cold ischemic time (n=20), and OLT group with 12 hr donor cold ischemic time (OLTn=20). Expression of mucins and GATA factors in bile ducts was examined by real-time polymerase chain reaction, immunohistochemistry, and immunoblotting. Bile was collected for biochemical analysis, and the histological changes associated with bile duct injury were evaluated.
In normal bile ducts, Muc1, Muc2, Muc3A, Muc4, and Muc6 mRNA were expressed, whereas Muc5AC mRNA was undetectable. The expression of Muc1, Muc3A, and Muc4 but not Muc2 and Muc6 at mRNA level in graft bile ducts decreased remarkably early after OLT. The decreased expression of Muc1 and Muc4 was further confirmed at protein level by immunohistochemistry and immunoblotting. Downregulation of Muc1 and Muc3A expression by prolonged cold ischemic time was significantly associated with the injury severity scores of large but not small bile ducts. Among six GATA factors, GATA3, GATA4, and GATA6 mRNA were expressed in normal bile ducts. GATA4 and GATA6 mRNA levels decreased significantly after OLT.
Downregulation of Muc1 and Muc3A expression by prolonged cold ischemic time may play a potential role in large bile duct injury early after OLT.
The high incidence of lung-damaging life-threatening respiratory infections in infants may be related to the immaturity of their immune systems. To determine whether lung immune features differ in early life compared with those in adulthood, whole lung as well as lung T lymphocyte and DC responses were investigated in BALB/c neonates versus adults. Higher expression of GATA-3 and rapid and sustained production of type 2 cytokines by lung explants after in vitro exposure to anti-CD3 was the hallmark of the neonatal period, suggestive of a Th2 bias. Neonatal lung GATA-3-producing cells were identified as CD3(+), CD4 and CD8 double-negative T lymphocytes, a subset found at a higher frequency in neonatal than adult lung. The neonatal lungs contained fewer conventional DCs, with a lower ratio of CD103(+) to CD11b(+) DCs, and a much lower number of plasmacytoid DCs in comparison with adult lungs. Yet, when stimulated in vivo by BCG, neonatal lung DCs matured and primed adult naïve CD4(+) T cells toward Th1 as efficiently as adult BCG-primed lung DCs. Conversely, both adult and neonatal BCG-primed lung DCs induced a Th2 cytokine response from neonatal naïve lymph node T cells, suggestive of an intrinsic feature of neonatal T lymphocytes.
Antipsychotic drugs (APDs) are commonly used to ease the symptoms of schizophrenia; however, these same drugs also have an effect on the human immune system. Our previous studies have shown that risperidone and clozapine effectively decrease the production of IFN-γ for CD4(+) T-cells in PBMC. In contrast, haloperidol causes an increase in the production of IFN-γ for CD4(+) T-cells in PBMC. In this study we show that risperidone and clozapine can reduce Th1 cell differentiation and T-bet expression. The differentiation of Th1 cells was reduced in clozapine or risperidone treated PBMC by inhibiting the phosphorylation of AKT but not STAT-4. Typical APD, haloperidol, had the opposite effect in regulating T cell differentiation when compared with atypical APDs including risperidone and clozapine. Haloperidol decreased the expression of GATA-3, a Th2-related transcription factor, by inhibiting NF-κB activation rather than STAT-6 phosphorylation and thus decreased Th2 differentiation. In addition, chronic risperidone and clozapine treatment reduces the IFN-γ producing CD4(+) T-cell population within PBMC. In conclusion, this study suggests that APDs do indeed regulate the body's immune response and therefore all APDs should have their own patent in regulating immune responses.
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.
It is currently unclear whether our classifications for T helper cell subtypes truly define stable lineages or rather they represent cells with a more flexible phenotype. This distinction is important for predicting the behavior of T helper cells during normal immune responses as well as in pathogenic conditions. Determining the mechanisms by which T helper cell lineage-defining transcription factors are expressed and subsequently regulate epigenetic and downstream gene regulatory events will provide insight into this complex question. Importantly, lineage-defining transcription factors that regulate epigenetic events have the potential to redefine the fate of the cell when they are expressed. In contrast, factors that regulate the events downstream of a permissive epigenetic environment will only have the capacity to modulate the underlying gene expression profile that is already established in that cell. Finally, mechanisms related to the antagonism versus cooperation between the lineage-defining factors for opposing T helper cell subsets will influence the characteristics of the cell. Here, we provide an overview of these topics by discussing epigenetic states in T helper cell subtypes as well as the mechanisms by which lineage-defining factors, such as T-bet, regulate gene expression profiles at both the epigenetic and general transcription level. We also examine some of what is known about the interplay between the T helper cell lineage-defining transcription factors T-bet, GATA3, Foxp3, Rorγt, and Bcl-6 and how this relates to the proper functioning of T helper cell subsets. Defining the mechanisms by which these factors regulate gene expression profiles will aid in our ability to predict the functional capabilities of T helper cell subsets.
Prostate cancer (PCA) is a clinically heterogeneous and often multifocal disease with a clinical outcome difficult to predict. A deeper knowledge of the molecular basis of the disease may lead to a better prediction of prognosis. Therefore, in this study we investigated the molecular basis of PCA by identifying potential tumor markers in laser-microdisected PCA tissues. Among a group of PCA patients, quantitative RT-PCR analysis was performed to compare the expression of 70 genes. These genes were selected from the results of two microarrays which investigated the gene expression profile differences between moderately or poorly differentiated prostate carcinoma glands and the corresponding normal glands. Among the genes examined, CDKN2A, GATA3, CREBBP, ITGA2, NBL1 and TGM4 were down-regulated in the prostate carcinoma glands compared to the corresponding normal glands, whereas TFF3, TMPRSS2 and ERG were up-regulated. Our findings indicate that these genes may play roles as tumor suppressor genes or oncogenes in PCA, and may serve as potential tumor markers and novel therapeutic targets.
Drug-induced liver injury is a growing concern for pharmaceutical companies and patients because numerous drugs have been linked to hepatotoxicity and it is the most common reason for a drug to be withdrawn. Flutamide rarely causes liver dysfunction in humans, and immune allergic reactions have been suggested in some cases. In this study, we investigated the mechanisms of flutamide-induced liver injury in BALB/c mice. Plasma alanine aminotransferase and aspartate aminotransferase levels were significantly increased 3, 6 and 9 h after flutamide (1500 mg kg⁻¹ , p.o.) administration. The biomarker for oxidative stress was not changed, but Th2-dominant immune-related factors, such as interleukin (IL)-4, IL-5, STAT6 and GATA-binding protein (GATA)-3, were induced in flutamide-administered mice. The pre-administration of monoclonal-IL-4 antibody suppressed the hepatotoxicity of flutamide. In addition, we investigated the effect of 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂; 10 µg per mouse, i.p.) administration on flutamide-induced acute liver injury. Coadministration of DK-PGD₂ and flutamide resulted in a significant increase in alanine aminotransferase and a remarkable increase of macrophage inflammatory protein-2. In conclusion, we demonstrated that flutamide-induced acute liver injury is mediated by Th2-dominant immune responses in mice.
A number of distinguished populations with manifold functions and various pathways of differentiation have been identified within T lymphocytes. The cells expressing CD4 coreceptor on their cell surface are the most varied group. Depending on changes in different tissue microenvironments induced by such cytokines as IL-4, IFN-γ, IL-10 or TGF-β, CD4+ T lymphocytes can differentiate into alternative subpopulations performing helper, regulatory/suppressor function (Th1, Th2, Th9, Th17, Th22, Tfh, iTreg and Tr1). In the direction of lineage differentiation of these lymphocytes, transcription factors play the key role. The most important of them are T-bet, GATA3, RORγt, FOXP3, AHR and c-Maf. A cytokine binding to a specific receptor activates the transcription factors, other DNA-binding proteins, and epigenetic alterations, enabling the transcription of proper genetic information. The plasticity of CD4+ cell differentiation seems to be in dynamic balance between initial commitment and flexibility of these cells in the face of a changing environment. Even more, phenotypical and functional borders between particular subpopulations have turned out to be fluent. Then, the influence of extrinsic factors on the activation of mechanisms responsible for conversion of CD4+ T lymphocytes into functional mature cells appears to be more complicated than was previously thought.
Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.
Th2 cell differentiation is critically influenced by transcription factor GATA-3 and by various cis-acting elements including enhancers, silencers and a locus control region (LCR) in the Th2 cytokine locus. Th2 LCR-deficient Th2 cells completely lost the expression of GATA-3 and the phosphorylation of STAT6. Histone 3 lysine 4 (H3-K4) was hypomethylated in the gata3 locus in these cells. GATA-3 and STAT6 bound several regulatory regions in the gata3 locus and transactivated the expression of the gata3 gene. These results suggest that Th2 differentiation program stimulates feed-forward regulation of gata3 gene expression.
DNA evolution models made invaluable contributions to comparative genomics, although it seemed formidable to include non-genomic features into these models. In order to build an evolutionary model of transcription networks (TNs), we had to forfeit the substitution model used in DNA evolution and to start from modeling the evolution of the regulatory relationships. We present a quantitative evolutionary model of TNs, subjecting the phylogenetic distance and the evolutionary changes of cis-regulatory sequence, gene expression and network structure to one probabilistic framework. Using the genome sequences and gene expression data from multiple species, this model can predict regulatory relationships between a transcription factor (TF) and its target genes in all species, and thus identify TN re-wiring events. Applying this model to analyze the pre-implantation development of three mammalian species, we identified the conserved and re-wired components of the TNs downstream to a set of TFs including Oct4, Gata3/4/6, cMyc and nMyc. Evolutionary events on the DNA sequence that led to turnover of TF binding sites were identified, including a birth of an Oct4 binding site by a 2nt deletion. In contrast to recent reports of large interspecies differences of TF binding sites and gene expression patterns, the interspecies difference in TF-target relationship is much smaller. The data showed increasing conservation levels from genomic sequences to TF-DNA interaction, gene expression, TN, and finally to morphology, suggesting that evolutionary changes are larger at molecular levels and smaller at functional levels. The data also showed that evolutionarily older TFs are more likely to have conserved target genes, whereas younger TFs tend to have larger re-wiring rates.
The role of Stat5 in maintaining adequate lactation was studied in Stat5a(-/-) mice expressing a conditionally suppressed transgenic STAT5 in their mammary glands. This system enables distinguishing STAT5's effects on lactation from its contribution to mammary development during gestation. Females were allowed to express STAT5 during their first pregnancy. After delivery, STAT5 levels were manipulated by doxycycline administration and withdrawal. In two lines of genetically modified mice, the absence of STAT5 expression during the first 10 days of lactation resulted in a decrease of 29% or 41% in newborn weight gain. The STAT5-dependent decrease in growth was recoverable, but not completely reversible, particularly when STAT5 expression was omitted for the first 4 days of lactation. Within the first 10 days of STAT5-omitted lactation, alveolar occupancy regressed by 50% compared to that measured at delivery. By Day 10, only 18% of the fat-pad area was involved in milk production. The alveolar regression caused by 4 days of STAT5 deficiency was reversible, but neonate growth remained delayed. STAT5 deficiency resulted in reduced estrogen receptor α and connexin 32 gene expression, accompanied by delayed induction of both anti- and pro-apoptotic Bcl-2 family members. An increase in Gata-3 expression may reflect an attempt to maintain alveolar progenitors. A decrease of 39% and 23% in WAP and α-lactalbumin expression, respectively, with no associated effects on β-casein, also resulted from lack of STAT5 expression in the first 10 days of lactation. This deficiency enhances the major effect of alveolar regression on delayed weight gain in newborns.
Allergic inflammation is due to a complex interplay between several inflammatory cells, including mast cells, basophils, lymphocytes, dendritic cells, eosinophils, and sometimes neutrophils. These cells produce multiple inflammatory mediators, including lipids, purines, cytokines, chemokines, and reactive oxygen species. Allergic inflammation affects target cells, such as epithelial cells, fibroblasts, vascular cells, and airway smooth muscle cells, which become an important source of inflammatory mediators. Sensory nerves are sensitized and activated during allergic inflammation and produce symptoms. Allergic inflammatory responses are orchestrated by several transcription factors, particularly NF-κB and GATA3. Inflammatory genes are also regulated by epigenetic mechanisms, including DNA methylation and histone modifications. There are several endogenous anti-inflammatory mechanisms, including anti-inflammatory lipids and cytokines, which may be defective in allergic disease, thus amplifying and perpetuating the inflammation. Better understanding of the pathophysiology of allergic inflammation has identified new therapeutic targets but developing effective novel therapies has been challenging. Corticosteroids are highly effective with a broad spectrum of anti-inflammatory effects, including epigenetic modulation of the inflammatory response and suppression of GATA3.
Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized.
Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA.
Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.
GATA3 has been identified as a master regulator of T helper cells, as well as being important for early thymic progenitors and T-cell commitment. However, Gata3 expression initiates already at the hematopoietic stem cell (HSC) level, implicating a potential role also in the regulation of HSCs. Herein we used a conditional Gata3 knockout strategy in which Gata3 expression was completely deleted from the earliest stage of embryonic hematopoietic development after emergence of HSCs from hemogenic endothelium. Through a detailed analysis of HSCs at the phenotypic and functional level, we demonstrate that steady-state levels of HSCs are normal in Gata3(fl/fl)Vav-Cre(tg/+) mice. Moreover, through long-term primary and secondary transplantation experiments, we also unequivocally demonstrate that Gata3 has a redundant role in post-transplantation HSC self-renewal.
Despite the importance of interleukin-13 (IL-13) in systemic sclerosis (SSc) and other fibrotic diseases, its mechanisms of action are not understood. We have reported that excessive amounts of IL-13 are produced by peripheral blood effector CD8+ T cells from patients with diffuse cutaneous SSc (dcSSc). The aim of the present study was to establish the molecular basis of IL-13 dysregulation in the pathogenesis of SSc.
Quantitative polymerase chain reaction analysis and intracellular staining were used to study the transcription factors that control naive peripheral blood CD8+ T cell differentiation into type 1 and type 2 cytokine-secreting cells. Intracellular staining revealed that GATA-3 levels in freshly isolated naive CD8+ T cells correlated with specific clinical manifestations. We therefore assessed the effects of GATA-3 inhibition on IL-13 production in CD8+ T cells from the SSc patients.
Freshly isolated naive peripheral blood CD8+ T cells expressed high levels of GATA-3 and failed to down-regulate IL-13 production when cultured under type 1-skewing conditions, but maintained adequate levels of interferon-γ production. Cellular GATA-3 levels were significantly higher in patients with dcSSc and early inflammatory disease. Silencing of GATA-3 with small interfering RNA significantly reduced IL-13 production by CD8+ T cells, demonstrating a causal relationship between GATA-3 and IL-13.
These results provide important new insights into SSc pathogenesis and suggest that increased GATA-3 expression in CD8+ T cells could be a highly relevant biomarker of immune dysfunction in patients with dcSSc. GATA-3 could be a novel therapeutic target for this currently incurable disease.
CD4 T(h) are critical for orchestrating adaptive immune responses. The expression of the transcription factor GATA3 (GATA-binding protein 3) is up-regulated or down-regulated during T(h)2 or T(h)1 cell differentiation, respectively. Furthermore, GATA3 is responsible for induction of T(h)2 differentiation and represses T(h)1 differentiation. In this review, we present an updated view on the molecular mechanisms through which GATA3 regulates T(h)1/T(h)2 differentiation. During T(h)2 cell differentiation, GATA3 directly binds to the T(h)2 cytokine gene locus at several regions and regulates expression. On the other hand, GATA3 inhibits T(h)1 cell differentiation by preventing up-regulation of IL-12 receptor β2 and STAT4 (signal transducer and activator of transcription 4) and neutralization of Runx3 (runt-related transcription factor 3) function through protein-protein interaction. GATA3 may also directly act on the Ifng gene. In summary, GATA3 serves as a transcriptional activator or repressor through direct action on transcriptional machinery and/or affecting chromatin remodeling at many critical loci encoding cytokines, cytokine receptors, signaling molecules as well as transcription factors that are involved in the regulation of T(h)1 and T(h)2 differentiation.
Allergic asthma is associated with airway eosinophilia, which is regulated by different T-effector cells. T cells express transcription factors T-bet, GATA-3, RORγt and Foxp3, representing Th1, Th2, Th17 and Treg cells respectively. No study has directly determined the relative presence of each of these T cell subsets concomitantly in a model of allergic airway inflammation. In this study we determined the degree of expansion of these T cell subsets, in the lungs of allergen challenged mice. Cell proliferation was determined by incorporation of 5-bromo-2'-deoxyuridine (BrdU) together with 7-aminoactnomycin (7-AAD). The immunohistochemical localisation of T cells in the lung microenvironments was also quantified. Local expression of cytokines, chemokines and receptor genes was measured using real-time RT-PCR array analysis in tissue sections isolated by laser microdissection and pressure catapulting technology. Allergen exposure increased the numbers of T-bet(+), GATA-3(+), RORγt(+) and Foxp3(+) cells in CD4(+)CD25(+) and CD4(+)CD25(-) T cells, with the greatest expansion of GATA-3(+) cells. The majority of CD4(+)CD25(+) T-bet(+), GATA-3(+), RORγt(+) and Foxp3(+) cells had incorporated BrdU and underwent proliferation during allergen exposure. Allergen exposure led to the accumulation of T-bet(+), GATA-3(+) and Foxp3(+) cells in peribronchial and alveolar tissue, GATA-3(+) and Foxp3(+) cells in perivascular tissue, and RORγt(+) cells in alveolar tissue. A total of 28 cytokines, chemokines and receptor genes were altered more than 3 fold upon allergen exposure, with expression of half of the genes claimed in all three microenvironments. Our study shows that allergen exposure affects all T effector cells in lung, with a dominant of Th2 cells, but with different local cell distribution, probably due to a distinguished local inflammatory milieu.
The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate-rich elements (AREs) present in the 3' untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3'UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.
Naïve CD4(+) T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4(+) cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4(+) T cells under the Th2 condition. Adenoviral transduction of naïve CD4(+) T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.
Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions.Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction.
Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.
Vitamin D has several reported immunomodulatory properties including the reduced generation of pro-inflammatory CD4+ T helper 1 (Th1) cells and the increase in levels of the anti-inflammatory Th2 subset. Less clear has been the impact of vitamin D on the pro-inflammatory Th17 subset, and whether and how vitamin D may preferentially drive the polarization of one of the T helper subsets.
Using human peripheral blood-derived mononuclear cells and mouse splenocytes and lymph node cells in culture, we examined whether and how vitamin D preferentially skews T cells towards the Th1, Th2 or Th17 subsets. Mice afflicted with the multiple sclerosis-like condition, experimental autoimmune encephalomyelitis (EAE), were examined in vivo for the relevance of the tissue culture-derived results.
We report that the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 {1,25(OH)2D3}, consistently generates human and murine Th2 cells in culture, frequently leaving unchanged the levels of Th1/Th17 cytokines. As a result, the ratio of Th2 to Th1 and Th17 is increased by 1,25(OH)2D3. The upregulation of Th2 to Th1 or Th17 subsets by 1,25(OH)2D3 is enabled by an increase of the GATA-3 transcription factor, which itself is promoted upstream by an elevation of the STAT6 transcription factor. In mice, the alleviation of EAE severity by 1,25(OH)2D3 is accompanied by elevation of levels of GATA-3 and STAT6. Significantly, the efficacy of 1,25(OH)2D3 in ameliorating EAE is completely lost in mice genetically deficient for STAT6, which was accompanied by the inability of 1,25(OH)2D3 to raise GATA-3 in STAT6 null lymphocytes.
These results of vitamin D promoting a Th2 shift through upstream GATA-3 and STAT6 transcription factors shed mechanistic understanding on the utility of vitamin D in MS.
To investigate the effect of low-level toluene inhalation on immune regulation in an allergic mouse model, C3H/HeN mice were exposed to 0, 5, 50, or 500ppm of toluene for 6h/day, 5 days/week for 3 or 6 weeks. For allergic mouse model, half of the mice in each group were immunized with ovalbumin (OVA). Allergic mice exposed to toluene for 3 weeks did not exhibit any changes in their plasma, lung or spleen samples. Although exposure to toluene alone for 6 weeks did not increase the number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, coexposure to 50ppm toluene and OVA increased the number of BAL cells. Histological changes and increased amounts of fibronectin were observed in the lungs of OVA-immunized, 50-ppm-toluene-exposed mice. Exposure to 500ppm significantly increased the expressions of transcription factors STAT3, STAT4 and STAT5a mRNAs in spleen. In spleens from the allergic mouse model, the expressions of STAT3, STAT4, STAT5a, STAT6, GATA3 and Foxp3 mRNAs were significantly enhanced following exposure to 50ppm toluene for 6 weeks, but the expression of T-bet mRNA was not increased. Regarding the Th1/Th2 balance, the expressions of IL-4 and IL-12 mRNAs were enhanced in the spleens of toluene-exposed mice. Total IgG1 antibody production in the plasma was significantly increased in the 50-ppm-toluene-exposed allergic mouse model. These results indicate that low-level toluene exposure might dysregulate the allergic responses to OVA in C3H/HeN mice.
Dendritic cells (DCs) are crucial to shape the adaptive immune response. Extensive in vitro manipulation reprograms T(H)2 and T(H)17 cell lines into T(H)1 cells, leading to the concept of CD4(+) T(H) cell subset plasticity. The conversion of memory T(H)17 cells into T(H)2 cells or vice versa remains to be clarified.
We examined the localization of T(H)17/T(H)2 cells in vivo, their cellular origin (T(H)2 vs T(H)17), and the underlying mechanisms that drive the generation of these double T(H) producers.
Antigen-loaded bone marrow-derived DCs (ovalbumin-DCs) were repeatedly administered locally (intratracheally) or systemically (intravenously) to naive mice to elicit chronic airway inflammation. Inflamed lungs and mediastinal lymph nodes were examined for the presence of IL-17(+)IL-13(+)IL-4(+)CD4(+) T cells that coexpressed retinoic acid receptor-related orphan receptor γt and GATA-3 (T(H)17/T(H)2).
We show that repetitive administration of inflammatory ovalbumin-DCs, locally or systemically, promoted the development of antigen-specific T(H)17/T(H)2 cells in lungs and mediastinal lymph nodes. Immunized mice had IgE-independent and steroid-resistant airway inflammation with a mixed neutrophil and eosinophil infiltration of the bronchoalveolar lavage fluid. Airway inflammatory signal regulatory protein α-positive DCs reprogrammed in vitro-generated T(H)17 but not T(H)2 cells, as well as lung effector T(H) cells, into T(H)17/T(H)2 cells.
We demonstrate the existence of T(H)17/T(H)2 cells that express GATA-3 in inflamed tissues and their T(H)17 origin. We further propose that repeated immunization with inflammatory DCs prevails on the route of DC administration to drive T(H)17/T(H)2-associated chronic lung inflammation.
A well-known association between vitamin D(3) and infection with Mycobacterium tuberculosis has previously been reported, but little is known regarding the underlying mechanisms. We have investigated how 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] affects the proinflammatory cytokine production induced by M. tuberculosis. Furthermore, we explored whether 1,25(OH)(2)D(3) influence the production of the protective antimycobacterial peptide cathelicidin. Upon in vitro stimulation with M. tuberculosis, 1,25(OH)(2)D(3) induced a dose-dependent down-regulation of IL-6, TNFα and IFNγ, while increasing the production of IL-10 in culture supernatant as well as cathelicidin mRNA expression. This effect on cytokine response was not due to modulation of T-helper cell differentiation, as T-bet, GATA3, Foxp3 and ROR-γt mRNA expression remained unaffected. Similarly, 1,25(OH)(2)D(3) did not affect suppressor of cytokine signaling (SOCS)1 and SOCS3 mRNA expression. The mechanism whereby 1,25(OH)(2)D(3) inhibited the proinflammatory cytokine response was through reduced expression of the pattern recognition receptors (PRR) - TLR2, TLR4, Dectin-1 and mannose receptor, whose mRNA and protein expression were both reduced. The suppression of PRRs could be restored by a VDR antagonist. Upon M. tuberculosis stimulation, 1,25(OH)(2)D(3) modulates the balance in cytokine production towards an anti-inflammatory profile by repression of TLR2, TLR4, Dectin-1 and mannose receptor expression, while increasing cathelicidin production. These two effects may have beneficial consequences, by reducing the collateral tissue damage induced by proinflammatory cytokines, while the antibacterial effects of cathelicidin are enhanced.
Hepatoid adenocarcinoma is a rare neoplasm, which has a striking morphologic similarity to hepatocarcinoma. It has been described in different organs, the most common are stomach, lung, and pancreas. In some cases, it is characterized by high serum levels of α-fetoprotein. This tumor has a pattern similar to the hepatocarcinoma. The typical features are a combination of histopathologic aspects of solid nests and trabecular structures of polygonal atypical cells with eosinophil and granular cytoplasm and immunohistochemical expression of α-fetoprotein and of carcinoembryonic antigen in half of cases. Here, we report the case of an old female patient affected by hepatoid adenocarcinoma of the ureter with ovarian, small intestine, and hepatic involvement. We discuss the clinical aspects, the morphologic features, and the immunoistochemical staining useful for differential diagnosis.
Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig-mouse cytoplasmic hybrids as a model to address nuclear-cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.
Haploinsufficiency of Gata3 causes hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome in mice and humans. Gata3 null mutation leads to early lethality around embryonic day (E)11.5, but catecholamine precursor administration can rescue Gata3 null mutants to E16.5. At E11.5, GATA3 deficiency results in the development of an empty otocyst with an endolymphatic duct. However, using rescued mice we found that some morphogenesis and neurosensory development is possible in the ear without Gata3. Extending previous studies, we find that at E16.5, Gata3 mutant inner ears can undergo partial morphogenesis and develop an endolymphatic duct, a utricular and saccular recess, and a shortened cochlear duct. In addition to the obvious morphogenic aberrations, these studies demonstrate that a subset of neurons develop and connect a fragmented sensory patch of MYO7A-positive hair cells to the vestibular nuclei of the brainstem. In situ hybridization studies reveal altered expression of several transcription factors relevant to ear development and we hypothesize that this may relate to the observed dysmorphia and restricted neurosensory development. While a cochlear duct can form, there is no concurrent cochlear neurosensory development, observations consistent with specific hearing defects encountered by HDR patients and mice with Gata3-associated expression alterations. Gata3 null mutant phenocopies the otic maldevelopment (cochlear duct formation in the absence of neurosensory development) seen in Foxg1cre mediated conditional deletion of microRNA processing enzyme, Dicer1. Finally, while GATA3 is expressed in the developing vestibulo-cochlear efferent (VCE) neurons, and its absence in the null mutants disrupts VCE projections to the ear, loss of GATA3 does not affect VCE progenitor cell migration.
Transplant glomerulitis, characterized by mononuclear cell infiltration of glomeruli, is likely to occur during clinical or subclinical antibody-mediated rejection.
To determine whether T-cell phenotype influences the clinical presentation of this pathologic condition, we used reverse transcription quantitative polymerase chain reaction to analyze expression of Treg cells (Foxp3), cytotoxic CD8 T cells (Granzyme B), Th1 cells (INF-γ,T Bet), Th2 cells (GATA3, IL-4), and Th17 pathway (IL-17). Our study included 20 renal transplant recipients exhibiting subclinical glomerulitis (SG) diagnosed after a routine 3-month posttransplant biopsy. Results were compared with those observed in 22 patients with normal routine biopsies at 3 months (N) and 17 patients with clinical glomerulitis occurring during early acute renal dysfunction within the first year after transplantation in a context of acute antibody-mediated rejection.
Our results show that expression of IL-4 mRNA was significantly higher in SG patients than in N patients (P = 0.02). Expression of IFN-γ was significantly higher in patients with clinical glomerulitis than in patients with SG (P<0.001) and was associated with a clinical expression of glomerulitis.
Our results suggest that the balance of Th1/Th2 is likely to differentiate clinical expression of transplant glomerulopathy. They also indicate that therapeutic approaches in cases of SG should be defined with caution and take into account transcriptional criteria.
Differentiation of sympathetic neurons is controlled by a group of transcription factors, including Phox2b, Ascl1, Hand2 and Gata3, induced by bone morphogenetic proteins (BMPs) in progenitors located in ganglion primordia at the dorsal aorta. Here, we address the function of the transcription factors AP-2β and AP-2α, expressed in migrating neural crest cells (NCC) and maintained in sympathetic progenitors and differentiated neurons. The elimination of both AP-2α and AP-2β results in the virtually complete absence of sympathetic and sensory ganglia due to apoptotic cell death of migrating NCC. In the AP-2β knockout only sympathetic ganglia (SG) are targeted, leading to a reduction in ganglion size by about 40%, which is also caused by apoptotic death of neural crest progenitors. The conditional double knockout of AP-2α and AP-2β in sympathetic progenitors and differentiated noradrenergic neurons results in a further decrease in neuron number, leading eventually to small sympathetic ganglion rudiments postnatally. The elimination of AP-2β also leads to the complete absence of noradrenergic neurons of the Locus coeruleus (LC). Whereas AP-2α/β transcription factors are in vivo not required for the onset or maintenance of noradrenergic differentiation, their essential survival functions are demonstrated for sympathetic progenitors and noradrenergic neurons.
GATA binding protein 3 (GATA3) is a key molecule regulating the balance in the ratio of type 1 helper T (Th1) cells to type 2 helper T (Th2) cells, which is thought to be indicative of the pathogenesis of allergic diseases such as asthma and atopic dermatitis. The aim of this study was to investigate the role of GATA3 in allergic skin inflammation. Transgenic (Tg) mice overexpressing human GATA3 (hGATA3) were produced by the microinjection of pCMV/hGATA3 constructs into fertilized mouse eggs. The hGATA3 gene was successfully expressed at the protein level in the lymph node and thymus of CMV/hGATA3 transfected cells and Tg mice. CMV/hGATA3 Tg mice showed a significant increase in the allergic skin inflammation response such as ear thickness, draining auricular lymph node (aLN) weight, epidermal thickness, inflammatory cell number and Th2 immunoglobulin (Ig) concentration compared to wild-type (WT) mice after phthalic anhydride (PA) treatment. Furthermore, the secretion of Th2 type cytokines was increased by PA treatment in CMV/hGATA3 Tg mice, while the secretion of Th1 type cytokine was suppressed under the same conditions. However, the increased levels of Th2 type cytokines in CMV/hGATA3 Tg mice were almost recovered by the down-regulation of GATA3 expression with D-pinitol treatment. Therefore, these findings suggest that GATA3 could be considered as a potential target regulating the mechanism responsible for the differences in allergic skin inflammation.
The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.
Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling and increased expression of a set of Th2-specific genes, including those encoding Th2 cytokines. IL-4-mediated STAT6 activation induces high levels of transcription of GATA3, a master regulator of Th2 cell differentiation, and enforced expression of GATA3 induces Th2 cytokine expression. However, it remains unclear whether the expression of other Th2-specific genes is induced directly by GATA3. A genome-wide unbiased chromatin immunoprecipitation assay coupled with massive parallel sequencing analysis revealed that GATA3 bound to 1279 genes selectively in Th2 cells, and 101 genes in both Th1 and Th2 cells. Simultaneously, we identified 26 highly Th2-specific STAT6-dependent inducible genes by DNA microarray analysis-based three-step selection processes, and among them 17 genes showed GATA3 binding. We assessed dependency on GATA3 for the transcription of these 26 Th2-specific genes, and 10 genes showed increased transcription in a GATA3-dependent manner, whereas 16 genes showed no significant responses. The transcription of the 16 GATA3-nonresponding genes was clearly increased by the introduction of an active form of STAT6, STAT6VT. Therefore, although GATA3 has been recognized as a master regulator of Th2 cell differentiation, many Th2-specific genes are not regulated by GATA3 itself, but in collaboration with STAT6.
We previously reported that Lgr4 has a critical role in the morphogenesis of kidney, but the detailed functions of Lgr4 in kidney development have not been elucidated. In contrast to Lgr4 null mice with 129Ola × C57BL/6J mixed background, C57BL/6J-backcrossed Lgr4 null mice (Lgr4(-/-)) showed the severe phenotype of embryonic lethality and also had dilated tubules in kidneys at E16.5. Based on quantitative RT-PCR and in situ hybridization, branching morphogenesis at E15.5 in the Lgr4(-/-) was arrested earlier, and both DBA-lectin staining and immunohistochemical analysis using Aqp3 antibodies showed that the ureteric bud (UB) of Lgr4(-/-) kidneys underwent premature differentiation. Furthermore, quantitative RT-PCR and histological analysis suggested that the impaired UB differentiation was caused by down-regulation of the Wnt pathway and Gata3 in the Lgr4(-/-) kidneys. We demonstrate here that Lgr4 has a novel function for maintaining the UB in an undifferentiated state.
Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.
Immune dysfunction is hallmark of patients with non-small cell lung carcinoma (NSCLC). The molecular mechanism involved in COX-2- and PGE2-mediated production of immunosuppressive cytokine IL-10 is not well-understood. Our study addresses the involvement of T cell downstream signalling intermediates, cytokines (IL-10 and IFN-γ) and their transcription factors (T-bet and GATA-3) in COX-2-mediated regulation of lymphocyte functions in NSCLC patients. In comparison to healthy individual, a marked decrease in lymphocyte proliferation to anti-CD3 MAb was observed in NSCLC patients by thymidine incorporation assay. Using flow cytometry, decrease in intracellular calcium release with increase in reactive oxygen species was observed in lymphocytes of NSCLC patients. These patients showed increased IL-10 and PGE2 with reduced IFN-γ production by ELISA. Results demonstrated defect in regulation of transcription factors T-bet and GATA-3 as analysed by Western blotting (WB), immunoprecipitation and EMSA. Overexpression of p-p38, p-ERK and COX-2 were observed with diminished p-JNK by WB. IL-10/IFN-γ levels were found to be differentially regulated via p38 and ERK mitogen-activated protein kinase (MAPK) pathways in cooperation with COX-2. Inhibition of these pathways using selective inhibitors lead to increased lymphocyte proliferative response to anti-CD3 MAb and IFN-γ production with decrease in IL-10 production. Studies showed involvement of ERK, p38 and COX-2 pathways in high IL-10 production, driven by lung tumour derived PGE2. The selective COX-2 inhibitor rofecoxib showed ability to alter the cytokine balance by affecting regulation of T-bet and GATA-3 transcription factors.
Regionalization of the central nervous system is controlled by local networks of transcription factors that establish and maintain the identities of neuroepithelial progenitor areas and their neuronal derivatives. The conserved cerebral Bauplan of vertebrates must result essentially from conserved patterns of developmentally expressed transcription factors. We have previously produced detailed molecular maps for the alar plate of prosomere 1 (the pretectal region) in chicken (Ferran et al., 2007, 2008, 2009). Here we compare the early molecular signature of the pretectum of two closely related avian species of the family Phasianidae, Coturnix japonica (Japanese quail) and Gallus gallus (chicken), aiming to test conservation of the described pattern at a microevolutionary level. We studied the developmental pretectal expression of Bhlhb4, Dbx1, Ebf1, Gata3, Gbx2, Lim1, Meis1, Meis2, Pax3, Pax6, Six3, Tal2, and Tcf7l2 (Tcf4) mRNA, using in situ hybridization, and PAX7 immunohistochemistry. The genoarchitectonic profile of individual pretectal domains and strata was produced, using comparable section planes. Remarkable conservation of the combinatorial genoarchitectonic code was observed, fundamented in a tripartite anteroposterior subdivision. However, we found that at corresponding developmental stages the pretectal region of G. gallus was approximately 30% larger than that of C. japonica, but seemed relatively less mature. Altogether, our results on a conserved genoarchitectonic pattern highlight the importance of early developmental gene networks that causally underlie the production of homologous derivatives in these two evolutionarily closely related species. The shared patterns probably apply to sauropsids in general, as well as to more distantly related vertebrate species.
Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.
Systemic lupus erythematosus (SLE) is a chronic, relapsing, and remitting disease affecting primarily African American females of child bearing age. Familial aggregation of this disease suggests that at least part of the susceptibility for this disease is genetic, although environmental and hormonal influences are also likely to play a role. Early studies of genetic susceptibility to SLE revealed several of the major histocompatibility complex molecules, namely HLA DR, to be linked to SLE. Meta-analysis of genome scans has yielded loci significant for lupus patients, one of which includes the MHC region. Regulatory T cells are immunoregulatory cells that modulate activated immune cells. These cells play a large role in homeostasis of the immune responses and maintenance of immunologic tolerance, i.e., prevention of autoimmunity. Decreased numbers of regulatory T cells have been described in many autoimmune diseases, including systemic lupus erythematosus. Autoantibody production in systemic lupus erythematosus and the resulting immune complex formation and complex deposition into tissues are arguably the central core of immune dysregulation leading to disease manifestations and symptoms. Inability of the immune system to recognize and inhibit autoreactive immune cells in this particular autoimmune disease may be the result of inappropriate numbers and function of regulatory T cells. This study aims to characterize the immune cell population in patients from our community suffering from systemic lupus erythematosus and to prove that these patients exhibit a unique cellular profile compared to healthy age, race and gender matched control subjects. Surprisingly, our findings demonstrate that patients from the local Mississippi area exhibit increased proportions of CD25(+) FoxP3(+) regulatory T cells and CD25(+) FoxP3(-) T cells (of CD45(+) CD3(+) CD4(+) helper T cells) as compared to healthy controls. HLA tissue-typing of these lupus patients revealed a prominent subgroup (~30%) of patients possessing the HLA DRB1*1503 allele. The investigation of this subgroup demonstrated regulatory T cell composition similar to that of the total lupus group and to that of the non-HLA DRB1*1503 subgroup. Genetic analysis for molecular gene expression levels of various lupus-associated genes by real-time PCR demonstrated a unique profile as compared to healthy controls. Increased gene expression of FoxP3 together with decreased gene expression levels of GATA3, TNFAIP3, and TNFSF4 suggest that variations in gene products compared to healthy controls may be playing a role in the immune cell dysregulation and disproportionate CD25(+) FoxP3(+) regulatory T cells.
To evaluate the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist on the induction of regulatory T cells (Tregs) in a murine model of allergic rhinitis.
Randomized controlled trial.
Animal study.
BALB/c mice that received ovalbumin sensitization and challenge served as the ovalbumin group (n = 6). Two separate groups of 6 mice received intragastric administration with PPAR-γ agonist pioglitazone (30 mg/kg/d) or pioglitazone plus PPAR-γ antagonist GW9662 (0.5 mg/d) before each ovalbumin challenge. The control group (n = 6) was treated with drug vehicle alone. Various allergic responses were assessed. Real-time polymerase chain reaction was performed to investigate the mRNA expression of forkhead box P3 (Foxp3), T-bet, and GATA-3. Flow cytometry was used to determine the percentage of Tregs.
Mice developed typical pathophysiological allergic rhinitis features after the ovalbumin challenge. The frequencies of sneezing and scratching were significantly decreased by pioglitazone treatment (P < .0001). Eosinophils infiltration and the levels of interleukin-5 and interferon-γ in nasal cavity lavage fluid and sera immunoglobulin E were also markedly decreased by pioglitazone (P < .001). The expression of Foxp3 mRNA and the population of Tregs were significantly increased by pioglitazone (P < .05). Cotreatment with GW9662 reversed the anti-inflammatory effects of pioglitazone. The effects of PPAR-γ agonist on Foxp3 mRNA expression and Tregs induction were abrogated by administration of GW9662.
PPAR-γ agonist attenuates upper airway allergic inflammation in a PPAR-γ-dependent fashion, and the beneficial effects of pioglitazone in airway allergic inflammation may be mediated by induction of Tregs.
Rainbow trout and Atlantic salmon interleukin-4/13A (IL-4/13A) genes were identified. They were found expressed at high level in thymus, gill, and skin, in concert with the transcription factor gene GATA-3. High expression levels of IL-4, IL-13, and GATA-3 were also detected in murine thymus, suggesting similar importance of the fish and mammalian homologues for early T cell development. In mammals, combined high expression of IL-4/13 and GATA-3 in tissues other than thymus is mostly indicative of Th2 responses. Th2-skewage may protect fish skin and gill from parasites and from damage by inflammatory Th1 and Th17 responses. The immune milieus of fish gill and skin are relevant to aquaculture, because these tissues are preferred sites for vaccine administration. The similarities between the immune milieus of fish gill and thymus may reflect an evolutionary relationship, since these tissues map close together lining the gill cavity. Expression patterns of IL-4/13A and interferon gamma (IFN-γ) in isolated trout gill cells and pronephrocytes were consistent with Th2 identity of IL-4/13A.
The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation.
We report on the mRNA levels of a panel of transcription factors in the kidney and spleen tissues, and in the cell populations from the blood, the spleen, and in the sorted kidney progenitor cells. The mRNA levels of cebpα, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1 were assessed in healthy goldfish as well as in fish challenged with two different pathogens, Aeromonas salmonicida A449 or Trypanosoma carassii. Spleen tissue from healthy goldfish showed higher expression of myeloid (cjun), erythroid (gata1) and lymphoid (gata3, pax5) transcription factors, and lower expression of the myeloid transcription factor cebpα when compared to that of kidney. Splenocytes and PBLs had significantly higher mRNA levels of the transcription factors involved in myeloid (pu.1, mafb, cjun, egr1, cebpa), erythroid (gata1, lmo2), and lymphoid pathways (gata3 and pax5) compared to sorted kidney R1 progenitor cells, while R1 progenitor cells had higher mRNA levels of early progenitor transcription factors (runx1 and cmyb). Furthermore, the R1 progenitor cells had higher mRNA levels of the transcription factors involved in early progenitor cells (egr1, gata2) and the lymphoid lineage progenitors (gata3, pax5) compared to those in kidney. The mRNA levels of the transcription factors (gata2, mafb, cjun, gata1, lmo2, gata3, and pax5) in R1 progenitor cells changed during cultivation; they were elevated in day 2 R1 cells and down-regulated by day 6 of cultivation, when compared to those of day 0 R1 cells. Treatment of day 2 R1 progenitor cells with rgCSF-1 resulted in an up-regulation of transcription factors important for myeloid cell development (cjun and egr1). Similarly, rgkitla up-regulated the expressions of myeloid (mafb, egr1 and cebpa) transcription factors. Changes in the expression of transcription factors in the R1 progenitor cells were related to the observed developmental processes of myeloid progenitor cells during cultivation or treatment with recombinant growth factors in vitro. We also observed differential expressions of the transcription factors in R1 progenitor cells following exposure of the goldfish to either prokaryotic (heat-killed A. salmonicida A449) or eukaryotic (T. carassii) pathogens.
Investigation of serotonergic neuronal activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Serotonergic neurons derived from embryonic stem cells (ESCs) could provide a platform for such studies and provide models for use in drug discovery. Here, we report enhancement of serotonergic differentiation using a genetic approach. Expression of Gata2 increased the yield of serotonergic neurons. Enhancement was only achieved when Gata2 was expressed under the control of the tissue-specific promoter of the transcription factor Nkx6.1. High levels of Gata2 expression in ESCs compromised pluripotency and induced non-neuronal differentiation. Combined directed expression of Gata2, proneural gene Mash1, and forkhead transcription factor Foxa2 further enhanced serotonergic neural differentiation, resulting in a 10-fold increase in serotonin content. These neurons were also capable of depolarization (KCl, 30 mM)-induced elevations of intracellular Ca(2+) . The presence of sonic hedgehog during differentiation produced a further modest increase in numbers (1.5-fold). Transgene expression did not influence the number of tyrosine hydroxylase positive neurons in the cultures after 20 days, implying that Gata2, Mash1, and Foxa2 modulate in vitro differentiation at a time beyond the decision-point for dopaminergic or nondopaminergic commitment. This study demonstrates that the directed expression of specific transcription factors enhances serotonergic neuron differentiation in vitro and highlights the importance of transgene expression at the right stage of ESC differentiation to effect the generation of a desired neural subtype.
Churg-Strauss syndrome (CSS) is a rare systemic vasculitis associated with eosinophilia and granuloma formation. The contribution of individual T-helper cell lineages in pathogenesis of CSS is unknown. We hypothesised that in CSS an imbalance of major effector T-cell subpopulations takes place, and is further influenced by the mode of treatment.
We investigated the immunophenotype, cytokine production and transcriptome profile in peripheral blood lymphocytes (PBL) from 19 patients with stable CSS (10 were treated with glucocorticoids alone (CSS/GC), 9 with steroids and other immunosuppressive drugs (CSS/IS)), and 13 healthy controls. Furthermore, serum IL-5 and CCR4-active chemokines (CCL17, CCL22) were measured in six patients with active disease and upon remission.
All CSS patients had decreased percentage of FoxP3+ regulatory T cells. In the CSS/GC group we found an increase in the Th17/Treg ratio and up-regulation of both Th2 and Th17 markers as evidenced by (1) over expression of Th2-related genes (GATA3, STAT6) in PBL, (2) elevated concentrations of serum IL-5 and CCL17, and (3) a concomitant increase in the number of Th17 cells, and secretion of IL-17A by stimulated PBL. The level of CCR4-active chemokines was increased in active-CSS, and correlated with blood eosinophilia. The combined treatment with steroids and other immunosuppressive drugs was associated with a significant decrease in both Th2-related chemokines and the number of Th17 cells.
Our results indicate that both Th2 and Th17 lineages are involved in the pathogenesis of CSS, while CCR4-active chemokines contribute to eosinophilia in the active disease. These phenomena are down regulated by immunosuppressive therapy.
The aim of this study was to investigate the ratio of the transcription factors T-bet/GATA-3 in patients with allergic asthma. Forty-seven individuals with allergic asthma and 47 healthy control individuals provided 5 ml of anticoagulated peripheral venous blood. Lymphocytes in peripheral blood were isolated by Ficoll and treated with phytohemagglutinin (PHA) at a final concentration of 100 mg/l for 48 h. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels were detected using an enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of both T-bet and GATA-3 were detected using reverse transcription polymerase chain reaction (RT-PCR). IFN-γ levels in the lymphocytes of asthmatic patients were lower than those of the control group (P<0.05) and were positively correlated with the ratio of T-bet/GATA-3. By contrast, IL-4 levels were significantly higher in asthmatic patients than in the control group (P<0.01) and were negatively correlated with the ratio of T-bet/GATA-3. In conclusion, the ratio of T-bet/GATA-3 can be used as an objective indicator of immune imbalance in patients with allergic asthma.
CD4+ T helper (Th) cells have been divided into different subsets as defined by their cytokine products and functions after their activation. CD4+ T cell subsets are continuously discovered and until now Th1, Th2, Th9, Th17, and regulatory T (Treg) cells have been almost unanimously recognized but yet not completely characterized. The selective production of cytokines by each of the subsets is probably the master key of the mechanisms of immune regulation. The cytokine milieu is extremely important on deciding the fate of T cells. Generally, more than one cytokine is needed for differentiating to a particular lineage and just recently it was shown that this status quo of commitment could be challenged. It is well known that cytokines bind to Type I/II cytokine receptors signaling via Janus kinases (JAKs) followed by activation of Signal Transducer and Activator of Transcription (STAT). STAT molecules work together with other transcription factors (Foxp3, RORgammat and RORalpha, T-bet, GATA3, Runx 1, NFAT, etc.) also controlled by cytokines, in modulating the Th phenotype and functions. In this review, we analyze the plasticity of Treg population focusing on the most recent discoveries on how microenvironmental cytokines refine/modify Treg phenotype and function, thus changing their fate.
To quantify and functionally characterize the intramyocardial T-cells in endomyocardial biopsies (EMBs) from patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM).
Expression of genes characterizing Th1 [interferon (IFN)γ, Tbet-1, Eomesodermin, interleukin (IL)-27], Th2 (IL-4, IL-5, GATA3), Th17 (IL-17), regulatory [regulatory T-cells (Treg); FoxP3, TGFβ, IL-10], anergic (GRAIL), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A), as well as of functional T-cell receptor Vbeta (TRBV) families were investigated in EMBs from AMC patients (n= 58) and DCM patients (n= 34) by pre-amplified real-time reverse transcription-polymerase chain reaction. These data were compared with EMBs from n= 19 controls. Expression of CD3d, CD3z, and TRBC (T-cell receptor beta constant region) were associated with the immunohistological diagnosis of inflammatory cardiomyopathy (DCMi). In EMBs from DCM patients with increased CD3d expression, significantly increased markers of Th1 (IFNγ, T-bet, Eomesodermin), regulatory T-cells (Treg; FoxP3, TGFβ), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A) were present, while no differential polarization of T-cells was found in EMBs form AMC patients. A differential dominance of distinct functional TRBV families was associated with different cardiotropic viruses: TRBV 11 and 24 with Parvovirus B19; TRBV4, 10 and 28 with human herpes virus type 6; and TRBV14 for Coxsackie virus, respectively.
The T-cell infiltrates in human DCMi are characterized by differential expression of functional T-cell markers indicating Th1, Treg, and CTLs, while no major role could be confirmed for Th17. The virus-associated differential TRBV dominance suggests an antiviral specificity of virus-induced T-cell responses in human DCMi.
Epithelial tumor cells transit to a mesenchymal state in response to extracellular cues, in a process known as epithelial-to-mesenchymal transition (EMT). The precise nature of these cues has not been fully defined, an important issue given that EMT is an early event in tumor metastasis. Here, we have found that a population of metastasis-prone mouse lung adenocarcinoma cells expresses Notch and Notch ligands and that the Notch ligand Jagged2 promotes metastasis. Mechanistically, Jagged2 was found to promote metastasis by increasing the expression of GATA-binding (Gata) factors, which suppressed expression of the microRNA-200 (miR-200) family of microRNAs that target the transcriptional repressors that drive EMT and thereby induced EMT. Reciprocally, miR-200 inhibited expression of Gata3, which reversed EMT and abrogated metastasis, suggesting that Gata3 and miR-200 are mutually inhibitory and have opposing effects on EMT and metastasis. Consistent with this, high levels of Gata3 expression correlated with EMT in primary tumors from 2 cohorts of lung adenocarcinoma patients. These findings reveal what we believe to be a novel Jagged2/miR-200-dependent pathway that mediates lung adenocarcinoma EMT and metastasis in mice and may have implications for the treatment of human epithelial tumors.
Expression of forkhead-box protein A1 (FOXA1), a transcription factor important for normal development of the prostate gland, is thought to be controlled by steroid hormones and GATA3. The aim of this study was to investigate the expression and potential role of FOXA1 and GATA3 transcription factors as prognostic factors in prostate cancer.
Expression of FOXA1, GATA3 and androgen receptor (AR) was retrospectively analysed by immunohistochemistry in a series of 80 primary tumours and 28 metastatic prostate cancers, including 15 matched paired samples. Nuclear AR expression did not significantly differ between primary and metastatic tumours. High-level nuclear FOXA1 expression was seen in 19% of primary and 89% of metastatic tumours (P < 0.0001). FOXA1 expression correlated positively with tumour size, extraprostatic extension, angiolymphatic invasion, AR and lymph node metastases at diagnosis, but did not correlate with age, T stage, Gleason score, presence of prostatic intraepithelial neoplasia or multifocality, seminal vesicle or perineural invasion, or surgical excision margin status. Expression of GATA3 was not seen in either normal epithelium or tumour.
Our preliminary analyses suggest that high-level FOXA1 expression is associated with the development of metastatic prostate cancer. If these data are confirmed, FOXA1 expression could be used to classify patients at higher risk for metastases.
We report the case of a young Emirati boy with HDR (Hypoparathyroidism, sensorineural Deafness, and Renal hypoplasia) syndrome due to the novel heterozygous deletion of two nucleotides (c.35_36delGC ) in exon 2 of the GATA3 gene. The patient developed hypocalcemia and hypomagnesemia at 3 weeks of age with high fractional excretion of magnesium, indicating renal magnesium loss. This is the first published report of hypomagnesemia in association with HDR syndrome.
Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme degrading the extracellular matrix and overexpressed by many tumors. Here, we documented the presence of MMP-2-specific CD4(+) T cells in tumor-infiltrating lymphocytes (TILs) from melanoma patients. Strikingly, MMP-2-specific CD4(+) T cells displayed an inflammatory T(H)2 profile, i.e., mainly secreting TNF-α, IL-4, and IL-13 and expressing GATA-3. Furthermore, MMP-2-conditioned dendritic cells (DCs) primed naïve CD4(+) T cells to differentiate into an inflammatory T(H)2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 degrades the type I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production. Active MMP-2, therefore, acts as an endogenous type 2 "conditioner" and may play a role in the observed prevalence of detrimental type 2 responses in melanoma.
A new subset of CD4(+) T cells, Th17, has been recently discovered independent from Th1/Th2 paradigm. The aim of this study was to investigate the effects of non-surgical periodontal therapy on the expression of Th17/Th1/Th2 cytokines and transcription factors, and Th17 cell vibration in Chinese chronic periodontitis patients.
The levels of Th17/Th1/Th2 cytokines (IL-17, IL-21/IFN-γ/IL-4) in gingival crevicular fluid from 30 chronic periodontitis patients before and after treatment were determined by ELISA. The expression of transcription factors (RORC, T-bet and GATA-3) in peripheral blood was measured by real-time PCR, and the levels of Th17 cells in CD4(+) T cells were determined by flow cytometry.
After treatment, the levels of IL-17 and IL-21 were down-regulated (P<0.05), and IL-4 was increased (P<0.05), but there were no differences in the level of IFN-γ (P>0.05). Correspondingly, the expression of RORC was decreased 1.99-fold (P<0.05), and GATA-3 was increased 1.76-fold (P<0.05). However, there were no differences in the level of T-bet (P>0.05). Moreover, the quantity of Th17 cells in peripheral blood was decreased (P<0.05), especially IL-17(+) IFN-γ(+) subgroup.
These results suggest that Th17 cells play a destructive role in the immune balance of periodontitis, and the effect of Th1 cells is not significant, while Th2 cells have a protective effect.
Transcription factor GATA-3 is vital for multiple stages of T cell and natural killer (NK) cell development, and yet the factors that directly regulate Gata3 transcription during hematopoiesis are only marginally defined. Here, we show that neither of the Gata3 promoters, previously implicated in its tissue-specific regulation, is alone capable of directing Gata3 transcription in T lymphocytes. In contrast, by surveying large swaths of DNA surrounding the Gata3 locus, we located a cis element that can recapitulate aspects of the Gata3-dependent T cell regulatory program in vivo. This element, located 280 kbp 3' to the structural gene, directs both T cell- and NK cell-specific transcription in vivo but harbors no other tissue activity. This novel, distant element regulates multiple major developmental stages that require GATA-3 activity.
Immune reconstitution inflammatory syndrome (IRIS) caused by mycobacterium in patients with AIDS is often experienced in clinical practice. There is, however, a paucity of data documenting the histopathological findings and the pathogenesis. We determined the immunopathological characteristics of IRIS associated with Mycobacterium parascrofulaceum infection in an AIDS patient. A patient presented with pulmonary lymphadenitis and involvement of the pulmonary lingular segment. Portions of the involved lymph nodes and lung were excised, and the immunological properties were analyzed by immunohistochemical assays. The histological characteristics of lymph nodes showed a caseous necrosis. Histopathologically, the pulmonary lesion was composed of exudative and proliferative lesions. CD4(+), CD8(+), CD57(+), and CD25(+)/FoxP3(+) cells were observed in both types of lesions. Clusters of CD20(+) cells and GATA3(+) cells were predominantly observed in exudative lesions, while T-bet(+) cells were dominant in proliferative lesions. ROR-γ(+) cells were also observed in exudative lesions. These results indicate that the cellular immunity to mycobacteria was recovering in the lung tissue. In M. parascrofulaceum pulmonary infection, the exudative lesion had characteristics of Th2 and Th17-type immunities. In contrast, the proliferative lesion had characteristics of Th-1 type immunity. Our data provide the first evidence to reveal the status of the axis of distinctive immunity in the process of granuloma formation caused by a mycobacterium-related infection.
Neurological manifestations are present in 5% to 30% of patients with Behçet's disease (BD). Neuro-Behçet's Disease (NBD) is hypothetically caused by T helper (Th) cells, which development is dependent on the expression of lineage-specific transcription factors. Cerebrospinal fluid (CSF) mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 were assessed in 18 NBD patients and 26 controls disease [16 noninflammatory neurological disease (NIND) and 10 headache attributed to Behçet's disease (HaBD)]. Expression of TBX21 (Th1), RORC (Th17) and Foxp3 (Treg) were increased in NBD patients compared to HaBD and NIND patients. EBI3 and Th2-associated GATA3 expressions were found to be decreased (P<0.0001 and P<0.0001) in NBD patients. Analysis of transcription factor ratios, revealed an increase in the RORC/FOXP3 and TBX21/GATA3 ratios in NBD patients (P<0.0001; P<0.0003). Our findings indicate that both Th1 and Th17 mRNA expressions involving a possible impairment of Treg cells. This might play a role in CSF-NBD inflammation, permitting activation of harmful T cell subpopulations. The TBX21/GATA3 and RORC/FOXP3 ratios dysregulations in NBD are consistent with those reported in other inflammatory diseases and indicating the plasticity existing between Th1, Th17 and Treg cells during inflammation.
This study was aimed to investigate the T cell (helper T cells) immune status in ITP patients and its relation with therapeutic response. 20 de novo ITP patients were enrolled (8 males, 12 females) with a median age of 41 (20 to 81). Real-time RT-PCR method was used to measure the gene expression of Th cells including T-bet, IFN-γ, GATA-3, TGF-β, Foxp3, IL-2, IL-4 in PBMNC of ITP patients before and after conventional dose of prednisone therapy [1 mg/(kg·d)] and in PBMNC of 20 normal controls. The results showed that T-bet, IFN-γ and IL-2 were significantly over-expressed in PBMNC of ITP patients before treatment compared with that in normal controls (p < 0.01), and compared with that before treatment, T-bet, IFN-γ, and IL-2 were markedly down-regulated in ITP patients after treatment. Before treatment, the expressions of Foxp3, TGF-β, GATA3 and IL-4 in ITP patients did not show difference from normal controls, while after treatment Foxp3 were more up-regulated than that before treatment (p < 0.05). After treatment, TGF-β expression showed a different pattern between old and young patients. TGF-β expression was down-regulated (p < 0.05) among ITP patients younger than 60, while up-regulated in older patients. It is concluded that there is an imbalance of Th1/Th2/Treg cytokines in ITP patients, which can be reversed by glucocorticoid treatment. The conventional dose of glucocorticoid may be regarded as effective therapy for de novo ITP patients, it may correlate with improvement of imbalance between Th1/The2/Treg cytokines.
Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.
GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.
The tyrosine kinase 2 variant rs34536443 has been established as a genetic risk factor for multiple sclerosis in a variety of populations. However, the functional effect of this variant on disease pathogenesis remains unclear. This study replicated the genetic association of tyrosine kinase 2 with multiple sclerosis in a cohort of 1366 French patients and 1802 controls. Furthermore, we assessed the functional consequences of this polymorphism on human T lymphocytes by comparing the reactivity and cytokine profile of T lymphocytes isolated from individuals expressing the protective TYK2(GC) genotype with the disease-associated TYK2(GG) genotype. Our results demonstrate that the protective C allele infers decreased tyrosine kinase 2 activity, and this reduction of activity is associated with a shift in the cytokine profile favouring the secretion of Th2 cytokines. These findings suggest that the rs34536443 variant effect on multiple sclerosis susceptibility might be mediated by deviating T lymphocyte differentiation toward a Th2 phenotype. This impact of tyrosine kinase 2 on effector differentiation is likely to be of wider importance because other autoimmune diseases also have been associated with polymorphisms within tyrosine kinase 2. The modulation of tyrosine kinase 2 activity might therefore represent a new therapeutic approach for the treatment of autoimmune diseases.
Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) which are located at the lowest end of an oxygen gradient within the bone marrow (BM). In this report, we investigated whether oxygen tension could affect NK cell differentiation from hematopoietic cells in vitro. We found that hypoxia led to an inhibition of differentiation in NK cells, and increased oxygen supply alleviated this inhibition and restored NK cell differentiation under hypoxic condition. Hypoxia-treated cells demonstrated reduced mRNA expression of transcription factors (TFs) that have important roles in NK cell differentiation, such as EOMES, T-bet, GATA-3 and ETS-1. Moreover, hypoxia-pretreated cells recovered mRNA expression of TFs when the oxygen tension was changed to normoxia. Our findings suggest that oxygen tension modulates in vitro differentiation of NK cells through the regulation of TF expression.
Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 μM), noradrenaline (30 μM), acetylcholine (30 μM) and dopamine (30 μM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 μM) and CGP55845 (1 μM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 μM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 μM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.
Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high level of sensitivity of mammalian hearing. The factors that influence prestin expression are still largely unknown. We studied the effects of the application of retinoic acid, a ligand of a nuclear receptor, and of butyric acid, an inhibitor of histone deacetylase activity, on the expression of mRNA of prestin and Gata-3 in the organotypic culture of the organ of Corti of newborn rats using RT-PCR. Application of retinoic acid at concentrations of 1-50 μM results in a dose-dependent expression decrease after two days in culture. Treatment with sodium butyrate (0.5-2 mM) elevated the expression of prestin and Gata-3. Statistically significant correlations between Gata-3 and prestin mRNA levels were observed under all conditions. The data indicate that retinoid nuclear transcription factors, GATA-3 and histone acetylation/deacetylation processes may have a regulatory role to play in prestin expression.
CD4(+) T cells are essential for defenses against pathogens and affect the functions of most cells involved in the immune response. Although CD4(+) T cells generally recognize peptide antigens bound to MHC-II molecules, important subsets are restricted by other MHC or MHC-like molecules, including CD1d-restricted "invariant" iNK T cells. This review discusses recently identified nodes in the transcriptional circuits that are involved in controlling CD4(+) T-cell differentiation, notably the commitment factor Thpok and its interplay with Runx transcriptional regulators, and focuses on how transcription factors acting upstream of Thpok, including Gata3, Tox and E-box proteins, promote the emergence of CD4-lineage-specific gene expression patterns.
A plasmacytoid variant of urothelial carcinoma has been recently recognized in the World Health Organization classification system. This is characterized by a discohesive growth of plasmacytoid cells with eccentric nuclei, extending in the bladder wall and often in the perivesical adipose tissue. Herein, we report the clinicopathologic, immunohistochemical, ultrastructural, and molecular features of a series of plasmacytoid urothelial carcinoma of the urinary bladder. Four bladder carcinomas characterized by epithelial cells with morphologic appearance resembling plasma cells were evaluated at the immunohistochemical, electron microscopic, and molecular genetic levels. Tumor cells stained with cytokeratins, epithelial membrane antigen, GATA-3 (endothelial transcription factor 3), CD15, p53, and p16. In addition, malignant cells strongly stained with CD138 in all the cases, whereas leukocyte common antigen and multiple myeloma 1/interferon regulatory factor 4 were completely negative, nor immunoreactivity was seen for either κ or λ light chains. The electron microscopic examination showed the presence of divergent squamous and glandular differentiation. At variance with conventional urothelial carcinoma, the analysis of exons 4-9 of TP53 gene revealed no alteration in all the 4 tumors tested, and this can be of value in choosing additional chemotherapy after surgery. Plasmacytoid carcinoma of the bladder is a tumor entity, which can be characterized by specific immunohistochemical markers, including positivity for GATA-3, and presents phenotypic and genotypic peculiarities.
The GATA3 gene is a potential tumour marker and putative tumour suppressor gene in breast cancer. Its expression is associated with better prognosis and disease free survival in breast cancer patients. We aimed to evaluate GATA3 transcriptome expression and mutation in breast carcinomas and correlate its expression with oestrogen receptor (ER), progesterone receptor (PR), lymph node (LN) status, tumour grade and c-erbB-2 expression. Twenty-two breast infiltrating ductal carcinomas and paired normal tissues were used in Branch DNA assay to detect GATA3 mRNA expression. Normalized data for GATA3 mRNA expression were grouped according to the ER, PR and LN status, tumour grade and c-erbB-2 expression of the tumours. Statistical significance was tested using t-test and ANOVA at 95% confidence interval level. Mutational analysis of GATA3 was performed by direct sequencing of the coding regions of GATA3 mRNA. Our findings showed that GATA3 gene were over-expressed and under-expressed by > 2 fold change in 12 and 4 tested samples, respectively. Eighty per cent of ER positive breast carcinomas were GATA3 positive. There was a statistically significant correlation between GATA3 expression and ER at 95% confidence interval level between the study groups. On the contrary, GATA3 expression was not statistically significant with PR, LN, tumour grade and c-erbB-2 expression in our study. In addition, we observed that there was no mutation in mRNA coding region in 16 breast carcinomas that showed GATA3 differential gene expression. Our preliminary results suggested that GATA3 is linked to the ER. This scenario suggests that GATA3 may play a crucial role in oestrogen receptor positive breast cancer patients. Whether GATA3 expression is involved in regulating tumour cell growth in oestrogen responsive breast cancer is a key question that remains to be answered.
Epigenetic alteration of the genome has been shown to provide palliative effects in mouse models of certain human autoimmune diseases. We have investigated whether chromatin remodeling could provide protection against autoimmune diabetes in NOD mice. Treatment of female mice during the transition from prediabetic to diabetic stage (18-24 weeks of age) with the well-characterized histone deacetylase inhibitor, trichostatin A effectively reduced the incidence of diabetes. However, similar treatment of overtly diabetic mice during the same time period failed to reverse the disease. Protection against diabetes was accompanied by histone hyperacetylation in pancreas and spleen, enhanced frequency of CD4(+) CD62L(+) cells in the spleen, reduction in cellular infiltration of islets, restoration of normoglycemia and glucose-induced insulin release by beta cells. Activation of splenic T lymphocytes derived from protected mice in vitro with pharmacological agents that bypass the antigen receptor or immobilized anti-CD3 antibody resulted in enhanced expression of Ifng mRNA and protein without altering the expression of Il4, Il17, Il18, Inos and Tnfa genes nor the secretion of IL-2, IL-4, IL-17 and TNF-α proteins. Consistently, expression of the transcription factor involved in Ifng transcription, Tbet/Tbx21 but not Gata3 and Rorgt, respectively, required for the transcription of Il4 and Il17, was upregulated in activated splenocytes of protected mice. These results indicate that chromatin remodeling can lead to amelioration of diabetes by using multiple mechanisms including differential gene transcription. Thus, epigenetic modulation could be a novel therapeutic approach to block the transition from benign to frank diabetes.
The evolution and therapeutic outcome of American tegumentary leishmaniasis (ATL) depend upon many factors, including the balance between Th1 and Th2 cytokines to control parasite multiplication and lesion extension. Other cytokines known for their role in inflammatory processes such as interleukin IL-17 or IL-18 as well as factors controlling keratinocyte differentiation and the inflammatory process in the skin, like the Notch system, could also be involved in the disease outcome. Notch receptors are a group of transmembrane proteins that regulate cell fate decisions during development and adulthood in many tissues, including keratinocyte differentiation and T-cell lineage commitment, depending on their activation by specific groups of ligands (Delta-like or Jagged).
To compare the in situ expression of Notch system proteins (receptors, ligands and transcriptional factors) and cytokines possibly involved in the disease outcome (IL-17, IL-18, IL-23 and transforming growth factor-β) in ATL cutaneous and mucosal lesions, according to the response to therapy with N-methyl glucamine.
Cutaneous and mucosal biopsies obtained from patients prior to therapy with N-methyl glucamine were analysed by immunohistochemistry and real-time polymerase chain reaction.
Notch receptors and Delta-like ligands were found increased in patients with ATL, particularly those with poor response to therapy or with mucosal lesions.
The increase of Notch receptors and Delta-like ligands in patients with a poor response to treatment suggests that these patients would require a more aggressive therapeutic approach or at least a more thorough and rigorous follow-up.
The transcription factors T-bet and GATA3 determine the differentiation of helper T cells into Th1 or Th2 cells, respectively. An altered ratio of their relative expression promotes the pathogenesis of certain immunological diseases, but whether this may also contribute to the pathogenesis of antibody-mediated rejection (ABMR) versus T cell-mediated rejection (TCMR) is unknown. Here, we characterized the intragraft expression of T-bet and GATA3 and determined the correlation of their levels with the presence of typical lesions of ABMR and TCMR. We found a predominant intraglomerular expression of T-bet in patients with ABMR, which was distinct from that in patients with TCMR. In ABMR, interstitial T-bet expression was typically located in peritubular capillaries, although the overall quantity of interstitial T-bet was less than that observed in TCMR. The expression of intraglomerular T-bet correlated with infiltration of CD4+ and CD8+ lymphocytes, which express T-bet, as well as intraglomerular CD68+ monocyte/macrophages, which do not express T-bet. The predominance of intraglomerular T-bet expression relative to GATA3 expression associated with poor response to treatment with bolus steroid. In summary, predominance of intraglomerular T-bet expression correlates with antibody-mediated rejection and resistance to steroid treatment.
Dysregulated Th subset responses, characterized by Th2-dominant allergic inflammation, are thought to be central to the pathogenesis of atopic dermatitis (AD). Glucosamine has been shown to have immunosuppressive properties, but its effect on AD has not been examined. In this study, the immunoregulatory effects of glucosamine, using dermatophagoides farinae (Df)-induced AD-like skin lesions in NC/Nga mice, were investigated. The clinical scores were reduced significantly by the treatment with glucosamine at 10 and 20 mg/day. Histological analysis of the skin also revealed that treatment of glucosamine at 10 and 20 mg/day significantly reduced the inflammatory cellular infiltrate, including mast cells and eosinophils. The levels of serum IgE and Th2 cytokines in spleen cells were reduced, whereas no significant change was detected in IFN-γ, a Th1 cytokine. To determine the mechanism associated with inhibition of the Th2 immune response, the effects of glucosamine on the selective differentiation pathway of the Th subset in vitro was examined in NC/Nga mice. The results showed that glucosamine suppressed the differentiation of naïve CD4(+) T cells to Th2 cells in vitro. On the basis of in vivo and in vitro results of the NC/Nga mice, the immunobiological effects of glucosamine on peripheral blood mononuclear cells from patients with AD were examined. The production of Th2 cytokines, such as IL-4 and IL-5, was significantly decreased after in vitro administration of glucosamine, which suggest that glucosamine might be a useful immunomodulatory agent for the treatment of human AD.
Mounting evidence from animal models has demonstrated that alterations in T-cell receptor (TCR) signaling alone can lead to dramatically skewed differentiation of naive T cells into T(H)2 cells, to T(H)2 effector functions, and to T(H)2-related diseases. There is significant potential relevance of these observations to human disease. Specifically, a number of immunodeficiencies associated with atopic disease might have atopy as a manifestation because of aberrant TCR signaling. It is therefore important to attempt to identify a role for defects in TCR signaling in the pathogenesis of common atopic diseases.
Th1 cells have been implicated as the causal agents in the pathogenesis of autoimmunity. SLE represents the classical prototype of systemic autoimmune disease. Copy number variations (CNVs) have been discovered to have phenotypic consequences and associate with various types of diseases. The current study aims to explore a possible association between CNVs of Th1 cell-related genes and the risk of SLE.
Genomic DNA and RNA from 532 SLE patients and 576 healthy controls were extracted. CNVs of Th1 cell-related genes (T-bet, Stat4, IL-12A, IL-12B, IFN-γ, IP-10 and CXCR3) as well as Th2 and Treg cell-related genes (c-Mef, GATA3, Foxp3, IL-6 and TGF-β) were examined, and mRNA levels of IL-12B and T-bet were examined.
Frequencies of IL-12B and T-bet CNVs in SLE patients were significantly higher than those in healthy controls. CNVs of IL-12B and T-bet had no synergistic contribution to SLE. The mRNA levels of IL-12B and T-bet in the samples with more than two copies of DNA were significantly higher than those with two copies of DNA.
CNVs of IL-12B and T-bet are associated with the risk of SLE.
To investigate the effect of Astragalus in regulating the imbalance between naive helper T cells (Th1/Th2) cytokines expression in patients with cervical cancer.
Thirty patients with cervical cancer received intravenous dripping with 20 mL of Astragalus Injection (AI, contained extract from 40 g of crude drug) per day for 1 week, peripheral blood sample was collected from patients separately before and after treatment for extract mononuclear cells by density gradient centrifugation. The positive percentages of CD4+ interferon-gamma (IFN-gamma ) cell and CD4+ interleukin-4 (IL-4) cell in total CD4+ cells were measured by flow cytometry; the contents of IFN-gamma, IL-4 in culture supernate were detected with ELISA; and the expressions of T-cell transcription factor T-cells (T-bet) and GATA-binding protein-3 (GATA-3) were determined by RT-PCR. The data were controlled by those get from 10 healthy persons.
CD4+ IFN-gamma positive cell percentage, T-bet mRNA expression level and concentration of IFN-gamma in supernate were significantly lower in patients than those in healthy control respectively, while CD4+ IL-4 positive percentage, level of GATA3 mRNA expression and IL-4 concentration in supernate were insignificantly different between the two groups. After AI treatment, the lowered parameters were up-regulated (P < 0.05), and no obvious change was observed in the CD4+ IL-4 positive cell associated parameters.
Th1/Th2 cell function imbalance existed in patients with cervical cancer, showing a Th2 predominant reaction mode; AI can regulate the imbalance, offset to Th1, thus to display its anti-tumor effect.
Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.
T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes.
Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P<0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.
Gastric adenocarcinoma is a major health problem world-wide, as this is the second most common cause of cancer death in the world. It has been estimated that infection by Helicobacter pylori cause at least half of the gastric cancers. Previously, we have demonstrated that H. pylori antigens directly activate NK cells to secrete IFN-γ. There is also a marked synergistic effect in NK cells stimulated with bacterial lysate and low levels of IL-12, a cytokine which is produced by macrophages and dendritic cells in the H. pylori-infected stomach. The present study was designed to investigate whether NK cells from gastric cancer patients display an altered ability to respond to components from H. pylori and other bacteria. The results show that NK cells from peripheral blood of gastric cancer patients have a severely suppressed ability to produce IFN-γ after stimulation with H. pylori lysate and the synthetic bacterial lipoprotein FSL-1. Furthermore, the synergistic effect of IL-12 and lysate is absent in gastric cancer patients, unless the concentration of IL-12 is increased 10-fold. We also demonstrate that there is a similar lack of IFN-γ production from NK cells isolated from the gastric mucosa of cancer patients. In addition, we propose that the observed suppression is due to tumour-derived TGF-β and that increased expression of the transcription factor GATA-3 may be responsible for the TGF-β induced suppression.
Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood.
In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipitation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression.
We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words).
GATA3 mutations cause HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome and, consistent with the presence of the second DiGeorge syndrome locus (DGS2) proximal to GATA3, distal 10p deletions often leads to HDR and DiGeorge syndromes. Here, we report on six Japanese patients with GATA3 abnormalities. Cases 1-5 had a normal karyotype, and case 6 had a 46,XX,del(10)(p15) karyotype. Cases 1-6 had two or three of the HDR triad features. Case 6 had no DiGeorge syndrome phenotype except for hypoparathyroidism common to HDR and DiGeorge syndromes. Mutation analysis showed heterozygous GATA3 mutations in cases 1-5, i.e., c.404-405insC (p.P135fsX303) in case 1, c.700T>C & c.708-709insC (p.F234L & p.S237fsX303) on the same allele in case 2, c.737-738insG (p.G246fsX303) in case 3, c.824G>T (p.W275L) in case 4, and IVS5+1G>C (splice error) in case 5. Deletion analysis of chromosome 10p revealed loss of GATA3 and preservation of D10S547 in case 6. The results are consistent with the previous finding that GATA3 mutations are usually identified in patients with two or three of the HDR triad features, and provide supportive data for the mapping of DGS2 in the region proximal to D10S547.
Human peripheral blood natural killer progenitors represent a flexible, heterogeneous population whose phenotype and function are controlled by their membrane-bound IL-15. Indeed, reciprocal membrane-bond IL-15 trans-presentation commits these cells into NK differentiation, while membrane-bound IL-15 stimulation with its soluble ligand (sIL-15Rα) triggers a reverse signal (pERK1/2 and pFAK) that modifies the developmental program of at least two subsets of PB-NKPs. This treatment generates: i) the expansion of an immature NK subset growing in suspension; ii) the appearance of an unprecedented adherent non-proliferative subset with a dendritic morphology co-expressing marker, cytokines and functions typical of myeloid dendritic cells (CD1a(+)/BDCA1(+)/IL-12(+)) and NK cells (CD3-/NKp46(+)/ CD56(+)/IFNγ(+)). The generation of these putative NK/DCs is associated to the rapid inhibition of negative regulators of myelopoiesis (the transcription factors STAT6 and GATA-3) followed by the transient upregulation of inducers of myeloid development, such as the transcription factors (PU.1, GATA-1) and the anti-apoptotic molecule (MCL-1).
Functional noradrenergic transmission requires the coordinate expression of enzymes involved in norepinephrine (NE) synthesis, as well as the norepinephrine transporter (NET) which removes NE from the synapse. Inflammatory cytokines acting through gp130 can suppress the noradrenergic phenotype in sympathetic neurons. This occurs in a subset of sympathetic neurons during development and also occurs in adult neurons after injury. For example, cytokines suppress noradrenergic function in sympathetic neurons after axotomy and during heart failure. The molecular basis for suppression of noradrenergic genes is not well understood, but previous studies implicated a reduction of Phox2a in cytokine suppression of dopamine beta hydroxylase. We used sympathetic neurons and neuroblastoma cells to investigate the role of Phox2a in cytokine suppression of NET transcription. Chromatin immunoprecipitation experiments revealed that Phox2a did not bind the NET promoter, and overexpression of Phox2a did not prevent cytokine suppression of NET transcription. Hand2 and Gata3 are transcription factors that induce noradrenergic genes during development and are present in mature sympathetic neurons. Both Hand2 and Gata3 were decreased by cytokines in sympathetic neurons and neuroblastoma cells. Overexpression of either Hand2 or Gata3 was sufficient to rescue NET transcription following suppression by cytokines. We examined expression of these genes following axotomy to determine if their expression was altered following nerve injury. NET and Hand2 mRNAs decreased significantly in sympathetic neurons 48 h after axotomy, but Gata3 mRNA was unchanged. These data suggest that cytokines can inhibit NET expression through downregulation of Hand2 or Gata3 in cultured sympathetic neurons, but axotomy in adult animals selectively suppresses Hand2 expression.
Selective breeding has been employed to improve resistance to infectious diseases in aquaculture and it is of importance to investigate the expression profiles of immune genes together with complement activity of Atlantic salmon with different genetic background in response to pathogens, in particular against Aeromonas salmonicida. This study examined acute phase products, and several central T cell cytokines and a transcription factor in different tissues, namely head kidney, spleen and liver, in two families of Atlantic salmon with high and low mortalities, after challenge by A. salmonicida. The results showed that the expression pattern of target genes differed in lymphoid and non-lymphoid organs in the two families. Generally, in lymphoid organs, higher expression of pro-inflammatory genes, such as TLR5M, TLR5S, GATA3, IFN-γ, IL-17D, as well as the pleiotropic cytokine gene IL-10 in the resistant family was observed at the same time point. One may speculate that a relatively high immune response is a pre-requisite for increased survival in a A. salmonicida challenge test. In addition, the resistant fish possessed higher complement activity pre-challenge compared to susceptible fish. Complement activity may be applied as an indicator in selective breeding for enhanced disease resistance.
recently, molecular subclassification of breast carcinomas has been proposed as a new prognostic parameter.
we classified 222 invasive breast carcinoma cases in 5 molecular subtypes by using tissue microarray (TMA) and immunohistochemistry methods. These subtypes were luminal A (estrogen receptor/ER and/or progesterone receptor/ PR positive), luminal B (ER and/or PR positive + HER2 positive), HER2-expressing type (ER and PR negative, HER2 positive), basal-like type (ER, PR and HER2 negative, positive with at least one of these myoepithelial markers: CK5/6, CK14, EGFR) and null type (ER, PR, HER2 and myoepithelial markers negative). We compared these subtypes according to their clinicopathological features and GATA3 expression.
luminal A was the most frequent subtype. According to overall survival rates, HER2-expressing and basal- like types had the worst prognosis, while luminal A had the best. However, luminal B had the worst prognosis according to disease free survival. Most of the squamous differentiated metaplastic carcinomas were basal-like type. Tubular and mucinous carcinomas were luminal A. Most basal-like tumors were grade III. The majority of grade I tumors were luminal A. GATA3 positivity was associated with low grade tumors and luminal A subtype.
molecular classification can be accepted as an independent prognostic factor for invasive breast carcinomas. GATA3 expression was associated with luminal A and low histological grade. However, it wasn't shown as an independent parameter.
Clinical data suggest that the estrogen receptor (ER) contributes to chemotherapeutic responsiveness. However, ER status alone is not consistently predictive. In this study, we used a microarray approach to find novel ER-related genes that predicted chemotherapy responses, with the hope of providing a robust multi-variable prediction method.
One hundred and ten patients with stages II and III breast cancer were included. They received four preoperative cycles of a weekly PCb (paclitaxel plus carboplatin) regimen. A total of 55 training cases were used for marker discovery and for identification of any ER-related genes that may have been associated with a chemotherapeutic response ("training cases"). The other 55 patients were available as an independent validation set ("validation cases") to test, using immunohistochemistry (IHC).
In the training set, 20 significantly differentially expressed genes were identified. Among these 20 genes, TFF1, ESR1, GATA3 and TFF3 were found to be ER-related. Among 55 independent validation cases, univariate analysis indicated that clinical variables and ER-related genes were all significantly associated with pCR. It was shown that the pCR rate was as high as 80% when these five factors were all negative. In contrast, these five factors were all positive in seven of nine chemo-resistant patients.
In conjunction with levels of ER-related genes, expression of ER protein may provide important predictive outcomes for responses to neoadjuvant chemotherapy and may allow for the identification of a subgroup of patients who could significantly benefit from chemotherapy (or who may be resistant to it).
We have previously shown that the in vitro embryonic development and the yield of viable calves were increased by using a two-step chemically defined medium for post-fertilization culture of bovine embryos. In this study, we explored the embryonic development and the temporal behavioral interaction of the genes involved in IFNτ gene expression and how they behave in an orchestrated manner to increase the developmental competence of IVF produced embryos by culturing in the chemically defined medium. Behavior of genes included ETS2, CDX2, GATA2, GATA3, OCT4 and NANOG was analyzed in early bovine IVF produced embryos, (from compact morulae to the blastocyst hatching stages), by semi- and relative quantitative PCR and compared between two in vitro culture (IVC) systems, two-step chemically defined medium and modified synthetic oviductal fluid (mSOF) containing 8 mg/mL, BSA. Early embryonic development was found to be better in two-step chemically defined culture system than that of mSOF as indicated by the increment of blastocyst yield, 33.1% in two-step culture system vs 18.8% in mSOF medium, and the blastocyst hatching, 52.3% in two-step culture system vs 33.5% in mSOF medium. Relative quantitative gene expression showed harmonic behavior in the two-step culture system rather than the culture in mSOF, IFNτ showed even increase throughout the embryonic development in the two-step culture medium while it decreased with blastocyst hatching in mSOF culture condition. Temporal dominance of OCT4 over all the transcription factors was found in regulation of IFNτ expression (the major factor of expression regulation but in inverse manner). However, ETS2, CDX2, GATA2 and GATA3 are potent IFNτ stimulator in cumulative manner but in case of OCT4 decrement. CDX2 directly related with IFNτ, but still under OCT4 dominance and also regulated by the subservient of OCT4 which is NANOG. In conclusion, this study confirmed our previous results about the usefulness of using the two-step chemically defined culture medium for increasing the developmental competence of IVF produced embryos and elucidated the dominance of OCT4 over the other genes implicated in regulation of IFNτ expression.
To better understand the immune response to highly pathogenic avian influenza virus, we compared expression of cytokines in chickens infected with avian influenza virus (A/Vietnam/1203/04) to that in uninfected chickens. Gene expression analyses revealed that influenza disseminated to multiple organs where immune responses could be identified. Among those cytokines influenced by influenza infection were the T helper type (Th)1-associated cytokines interleukin (IL)12 and interferon γ. In addition, a corresponding downregulation of the intracytoplasmic factor GATA3 was identified, whereas the Th2 cytokines IL4 and IL10 did not appear to be impacted by the infection. The inflammatory cytokine IL6 also appeared to be highly upregulated along with type 1 and type 3 interferon. Together, these data indicate that a strong inflammatory and Th1 response occurs after highly pathogenic avian influenza infection in the chicken that has implications for strategies that target the immune system for improving resistance to avian influenza.
Early thymocytes possess multilineage potential, which is progressively restricted as cells transit through the double-negative stages of T-cell development. DN1 cells retain the ability to become natural killer cells, dendritic cells, B cells, and myeloid cells as well as T cells, but these options are lost by the DN3 stage. The Notch1 signaling pathway is indispensable for initiation of the T-cell lineage and inhibitory for the B-cell lineage, but the regulatory mechanisms by which the T-cell fate is locked in are largely undefined. Previously, we discovered that the E-protein transcription factor HEBAlt promoted T-cell specification. Here, we report that HEB(-/-) T-cell precursors have compromised Notch1 function and lose T-cell potential. Moreover, reconstituting HEB(-/-) precursors with Notch1 activity enforced fidelity to the T-cell fate. However, instead of becoming B cells, HEB(-/-) DN3 cells adopted a DN1-like phenotype and could be induced to differentiate into thymic NK cells. HEB(-/-) DN1-like cells retained GATA3 and Id2 expression but had lower levels of the Bcl11b gene, a Notch target gene. Therefore, our studies have revealed a new set of interactions between HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes.
Obesity causes insulin resistance in target tissues - skeletal muscle, adipose tissue, liver and the brain. Insulin resistance predisposes to type-2 diabetes (T2D) and cardiovascular disease (CVD). Adipose tissue inflammation is an essential characteristic of obesity and insulin resistance. Neuronatin (Nnat) expression has been found to be altered in a number of conditions related to inflammatory or metabolic disturbance, but its physiological roles and regulatory mechanisms in adipose tissue, brain, pancreatic islets and other tissues are not understood.
We identified transcription factor binding sites (TFBS) conserved in the Nnat promoter, and transcription factors (TF) abundantly expressed in adipose tissue. These include transcription factors concerned with the control of: adipogenesis (Pparγ, Klf15, Irf1, Creb1, Egr2, Gata3); lipogenesis (Mlxipl, Srebp1c); inflammation (Jun, Stat3); insulin signalling and diabetes susceptibility (Foxo1, Tcf7l2). We also identified NeuroD1 the only documented TF that controls Nnat expression. We identified KEGG pathways significantly associated with Nnat expression, including positive correlations with inflammation and negative correlations with metabolic pathways (most prominently oxidative phosphorylation, glycolysis and gluconeogenesis, pyruvate metabolism) and protein turnover. 27 genes, including; Gstt1 and Sod3, concerned with oxidative stress; Sncg and Cxcl9 concerned with inflammation; Ebf1, Lgals12 and Fzd4 involved in adipogenesis; whose expression co-varies with Nnat were identified, and conserved transcription factor binding sites identified on their promoters. Functional networks relating to each of these genes were identified.
Our analysis shows that Nnat is an acute diet-responsive gene in white adipose tissue and hypothalamus; it may play an important role in metabolism, adipogenesis, and resolution of oxidative stress and inflammation in response to dietary excess.
Cho-kyung-jong-ok-tang (CKJOT) is a traditional Korean herbal formula specifically used for female infertility including unexplained recurrent pregnancy loss (RPL).
The present study aims to evaluate the effects of CKJOT on mouse natural killer (NK) cells to address the possible immunological basis of protective effects of this herbal medicine on unexplained RPL.
NK cells isolated from spleens of 6-week-old C57BL/6 mice were differentiated into NK0, NK1, and NK2 cells in the presence of various concentrations of CKJOT-extract. Apoptotic cell number, level of intracellular cytokines, and expression of cytokine-related transcription factors were measured.
CKJOT had little effect in improving viability of NK0, NK1, and NK2 cells. However, CKJOT addition during NK cell differentiation suppressed the production of interferon-gamma (IFN-γ), and enhanced that of interleukin-5, in the NK1 and NK2 subsets, respectively. T-bet, a transcription factor associated with IFN-γ expression was down-regulated; while Th2 linked transcription factors (STAT6 and GATA3) were up-regulated especially with 100 μg/mL treatment of CKJOT.
The type 2 shift in NK cell-secreted cytokines induced by CKJOT in mouse NK cells may explain the protective effect associated with its traditional use in unexplained RPL.
The T-cell functions of a proliferation-inducing ligand (APRIL, also known as TNFSF13) remain largely undefined. We previously showed that APRIL suppressed Th2 cytokine production in cultured CD4(+) T cells and Th2 antibody responses. Here we show that APRIL suppresses allergic lung inflammation, which is associated with diminished expression of the transcription factor c-maf. Mice deficient in the April gene (April(-/-) mice) had significantly aggravated lung inflammation compared with WT mice in the ovalbumin-induced allergic lung inflammation model. Likewise, blockade of APRIL in WT mice by the APRIL-receptor fusion protein, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)-Ig, enhanced lung inflammation. Transfer of APRIL-sufficient, ovalbumin-specific, TCR-transgenic CD4(+) T (OT-II) cells to April(-/-) mice restored the suppressive effect of APRIL on lung inflammation. Mechanistically, the expression of the Th2 cytokine transcription factor c-maf, but not GATA-3, was markedly enhanced in April(-/-) CD4(+) T cells at the RNA and protein level and under non-polarizing (Th neutral, ThN) and Th2-polarizing conditions. Since c-maf transactivates the IL-4 gene, the increased c-maf expression in April(-/-) mice readily explains increased Th2 cytokine production. Independent of its effect on IL-4, APRIL suppressed IL-13 expression. APRIL thus may regulate lung inflammation in a dual way, by acting on c-maf expression and by directly controlling IL-13 production.
Umbilical cord tissue is gaining attention as a novel source of multipotent stem cells because it is easily obtainable, ethically acceptable and the cells are immunologically naïve. In this study, we have isolated and characterized a new cell type expressing MUCIN1 (CD227) from human umbilical cord lining which we termed MUCIN-expressing Cord Lining Epithelial Cell (CLEC-muc). We found that CLEC-muc is highly proliferative and had significant clonogenic ability. These cells express embryonic stem cell markers OCT-4, NANOG, SSEA-4, REX1 and SOX2. Despite the abundant expression of epithelial cell marker MUCIN1 and cytokeratins, this population is also positive to the mesenchymal stem cell (MSC) marker CD166. CLEC-muc is unique in p63 expression that shuttles from the cytoplasm to the nucleus over time in culture. To understand p63 regulation and function in CLEC-muc, cells were treated with BMP4, a potent morphogen that plays a role in epidermal differentiation via p63 upregulation in ES cell and subsequent analyses were done. We found that BMP4 does not alter cytoplasmic expression of p63 that promotes cell proliferation. However, it increases nuclear p63 expression together with several other epithelial-associated genes such as GATA3, JAGGED1, NOTCH1, HES1 and IKKα. BMP4 has also been found to weakly induce deltaNp63 expression in CLEC-muc. Our results suggest that CLEC-muc is a novel stem cell-like population that can be further differentiated by BMP4 to generate specific cell-types probably destined to form non-keratinized epithelia.
To study the actions of transcription factors, T-bet and GATA-3, and their relevant signal transduction pathways on the immune-related pathogenesis with chronic aplastic anemia (CAA), and to investigate the immunological regulation mechanism of Shengxue Mixture (SXM) in regulating levels of Th cell imbalance, transcriptional factor and relevant signal pathways.
All CAA patients selected from Yueyang Hospital of Shanghai University of traditional Chinese medicine were equally randomized into the treated group and the control group, 20 patients in each group, and 20 healthy persons were selected as normal group, the former was treated with SXM according to patients' syndrome patterns, namely, SXM-1 was given to patients of Pi-Shen yang-deficiency pattern, and SXM-2 to those of Pi-Shen yin-deficiency pattern. Patients in the control group were treated with cyclosporin A (CsA). The mRNA expressions of T-bet, GATA-3, signal transducers and activators of transcription 4 (STAT4) and 6 (STAT6) in peripheral blood mononuclear cell (PBMNC) of patients were determined using real-time fluorescent quantitation polymerase chain reaction before and after treatment, meantime, the Th1/Th2 proportion in peripheral blood, and levels of IFN-gamma, IL-12 and IL-4 in PBMNC-cultured supernatant were detected by flow cytometry and enzyme linked immunosorbent assay.
The mRNA expressions of PBMNC T-bet and STAT4, ratios of T-bet/GATA-3, Th1 proportion and Th1/Th2 ratio, levels of IFN-gamma and IL-12 in PBMNC-cultured supernatant were all significantly higher in CAA patients than in healthy controls (P < 0.01), which were lowered after treatment but didn't reach the normal range (all P < 0.01), excepting for IL-12 level. Comparisons of the changes between the two treated groups showed insignificant difference (P > 0.05). While the difference between patients and healthy persons in terms of GATA-3, STAT6, Th2 proportion, and IL-4 were insignificant (P > 0.05), either before or after treatment.
Abnormal activation of IFN-gamma/T-bet and IL-12/ STAT4 pathways, as well as Th1/Th2 balance deviating to Th1 excursion play vital roles in the immunological pathogenesis of CAA. SXM and CsA could lower the aforesaid abnormal activation and correct Th1 hyper-polarization, so as to alleviate the over-activated cell-mediated immunity to eliminate hematopoietic depression in CAA patients.
Florfenicol has been shown to possess anti-inflammatory activity. However, its possible use for asthma has not yet been studied. First we investigated the anti-inflammatory properties of florfenicol using mice asthma model. BALB/c mice were immunized and challenged by ovalbumin. Treatment with florfenicol caused a marked reduction in inflammatory cells and three Th2 type cytokines in the bronchoalveolar lavage fluids of mice. The levels of ovalbumin-specific IgE and airway hyperresponsiveness were significantly altered after treatment with florfenicol. Histological studies using H&E and AB-PAS staining demonstrate that florfenicol substantially inhibited ovalbumin-induced inflammatory cells infiltration in lung tissue and goblet cell hyperplasia in the airway. These results were similar to those obtained with dexamethasone treatment. We then investigated which signal transduction mechanisms could be implicated in florfenicol activity. Our results suggested that the protective effect of florfenicol was mediated by the inhibition of the p38 MAPK-mediated phosphorylation of GATA 3.
Oral lichen planus (OLP) is a T-cell-mediated chronic inflammatory oral disease of unknown aetiology. Imbalanced cytokine production by Th1 and Th2 probably contributes to the pathogenesis of OLP. Growing evidence has suggested that two Th1/Th2-specific transcription factors, T-bet and GATA-3, may play a critical role in the development of Th1 and Th2 immunity. The aim of the present study was to investigate the mRNA expressions of T-bet and GATA-3 in the peripheral blood mononuclear cells of OLP subjects, and their expression patterns in relation to several clinical features.
Expressions of T-bet and GATA-3 mRNA in peripheral blood mononuclear cells isolated from twenty-eight OLP subjects and sixteen controls were detected by real-time reverse transcription-polymerase chain reaction.
When OLP subjects were regarded as a whole group, T-bet mRNA level and the ratio of T-bet/GATA-3 mRNA in OLP subjects were significantly higher (P<0.05) than those in controls. When the OLP subjects were divided according to different clinical forms, genders or age groups, T-bet, but not GATA-3, mRNA levels in reticular (P<0.01), female (P<0.05) and elder (age>55, P<0.05) OLP patients were significantly higher than those in control subjects. T-bet/GATA-3 mRNA ratio only in reticular (P<0.05) OLP subjects was significantly higher than that in control subjects.
The results implicate a predominant role of Th1-type immune response in pathogenesis of OLP. Different gene expressions of T-bet in different clinical features may indicate different immunoregulatory mechanisms of OLP.
In mammals, IL-21 is a common γ chain cytokine produced by activated CD4(+) T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4-72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8β, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.
Regulatory T cells (Tregs) have been suggested to be deeply associated with immune tolerance and long-term graft survival in transplantation. Some recipients with stable graft function (ST) could possibly minimize immunosuppression during the maintenance period. However, effective assays for assessing the suitability of patients have yet to be established. The purpose of this study was to elucidate the clinical relevance of Treg-related gene expression such as forkhead box P3 (Foxp3) in peripheral blood after renal transplantation.
Several key molecules related to the function of immune cells such as Treg, including Foxp3, transforming growth factor-β, cytotoxic T-lymphocyte antigen-4, chemokine receptor 7, toll-like receptor 4, granzyme B, T-bet, GATA3, RORC, α1,2-mannosidase, and proteasome subunit β 10 were examined in the peripheral blood of 272 renal transplant recipients by quantitative real-time reverse-transcriptase polymerase chain reaction. The expression levels were compared between recipients with chronic rejection and ST.
Foxp3 messenger RNA (mRNA) levels were reduced immediately after transplantation and gradually recovered. Pretransplantation levels were closely correlated with 1 year posttransplantation levels. Recipients with chronic rejection had significantly lower levels of Foxp3, chemokine receptor 7, and granzyme B mRNA, and higher levels of toll-like receptor 4 and proteasome subunit β 10 mRNA compared with those with ST, although Foxp3 was the most relevant marker.
There is a possibility that monitoring mRNA expression levels of Treg-related molecules in peripheral blood might offer useful information on patient selection and early detection of rejection when immunosuppression minimization strategy is implemented in renal transplantation.
GATA3 is a member of the GATA family of transcription factors. Heterozygous GATA3 abnormalities are associated with hypoparathyroidism, sensorineural deafness, and renal abnormality (HDR syndrome). However, this triad of symptoms does not occur in all HDR patients and other clinical features may be present in some cases. We report the clinical phenotypes and the molecular analysis of GATA3 in five Japanese HDR patients, including two familial cases. All five patients had hypoparathyroidism and sensorineural deafness, however renal abnormalities were absent in four patients. In addition, two patients with different mutations of GATA3 had female genital tract abnormalities. Sequence analysis of GATA3 demonstrated three novel (R262G, c1063delC and C318) and two reported mutations (c.432insG and c.1051-1G>T). Transient transfection assay using the GATA3 activating reporter system revealed that the transactivating activity of the R262G, c.1063delC, C318S and c.432insG mutants were markedly decreased, indicating that all four mutations are loss-of-function. In conclusion, this study reiterates the clinical variability in HDR syndrome and identifies three novel mutations of GATA3.
Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell death. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), the remaining cells of the organ of Corti are transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, except for the apex and few fibers in the base. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.
Peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists have been shown to be involved in the regulation of allergic inflammatory responses. The molecular mechanisms by which PPAR-gamma activation inhibits the inflammatory process have not been well understood.
BALB/c mice received ovalbumin (OVA) sensitization followed by OVA intranasal challenge. Mice in the treatment group received intragastric administration with pioglitazone (PIO; 30 mg/kg) before each OVA challenge. Various allergic responses were then assessed.
The frequencies of sneezing and nose-scratching and eosinophil infiltration decreased significantly in the PIO treatment group compared with the OVA group (p < 0.05). The PIO treatment also showed that the levels of nasal cavity lavage fluid interleukin (IL)-5 and sera OVA-specific immunoglobulin E (IgE) were markedly reduced (p < 0.05). PIO significantly increased the expression of Foxp3 mRNA (p < 0.05) and induced production of regulatory T lymphocyte (p < 0.01) compared with the OVA group.
Given the potent effectiveness shown by PIO, we conclude that PPAR-gamma agonists deserve investigation as potential therapies for human allergic upper airway inflammation.
periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown.
Balb/C mice were submitted to the co-induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL [induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges], and evaluated regarding PD and AL severity, immune response [cytokine production at periodontal tissues, and T-helper transcription factors in submandibular lymph nodes (LNs)] and infection parameters.
PD/AL co-induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, interferon-γ, IL-17A, receptor activator of nuclear factor κ-light-chain-enhancer of activated B cells ligand and matrix metalloproteinase (MMP)-13 levels in periodontal tissues, while IL-4 and IL-10 levels were unaltered by AL co-induction. AL co-induction also resulted in upregulated T-bet and related orphan receptor γ and downregulated GATA3 levels expression in submandibular LNs when compared with PD group.
our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.
Acute ethanol intoxication has the potential to alter immune reactivity by various pathways. The aim of this study was to investigate T-helper cell subsets transcription factors and cytokines in human peripheral blood mononuclear cells (PBMCs) following a single dose of lipopolysaccharide (LPS) with or without ethanol exposure.
Human PBMCs were cultured in the presence of 100 mM ethanol and/or 100 ng/ml LPS for various time periods (1, 3, 8, and 24 hours) and analyzed for the kinetics of gene expression by quantitative real-time PCR of selected transcription factors (T-bet, GATA3, Foxp3, and RORγt) and cytokines (TNF-α, IL-6, IL-10, and IFN-γ). The proportion of Th17 and Treg cells was identified 24 hours after treatment with ethanol and LPS by multiparameter flow cytometry. Viability and amount of dead cells were analyzed after 24 and 48 hours by MTT assay and flow cytometry.
  Following LPS challenge, gene expression of Foxp3 increased, whereas RORγt decreased after 3 hours, GATA3 decreased within 1 hour, whereas expression of T-bet did not change at any time. Gene expression of TNF-α, interferon-γ (IFN-γ), and IL-6 peaked after 3 hours, expression of IL-10 peaked after 8 hours. Ethanol suppressed the LPS-induced gene expression of Foxp3, RORγt, and T-bet after 8 hours, expression of TNF-α and IFN-γ was also suppressed after 3 and 8 hours. Markers of inflammation including TNF-α and IL-1β in supernatant of PBMCs were significantly decreased, while levels of IL-10 and IL-6 remained unchanged following ethanol exposure. Furthermore, ethanol-treated cells alone or in combination with LPS had significantly fewer IL-17- and IFN-γ-secreting CD4+ T cells but constant proportion of Treg cells when compared to control cells. Proliferation and viability of the cells were not influenced under these conditions.
Alcohol interferes with the kinetics of Foxp3, RORγt, and T-bet gene expression and the production of TNF-α and IL-1ß and influences the balance of Treg/Th17 cells following LPS exposure.
GATA-3 is a master regulator of T helper type 2 (T(H)2) differentiation. However, the molecular basis of GATA-3-mediated T(H)2 lineage commitment is poorly understood. Here we identify the DNase I-hypersensitive site 2 (HS2) element located in the second intron of the interleukin 4 locus (Il4) as a critical enhancer strictly controlled by GATA-3 binding. Mice lacking HS2 showed substantial impairment in their asthmatic responses and their production of IL-4 but not of other T(H)2 cytokines. Overexpression of Gata3 in HS2-deficient T cells failed to restore Il4 expression. HS2 deletion impaired the trimethylation of histone H3 at Lys4 and acetylation of histone H3 at Lys9 and Lys14 in the Il4 locus. Our results indicate that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci.
The study was aimed to explore the effects of T-bet (T-box expressed in T cell), GATA-3(GATA binding protein 3) and relevant signal transduction pathways on the immune-related pathogenesis of chronic aplastic anemia (CAA), and to investigate the immunological regulation mechanism in the treatment of CAA by using cyclosporine A (CsA) at the level of Th cell imbalance, transcriptional factors, and relevant signal pathways. The real-time fluorescent quantitative polymerase chain reaction (real-time FQ-PCR) was used to determine the mRNA expression of T-bet, GATA-3, signal transducers and activators of transcription 4 (STAT4) and signal transducers and activators of transcription 6 (STAT6) in peripheral blood mononuclear cell (PBMNC) of CAA patients before and after treatment with CsA; the flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA) were used to determine the Th1/Th2 proportion in peripheral blood, and levels of IFN-γ, IL-12 and IL-4 in PBMNC-cultured supernatant. Healthy people were included to test the above indexes. The results showed that the mRNA expression of PBMNC T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion, Th1/Th2 ratio and levels of IFN-γ and IL-12 in PBMNC-cultured supernatant of CAA patients were significantly higher than those of healthy people (p < 0.01). After treating with CsA for 6 months of CsA treatment, expression of T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion, IFN-γ and IL-12 levels were lower than before, however, the expression of T-bet, STAT4, T-bet/GATA-3 ratio, Th1 proportion and IFN-γ had not been reduced to normal state. Compared to healthy people, no significant difference existed in the mRNA expression of GATA-3, STAT6, Th2 proportion, as well as level of IL-4 before and after treatment (p>0.05). It is concluded that the abnormal activation of IFN-γ/T-bet and IL-12/STAT4 pathways, as well as Th balance deviating to Th1 excursion play vital roles in the immunological pathogenesis of AA. CsA lowers the abnormal activation of IFN-γ/T-bet and IL-12/STAT4 pathways to correct Th1 hyperpolarization, which may reduce the abnormally activated cell-mediated immunity and relax hematopoietic depression of AA patients.
HDR syndrome is an autosomal dominant disorder characterized by hypoparathyroidism, sensorineural deafness, and renal anomaly caused by mutation of the GATA3 gene located at chromosome 10p15. We report the case of a neonate with HDR syndrome and a novel GATA3 mutation. We performed genetic and functional analysis of GATA3 in this patient and identified a novel heterozygous 1516G> C missense mutation in exon 5, resulting in a cysteine-to-serine substitution at codon 321 (Cys321Ser). Mutated and wild-type GATA3 proteins were expressed at a similar level in vitro, indicating that the mutated GATA3 protein was stable. Luciferase assay revealed that the Cys321Ser-mutated GATA3 lacked transactivation activity due to loss of DNA-binding activity as confirmed by gel shift assay. Moreover, mutated GATA3 exerted a dominant-negative effect over the transactivation activity of wild-type GATA3. These findings indicate that not only haploinsufficiency of GATA3 but also the dominant-negative effect of Cys321Ser-mutated GATA3 might have been responsible for the HDR syndrome phenotype of our patient.
Asthma is a chronic inflammatory disorder in which Th2, Th1 and suppressive T cells (Tregs) play a role. The transcription factor FoxP3 plays a role in Treg differentiation while T-bet is important for Th1 and GATA-3 for Th2 differentiation from naïve T cells. Recent data show that age-related deregulation of Treg cells is a mechanism of senescence affecting several chronic diseases. It is crucial to understand the behaviour of these cell populations in asthma for elderly patients.
To evaluate FoxP3, GATA-3 and T-bet gene expression under basal conditions and after in vitro activation in a group of elderly asthmatic compared with age-matched healthy individuals.
Thirty-two elderly asthmatics and 17 healthy elderly individuals were selected. Serum total IgE was measured, and peripheral blood mononuclear cells (PBMCs) were isolated and stimulated in vitro with anti-CD3/anti-CD28, followed by mRNA isolation. After reverse transcription, real-time quantitative PCR was performed and relative quantification was determined 2(-ΔΔCt)(2(-ΔΔCt) method).
The mean values and standard deviation of FoxP3, GATA-3 and T-bet relative expression for control vs. asthma were 10.2±6.8 vs. 4.8±3.8, 2.4±2.9 vs. 1.7±0.9 and 3.3±2.1 vs. 2.1±1.5, respectively. Healthy individuals showed significantly higher expression of FoxP3 and T-bet; asthmatics had a lower T-bet/GATA-3 ratio, higher serum IgE and a positive significant correlation between total IgE and GATA-3 expression.
Elderly asthmatic patients have lower FoxP3 mRNA expression in PBMC, which can be associated with the sustained inflammatory process and with the decreased immune tolerance by Treg cells. The T-bet deficiency and the correlation of GATA-3 expression with the increase of IgE are characteristics of long-lasting asthma. Changes related to the immunosenescence process could provide an explanation for the minor differences observed between the groups. It is important to clarify persistent modifications in long-lasting asthma in the elderly and adequate future therapeutic approaches.
Over the past years, increasing numbers of distinct subsets have been discovered and identified for a T lymphocytes' entity. Differentiation and function of each T cell subset are controlled by a specific master transcription factor. Importantly, Runt-related transcription factors, particularly Runx1 and Runx3, interplay with these master regulators in various aspects of T cells' immunity. In this review article, we first explain roles of Th-Pok and Runx3 in differentiation of CD4 versus CD8 single positive cells, and later focus on cross-regulation of Th-Pok and Runx3 and their relationship with other factors such as TCR strength. Next, we provide evidences for the direct interplay of Runx1/3 with T-bet and GATA3 during Th1 versus Th2 commitment to activate or silence transcription of signature cytokine genes, IFNγ and IL4. Lastly, we explain feed-forward relationship between Runx1 and Foxp3 and discuss roles of Runx1 in regulatory T cells' suppressive activity. This review highlights an essential importance of Runx molecules in controlling various T cell subsets' differentiation and functions through molecular interplay with the master transcription factors in terms of protein-protein interaction as well as regulation of gene expression.
The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. GATA3 in particular is necessary for mammary gland maturation, and its loss has been implicated in breast cancer development. Our goal was to validate the ability of GATA3 expression to predict survival in breast cancer patients. Protein expression of GATA3 was analyzed on a high-density tissue microarray consisting of 242 cases of breast cancer. We associated GATA3 expression with patient outcomes and clinicopathologic variables. Expression of GATA3 was significantly increased in breast cancer, in situ lesions, and hyperplastic tissue compared with normal breast tissue. GATA3 expression decreased with increasing tumor grade. Low GATA3 expression was a significant predictor of disease-related death in all patients, as well as in subgroups of estrogen receptor-positive or low-grade patients. In addition, low GATA3 expression correlated with increased tumor size and estrogen and progesterone receptor negativity. GATA3 is an important predictor of disease outcome in breast cancer patients. This finding has been validated in a diverse set of populations. Thus, GATA3 expression has utility as a prognostic indicator in breast cancer.
The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck(-/-) Th1 skewed cells have normal expression of T-bet and produce IFN-γ at WT levels. However, there is a 3-fold increase in IL-10 producing cells in the mutant cultures. These cells do not have elevated levels of IL-4, GATA3, IL-17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3(+) T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL-10 in the lck(-/-) Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L(+) (naïve) cells are similar to WT cells. Furthermore, this IL-10 expression appears to be dependent on IL-12 and correlates with elevated c-Maf. These data highlight a previously unappreciated role for Lck in regulating IL-10 in Th1 cells.
Previously, we found that gene expression in histologically normal breast epithelium (NlEpi) from women at high breast cancer risk can resemble gene expression in NlEpi from cancer-containing breasts. Therefore, we hypothesized that gene expression characteristic of a cancer subtype might be seen in NlEpi of breasts containing that subtype.
We examined gene expression in 46 cases of microdissected NlEpi from untreated women undergoing breast cancer surgery. From 30 age-matched cases [15 estrogen receptor (ER)+, 15 ER-] we used Affymetryix U133A arrays. From 16 independent cases (9 ER+, 7 ER-), we validated selected genes using quantitative real-time PCR (qPCR). We then compared gene expression between NlEpi and invasive breast cancer using four publicly available data sets.
We identified 198 genes that are differentially expressed between NlEpi from breasts with ER+ (NlEpiER+) compared with ER- cancers (NlEpiER-). These include genes characteristic of ER+ and ER- cancers (e.g., ESR1, GATA3, and CX3CL1, FABP7). qPCR validated the microarray results in both the 30 original cases and the 16 independent cases. Gene expression in NlEpiER+ and NlEpiER- resembled gene expression in ER+ and ER- cancers, respectively: 25% to 53% of the genes or probes examined in four external data sets overlapped between NlEpi and the corresponding cancer subtype.
Gene expression differs in NlEpi of breasts containing ER+ compared with ER- breast cancers. These differences echo differences in ER+ and ER- invasive cancers. NlEpi gene expression may help elucidate subtype-specific risk signatures, identify early genomic events in cancer development, and locate targets for prevention and therapy.
We hypothesised that differences in microRNA expression profiles contribute to the contrasting natural history and clinical outcome of the two most common types of malignant germ cell tumour (GCT), yolk sac tumours (YSTs) and germinomas.
By direct comparison, using microarray data for paediatric GCT samples and published qRT-PCR data for adult samples, we identified microRNAs significantly up-regulated in YSTs (n = 29 paediatric, 26 adult, 11 overlapping) or germinomas (n = 37 paediatric). By Taqman qRT-PCR we confirmed differential expression of 15 of 16 selected microRNAs and further validated six of these (miR-302b, miR-375, miR-200b, miR-200c, miR-122, miR-205) in an independent sample set. Interestingly, the miR-302 cluster, which is over-expressed in all malignant GCTs, showed further over-expression in YSTs versus germinomas, representing six of the top eight microRNAs over-expressed in paediatric YSTs and seven of the top 11 in adult YSTs. To explain this observation, we used mRNA expression profiles of paediatric and adult malignant GCTs to identify 10 transcription factors (TFs) consistently over-expressed in YSTs versus germinomas, followed by linear regression to confirm associations between TF and miR-302 cluster expression levels. Using the sequence motif analysis environment iMotifs, we identified predicted binding sites for four of the 10 TFs (GATA6, GATA3, TCF7L2 and MAF) in the miR-302 cluster promoter region. Finally, we showed that miR-302 family over-expression in YST is likely to be functionally significant, as mRNAs down-regulated in YSTs were enriched for 3' untranslated region sequences complementary to the common seed of miR-302a~miR-302d. Such mRNAs included mediators of key cancer-associated processes, including tumour suppressor genes, apoptosis regulators and TFs.
Differential microRNA expression is likely to contribute to the relatively aggressive behaviour of YSTs and may enable future improvements in clinical diagnosis and/or treatment.
Elucidation of early life factors is critical to understand the development of allergic diseases, especially those manifesting in early life such as food allergies and atopic dermatitis. Cord blood IgE (CBIgE) is a recognized risk factor for the subsequent development of allergic diseases. In contrast with numerous genetic studies of total serum IgE in children and adults, limited genetic studies on CBIgE have been conducted.
To test the associations between functional or tagging single nucleotide polymorphisms (SNPs) in genes involved in the T(H)1/T(H)2 pathway and CBIgE in a large US inner-city birth cohort.
CBIgE, measured by Phadia ImmnunoCAP, was analyzed as a continuous and a binary variable. The association of each SNP with the 2 outcomes was tested using tobit and logistic regression models, respectively, with adjustment for pertinent covariates, ancestral proportion, and multiple testing. Ethnic heterogeneity and gene-gene interactions were also explored.
Three SNPs (rs1800925, rs2069743, and rs1295686) in the IL13 gene were significantly associated with CBIgE concentration (P ≤ 6 × 10(-4), FDR-corrected P < .05). These SNPs jointly influenced CBIgE in a dose-response manner (P for trend = 9 × 10(-8)). Significant associations also were observed for SNPs in the IL-13 receptor α1 (rs5956080) and signal transducer and activator of transcription 6 (rs11172106) genes. Ethnicity-specific genetic effects were observed for SNPs in the IL5 and GATA3 genes. Several gene-gene interactions (including IL13-IL4 receptor and IL13-signal transducer and activator of transcription 6 interactions) were detected in relation to CBIgE.
Our data demonstrated that multiple SNPs were individually and jointly associated with CBIgE, with evidence of gene-gene interactions and ethnic heterogeneity. These findings suggest that genetic regulation of IgE may begin in utero.
To identify susceptibility loci for classical Hodgkin's lymphoma (cHL), we conducted a genome-wide association study of 589 individuals with cHL (cases) and 5,199 controls with validation in four independent samples totaling 2,057 cases and 3,416 controls. We identified three new susceptibility loci at 2p16.1 (rs1432295, REL, odds ratio (OR) = 1.22, combined P = 1.91 × 10(-8)), 8q24.21 (rs2019960, PVT1, OR = 1.33, combined P = 1.26 × 10(-13)) and 10p14 (rs501764, GATA3, OR = 1.25, combined P = 7.05 × 10(-8)). Furthermore, we confirmed the role of the major histocompatibility complex in disease etiology by revealing a strong human leukocyte antigen (HLA) association (rs6903608, OR = 1.70, combined P = 2.84 × 10(-50)). These data provide new insight into the pathogenesis of cHL.
The Wnt pathway transcription factor T cell factor 1 (TCF-1) plays essential roles in the control of several developmental processes, including T cell development in the thymus. Although previously regarded as being required only during early T cell development, recent studies demonstrate an important role for TCF-1 in T helper 2 (Th2) cell polarization. TCF-1 was shown to activate expression of the Th2 transcription factor GATA-binding protein 3 (GATA3) and thus to promote the development of IL-4-producing Th2 cells independent of STAT6 signaling. In this study, we show that TCF-1 is down-regulated in human naive CD4(+) T cells cultured under Th2-polarizing conditions. The down-regulation is largely due to the polarizing cytokine IL-4 because IL-4 alone is sufficient to substantially inhibit TCF-1 expression. The IL-4-induced suppression of TCF-1 is mediated by STAT6, as shown by electrophoretic mobility shift assays, chromatin immunoprecipitation, and STAT6 knockdown experiments. Moreover, we found that IL-4/STAT6 predominantly inhibits the shorter, dominant-negative TCF-1 isoforms, which were reported to inhibit IL-4 transcription. Thus, this study provides a model for an IL-4/STAT6-dependent fine tuning mechanism of TCF-1-driven T helper cell polarization.
Somatic cell nuclear transfer (NT)-specific effects on postblastocyst early cattle embryogenesis were investigated by comparison to in vitro-produced (IVP) embryos grown under identical conditions to embryonic days (E) 14 and 15. Recipient effects were excluded by transferring mixed batches of NT and IVP embryos into each cow. Embryo recovery rates, proportions with an epiblast and embryo, as well as epiblast dimensions did not differ between NT and IVP embryos. A developmental expression profile was determined for nine trophoblast markers, two inner cell mass (ICM)/epiblast markers, and E-cadherin at nine time points between E7 and E26, providing a molecular gene signature assay for developmental progression. Gene expression levels for these genes (Cdx2, Elf5, Mash2, Ifn-tau, Furin, Kunitz1, Pag11, Gata3, Oct4 and Ifitm3) were equal in NT and IVP embryos of equivalent length. Furthermore, the average residual deviation of all 10 genes did not differ significantly suggesting an overall "normality" in gene expression of E14/15 NT embryos. The absence of NT-specific defects during the second, highly selective, week of cattle embryogenesis is interpreted as supportive for the view that NT-associated defects are predominantly of an epigenetic nature.
GATA family transcription factors play multiple vital roles in hematopoiesis in many cell lineages, and in particular, T cells require GATA-3 for execution of several developmental steps. Transcriptional activation of the Gata3 gene is observed throughout T-cell development and differentiation in a stage-specific fashion. GATA-3 has been described as a master regulator of T-helper 2 (Th2) cell differentiation in mature CD4(+) T cells. During T-cell development in the thymus, its roles in the CD4 versus CD8 lineage choice and at the β-selection checkpoint are the best characterized. In contrast, its importance prior to β-selection has been obscured both by the developmental heterogeneity of double negative (DN) 1 thymocytes and the paucity of early T-lineage progenitors (ETPs), a subpopulation of DN1 cells that contains the most immature thymic progenitors that retain potent T-lineage developmental potential. By examining multiple lines of in vivo evidence procured through the analysis of Gata3 mutant mice, we have recently demonstrated that GATA-3 is additionally required at the earliest stage of thymopoiesis for the development of the ETP population. Here, we review the characterized functions of GATA-3 at each stage of T-cell development and discuss hypothetical molecular pathways that mediate these functions.
Lymphopoiesis generates mature B, T, and NK lymphocytes from hematopoietic stem cells via a series of increasingly restricted developmental intermediates. The transcriptional networks that regulate these fate choices are composed of both common and lineage-specific components, which combine to create a cellular context that informs the developmental response to external signals. E proteins are an important factor during lymphopoiesis, and E2A in particular is required for normal T- and B-cell development. Although the other E proteins, HEB and E2-2, are expressed during lymphopoiesis and can compensate for some of E2A's activity, E2A proteins have non-redundant functions during early T-cell development and at multiple checkpoints throughout B lymphopoiesis. More recently, a role for E2A has been demonstrated in the generation of lymphoid-primed multipotent progenitors and shown to favor their specification toward lymphoid over myeloid lineages. This review summarizes both our current understanding of the wide-ranging functions of E proteins during the development of adaptive lymphocytes and the novel functions of E2A in orchestrating a lymphoid-biased cellular context in early multipotent progenitors.
Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.
Polycomb group (PcG) and trithorax group (TrxG) complexes exert opposing effects on the maintenance of the transcriptional status of the developmentally regulated Hox genes. In this study, we show that activation of STAT6 induces displacement of the PcG complex by the TrxG complex at the upstream region of the gene encoding GATA3, a transcription factor essential for T helper type 2 (Th2) cell differentiation. Once Th2 cells differentiate, TrxG complex associated with the TrxG component Menin binds to the whole GATA3 gene locus, and this binding is required for the long-term maintenance of expression of GATA3 and Th2 cytokine. Thus, STAT6-mediated displacement of PcG by the TrxG complex establishes subsequent STAT6-independent maintenance of GATA3 expression in Th2 cells via the recruitment of the Menin-TrxG complex.
Naturally occurring regulatory T (nTreg) cells express Foxp3 and were originally discovered as immune suppressors critical for self-tolerance and immune homeostasis. Through yet-to-be-defined mechanisms, nTreg cells were recently shown to convert into proinflammatory cells. Particularly, attenuation of Foxp3 expression led to Th2 conversion of nTreg cells in vivo. In this paper, we demonstrated an nTreg-specific mechanism controlling their Th2 conversion. We found that wild-type nTreg cells expressing reduced levels of Foxp3 but not those expressing no Foxp3 produced the Th2 cytokine IL-4. Intriguingly, IL-4 production by converted nTreg cells is required for Th2 differentiation of coexisting naive CD4 T cells in vivo, suggesting that Th2 conversion of nTreg cells might be critical for directing Th2 immune responses. Th2 conversion of nTreg cells was not due to their inability to become Th1 cells, because IFN-γ was produced by Foxp3-low-expressing cells when IL-4/STAT-6 signaling was abrogated. Surprisingly, however, unlike naive CD4 T cells whose IL-4 production is dependent on STAT-6, Foxp3-low-expressing cells generated IL-4 independent of STAT-6, indicating an intrinsic mechanism that favors nTreg-to-Th2 differentiation. Indeed, compared with naive CD4 T cells, nTreg expressed elevated levels of GATA-3 independent of STAT-6. And GATA-3 was required for nTreg-to-Th2 conversion. Foxp3 may account for this GATA-3 upregulation in nTreg cells, because ectopic expression of Foxp3 preferentially promoted GATA-3 but not T-bet expression. Thus, we have identified an intrinsic mechanism that imposes a Th2/Th1 imbalance and predisposes Foxp3-expressing cells to IL-4 production independent of STAT-6 signaling.
The imbalance between regulatory T cells (Treg) and effector T cells is important for maintaining of psoriasis vulgaris. FOXP3 is a master control transcription factor for the development and function of Tregs and is critical for transcriptional repression. Tacrolimus is effective in treatment of psoriasis vulgaris. Data show that tacrolimus has multiple impacts on FOXP3, but the exact pharmacological mechanism of tacrolimus on FOXP3 have yet to be elucidated. We herein suggest the bidirectional immunoregulation of tacrolimus on FOXP3. High concentration of tacrolimus renders the cooperation of NFAT with STAT6 and NF-κB to activate GATA3 transcription. On the contrary, low concentration of tacrolimus results in higher nucleus level of NFAT, which directly binds to FOXP3 enhancer and/or cooperates with Smad3 to activate FOXP3 transcription. Further studies using loss of function and over-expression methods are needed to determine the detailed molecules involved in this bidirectional immunoregulation of tacrolimus on FOXP3.
Dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. We exploited the zebrafish (Danio rerio) gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. This organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components.
Fish were maintained with either zinc supplemented water (4.0 μM) and diet (2023 mg zinc kg-1) or water and diet containing Zn2+ at 0.25 μM and 233 mg zinc kg-1, respectively. Gill tissues were harvested at five time points (8 hours to 14 days) and transcriptome changes analysed in quintuplicate using a 16 K microarray with results anchored to gill Zn2+ influx and whole body nutrient composition (protein, carbohydrate, lipid, elements). The number of regulated genes increased up to day 7 but declined as the fish acclimated. In total 525 genes were regulated (having a fold-change more than 1.8 fold change and an adjusted P-value less than 0.1 which is controlling a 10% False discovery rate, FDR) by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. Many genes displayed cyclic expression, typical for homeostatic control mechanisms. Annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". A suite of genes linked to "development" were also statistically overrepresented. More specifically, early regulation of genes was linked to a few key transcription factors (e.g. Mtf1, Jun, Stat1, Ppara, Gata3) and was followed by hedgehog and bone morphogenic protein signalling.
The results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. This provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status. The study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation.
The inflammatory cytokine interleukin (IL)-17 is involved in the pathogenesis of allergic diseases. However, the identity and functions of IL-17-producing T cells during the pathogenesis of allergic diseases remain unclear. Here, we report a novel subset of T(H)2 memory/effector cells that coexpress the transcription factors GATA3 and RORγt and coproduce T(H)17 and T(H)2 cytokines. Classical T(H)2 memory/effector cells had the potential to produce IL-17 after stimulation with proinflammatory cytokines IL-1β, IL-6, and IL-21. The number of IL-17-T(H)2 cells was significantly increased in blood of patients with atopic asthma. In a mouse model of allergic lung diseases, IL-17-producing CD4(+) T(H)2 cells were induced in the inflamed lung and persisted as the dominant IL-17-producing T cell population during the chronic stage of asthma. Treating cultured bronchial epithelial cells with IL-17 plus T(H)2 cytokines induced strong up-regulation of chemokine eotaxin-3, Il8, Mip1b, and Groa gene expression. Compared with classical T(H)17 and T(H)2 cells, antigen-specific IL-17-producing T(H)2 cells induced a profound influx of heterogeneous inflammatory leukocytes and exacerbated asthma. Our findings highlight the plasticity of T(H)2 memory cells and suggest that IL-17-producing T(H)2 cells may represent the key pathogenic T(H)2 cells promoting the exacerbation of allergic asthma.
Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.
Preplacodal ectoderm arises near the end of gastrulation as a narrow band of cells surrounding the anterior neural plate. This domain later resolves into discrete cranial placodes that, together with neural crest, produce paired sensory structures of the head. Unlike the better-characterized neural crest, little is known about early regulation of preplacodal development. Classical models of ectodermal patterning posit that preplacodal identity is specified by readout of a discrete level of Bmp signaling along a DV gradient. More recent studies indicate that Bmp-antagonists are critical for promoting preplacodal development. However, it is unclear whether Bmp-antagonists establish the proper level of Bmp signaling within a morphogen gradient or, alternatively, block Bmp altogether. To begin addressing these issues, we treated zebrafish embryos with a pharmacological inhibitor of Bmp, sometimes combined with heat shock-induction of Chordin and dominant-negative Bmp receptor, to fully block Bmp signaling at various developmental stages. We find that preplacodal development occurs in two phases with opposing Bmp requirements. Initially, Bmp is required before gastrulation to co-induce four transcription factors, Tfap2a, Tfap2c, Foxi1, and Gata3, which establish preplacodal competence throughout the nonneural ectoderm. Subsequently, Bmp must be fully blocked in late gastrulation by dorsally expressed Bmp-antagonists, together with dorsally expressed Fgf and Pdgf, to specify preplacodal identity within competent cells abutting the neural plate. Localized ventral misexpression of Fgf8 and Chordin can activate ectopic preplacodal development anywhere within the zone of competence, whereas dorsal misexpression of one or more competence factors can activate ectopic preplacodal development in the neural plate. Conversely, morpholino-knockdown of competence factors specifically ablates preplacodal development. Our work supports a relatively simple two-step model that traces regulation of preplacodal development to late blastula stage, resolves two distinct phases of Bmp dependence, and identifies the main factors required for preplacodal competence and specification.
The classification of breast cancer into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemopreventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα, FOXA1, and GATA-3. FOXA1 is associated with luminal type A cancers, while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups, there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall, the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1-/GATA-3+ in the late stage. Thus, PyMT model serves as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors, indicating delay in tumor progression. Although these tumors were histologically similar to those in controls, there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies, PEITC depleted AldeFluor-positive putative stem/progenitor cells, which may partly be responsible for the delay in tumor initiation.
The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.
TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1-expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1-LMO-Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.
Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors.
The aim of this study was to assess the balance of CD4(+)T cell populations in relapsing-remitting MS.
Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers.
In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4(+)CD25(hi)Treg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005).
Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4(+)phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.
A large number of hyposerotonergic genetic models have been generated over the past few years. Serotonin (5-HT) depletion has been obtained via targeting of genes involved in 5-HT synthesis (Tph1 and Tph2), specification and determination of the 5-HT phenotype during development (GATA3, Pet1, and Lmx1b), and 5-HT storage or clearance (Vmat2 and SERT). Here we review these various models from a developmental perspective, beginning with a description of the sources of 5-HT during development. We then summarize the neurological and behavioral alterations that have been observed in the genetic hyposerotonergic models. Although these models appear to have normal brain development and do not exhibit any gross morphological defects, problems in somatic growth and physiological functions have been observed. Abnormal adult behavior is also seen, although whether it results from depletion of 5-HT during development or functional 5-HT deficiencies in adult life remains unclear. Evidence from these hyposerotonergic models suggests that the developing brain may not need 5-HT for the establishment of general organization and structure. However, central 5-HT appears to be necessary for postnatal body growth, maturation of respiratory and vegetative control, and possibly for the development of normal adult behavior.
CD1d-restricted NKT cells include CD4(+) and DN subsets, with an additional CD8(+) subset that is present in humans but not in mice. The molecular regulation of CD4/CD8 expression by NKT cells, and differentiation of these NKT-cell subsets, is poorly understood. The transcription factors GATA3 and ThPOK regulate lineage commitment of conventional MHC class II-restricted CD4(+) T cells; however, their role in CD4/CD8 expression by CD1d-restricted NKT cells is less clear. A new study in this issue of the European Journal of Immunology demonstrates a key role for ThPOK in differentiation of NKT-cell subsets. This study reveals that GATA3 and ThPOK are necessary for the development of CD4(+) NKT cells. Furthermore, ThPOK-deficient mice generate an unusual population of CD8(+) NKT cells, which are absent in control mice. This study sheds new light on the underlying molecular events leading to the emergence of distinct NKT-cell subsets.
Lymphocytic colitis (LC) is a disease of unknown aetiology. Among other pathogenetic possibilities, an abnormal reaction to a luminal antigen has been discussed. To clarify this fact, we characterized the inflammatory infiltrate in LC and compared it with the Th1 response-related coeliac disease (CD). Biopsies from 10 LC and 10 CD patients were analyzed by immunohistochemistry for detection of T-bet, the master regulator of Th1 response and its antagonist GATA-3 in T cells employing double labellings. In LC, 10-20% of intraepithelial lymphocytes (IEL) expressed GATA-3 and the remaining T-bet, whereas in CD, all IEL were T-bet-positive. The T cells in the lamina propria of LC (65-70% CD4+; 30-35% CD8+) showed a mixed expression pattern of T-bet and GATA-3. The majority of the CD4+ T cells were GATA-3+, while T-bet and GATA-3 were expressed at a similar frequency by the CD8+ T cells. Most of the T cells in the lamina propria of CD specimens were CD4+, showing a predominant T-bet expression. Also, most of the CD8+ lamina propria T cells in CD were T-bet+. We conclude that in contrast to CD, which exhibits immunophenotypical features of a Th1-response, LC shows features of a mixed Th1/Th2 immune response.
Allergic airway inflammation is a disease in which T helper 2 (Th2) cells have a critical function. The molecular mechanisms controlling Th2 differentiation and function are of paramount importance in biology and immunology. Recently, a network of PB1-containing adapters and kinases has been shown to be essential in this process owing to its function in regulating cell polarity and the activation of critical transcription factors. Here, we show in vivo data showing that T-cell-specific NBR1-deficient mice show impaired lung inflammation and have defective Th2 differentiation ex vivo with alterations in T-cell polarity and the selective inhibition of Gata3 and nuclear factor of activated T c1 activation. These results establish NBR1 as a novel PB1 adapter in Th2 differentiation and asthma.
Schnurri (Shn) is a large zinc-finger containingprotein, which plays a critical role in cell growth, signal transduction and lymphocyte development. There are three orthologues (Shn-1, Shn-2 and Shn-3) in vertebrates. In Shn-2-deficient mice, the activation of NF-kappaB in CD4 T cells is upregulated and their ability to differentiate into Th2 cells is enhanced in part through the increased expression of GATA3. Shn-2 is found to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit the NF-kappaB-driven promoter activity. In addition, Th2-driven allergic airway inflammation was enhanced in Shn-2-deficient mice. Therefore, Shn-2 appears to negatively control the differentiation of Th2 cells and Th2 responses through the repression of NF-kappaB function. Memory Th1/Th2 cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. The expression levels of CD69 and the number ofapoptotic cells are selectively increased in Shn-2-deficient Thl/Th2 cells when they are transferred into syngeneic host animals, in which memoryh Th1/Th2 cells are generated within a month. In addition, an increased susceptibility to apoptotic cell death is also observed in vitro accompanied with the increased expression of FasL, one of the NF-kappaB-dependent genes. Th2 effector cells overexpressing the p65 subunit of NF-kappaB demonstrate a decreased cell survival particularly in the lymph node. These results indicate that Shn-2-mediated repression of NF-kappaB is required for cell survival and the successful generation of memory Th1/Th2 cells. This may point to the possibility that after antigen clearance the recovery of the quiescent state in effector Th cells is required for the generation of memory Th cells. A repressor molecule Shn-2 plays an important role in this process.
The lmo2 gene is a specific oncogene in T-cell leukemia, for its ectopic expression causes both increased pro-T-cell proliferation and differentiation arrest, leading to the onset of leukemia. Notably, DeltaEF1 (also known as ZEB1), a member of zinc finger-homeodomain family transcription factor, also exhibits crucial function in promoting T-cell differentiation. In this study, we found that DeltaEF1 was positively regulated by T-lineage-specific transcriptional regulator GATA3, while ectopically expressed LMO2 targeted to DeltaEF1 promoter by interaction with GATA3 and inhibited DeltaEF1 expression in transcriptional level. Moreover, LMO2 interacted with the N-terminal zinc finger domain of DeltaEF1 protein and inhibited its positive transcriptional regulatory function by this interaction. Taken together, our findings revealed that ectopically expressed LMO2 impaired the function of DeltaEF1 in both transcriptional and protein levels and identified DeltaEF1 as a novel pathogenic target of LMO2 in T-cell leukemia.
Primary bladder adenocarcinoma accounts for 0.5-2% of all malignant bladder tumours. Literature data indicate the bladder as the second most common site of metastatic genitourinary tumours, with the kidney as the most frequent location. Secondary tumours of the bladder account for about 2.3% of all bladder malignancies encountered in surgical specimens. Herein, we describe an adenocarcinoma deeply infiltrating the bladder wall, with no morphologic features of transitional cell carcinoma, in a patient with a previous diagnosis of primary lung adenocarcinoma, mixed subtype. In this case, the use of a limited immunohistochemical panel including napsin A, a recently described highly sensitive marker for lung adenocarcinoma, GATA3 and S100P, two novel markers of urothelial differentiation, was of crucial importance in differentiating between lung adenocarcinoma metastatic to the bladder and primary bladder adenocarcinoma.
Churg-Strauss syndrome (CSS) is characterized by systemic vasculitis and blood and tissue eosinophilia. Blood eosinophilia correlates with disease activity, and activated T cells from CSS patients are predominantly T helper 2 (Th2). Interleukin (IL)-25 has been shown to link innate and adaptive immunity by enhancing Th2 cytokine production. We sought to determine the involvement of IL-25 and its receptor IL-17RB in the pathogenesis of CSS. We found increased levels of IL-25 in the serum of active CSS patients (952 ± 697 vs 75 ± 49 pg/mL in inactive patients and 47 ± 6 pg/mL in healthy donors). IL-25 was correlated with disease activity and eosinophil level. Eosinophils were the main source of IL-25, whereas activated CD4(+) memory T cells were the IL-17RB-expressing cells in CSS. IL-25 enhanced the production of IL-4, IL-5, and IL-13 by activated peripheral blood mononuclear cells. IL-25 and IL-17RB were observed within the vasculitic lesions of patients with CSS, and IL-17RB colocalized with T cells. Increased expression of IL-17RB, tumor necrosis factor receptor-associated factor 6, and JunB in vasculitic lesions of CSS underscored the IL-25-mediated activation, whereas up-regulation of GATA3 and IL-10 supported Th2 differentiation. Our findings suggest that eosinophils, through the production of IL-25, exert a critical role in promoting Th2 responses in target tissues of CSS.
High rates of embryonic, fetal, or placental abnormalities have consistently been observed in bovine cloning. Segregation of inner cell mass (ICM) and trophectoderm (TE) lineages in early embryos is an important process for fetal and placental formation. In mouse embryos, differentiation of ICM and TE is regulated by various transcription factors, such as OCT-4, CDX2, and TEAD4, but molecular mechanisms that regulate differentiation in bovine embryos remain unknown. To clarify gene transcripts involved in segregation of ICM and TE lineages in bovine embryos, we examined the relative abundances of OCT-4, CDX2, TEAD4, GATA3, NANOG, and FGF4 transcripts in blastocyst embryos derived from in vitro fertilization (IVF). Furthermore, transcript levels of such genes in bovine embryos derived from somatic cell nuclear transfer (NT-SC) and in vivo (Vivo) were also compared. OCT-4, NANOG, and FGF4 transcript levels in IVF embryos were significantly higher in ICM than in TE. In contrast, the CDX2 transcript level was lower in ICM than in TE. In NT-SC embryos at the blastocyst stage, transcript levels of all genes except CDX2 were lower than that in Vivo embryos. In the elongated stage, expression levels of the six genes did not differ between NT-SC and Vivo embryos. We observed aberrant expression patterns of various genes involved in segregation of ICM and TE lineages in bovine NT-SC embryos. These results raise the possibility that abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage.
Childhood acute lymphoblastic leukemia (ALL) has been hypothesized to have an infection- and immune-related etiology. The lack of immune priming in early childhood may result in abnormal immune responses to infections later in life and increase ALL risk.
The current analyses examined the association between childhood ALL and 208 single-nucleotide polymorphisms (SNP) of 29 adaptive immune function genes among 377 ALL cases and 448 healthy controls. Single SNPs were analyzed with a log-additive approach using logistic regression models adjusted for sex, age, Hispanic ethnicity, and race. Sliding window haplotype analyses were done with haplotypes consisting of 2 to 6 SNPs.
Of the 208 SNPs, only rs583911 of IL12A, which encodes a critical modulator of T-cell development, remained significant after accounting for multiple testing (odds ratio for each copy of the variant G allele, 1.52; 95% confidence interval, 1.25-1.85; P = 2.9 x 10(-5)). This increased risk was stronger among firstborn children of all ethnicities and among non-Hispanic children with less day care attendance, consistent with the hypothesis about the role of early immune modulation in the development of childhood ALL. Haplotype analyses identified additional regions of CD28, FCGR2, GATA3, IL2RA, STAT4, and STAT6 associated with childhood ALL.
Polymorphisms of genes on the adaptive immunity pathway are associated with childhood ALL risk.
Results of this study support an immune-related etiology of childhood ALL. Further confirmation is required to detect functional variants in the significant genomic regions identified in this study, in particular for IL12A.
While most CD4(+) T cells are MHC class II-restricted, a small subset, including the CD1d-restricted 'invariant' NKT (iNKT) cells, are selected on non-classical MHC-I or MHC-I-like molecules. We previously showed that the sequential activity of two zinc finger transcription factors, Gata3 and Thpok, promotes the differentiation of conventional, MHC II-restricted thymocytes into CD4(+) T cells. In the current study, we show that a Gata3-Thpok cascade is required for the differentiation of CD4(+) iNKT cells. Gata3 is required for iNKT cells to express Thpok, whereas Thpok is needed for proper NKT cell differentiation, and notably for NKT cells to maintain CD4 and terminate CD8 expression. These findings identify the sequential activity of Gata3 and Thpok as a hallmark of CD4(+) T-cell differentiation, regardless of MHC restriction.
GATA transcription factors have been implicated in controlling adipogenesis in Drosophila and in mammals. In mammals, both GATA2 and GATA3 have been shown to be present in preadipocytes, and their silencing allows the onset of adipogenesis. Overexpression of GATA proteins blocks adipogenesis in cellular assays. GATA factors have been found to operate through recruiting cofactors of the Friend of GATA (FOG) family. FOG proteins, in turn, recruit co-regulators, including C-terminal binding proteins (CTBPs). We have investigated whether FOGs and CTBPs influence adipogenesis. We found that both FOG1 and FOG2 are expressed in cells prior to adipogenesis but are down-regulated as adipogenesis proceeds. Overexpression of FOG1 or FOG2 interferes with adipogenesis. Mutant versions of FOG2 unable to bind CTBP or GATA proteins are impaired in their inability to inhibit adipogenesis. Finally, a mutant version of GATA2, unable to associate with FOGs, also displays abnormal activity and causes enhanced cell proliferation. These results implicate FOGs and CTBPs as partners of GATA proteins in the control of adipocyte proliferation and differentiation.
The transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional Gata3 elimination. Inactivation of Gata3 in embryonic DBH-expressing neurons elicits a strong reduction in neuron numbers due to apoptotic cell death and reduced proliferation. No selective effect on noradrenergic gene expression (TH and DBH) was observed. Interestingly, Gata3 elimination in DBH-expressing neurons of adult animals also results in a virtually complete loss of sympathetic neurons. In the Gata3-deficient population, the expression of anti-apoptotic genes (Bcl-2, Bcl-xL, and NFkappaB) is diminished, whereas the expression of pro-apoptotic genes (Bik, Bok, and Bmf) was increased. The expression of noradrenergic genes (TH and DBH) is not affected. These results demonstrate that Gata3 is continuously required for maintaining survival but not differentiation in the sympathetic neuron lineage up to mature neurons of adult animals.
The differentiation of CD4(+) T cells into the Th2 subset is controlled by the transcription factor GATA-3. GATA-3 is both necessary and sufficient for Th2 differentiation and works through the induction of chromatin remodeling at the Th2 effector cytokine loci. We show in this study that IL-4 stimulation induces GATA-3 mRNA upregulation, but the level of GATA-3 protein induced is insufficient for Th2 differentiation. The levels of GATA-3 protein and Th2 differentiation are enhanced by concomitant TCR signaling through the PI3K/mammalian target of rapamycin pathway. The PI3K-mediated increase in GATA-3 protein occurs without increasing the GATA-3 mRNA level. Rather, TCR signaling through PI3K specifically enhances the translation rate of GATA-3 without affecting the protein stability. Importantly, this role of TCR signaling is independent of the effects of TCR signaling in T cell survival and expansion. Thus, TCR signaling through PI3K may play a critical role in Th2 differentiation by the specific enhancement of GATA-3 translation.
To investigate the inhibitory effects and mechanism of apigenin (APG) on dominant response of Th2 cells in asthma model of mice.
Thirty-two 6-week-old healthy BALB/c mice, SPF grade, were randomly divided into four groups equally, the normal control group (A), the asthma model group (B), and the two APG groups (C and D) consisted of asthma model mice treated respectively with high-dose (20 mg/kg per day) and low-dose (2 mg/kg per day) APG given by dissolving in 1% dimethyl sulphoxide via intraperitoneal injection. The murine asthma model was established by ovalbumin (OVA) sensitization and provocation. Twenty-four hours after the last airway provocation, acetylcholine (Ach) was administered via caudalis vein for measuring airway resistance by pulmonary function detector; levels of IL- 4 and IL-13 in bronchoalveolar lavage fluid (BALF) and total IgE in serum were determined by enzyme-linked immunosorbent assay (ELISA); total and differential cell counts in BALF were measured by light microscopy; the airway inflammatory infiltration was detected by haematoxylin and eosin (HE) staining; and the signal transducer and activator of transcription 3 (GATA-3) in the lung tissue was determined by Western blot analysis.
As compared with Group A, the airway hyper-reactivity, airway inflammation, cell count and eosinophil percentage in BALF, levels of total serum IgE and BALF IL-4 and IL-13, and GATA-3 protein expression in the lung tissue were significantly increased in Group B (P < 0.05). As compared with Group B, all the above-mentioned indices in Group C and D were lower, showing respective significant difference (P < 0.05), and significant difference was also shown between the two APG treated groups (P < 0.05).
APG could reduce the airway inflammation and hyper-reactivity by down-regulating the expressions of pulmonary GATA-3 and Th, cytokines, which is a potential drug for asthma therapy.
Belatacept is thought to disrupt the interaction between CD80/86 and CD28, thus preventing T-cell activation by blocking the co-stimulatory second signal. However, the consequences on the T-cell profile in human renal transplant cases have not been determined.
In this study, we analysed intra-graft levels of the mRNAs for Treg (FOXP3), cytotoxic CD8 T cells (Granzyme B), Th1 (INFγ, Tbet), Th2 (GATA3) and Th17 (RORγt and IL-17) in protocol biopsies obtained 12 months after renal transplantation in recipients treated with Belatacept or calcineurin inhibitor (CNI).
Only the intra-graft abundance of FOXP3 mRNA was significantly lower (P < 0.001) in the Belatacept group than the CNI group. Conclusions. These results are in agreement with in vitro data suggesting that CD28 is a major co-stimulatory signal of both Tregs development and peripheral homeostasis but contrast with clinical trials showing a better 1-year graft function and a lower incidence of chronic allograft nephropathy in patients receiving Belatacept than patients treated with CNI. They suggest that immune benefits induced by Belatacept are not mediated by Treg expansion and that FOXP3 is not by itself a prognostic marker of long-term graft function in a non-inflammatory context. These results have to be, however, considered as preliminary since the size of our study population is limited.
The transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T-cell transcription factor GATA3 is observed in B cell-derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidate the regulation and function of this factor in HL. We demonstrate binding of NFκB and Notch-1, 2 factors with deregulated activity in HL to GATA3 promoter elements. Interference with NFκB and Notch-1 activity led to decreased GATA3 expression, indicating a dependency of deregulated GATA3 expression on these transcription factors. Down-regulation of GATA3 in HL cell lines demonstrated its role in the regulation of IL-5, IL-13, STAT4, and other genes. A correlation between GATA3 and IL-13 expression was confirmed for HRS cells in HL tissues. Thus, GATA3 shapes the cytokine expression and signaling that is typical of HL. Conclusively, aberrant GATA3 expression in HRS cells is stimulated by the deregulated constitutive activity of NFκB and Notch-1, indicating a complex network of deregulated transcription factors in these cells. GATA3 activity significantly contributes to the typical cytokine secretion of and signaling in HRS cells, which presumably plays an essential role in HL pathogenesis.
The clinical impact of FLT3-internal tandem duplications (ITDs), an adverse prognostic marker in adults aged < 60 years with primary cytogenetically normal acute myeloid leukemia (CN-AML), requires further investigation in older patients. In CN-AML patients aged ≥ 60 years treated on Cancer and Leukemia Group B frontline trials, we found that FLT3-ITD remained associated with shorter disease-free survival (P < .001; hazard ratio = 2.10) and overall survival (P < .001; hazard ratio = 1.97) in multivariable analyses. This impact on shorter disease-free survival and overall survival was in patients aged 60-69 (P < .001, each) rather than in those aged ≥ 70 years. An FLT3-ITD-associated gene-expression signature revealed overexpression of FLT3, homeobox genes (MEIS1, PBX3, HOXB3), and immunotherapeutic tar-gets (WT1, CD33) and underexpression of leukemia-associated (MLLT3, TAL1) and erythropoiesis-associated (GATA3, EPOR, ANK1, HEMGN) genes. An FLT3-ITD-associated microRNA-expression signature included overexpressed miR-155 and underexpressed miR-144 and miR-451. FLT3-ITD identifies older CN-AML patients with molecular high risk and is associated with gene- and microRNA-expression signatures that provide biologic insights for novel therapeutic approaches.
The generation of specific T lymphocyte subsets is under the strict control of specific transcription factors, as this has been shown by knockout experiments in mice. Here, we show that siRNAs that specifically target the transcription factor Gata3 (which is required for the development of T helper 1 cells) or T-Bet (which is required for the development of T helper 2 cells) can be effective in vivo. Thus, the intraperitoneal injection of siRNAs specific for Gata3 or t-Bet leads to the specific depletion of their target gene products in vivo, in the spleen and in the lymph nodes of mice. The immunomodulatory action of these siRNAs was validated in a model of anti-tumor vaccination in which colorectal cancer cells that succumb to anthracyclin-induced immunogenic cell death were injected subcutaneously into one flank, in the absence of any adjuvant and live tumor cells were injected simultaneously in the opposite flank of immunocompetent mice. In this setting, the siRNA targeting t-Bet was able to accelerate tumor growth while the siRNA targeting Gata3 significantly reduced the proliferation of cancer cells in vivo. These effects were dependent on the immune response elicited by dying tumor cells because both siRNAs failed to modulate the growth of tumors in non-vaccinated mice. The immune response-dependent anticancer effect of the Gata3-specific siRNA was not due to the induction of class I interferons and could be fully abolished by co-injection of t-Bet-specific siRNA. These results demonstrate the possibility to use siRNAs for immunomodulaton in vivo and illustrate the antagonistic implication of distinct T helper populations in anti-cancer immune responses.
This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.
We demonstrated previously that GATA-3 overexpression markedly enhanced allergen-induced airway inflammation and airway remodelling, including subepithelial fibrosis, and smooth muscle cell hyperplasia, in transgenic mice.
Because cysteinyl leukotrienes (cysLTs) have been shown to be involved in such structural changes, the effects of a specific cysLT1 receptor antagonist, montelukast, were evaluated in a mouse model of chronic asthma.
GATA-3-overexpressing mice and wild-type Balb/c mice were sensitized and repeatedly challenged by ovalbumin (OVA) or saline. The effects of montelukast on the development of airway remodelling were compared between the two mouse genotypes.
CysLTs in the lung were increased after repeated allergen challenges, and significantly enhanced in GATA-3-overexpressing mice. The enhanced cysLT levels were accompanied by the development of eosinophilia, smooth muscle cell hyperplasia, and increased stromal cell-derived factor-1 gene expression with a small increase in pro-collagen gene expression in OVA-challenged GATA-3-overexpressing mice, but not in wild-type mice. Montelukast significantly decreased lung cysLT levels and inhibited the GATA-3-overexpression-related airway remodelling, potently preventing smooth muscle cell hyperplasia, but partially suppressed the increased pro-collagen gene expression and eosinophilic inflammation. Increases in the levels of IL-4, IL-5, IL-13, and eotaxin in bronchial lavage and TGF-β gene expression in the lungs were induced by OVA in both mouse genotypes. Montelukast treatment also significantly reduced these levels to the levels seen after saline challenges in GATA-3-overexpressing mice.
Montelukast efficaciously prevented airway inflammation and remodelling in a GATA-3-overexpression antigen challenge mouse model by decreasing the cysLT-driven Th2 cytokine cycle of amplification of airway pathologies.
GATA-binding protein-3 (GATA-3) regulates the T helper type 2 (Th2) cytokine locus through induction of chromatin remodelling. However, the molecular mechanism for this is poorly understood. To understand this mechanism better, we screened GATA-3 interacting proteins using affinity purification and mass spectrometry. We found that GATA-3 bound to metastasis-associated protein 2 (MTA-2), a component of the NuRD chromatin remodelling complex. GATA-3 and MTA-2 in turn bound to several regulatory regions of the Th2 cytokine locus and the ifng promoter. Cell transfection assay showed that MTA-2 acted as an antagonist with GATA-3 in the expression of Th2 cytokines, but co-operated with GATA-3 in the repression of the ifng gene expression. These results suggest that GATA-3 interacts with MTA-2 to co-ordinately regulate Th2 cytokine and ifng loci during T helper cell differentiation.
The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71-72%) but low identities to mammalian T-bet genes (41-42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73-88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial (Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-gamma and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression, may allow novel future control measures against disease in fish to be developed.
To detect the expression of interleukin (IL)-18 of the peripheral blood cells and IL-18 receptor alpha chain (IL-18Ralpha) on the surface of CD(3)(+) cells in patients newly diagnosed as immune thrombocytopenia (ITP) before medication and to explore the roles of IL-18 and IL-18Ralpha in the development of ITP.
Eighteen out-patients or inpatients with acute ITP accepting treatment in Qilu Hospital were enrolled in this study and 15 matching healthy subjects were taken as control. Plasma IL-18 level was detected with enzyme linked immunosorbent assay (ELISA), the expression of IL-18Ralpha on CD(3)(+) lymphocytes and total lymphocytes were measured with flow cytometry;T-bet and GATA-3 mRNA were measured with reverse transcriptase polymerase chain reaction (RT-PCR).
The expression of IL-18 in acute ITP plasma was (468.57 + or - 141.62) ng/L and IL-18Ralpha on the surface of CD(3)(+) cells and lymphocytes were (8.50 + or - 3.16)% and (9.16 + or - 2.98)% respectively. The levels of IL-18 and IL-18Ralpha were increased in active ITP patients as compared with those in the controls (P < 0.05). The levels of IL-18 mRNA (0.12 + or - 0.02) and T-bet mRNA (0.07 + or - 0.02) were significantly increased in patients with active ITP as compared with those in the controls (P < 0.05), while GATA-3 mRNA (0.0039 + or - 0.0014) were significantly decreased in patients with active ITP (P < 0.05). The balance between T-bet and GATA-3 was significantly disturbed in ITP.
Through the variation of the levels of gene and protein, our study showed that IL-18 and IL-18Ralpha might upregulate the expression of Th1-cytokines in ITP patients. It is also suggested that IL-18 has potential association with the development of ITP. Especially, it may provide a new treatment method for ITP by regulating the ratio of T-bet and GATA-3 and resuming the balance of Th1/Th2.
Many functions of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) have been defined, but relatively little is known about the biology of an alternative mTOR complex, mTORC2. We showed that conditional deletion of rictor, an essential subunit of mTORC2, impaired differentiation into T helper 1 (Th1) and Th2 cells without diversion into FoxP3(+) status or substantial effect on Th17 cell differentiation. mTORC2 promoted phosphorylation of protein kinase B (PKB, or Akt) and PKC, Akt activity, and nuclear NF-kappaB transcription factors in response to T cell activation. Complementation with active Akt restored only T-bet transcription factor expression and Th1 cell differentiation, whereas activated PKC-theta reverted only GATA3 transcription factor and the Th2 cell defect of mTORC2 mutant cells. Collectively, the data uncover vital mTOR-PKC and mTOR-Akt connections in T cell differentiation and reveal distinct pathways by which mTORC2 regulates development of Th1 and Th2 cell subsets.
In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr' (KA') and V3 interneurons. We show here that the differentiation of the correct number of KA' cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA' cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA' cells of the lateral floor plate and the KA' cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA' cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA' cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA' and KA' cells.
T cell differentiation determines susceptibility and resistance to experimental cutaneous leishmaniasis, yet mixed T1/Th2 responses characterize the clinical spectrum of human infection with Leishmania (Viannia) species.
To discern the interrelationship of T cell differentiation and outcome of human infection, we examined factors that regulate T cell differentiation and Th1/Th2 cytokine responses in asymptomatic infection, active and historical chronic and recurrent cutaneous leishmaniasis. T-bet, GATA-3, Foxp3, and cytokine gene expression were quantified by real-time polymerase chain reaction and correlated with interleukin 2, interferon gamma, tumor necrosis factor alpha, interleukin 4, interleukin 13, and interleukin 10 secretion during in vitro response to live Leishmania panamensis.
Higher GATA-3 expression than T-bet expression occurred throughout the 15 days of coculture with promastigotes; however, neither transcription nor secretion of interleukin 4 was detected. A sustained inverse correlation between GATA-3 expression and secretion of proinflammatory cytokines interferon gamma and tumor necrosis factor alpha was observed in asymptomatic infection. In contrast, higher T-bet expression and a higher ratio of T-bet to GATA-3 characterized active recurrent disease. Down-regulation of T-bet and GATA-3 expression and increased interleukin 2 secretion, compared with control subjects, was directly correlated with Foxp3 expression and interleukin 13 secretion in chronic disease.
Regulation of the inflammatory response rather than biased Th1/Th2 response distinguished asymptomatic and recalcitrant outcomes of infection with Leishmnania viannia species.
To evaluate the direct effects of Cyperi Rhizoma (CR), a plant water extract, on Th1/Th2 lineage development in vitro, this study was conducted. Sorted CD4(+) T cells obtained from the splenocytes of BALB/c mice were activated with anti-CD3/anti-CD28 and then cultured in medium that contained CR medium under Th1 inducing or Th2 inducing conditions. Subsequently, IFN-gamma or IL-4 secreting cells were quantitated using flow cytometry analysis. In addition, IFN-gamma and IL-4 protein secretions were detected by ELISA analysis, after which, IFN-gamma and T-bet transcripts, key players in the Th1 immune function, and also, IL-4 and GATA-3, which are primary components in the Th2 immune mechanism, were quantitated by real-time RT-PCR. CR had no mitogenic effects on un-stimulated CD4(+) T cells, however, it increased the CD4(+) T cell population. Th1/Th2 polarization experiments revealed that CR enhanced IFN-gamma secretion in Th1 cells, but reduced the IL-4 in Th2 cells, and this occurred in a dose-dependent manner and showed significances. In addition, under Th1/Th2 skewed conditions, the transcription levels of IFN-gamma and T-bet were considerably increased, while the expressions of IL-4 and GATA-3 were relatively decreased with CR treatment. These findings suggest that CR enhances Th1 lineage development by increasing Th1 specific cytokine expression and secretion and reduces Th2 lineage development by repressing Th2 specific cytokine productions. Therefore, CR extract may be useful for preventing the onset of allergies or improving allergic symptoms.
The clinical symptomatology in the X-linked Wiskott-Aldrich syndrome (WAS), a combined immunodeficiency and autoimmune disease resulting from WAS protein (WASp) deficiency, reflects the underlying coexistence of an impaired T helper 1 (TH1) immunity alongside intact TH2 immunity. This suggests a role for WASp in patterning T(H) subtype immunity, yet the molecular basis for the TH1-TH2 imbalance in human WAS is unknown. We have discovered a nuclear role for WASp in the transcriptional regulation of the TH1 regulator gene TBX21 at the chromatin level. In primary TH1-differentiating cells, a fraction of WASp is found in the nucleus, where it is recruited to the proximal promoter locus of the TBX21 gene, but not to the core promoter of GATA3 (a TH2 regulator gene) or RORc (a TH17 regulator gene). Genome-wide mapping demonstrates association of WASp in vivo with the gene-regulatory network that orchestrates TH1 cell fate choice in the human TH cell genome. Functionally, nuclear WASp associates with H3K4 trimethyltransferase [RBBP5 (retinoblastoma-binding protein 5)] and H3K9/H3K36 tridemethylase [JMJD2A (Jumonji domain-containing protein 2A)] proteins, and their enzymatic activity in vitro and in vivo is required for achieving transcription-permissive chromatin dynamics at the TBX21 proximal promoter in primary differentiating TH1 cells. During TH1 differentiation, the loss of WASp accompanies decreased enrichment of RBBP5 and, in a subset of WAS patients, also of filamentous actin at the TBX21 proximal promoter locus. Accordingly, human WASp-deficient TH cells, from natural mutation or RNA interference-mediated depletion, demonstrate repressed TBX21 promoter dynamics when driven under TH1-differentiating conditions. These chromatin derangements accompany deficient T-BET messenger RNA and protein expression and impaired TH1 function, defects that are ameliorated by reintroducing WASp. Our findings reveal a previously unappreciated role of WASp in the epigenetic control of T-BET transcription and provide a new mechanism for the pathogenesis of WAS by linking aberrant histone methylation at the TBX21 promoter to dysregulated adaptive immunity.
Activation of naive CD8 T cells in vitro in the presence of IL-4 induces type 2 cytokine expression, loss of CD8 expression, and reduced cytolytic potential. This represents a major shift from the canonical phenotype of effector CD8 T cells. It has not been established, however, whether IL-4 can induce comprehensive type 2 cytokine expression by CD8 T cells in vivo, nor whether the effects of IL-4 on type 2 cytokine production by CD8 T cells can be inhibited by IFN-gamma. Furthermore, disparate results have been reported regarding the anti-tumor ability of type 2 polarized effector CD8 T cells, and the effects of IFN-gamma in this respect remain unknown. To address these questions, wild-type or IFN-gamma-deficient OVA-specific CD8(+) T cells were activated in RAG-2(-/-) gamma c(-/-) recipients with control or IL-4-expressing OVA(+) tumor cells, and then transferred to secondary recipients for tumor challenge. Tumor-derived IL-4 induced the expression of type 2 cytokines and the transcription factor GATA-3 by responding CD8 T cells while reducing their CD8 coreceptor expression and ability to eliminate a secondary tumor challenge. Each of these effects of IL-4 was exaggerated in IFN-gamma-deficient, compared with wild-type, CD8 T cells. The results demonstrate that endogenous IFN-gamma counteracts the induction of type 2 cytokines and the downregulation of both CD8 coreceptor levels and the anti-tumor response in CD8 T cells exposed to IL-4 during activation in vivo. These findings may explain the anomalies in the reported functional phenotype of type 2 polarized CD8 T cells.
T helper 2 cells regulate inflammatory responses to helminth infections while also mediating pathological processes of asthma and allergy. IL-4 promotes Th2 development by inducing the expression of the GATA3 transcription factor, and the Th2 phenotype is stabilized by a GATA3-dependent autoregulatory loop. In this study, we found that type I IFN (IFN-alpha/beta) blocked human Th2 development and inhibited cytokine secretion from committed Th2 cells. This negative regulatory pathway was operative in human but not mouse CD4(+) T cells and was selective to type I IFN, as neither IFN-gamma nor IL-12 mediated such inhibition. IFN-alpha/beta blocked Th2 cytokine secretion through the inhibition of GATA3 during Th2 development and in fully committed Th2 cells. Ectopic expression of GATA3 via retrovirus did not overcome IFN-alpha/beta-mediated inhibition of Th2 commitment. Thus, we demonstrate a novel role for IFN-alpha/beta in blocking Th2 cells, suggesting its potential as a promising therapy for atopy and asthma.
Lacrimal gland enlargement (LGE) is one of the characteristics of Mikulicz's disease (MD). Recently, marked serum IgG4 elevation and infiltration of IgG4-positive plasmacytes in the enlarged exocrine glands have been reported in MD patients. However, little is known about the role of CD4+ T cells and their cytokines in IgG4-related diseases. The aim of this study was to evaluate the characteristics of CD4+ T cells in patients with IgG4-related diseases.
We investigated the clinical characteristics of 9 patients with LGE and elevated serum IgG4 levels (named IgG4-related LGE). We also examined mRNA expression of cytokines and transcription factors of peripheral blood CD4+ T cells in patients with IgG4-related LGE.
All patients with IgG4-related LGE showed elevated serum IgE levels. In addition, 5 of 9 patients with IgG4-related LGE exhibited eosinophilia and asthma-like symptoms. In patients with IgG4-related LGE, mRNA expression of IL-4, IL-5, IL-10 and GATA-3 but not IFN-gamma or T-bet was enhanced on CD4+ T cells compared with that in healthy controls.
Th2 cells may be involved in the pathogenesis of IgG4-related diseases.
Special AT-rich binding protein 1 (SATB1) is a global chromatin organizer and a transcription factor regulated by interleukin-4 (IL-4) during the early T helper 2 (Th2) cell differentiation. Here we show that SATB1 controls multiple IL-4 target genes involved in human Th cell polarization or function. Among the genes regulated by SATB1 is that encoding the cytokine IL-5, which is predominantly produced by Th2 cells and plays a key role in the development of eosinophilia in asthma. We demonstrate that, during the early Th2 cell differentiation, IL-5 expression is repressed through direct binding of SATB1 to the IL-5 promoter. Furthermore, SATB1 knockdown-induced up-regulation of IL-5 is partly counteracted by down-regulating GATA3 expression using RNAi in polarizing Th2 cells. Our results suggest that a competitive mechanism involving SATB1 and GATA3 regulates IL-5 transcription, and provide new mechanistic insights into the stringent regulation of IL-5 expression during human Th2 cell differentiation.
This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587+/-14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.
Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC.
We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential.
We identified a unique pathway signature with three major biological alterations-loss of normal renal function, down-regulated metabolism, and immune activation-which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development.
ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have identified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology.
FOXA1, estrogen receptor alpha (ERalpha) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERalpha and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERalpha expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERalpha and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERalpha expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERalpha-positive breast cancers, at least in part, through its control of ERalpha expression.
GATA-3, a transcription factor, participates in regulating cell development, proliferation, and death. This study was aimed at evaluating the roles of GATA-3 in protecting osteoblasts against oxidative stress-induced apoptotic insults and their possible mechanisms. Pretreatment with nitric oxide (NO) for 24 hours protected osteoblasts, prepared from neonatal rat calvaria, against oxidative stress-induced apoptotic insults. Such protection involved enhancement of Bcl-X(L) messenger mRNA and protein syntheses and the translocation of this antiapoptotic protein from the cytoplasm to mitochondria. GATA-3 was detected in rat osteoblasts, and GATA-3-specific DNA-binding elements exist in the promoter region of the bcl-x(L) gene. NO preconditioning attenuated oxidative stress-caused suppression of GATA-3 mRNA and protein synthesis and the translocation of this transcription factor from the cytoplasm to nuclei. Application of GATA-3 small interfering siRNA into osteoblasts decreased the levels of this transcription factor and simultaneously inhibited Bcl-X(L) mRNA synthesis. Pretreatment with NO lowered the oxidative stress-caused alteration in the binding of GATA-3 to its specific DNA motifs. Oxidative stress-inhibited Runx2 mRNA expression, but NO preconditioning decreased such inhibition. NO pretreatment time-dependently enhanced the association of GATA-3 with Runx2. Knocking down the translation of GATA-3 using RNA interference significantly decreased the protection of NO preconditioning against oxidative stress-induced alterations of cell morphologies, DNA fragmentation, and cell apoptosis. In comparison, overexpression of GATA-3 could promote NO preconditioning-involved Bcl-X(L) expression and cell survival. Therefore, this study shows that GATA-3 plays critical roles in mediating survival signals in osteoblasts, possibly through upregulating bcl-x(L) gene expression.
The nature and differentiation of regulatory CD8(+)CD28(-) T cells are poorly understood. In this study, we demonstrate that native Ag trichosanthin (Tk), a highly purified linear peptide isolated from a Chinese medicinal herb, is able to induce strong suppression of OVA-specific lymphoproliferation at low concentrations via activation of IL-4/IL-10-secreting CD8(+)CD28(-) regulatory T cells (Tregs). To elucidate the underlying mechanisms, we firstly identified two types of mouse inbred strains, high susceptible (HS) and low susceptible, for the Tk-related suppression. They are H-2(d) (or H-2(b)) and H-2(k), respectively. The suppression is evoked only if bone marrow-derived dendritic cells (BDCs) instead of purified T cells are treated with Tk in an OVA-specific T-BDC interaction. Moreover, a special pattern of cytokine/transcription factors (IL-4(+)IL-10(+)IFN-gamma(-)Gata3(+)T-bet(-)) during suppressed OVA-specific T cell proliferation was observed in HS C57BL/6 but not in low-susceptible C3H/He mice. Consistently, the percentage of CD8(+)CD28(-) Tregs preferentially expanded from 5.5 to 26.1% in the presence of Tk, an occurrence that was also detected only in HS C57BL/6 mice. These expanded Tregs were able to induce a strong inhibition of one-way MLCs, which indicated that the Tk-induced hyporeaction and the activation of CD8(+)CD28(-) Tregs might be under the influence of different genetic backgrounds. Additionally, obvious alterations of phenotypic parameters of BDCs after Tk stimulation were also identified, including enhanced production of IL-10, decreased secretion of IL-12, and detection of Jagged1, a Notch ligand on BDCs. Collectively, our data suggest that the changed APC-related factors are essential, at least in part, for the activation and differentiation of Tk-induced CD8(+)CD28(-) Tregs.
Protein kinase C-theta (PKC-theta) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-theta may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-theta in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-theta(-/-) and PKC-theta(+/+) wild-type mice with Toxoplasma gondii. The PKC-theta(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-theta(-/-) mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-theta(-/-) mice, paralleled by the diminished GATA3 expression of PKC-theta(-/-) CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP-1, and MAPK pathways in T. gondii-infected PKC-theta(-/-) mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-theta(-/-) mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-theta in T cells. These findings imply that PKC-theta inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.
Heterozygous mutations of GATA3, which encodes a dual zinc-finger transcription factor, cause hypoparathyroidism with sensorineural deafness and renal dysplasia. Here, we have investigated the role of GATA3 in parathyroid function by challenging Gata3+/- mice with a diet low in calcium and vitamin D so as to expose any defects in parathyroid function. This led to a higher mortality among Gata3+/- mice compared with Gata3+/+ mice. Compared with their wild-type littermates, Gata3+/- mice had lower plasma concentrations of calcium and parathyroid hormone (PTH) and smaller parathyroid glands with a reduced Ki-67 proliferation rate. At E11.5, Gata3+/- embryos had smaller parathyroid-thymus primordia with fewer cells expressing the parathyroid-specific gene glial cells missing 2 (Gcm2), the homolog of human GCMB. In contrast, E11.5 Gata3-/- embryos had no Gcm2 expression and by E12.5 had gross defects in the third and fourth pharyngeal pouches, including absent parathyroid-thymus primordia. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays showed that GATA3 binds specifically to a functional double-GATA motif within the GCMB promoter. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and, with GCM2/B, forms part of a transcriptional cascade in parathyroid development and function.
GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4(+) cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4(+) T cells, decreased expression of GATA-3 and Th2 cytokines. In primary T cells, c-Myb allows GATA-3 to autoactivate its own expression, an event that requires the direct interaction of c-Myb and GATA-3 on their respective binding sites in promoter of GATA-3. Immunoprecipitation revealed that the c-Myb/GATA-3 complex contained Menin and mixed lineage leukemia (MLL). MLL recruitment into the c-Myb-GATA-3-Menin complex was associated with the formation Th2 memory cells. That MLL-driven epigenetic changes were mechanistically important for this transition was suggested by the fact that silencing c-Myb significantly decreased the methylation of histone H3K4 and the acetylation of histone H3K9 at the GATA-3 locus in developing Th2 and CD4(+) effector/memory cells. Therefore, c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and, with the recruitment of MLL, Th2 cell maturation in primary human peripheral blood T cells.
The notion that family support may buffer individuals under adversity from poor outcomes has been theorized to have important implications for mental and physical health, but little is known about the biological mechanisms that explain these links. We hypothesized that adults who grew up in low socioeconomic status (SES) households but who experienced high levels of maternal warmth would be protected from the pro-inflammatory states typically associated with low SES. A total of 53 healthy adults (aged 25-40 years) low in SES early in life were assessed on markers of immune activation and systemic inflammation. Genome-wide transcriptional profiling also was conducted. Low early-life SES individuals who had mothers, who expressed high warmth toward them, exhibited less Toll-like receptor-stimulated production of interleukin 6, and reduced bioinformatic indications of pro-inflammatory transcription factor activity (NF-κB) and immune activating transcription factor activity (AP-1) compared to those who were low in SES early in life but experienced low maternal warmth. To the extent that such effects are causal, they suggest the possibility that the detrimental immunologic effects of low early-life SES environments may be partly diminished through supportive family climates.
It was hypothesized that the suppressive effect of diosgenin (1) on the intestinal T helper (Th)2 responses is associated with an enhancement of the regulatory T-cell immunity. Ovalbumin (OVA)-sensitized BALB/c mice were gavaged daily with 1 and received repeatedly oral OVA challenges to induce intestinal allergic responses. The expression of Th2- and Treg-related cytokines and transcription factors was examined by immunohistochemical staining and RT-PCR. Administration of 1 markedly attenuated the intestinal expression of interleukin (IL)-4 and GATA3. In addition, administration of 1 reversed the diminished density of intestinal Foxp3(+) cells induced by OVA oral challenges and enhanced the expression of IL-10 by Foxp3(+) cells markedly. These results suggest that the suppressive effect of 1 on allergen-induced intestinal Th2 responses is closely associated with an up-regulation of the regulatory T-cell immunity in the inflammatory site.
Immune features of infants with food allergy have not been delineated.
We sought to explore the basic mechanisms responsible for food allergy and identify biomarkers, such as skin prick test (SPT) responses, food-specific IgE levels, and mononuclear cell responses, in a cohort of infants with likely milk/egg allergy at increased risk of peanut allergy.
Infants aged 3 to 15 months were enrolled with a positive SPT response to milk or egg and either a corresponding convincing clinical history of allergy to milk or egg or moderate-to-severe atopic dermatitis. Infants with known peanut allergy were excluded.
Overall, 512 infants (67% male) were studied, with 308 (60%) having a history of a clinical reaction. Skin test responses, detectable food-specific IgE, or both revealed sensitization as follows: milk, 78%; egg, 89%; and peanut, 69%. SPT responses and food-specific IgE levels were discrepant for peanut (15% for IgE > or = 0.35 kU(A)/L and negative SPT response vs 8% for positive SPT response and IgE <0.35 kU(A)/L, P = .001). Mononuclear cell allergen stimulation screening for CD25, cytokine-inducible SH2-containing protein (CISH), forkhead box protein 3 (FOXP3), GATA3, IL10, IL4, IFNG, and T-box transcription factor (TBET) expression by using casein, egg white, and peanut revealed that only allergen-induced IL4 expression was significantly increased in those with clinical allergy to milk (compared with nonallergic subjects) and in those sensitized to peanut, despite the absence of an increase in GATA3 mRNA expression.
Infants with likely milk/egg allergy are at considerably high risk of having increased peanut-specific IgE levels (potential allergy). Peanut-specific serum IgE levels were a more sensitive indicator of sensitization than SPT responses. Allergen-specific IL4 expression might be a marker of allergic risk. Absence of an increase in GATA3 mRNA expression suggests that allergen-specific IL-4 might not be of T-cell origin.
Paclitaxel, a diterpenoid isolated from the bark of the Taxus cuspidate cv. Nana, was evaluated for its adjuvant effect on the immune responses in a mouse model. Fifty-six mice were randomly distributed into seven groups with 8 mice in each. Animals were subcutaneously immunized on days 1 and 21 with 100microg of paclitaxel, 10microg of ovalbumin (OVA), OVA with paclitaxel (50, 100 or 200microg) or with aluminum hydroxide (alum). Two weeks after the primary and boost immunizations, blood samples were collected for measurement of serum antibodies. Splenocytes were separated for detection of lymphocyte proliferation in responses to concanavalin A (Con A), lipopolysaccharide (LPS) and OVA, and mRNA expression of Th1 cytokines (IFN-gamma and IL-12), Th2 cytokines (IL-10 and IL-5) and transcription factors T-bet/GATA-3 (Th1/Th2 switcher). Results showed that coadministration of OVA with paclitaxel induced significantly higher IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM responses than when OVA was used alone. In addition, up-regulated T-bet/GATA-3 together with significantly increased mRNA expression of IL-4, IL-10, IFN-gamma and IL-12 by splenocytes, as well as the proliferative responses of splenocytes to Con A, LPS and OVA were observed in paclitaxel-adjuvanted groups. Incubation of a murine macrophage-like cell line with paclitaxel significantly increased TNF-alpha and -10 released from the cells and expression of microRNAs such as miR-155, miR-147, miR-146a and miR-132. Therefore, paclitaxel activated both Th1 and Th2 responses. Considering its unique adjuvant effect demonstrated in this study and a safe record clinically used as an antineoplastic agent, paclitaxel could be an ideal adjuvant candidate when mixed Th1/Th2 immune responses are needed.
Divisions of polarised blastomeres that allocate polar cells to outer and apolar cells to inner positions initiate the first cell fate decision in the mouse embryo. Subsequently, outer cells differentiate into trophectoderm while inner cells retain pluripotency to become inner cell mass (ICM) of the blastocyst. Elimination of zygotic expression of trophectoderm-specific transcription factor Cdx2 leads to defects in the maintenance of the blastocyst cavity, suggesting that it participates only in the late stage of trophectoderm formation. However, we now find that mouse embryos also have a maternally provided pool of Cdx2 mRNA. Moreover, depletion of both maternal and zygotic Cdx2 from immediately after fertilization by three independent approaches, dsRNAi, siRNAi and morpholino oligonucleotides, leads to developmental arrest at much earlier stages than expected from elimination of only zygotic Cdx2. This developmental arrest is associated with defects in cell polarisation, reflected by expression and localisation of cell polarity molecules such as Par3 and aPKC and cell compaction at the 8- and 16-cell stages. Cells deprived of Cdx2 show delayed development with increased cell cycle length, irregular cell division and increased incidence of apoptosis. Although some Cdx2-depleted embryos initiate cavitation, the cavity cannot be maintained. Furthermore, expression of trophectoderm-specific genes, Gata3 and Eomes, and also the trophectoderm-specific cytokeratin intermediate filament, recognised by Troma1, are greatly reduced or undetectable. Taken together, our results indicate that Cdx2 participates in two steps leading to trophectoderm specification: appropriate polarisation of blastomeres at the 8- and 16-cell stage and then the maintenance of trophectoderm lineage-specific differentiation.
Previous studies have demonstrated that the proteasome inhibitor, lactacystin, suppresses cytokine production and induction of other inflammatory mediators by LPS-stimulated macrophages. The purpose of the present studies was to determine the effect of lactacystin upon the function of activated human Jurkat T cells and murine splenocytes. Lactacystin treatment suppressed interleukin (IL)-2, interferon (IFN)gamma, and IL-13 production similarly in both activated Jurkat cells and primary splenocytes. Interestingly, lactacystin produced differential effects on IL-4 transcription in the two models. While lactacystin inhibited IL-4 mRNA transcription in primary splenocytes, it induced IL-4 mRNA in a concentration-dependent manner in Jurkat cells. The increase in IL-4 mRNA levels by lactacystin did not correlate with increases in T(H2)-specific transcription factors, avian musculoaponeurotic fibrosarcoma AS42 oncogene homolog (c-maf) or GATA binding protein 3 (GATA-3). In addition, the binding of both GATA-3 and t-bet to their respective response elements was essentially unchanged by lactacystin treatment in both splenocytes and Jurkat T cells, suggesting the induction of IL-4 is due to other mechanisms. Collectively, the current studies suggest proteasomal activity has differential effects on IL-4 transcription in activated Jurkat cells and primary splenocytes.
Chromosome 10p terminal deletions have been associated with DiGeorge phenotype, and within the same genomic region haploinsufficiency of GATA3 causes the HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia). We have performed detailed molecular analysis of four patients with partial overlapping 10p deletions by using FISH-mapping, array-CGH, and custom-designed high-resolution oligonucleotide array. All four patients had mental retardation and speech impairment and three of them showed variable signs of HDR syndrome. In addition, two patients had autistic behaviors and had similar dysmorphic features giving them a striking physical resemblance. A review of the literature identified 10 previously published cases with similar 10p deletions and reliable molecular or molecular cytogenetic mapping data. The combined information of present and previous cases suggests that partial deletions of 10p14-p15 represent a syndrome with a distinct and more severe phenotype than previously assumed. The main characteristics include severe mental retardation, language impairment, autistic behavior, and characteristic clinical features. A critical region involved in mental retardation and speech impairment is defined within 1.6 Mb in 10p15.3. In addition, deletion of 4.3 Mb within 10p14 is associated with autism and characteristic clinical findings.
Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).
We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.
We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.
Genetic and epigenetic programming of T helper (Th) cell subsets during their polarization from naive Th cells establishes long-lived memory Th cells that stably maintain their lineage signatures. However, whether memory Th cells can be redifferentiated into another Th lineage is unclear. In this study, we show that Ag-specific memory Th cells were redifferentiated into Foxp3(+) T cells by TGF-beta when stimulated in the presence of all-trans retinoic acid and rapamycin. The "converted" Foxp3(+) T cells that were derived from Th2 memory cells down-regulated GATA-3 and IRF4 and produced little IL-4, IL-5, and IL-13. Instead, the converted Foxp3(+) T cells suppressed the proliferation and cytokine production of Th2 memory cells. More importantly, the converted Foxp3(+) T cells efficiently accumulated in the airways and significantly suppressed Th2 memory cell-mediated airway hyperreactivity, eosinophilia, and allergen-specific IgE production. Our findings reveal the plasticity of Th2 memory cells and provide a strategy for adoptive immunotherapy for the treatment of allergic diseases.
Allergic asthma is associated with eosinophilic inflammation in the airways. Animal models commonly used to elucidate allergic inflammation mechanisms include BALB/c and C57BL/6 mice. Our aim was to evaluate lung eosinophilia and the corresponding Th1/Th2 balance in the two strains after allergen exposure. BALB/c and C57BL/6 mice were subjected to ovalbumin-induced allergic airway inflammation using BrdU to label newly produced cells. The numbers of new eosinophils were evaluated by differential cell count and immunocytochemistry (MBP+BrdU+). Proliferation rate of lung eosinophils was measured by analysis of CD45+CCR3+BrdU+ cells by FACS. Distribution of newly produced eosinophils in the lung and the Th1/Th2 (CD4+T-bet+/CD4+GATA-3+) balance was evaluated by immunohistochemistry. Allergen challenge with ovalbumin induced comparable eosinophilia in bone marrow (BM), blood and lung tissue in both strains of mice compared to phosphate-buffered saline controls, which was confirmed by immunocytochemistry. There was a small increase in the number of lung MBP+BrdU(-) eosinophils in C57BL/6 mice compared to BALB/c mice, which suggests a basal increase in this strain following sensitization. While there was no difference in eosinophilic proliferation in the lung, the distribution of the newly produced eosinophils differs between the two strains. BALB/c mice showed staining primarily around vessels and airways, whereas C57BL/6 mice showed a more even distribution in the lung tissue. No difference in the Th1/Th2 balance was observed between two strains. This study shows that there is a difference in the distribution of eosinophils in the lung between the C57BL/6 and BALB/c mice, but no difference in eosinophil production or Th1/Th2 balance.
T helper cell differentiation is controlled by a network of transcription factors. In this issue of Immunity, Yagi et al. (2010) describe the opposing effects of transcription factors Runx3 and GATA3 in the network promoting T helper 1 cell development.
In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggesting that there are more factors working in the trophoblast lineage. Here, we report that the transcription factor Tcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that the molecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating that they are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish the extraembryonic trophoblast maintenance program in murine embryos.
The transcription factor GATA3 is crucial for the differentiation of naive CD4(+) T cells into T helper 2 (Th2) cells. Here, we show that deletion of Gata3 allowed the appearance of interferon-gamma (IFN-gamma)-producing cells in the absence of interleukin-12 (IL-12) and IFN-gamma. Such IFN-gamma production was transcription factor T-bet independent. Another T-box-containing transcription factor Eomes, but not T-bet, was induced both in GATA3-deficient CD4(+) T cells differentiated under Th2 cell conditions and in Th2 cells with enforced Runx3 expression, contributing to IFN-gamma production. GATA3 overexpression blocked Runx3-mediated Eomes induction and IFN-gamma production, and GATA3 protein physically interacted with Runx3 protein. Furthermore, we found that Runx3 directly bound to multiple regulatory elements of the Ifng gene and that blocking Runx3 function in either Th1 or GATA3-deficient "Th2" cells results in diminished IFN-gamma production by these cells. Thus, the Runx3-mediated pathway, actively suppressed by GATA3, induces IFN-gamma production in a STAT4- and T-bet-independent manner.
Naïve CD4(+) T cells are educated in the thymus to survey the periphery for cognate antigen while ignoring self or commensal antigens. T cell antigen receptor (TCR) cross-linking initiates signaling cascades that integrate information from co-stimulatory receptors and locally available cytokines to chart the course of inflammation. The dynamic composition of transcription factors acting within a given T cell drive clonal expansion and specify differentiation into a growing array of effector and regulatory T cell subsets. The classical gamma-interferon (IFNgamma)-secreting T helper type (Th)-1 and IL-4-producing Th2 cell subsets utilize T-bet or GATA3 as master lineage regulators. It is now understood that naïve T cells also differentiate into pro-inflammatory Th17 or tissue-protective inducible T regulatory (iTreg) cells under the respective guidance of RORgammat or FOXP3. Emerging data highlight the reoccurring theme that these Th17 and iTreg master regulators prescribe T cell lineage commitment through interactions with each other, as well as with a broader network of auxiliary transcription factors.
To investigate the effects of garlicin on the expression of transcription factors T-bet and GATA-3 in blood lymph cells of experimental rats with allergic rhinitis.
Thirty healthy SD rats were randomly divided into 3 groups, with 10 rats for each. Ten rats (pharmacological group) were sensitized and intranasally challenged by ovalbumin (OVA), aluminium hydroxide hydrate gel and Bordetella pertussis (B. pertussis) inactive microorganism suspension adjuvants, as the allergic rhinitis models, and then injection of garlicin (0.4 ml) intraperitoneally per day for 10 days. Ten rats in the control group were immunized as the pharmacological group, and then injection of physiological saline as equal volume as garlicin. Ten rats in the negative control group were investigated using physiological saline only. Plasma and lymph cells were separated from 2 ml blood which was extracted from rat heart. Enzyme linked immunosorbent assay (ELISA) was utilized to detect the levels of plasma IL-4 and IFN-γ, and RT-PCR was utilized to detect the expression of T-bet and GATA-3.
The levels of IL-4 (x(-) ± s) were (6.292 ± 1.734), (14.252 ± 1.971), (4.916 ± 0.600) pg/ml, respectively, and the levels of IFN-γ were (24.338 ± 2.375), (12.364 ± 1.749), (16.136 ± 2.012) pg/ml, respectively, among the pharmacological, control and negative control groups. The plasma level of IL-4 in the pharmacological group rats was lower than that of control group rats (t = 4.23, P < 0.05), while there was no significant difference between the pharmacological and negative control group rats (t = 1.01, P > 0.05). The plasma level of IFN-γ was increased significantly in the pharmacological group rats when compared with the value of control group rats (t = 4.61, P < 0.05) and with the negative control group (t = 5.79, P < 0.05), respectively. There was no significant difference (t = 1.17, P > 0.05) in plasma level of IFN-γ in the control group rats when compared with the negative control group. Relative quantization of expression of GATA-3 (median) was 0.826, 1.029 and 0.474, and those of T-bet (median) were 1.245, 0.280 and 0.544, respectively, among the pharmacological, control and negative control groups. There were significant difference among 3 groups (chi² = 6.599, P = 0.019; chi² = 9.884, P = 0.007, respectively). Relative quantization of expression of GATA-3 in the control group rats was significantly higher than that in the negative control group and pharmacological group rats (chi² value were 10.113 and 2.384, respectively, all P < 0.05), and that of GATA-3 in the pharmacological group rats was increased as compared to the negative control group rats (chi² = 3.784, P < 0.05). The level of expression of T-bet in pharmacological group rats was elevated markedly with a significant difference as compared to both control (chi² = 16.021, P < 0.05) and negative control groups (chi² = 14.103, P < 0.05).
Garlicin may upregulate the expression of T-bet dramatically and downregulate the expression of transcription factor GATA-3 in the experimental models of allergic rhinitis.
Although cell-to-cell variability has been recognized as an unavoidable consequence of stochasticity in gene expression, it may also serve a functional role for tuning physiological responses within a cell population. In the immune system, remarkably large variability in the expression of cytokine genes has been observed in homogeneous populations of lymphocytes, but the underlying molecular mechanisms are incompletely understood. Here, we study the interleukin-4 gene (il4) in T-helper lymphocytes, combining mathematical modeling with the experimental quantification of expression variability and critical parameters. We show that a stochastic rate-limiting step upstream of transcription initiation, but acting at the level of an individual allele, controls il4 expression. Only a fraction of cells reaches an active, transcription-competent state in the transient time window determined by antigen stimulation. We support this finding by experimental evidence of a previously unknown short-term memory that was predicted by the model to arise from the long lifetime of the active state. Our analysis shows how a stochastic mechanism acting at the chromatin level can be integrated with transcriptional regulation to quantitatively control cell-to-cell variability.
Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.
Beta-carbolines (BCs) are potential endogenous and exogenous neurotoxins that may contribute to the pathogenesis of Parkinson's disease. However, we recently demonstrated protective and stimulatory effects of 9-methyl-BC (9-me-BC) in primary dopaminergic culture. In the present study, treatment with 9-me-BC unmasked a unique tetrad of effects. First, tyrosine hydroxylase (TH) expression was stimulated in pre-existing dopa decarboxylase immunoreactive neurons and several TH-relevant transcription factors (Gata2, Gata3, Creb1, Crebbp) were up-regulated. Neurite outgrowth of TH immunoreactive (THir) neurons was likewise stimulated. The interaction with tyrosine kinases (protein kinase A and C, epidermal growth factor-receptor, fibroblast growth factor-receptor and neural cell adhesion molecule) turned out to be decisive for these observed effects. Second, 9-me-BC protected in acute toxicity models THir neurons against lipopolysaccharide and 2,9-dime-BC(+) toxicity. Third, in a chronic toxicity model when cells were treated with 9-me-BC after chronic rotenone administration, a pronounced regeneration of THir neurons was observed. Fourth, 9-me-BC inhibited the proliferation of microglia induced by toxin treatment and installed an anti-inflammatory environment by decreasing the expression of inflammatory cytokines and receptors. Finally, 9-me-BC lowered the content of alpha-synuclein protein in the cultures. The presented results warrant the exploration of 9-me-BC as a novel potential anti-parkinsonian medication, as 9-me-BC interferes with several known pathogenic factors in Parkinson's disease as outlined above. Further investigations are currently under way.
Interleukin-13 (IL-13) is recognized to be a key modulator in the pathogenesis of Th2-induced allergic inflammation. Transcription factors GATA3 and NFAT1 have been both implicated in the regulation of Th2 cytokines. We previously demonstrated the GATA3-NFAT1 association during human T cell activation. However, the function of the GATA3-NFAT1 complex in Th2 cytokines regulation is still unknown. Small interference RNA (siRNA) was constructed to knock down GATA3 expression in Hut-78 cells to investigate the possible role of GATA3-NFAT1 complex in IL-13 transcription.
Cells were stimulated with anti-CD3 plus anti-CD28 antibodies to mimic in vivo antigen-mediated co-stimulation; the expression of IL-13 mRNA was determined by real-time PCR; chromation immunoprecipitation (CHIP) assay was employed to investigate the NFAT1 binding to IL-13 promoter.
GATA3 siRNA suppressed the expression of GATA3 both in mRNA and protein levels in Hut-78 cells. The binding of NFAT1 to IL-13 promoter was inhibited by GATA3 siRNA in activated T cells, which was followed by the reduction of IL-13 transcription.
GATA3-NFAT1 complex may play an important role in the regulation of IL-13 transcription in human T cells.
The severity of asthma after puberty is higher in women than in men. Increased numbers of eosinophils in the airways of female mice after antigen challenge was associated with increased levels of T helper (Th)2 cytokines at the site of inflammation, and in human and mouse studies, the profile of cytokines produced by immune cells from women showed greater Th2 predominance. The aim of this study was to investigate gender differences in the development of Th2 immune responses.
Male and female C57BL/6 mice were sensitized with ovalbumin. Cells prepared from bronchial lymph nodes were cultured in the absence or presence of ovalbumin. Cytokine concentrations in the culture supernatants were measured, and IL-5 and GATA-binding protein 3 (GATA-3) gene expression were evaluated. T-cell subsets were analysed using specific surface markers.
The concentrations of IL-4, IL-5, IL-13 and IL-10, but not interferon-gamma or transforming growth factor-beta(1), were higher in cell supernatants from female mice than in those from male mice. IL-5 and GATA-3 gene expressions were higher in cells from women than in cells from men. The numbers of CD3(+)CD4(+)T1/ST2(+) cells, but not CD3(+)CD4(+) or CD4(+)CD25(+) cells, were significantly higher in cells from women than in cells from men.
Greater antigen-induced Th2 cytokine production by bronchial lymph node cells from female mice was associated with enhanced Th2 cell differentiation and increased expression of the Th2-specific transcription factor, GATA-3.
During the development of immune responses to pathogens, self-antigens, or environmental allergens, naive CD4(+) T cells differentiate into subsets of effector cells including Th1, Th2, and Th17 cells. The differentiation into these subsets is controlled by specific transcription factors. The activity of these effector cells is limited by nTregs and iTregs, whose differentiation and maintenance are dependent on the transcription factor Foxp3. The regulation of autoimmune diseases mediated by Th1 and Th17 cells by Tregs has been studied and reviewed extensively. However, much less has been presented about the interplay between Tregs and Th2 cells and their contribution to allergic disease. In this perspective, we discuss the regulation of Th2 cells by Tregs and vice versa, focusing on the interplay between the IL-4-activated STAT6/GATA3 pathway and Foxp3.
Psoriasis is considered as a T cell-mediated autoimmune disease, and the Th1 response has been established as the major immune agent in its pathomechanisms. The relative expression of Th1 and Th2 transcription factors, T-bet and GATA-3, resulting in a swing in the Th1/Th2 pendulum, has been implicated in a number of immunological diseases. However, their expression and correlation with psoriasis has not yet been studied. Our aim was to evaluate the expression of T-bet and GATA-3 in psoriatic patients and determine their correlation with serum levels of IFN-gamma and IL-4. Sera, peripheral blood mononuclear cells (PBMCs) and skin lesions were taken from 23 patients with psoriasis vulgaris. Serum levels of IFN-gamma and IL-4 were measured by ELISA. T-bet and GATA-3 mRNA expression in PBMCs was analyzed by RT-PCR. Lesional expression and distribution of CD4, CD8, T-bet and GATA-3 were assessed by immunohistochemistry. Blood and skin samples of healthy individuals served as controls. A markedly higher IFN-gamma and lower IL-4 concentration in the serum of psoriatic patients was found. A significantly higher expression of T-bet mRNA and a lower expression of GATA-3 mRNA in PBMCs, and consequently, a much higher T-bet/GATA-3 ratio in patients than in controls were shown. T-bet mRNA expression was strongly correlated with serum IFN-gamma secretion in patients; furthermore, the correlation between T-bet/GATA-3 ratio and IFN-gamma/IL-4 ratio was revealed. We also observed a significant increase in CD4+ cells and T-bet positive cells in psoriatic lesions. These results suggested that T-bet and GATA-3 might be regulator genes for psoriasis via the Th1/Th2 balance, and the Th1-specific transcription factor, T-bet, may play an important role in the development and maintenance of psoriasis.
Naive T-helper (Th) cells differentiate into distinct lineages including Th1, Th2, Th17 and regulatory T (Treg) cells. Each of these Th-lineages has specific functions in immune defense and T cell homeostasis. Th cell fate decisions and commitment are dependent on the kind and strength of T cell stimulation and the subsequent gene expression profiles. Our analysis targeted the identification of new regulatory transcription factor binding sites (TFBSs) in the promoter regions of up- and down-regulated genes in Treg cell differentiation and lineage maintenance. For this approach we compared different gene groups from global gene expression studies with background models of randomly selected genes to identify significantly overrepresented TFBSs. Results of our analysis suggest that Ets and IRF family members contribute to the regulation of the initial induction of Treg cells. Furthermore, we identified the overrepresented TFBS-pairs Runx-NFAT and GATA3-Foxp3 in Treg specific genes and Foxp3 dependent genes, respectively. Interestingly, previous studies have observed functional interactions of both TFBS-pairs in T cells. This study provides a starting point for further investigations to elucidate the transcriptional network in Treg cells.
The Src family kinase Lck has been shown to be crucial in T cell signaling and development. However, its role in Th effector functions is not well understood. Lck has previously been shown to play a role in the cytokine expression of Th2 cells, but the mechanism by which Lck influences Th2 effector functions is unknown. Using a mouse model, we report that Lck is important in regulating the expression of IL-4 in Th2 skewed cells but is not as necessary for the expression of Th2 cytokines IL-5, IL-10, and IL-13. Furthermore, in the absence of Lck, T-bet and GATA-3 expression is aberrant. Moreover, this atypical expression pattern of T-bet and GATA-3 correlates with increased histone 3 acetylation at the Ifng locus and production of the Th1 cytokine IFN-gamma. We find overexpression of GATA-3 restores IL-4 expression in lck(-/-) Th2 cells; this indicates that the decreased IL-4 expression is due in part to reduced amounts of GATA-3. Taken together, these data imply that Lck mediates Th2 differentiation through effects on T-bet and GATA-3.
Candida species are the most common source of nosocomial invasive fungal infections. Previous studies have indicated that T-helper immune response is the critical host factor for susceptibility to Candida infection. The transcription factor GATA-3 is known as the master regulator for T-helper type 2 (Th2) differentiation. We therefore investigated the role of GATA-3 in the host defense against systemic Candida infection using GATA-3-overexpressing transgenic mice. The survival of GATA-3-overexpressing mice after Candida infection was significantly lower than that of wild-type mice. Candida outgrowth was significantly increased in the kidneys of GATA-3-overexpressing mice, compared with wild-type mice. The levels of various Th2 cytokines, including interleukin-4 (IL-4), IL-5, and IL-13, were significantly higher while the level of Th1 cytokine gamma interferon was significantly lower in the splenocytes of GATA-3-overexpressing mice after Candida infection. Recruitment of macrophages into the peritoneal cavity in response to Candida infection and their phagocytic activity were significantly lower in GATA-3-overexpressing mice than in wild-type mice. Exogenous administration of gamma interferon to GATA-3-overexpressing mice significantly reduced Candida outgrowth in the kidney and thus increased the survival rate. Administration of gamma interferon also increased the recruitment of macrophages into the peritoneal cavity in response to Candida infection. These results indicate that overexpression of GATA-3 modulates macrophage antifungal activity and thus enhances the susceptibility to systemic Candida infection, possibly by reducing the production of gamma interferon in response to Candida infection.
The role of T regulatory cells in spontaneous recovery from cow's milk allergy (CMA) is unclear. We investigated the mRNA expression of 12 T-cell markers and the protein expression of CD4, CD25, CD127, FoxP3 after in vitro beta-lactoglobulin stimulation of peripheral blood mononuclear cells from children with persisting CMA (n=16), early recovery (n=20) or no atopy (n=21). Artificial neural networks with exhaustive search for all marker combinations revealed that markers FoxP3, Nfat-C2, IL-16 and GATA-3 distinguished patients with persisting CMA most accurately from other study groups. FoxP3 mRNA expression following beta-lactoglobulin stimulation was highest in children with persisting CMA. Also the FoxP3 intensity in CD4(+) CD25(high)CD127(low) cells was higher in children with CMA compared with non-atopic children. The expression profile of both Th2- and T regulatory cell-related genes thus reflects the clinical activity of CMA. Tolerance, in contrast, is not characterized by activation of circulating T regulatory cells.
A dramatic decrease in circulating lymphocyte number is regularly described after septic shock. However, it is unknown how early this alteration develops after diagnosis of shock and if it remains stable over time. Twenty-one septic shock patients with no comorbidities were included within 2 h after the beginning of vasopressive treatment. Flow cytometry phenotyping of circulating leukocyte subpopulations and quantitative real-time polymerase chain reaction of T-bet, GATA-3, FOXP3, and RORγ mRNA were performed in patients from the diagnosis of shock and every 6 h during the subsequent 48 h. From their admission in the intensive care unit, patients present with major alterations of circulating leukocyte count (leukocytosis, neutrophilia, and major lymphopenia). The numbers of every lymphocyte subpopulations (T, B, and natural killer cells) were diminished. Gene expression analysis of transcription factors specific for TH1, TH2, CD4CD25 regulatory, and TH17 lymphocytes showed a severe decrease in comparison with healthy individuals' values. These alterations remain stable during the first 48 h after inclusion in the protocol despite early and aggressive resuscitation and antibiotherapy administered in patients. At the time of diagnosis of shock and admission in the intensive care unit, septic patients already present with severe lymphopenia involving every lymphocyte subsets including CD4 T-cell subpopulations. No significant variation could be detected within the first 48 h. This should be taken into account in the forthcoming clinical trials testing immunomodulating therapies in septic shock patients.
Loss of transforming growth factor-beta receptor III (TbetaRIII) correlates with loss of transforming growth factor-beta (TGF-beta) responsiveness and suggests a role for dysregulated TGF-beta signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TbetaRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TbetaRIII expression in human cells. We localize GATA3's binding to a 10-bp region of the TbetaRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TbetaRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TbetaRIII expression during ccRCC progression.
Gastric cancer is a serious public health cancer and causes nearly 1 million deaths a year worldwide. Th1 cells play critical roles in orchestrating the adaptive immune responses against gastric cancer. T-bet, a member of the T-box family of transcription factors, is the Th1 master regulator and up-regulated during Th1 differentiation. Polymorphisms have also been shown to exist in T-bet. Some reports indicated that some tumours were associated with the drift of Th1 and Th2. In the present work, we investigated the drift of Th1/Th2 by detecting the expression levels of T-bet, IFN-gamma, IL-4, and GATA-3 in peripheral blood mononuclear cell of gastric cancer patients by real-time PCR, explored the relationship between the polymorphism of T-bet gene and drift of Th1/Th2 by gene sequence, western blot, and gene transfection. Our results indicated that a predominant Th2 phenotype was existence. T-bet gene mutations may be associated with Th2-dominated condition in gastric cancers.
To date there is little information on the inflammatory profiles of patients suffering from persistent (PER) and intermittent allergic rhinitis (IAR). Also, it is not clear whether differences exist in eosinophilic inflammation and/or T-helper cell sub-populations and their markers. The aim of this study was to primarily evaluate the inflammatory profiles of patients with moderate/severe PER and IAR.
Inferior nasal turbinate tissue was obtained from 12 PER, 12 IAR and 12 nonallergic nonrhinitic (control) patients, and symptoms (visual analogue scales, VAS) and impairment of life was monitored. All tissues were assessed for eosinophil and mast cell numbers by immunohistochemistry; IL-5, ECP and IgE concentrations by immunoassay; mRNA for transcription factors GATA-3, T-bet, FOXP3 and RORc by quantitative real-time polymerase chain reaction; and IgE-induced release of LTC(4)/D(4)/E(4) and PGD(2)in vitro.
Eosinophils and mast cells were significantly increased in patients with PER and patients with IAR compared to control subjects; by patients with PER demonstrating even significantly greater increase of both cell types than patients with IAR. Similarly, ECP IL-5, GATA-3 mRNA expression and IgE-induced release of LTC4/D(4)/E(4) and PGD(2) from mast cells were significantly increased in patients with PER compared to patients with IAR. In contrast, the expression of T-bet, FOXP3 or RORc mRNA was not significantly different in the PER, IAR or control patients.
The findings from the present study suggest that PER is characterized by a significantly greater eosinophilic and predominantly Th2 cell-mediated nasal inflammatory profile compared to IAR.
GATA3, a transcription factor that regulates T lymphocyte differentiation and maturation, is exclusively expressed in early stage well differentiated breast cancers but not in advanced invasive cancers. However, little is understood regarding its activity and the mechanisms underlying this differential expression in cancers. Here, we employed GATA3-positive, non-invasive (MCF-7) and GATA3-negative, invasive (MDA-MB-231) breast cancer cells to define its role in the transformation between these two distinct phenotypes. Ectopic expression of GATA3 in MDA-MB-231 cells led to a cuboidal-like epithelial phenotype and reduced cell invasive activity. These cells also increased E-cadherin expression but decreased levels of vimentin, N-cadherin, and MMP-9. Further, MDA-MB-231 cells expressing GATA3 grew smaller primary tumors without metastasis compared with larger metastatic tumors derived from control MDA-MB-231 cells in xenografted mice. GATA3 was found to induce E-cadherin expression through binding GATA-like motifs located in the E-cadherin promoter. Blockade of GATA3 using small interfering RNA gene knockdown in MCF-7 cells triggered fibroblastic transformation and cell invasion, resulting in distant metastasis. Studies of human breast cancer showed that GATA3 expression correlated with elevated E-cadherin levels, ER expression, and long disease-free survival. These data suggest that GATA3 drives invasive breast cancer cells to undergo the reversal of epithelial-mesenchymal transition, leading to the suppression of cancer metastasis.
Inflammatory bowel disease is characterized with uncontrolled immune response in inflamed mucosa, with dominance of Th1 cells. Recently, all-trans retinoic acid has been shown that can lead T-cell response by suppressing Th17 development via retinoic acid receptor (RAR), but it is still unknown whether all-trans retinoic acid can modulate Th1 response of inflammatory bowel disease. In the experiment, we investigated the effect of all-trans retinoic acid on trinitrobenzene sulfonic acid (TNBS)-induced murine colitis, and the possible mechanism. Mice were intraperitoneally treated daily with all-trans retinoic acid (the agonist of RAR-alpha) or LE135 (the antagonist of RAR-alpha) or medium, and sacrificed 6 days later. Colon was collected for histological analysis and myeloperoxidase (MPO) activity measurement. Lamina propria mononuclear cells (LPMCs) were isolated, cultured, and assayed for the expressions of T-bet and GATA-3 by the use of Western blot and for cytokine levels by the use of ELISA. All-trans retinoic acid treatment inhibited inflammatory responses as shown by lower histological inflammatory scores and MPO activity, compared with LE135 and medium groups. Furthermore, in LPMCs culture supernatants, the levels of Th1 cytokines (INF-gamma, IL-12, and TNF-alpha) were decreased while those of Th2 cytokines (IL-4 and IL-10) were increased significantly in all-trans retinoic acid-treated mice. In addition, T-bet expression in LPMCs was inhibited and GATA-3 expression was up-regulated in all-trans retinoic acidtreated mice. On the contrary, LE135 showed the reverse effects in colon inflammation and cytokine profile. By shifting Th1 to Th2 profile in inflamed mucosa, all-trans retinoic acid down-regulates inflammatory response and ameliorates acute TNBS-induced colitis, which suggests the ligand of RAR-alpha-based pharmaceutical strategies for managing inflammatory bowel disease.
Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.
Although allogeneic bone marrow transplantation has been shown to prevent autoimmune diabetes in heavily irradiated nonobese diabetic (NOD) mice, a similar procedure is not suitable for the treatment of patients with type 1 diabetes because of associated severe side effects. Therefore, we evaluated whether mouse newborn blood (NBB), equivalent to human umbilical cord blood, could be used for diabetes prevention without recipient preconditioning. To test this hypothesis, unconditioned, prediabetic female NOD mice were given a single injection of whole NBB derived from the allogeneic diabetes-resistant mouse strain C57BL/6. Transfusion of allogeneic NBB but not adult blood prevented diabetes incidence in a majority of treated mice for a prolonged period of time. This was accompanied by the release of insulin in response to a challenge with glucose. Invasive cellular infiltration of islets was also substantially reduced in these mice. Although NBB transfusion induced a low level of hematopoietic microchimerism, it did not strictly correlate with amelioration of diabetes. Induction of genes implicated in diabetes, such as Il18, Tnfa, and Inos but not Il4, Il17 or Ifng, was repressed in splenocytes derived from protected mice. Notably, expression of the transcription factor Tbet/Tbx21 but not Gata3 or Rorgt was upregulated in protected mice. These data indicate that allogeneic NBB transfusion can prevent diabetes in NOD mice associated with modulation of selected cytokine genes implicated in diabetes manifestation. The data presented in this study provide the proof of principle for the utility of allogeneic umbilical cord blood transfusion to treat patients with autoimmune diabetes.
We have known since 1991 how to induce naive CD4 T cells to differentiate in vitro into Th2 cells and, over the ensuing years, a comprehensive picture of the molecules involved in this important process has emerged. GATA3 and STAT5 are both essential for in vitro differentiation, stimulating naive cells through a process involving induction, which is T-cell receptor (TCR) dependent but interleukin (IL)-4 independent, and commitment, which is IL-4 dependent. Th2 differentiation in vivo appears more complex. GATA3 and probably STAT5 are required in vivo but, at least for certain helminth infections, the IL-4/IL-4Ra/STAT6 pathway is dispensable. The role of thymic stromal lymphopoietin and of low TCR signal strength and the participation of basophils in establishing a Th2-baising in vivo environment have achieved considerable attention. Here I discuss the major players in Th2 differentiation particularly as they may exert their effects in vivo.
Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1 expression without amplification. Silencing of Cyclin D1 expression was previously found to trigger differentiation of neuroblastoma cells. Overexpression of Cyclin D1 is therefore one of the most frequent mechanisms with a postulated function in neuroblastoma pathogenesis. The cause for the Cyclin D1 overexpression is unknown. Here we show that Cyclin D1 overexpression results from transcriptional upregulation. To identify upstream regulators, we searched in mRNA profiles of neuroblastoma tumor series for transcription factors with expression patterns correlating to Cyclin D1. GATA3 most consistently correlated to Cyclin D1 in four independent data sets. We identified a highly conserved GATA3 binding site 27 bp upstream of the Cyclin D1 transcriptional start. Chromatin immune precipitation confirmed binding of GATA3 to the Cyclin D1 promoter. Overexpression of GATA3 induced Cyclin D1 promoter activity, which decreased after site-directed mutagenesis of the GATA3 binding site in the Cyclin D1 promoter. Silencing of GATA3 resulted in reduced Cyclin D1 promoter activity and reduced Cyclin D1 mRNA and protein levels. Moreover, GATA3 silencing caused differentiation that was similar to that caused by Cyclin D1 inhibition. These finding implicate GATA3 in Cyclin D1 overexpression in neuroblastoma.
Environmental factors, including the intrauterine environment, can influence the risk of allergy development. In the present study, we investigated whether lifestyle and parental allergen sensitization status are reflected at gene expression level in the intrauterine environment.
  mRNA expression of 17 genes was determined by means of quantitative real-time PCR in term placenta of 36 families participating in the ALADDIN study (Assessment of Lifestyle and Allergic Disease During Infancy). Data were analysed using a linear regression model to estimate the influence of lifestyle and parental allergen sensitization on the relative mRNA expression levels. Immunohistochemistry on placenta biopsies was used to verify protein expression.
Significant differences in mRNA expression levels were detected at the foetal side of the placenta, where CD14 was expressed at higher levels in placentas from families living on a farm compared to not living on a farm, and IL-12(p40) was expressed at lower levels when the father was sensitized compared to nonsensitized. At the maternal side of the placenta, higher expression of STAT4 and lower expression of GATA3 were detected in families with sensitized compared to nonsensitized mothers, and IL-12(p40) was lower expressed when the families were living on a farm compared to not living on a farm. Immunohistochemistry performed for STAT4 and GATA3 showed that protein and mRNA levels correlated well.
Living on a farm and parental allergen sensitization are reflected in the intrauterine environment at the gene expression level.
Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities.With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies,as well as to define tumour-specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef-8 BeadChip from Illumina.Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5-fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3,PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real-time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery.
Estrogen receptor (ER)alpha is a critical target of therapeutic strategies to control the proliferation of hormone-dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. We have established the cellular mechanism by which indole-3-carbinol (I3C), a promising anticancer phytochemical from Brassica vegetables, ablates ERalpha expression, and we have uncovered a critical role for the GATA3 transcription factor in this indole-regulated cascade. I3C-dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ERalpha protein. I3C inhibits endogenous binding of ERalpha with the 3'-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ERalpha promoter, leading to a loss of GATA3 and ERalpha expression. Ectopic expression of GATA3 has no effect on I3C-induced ERalpha protein degradation but does prevent I3C inhibition of ERalpha promoter activity, demonstrating the importance of GATA3 in this I3C-triggered cascade. Our preclinical results implicate I3C as a novel anticancer agent in human cancers that coexpress ERalpha, GATA3, and AhR, a combination found in a large percentage of breast cancers but not in other critical ERalpha target tissues essential to patient health.
Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4(+) T-cell responses remains unclear. In the present study, CD4(+) T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture (CLP)) were analyzed in vitro. CD4(+)CD62L(+) T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4(+)CD62L(+) T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the T(H)1 or T(H)2 lineage. Repressive histone methylation marks were also evident at promoter regions for the T(H)1 cytokine IFN-gamma and the T(H)2 transcription factor GATA-3 in naïve CD4(+) T cells from CLP mice. These results provide evidence that CD4(+) T-cell subsets from post-septic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.
In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H)2) specific transcription factor that regulates production of T(H)2 cytokines and functions as T(H)2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+) T cells, suggesting that SATB1 influences T(H)2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H)2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H)2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H)2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H)2 cells in a Wnt-dependent manner. SATB1 coordinates T(H)2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates T(H)2 lineage commitment by mediating Wnt/beta-catenin signalling. This report identifies a new global transcription factor involved in beta-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.
Prenatal exposures to immunogenic stimuli, such as bacterial LPS, have shown to influence the neonatal immune system and lung function. However, no detailed analysis of the immunomodulatory effects of LPS on postnatal T helper cell differentiation has been performed. Using a rat model, we investigated the effect of prenatal LPS exposure on postnatal T cell differentiation and experimental allergic airway disease. Pregnant rats were injected with LPS on day 20 and 21 (term = 22 days). Some of the offspring were sensitized and challenged with ovalbumin. Positive control animals were placebo exposed to saline instead of LPS, whereas negative controls were sensitized with saline. Expression of T cell-related transcription factors and cytokines was quantified in the lung, and airway hyperresponsiveness was measured. Prenatal LPS exposure induced a T helper 1 (T(H)1) immune milieu in the offspring of rats [i.e., increased T-bet and T(H)1 cytokine expression while expression of T(H)2-associated transcription factors (GATA3 and STAT6) and cytokines was decreased]. Prenatal LPS exposure did not trigger T(H)17 cell differentiation in the offspring. Furthermore, prenatal LPS exposure reduced ovalbumin-induced (T(H)2-mediated) airway inflammation, eosinophilia, and airway responsiveness. Thus, in utero exposure to endotoxin promotes a T(H)1 immune environment, which suppresses the development of allergic airway disease later in life.
Single hematopoietic cells from carp (Cyprinus carpio) kidney were seeded to each well of 96-well plates and cultured in the presence of a supporting cell layer and conditioned media (CM). The CM were obtained from bulk-cultured carp hematopoietic cells, in which T and macrophage-lineage cells rapidly proliferated as previously reported. After 2-3 weeks, colony formation was found in 0-4 wells of each plate. Three different morphological types of colonies were observed: "type I colonies", "type II colonies" and "mixed-type colonies". Type I colony cells were interpreted as composed by macrophage-lineage cells, since they expressed a specific macrophage marker, M-CSFR/csf1r gene, and most of them phagocytosed latex particles. Type II colony cells were interpreted as composed by T lineage cells, since they expressed several T cell marker genes including gata3, lck and TCRbeta, but did not engulf latex particles. Mixed-type colonies were interpreted as composed by both macrophages and T lineage cells. They expressed not only the M-CSFR gene but also a T cell marker gene, gata3, but not other T cell markers, such as lck and TCRbeta. These results indicated that the mixed-type colonies were developed from immature common progenitors of macrophage and T cell. In contrast, type I and type II colonies were developed from more mature and mono-potent progenitors of macrophage and T cell, respectively.
To study the expression of transcriptional factors T-bet and GATA3 mRNA and the levels of IFN-gamma and IL-4 in blood in children with acute idiopathic thrombocytopenic purpura (ITP) and investigate the tendency of polarization of Th1/Th2 in children with ITP.
Blood T-bet and GATA3 mRNA expression were examined using RT-PCR and plasma IFN-gamma and IL-4 levels were measured using EIASA in children with acute ITP in acute (n=30) and remission stages (n=28). Twenty healthy children served as the controls.
Blood T-bet mRNA expression and IFN-gamma levels in children with ITP in the acute stage were markedly higher than those in the remission stage and controls (p<0.01). In contrast, blood GATA3 mRNA expression and IL-4 levels in children with ITP in the acute stage were significantly lower than those in the remmission stage and controls (p<0.01).
The high expression of T-bet and IFN-gamma and the low expression of GATA3 and IL-4 indicate the existence of Th1 polarization in children with acute ITP. This might be related to the regulation of T-bet and GATA3.
Aurora-A is a proto-oncogenic mitotic kinase that is frequently overexpressed in human epithelial malignancies including in breast and ovarian cancers. The mechanism of transcriptional upregulation of Aurora-A in human breast cancer is not yet elucidated. We report herein that Aurora-A transcription is positively regulated by GATA-3 in response to estrogen in estrogen receptor α (ERα)-positive cells. Transient expression of aurora-A promoter deletion mutants in luciferase constructs identified a GATA binding sequence motif as a functional regulatory element in ERα-positive breast cancer cells. Electrophoretic mobility shift assay identified the binding of regulatory proteins to the GATA element. Anti-GATA-3 antibody generated a supershifted complex. Recruitment of GATA-3 to the aurora-A promoter was verified by chromatin immunoprecipitation analysis with GATA-3 antibody. Ectopic expression of GATA-3 resulted in elevated expression of Aurora-A in both ERα-positive and negative cells while siRNA-mediated silencing led to downregulation of endogenous Aurora-A in ERα-positive cells. Estrogen treatment of ERα-positive cells induced increased Aurora-A expression with enhanced recruitment of GATA-3 to the aurora-A promoter. Finally, in the ACI rat model of estrogen-induced breast cancer, known to be associated with elevated Aurora-A expression, we observed increased expression of GATA-3 in preinvasive and invasive mammary epithelial cells exposed to prolonged estrogen treatment and in developing breast tumors. These results demonstrate a direct positive role of estrogen in regulating Aurora-A expression through activation of the ERα-GATA-3 signaling cascade and suggest that this pathway may be critical in the origin of estrogen-stimulated sporadic breast cancer.
The aryl hydrocarbon receptor (AhR) is part of a signaling system that is mainly triggered by xenobiotic agents. Increasing evidence suggests that AhR may regulate immunity to infections. To determine the role of AhR in the outcome of toxoplasmosis, we used AhR-/- and wild-type (WT) mice. Following an intraperitoneal infection with Toxoplasma gondii (T. gondii), AhR-/- mice succumbed significantly faster than WT mice and displayed greater liver damage as well as higher serum levels of tumor necrosis factor (TNF)-alpha, nitric oxide (NO), and IgE but lower IL-10 secretion. Interestingly, lower numbers of cysts were found in their brains. Increased mortality was associated with reduced expression of GATA-3, IL-10, and 5-LOX mRNA in spleen cells but higher expression of IFN-gamma mRNA. Additionally, peritoneal exudate cells from AhR-/- mice produced higher levels of IL-12 and IFN-gamma but lower TLR2 expression than WT mice. These findings suggest a role for AhR in limiting the inflammatory response during toxoplasmosis.
To explore the potential role of thymic stromal lymphopoietin (TSLP) and its downstream molecules in the development of ocular allergic inflammation using a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC).
BALB/c mice were topically challenged with SRW pollen after they were sensitized with SRW in the footpad. After the last SRW challenge, the corneal epithelium, conjunctiva, and cervical lymph nodes were harvested for total RNA extraction and gene expression by RT and real-time PCR, and whole eye globes were collected to make cryosections for immunohistochemical staining.
Repeated topical challenges with SRW allergen generated typical signs of AC in mice. Compared with the untreated controls, TSLP mRNA expression and immunoreactivity were significantly increased in the corneal and conjunctival epithelia of SRW-induced AC mice. CD11c(+) and OX40L(+) immunoreactive cells largely infiltrated the conjunctiva with increased mRNA levels of CD11c, TSLPR, and OX40L detected in the corneal epithelium, conjunctiva, and cervical lymph nodes. CD4(+) Th2 cell infiltration was evidenced by increased levels of mRNA and immunoreactivity of CD4, IL-4, IL-5, and IL-13 in the ocular surface, mainly in the conjunctiva, accompanied by increased expression of OX40, STAT6, and GATA3, in AC mice. The maturation of immature DCs was observed with the use of TSLP containing conditioned media from corneal epithelial cultures exposed to polyI:C, which stimulates TSLP production.
This study provides new findings regarding the role of local mucosal epithelial cells in the initiation of ocular allergic inflammation by producing a novel proallergic cytokine, TSLP, which activates dendritic cells to prime Th2 differentiation and allergic inflammation through the TSLP-TSLPR and OX40L-OX40 signaling pathway.
The Ca(2+) dependent transcription factor family known as nuclear factor of activated T cells (NFAT) has been shown to be important in T-cell immune responses. Because NFAT proteins have a weak DNA-binding capacity, they cooperate with other transcription factors at composite sites within the promoters of target genes. Recently, NFAT was shown to also be important for the induction of specific genetic programs that guide the differentiation and effector or regulatory activities of CD4(+) T helper subsets via the transcriptional regulation of their lineage-specific transcription factors, specifically T-bet (Th1), Gata3 (Th2), RORgammat (Th17), and Foxp3 (iTregs). In addition, the NFAT family governs the transcription of several signature cytokines, including their cytokine receptors. Subsequently, the integration of these complex intracellular signal transduction cascades is considered to critically determine the crosstalk between the T-cell receptor and receptors that are activated by both the adaptive and innate immune systems to determine pathways of T helper cell differentiation and function. Here, we carefully review the critical role of the established transcriptional partners and functional outcomes of these NFAT interactions in regard to the effector responses of these clinically relevant CD4(+) T helper subsets.
To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage.
We performed immunohistochemical analyses of 12 proteins [CD44, CD24, ALDH1, vimentin, osteonectin, EPCR, caveolin 1, connexin 43, cytokeratin 18 (CK18), MUC1, claudin 7, and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only, 135 cases of IDC with ductal carcinoma in situ (DCIS), 35 cases of DCIS with microinvasion, and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry.
CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors, especially in luminal A subtype. The expression of vimentin, osteonectin, connexin 43, ALDH1, CK18, GATA3, and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells.
Our findings suggest that in breast cancer, the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage.
The normal counterparts of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) have not been accurately identified. We immunohistochemically analyzed 10 PTCL-NOS cases to examine the expression of the master regulators of T-cell differentiation and of surface antigens, including chemokine receptors. All cases were positive for the master regulator of helper T cells (Th-POK) and the marker of effector T cells (CD45RO). Three cases each were positive for T-Bet and GATA3, which are master regulators of helper T cells (T(H) ) type 1 (T(H)1) and 2 (T(H)2), respectively. Two cases were positive for the surface antigens of central memory (Tcm) (CCR7 and CD62L), and 1 case was positive for follicular helper T-cell (TFH) phenotype (BCL6, CXCL13, and PD-1). The remaining case was negative for all markers of effector T(H) subtypes. These results suggest the postulated normal counterparts of PTCL-NOS identified in 9 of the 10 cases consist of T(H)1, T(H)2, TCM, and TFH.
S-nitrosoglutathione (GSNO) is a physiological nitric oxide molecule which regulates biological activities of target proteins via s-nitrosylation leading to attenuation of chronic inflammation. In this study we evaluated the therapeutic efficacy of GSNO in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Oral administration of GSNO (0.5 or 1.0 mg/kg) reduced disease progression in chronic models (SJL and C57BL/6) of EAE induced with PLP((139-151)) or MOG((35-55)) peptides, respectively. GSNO attenuated EAE disease by reducing the production of IL17 (from Th(i) or Th17 cells) and the infiltration of CD4 T cells into the central nervous system without affecting the levels of Th1 (IFN gamma) and Th2 (IL4) immune responses. Inhibition of IL17 was observed in T cells under normal as well as Th17 skewed conditions. In vitro studies showed that the phosphorylation of STAT3 and expression of ROR gamma, key regulators of IL17 signaling, were reduced while phosphorylation of STAT4 or STAT6 and expression of T-bet or GATA3 remained unaffected, suggesting that GSNO preferentially targets Th17 cells. Collectively, GSNO attenuated EAE via modulation of Th17 cells and its effects are independent of Th1 or Th2 cells functions, indicating that it may have therapeutic potential for Th17-mediated autoimmune diseases.
IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-beta directs mouse CD4(+)CD25(-)CD62L(+) T cells to commit to inflammatory IL-9 producing CD4(+) T cells.
Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells. In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4(+)IL-9(+) T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNgamma or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production.
Taken together these data show both the differences and similarities between mouse and human CD4(+)IL9(+) T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4(+) T cells to antigen.
The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.
Current T cell differentiation models invoke separate T helper 2 (Th2) and Th1 cell lineages governed by the lineage-specifying transcription factors GATA-3 and T-bet. However, knowledge on the plasticity of Th2 cell lineage commitment is limited. Here we show that infection with Th1 cell-promoting lymphocytic choriomeningitis virus (LCMV) reprogrammed otherwise stably committed GATA-3(+) Th2 cells to adopt a GATA-3(+)T-bet(+) and interleukin-4(+)interferon-gamma(+) "Th2+1" phenotype that was maintained in vivo for months. Th2 cell reprogramming required T cell receptor stimulation, concerted type I and type II interferon and interleukin-12 signals, and T-bet. LCMV-triggered T-bet induction in adoptively transferred virus-specific Th2 cells was crucial to prevent viral persistence and fatal immunopathology. Thus, functional reprogramming of unfavorably differentiated Th2 cells may facilitate the establishment of protective immune responses. Stable coexpression of GATA-3 and T-bet provides a molecular concept for the long-term coexistence of Th2 and Th1 cell lineage characteristics in single memory T cells.
RNAi is the process of sequence-specific, post-transcriptional/transcriptional gene silencing through siRNA. RNAi is a popular method of controlling gene expression and has potential in the development of drugs for several diseases, such as various types of cancer and viral infections. Gene therapy for asthma has already been developed and has demonstrated promising results in animal models. Recent progress in delivering siRNA to the respiratory system has also improved the therapeutic feasibility of RNAi for asthma. In this review, the mechanism, as well as the general strategies and problems associated with the application of RNAi in vivo, are discussed, focusing on the possibility of applying RNAi to alleviate airway inflammation in allergic asthma. Data from studies with siRNA against molecules involved in allergic responses are summarized, and the potential and limitations of RNAi as a novel therapeutic approach are discussed.
Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H2O2, NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.
Estrogen receptor alpha (ERalpha)-positive breast cancers that co-express transcription factors GATA-3 and FOXA1 have a favorable prognosis. These transcription factors form an autoregulatory hormonal network that influences estrogen responsiveness and sensitivity to hormonal therapy. Disruption of this network may be a mechanism whereby ERalpha-positive breast cancers become resistant to therapy. The transcription factor T-bet is a negative regulator of GATA-3 in the immune system. In this study, we report that insulin increases the expression of T-bet in breast cancer cells, which correlates with reduced expression of GATA-3, FOXA1, and the ERalpha:FOXA1:GATA-3 target gene GREB-1. The effects of insulin on GATA-3 and FOXA1 could be recapitulated through overexpression of T-bet in MCF-7 cells (MCF-7-T-bet). Chromatin immunoprecipitation assays revealed reduced ERalpha binding to GREB-1 enhancer regions in MCF-7-T-bet cells and in insulin-treated MCF-7 cells. MCF-7-T-bet cells were resistant to tamoxifen in the presence of insulin and displayed prolonged extracellular signal-regulated kinase and AKT activation in response to epidermal growth factor treatment. ERalpha-positive cells with intrinsic tamoxifen resistance as well as MCF-7 cells with acquired tamoxifen and fulvestrant resistance expressed elevated levels of T-bet and/or reduced levels of FOXA1 and GATA-3. Analysis of publicly available databases revealed ERalpha-positive/T-bet-positive breast cancers expressing lower levels of FOXA1 (P = 0.0137) and GATA-3 (P = 0.0063) compared with ERalpha-positive/T-bet-negative breast cancers. Thus, T-bet expression in primary tumors and circulating insulin levels may serve as surrogate biomarkers to identify ERalpha-positive breast cancers with a dysfunctional hormonal network, enhanced growth factor signaling, and resistance to hormonal therapy.
CD4 T helper 2 (Th2) cells have critical functions in immune responses against extracellular parasites and are involved in asthma and other allergic diseases. The differentiation of naïve CD4 T cells into Th2 cells is initiated from T-cell receptor and cytokine-mediated signaling followed by upregulation of GATA3 and activation of signal transducer and activator of transcription 5 (STAT5), two indispensable events for this differentiation process. In this review, regulation of GATA3 expression and STAT5 activation and functions of these two transcription factors in inducing the expression of Th2 cytokines, cytokine receptors as well as epigenetic modification at Th2 cytokine locus are summarized. Furthermore, I present positive and negative regulatory networks important for Th2 cell commitment, selective growth of committed Th2 cells and suppression of alternative lineage fates. Finally, the difference between in vitro and in vivo Th2 differentiation is discussed.
Promoter DNA methylation of CpG islands is an important epigenetic mechanism in cancer development. We have characterized the promoter methylation profile of 82 genes in three prostate cancer cell lines (LNCaP, PC3, and DU145) and two normal prostate cell lines (RWPE1 and RWPE2). The methylation pattern was analyzed using a Panomics gene array system that consists of immobilized probes of known gene promoters on a nitrocellulose membrane. Methylation binding protein-purified methylated DNA was hybridized on the membrane and detected by the chemiluminescence method. We analyzed methylation profile in normal (RWPE1) versus cancerous cells and androgen receptor (AR)-sensitive (LNCaP) versus AR-negative cells (DU145 and PC3). Our study shows that >50% of the genes were hypermethylated in prostate cancer cells compared with 13% in normal cell lines. Among these were the tumor suppressor (RB, TMS1, DAPK, RBL1, PAX6, and FHIT), cell cycle (p27KIP1 and CDKN2A), transporters (MDR1, MLC1, and IGRP), and transcription factor (STAT1, CIITA, MYOD, and NPAT) genes. Relative methylation pattern shows that most of these genes were methylated from 5-fold to >10-fold compared with the normal prostate cells. In addition, promoter methylation was detected for the first time in target genes such as RIOK3, STAT5, CASP8, SRBC, GAGE1, and NPAT. A significant difference in methylation pattern was observed between AR-sensitive versus AR-negative cancer cells for the following genes: CASP8, GPC3, CD14, MGMT, IGRP, MDR1, CDKN2A, GATA3, and IFN. In summary, our study identified candidate genes that are methylated in prostate cancer.
The Th2 cytokine gene locus has emerged as a remarkable example of coordinated gene expression, the regulation of which seems to be rooted in an extensive array of cis-regulatory regions. Using a hypothesis-generating computational approach that integrated multispecies (n = 11) sequence comparisons with algorithm-based transcription factor binding-site predictions, we sought to identify evolutionarily conserved noncoding regions (ECRs) and motifs shared among them, which may underlie coregulation. Twenty-two transcription factor families were predicted to have binding sites in at least two Th2 ECRs. The ranking of these shared motifs according to their distribution and relative frequency pointed to a regulatory hierarchy among the transcription factor families. GATA sites were the most prevalent and widely distributed, consistent with the known role of GATA3 as a Th2 master switch. Unexpectedly, sites for ETS-domain proteins were also predicted within several Th2 ECRs and the majority of these sites were found to support Ets-1 binding in vitro and in vivo. Of note, the expression of all three Th2 cytokines (IL-5, -13, and -4) was significantly and selectively decreased in Th2 cells generated from Ets-1-deficient mice. Collectively, these data suggest that Ets-1 contributes to Th2 cytokine gene regulation by interacting with multiple cis-regulatory regions throughout the Th2 locus.
To study the mechanism of baicalin on the cytokines of Th1/Th2 in murine model of asthma.
The murine model of asthma was induced by OVA. Different doses of baicalin were orally administered to the mice respectively. The spleen cells were cultured 3 days for the measurement of IFN-gamma, IL-4, IL-5 and IL-10 by ELISA. After 2 days of culture, the spleen cells were treated with Trizol for extraction of total RNA. The gene expressions of T-bet, GATA-3 and STAT-6 were analyzed by RT-PCR.
The treatment with baicalin obviously decreased the production of IL-4 and IL-5 and the gene expression of GATA-3, STAT-6, but increased the production of IL-10.
Baicalin may modulate the Th1/Th2 balance mainly by altering the gene expressions of GATA-3 and STAT-6 in vivo and increasing the production of IL-10.
Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.
Patients with Wiskott-Aldrich syndrome (WAS) have numerous immune cell deficiencies, but it remains unclear how abnormalities in individual cell types contribute to the pathologies of WAS. In T cells, the WAS protein (WASp) regulates actin polymerization and transcription, and plays a role in the dynamics of the immunologic synapse. To examine how these events influence CD4 function, we isolated the WASp deficiency to CD4(+) T cells by adoptive transfer into wild-type mice to study T-cell priming and effector function. WAS(-/-) CD4(+) T cells mediated protective T-helper 1 (Th1) responses to Leishmania major in vivo, but were unable to support Th2 immunity to Nippostrongylus brasiliensis or L major. Mechanistically, WASp was not required for Th2 programming but was required for Th2 effector function. WAS(-/-) CD4(+) T cells up-regulated IL-4 and GATA3 mRNA and secreted IL-4 protein during Th2 differentiation. In contrast, cytokine transcription was uncoupled from protein production in WAS(-/-) Th2-primed effectors. WAS(-/-) Th2s failed to produce IL-4 protein on restimulation despite elevated IL-4/GATA3 mRNA. Moreover, dominant-negative WASp expression in WT effector T cells blocked IL-4 production, but had no effect on IFNgamma. Thus WASp plays a selective, posttranscriptional role in Th2 effector function.
Serum pro-inflammatory cytokine levels are frequently elevated in the acute phase of pediatric inflammatory bowel disease (IBD). Because the role of pro-inflammatory cytokine in the acute phase of pediatric IBD has not been well investigated, the serum levels of pro-inflammatory cytokines and the expression of Th1 and Th2 signaling molecules in mucosa from the acute phase of pediatric IBD were examined.
Twenty children with ulcerative colitis (UC; mean age, 9.95 ± 4.10 years) and 12 with Crohn's disease (CD; mean age, 10.0 ± 4.90 years) were enrolled for the serum cytokine (interleukin [IL]-4, IL-5, IL-6, tumor necrosis factor-α, tumor growth factor-β1, and interferon-γ) assay. Expression of T-helper cell 1 (Th1) (T-box expressed in T cells: T-bet and signal transducer and activator of transcription-4: STAT-4) and Th2 (GATA-3 and STAT-6) signaling molecules was examined on real-time polymerase chain reaction using mucosal samples from eight children in the acute phase of UC, eight with CD and eight controls.
Significant elevation of serum IL-4 and IL-6 levels was detected at the acute phase of pediatric UC and CD compared with levels at remission (P < 0.05 in each). The mucosal expression of GATA-3 and STAT-4 was significantly enhanced in the acute phase of pediatric UC compared with normal mucosa. No significant difference was observed in the expression of all examined molecules in the acute phase of pediatric CD.
IL-4 and its signaling molecule GATA-3, as well as the Th1 signaling molecule STAT-4, are involved in the pathogenesis of acute phase of pediatric UC.
Based on observations that mutations of GATA-3 are responsible for the HDR-syndrome (hypoparathyroidism, deafness, renal defects) and that GATA-transcription factors have an important role to play in inner ear development, we hypothesized that these transcription factors may be involved in regulatory changes of prestin transcription. To prove this, we examined in parallel the expression of mRNA of prestin and Gata-3,-2 and Gata-1 in the organ of Corti during early postnatal development of rats and in organotypic cultures. Remarkable relations are observed between prestin and Gata-3,-2 expression in organ of Corti preparations in vivo and in vitro: (i) Gata-3,-2 expression display similar apical-basal gradients as prestin mRNA levels. (ii) The prestin expression increases between postnatal day two and postnatal day eight by a factor of about four in the apical and middle segments and by a factor of two in the basal part. Highly significant Pearson correlation coefficients were observed between Gata-3,-2 mRNA and prestin levels when the data were evaluated by regression analyses. (iii) Parallel changes of prestin mRNA and Gata-3,-2 mRNA levels were observed in response to thyroid hormone and to gemfibrozil application. These observations suggest a regulatory role played by the Gata-3,-2 transcription factors in prestin expression.
Acute allograft rejection (AR) remains a major problem in solid organ transplantation. The pivotal mechanism hinges on alloantigen recognition by recipient T helper (T(h)) cells that differentiate into T(h)1 and T(h)2. This study investigated the association of mRNA levels of the transcription factors T-box expressed in T cells and GATA-binding protein 3 with the development of T(h)1/T(h)2-directed immune responses. We investigated the expression of T-bet and GATA-3 mRNA levels and the protein levels of their marker cytokines interleukin (IL)-2 and IL-4 in orthotopically transplanted rat lungs during AR. We observed a nonsignificant increase in T-bet expression following allografting at days 3 and 5 but there was a significant reduction in GATA-3 expression on day 5 compared with controls. The ratio of T-bet to GATA-3 expression showed a trend to increase at day 3 following allografting reaching significance at 5 days. These changes were associated with a significant increase in the expression of IL-2 over IL-4 on days 3 and 5. This study suggests that T(h)1 responses play a major role during AR in the rat lung, and that this differentiation can be monitored by measuring mRNA levels of T-bet and GATA-3.
Besides being mandatory in the metabolic system, adipokines like leptin directly affect immunity. Leptin was found to be necessary in T helper 1 (Th1)-dependent inflammatory processes, whereas effects on Th2 cells are rarely understood. Here, we focused on leptin in T-helper cell polarization and in Th2-mediated intestinal inflammation in vivo. The induction of cytokine-producing Th1 or Th2 cells from naive CD4(+) T cells under polarizing conditions in vitro was generally decreased in cells from leptin-deficient ob/ob mice compared with wild-type mice. To explore the in vivo relevance of leptin in Th2-mediated inflammation, the model of oxazolone-induced colitis was employed in wild-type, ob/ob, and leptin-reconstituted ob/ob mice. Ob/ob mice were protected, whereas wild-type and leptin-reconstituted ob/ob mice developed colitis. The disease severity went in parallel with local production of the Th2 cytokine IL-13. A possible explanation for the protection of ob/ob mice in Th1- as well as in Th2-dependent inflammation is provided by a decreased expression of the key transcription factors for Th1 and Th2 polarization, T-bet and GATA-3, in naive ob/ob T cells. In conclusion, these results support the regulatory function of the adipokine leptin within T-cell polarization and thus in the acquired immune system and support the concept that there is a close interaction with the endocrine system.
Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.
To evaluate the role of interleukin (IL)-18 and IL-18 receptor (IL-18R) in the predominant Th1 type cytokine response in patients with immune thrombocytopenia (ITP).
Fifteen patients with active phase ITP, eighteen in remission and thirteen healthy controls were enrolled in this study. T-bet and GATA-3 mRNA levels in peripheral blood mononucleated cells (PBMNC) were measured by reverse transcriptase polymerase chain reaction (RT-PCR); the plasma IL-18 level by enzyme linked immunosorbent assay (ELISA), the expression of IL-18R on CD3(+) lymphocytes and total lymphocytes by flow cytometry(FCM).
The T-bet mRNA levels in patients with active phase ITP was 3.572 fold as much as that in the controls (P < 0.05), while the GATA-3 mRNA levels were 0.378 fold of that in controls (P < 0.05). The levels of plasma IL-18 and IL-18R on CD3(+) lymphocytes were significantly increased in active phase ITP than in remission phase and controls. There was no difference in ratio of T-bet/GATA-3 between remitted ITP and controls and so was for T-bet mRNA, GATA-3 mRNA, plasma IL-18 and IL-18R on CD3(+) lymphocytes.
ITP as a disease of Th1-dominant response there is an unbalance between T-bet and GATA-3 in its active phase; IL-18 and IL-18R being upregulated.
We report on a Japanese girl with HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome who developed diabetes mellitus (DM) at three years of age (blood glucose 713 mg/dL, HbA(1c) 8.0%) in the absence of anti-glutamic acid decarboxylase autoantibodies. Mutation analysis revealed a de novo heterozygous two base pair deletion at exon 6 of the GATA3 gene (c.1200_1201delCA; p.H400fsX506). GATA3 expression was identified by PCR amplification for human pancreas cDNA, and mouse Gata3 was weekly but unequivocally expressed in pancreatic beta cells. The results, in conjunction with the previous findings indicating the critical role of GATA3 in lymphocyte function, GATA3 haploinsufficiency may affect the function of beta cells and/or lymphocytes, leading to the development of DM in relatively exceptional patients with high susceptibility to DM.
The Oncotype DX assay is one of the molecular tests that provide predictive and prognostic information to breast cancer patients with estrogen receptor (ER)-positive and node-negative disease. This study evaluates the association of Forkhead-box protein A1 (FOXA1) and GATA-binding protein 3 (GATA3) expressions with Oncotype DX recurrences scores in 77 cases of patients with ER-positive node-negative breast carcinomas diagnosed at Indiana University. The data were correlated with patient age, tumor size, histologic type, Scarff-Bloom-Richardson score, histologic grade, and progesterone receptor status. The median FOXA1 and GATA3 scores were 240 and 200, respectively. The Oncotype DX recurrence scores were low in 57%, intermediate in 30%, and high in 13% of cases. FOXA1 expression correlated negatively with Oncotype DX recurrence scores (P=0.004), and histologic type (P=0.0004). Oncotype DX recurrences score also correlated negatively with progesterone receptor (P=0.035) with 100% of progesterone receptor-negative cases having high or intermediate Oncotype DX scores. FOXA1 and GATA3 expressions correlated positively (P=0.014). The correlation between FOXA1 expression and Oncotype DX recurrence scores remained significant after adjusting for multiple comparisons and controlling for confounders such as histological type, grade, and progesterone receptor. A statistically significant correlation between the Oncotype DX recurrence scores and FOXA1 expression in our diverse cohort of ER-positive breast cancer patients was observed. We propose that this may represent a more cost-effective strategy to further risk stratify patients with good prognosis in whom chemotherapy may be omitted. To confirm these findings, further studies in a larger cohort of patients are warranted.
IL-23/IL-17 axis is an important regulator in various inflammatory diseases. However, the role of IL-23 in allergic airway inflammation is not well understood. In this study, we show that in an allergen-induced asthma model, mice with transgenic overexpression of IL-23R exhibited increased airway infiltration of eosinophils and Th2 cytokine production, whereas those deficient in IL-23 displayed reduced airway inflammation. In vitro, IL-23-IL-23R signaling promoted GATA-3 expression and enhanced Th2 cytokine expression. Conversely, in the absence of this signal, Th2 cell differentiation was partially inhibited. Therefore, IL-23 signaling may regulate allergic asthma through modulation of Th2 cell differentiation.
Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors.
Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4(+) helper T cells during Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcription factor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN-gamma and IL-4 are also regulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well as Th1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3 in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4(+) cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factor that influences the manifestation of CD4(+) cells as the Th1 or Th2 phenotypes.
Haploinsufficiency for the transcription factor GATA3 leads to hearing loss in humans. It is expressed throughout the auditory sensory epithelium (SE). In the vestibular organs, GATA3 is limited to the striola reversal zone of the utricle. Stereocilia orientation shifts 180 degrees at this region, which contains morphologically distinct type-I hair cells. The striola is conserved in all amniotes, its function is unknown, and GATA3 is the only known marker of the reversal zone. To identify downstream targets of GATA3 that might point to striolar function, we measured gene expression differences between striolar and extra-striolar SE. These were compared with profiles after GATA3 RNAi and GATA3 over-expression. We identified four genes (BMP2, FKHL18, LMO4, and MBNL2) that consistently varied with GATA3. Two of these (LMO4 and MBNL2) were shown to be direct targets of GATA3 by ChIP. Our results suggest that GATA3 impacts WNT signaling in this region of the sensory macula.
Rejection of transplanted liver occurs when the host generates alloantigen-reactive T cells, and CD4(+) T-cell subsets, including Th1, Th2, T17 and iTreg, could be involved in changing the dynamics of graft rejection onset. In the current immunosuppressive strategies, rejection is treated as an undifferentiated process that is uniform, which results in the failure of tolerance induction. Here, we established a rejection model to observe the reciprocal interaction of CD4(+) T-cell subsets in the complex networks of the immune system.
Orthotopic liver transplantation (OLT) from male inbred Lewis rats (n=15) to male inbred Brown Norway (BN) rats was performed by Kamada's two-cuff technique without reconstruction of the hepatic artery. OLT from BN to BN rats (n=5) was performed as a control. Recipients were sacrificed on postoperative days 3, 5, 7, 9, 11, 13 and 15. Recipient spleens and grafts were harvested, fixed in 10% neutral formalin, and embedded in paraffin. Meanwhile, hematoxylin and eosin and immunohistochemical staining was done, acute rejection was graded by the Banff scheme, and the number of T-bet(+), GATA-3(+), RORgammat(+) and FOXP3(+) cells in the spleen and grafts were counted.
In recipient spleens, the T-bet(+) and RORgammat(+) cells were increased more significantly in the mild acute rejection (AR) group than in the control group (P=0.016, P=0.009, respectively), while both cell types were decreased in the moderate AR group. Compared with the control group, the RORgammat(+) cells did not differ significantly in the severe AR group, while the T-bet(+) cells were significantly decreased (P=0.465, P=0.009, respectively). The GATA-3(+) cells were significantly decreased in the mild AR group compared with the control group (P=0.009). With regard to the FOXP3(+) cells, there was no significant difference between the control and mild AR groups (P=0.754), while they were significantly decreased in the moderate and severe AR groups (P=0.028, P=0.009, respectively). The ratio of T-bet(+)/GATA-3(+) cells was more associated with AR than was the RORgammat(+)/FOXP3(+) cell ratio in the early stage. In the graft, the T-bet(+) and RORgammat(+) cells were significantly increased in mild, moderate and severe AR groups compared with the control group (P<0.009). The expression of GATA-3(+) cell was not significantly increased in the mild AR group compared with the control group, while it was significantly increased in the moderate and severe AR groups (P=0.028, P=0.009, respectively). Concerning the FOXP3(+) cells, no significant difference was found between the control and mild AR groups (P=0.347), while they were significantly increased in the moderate and severe AR groups (P<0.009).
Our study revealed the dynamic changes of T-bet(+), GATA-3(+), RORgammat(+) and FOXP3(+) cells in the spleen and grafts of recipient rats. It seems that the ratio of T-bet(+)/GATA-3(+) cells was more associated with AR than was RORgammat(+)/FOXP3(+) cells in the early stage of rejection, while the ratio of RORgammat(+)/FOXP3(+) cells was associated more with the later but more persistent stage of rejection. This study could make a contribution to the optimal selection of immunosuppressive regimens according to the dynamic changes in CD4(+) T-cell subsets at different stages of rejection.
During development, compartmentalization of an early embryonic structure produces blocks of cells with distinct properties and developmental potentials. The auditory and vestibular components of vertebrate inner ears are derived from defined compartments within the otocyst during embryogenesis. The vestibular apparatus, including three semicircular canals, saccule, utricle, and their associated sensory organs, detects angular and linear acceleration of the head and relays the information through vestibular neurons to vestibular nuclei in the brainstem. How the early developmental events manifest vestibular structures at the molecular level is largely unknown. Here, we show that LMO4, a LIM-domain-only transcriptional regulator, is required for the formation of semicircular canals and their associated sensory cristae. Targeted disruption of Lmo4 resulted in the dysmorphogenesis of the vestibule and in the absence of three semicircular canals, anterior and posterior cristae. In Lmo4-null otocysts, canal outpouches failed to form and cell proliferation was reduced in the dorsolateral region. Expression analysis of the known otic markers showed that Lmo4 is essential for the normal expression of Bmp4, Fgf10, Msx1, Isl1, Gata3, and Dlx5 in the dorsolateral domain of the otocyst, whereas the initial compartmentalization of the otocyst remains unaffected. Our results demonstrate that Lmo4 controls the development of the dorsolateral otocyst into semicircular canals and cristae through two distinct mechanisms: regulating the expression of otic specific genes and stimulating the proliferation of the dorsolateral part of the otocyst.
Extensive studies have demonstrated that Th1 type immunity is predominant in pre-eclampsia, but there is little concern with regard to the intracellular mechanisms behind this initial T-cell polarization. In this study, we investigated whether the imbalance of the T-cell transcription factors contributes to it.
A total of 15 pre-eclamptic patients and 15 healthy pregnant women were enrolled in this study. The expression levels of transcription factors for Th1 (T-bet), Th2 (GATA3), Th17 (RORc) and Treg (FOXP3) cells, together with the Th1/Th2 status, were simultaneously investigated in both peripheral blood mononuclear cells (PBMCs) and decidua.
The expression levels of FOXP3 mRNA were decreased in both PBMCs and decidua from pre-eclamptic patients compared with healthy pregnant women (P < 0.05), and T-bet mRNA and RORc mRNA were significantly increased (P < 0.05), while Th1/Th2 balance shifted toward the Th1 immunity. Furthermore, there was a negative correlation between FOXP3 mRNA and Th1 cells (P < 0.05), and the expression level of T-bet mRNA correlated strongly with Th1 cells (P < 0.05).
Decreased expression of FOXP3 mRNA and increased expression of T-bet mRNA may contribute to Th1 type immunity predominant in pre-eclampsia.
An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life ('hygiene hypothesis'). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection.
The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their T(Helper) cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments.
It was shown that A. lwoffii F78 induced a T(H)1-polarizing program in human dendritic cells which led to T(H)1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects.
We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a T(H)1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects.
Periodontitis is a widespread, complex inflammatory disease of the mouth, which results in a loss of gingival tissue and alveolar bone, with aggressive periodontitis (AgP) as its most severe form. To identify genetic risk factors for periodontitis, we conducted a genome-wide association study in German AgP patients. We found AgP to be strongly associated with the intronic SNP rs1537415, which is located in the glycosyltransferase gene GLT6D1. We replicated the association in a panel of Dutch generalized and localized AgP patients. In the combined analysis including 1758 subjects, rs1537415 reached a genome-wide significance level of P= 5.51 x 10(-9), OR = 1.59 (95% CI 1.36-1.86). The associated rare G allele of rs1537415 showed an enrichment of 10% in periodontitis cases (48.4% in comparison with 38.8% in controls). Fine-mapping and a haplotype analysis indicated that rs1537415 showed the strongest association signal. Sequencing identified no further associated variant. Tissue-specific expression analysis of GLT6D1 indicated high transcript levels in the leukocytes, the gingiva and testis. Analysis of potential transcription factor binding sites at this locus predicted a significant reduction of GATA-3 binding affinity, and an electrophoretic mobility assay indicated a T cell specific reduction of protein binding for the G allele. Overexpression of GATA-3 in HEK293 cells resulted in allele-specific binding of GATA-3, indicating the identity of GATA-3 as the binding protein. The identified association of GLT6D1 with AgP implicates this locus as an important susceptibility factor, and GATA-3 as a potential signaling component in the pathophysiology of periodontitis.
How naive CD4(+) T cells commit to the T helper type 2 (T(H)2) lineage is poorly understood. Here we show that the basic helix-loop-helix transcription factor Dec2 was selectively expressed in T(H)2 cells. CD4(+) T cells from Dec2-deficient mice showed defective T(H)2 differentiation in vitro and in vivo in an asthma model and in response to challenge with a parasite antigen. Dec2 promoted expression of interleukin 4 (IL-4), IL-5 and IL-13 during early T(H)2 differentiation and directly bound to and activated transcription of genes encoding the transcription factors JunB and GATA-3. As GATA-3 induces Dec2 expression, our findings also indicate a feed-forward regulatory circuit during T(H)2 differentiation.
In the adult mouse, distinct morphological and transcriptional differences separate stomach from intestinal epithelium. Remarkably, the epithelial boundary between these two organs is literally one cell thick. This discrete junction is established suddenly and precisely at embryonic day (E) 16.5, by sharpening a previously diffuse intermediate zone. In the present study, we define the dynamic transcriptome of stomach, pylorus, and intestinal tissues between E14.5 and E16.5. We show that establishment of this boundary is concomitant with the induction of over a thousand genes in intestinal epithelium, and these gene products provide intestinal character. Hence, we call this process intestinalization. We identify specific transcription factors (Hnf4 gamma, Creb3l3, and Tcfec) and examine signaling pathways (Hedgehog and Wnt) that may play a role in this process. Finally, we define a unique expression domain at the pylorus itself and detect novel pylorus-specific patterns for the transcription factor Gata3 and the secreted protein nephrocan.
To investigate the role of transcription factors T-bet and GATA-3 in the pathogenesis of systemic lupus erythematosus (SLE).
The expression of T-bet and GATA-3 mRNA in the peripheral blood mononuclear cells (PBMCs) of 60 patients with SLE and 20 normal control subjects were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).
Compared with the normal controls, T-bet mRNA expression decreased whereas GATA-3 mRNA expression increased significantly in patients with SLE, active SLE and inactive SLE.
The high expression of GATA-3 promotes the immature T cells to differentiate into Th2 cells, induces Th2 cells to secrete the cytokines IL-6 and IL-10, which, along with the low expression of Th1 as a result of T-bet expression inhibition, causes B cell activation to produce a large quantity of autoantibodies, leading finally to multiple organ injuries. Effective regulation of GATA-3 expression may provide a new clue for the treatment of SLE.
Our previous studies illustrated that berberine inhibited adipogenesis in murine-derived 3T3-L1 preadipocytes and human white preadipocytes. In this study, the effects of berberine on the adipogenesis of high-fat diet-induced obesity (FD) or normal diet (ND) mice and possible transcriptional impact are investigated. The results demonstrated that in FD mice, berberine reduced mouse weight gain and food intake and serum glucose, triglyceride, and total cholesterol levels accompanied with a down-regulation of PPARgamma expression and an up-regulation of GATA-3 expression. Berberine had no adverse effects on ND mice. These encouraging findings suggest that berberine has excellent pharmacological potential to prevent obesity.
SAMP1/Yit mice develop spontaneous, segmental, transmural ileitis recapitulating many features of Crohn's disease (CD). The ileitic phenotype may have arisen during crosses of SAMP1 mice selected for the presence of skin lesions. We hereby describe that the original SAMP1 strain similarly develops ileitis. Our aim was to characterize the histopathological and immunological features of this model and assess its responsiveness to standard inflammatory bowel disease (IBD) therapy.
The time course of histopathological features of ileitis was assessed. Immune compartments were characterized by flow cytometry. Ileal cytokine profiles and transcription factors were determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). Finally, response to corticosteroid therapy and its effect on immune compartments and cellularity was evaluated.
Histological features and time course of disease were conserved, compared to those reported in SAMP1/Yit strains, with similar expansion of CD19+, CD4+, and CD8+ effector (CD44(high) CD62L(low)), and central memory lymphocytes (CD44(high)CD62L(high)). However, different from SAMP1/YitFc mice, analysis of ileal cytokine profiles revealed initial T(H)1 polarization followed by T(H)2-polarized profile accompanied by prominent eosinophilia during late disease. Lastly, corticosteroids attenuated ileitis, resulting in decreased lymphocyte subsets and cellularity of compartments.
Here we report that the ileitic phenotype of SAMP1-related strains was already present in the original SAMP1 strain. By contrast, the cytokine profile within the terminal ilea of SAMP1 is distinct from the mixed T(H)1/T(H)2 profile of SAMP1/YitFc mice during late disease, as it shows predominant T(H)2 polarization. Dissemination of these strains may advance our understanding of CD pathogenesis, which in 60% of patients involves the terminal ileum.
The identification of the organisation and dynamics of molecular pathways is crucial for the understanding of cell function. In order to reconstruct the molecular pathways in which a gene of interest is involved in regulating a cell, it is important to identify the set of genes to which it interacts with to determine cell function. In this context, the mining and the integration of a large amount of publicly available data, regarding the transcriptome and the proteome states of a cell, are a useful resource to complement biological research.
We describe an approach for the identification of genes that interact with each other to regulate cell function. The strategy relies on the analysis of gene expression profile similarity, considering large datasets of expression data. During the similarity evaluation, the methodology determines the most significant subset of samples in which the evaluated genes are highly correlated. Hence, the strategy enables the exclusion of samples that are not relevant for each gene pair analysed. This feature is important when considering a large set of samples characterised by heterogeneous experimental conditions where different pools of biological processes can be active across the samples. The putative partners of the studied gene are then further characterised, analysing the distribution of the Gene Ontology terms and integrating the protein-protein interaction (PPI) data. The strategy was applied for the analysis of the functional relationships of a gene of known function, Pyruvate Kinase, and for the prediction of functional partners of the human transcription factor TBX3. In both cases the analysis was done on a dataset composed by breast primary tumour expression data derived from the literature. Integration and analysis of PPI data confirmed the prediction of the methodology, since the genes identified to be functionally related were associated to proteins close in the PPI network. Two genes among the predicted putative partners of TBX3 (GLI3 and GATA3) were confirmed by in vivo binding assays (crosslinking immunoprecipitation, X-ChIP) in which the putative DNA enhancer sequence sites of GATA3 and GLI3 were found to be bound by the Tbx3 protein.
The presented strategy is demonstrated to be an effective approach to identify genes that establish functional relationships. The methodology identifies and characterises genes with a similar expression profile, through data mining and integrating data from publicly available resources, to contribute to a better understanding of gene regulation and cell function. The prediction of the TBX3 target genes GLI3 and GATA3 was experimentally confirmed.
The alpha7 nicotinic acetylcholine receptor (nAChR) was recently described as an anti-inflammatory target in both macrophages and T cells. Its expression by immune cells may explain the epidemiological data claiming a negative link between cigarette smoking and several inflammatory diseases. In this study, we determined the immunological effects of alpha7 nAChR activation by nicotine. Our results indicate that the alpha7 nAChR is expressed on the surface of CD4(+) T cells and that this expression is up-regulated upon immune activation. Nicotine reduced T cell proliferation in response to an encephalitogenic Ag, as well as the production of Th1 (TNF-alpha and IFN-gamma) and Th17 cytokines (IL-17, IL-17F, IL-21, and IL-22). IL-4 production was increased in the same setting. Attenuation of the Th1 and Th17 lineages was accompanied by reduced T-bet (50%) and increased GATA-3 (350%) expression. Overall, nicotine induced a shift to the Th2 lineage. However, alpha7(-/-)-derived T cells were unaffected by nicotine. Furthermore, nicotine reduced NF-kappaB-mediated transcription as measured by IL-2 and IkappaB transcription. In vivo, administration of nicotine (2 mg/kg s.c.) suppressed the severity of CD4(+) T cell-mediated disease experimental autoimmune encephalomyelitis. alpha7(-/-) mice were refractory to nicotine treatment, although disease severity in those animals was reduced, due to impairment in Ag presentation. Accordingly, CD4(+) and CD11b(+) cells infiltration into the CNS, demyelination, and axonal loss were reduced. Our data implicate a role for the alpha7 nAChR in immune modulation and suggest that alpha7 nAChR agonists may be effective in the treatment of inflammatory disorders.
Allergic asthma is an inflammatory disease of the lung characterized by abnormal T helper-2 (T(H)2) lymphocyte responses to inhaled antigens. The molecular mechanisms leading to the generation of T(H)2 responses remain unclear, although toll-like receptors (TLRs) present on innate immune cells play a pivotal role in sensing molecular patterns and in programming adaptive T cell responses. Here we show that in vivo activation of TLR4 by house dust mite antigens leads to the induction of allergic disease, a process that is associated with expression of a unique subset of small, noncoding microRNAs. Selective blockade of microRNA (miR)-126 suppressed the asthmatic phenotype, resulting in diminished T(H)2 responses, inflammation, airways hyperresponsiveness, eosinophil recruitment, and mucus hypersecretion. miR-126 blockade resulted in augmented expression of POU domain class 2 associating factor 1, which activates the transcription factor PU.1 that alters T(H)2 cell function via negative regulation of GATA3 expression. In summary, this study presents a functional connection between miRNA expression and asthma pathogenesis, and our data suggest that targeting miRNA in the airways may lead to anti-inflammatory treatments for allergic asthma.
FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, suppresses immune responses by inhibiting T-cell migration into target tissues; however, it does not alter T-cell functions. In this study, we investigated the biological effects of FTY720 on NKT cells. Unlike T cells, FTY720 suppressed the production of IL-4, IFN-gamma, IL-10, and IL-13 by NKT cells through the S1P1 receptor (S1P(1)). Moreover, FTY720 also inhibited the expression of T-bet and GATA-3 of NKT cells in the presence of TCR engagement. However, it did not inhibit NKT cell migration in vitro or in vivo. In a K/BxN serum transfer arthritis model, FTY720 suppressed arthritis in B6, but not in CD1d(-/-) mice. Moreover, the adoptive transfer of control NKT cells restored arthritis in CD1d(-/-) mice, whereas FTY720-pretreated NKT cells did not. The number of NKT cells in the joints of B6 mice given FTY720 was similar to that in the joints of untreated B6 mice, whereas the production of IL-4 and IFN-gamma was reduced in the FTY720-treated B6 mice. Taken together, these data show that FTY720 suppresses cytokine production in NKT cells through S1P(1), but not NKT cell migration. Thus, FTY720 may be useful in the treatment of NKT cell-promoted immune diseases.
Type I diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells, and effective treatment of the disease might require rescuing beta cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional beta cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-beta-dependent emergence of autoantigen-specific regulatory T cells and recovery of beta cell function in the diabetic recipients.
Foxp3(+) regulatory T cells (Tregs) are crucial for preventing autoimmunity. We have demonstrated that depletion of Foxp3(+) Tregs results in the development of a scurfy-like disease, indicating that Foxp3(-) effector T cells are sufficient to induce autoimmunity. It has been postulated that nonfunctional Tregs carrying potentially self-reactive T cell receptors may contribute to scurfy (sf) pathogenesis due to enhanced recognition of self. Those cells, however, could not be identified in sf mutants due to the lack of Foxp3 protein expression. To address this issue, we crossed the natural sf mouse mutant with bacterial artificial chromosome transgenic DEREG (depletion of regulatory T cells) mice. Since DEREG mice express GFP under the control of an additional Foxp3 promoter, those crossings allowed proving the existence of "would-be" Tregs, which are characterized by GFP expression in the absence of functional Foxp3. Sf Tregs lost their in vitro suppressive capacity. This correlated with a substantial reduction of intracellular cAMP levels, whereas surface expression of Treg markers was unaffected. Both GFP(+) and GFP(-) sf cells produced high amounts of Th2-type cytokines, reflected also by enhanced Gata-3 expression, when tested in vitro. Nevertheless, sf Tregs could be induced in vitro, although with lower efficiency than DEREG Tregs. Transfer of GFP(+) sf Tregs, in contrast to GFP(-) sf T cells, into RAG1-deficient animals did not cause the sf phenotype. Taken together, natural and induced Tregs develop in the absence of Foxp3 in sf mice, which lack both suppressive activity and autoreactive potential, but rather display a Th2-biased phenotype.
Interleukin (IL)-17-producing T helper (Th17) cells play a critical role in the pathophysiology of several autoimmune disorders. The differentiation of Th17 cells requires the simultaneous presence of an unusual combination of cytokines: IL-6, a proinflammatory cytokine, and transforming growth factor (TGF) beta, an antiinflammatory cytokine. However, the molecular mechanisms by which TGF-beta exerts its effects on Th17 cell differentiation remain elusive. We report that TGF-beta does not directly promote Th17 cell differentiation but instead acts indirectly by blocking expression of the transcription factors signal transducer and activator of transcription (STAT) 4 and GATA-3, thus preventing Th1 and Th2 cell differentiation. In contrast, TGF-beta had no effect on the expression of retinoic acid receptor-related orphan nuclear receptor gammat, a Th17-specific transcription factor. Interestingly, in Stat-6(-/-)T-bet(-/-) mice, which are unable to generate Th1 and Th2 cells, IL-6 alone was sufficient to induce robust differentiation of Th17 cells, whereas TGF-beta had no effect, suggesting that TGF-beta is dispensable for Th17 cell development. Consequently, BALB/c Stat-6(-/-)T-bet(-/-) mice, but not wild-type BALB/c mice, were highly susceptible to the development of experimental autoimmune encephalomyelitis, which could be blocked by anti-IL-17 antibodies but not by anti-TGF-beta antibodies. Collectively, these data provide evidence that TGF-beta is not directly required for the molecular orchestration of Th17 cell differentiation.
Upon detection of antigen, CD4(+) T helper (Th) cells can differentiate into a number of effector types that tailor the immune response to different pathogens. Alternative Th1 and Th2 cell fates are specified by the transcription factors T-bet and GATA-3, respectively. Only a handful of target genes are known for these two factors and because of this, the mechanism through which T-bet and GATA-3 induce differentiation toward alternative cell fates is not fully understood. Here, we provide a genomic map of T-bet and GATA-3 binding in primary human T cells and identify their target genes, most of which are previously unknown. In Th1 cells, T-bet associates with genes of diverse function, including those with roles in transcriptional regulation, chemotaxis and adhesion. GATA-3 occupies genes in both Th1 and Th2 cells and, unexpectedly, shares a large proportion of targets with T-bet. Re-complementation of T-bet alters the expression of these genes in a manner that mirrors their differential expression between Th1 and Th2 lineages. These data show that the choice between Th1 and Th2 lineage commitment is the result of the opposing action of T-bet and GATA-3 at a shared set of target genes and may provide a general paradigm for the interaction of lineage-specifying transcription factors.
The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1(lck-/-)). Although deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1(lck-/-) T cells have a lower activation threshold than wild-type T cells. When TCR engagement is limiting, Sfpi1(lck-/-) T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild-type cells. We show that PU.1 modulates the levels of TCR expression in CD4(+) T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3-dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4(+) T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold, and increased homogeneity in Th2 populations.
It has been reported that the botanical alkaloids, berberine and evodiamine inhibit mouse preadipocyte 3T3-L1 differentiation. The aim of this study was to investigate the effect and transcriptional impact of berberine and evodiamine individually and in combination on human white preadipocyte (HWP) differentiation. We have shown that treatment with 8 microM berberine or 4 microM evodiamine resulted in a major inhibition of HWP differentiation accompanied by up-regulation of both GATA binding protein 2 and 3 (GATA-2 and GATA-3) mRNA and protein expression, suggesting that both compounds may have excellent potential as agents to prevent obesity.
The present study was conducted on CD4(+) T cells, isolated from wild type (WT) and PPARalpha(null) mice, in order to assess the mechanism of action of docosahexaenoic acid (DHA), an n-3 fatty acid, in the modulation of two transcription factors, i.e., T-bet and GATA-3, implicated in T-cell differentiation towards, respectively, T(H)1 and T(H)2 phenotype. The T-cells from PPARalpha(null) mice secreted higher IFN-gamma and lower IL-4 concentrations than WT T-cells. Furthermore, the deletion of PPARalpha gene in T-cells resulted in the upregulation of T-bet and downregulation of GATA-3 both at mRNA and protein levels. DHA exerted not only an inhibitory effect on T-cell proliferation, but also diminished IFN-gamma and stimulated IL-4 secretions in both cell types. DHA also downregulated T-bet and upregulated GATA-3 both at transcription and protein levels. Though the T-cells from PPARalpha(null) mice expressed higher p38 phosphorylation than WT T-cells, DHA diminished the MAP kinase phosphorylation (p38 and ERK1/2) in both the cell types. The pharmacological inhibitors of MAP kinases also downregulated T-bet and upregulated GATA-3 in T-cells. Altogether, these results demonstrate that DHA, via its action on MAP kinases, modulates the expression of transcription factors. These results also explain the mechanism of action of this fatty acid on T-cell differentiation in disease and health.
There is increasing evidence that the numerous mechanisms that regulate cell differentiation during normal development are also involved in tumorigenesis. In breast cancer, differentiation markers expressed by the primary tumor are routinely profiled to guide clinical decisions. Indeed, numerous studies have shown that the differentiation profile correlates with the metastatic potential of tumors. The transcription factor GATA3 has emerged recently as a strong predictor of clinical outcome in human luminal breast cancer. In the mammary gland, GATA3 is required for luminal epithelial cell differentiation and commitment, and its expression is progressively lost during luminal breast cancer progression as cancer cells acquire a stem cell-like phenotype. Importantly, expression of GATA3 in GATA3-negative, undifferentiated breast carcinoma cells is sufficient to induce tumor differentiation and inhibits tumor dissemination in a mouse model. These findings demonstrate the exquisite ability of a differentiation factor to affect malignant properties, and raise the possibility that GATA3 or its downstream genes could be used in treating luminal breast cancer. This review highlights our recent understanding of GATA3 in both normal mammary development and tumor differentiation.
Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4(+) T cell immunity, which correlates with the presence of a predominant GATA-3(+) tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4(+) T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.
We first evaluated the influence of IL-12 and IL-27, as single agents and in association, on the polarization of CEA-specific Th2 CD4(+) T cell clones from a pancreatic cancer patient. We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal. Second, we evaluated the effect of the combined treatment on polyclonal CEA-specific CD4(+) T cells in short-time re-stimulation assays. In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity.
Collectively, our results demonstrate that tumor antigen specific Th2 CD4(+) T cells in pancreatic cancer are endowed with functional plasticity. Hence, loco-regional cytokines delivery or targeted therapy based on antibodies or molecules directed to the tumor stroma might improve anti-tumor immunity and ameliorate fibrosis, without systemic toxicity.
A member of winged-helix/forkhead transcription factors, Foxp1, is expressed in the developing spinal cord during mouse embryogenesis. To shed light on the potential role of Foxp1 in neurons of the developing spinal cord, we investigated the detailed expression pattern of Foxp1 between embryonic day (E) 9.5 and E17.5. At E10.25, some postmitotic neurons with strong expression of Foxp1 (Foxp1(high)) were first detected in the ventral half of the brachial spinal cord. By E11.5, Foxp1(high) neurons increased in the ventral spinal cord at the limb levels. All of Foxp1(high) neurons at the limb levels were Islet2(+)/Lhx3(-) motor neurons (MNs) of the lateral motor column and some neurons that expressed Foxp1 weakly (Foxp1(low)) at the thoracic level were MNs of the preganglionic motor column. Between E12.5 and E17.5, Foxp1(low) neurons were also observed in the intermediate zone throughout the ventral spinal cord, all of which were Pax2(+), En1(+), Evx1(-), Chx10(-), Gata3(-), and Lhx3(-) V1 interneurons. Interestingly, no colocalization of Foxp1 with Lhx3 was observed in the developing spinal cord. In addition, overexpression of Foxp1 markedly attenuated the endogenous expression of Lhx3 in a neuroendocrine cell line. Chromatin immunoprecipitation assays in a neuronal cell line and E13.5 spinal cords revealed an interaction between Foxp1 and the consensus motif in the Lhx3 promoter. These results suggest that Foxp1 may play some important roles in the determination of neuronal fates of the ventral spinal cord, possibly through the suppression of Lhx3 expression.
To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice.
A number of methods have been established for identifying sentinel nodes (SNs). In the present study, we attempted to clarify the immunological status of SNs with or without micrometastasis in breast cancer patients. SNs were identified by the dye- and gamma probe-guided method. Total RNA was extracted from the SNs, and the expression of T-BET, GATA-3, and FOXP3 were evaluated using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Micrometastasis was identified as microscopically negative but positive by RT-PCR specific for mammaglobin. Of 88 patients, 17 (19.3%) showed positive metastasis in SNs (pN1, 14; pN2, 3). Of the 71 metastasis-negative SNs, 11 showed positive bands on RT-PCR specific for mamma-globin [pN0(mol+)]. There was no significant correlation among clinicopathological features with or without micrometastasis. Immunological parameters were compared among the 60 pN0, 11 pN0(mol+), and 17 pN1-2. Although T-BET expression was higher in pN0(mol+) than pN0, FOXP3 expression was also higher in pN0(mol+) than pN0. In pN1-2, T-BET expression decreased compared with pN0(mol+), but FOXP3 expression did not. On the other hand, GATA-3 expression inversely increased in pN1-2 compared with pN0(mol+). In patients with breast cancer, micrometastasis can stimulate Th1 response in SNs. However, the Treg cell response is also induced at the micrometastasis level and persists during the progression of metastasis in SNs. Then, the shift in the Th1/Th2 balance may preferentially lean toward Th2 responses in pN1-2 SNs and suppress antitumor immune responses. Micrometastasis [pN0(mol+)] is a status immunologically distinguishable from pN0 and pN1-2.
We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKBbeta, a dramatic increase in Akt1/PKBalpha protein and relocation of Akt1/PKBalpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome.
To observe the therapeutic effect of RelB-silenced dendritic cells (DCs) in experimental autoimmune myasthenia gravis (EAMG), and further to investigate the mechanism of this immune system therapeutic.
(1) RelB-silenced DCs and control DCs were prepared and the supernatants were collected for IL-12p70, IL-6, and IL-23 measurement by ELISA. (2) RelB-silenced DCs and control DCs were co-cultured with AChR-specific T cells, and the supernatant was collected to observe the IL-17, IFN-gamma, IL-4 production. (3) EAMG mice with clinical scores of 1 to 2 were collected and enrolled randomly into the RelB-silenced DC group or the control DC group. RelB-silenced DCs or an equal amount of control DCs were injected intravenously on days 0, 7, and 14 after enrollment. Clinical scores were evaluated every other day. Twenty days after allotment, serum from individual mice was collected to detect serum concentrations of anti-mouse AChR IgG, IgG1, IgG2b, and IgG2c. The splenocytes were isolated for analysis of lymphocyte proliferative responses, cytokine (IL-17, IFN-gamma, IL-4) production, and protein levels of RORgammat, T-bet, GATA-3, and FoxP3 (the special transcription factors of Th17, Th1, Th2, and Treg, respectively).
(1) RelB-silenced DCs produced significantly reduced amounts of IL-12p70, IL-6, and IL-23, as compared with control DCs. (2) RelB-silenced DCs spurred on the CD4(+) T cells from Th1/Th17 to the Th2 cell subset in the co-culture system. (3) Treatment with RelB-silenced DCs effectively reduced myasthenic symptoms and levels of serum anti-acetylcholine receptor autoantibody in mice with ongoing EAMG. Th17-related markers (RORgammat, IL-17) and Th1-related markers (T-bet, IFN-gamma) were downregulated, whereas Th2 markers (IL-4 and GATA3) and Treg marker (FoxP3) were upregulated.
RelB-silenced DCs were able to create a particular cytokine environment that was absent of inflammatory cytokines. RelB-silenced DCs provide a practical means to normalize the differentiation of the four T-cell subsets (Th17, Th1, Th2, and Treg) in vivo, and thus possess therapeutic potential in Th1/Th17-dominant autoimmune disorders such as myasthenia gravis.
Cytohesin binder and regulator (Cybr) is known to regulate leukocyte adhesion and migration. However, its function in T-cells is poorly understood. Here, we investigated the role of Cybr in CD4(+) T-cell function and host immunity. Cybr inhibited p38 phosphorylation following CD4(+) T-cell stimulation. Since p38 regulates the expression of T-box expressed in T-cells (T-bet) but not GATA binding protein 3 (GATA-3) in T-cells, Cybr decreased the expression of T-bet and IFN-gamma in CD4(+) T-cells. Moreover, we found that host immunity against Listeria infection and IFN-gamma production in blood were significantly compromised in Cybr-overexpressing transgenic mice. In summary, our data suggest that Cybr represses the expression of T-bet and IFN-gamma via an inhibition of p38 in T-cells and consequently reduces host resistance to bacterial infection in mice.
The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown.
The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knock-down was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide microarrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGFbeta signalling and stress response.
Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation.
The zinc-finger transcription factors Gata3 and ThPOK have both been implicated in positive selection of double positive (DP) thymocytes towards the CD4 lineage. As in the absence of Gata3, expression of ThPOK is lacking, Gata3 may directly regulate ThPOK expression. As ThPOK failed to promote CD4(+) lineage differentiation of Gata3-deficient cells, ThPOK cannot be the only Gata3 target gene essential for the induction of the CD4(+) lineage program. Therefore, it is conceivable that Gata3 is essential for selected DP T cells to reach the developmental stage at which ThPOK expression is induced. Here, we show that Gata3 overexpression does not affect ThPOK expression levels in DP or CD4(+) thymocytes, providing evidence that Gata3 does not directly regulate ThPOK. To identify additional target genes that clarify Gata3 function at the DP thymocyte stage, we performed gene expression profiling assays in wild-type mice and transgenice mice with enforced expression of Gata3, in the presence or absence of the MHC class II-restricted DO11.10 TCR. We found that Gata3 expression in DP cells undergoing positive selection was associated with downregulation of the V(D)J-recombination machinery genes Rag1, Rag2 and TdT. Moreover, Gata3 overexpression was associated with downregulation of many signaling molecules and the induction of modulators of TCR signaling, including Ctla-4 and thrombospondin 2. Together with our previous finding that Gata3 reduces expression of CD5, a negative regulator of TCR signaling, and upregulates TCR expression, these findings indicate that Gata3 in DP cells mainly functions to (i) terminate TCRalpha gene rearrangement, and (ii) regulate TCR signal intensity or duration in cells undergoing positive selection towards the CD4 lineage.
Meta-analyses of microarray data indicate that GATA3 is co-expressed with estrogen receptor alpha (ER) in breast cancer cells. While the significance of this remains unclear, it is thought that GATA3 may serve as a prognostic indicator in breast tumors and may play a role in ER signaling. Recently, reciprocal regulation of GATA3 and ER transcription was demonstrated, suggesting that control of their expression is intertwined. We sought to determine whether GATA3 and ER expression was also coordinately regulated at other levels. Unlike ER, GATA3 was not under epigenetic control and was not re-expressed in the presence of DNMT or HDAC inhibitors in ER/GATA3-negative cells. However, like ER, these inhibitors decreased GATA3 expression in ER/GATA3-positive cell lines. We have previously reported that ER mRNA stability is increased through binding of the RNA-binding protein HuR/ELAV1 to the 3'untranslated region (UTR) and that DNMT and HDAC inhibitors reduce ER expression by altering this interaction. Biotin pull-down assays using a biotinylated GATA3 RNA probe confirmed that HuR also binds to the GATA3 3'UTR. Inhibition of HuR using siRNA probes decreased GATA3 mRNA, mRNA stability and protein expression, indicating that HuR plays a role in regulating GATA3 expression. Inhibition of either HuR or GATA3 reduced cell growth of MCF7 cells. Based on our findings, it is clear that coordinate regulation of ER and GATA3 occurs, however differences do exist. These findings may aid in identification of new targets that control cell growth of breast cancer cells.
The hypoparathyroidism, deafness, renal dysplasia (HDR) syndrome is caused by mutations in the gene encoding GATA3, which belongs to a family of dual zinc-finger transcription factors that have a role in vertebrate embryonic development.
The aim of the study was to identify the GATA3 mutation in a HDR patient and determine its functional consequences. PATIENT AND DESIGN: A patient with HDR was studied after approval from the local ethical committee. Leukocyte DNA was used with GATA3-specific primers for PCR amplification, and the DNA sequences of the PCR products were determined. Wild-type and mutant GATA3 constructs were transfected into COS-7 cell, and their functions were assessed by Western blot analysis, immunocytochemistry, EMSAs, luciferase reporter assays, and three-dimensional modeling.
A novel missense mutation, Thr272Ile, in zinc finger 1 (ZnF1) of GATA3 was identified. Western blot analysis and immunofluorescence revealed that the mutation did not affect nuclear localization of GATA3. However, EMSAs showed it to reduce DNA binding affinity, but not stability, and yeast two-hybrid assays demonstrated that the mutant GATA3 resulted in a loss of interaction with ZnF1 and ZnF6 of the cofactor FOG2. The mutant GATA3 significantly reduced luciferase reporter activity by more than 65% (P < 0.001), and three-dimensional modeling indicated that the functional abnormalities may be due to a loss of Thr272 polar side chain interaction with Leu268.
A novel missense HDR-associated GATA3 mutation, Thr272Ile, has been identified and shown to result in reduced DNA binding, a partial loss of FOG2 interaction, and a decrease in gene transcription.
The transcription factor GATA-3 was recently identified as a master regulator in the specification of the inner root sheath. Additionally, it seems to play a role in skin barrier physiology. p63 binds and transactivates the GATA-3 promoter. While the expression profile of GATA-3 is delineated for the mouse, little is known about its expression in the adult human hair follicle and no studies are published about its distribution during human cutaneous embryogenesis.
We examined samples from embryonic, fetal and adult human skin for the expression of GATA-3 using immunohistochemistry.
GATA-3 is expressed late during human skin development. Its expression pattern is comparable to the mouse and confined to the Huxley layer and inner root sheath cuticle but sparing the Henle layer. In addition, GATA-3 localizes to the spinous cell layer of the interfollicular epidermis.
From the described expression pattern, it is highly probable that GATA-3 plays a role in follicular and epidermal morphogenesis. What the anatomically confined expression of GATA-3 to the spinous layer means biologically for the physiology of the skin is still unclear. Likewise, it still needs to be shown if GATA-3 could be exploited in the diagnosis of adnexal neoplasms.
Umbilical cord blood (UCB) is enriched with transplantable CD34+ cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34-lineage- cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34-lineage- cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.
CD34-lineage- cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-gamma, GM-CSF, TNF-alpha and CCL3/MIP-1alpha, and cytolytic activity against NK-sensitive tumour cell targets. CD34-lineage- cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34-lineage- cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)-CD56+CD16-/+ phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.
This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.
The aim of this study is to elucidate the expression patterns of GATA transcription factors in neuroblastoma and the developing sympathetic nervous system (SNS).
GATA-2, -3 and -4 and their cofactor friend-of-GATA (FOG)-2 were investigated in primary neuroblastoma by immunohistochemistry, real-time RT-PCR (n=73) and microarray analysis (n=251). In addition, GATA-2, -3 and FOG-2 expression was determined by northern-blot hybridisation. In the developing murine SNS, Gata-4 and Fog-2 were examined by immunohistochemistry.
Although Gata-2, -3 and Fog-2 are expressed in the developing nervous system, Gata-4 was not detected. In contrast, protein expression of all factors was observed in human neuroblastoma. Northern-blot hybridisation and real-time RT-PCR suggested specific expression patterns of the four genes in primary neuroblastoma, but did not show unequivocal results. In the large cohort examined by microarrays, a significant association of GATA-2, -3 and FOG-2 expression with low-risk features was observed, whereas GATA-4 mRNA levels correlated with MYCN-amplification.
The transcription factors GATA-2 and -3, which are essential for normal SNS development, and their cofactor FOG-2 are downregulated in aggressive but not in favourable neuroblastoma. In contrast, upregulation of GATA-4 appears to be a common feature of this malignancy and might contribute to neuroblastoma pathogenesis.
During early mammalian development, genesis of the first two cell lineages, inner cell mass (ICM) and trophectoderm (TE), is dependent upon functions of key transcription factors that are expressed in a regulated and spatially restricted fashion. In this study, we demonstrate that during early mouse development, mRNA expression of transcription factor GATA3 is induced at the 4-cell stage and is consistently present during pre-implantation embryonic development. Interestingly, at the blastocyst stage, Gata3 mRNA is selectively up-regulated within the TE lineage, and GATA3 protein is abundantly present only in the TE but not in the ICM. Using mouse trophoblast stem cells (TS cells) as a model, we found that, knockdown of GATA3 by RNA interference (RNAi) down-regulates expression of caudal-type homeobox 2 (CDX2), a key regulator of the TE lineage. Chromatin immunoprecipitation (ChIP) analyses revealed that, in TS cells, GATA3 directly regulates Cdx2 transcription from a conserved GATA motif at the intron 1 region of the Cdx2 locus. ChIP analyses with mouse blastocysts also detected GATA3 occupancy at intron 1 of the Cdx2 locus. In addition, down-regulation of GATA3 in pre-implantation mouse embryos reduces Cdx2 expression and inhibits morula to blastocyst transformation. Our results indicate a novel function of GATA3, in which it is selectively expressed in TE, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation.
Chlamydial Inclusion membrane proteins (Incs), are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT) Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further.
A total of 14 CT-positive fertile, 18 CT-positive infertile women and 25 uninfected controls were enrolled in this study. CD8 depleted, CD4 enriched cervical cells were isolated and upon stimulation with IncB and IncC, modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, IL-23, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF) and T cell lineage regulating transcription factors T-Bet and GATA3 was determined by real-time reverse-transcriptase (RT)-PCR and ELISA.
Significant higher expression (P < 0.05) of Interferon-gamma, IL-12, IL-23 and GM-CSF were found in Inc-stimulated CD4 enriched cervical cells of CT-positive fertile women and contrastingly high IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found in CT-positive infertile women. Positive correlation (P < 0.05) was found between Interferon-gamma and T-Bet levels in CT-positive fertile women and IL-4 mRNA and GATA3 levels in CT-positive infertile patients upon IncB and IncC stimulation.
Overall our data shows that CT IncB and IncC are able to upregulate expression of cytokines, namely interferon-gamma, IL-12, IL-23 and GM-CSF in CT-positive fertile women while expression of IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 were upregulated in CT-positive infertile women. Our study also suggests that Incs are able to modulate expression of T cell lineage determinants indicating their involvement in regulation of immune cells.
The mature alphabeta T cell population is divided into two main lineages that are defined by the mutually exclusive expression of CD4 and CD8 surface molecules (coreceptors) and that differ in their MHC restriction and function. CD4 T cells are typically MHC-II restricted and helper (or regulatory), whereas CD8 T cells are typically cytotoxic. Several transcription factors are known to control the emergence of CD4 and CD8 lineages, including the zinc finger proteins Thpok and Gata3, which are required for CD4 lineage differentiation, and the Runx factors Runx1 and Runx3, which contribute to CD8 lineage differentiation. This review summarizes recent advances on the function of these transcription factors in lineage differentiation. We also discuss how the "circuitry" connecting these factors could operate to match the expression of the lineage-committing factors Thpok and Runx3, and therefore lineage differentiation, to MHC specificity.
As previously shown, higher levels of NOTCH1 and increased NF-kappaB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency (than expected by chance) of NF-kappaB-binding sites (BS), including potentially novel NF-kappaB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappaB, and other important TFs on more primitive HSC sets.
Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.
To investigate the expression of Th1/Th2 transcription factors and cytokines in peripheral blood of patients with esophageal squamous cell carcinoma (ESCC) during radiotherapy to provide an evidence for using traditional Chinese medicine in anti-tumor immunotherapy.
Sixty patients with ESCC undergoing radiotherapy were randomly and equally assigned to the tested group (treated with combined treatment of Aidi Injection) and the control group (treated with radiotherapy alone). A group consisted of 20 healthy persons was set up meanwhile as the normal control. Patients' expressions of Th1 and Th2 type transcription factors and cytokines were detected before and after radiotherapy, the expression of T-bet and GATA-3 in peripheral blood monoclear cells (PBMCs) were detected by Real-time quantitative PCR (RT-PCR), and plasma expressions of interferon gamma (IFN-gamma), interleukin 2, 4 and 10 (IL-2, IL-4, IL-10) were detected by enzyme-linked immunosorbent assay (ELISA).
Compared with the healthy control group, the expressions of Th1 type transcription factor T-bet and cytokines IFN-gamma, IL-2 in ESCC patients were significantly lower (P < 0.01), while expressions of Th2 type transcription factor GATA-3 and cytokines IL-4, IL-10 were significantly higher (P < 0.01). These changes were exacerbated significantly after radiotherapy in the control group (P < 0.01), but only showed a slight change in the tested group with statistical insignificance (P > 0.05).
Radiotherapy can not reverse the Th1/Th2 shift presented in ESCC patients, while the combined intravenous dripping of Aidi Injection during radiotherapy can inhibit it effectively. The combined therapy could elevate the immune function in organism, enhance the radiosensitivity, and attenuate the toxic-adverse effect of radiotherapy.
During the development of the inner ear, auditory and vestibular ganglion neurons are generated in a highly regulated sequential process. First, neuroblasts are specified, delaminate from the epithelium of the otocyst, and migrate to form the auditory-vestibular ganglion (AVG). These neuroblasts then undergo proliferation and differentiate into afferent neurons of the auditory and vestibular ganglia. The zinc finger transcription factor Gata3 has been shown to play a role in cell proliferation and differentiation in various regions of the inner ear. Here we profile the spatiotemporal expression pattern of Gata3 in the developing auditory and vestibular ganglia of the chick embryo. Gata3 is expressed in a distinct population of sensorineural precursor cells within the otic epithelium, but is not expressed in migrating or proliferating neuroblasts. Following terminal mitosis, Gata3 expression is restricted to very few cells in the auditory ganglion and is not expressed in any cells of the vestibular ganglion. Gata3 expression levels then increase in auditory neurons as they mature. The increase of Gata3 in auditory ganglion neurons is accompanied by decreased expression of NeuroD. Our results suggest that Gata3 may be specifically involved in the differentiation of auditory ganglion neurons.
Expression of T1ST2, the IL-33R, by Th2 cells requires GATA3. Resting Th2 cells express little GATA3, which is increased by IL-33 and a STAT5 activator, in turn increasing T1ST2 from its low-level expression on resting Th2 cells. Th2 cells that have upregulated T1ST2 produce IL-13, but not IL-4, in response to IL-33 plus a STAT5 activator in an antigen-independent, NF-kappaB-dependent, cyclosporin A (CsA)-resistant manner. Similarly, Th17 cells produce IL-17A in response to IL-1beta and a STAT3 activator and Th1 cells produce IFNgamma in response to IL-18 and a STAT4 inducer. Thus, each effector Th cell produces cytokines without antigenic stimulation in response to an IL-1 family member and a specific STAT activator, implying an innate mechanism through which memory CD4 T cells are recruited by an induced cytokine environment.
The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.
The differentiation of activated CD4(+) T cells into the T helper type 1 (T(H)1) or T(H)2 fate is regulated by cytokines and the transcription factors T-bet and GATA-3. Whereas interleukin 12 (IL-12) produced by antigen-presenting cells initiates the T(H)1 fate, signals that initiate the T(H)2 fate are not completely characterized. Here we show that early GATA-3 expression, required for T(H)2 differentiation, was induced by T cell factor 1 (TCF-1) and its cofactor beta-catenin, mainly from the proximal Gata3 promoter upstream of exon 1b. This activity was induced after T cell antigen receptor (TCR) stimulation and was independent of IL-4 receptor signaling through the transcription factor STAT6. Furthermore, TCF-1 blocked T(H)1 fate by negatively regulating interferon-gamma (IFN-gamma) expression independently of beta-catenin. Thus, TCF-1 initiates T(H)2 differentiation of activated CD4(+) T cells by promoting GATA-3 expression and suppressing IFN-gamma expression.
Nasal polyposis has different etiologies in Western and Eastern countries. Furthermore, its pathogenesis is still poorly understood.
To determine the T-cell phenotypes involved in nasal polyposis in Chinese patients.
Twenty-four Chinese patients with nasal polyps were studied. CD4, CD8, Foxp3, and interleukin (IL) 17 were analyzed by immunohistochemical staining. Expression of T-bet, GATA-3, Foxp3, and RORgammat mRNA was detected by real-time polymerase chain reaction. The levels of T-cell cytokines (IL-4, IL-5, interferon [IFN] gamma, IL-10, IL-17, and transforming growth factor [TGF] beta) were determined using enzyme-linked immunosorbent assay, and serum immunoglobulin (Ig) E levels were measured using the UNICAP system.
Increased expression of CD4+ and CD8+ and decreased expression of Foxp3 and IL-17 were detected in nasal polyps compared with control tissue. Furthermore, expression of T-bet and GATA-3 mRNA was upregulated, whereas Foxp3 mRNA expression was markedly downregulated. Furthermore, increased levels of IFN-gamma, IL-4 and IL-5 and decreased levels of IL-10 and TGF-beta were found in nasal polyps. There was no association between Staphylococcus aureus exotoxin (SAE)-specific IgE and T regulatory cell (Treg) insufficiency in nasal polyps.
Our findings demonstrate that excessive infiltration of CD4+ and CD8+ T cells in nasal polyps may be associated with expression of Foxp3+ by Tregs but not with SAEs in Chinese patients.
We sought to determine whether the levels of expression of 17 candidate genes were associated with locoregional control after breast-conserving treatments of early-stage breast cancers in young, premenopausal women.
Gene expression was measured by using RT-PCR in the breast tumors of a series of 53 young (younger than 40 years), premenopausal patients. All treatments consisted of primary breast-conserving surgery followed by whole-breast radiotherapy (+/- regional lymph nodes) with or without systemic treatments (chemotherapy +/- hormone therapy). The median follow-up was 10 years.
The 10-year locoregional control rate was 70% (95% CI, 57% to 87%). In univariate analysis, no clinical/pathologic prognostic factors were found to be significantly associated with decreased locoregional control. Expression of three genes was found to be significantly associated with an increased locoregional recurrence rate: low estrogen-receptor beta, low aromatase, and high GATA3. Two others were associated with only a trend (P < 0.10): low HER1 and SKP2. In multivariate analysis, only the absence of aromatase was significantly associated with an increased locoregional recurrence rate (P = 0.003; relative risk = 0.49; 95% CI 0.29 to 0.82).
Recent data give credit to the fact that breast cancer in young women is a distinct biologic entity driven by special oncogenic pathways. Our results highlight the role of estrogen-signaling pathways (mainly CYP19/aromatase, GATA3, and ER-beta) in the risk of locoregional recurrence of breast cancer in young women. Confirmation in larger prospective studies is needed.
During vertebrate lens development, the anterior, ectoderm-derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA-3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA-3 deficiency impairs lens development. Interestingly, expression of E-cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of gamma-crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell-cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA-3 is essential for lens cells differentiation and proper cell cycle control.
Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor, the expression of which correlates with negative prognosis in breast cancer. ERRalpha shares functional features with the estrogen receptor alpha (ERalpha) and its activity is modulated by the ERBB2 signaling pathway. Using genome-wide binding sites location analyses in ERalpha-positive and ERalpha-negative breast cancer cell lines, we show that ERRalpha and ERalpha display strict binding site specificity and maintain independent mechanisms of transcriptional activation. Nonetheless, ERRalpha and ERalpha coregulate a small subset of common target genes via binding either to a dual-specificity binding site or to distinct cognate binding sites located within the extended promoter region of the gene. Although ERRalpha signaling in breast cancer cells is mostly independent of ERalpha, the small fraction of common ERRalpha/ERalpha targets comprises genes with high relevance to breast tumor biology, including genes located within the ERBB2 amplicon and GATA3. Finally, unsupervised hierarchical clustering based on the expression profiling of ERRalpha direct target genes in human breast tumors revealed four main clusters that recapitulate established tumor subtypes. Taken together, the identification and functional characterization of the ERRalpha transcriptional network implicate ERRalpha signaling as a determinant of breast cancer heterogeneity.
To investigate the expression of transcription factors (TF) T-bet and GATA-3 mRNA in peripheral blood mononuclear cells and its correlation with immune status in esophageal cancer patients.
Sixty patients were divided into two groups according to the clinical data: group A consisting of stage I and II, group B including stage III and IV. The gene expression of T-bet and GATA-3 in 60 esophageal cancer patients and 30 healthy controls was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of IFN-gamma and IL-4 was measured by enzyme linked immunosorbent assay (ELISA).
Expression of T-bet mRNA in esophageal cancer patients (stage I and II: 0.27 +/- 0.05 ng/L, stage III and IV: 0.12 +/- 0.02 ng/L) was significantly lower than that in the healthy controls (1.35 +/- 0.14 ng/L), but the expression of GATA-3 mRNA in esophageal cancer patients (stage I and II: 0.45 +/- 0.06, stage III and IV: 0.55 +/- 0.03) was significantly higher than that in the healthy controls (0.09 +/- 0.10). The plasma level of Th1 cytokine IFN-gamma in the patients [stage I and II: (12.12 +/- 1.48) ng/L, stage III and IV: (8.44 +/- 0.90) ng/L] was significantly lower than that in the healthy controls, while the level of Th2 cytokine IL-4 in the patients [stage I and II: (18.64 +/- 0.77) ng/L, stage III and IV: (25.28 +/- 2.02) ng/L] was significantly higher than that in the healthy controls. However, neither in the expression of T-bet and GATA-3, nor in the plasma level of IFN-gamma and IL-4, showed a significant difference between group A and B.
In the peripheral blood of esophageal cancer patients, the expression of T-bet decreased, while GATA-3 increased, Th1/Th2 balance is broken, and the Th2 is dominant. T-bet and GATA-3 play a part role in the regulation of Th1/Th2 balance.
CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.
Expression of interferon-tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT-PCR analysis between bovine trophoblast CT-1 and Mardin-Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT-1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (-631 to +59 bp) of bovine IFNT gene (bIFNT, IFN-tau-c1), over-expression of GATA2/GATA3 did not affect the transcription of bIFNT-reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up-regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT-1 cells, endogenous bIFNT gene transcription was up-regulated by over-expression of GATA2 or GATA3, but down-regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast-specific regulation of bIFNT gene transcription.
We have previously described an analog peptide of type II collagen (CII) that can suppress collagen-induced arthritis (CIA). This analog peptide represents CII(245-270), the immunodominant epitope of CII, but with substitutions at 260, 261, and 263 - CII(245-270) (A(260), B(261), and N(263)) (A9). To elucidate the mechanisms responsible for suppression, we used mice transgenic for a collagen-specific T cell receptor (TCR). When we found that APCs pulsed with A9 failed to induce T cell phosphorylation of TCR-zeta and ZAP-70, we explored alternative signaling pathways. We determined that A9 instead induced phosphorylation of spleen tyrosine kinase (Syk). The importance of Syk was confirmed by the use of chemical Syk inhibitors, which blocked both cytokine secretion and activation of GATA-3 mediated by peptide A9. In summary, T cells use an alternative pathway in response to A9 that involves Syk. This novel T cell pathway may represent an important means for altering T cell phenotypes.
V(D)J recombination involves the stepwise assembly of B and T cell receptor genes as lymphocytes progress through the early stages of development. While the mechanisms that restrict each step in recombination to its appropriate developmental stage are largely unknown, they share many of the components that regulate transcription. For example, enhancer-dependent modifications in histone acetylation and methylation are essential for both germline transcription and rearrangement of antigen receptor genes. Promoters positioned proximal to individual D and J gene segments in Tcra, Tcrb, Tcrd, IgH, and Igk also contribute to antigen receptor gene assembly, though their effects appear more localized than those of enhancers. Tcrb assembly initiates with D-to-J joining at each of the two D-J-C gene segment clusters in DN1/2 thymocytes. DJ joints are fused with Vbeta elements to complete Tcrb recombination in DN3 cells. We have previously shown that Dbeta2 is flanked by upstream and downstream promoters, with the 5' promoter being held inactive until D-to-J recombination deletes the NFkappaB-dependent 3' promoter. We now report that activity of the 5' promoter reflects a complex interplay among Runx1, GATA-3, and E47 transcription factors. In particular, while multiple E47 and Runx1 binding sites clustered near the Dbeta2 5'RS and overlapping inr elements define the core 5'PDbeta2, they act in concert with an array of upstream GATA-3 sites to overcome the inhibitory effects of a 110bp distal polypurine.polypyrimidine (R.Y) tract. The dependence of 5'PDbeta2 on E47 is consistent with the reported role of E proteins in post-DN1 thymocyte development and V-to-DJbeta recombination.
GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage and differentiation of T helper type 2 cells. We have identified and characterized the full-length Atlantic salmon GATA-3 cDNA (3074bp), having two zinc finger domains which are fully conserved within teleosts and higher vertebrates. RT-PCR analysis revealed that the Atlantic salmon GATA-3 (AsGATA-3) is strongly expressed in gills, thymus, and brain. Moreover, the involvement of GATA-3 in Atlantic salmon immune response was demonstrated by investigating the early time dependent expression profile of GATA-3 in spleen and head kidney following intraperitoneal injection of live Aeromonas salmonicida, LPS, and beta-glucan. Furthermore, we have determined 1.9kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites and the AsGATA-3 promoter is a TATA-less promoter. Activities of presumptive regulatory regions of this gene were assessed by transfecting different 5' deletion constructs and the result showed the basal promoter and positive transcriptional regulator activity of AsGATA-3 gene is determined by sequences located between +58 and -199bp upstream of the transcriptional start site (TSS). This study provides further insights into the transcriptional regulation of AsGATA-3.
Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double-negative 1 (DN1; CD44(+) CD25(-)) thymocytes to the DN2 (CD44(+) CD25(+)) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were dramatically elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44(-) CD25(+)) stage, which was accompanied by enhanced Notch1 expression in T-cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased, whereas GATA3 levels increased in Id1 transgenic thymocytes. When overexpressed, GATA3 was able to upregulate Deltex1 transcription. Thus, T-cell commitment may be controlled by the interplay among E proteins, Gfi1b, and GATA3 transcription regulators, which influence Notch function through the expression of Deltex1.
T helper type 2 (T(H)2) bias, which is the propensity of naive CD4(+) T cells to differentiate into interleukin 4 (IL-4)-secreting T(H)2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T(H)2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T(H)2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T(H)2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4(+) T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T(H)2 bias.
Determining binding sites of transcription factors is important for understanding the transcriptional control of target genes. Although a transcription factor GATA3 plays a pivotal role in Th2 lymphocyte development, its physiological role is not clearly defined because the target genes remain largely unknown. In this study, we modified chromatin immunoprecipitation (ChIP), and isolated 121 GATA3 binding sites and 83 different annotated target genes. Re-ChIP analysis using anti-GATA3 and anti-RNA polymerase II mAbs and chromosome conformation capture assay demonstrate that GATA3-bound fragments interact with basal transcriptional units of target genes. GATA3 regulation of target genes under the control of binding fragments was confirmed by reporter assay and quantification of target gene mRNA expression in the presence of GATA inhibitor or short interfering RNA against GATA3. These data demonstrate that GATA3 binds to regulatory elements and controls target gene expression through physical interaction with core promoter regions.
Stemucronatoside L (SML), isolated from Stephanotis mucronata, could suppress the activation of T cells in vitro. However, the mechanisms responsible for its immunosuppressive activity remain poorly understood. The purpose of this study was to investigate whether SML could suppress Th1/Th2 immune responses and to characterize the cellular mechanisms involved. Effects of SML on T-lymphocyte subsets and the production of Th1 cytokines IL-2 and IFN-gamma, and Th2 cytokines IL-4 and IL-10 from ConA-stimulated mice splenocytes were detected by flow-cytometric analysis and ELISA method, respectively. Furthermore, effects of SML on mRNA expression level of Th1/Th2 cytokines and transcription factors T-bet and GATA-3 were evaluated by RT-PCR analysis. SML not only significantly decreased the percentage of CD4(+) T cells and the CD4(+)/CD8(+) ratio, but reduced the production of Th1/Th2 cytokines in a concentration-dependent manner. The mRNA expression levels of Th1/Th2 cytokines and transcription factors (T-bet and GATA-3) were also suppressed by SML. These results suggested that SML could simultaneously inhibit Th1/Th2 immune responses by suppressing gene expression of Th1/Th2 cytokines and transcription factors.
The expression of additional genes, other than oestrogen receptor (ER), may be important to the hormone-responsive phenotype of breast cancer. Microarray analyses have revealed that forkhead box A1 (FOXA1) and GATA binding protein 3 (GATA-3) are expressed in close association with ERalpha, both encoding for transcription factors with a potential involvement in the ERalpha-mediated action in breast cancer. The purpose of this study was to explore if the expression of FOXA1 and GATA-3 may provide an opportunity to stratify subsets of patients that could have better outcome, among the ERalpha-negative/poor prognosis breast cancer group.
We evaluate FOXA1 and GATA-3 expression in 249 breast carcinomas by immunohistochemistry, associating it with breast cancer molecular markers, clinicopathological features and patient's survival. The clinicopathological features and immunohistochemical markers of the tumours were compared using the chi-square test and ANOVA. Disease-free survival was analysed through Kaplan-Meier survival curves and Cox regression.
FOXA1 expression was demonstrated in 42% of invasive carcinomas, while GATA-3 was detected in 48% of the cases. FOXA1 expression was inversely associated with tumour size, Nottingham Prognostic Index, histological grade, lymph vascular invasion, lymph node stage and human epidermal growth factor receptor-2 (HER-2) overexpression, while GATA-3 expression showed inverse association with histological grade and HER-2. Both FOXA1 and GATA-3 were directly associated with ERalpha and progesterone receptor. Among FOXA1-positive tumours, 83.1% are comprised in the luminal A subtype, similar to GATA-3 where 87.7% of positive tumours were classified within this molecular subtype. In the subset of ERalpha-negative patients, those who were FOXA1-negative had a 3.61-fold increased risk of breast cancer recurrence when compared with the FOXA1-positive.
FOXA1 was a significant predictor of good outcome in breast cancer, whereas GATA-3 was an important luminal marker. The expression of FOXA1 may be used for risk stratification among ERalpha-negative patients.
In the present study, we investigated the anti-allergic activity of a glycoprotein isolated from Dioscorea batatas Decne (DBD glycoprotein, 30 kDa) on ovalbumin (OVA, 100 microg ml(-1))-induced T helper (Th) type-2 response in primary cultured mouse lymphocytes. Our results revealed that the DBD glycoprotein (200 microg ml(-1)) significantly attenuated the expressions of interleukin (IL)-4 and IL-10, whereas enhanced the expression of interferon (IFN)-gamma in OVA (100 microg ml(-1))-treated primary cultured lymphocytes. We also found that the DBD glycoprotein has inhibitory effects on phosphorylations of signal transducer and activator of transcription (STAT)-6, p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK in primary cultured lymphocytes. Interestingly, the DBD glycoprotein suppressed the transcriptional activation of GATA-binding protein 3 (GATA-3), whereas it enhanced the activity of t-box expressed in T cells (T-bet) in OVA-stimulated lymphocytes. The results from these experiments indicated that the DBD glycoprotein inhibits expressions of IL-4 and IL-10 through modulation of GATA-3, STAT-6, p44/42 MAPK and p38 MAPK in mouse lymphocytes. Therefore, we speculated that the DBD glycoprotein might be one component for preparation of nutraceutical health supplements for prevention of Th2 cell response-related immune disorder.
CD4 T cells can be primarily polarized to differentiate into Th1 or Th2 cells. CD44 is a marker of T cell activation and a property of long-lived memory cells and implicated in cell migration, activation, and differentiation. To date, whether CD44 has a role in regulating Th1-Th2 differentiation has not been determined. In this study, we compared Th1 and Th2 responses in wild-type and CD44-deficient mice in response to sheep RBC and chicken OVA, as well as examined Th1-Th2 differentiation in vivo and in vitro from CD44-sufficient and CD44-deficient naive CD4 T cells. We observed that deficiency of CD44 tended to inhibit Th1 while promoting Th2 differentiation. Furthermore, chimeric studies suggested that CD44 expression by CD4 T cells was essential for such Th2 bias. The regulation by CD44 occurred at the transcription level leading to up-regulated GATA3 and down-regulated T-bet expression in activated CD4 T cells. We also noted that CD44-deficiency could modify the state of dendritic cell subsets to induce a Th2-biased development. Results presented in this study demonstrate for the first time that CD44 participates in the regulation of Th1-Th2 differentiation.
Understanding immune responses of white-tailed deer (WTD) to infection with Mycobacterium bovis provides insight into mechanisms of pathogen control and may provide clues to development of effective vaccine strategies. WTD were vaccinated with either M. bovis BCG strain Pasteur or BCG strain Danish. Both vaccinees and unvaccinated controls were subsequently inoculated with virulent M. bovis via the intratonsillar route. Real-time PCR was used to assess T-cell mRNA expression in peripheral blood leukocytes (PBL) from animals following vaccination and infection. Recall T-cell responses were measured by assessing the relative expression of gamma interferon (IFN-gamma), T-cell-specific T-box transcription factor (Tbet), interleukin 12p40 (IL-12p40), IL-12p35, IL-23p19, FoxP3, IL-17, and GATA3 in PBL stimulated in vitro with purified protein derivative (PPD) of M. bovis or a recombinant fusion protein, ESAT6-CFP10. Animals vaccinated with BCG Danish expressed more IFN-gamma and Tbet than either BCG Pasteur-vaccinated animals or unvaccinated controls. BCG Pasteur-vaccinated animals expressed more GATA3 than either group. After infection, unvaccinated controls expressed more Tbet and IL-12p40 than vaccinated animals. BCG Pasteur-vaccinated animals expressed more GATA3 than either the unvaccinated controls or the BCG Danish-vaccinated animals after infection. Animals were divided into pathology groups to correlate gene expression with severity of pathology. Animals in the visible lesion group expressed more Tbet and IFN-gamma than animals that were culture negative, while Tbet and IFN-gamma expression in the culture-positive, no-visible-lesion group was intermediate. GATA3 expression inversely correlated with pathology. Overall, expression of immune response genes correlated more closely with pathology than vaccination treatment.
The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators.
Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression.
ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without a mutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia.
In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation.
A region of the VWF promoter between -2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.
To study glaucoma infiltration surgery induced the anterior chamber associated immune deviation (ACAID) by with or without anterior chamber (AC) implantation of foreign epithelium removed corneal graft in mice.
In this study, fifty Wistar mice were randomly divided into five groups (ten in each group) and additional five SD and one Wistar mouse were used as the providers of epithelium removed corneal grafts to induce ACAID as follows: Group A: corneal grafts from Wistar mouse were implanted into the AC 1 week after the infiltration surgery. Group B: the spleen cells were injected into the nape of neck 1 week after the glaucoma infiltration surgery. In group C, D, and E, the corneal grafts from SD mice were implanted into the AC at 1 week, 4 week, and 8 week after the glaucoma infiltration surgery, respectively. To induce the delayed type hypersensitivity (DTH), spleen cells were injected into the right ear pinna 1 week after the neck injection in group B, 2 weeks after the AC implantation of corneal grafts in other groups. At the same time points as the induction of DTH, heart blood was collected to detect the concentration of IL-4 and IL-10. The spleens were removed to evaluate the expression of the GATA-3 mRNA by RT-PCR. The eyeballs were enucleated and used to evaluate the histopathological changes.
(1) The DTH were found in group B, C, D, and E with corneal grafts of SD, but not in group A with that of Wistar. (2) The serum concentrations of IL-4 and IL-10 were statistically different in group C, D, and E (F = 49.124, 6.336; P < 0.01). The concentrations of IL-4 and IL-10 were significantly (P = 0.002) lower in group D than those in group C and E (3.759 +/- 0.250 vs 5.916 +/- 0.500 or 4.566 +/- 0.518, and 17.170 +/- 3.943 vs 44.447 +/- 17.167 or 35.643 +/- 21.233 microg/L, n = 10, respectively), which showed a biphase response fluctuation of ILs. (3) The GATA-3 mRNA was obviously up-regulated in group D and E. (4) The exudation and a few inflammatory cells in the AC were observed in group C, but not in group D and E.
The eye with the glaucoma infiltration surgery results in a temporary incapable of inducing ACAID, which is gradually recovered following the diminish of intraocular inflammation and the increase of levels of IL4, IL-10 and GATA-3 mRNA.
The loss of expression of the transcription factor GATA3 in breast tumors has been linked to aggressive tumor development and poor patient survival. In the present work, we address potential roles for GATA3 in breast tumor lung metastasis and progression. Using an aggressive breast cancer cell line, which metastasizes specifically to the lung, we show that GATA3 expression results in reduced tumor outgrowth in the mammary fat pad and lower lung metastatic burden in nude mice. Specifically, GATA3 expression inhibits breast cancer cell expansion inside the lung parenchyma. This phenotype correlates with the ability of GATA3 to negatively regulate the expression of several genes that promote breast cancer lung metastasis (ID1/-3, KRTHB1, LY6E and RARRES3). Conversely, the expression of genes encoding known inhibitors of lung metastasis (DLC1 (deleted in liver cancer 1) and PAEP (progestagen-associated endometrial protein)) is upregulated by GATA3. These data correlate with microarray data from human breast cancer patients, showing a strong correlation between high GATA3 expression and absence of metastases specifically to the lungs. We conclude that GATA3 inhibits primary breast tumor outgrowth and reduces lung metastatic burden by regulating key genes involved in metastatic breast tumor progression.
The role of allergy in the pathophysiology of otitis media with effusion (OME) remains unclear. We evaluated the role of allergy and the Th1/Th2 balance in OME patients.
Middle ear fluid was collected from 46 OME patients who had a ventilating tube inserted, and expression of GATA3, T-bet, IL-4, and IFN-gamma mRNA was assessed by RT-PCR. Expression of transcription factors and cytokines was compared in patients with and without allergy.
The levels of GATA3 and T-bet mRNA in effusion fluid correlated positively with the levels of IL-4 and IFN-gamma mRNA, respectively. However, the allergy and non-allergy groups showed no significant differences in expression of any of these transcription factors and cytokines. The T-bet:GATA3 ratio was higher in the non-allergy than in the allergy group, but the difference was not statistically significant.
Although we observed a significant positive correlation between GATA3 and IL-4 mRNA levels, we cannot conclude that OME with allergy is related to a Th-2 driven immune response.
Many animals are capable of changing gait with speed of locomotion. The neural basis of gait control and its dependence on speed are not fully understood. Mice normally use a single "trotting" gait while running at all speeds, either over ground or on a treadmill. Transgenic mouse mutants in which the trotting is replaced by hopping also lack a speed-dependent change in gait. Here we describe a transgenic mouse model in which the V2a interneurons have been ablated by targeted expression of diphtheria toxin A chain (DTA) under the control of the Chx10 gene promoter (Chx10::DTA mice). Chx10::DTA mice show normal trotting gait at slow speeds but transition to a galloping gait as speed increases. Although left-right limb coordination is altered in Chx10::DTA mice at fast speed, alternation of forelegs and hindlegs and the relative duration of swing and stance phases for individual limbs is unchanged compared with wild-type mice. The speed-dependent loss of left-right alternation is recapitulated during drug-induced fictive locomotion in spinal cords isolated from neonatal Chx10::DTA mice, and high-speed fictive locomotion evoked by caudal spinal cord stimulation also shows synchronous left-right bursting. These results show that spinal V2a interneurons are required for maintaining left-right alternation at high speeds. Whether animals that generate galloping or hopping gaits, characterized by synchronous movement of left and right forelegs and hindlegs, have lost or modified the function of V2a interneurons is an intriguing question.
An imbalance of Th1 and Th2 cytokines has been reported in MCNS. Interleukin-13 (IL-13: Th2 cytokine) has been implicated in the pathogenesis of MCNS, but Th1/Th2 regulators such as T-bet (Th1-specific transcription factor) and GATA-3 (Th2-specific transcription factor) have not been examined.
We isolated PBMC from 25 patients with MCNS during nephrosis and remission phases, from 17 nephrotic patients with membranous nephropathy (MN), and from 25 healthy subjects. We measured mRNA expression levels of T-bet, GATA-3, Stat5A (regulator of Th2 priming), IFN-gamma (Th1 cytokine), IL-2 (Th1 cytokine and activator of Stat5), IL-4 (Th2 cytokine), and IL-13 in PBMC, using real-time RT-PCR.
GATA-3, Stat5A, and IL-13 mRNA expression levels were higher in the nephrotic MCNS group compared to the others. IL-2 mRNA expression levels were higher in nephrotic patients with MCNS and MN than in MCNS patients in remission and healthy controls. There were no differences in mRNA expression levels of T-bet, IFN-gamma, and IL-4 between MCNS and MN patients and healthy controls.
This study is the first to reveal increased mRNA expression levels of GATA-3 and Stat5A in PBMC from MCNS patients in nephrosis. This study also supports recent findings suggesting the role of IL-13 in the development of MCNS. A predominant Th2 type of T cell activation may be involved in the pathogenesis of MCNS.
Two members of winged-helix/forkhead transcription factors, Foxp1 and Foxp2, are expressed in the developing and adult CNS, including the striatum, cerebral cortex, and thalamus. In a previous study, we have demonstrated that Foxp1 is expressed in a subpopulation of V1 interneurons in addition to motor neurons of the spinal cord during mouse embryogenesis. However, the detailed expression pattern of Foxp2 and its relationship with Foxp1 in the developing spinal cord remains to be elucidated. To shed light on the potential roles of Foxp1 and Foxp2 in the developing spinal cord, we characterized Foxp2-expressing cells during mouse embryogenesis. At embryonic day (E) 11.0, Foxp2-expressing cells were first observed in the ventral spinal cord, which were Pax6(-), p27(+), and neuron-specific class III beta-tubulin(+) postmitotic neurons. Between E13.5 and E15.5, high expression of Foxp2 was observed in both medial and lateral parts of the ventral spinal cord. Double-immunofluorescence staining for Foxp2 with some homeodomain transcription factors revealed that Foxp2-expressing neurons were Pax2(+), En1(+), Evx1(-), Chx10(-), Gata3(-), and Lhx3(-) V1 interneurons in the intermediate zone throughout the ventral spinal cord, indicating that Foxp2-expressing neurons were also V1 interneurons with the same phenotypes as Foxp1-expressing interneurons. In addition, neither Foxp1 nor Foxp2 was expressed in ventral calbindin(+) Renshaw cells. However, Foxp2 did not colocalize with Foxp1 in interneurons of the ventral spinal cord. These findings suggest that Foxp1 and Foxp2 are expressed in the distinct subsets of V1 interneurons that belong to non-Renshaw cells in the ventral spinal cord during embryogenesis. Thus, Foxp1 and Foxp2 may be involved in the determination of the cell type identities during late embryogenesis: the classes of neurotransmitters and the functional subtypes of non-Renshaw cells, such as Ia and Ib inhibitory interneurons.
To explore the relationship between GATA-3 and ICAM-1 in patients with allergic rhinitis.
The expression of GATA-3 was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in 37 patients with allergic rhinitis and 12 controls. ICAM-1 was determined by enzyme-linked immunosorbent assay.
The relative density ratio of GATA-3 to GAPDH in AR patients was 0.632 +/- 0.121, and in control group was 0.131 +/- 0.024. The concentrations of ICAM-1 were (237.5 +/- 37.3) pg/mg and (121.7 +/- 22.4) pg/mg in the two groups respectively. The expression of GATA-3 was positively correlated to the expression of ICAM-1 in patients with allergic rhinitis.
The overexpression of GATA-3 in patients with allergic rhinitis was related to the increase of ICAM-1. GATA-3 may play an important role in the pathogenesis of allergic rhinitis.
Sauchinone, a lignan compound isolated from the root of Saururus chinensis, has been recently demonstrated to exhibit anti-inflammatory activity via the suppression of NF-kB p65 activity in vitro. In an effort to evaluate the in vivo anti-inflammatory function of sauchinone, we have evaluated the effects of sauchinone on allergen-induced airway inflammation using a murine model of allergic asthma. We observed that marked eosinophilic and lymphocyte infiltration in the BAL fluid were suppressed to a significant degree by sauchinone, and that mucus-secreting goblet cell hyperplasia and collagen deposition in the airways were also ameliorated by administration of sauchinone treatment. Moreover, gene expression of the inflammatory cytokines, IL-13, and IL-5 and eotaxin in the lung, and IL-5 in the draining lymph node were significantly decreased in sauchinone-treated mice. We demonstrated that sauchinone repressed Th2 cell development in vitro and IL-4 production by Th2 cells, and also inhibited GATA-3-mediated IL-5 promoter activity in a dose-dependent manner. Collectively, sauchinone ameliorated allergen-induced airway inflammation, in part, by repressing GATA-3 activity for Th2 cell development, indicating the possible therapeutic potential of sauchinone in airway inflammatory diseases including allergic asthma and rhinitis.
We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11c(high)CD8 alpha(high)CD11b(low) dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 microg) and Flt3-L (3 microg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-gamma, eosinophilia and substantially increased IL-10 and the number of CD4(+)CD25(+) Forkhead winged helix transcription factor box P3 (Foxp3(+)) IL-10(+) T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-gamma levels, and decreased BALF IL-10 levels and the number of CD4(+)CD25(+)Foxp3(+)IL-10(+) T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4(+)CD25(+) T cells isolated from lungs of Flt3-L-treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4(+)CD25(+) T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4(+)CD25(+)Foxp3(+)IL-10(+)ICOS(+) T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.
The purpose of this study is to determine the inhibitory effect of a glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein, 75 kDa) on di(2-ethylhexyl) phthalate (DEHP)-induced differentiation of T helper (Th) type 2 cells in T lymphocytes separated from mice. This experiment evaluated the activities of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), transcription factors [signal transducer and activator of transcription (STAT)-6 and GATA-binding protein 3 (GATA3)], and Th2 cell-related cytokine [interleukin-4 (IL-4)] using immunoblotting and reverse transcription-polymerase chain reaction. Our results revealed that the CTB glycoprotein in the presence of DEHP inhibits the translocation of PKC from cytosol to membrane and the phosphorylation of p44/42 MAPK in primary cultured T lymphocytes. We also found that the CTB glycoprotein (100 microg/ml) has suppressive effects on transcriptional activation of STAT6, GATA3, and on the expression level of IL-4 in DEHP-treated T lymphocytes. The phosphorylation of STAT6 and GATA3 were collectively blocked by treatment with PKC inhibitor (staurosporine) as well as p44/42 MAPK inhibitor (PD 98059), respectively. The results from these experiments indicated that the CTB glycoprotein inhibits IL-4 expression, not IL-10 expression, on the stage of Th2 cell differentiation induced by DEHP in T lymphocytes. Hence, we speculate that the CTB glycoprotein has a strong inhibitory ability for expressions of allergy-related cytokines indirectly caused by DEHP in Th2 cell differentiation of the primary cultured mouse T lymphocytes.
The immunological mechanisms responsible for the immunomodulatory and anti-allergic effects of probiotic bacteria are still poorly defined. The combined effects of mixtures of different species of probiotic bacteria have been explored only in part. The present study describes the immunomodulatory activity of the VSL#3 probiotic preparation in in vitro and in vivo systems.
The activation and cytokine production by in vitro probiotic-stimulated bone-marrow dendritic cells (BM-DCs) and spleen cells isolated from naïve or Par j 1-sensitized mice were investigated. Mice were intranasally administered a sonicate preparation of VSL#3 before immunization with rPar j 1. Serum antibody levels and cytokine expression in the lung were determined.
Both live and sonicated VSL#3 preparations induced maturation and cytokine production by BM-DCs. Cytokine production by spleen cells from naïve or Par j 1-sensitized mice was modulated by the probiotic preparations towards a Treg/Th0 profile, characterized by increased IL-10 and IFN-gamma production. In vivo prophylactic treatment with VSL#3 induced a significant reduction of serum specific IgG1. At lung level, VSL#3 pre-treatment remarkably reduced IL-13 and IL-4 mRNA expression and increased IL-10 expression.
The VSL#3 preparations have not only the capacity to bias primary immune responses towards a Treg/Th0-type profile, but also to modify in the same way the functional characteristics of established in vitro Th2 responses. In vivo studies on a mouse model of Par j 1 sensitization indicate that the prophylactic intranasal treatment with probiotic bacteria is able to modulate the development of Th2-biased responses.
GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.
In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-kappaB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-alpha. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.
Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.
Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a "tumorigenic" signature defined using CD44(+)/CD24(-) breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.
Histone H3 lysine 4 methylation (H3K4me) has been proposed as a critical component in regulating gene expression, epigenetic states, and cellular identities1. The biological meaning of H3K4me is interpreted by conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states. The dysregulation of PHD fingers has been implicated in several human diseases, including cancers and immune or neurological disorders. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the carboxy-terminal PHD finger of PHF23 or JARID1A (also known as KDM5A or RBBP2), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukaemias, generated potent oncoproteins that arrested haematopoietic differentiation and induced acute myeloid leukaemia in murine models. In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukaemogenesis. Mutations in PHD fingers that abrogated H3K4me3 binding also abolished leukaemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1 and Pbx1), and enforced their active gene transcription in murine haematopoietic stem/progenitor cells. Mechanistically, NUP98-PHD fusions act as 'chromatin boundary factors', dominating over polycomb-mediated gene silencing to 'lock' developmentally critical loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukaemia stem cells. Collectively, our studies represent, to our knowledge, the first report that deregulation of the PHD finger, an 'effector' of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during mammalian development.
Autism spectrum disorder (ASD) is a disease of neuro-developmental origin of uncertain etiology. The current understanding is that both genetic and environmental factors contribute to the development of ASD. Exposure to valproate, thalidomide and alcohol during gestation are amongst the environmental triggers that are associated with the development of ASD. These teratogens may disturb the ontogeny of the brain by altering the expression pattern of genes that regulate the normal development of the brain. In this study, a neuron-like PC-12 cell model was used to examine the effects of these compounds on the binding potential of 50 different transcription factors to understand the molecular mechanism/s that may be involved in the teratogenesis caused by these agents. Cells in culture were treated with low or high concentrations of teratogens within a range that are reported in the blood of individuals. A pronounced increase in GATA transcription factor binding was observed for all three teratogens. Furthermore, Western blot analysis showed that GATA-3 level in the nuclear fractions was enhanced by each of the three teratogens. Results suggest that altered gene expression pattern due to heightened GATA-3 activities in the fetral brains following exposure to these teratogens may contribute to the development of ASD.
The molecular requirements for invariant Valpha14-bearing natural killer T cells (iNKT) in the thymus are poorly understood. A minute population of approximately 500 newly selected CD69(+)CD24(+) stage 0 (ST0) iNKT cells gives rise to approximately 100 times more CD44(neg/lo)CD24(-) stage 1 (ST1) cells, which then generate similar frequencies of CD44(hi)CD24(-) stage 2 (ST2) and mature iNKT cells. Although the increased number of ST1 compared with ST0 cells indicates the initiation of a proliferation wave in the very early stages of iNKT cell development, details about the controlling mechanism are currently lacking. Here, we show that the transcription factor c-Myc is required for iNKT cell development. Conditional ablation of c-Myc in double-positive thymocytes specifically impacted iNKT but not conventional T cell development. Within the iNKT population, a progressive reduction of iNKT cells was observed starting at ST1 (approximately 50-fold) and ST2 (approximately 350-fold), with a complete lack of mature cells in thymus, spleen, and liver. ST0/ST1 c-Myc-deficient iNKT cells showed reduced proliferation. In contrast, annexin V staining did not reveal increased apoptosis, and transgenic overexpression of BCL-2 did not rescue iNKT cell development in c-Myc-deficient mice. Moreover, expression of known iNKT differentiation factors such as Plzf and Gata3 was not dramatically altered. These, findings provide compelling evidence that c-Myc mediates an intrathymic proliferation wave immediately after agonist selection of iNKT cells and illustrate the importance of this expansion for the generation of mature iNKT cells in vivo.
Transplant glomerulopathy (TGP) appears to be a pathogenic feature of chronic antibody-mediated rejection, but the pathogenesis of this histologic entity is still poorly understood. Previous studies suggest the involvement of lymphocytes but the phenotypes of these cells have never been analyzed. Here, we report the first study of mRNAs for specific markers of CD4+ T cells including Th1 (T-bet and INFgamma), Th2 (IL4 and GATA3), Treg (Foxp3) and Th17 (IL-17 and RORgammat) subsets, cytotoxic CD8 T cells (Granzyme B) and B-cell markers (CD20) in renal biopsies from renal transplant recipients suffering interstitial fibrosis and tubular atrophy (IF/TA) with or without TGP but with a similar inflammatory score and controls including transplant recipients with normal renal function. Only INFgamma, T-bet (both functionally defined markers of Th1 CD4 T cells) and granzyme B (a CD8 cytotoxic marker) were significantly more strongly expressed in patients with TGP than in patients without TGP and normal controls. These results indicate a role of an active T-mediated inflammatory and cytotoxic process in the pathogenesis of TGP.
T regulatory (Treg) cells have been detected in periodontitis lesions, and forkhead box P3 (Foxp3) expression has been negatively correlated to receptor activator of nuclear factor-kappa B ligand (RANKL). The aim of this study was to correlate T-helper type 1 (Th1), Th2, Th17 and Treg transcription factor expressions, in gingival tissues from patients undergoing active periodontal tissue destruction, with bone loss-associated cytokines.
In 10 chronic periodontitis patients undergoing disease progression, the mRNA expressions of T-bet, GATA-3, Foxp3, RORC2, interleukin (IL)-1beta, IL-10, IL-17, RANKL, interferon (IFN)-gamma and transforming growth factor (TGF)-beta1 were quantified using real-time reverse transcription-polymerase chain reaction. The levels of these markers were compared between active and inactive periodontal lesions.
In active periodontal lesions, Foxp3, T-bet, RANKL, IL-17, IL-1beta and IFN-gamma were significantly over-expressed compared with inactive lesions. The expression of IFN-gamma was the highest within the active periodontal lesions, similar to that of TGF-beta1 within the inactive ones. There was a positive correlation between RANKL and IL-17. Additionally, RANKL and IL-17 were positively correlated with RORC2, but no correlation was detected with Foxp3.
These results lead us to speculate that Foxp3(+) cells that do not have a regulatory function might have a role in the pathogenesis of active periodontal lesions by down-regulating TGF-beta1 and IL-10 synthesis that lead to the over-expression of Th17-associated cytokines RANKL and IL-17.
Mammary epithelia are composed of luminal and myoepithelial/basal cells whose neoplastic transformations lead to distinct types of breast cancers with diverse clinical features. We report that mice deficient for the CDK4/6 inhibitor p18(Ink4c) spontaneously develop ER-positive luminal tumors at a high penetrance. Ink4c deletion stimulates luminal progenitor cell proliferation at pubertal age and maintains an expanded luminal progenitor cell population throughout life. We demonstrate that GATA3 binds to and represses INK4C transcription. In human breast cancers, low INK4C and high GATA3 expressions are simultaneously observed in luminal A type tumors and predict a favorable patient outcome. Hence, p18(INK4C) is a downstream target of GATA3, constrains luminal progenitor cell expansion, and suppresses luminal tumorigenesis in the mammary gland.
Breast cancers are traditionally divided into hormone-receptor positive and negative cases. This classification helps to guide patient management. However, a subgroup of hormone-receptor positive patients relapse irrespective of hormonal therapy. Gene expression profiling has classified breast tumours into five major subtypes with significant different outcome. The two luminal subtypes, A and B, show high expression of ESR1, GATA3 and FOXA1 genes. Prognostic biomarkers for oestrogen receptor (ER)-positive cases include progesterone receptor (PR) and androgen receptor (AR), and proteins related to proliferation or apoptotic resistance. The aim of this study was to identify the best predictors of success of hormonal therapy.
By immunohistochemistry we studied 10 markers in a consecutive series of 832 cases of breast carcinoma treated at the Paoli-Calmettes Institute from 1990 to 2002 and deposited onto tissue microarrays (TMA). These markers were luminal-related markers ER, PR, AR, FOXA1 and GATA3 transcription factors, proliferation-related Ki67 and CCND1, ERBB2, anti-apoptotic BCL2 and P53. We also measured vascular peritumoural invasion (VPI), size, grade and lymph node involvement. For 143 cases, gene expression profiles were available. Adjuvant chemotherapy and hormonal therapy were given to high- and low-risk patients, respectively. The 162 events observed and taken into account were metastases.
Molecular expression of the 10 parameters and subtype with ER status were strongly correlated. Of the 67 luminal A cases of this series, 63 were ER-positive. Multivariate analyses showed the highly significant prognostic value of VPI (hazard ratio (HR) = 2.47), Ki67 (HR = 2.9), P53 (HR = 2.9) and GATA3 (HR = 0.5) for the 240 patients who received hormonal therapy.
A panel of three antibodies (Ki67, P53 and GATA3) associated with VPI can significantly improve the traditional prognosticators in predicting outcome for ER-positive breast cancer patients receiving hormonal therapy.
It is known that a number of transcription factors are key regulators in the complex process of adipocyte differentiation including peroxisome proliferator activated receptor gamma (PPARgamma) and the CCAAT enhancer binding protein alpha (C/EBPalpha). Studies have demonstrated that in pre-adipocyte 3T3-L1 cells constitutive expression of the DNA binding proteins GATA-2 and GATA-3 results in protein/protein interactions with C/EBPalpha resulting in down regulation of PPARgamma and subsequent suppressed adipocyte differentiation with cells trapped at the pre-adipocyte stage. Thus it appears that GATA-2 and GATA-3 are of critical importance in regulating adipocyte differentiation through molecular interactions with PPARgamma and C/EBPalpha. Recent reports suggest that berberine, an isoquinoline derivative alkaloid isolated from many medicinal herbs prevents differentiation of 3T3-L1 cells via a down regulation of PPARgamma and C/EBPalpha expression. The aim of this study was to determine the effect of berberine on GATA-2 and 3 gene and protein expression levels during differentiation of 3T3-L1 cells. MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was used to detect the cytotoxic effects of berberine on the viability of 3T3-L1 cells during proliferation and differentiation. Differentiation of 3T3-L1 cells was monitored by Oil Red O staining and RT-PCR of PPARgamma and C/EBPalpha and the expression of GATA-2 and 3 was determined by RT-PCR and Western Blot. Results show that following treatment with 8microM berberine the mRNA and protein expression levels of GATA-2 and 3 were elevated and accompanied by inhibited adipocyte differentiation. These results may lead to the use of berberine to target the induction of specific genes such as GATA-2 and GATA-3 which affect adipocyte differentiation.
To evaluate the potential usefulness of a multivariable model, established mainly upon peripheral T-cell subsets, Th1/Th2, T-bet and GATA-3 gene expressions as well as TCM and Western medical diagnostic criteria, in predicting the selection of immunosuppressive therapy (IST) or androgens in treating patients with aplastic anemia (AA).
Peripheral blood T cell subsets in 85 patients with AA were serially analyzed by flow cytometry before and after treatment, and their T-bet and GATA-3 gene expressions were assessed meantime by Real-time PCR. Then analysis of Logistic regression equation and ROC curves were performed based on the cases responding to IST or androgens.
(1) According to the logistic equation and ROC curve of SPSS, setting the false positive rate as 0.10, the P value was 0.832. When P> or =0.832, patients were judged in the immunosuppressive dominant state, IST should be applied; when P<0.832, it means patients in the bone marrow failure dominant state, androgens should be added. (2) A novel theory is raised by the above-mentioned analysis, which indicated that the genesis and development process of AA could be divided in 2 stages, the abnormal immune dominant stage and the bone marrow failure dominant stage. For treatment of patients in the two stages, IST and androgens is the preference respectively.
The multivariable model could be used for indicating which stage the AA patient is in, the abnormal immune stage or the bone marrow failure stage, and thus to guide the proper selection of IST or androgens in clinical practice.
Interaction of ICOS with its ligand is essential for germinal center formation, T cell immune responses, and development of autoimmune diseases. Human ICOS deficiency has been identified worldwide in nine patients with identical ICOS mutations. In vitro studies of the patients to date have shown only mild T cell defect. In this study, we report an in-depth analysis of T cell function in two siblings with novel ICOS deficiency. The brother displayed mild skin infections and impaired Ig class switching, whereas the sister had more severe symptoms, including immunodeficiency, rheumatoid arthritis, inflammatory bowel disease, interstitial pneumonitis, and psoriasis. Despite normal CD3/CD28-induced proliferation and IL-2 production in vitro, peripheral blood T cells in both patients showed a decreased percentage of CD4 central and effector memory T cells and impaired production of Th1, Th2, and Th17 cytokines upon CD3/CD28 costimulation or PMA/ionophore stimulation. The defective polarization into effector cells was associated with impaired induction of T-bet, GATA3, MAF, and retinoic acid-related orphan nuclear hormone receptor (RORC). Reduced CTLA-4(+)CD45RO(+)FoxP3(+) regulatory T cells and diminished induction of inhibitory cell surface molecules, including CTLA-4, were also observed in the patients. T cell defect was not restricted to CD4 T cells because reduced memory T cells and impaired IFN-gamma production were also noted in CD8 T cells. Further analysis of the patients demonstrated increased induction of receptor activator of NF-kappaB ligand (RANKL), lack of IFN-gamma response, and loss of Itch expression upon activation in the female patient, who had autoimmunity. Our study suggests that extensive T cell dysfunction, decreased memory T cell compartment, and imbalance between effector and regulatory cells in ICOS-deficient patients may underlie their immunodeficiency and/or autoimmunity.
IL-17A is produced from Th17 cells, and is involved in many autoimmune and inflammatory diseases. IL-13R has not previously been reported to be functionally expressed on T cells; however, we found that purified BALB/c CD4(+) cells polarized to Th17 with TGF-beta, IL-6, and IL-23 have increased mRNA and protein expression of IL-13R alpha1 and mRNA expression of IL-4R alpha compared with Th0, Th1, or Th2 polarized cells. The addition of IL-13 at Th17 polarization negatively regulated IL-17A and IL-21 expression, and reduced the number of CD4(+) T cells producing IL-17A. Further, adding IL-13 at the time of Th17 cell restimulation attenuated IL-17A expression. CD4(+) Th17 polarized cells from IL-4 knockout (KO) mice also had IL-13-induced inhibition of IL-17A production, but this was not observed in IL-4R KO and STAT6 KO mice. Addition of IL-13 at polarization increased IL-13R expression in wild-type Th17 cells. Further, IL-13 administration during Th17 polarization down-regulated retinoic acid-related-gammaT, the transcription required for Th17 development; increased STAT6 phosphorylation, and up-regulated GATA3, the transcription factor activated during the development of Th2 cells. This IL-13-mediated effect was specific to Th17 cells as IL-13 neither decreased IFN-gamma expression by Th1 cells nor affected Th2 cell production of IL-4. Collectively, we have shown that Th17 cells express a functional IL-13R and that IL-13 negatively regulates IL-17A and IL-21 production by decreasing retinoic acid-related-gammaT expression and while increasing phosphorylation of STAT6 and GATA3 expression. Therefore, therapeutic intervention inhibiting IL-13 production could have adverse consequences by up-regulating Th17 inflammation in certain disease states.
The relationship of T follicular helper (TFH) cells to other T helper (Th) subsets is controversial. We find that after helminth infection, or immunization with helminth antigens, reactive lymphoid organs of 4get IL-4/GFP reporter mice contain populations of IL-4/GFP-expressing CD4(+) T cells that display the TFH markers CXCR5, PD-1, and ICOS. These TFH cells express the canonical TFH markers BCL6 and IL-21, but also GATA3, the master regulator of Th2 cell differentiation. Consistent with a relationship between Th2 and TFH cells, IL-4 protein production, reported by expression of huCD2 in IL-4 dual reporter (4get/KN2) mice, was a robust marker of TFH cells in LNs responding to helminth antigens. Moreover, the majority of huCD2/IL-4-producing Th cells were found within B cell follicles, consistent with their definition as TFH cells. TFH cell development after immunization failed to occur in mice lacking B cells or CD154. The relationship of TFH cells to the Th2 lineage was confirmed when TFH cells were found to develop from CXCR5(-) PD-1(-) IL-4/GFP(+) CD4(+) T cells after their transfer into naive mice and antigen challenge in vivo.
Differentiation of T(H)1 and T(H)2 effector cells proceeds through several phases: First, naïve CD4(+) precursor cells are instructed to differentiate as appropriate to optimally fight the infectious threat encountered. This process is governed by the IL12 and IL4 cytokines, as well as by signaling through the Notch receptor. In response to these signals, transcription is initiated of lineage specific cytokine genes including the Ifngamma and Il4 genes as well as of genes encoding transcriptional regulators, such as T-bet and Gata3. The respective differentiation programs are reinforced by both positive and negative feedback mechanisms. Furthermore, epigenetic modifications of the lineage specific genes result in the emergence of regulatory elements, which control high level lineage restricted expression by both intrachromosomal and interchromosomal associations. Together, these mechanisms ensure stable inheritance of the differentiated fate in the numerous progeny of the original naïve CD4(+) T cells.
To characterise fish haematopoietic stem/progenitor cells, it is necessary to develop a culture system that supports proliferation and differentiation of these cells. In the present study, we established cell lines from various tissues of carp (Cyprinus carpio) and ginbuna (Carassius auratus langsdorfii). By using these cell lines, we developed a culture system in which carp haematopoietic cells proliferated and were successively passaged. Cell lines from carp thymus (KoT), carp fin (KoF1) and ginbuna thymus (GTS6 and GTS9) were newly established. In addition to these cell lines, ginbuna fin (CFS) cell lines were also used as feeder layers. Kidney haematopoietic cells co-cultured with these feeder layers proliferated rapidly and were passaged over 20 times for more than 60 days. To characterise the proliferating cells, expression of marker genes for blood cell development were analysed. In the primary culture, marker genes for myeloid/erythroid progenitors (gata1), haematopoietic stem cells (gata2), neutrophils (mpx/mpo), B-cells (IgH) and T-cells (lck, TCRbeta and gata3) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Expression of most of the genes disappeared after the third passage, only T-cell marker genes were highly expressed after passages. These results indicate that multiple blood cells developed in the primary culture and then T-cell lineages dominantly proliferated after several passages.
Transcription factor GATA2 is expressed in numerous mammalian tissues, including neural, hematopoietic, cardiovascular and urogenital systems, and yet it plays important roles in the regulation of tissue-restricted gene expression. The Gata2 gene itself is also under stringent tissue-specific control and multiple cis-regulatory domains have been identified in the Gata2 locus. In this study we sought out and then examined in detail the domains that regulate Gata2 in the midbrain. We identified two discrete domains in the Gata2 promoter that direct midbrain expression; these distal 5H and proximal 2H regulatory domains are located 3.0 and 1.9 kbp, respectively, upstream of the transcriptional initiation site. Importantly, both domains contain GATA factor binding sites. Our analyses further revealed that GATA2 is essential for Gata2 gene expression in the midbrain, whereas GATA3 is not. Both the 2H and 5H domains have the independent ability to activate Gata2 gene expression in the midbrain superior colliculus, whereas the distal-5H domain is additionally capable of activating Gata2 transcription in the inferior colliculus. These results demonstrate that two distinct regulatory domains contribute to the Gata2 gene expression in the mouse midbrain and that Gata2 midbrain transcription is under positive autoregulation.
T helper 1 (Th1)-Th2 cell balance is key to host defense and its dysregulation has pathophysiological consequences. Basophils are important in Th2 cell differentiation. However, the factors controlling the onset and extent of basophil-mediated Th2 cell differentiation are unknown. Here, we demonstrate that Lyn kinase dampened basophil expression of the transcription factor GATA-3 and the initiation and extent of Th2 cell differentiation. Lyn-deficient mice had a marked basophilia, a constitutive Th2 cell skewing that was exacerbated upon in vivo challenge of basophils, produced antibodies to a normally inert antigen, and failed to appropriately respond to a Th1 cell-inducing pathogen. The Th2 cell skewing was dependent on basophils, immunoglobulin E, and interleukin-4, but was independent of mast cells. Our findings demonstrate that basophil-expressed Lyn kinase exerts regulatory control on Th2 cell differentiation and function.
Missing data pose one of the greatest challenges in the rigorous evaluation of biomarkers. The limited availability of specimens with complete clinical annotation and quality biomaterial often leads to underpowered studies. Tissue microarray studies, for example, may be further handicapped by the loss of data points because of unevaluable staining, core loss, or the lack of tumor in the histospot. This paper presents a novel approach to these common problems in the context of a tissue protein biomarker analysis in a cohort of patients with breast cancer. Our analysis develops techniques based on multiple imputation to address the missing value problem. We first select markers using a training cohort, identifying a small subset of protein expression levels that are most useful in predicting patient survival. The best model is obtained by including both protein markers (including COX6C, GATA3, NAT1, and ESR1) and lymph node status. The use of either lymph node status or the four protein expression levels provides similar improvements in goodness-of-fit, with both significantly better than a baseline clinical model. Using the same multiple imputation strategy, we then validate the results out-of-sample on a larger independent cohort. Our approach of integrating multiple imputation with each stage of the analysis serves as an example that may be replicated or adapted in future studies with missing values.
Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients.
The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated.
Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated.
T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance.
The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.
Multiple sclerosis (MS) is a devastating CNS disease of unknown origin. Multiple factors including genetic background, infection, and psychological stress affect the onset or progression of MS. Theiler's murine encephalomyelitis virus (TMEV) infection is an animal model of MS in which aberrant immunity leads to viral persistence and subsequently results in demyelination that resembles MS. Here, we examined how stress during acute TMEV infection altered virus-specific cell mediated responses. Using immunodominant viral peptides specific for either CD4(+) or CD8(+) T cells, we found that stress reduced IFN-gamma producing virus-specific CD4(+) and CD8(+) T cells in the spleen and CD8(+) T cells CNS. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus or viral peptides, indicated that stress decreased both type 1 and type 2 responses. Glucocorticoids were implicated in the decreased T cell function as the effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone. As T cells mediate viral clearance in this model, our data support the hypothesis that stress-induced immunosuppression may provide a mechanism for enhanced viral persistence within the CNS.
IFN-lambda1 (IL-29) plays a novel, emerging role in the inhibition of human Th2 responses. Here, we demonstrate that both naive and memory human CD4(+) T cells express mRNA for the IFN-lambda1-specific receptor, IL-28Ralpha, and are responsive to IFN-lambda1. Expression of Th2 cytokines (IL-4 and IL-13) was suppressed in naive and memory CD4(+) T cells by IFN-lambda1, without affecting their proliferation. Further, acquisition of IL-4Ralpha expression after stimulation was inhibited by IFN-lambda1, as was GATA3 expression. Finally, IFN-lambda1 diminished the change in cell-surface phenotype that accompanies differentiation of "central memory" T cells into "effector memory" T cells. Taken together, our data describe unique immunomodulatory effects of IFN-lambda1 and identify novel mechanisms for the reduction of existing Th2 responses and the regulation of new ones, in circulating naive and memory CD4(+) T cells.
In order to analyze the effect of vitamin E on Th1 and Th2 cytokine production, porcine peripheral blood mononuclear cells (PBMC) were isolated from healthy pigs (n=8) and cultured with either 0, 10, 50, or 100muM of vitamin E (alpha-tocopherol). PBMC were stimulated with PHA for either, 24h to determine: (a) the concentration of tocopherol incorporated into the cell membrane, (b) cytokine production and (c) Th1 and Th2 regulators gene expression; or 72h to determine the proliferation of PBMC. Vitamin E was incorporated into the PBMC in a dose dependent manner, giving as a result a high proliferation of cells irrespective of the dose of vitamin E used. Regarding cytokine production, vitamin E consistently decreases the mRNA expression and the percentage of cells producing IL-10. Vitamin E did not influence the production of IFN-gamma but the lowest level of vitamin E (10muM) was sufficient to maximally increase the proportion of cells producing IL-2, to diminish IL-4, and discreetly increase the mRNA expression of TBX21 vs. GATA3. In conclusion, our results revealed that vitamin E is able to suppress IL-10 production and to influence the production of IL-2, IL-4, and maybe TBX21. Vitamin E clearly has immunomodulatory effects, though further work in vivo to determine the physiological nature of these effects is warranted.
Apigenin, a dietary plant-flavonoid has shown anti-inflammatory and anticancer properties, however the molecular basis of this effect remains to be elucidated. Thus we elucidated to anti-allergic effect of apigenin in ovalbumin (OVA)-induced asthma model mice. The OVA-induced mice showed allergic airway reactions. It included an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid, an increase in inflammatory cell infiltration into the lung around blood vessels and airways, airway luminal narrowing, and the development of airway hyper-responsiveness (AHR). The administration of apigenin before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that apigenin plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of apigenin in terms of its effects in a murine model of asthma.
Sinomenine has been used to treat autoimmune diseases for centuries. However, the mechanism underlying its therapeutic effects remains unknown. Increasing recognition of the importance of the Th1/Th2 imbalance in nephritis has raised the questions of whether there is a Th1/Th2 imbalance in patients with mesangial proliferative nephritis (MsPGN) and whether sinomenine can modulate the Th1/Th2 imbalance. In this study, 25 MsPGN patients were treated with sinomenine and followed for 3 months. The expression of T-bet and GATA-3 mRNA in peripheral blood mononuclear cells (PBMCs) and the serum levels of interferon-gamma (IFN-gamma), interleukin (IL)-4, and IL-10 were studied at month 0, month 1, and month 3. The intra-renal expression of T-bet and GATA-3 was studied via immunohistochemistry. Results reveal that PBMCs from MsPGN patients expressed high levels of T-bet mRNA and low levels of GATA-3 mRNA, and the T-bet/GATA-3 ratio in MsPGN patients was significantly higher than that in healthy donors. Meanwhile, MsPGN patients were found to have simultaneously elevated IFN-gamma values and decreased IL-10 values. Immunohistochemistry revealed increased T-bet and decreased GATA-3 expression in renal tissues from MsPGN patients. Moreover, sinomenine was found to cause a decrease in T-bet mRNA expression, resulting in a drop in the T-bet/GATA-3 ratio. Sinomenine was also found to elicit a decrease in the serum levels of IFN-gamma. These results suggest that a shift toward the Th1 pathway of Th cell activation occurs in MsPGN patients, and that sinomenine has the potential to counter this shift in the Th1/Th2 balance and thereby produce therapeutic effects.
Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. Here, we provide a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. We further demonstrate that LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord.
To evaluate the effect of fumigating with Yinxieling (YXL) in treating patients with psoriasis vulgaris and its influence on T-bet and GATA-3 protein expressions in peripheral blood monocyte (PBMC).
Western blot method was employed to detect the T-bet and GATA-3 protein expressions in PBMC of 30 psoriasis vulgaris patients before and after they received fumigation therapy with YXL, also in 25 healthy persons for controls. The therapeutic efficacy was observed and the relationship between PASI scores and levels of T-bet and GATA-3 analyzed.
After treatment, 12 out of the 30 patients were cured, 9 were markedly effective, 8 effective and 1 unchanged, the cure rate being 40.0% and the effective rate 96.7%. Level of T-bet expression in PBMC of patients was 1.7917 +/- 0.3840, which was higher than that of healthy persons (0.8860 +/- 0.1486, P < 0.01), but the GATA3 expression was lower than that in control (0.8777 +/- 0.3114 vs. 1.2384 +/- 0.1783, P < 0.01). However, the two indexes were restored after fumigation to 1.3410 +/- 0.3642 and 1.0883 +/- 0.2435 respectively, showing significant difference to those before fumigation (P < 0.01). Correlation analysis showed that PASI score was positively correlated with level of T-bet expression (r = 0.7448, P < 0.01) and negatively correlated with level of GATA-3 expression (r = -0.8291, P < 0.01).
Fumigation therapy is effective in treating psoriasis vulgaris, its mechanism is possibly by way of modulating the equilibrium of the transcription factors T-bet and GATA-3 protein expressions in PBMC, and rectifying the immune abnormality of Th1/Th2 subsets imbalance.
Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3(+)CD25(+) T cells, an effect that was reversible by IFN-alpha treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3(+)CD25(+) T cells is dependent on TGF-beta but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3(+)CD25(+) T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3(+)CD25(+) T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-alpha treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.
Naturally occurring regulatory T cells have been associated with long-term allograft survival. We investigated whether gene transcripts of Treg-related molecules are upregulated or downregulated in kidney transplant recipients with different clinical outcomes and may serve as markers of operative tolerance.
Expression levels of transcription factor (forkhead box P3 [FOXP3], t-bet, and GATA3), regulatory molecule (cytotoxic T-lymphocyte antigen-4, glucocorticoid-induced tumor necrosis factor receptor-related protein, tribbles protein-1, and transforming growth factor-beta), and chemokine receptor (CCR7 and CXCR4) genes were measured in kidney graft recipients with long-term (> or = 9 years) stable renal function (LTS) or chronic rejection (ChrRx). Patients on dialysis and healthy individuals served as controls.
The level of FOXP3 transcripts was lower in ChrRx patients than in LTS patients (P<0.01). The highest transforming growth factor-beta transcripts were observed in ChrRx and the highest CCR7 and CXCR4 transcripts were observed in LTS patients. In LTS patients, FOXP3 gene expression was associated with CXCR4 gene expression (P=0.015). FOXP3 and CCR7 transcript levels were higher in LTS patients without calcineurin inhibitor therapy than in LTS patients with calcineurin inhibitors.
Our results suggest that high expression of FOXP3 and chemokine receptor genes in LTS patients are possible indicators of a regulatory process that contributes to long-term allograft acceptance. Markers that were increased in LTS patients were found to be decreased in ChrRx patients, suggesting that rejection may partly be the result of a lack of this regulatory process. FOXP3 and CCR7 and CXCR4 transcripts might be used as markers to distinguish patients who developed long-term allograft acceptance from patients who are prone to ChrRx.
Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists have been shown to have a therapeutic benefit in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). In this study, we investigated the mechanism by which the PPARalpha agonist gemfibrozil induces immune deviation and protects mice from EAE. We demonstrated that treatment with gemfibrozil increases expression of the Th2 transcription factor GATA-3 and decreases expression of the Th1 transcription factor T-bet in vitro and directly ex vivo. These changes correlated with an increase in nuclear PPARalpha expression. Moreover, the protective effects of PPARalpha agonists in EAE were shown to be partially dependent on IL-4 and to occur in a receptor-dependent manner. PPARalpha was demonstrated, for the first time, to regulate the IL-4 and IL-5 genes and to bind the IL-4 promoter in the presence of steroid receptor coactivator-1, indicating that PPARalpha can directly transactivate the IL-4 gene. Finally, therapeutic administration of PPARalpha agonists ameliorated clinically established EAE, suggesting that PPARalpha agonists may provide a treatment option for immune-mediated inflammatory diseases.
Chronic urticaria (CU), defined as the recurring incidence of wheals with or without angioedema for more than 6 weeks, is a common disorder associated with mast cell activation, degranulation, and histamine release. Considering the association between the CRTH2 gene and mast cells, we investigated the association of this gene polymorphism with the CU phenotype and antihistamine drug requirement in patients with CU.
Two groups consisting of 384 patients with CU and 231 patients as normal controls (NCs) were enrolled from the Department of Allergy and Rheumatology, Ajou University Hospital, Suwon, Korea. Two polymorphisms of the CRTH2 gene, -466T>C and -129C>A were genotyped using primer extension methods.
No significant differences were detected in the genotype and allele frequencies of the two CRTH2 polymorphisms between the CU and NC groups, and no significant associations were observed with clinical parameters, such as atopy status, serum total IgE, prevalence of autoantibodies and duration of CU. However, CU patients with homozygous TT genotypes had significantly higher dose requirements of antihistamines to control the CU symptoms (164.56 +/- 115.62 vs 137.38 +/- 90.15 loratadine equivalents, mg/week) than those with the CT and CC genotypes (p = 0.025). The luciferase activity was significantly enhanced in the construct containing CRTH2 466C compared with the -466T-containing construct (p < 0.001). Co-transfection experiments with GATA-3 (300 ng) and the -466T and -466C CRTH2 alleles revealed that the CRTH2 -466T allele produced a greater increase in induction of luciferase activity than the -466C allele (p < 0.001).
The CRTH2 -466T>C gene polymorphism may not affect on the phenotype of CU, but contributes to the required dose of antihistamines in patients with CU.
The maintenance of memory CD4 T cells is crucial for the establishment of immunological memory. The Polycomb (PcG) and Trithorax group (TrxG) genes control key developmental regulators such as the homeobox genes, and these two antagonize each other in the same developmental processes. Recently, PcG gene Bmi1 has been found to control memory Th1/Th2 cell survival and TrxG gene MLL is to control the maintenance of memory Th2 cell function selectively. Therefore, in memory CD4 T cells, PcG and TrxG genes appear to control distinct processes in a distinct manner, which indicates a novel regulatory feature of the PcG/TrxG genes.
The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2-driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4(+) T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2-polarizing concentrations of ET and CyaA selectively inhibit TCR-dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR-signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.
Oral intake of protein leads to tolerance through the induction of regulatory T cells (Tr cells) in mesenteric lymph nodes (MLNs). Here we show that the inhibition of cyclooxygenase-2 (COX-2) in vivo suppressed oral tolerance and was associated with enhanced differentiation of interleukin (IL)-4-producing T cells and reduced Foxp3(+) Tr-cell differentiation in MLN. As a result, the functional suppressive capacity of these differentiated mucosal T cells was lost. IL-4 was causally related to loss of tolerance as treatment of mice with anti-IL-4 antibodies during COX-2 inhibition restored tolerance. Dendritic cells (DCs) in the MLN differentially expressed COX-2 and reductionist experiments revealed that selective inhibition of the enzyme in these cells inhibited Foxp3(+) Tr-cell differentiation in vitro. Importantly, the inhibition of COX-2 in MLN-DC caused increased GATA-3 expression and enhanced IL-4 release by T cells, which was directly related to impaired Tr-cell differentiation. These data provide crucial insights into the mechanisms driving de novo Tr-cell induction and tolerance in the intestine.
Breast cancer is a heterogeneous disease and classification is important for clinical management. At least five subtypes can be identified based on unique gene expression patterns; this subtype classification is distinct from the histopathological classification. The transcription factor network(s) required for the specific gene expression signature in each of these subtypes is currently being elucidated. The transcription factor network composed of the oestrogen (estrogen) receptor alpha (ERalpha), FOXA1 and GATA3 may control the gene expression pattern in luminal subtype A breast cancers. Breast cancers that are dependent on this network correspond to well-differentiated and hormone-therapy-responsive tumours with good prognosis. In this review, we discuss the interplay between these transcription factors with a particular emphasis on FOXA1 structure and function, and its ability to control ERalpha function. Additionally, we discuss modulators of FOXA1 function, ERalpha-FOXA1-GATA3 downstream targets, and potential therapeutic agents that may increase differentiation through FOXA1.
Decreased infiltration of Foxp3+ T regulatory cell (Treg) is considered to be critical for the Th1/Th2 dysregulation of nasal polyps, while the cellular mechanism underlying Foxp3+ Treg insufficiency is currently not well defined.
We attempted to investigate the tissue expression of phosphorylated mammalian target of rapamycin (pmTOR) and infiltration of Foxp3+ Tregs in 28 nasal polyps and 16 controls by histological staining. We also evaluated the effects of blocking the mTOR signaling pathway with rapamycin on T cell phenotype selection and Foxp3+CD4+ Tregs expansion in a tissue culture system.
Significantly increased infiltration of pmTOR+ inflammatory cells and decreased infiltration of Foxp3+CD4+ Tregs into nasal polyps was observed, with an inverse association. In the tissue culture system, we detected significantly elevated Foxp3 expression and IL-10 production, as well as an increased percentage of Foxp3+ Tregs in nasal polyps after blocking the mTOR signaling pathway with rapamycin.
Here we demonstrate for the first time that the mTOR signaling pathway is associated with Foxp3+ Tregs insufficiency in nasal polyps. Inhibition of the mTOR signaling pathway may be helpful for enhancement of Foxp3+ Treg expansion, as well as modulation of T cell phenotype imbalances in nasal polyps.
Human GATA3 haploinsufficiency leads to HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome. The development of a specific subset of organs in which this transcription factor is expressed appears exquisitely sensitive to gene dosage. We report on a 14-year-old patient with symptomatic hypoparathyroidism, sensorineural bilateral deafness, unilateral renal dysplasia, bilateral palpebral ptosis, and horizontal nystagmus. Fundoscopy displayed symmetrical pseudopapilledema, and brain CT scan revealed basal ganglia calcifications. FISH analysis did not disclose any microdeletion in the 22q11.2 or 10p14 regions. GATA3 mutation analysis identified a heterozygous deletion of GG nucleotides at codon 36 and 37 (c.108_109delGG) in exon 2 causing a frameshift with a premature stop codon after a new 15-aminoacid sequence. Restriction endonuclease analysis performed in parents was negative. Our patient carries a novel "de novo" GATA3 mutation, providing further evidence that HDR syndrome is caused by haploinsufficiency of GATA3, which may be responsible for a complex neurologic picture besides the known triad.
Rheumatoid arthritis is associated with the infiltration of T helper cells into the joints. It is unclear whether interferon-gamma (IFNgamma)-producing Th1 cells or the novel T helper subset, interleukin-17 (IL-17)-producing Th17 cells, are the pathogenic mediators of joint inflammation in chronic nonautoimmune arthritis. Therefore, this study was aimed at examining whether the Th2-specific transcription factor GATA-3 can regulate arthritis, in an experimental murine model, by modulating Th1 and/or Th17 cell polarization.
Arthritis was induced with methylated bovine serum albumin (mBSA) in both wild-type and CD2 T cell-specific GATA-3 (CD2-GATA-3)-transgenic mice. At days 1 and 7 after the induction of arthritis, knee joints were scored macroscopically for arthritis severity and for histologic changes. Single-cell suspensions were generated from the spleens, lymph nodes, and inflamed knee joints. Cytokine expression by CD4+ T cells was determined using flow cytometry, and IL-17 expression in the inflamed knee joints was determined by enzyme-linked immunosorbent assay. Analyses of gene expression were performed for Th17-associated factors.
Wild-type mice developed severe joint inflammation, including massive inflammatory cell infiltration and bone erosion that increased significantly over time, reaching maximal arthritis scores at day 7. In contrast, only mild joint inflammation was observed in CD2-GATA-3-transgenic mice. This mild effect was further accompanied by systemic and local reductions in the numbers of IL-17+IFNgamma- and IL-17+IFNgamma+, but not IL-17-IFNgamma+, CD4+ T cells, and by induction of Th2 cytokine expression. Moreover, GATA-3 overexpression resulted in reduced gene expression of the Th17-associated transcription factor retinoic acid-related orphan receptor gammat.
These results indicate that enforced GATA-3 expression protects against severe joint inflammation and bone erosion in mice, accompanied by reduced differentiation of Th17 cells, but not Th1 cells, during mBSA-induced arthritis.
Expression of heat shock proteins has been described in different tissues relevant to human reproduction, including placenta. AlphaB-crystallin is a member of the small heat shock protein family (sHsp) exerting biologically important chaperon functions.
Immunofluorescence; immunoblot analysis; quantitative real-time-PCR; CpG island methylation analysis.
In this study, we once again describe the expression of alphaB-crystallin in the stroma of the placental villi and in the cytoplasm of decidual cells by immunofluorescence. In contrast, Hsp27--another sHsp family member--was detected exclusively in the syncytiotrophoblast layer. This varying expression pattern provides additional support to earlier reports of functional differences between both proteins. Semi-quantitative immunoblot analysis of placenta tissue specimens (n = 6) revealed Hsp27 expression exceeding that of alphaB-crystallin, albeit with interindividual variations. Inter-individual alphaB-crystallin expression variations were confirmed by quantitative RT-PCR. CpG island methylation was ruled out as the underlying cause for the inter-individual alphaB-crystallin expression variations. However, the expression extent of GATA3, which is a transcription factor with corresponding elements within the alphaB-crystallin gene (CRYAB) promoter, paralleled that of alphaB-crystallin. We demonstrated remarkable GATA3 expression in placental tissue, exceeding that of other endocrine organs.
We can conclude that the differential expression patterns of alphaB-crystallin and Hsp27 indicate functional differences between these highly related proteins in placental tissues.
Gamma-secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. In asthma, a subset of CD4(+) T cells is believed to initiate and perpetuate the disease.
The aim of this study was to evaluate the therapeutic potential of GSI against allergic asthma.
GSI was administered to an ovalbumin-sensitized mouse via an intranasal route at the time of ovalbumin challenge.
The administration of GSI inhibits asthma phenotypes, including eosinophilic airway inflammation, goblet cell metaplasia, methacholine-induced airway hyperresponsiveness, and serum IgE production. GSI treatment of bronchoalveolar lavage cells stimulated via TCR or non-TCR pathways led to a decrease in Th2 cytokine production with a concomitant increase in Th1 cytokine secretion. Expression of Hes-1, a target of Notch signaling, was down-regulated in conjunction with a reduction of Notch intracellular domain and GATA-3 levels after GSI treatment of bronchoalveolar lavage cells. GSI treatment resulted in an inhibition of NF-kappaB activation, and combined treatment with GSI and an NF-kappaB inhibitor augmented IFN-gamma production in a synergistic manner.
These data suggest that GSI directly regulates Th1 and Th2 responses in allergic pulmonary inflammation through a Notch signaling-dependent pathway and that GSI is of high therapeutic value for treating asthma by inhibiting airway inflammatory responses.
Among Tissue kallikrein genes (KLKs), KLK1 is abundantly expressed in human skin. Although its putative promoter is known to have various cis-elements, they have not been functionally tested. In the present study, the regulation mechanism of KLK1 promoter supporting such abundant expression was examined. Luciferase assay targeting the KLK1 promoter (nucleotide -1153/+40 from the major transcriptional start site) was performed on NHEK human keratinocyte. -954/-855, -428/-236, and -100/+40 had the induction activity. The motif search program failed to find unique binding motifs in -428/-236, whereas both -954/-855 and -100/+40 had a unique GATAs binding motif. Electrophoretic mobility shift assay (EMSA) and DNA footprinting confirmed the binding of NHEK nuclear protein to these motifs that were supershifted by anti-GATA3 antibody. Among GATA isoforms, GATA3 alone could be amplified in RNA obtained from NHEK. Moreover, introduction of GATA3 into fibroblastic NIH3T3 cells enhanced the activity of KLK1 promoter containing -954/+40, while that of GATA3 dominant negative mutant to NHEK cells impaired the same promoter's activity. Thus, GATA3 was found to bind the site located at -954/-855 and to be a key regulator of abundant KLK1 expression in human keratinocyte.
To study the mechanism of Ma Xing Shi Gan decoction on the cytokines of Th1/Th2 in a murine model of asthma.
The murine model of asthma was induced by OVA. Various doses of Ma Xing Shi Gan decoction were orally administered to the mice respectively. The spleen cells were cultured 3 days for measurement of IFN-gamma, IL4 and IL-5 by ELISA. After 2 days' culture, the spleen cells were treated with Trizol for extraction of total RNA. The gene expression of T-bet, GATA-3 and STAT-6 were analyzed by RT-PCR.
After the treatment with Ma Xing Shi Gan decoction, the production of IL-4 and IL-5 and the gene expression of GATA-3, STAT-6 obviously decreased.
Ma Xing Shi Gan decoction may modulate Thl/Th2 balance by suppressing the gene expression of GATA-3 and STAT-6 in vivo.
There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.
Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.
The identification of novel genes by high-throughput studies of complex diseases is complicated by the large number of potential genes. However, since disease-associated genes tend to interact, one solution is to arrange them in modules based on co-expression data and known gene interactions. The hypothesis of this study was that such a module could be a) found and validated in allergic disease and b) used to find and validate one ore more novel disease-associated genes.
To test these hypotheses integrated analysis of a large number of gene expression microarray experiments from different forms of allergy was performed. This led to the identification of an experimentally validated reference gene that was used to construct a module of co-expressed and interacting genes. This module was validated in an independent material, by replicating the expression changes in allergen-challenged CD4+ cells. Moreover, the changes were reversed following treatment with corticosteroids. The module contained several novel disease-associated genes, of which the one with the highest number of interactions with known disease genes, IL7R, was selected for further validation. The expression levels of IL7R in allergen challenged CD4+ cells decreased following challenge but increased after treatment. This suggested an inhibitory role, which was confirmed by functional studies.
We propose that a module-based analytical strategy is generally applicable to find novel genes in complex diseases.
Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common solid tumor in childhood. By microarray expression analysis (Affymetrix HU133A) important players in the noradrenalin biosynthesis pathway (DBH, DDC, GATA2, GATA3, PHOX2A, PHOX2B, SLC6A2 SLC18A1 and TH) were found to be among the top ranked genes in showing lower expression in unfavorable NB tumor types as compared to favorable ones. By quantitative PCR with TaqMan, this result was significantly verified for all transcripts (p<0.05, one-tailed) in a new set of 11 primary NB tumors (5 favorable vs. 6 unfavorable). PHOX2A, a downstream target of Phox2b, was found to be the sixth ranked gene from the microarray gene list. Since the PHOX2A gene is localized in a tumor suppressor candidate region at 11q, we screened this gene for mutations by DNA sequencing in 47 tumors of different stages. However, no critical changes were found that could support its role in tumor development or progression. Overall, the findings in this study either suggest that expression of this pathway could be a predictive differentiation marker of NB tumors, or our results could also imply that the noradrenalin biosynthesis pathway is involved in tumor pathogenesis.
The cytidine deaminases APOBEC3G and APOBEC3F exert anti-HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1) and 2 (Th2) effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti-interferon-gamma antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-gamma contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif.
Heterozygous somatic mutations of the transcription factor, GATA-3, have recently been reported in approximately 5% breast of tumors unselected for family history. We sequenced the GATA-3 gene in 55 breast tumors from women with familial breast cancer, and found seven heterozygous somatic mutations, all in non-BRCA1/2 cases in which the frequency was 22%. In contrast, we found mutations of GATA-3 in only 4% of 81 sporadic tumors analysed. It is possible that GATA3 mutations occur earlier in the evolution of BRCAx tumors, compared to BRCA1, BRCA2 or sporadic tumors, and are therefore easier to detect by direct sequencing in the presence of some stromal contamination.
To investigate the relationship between the dendritic cell (DC) subsets and transcriptive factors, T-bet, GATA-3, and immune imbalance in acquired severe aplastic anemia (SAA).
The DC1 (HLA-DR+Lin-CD11c+) and DC2 (HLA-DR+Lin-CD123+) in peripheral blood mononuclear cells (PBMNC) were measured with flow cytometry (FCM), the expressions of T-bet mRNA and GATA-3 mRNA in PBMNC with semiquantitative RT-PCR and the plasma level of IFN gamma and IL-4 with ELISA in 29 SAA patients and 16 healthy controls.
The percentages of DC1 in PBMNC were (0.44 +/- 0.24)% and (0.73 +/- 0.30)% in untreated and recovered SAA patients respectively, both were higher than that in controls (0.29 +/- 0.10)% (P < 0.05). The percentage of DC2 in the untreated cases was lower than that of recovered ones or controls [(0.18 +/- 0.14)% vs (0.28 +/- 0.20)% and (0.29 +/- 0.13)%] (P < 0.05). DC1/DC2 ratios were 3.45 +/- 2.71 and 2.90 +/- 0.95 in untreated and recovered groups respectively, both were higher than that in controls (1.15 +/- 0.56) (P < 0.05). No statistic difference in DC1/DC2 ratio was found between untreated and recovered patients (P < 0.05). The relative mRNA expression levels of transcriptive factor T-bet were 0.37 +/- 0.07, 0.20 +/- 0.07 and 0.17 +/- 0.05 in the above 3 groups, respectively, untreated group being higher than that of recovered group or healthy controls (P < 0.05). There was no statistic difference of GATA-3 expression among the 3 groups (P > 0.05). T-bet/GATA-3 ratio was 0.72 +/- 0.13 in untreated group, being higher than that of recovered group (0.33 +/- 0.08) or controls (0.35 +/- 0.11). The plasma level of IFN gamma in the untreated group was (50.9 +/- 1.1) ng/L, which was higher than that of recovered group [(49.7 +/- 0.9) ng/L] or controls [(49.7 +/- 0.7) ng/L]. There was significant positive correlations between T-bet and DC1/DC2 ratio (r = 0.445, P < 0.01), as well as between T-bet and IFN gamma (r = 0.402, P < 0.01).
Either DC1/DC2 or T-bet/GATA-3 ratio might become an index to estimate immune imbalance. High-expressed T-bet was related to the progress of SAA. In patients with SAA, DC1/DC2 ratio returns to normal range later than that of routine blood test does, indicating that immunosuppressive therapy should not be withdrawn too earlier.
Recent research has uncovered complex transcription factor networks that control the processes of T-cell development and differentiation. RUNX (runt-related transcription factor) proteins are among the many factors that have crucial roles in these networks. In this Review, we examine the mechanisms by which RUNX complexes act together with other transcription factors, such as Th-POK (T-helper-inducing POZ/Kruppel-like factor) and GATA-binding protein 3 (GATA3) in determining the CD4/CD8 lineage choice of developing thymocytes. In addition, we discuss evidence indicating that RUNX complexes are also involved in the differentiation of effector T-cell subsets and that the molecular mechanisms by which RUNX proteins regulate T-cell fate decisions are conserved between the thymus and periphery.
In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.
NKT cells comprise a rare regulatory T cell population of limited TCR diversity, with most cells using a Valpha14 Jalpha18 TCR. These cells exhibit a critical dependence on the signaling adapter molecule, signaling lymphocytic activation molecule-associated protein (SAP), for their ontogeny, an aspect not seen in conventional alphabeta T cells. Prior studies demonstrate that SAP enhances TCR-induced activation of NF-kappaB in CD4(+) T cells. Because NF-kappaB is required for NKT cell development, SAP might promote the ontogeny of this lineage by signaling to NF-kappaB. In this study, we demonstrate that forced expression of the NF-kappaB target gene, Bcl-x(L), or inhibitory NF-kappaB kinase beta, a catalytic subunit of the IkappaB kinase complex essential for NF-kappaB activation, fails to restore NKT cell development in sap(-/-) mice, suggesting that SAP mediates NKT cell development independently of NF-kappaB. To examine the role of SAP in NKT cell function, we generated NKT cells in sap(-/-) mice by expressing a transgene encoding the Valpha14 Jalpha18 component of the invariant TCR. These cells bound alpha-galactosylceramide-loaded CD1d tetramers, but exhibited a very immature CD24(+)NK1.1(-) phenotype. Although sap(-/-) tetramer-reactive cells proliferated in response to TCR activation, they did not produce appreciable levels of IL-4 or IFN-gamma. The reduction in cytokine production correlated with the near absence of GATA-3 and T-bet, key transcription factors regulating cytokine expression and maturation of NKT cells. Ectopic expression of GATA-3 partially restored IL-4 production by the NKT cells. Collectively, these data suggest that by promoting GATA-3 and T-bet expression, SAP exerts control over NKT cell development and mature NKT cell cytokine production.
Many advances in our understanding of the molecules that regulate the development, differentiation and function of T cells have been made over the past few years. One important regulator of T-cell differentiation is the transcription factor GATA-binding protein 3 (GATA3). Although the main function of GATA3 is to act as a master transcription factor for the differentiation of T helper 2 (T(H)2) cells, new research has helped to uncover crucial functions of GATA3 in T cells that go beyond T(H)2-cell differentiation and that are important at earlier stages of haematopoietic and lymphoid-cell development. This Review focuses on the functions of GATA3 from early thymocyte development to effector T-cell differentiation. In addition, we discuss the interactions between GATA3 and other transcription factors and signalling pathways, and highlight the functional significance of the GATA3 protein structure.
There is growing evidence that hypoxia-inducible transcription factors are involved in the pathophysiology of asthma. Hypoxia-inducible factor-1alpha (HIF-1alpha) in particular controls the expression of many hypoxia regulated genes, but whether HIF-1alpha directly contributes to allergen-driven immune responses is not known.
Partially HIF-1alpha-deficient mice (HIF-1alpha(+/-)) or wild-type littermate controls were used in all experiments. Spleen CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and cytokine secretion was measured in vitro. Mice were sensitized by intraperitoneal injection of ovalbumin (Ova) plus alum, and then challenged by intranasal Ova followed by bronchoalveolar lavage (BAL) and isolation of spleen cells. BAL cells were counted and the differential determined using cytospin, and splenocytes were incubated with Ova to measure recall cytokine production.
Interferon-gamma secretion was significantly higher in anti-CD3 plus anti-CD28 stimulated CD4+ T cells obtained from HIF-1alpha(+/-) mice compared to wild-type controls. HIF-1alpha(+/-) mice were protected from lung eosinophilia 72 h after allergen challenge, in association with enhanced secretion of interferon-gamma in recall responses of splenocytes.
HIF-1alpha contributes to allergic immune responses and lung eosinophilia in a mouse model of asthma.
Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.
Ikaros, a hematopoietic transcription factor, has well defined effects on early lymphocyte development in the bone marrow and thymus. In this study we demonstrate that Ikaros is a positive regulator of Th2 cytokine gene expression in peripheral T cells. CD4+ T cells from naive Ikaros(null) mice cultured under Th2-skewing conditions express the Th1 cytokine IFN-gamma and have reduced IL-4, IL-5, and IL-13 expression. Ikaros directly associates with several Th2 locus regulatory regions in naive CD4+ T cells. The decreased ability to express Th2 cytokines in Ikaros(null)T cells corresponds with histone 3 hypoacetylation across the Th2 cytokine locus as well as decreased GATA3 and cMaf and increased T-bet and STAT1 expression. These data support a model whereby Ikaros directly activates Th2 gene expression by promoting local chromatin accessibility during CD4+ T cell differentiation and also acts indirectly to regulate expression of Th2- and Th1-specific transcription factors.
Glucocorticoid (GC) immunosuppression and anti-inflammatory action involve the regulation of several transcription factors (TFs). GCs inhibit the acute production of T-helper (Th) 1 and Th2 cytokines but ultimately favor a shift toward Th2 phenotype. GCs inhibit the transcriptional activity of T-bet Th1 TF by a transrepression mechanism. Here we analyze GC regulation of GATA-3, the master driver of Th2 differentiation. We found that GCs inhibit GATA-3 transcriptional activity. We demonstrate that this mechanism does not involve physical interaction between the glucocorticoid receptor (GR) and GATA-3 or reduction of GATA-3 binding to DNA, as described previously for T-bet. Instead, GCs inhibit GATA-3 activity by inhibition of p38 mitogen-activated protein kinase induced GATA-3 phosphorylation. GCs also inhibit GATA-3 mRNA and protein expression. Finally, GATA-3 inhibition affects the interleukin-5 gene, a central Th2 cytokine. The IC(50) of dexamethasone is 10 nM with a maximum effect at 100 nM. All inhibitory actions were blocked by the GR antagonist RU38486 (1 uM), proving the specificity of GR action. In view of the crucial role of GATA-3 in T-cell differentiation and inflammation, we propose that the mechanism of GATA-3 inhibition compared with that in T-bet may have relevant implications in understanding and modulating the anti-inflammatory and Th-regulatory properties of GCs.
Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation.
Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10).
Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CKbeta8/CCL23, and osteoprotegerin, which were significantly higher than in control.
Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.
Metanephric kidney induction critically depends on mesenchymal-epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of beta-catenin function in the nephric duct and demonstrate that the beta-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.
GATA transcription factors are important regulators of tissue-specific gene expression during development. GATA2 and GATA3 have been implicated in the regulation of trophoblast-specific genes. However, the regulatory mechanisms of GATA2 expression in trophoblast cells are poorly understood. In this study, we demonstrate that Gata2 is transcriptionally induced during trophoblast giant cell-specific differentiation. Transcriptional induction is associated with displacement of GATA3-dependent nucleoprotein complexes by GATA2-dependent nucleoprotein complexes at two regulatory regions, the -3.9- and +9.5-kb regions, of the mouse Gata2 locus. Analyses with reporter genes showed that, in trophoblast cells, -3.9- and +9.5-kb regions function as transcriptional enhancers in GATA motif independent and dependent fashions, respectively. We also found that knockdown of GATA3 by RNA interference induces GATA2 in undifferentiated trophoblast cells. Interestingly, three other known GATA motif-dependent Gata2 regulatory elements, the -1.8-, -2.8-, and -77-kb regions, which are important to regulate Gata2 in hematopoietic cells are not occupied by GATA factors in trophoblast cells. These elements do not show any enhancer activity and also possess inaccessible chromatin structure in trophoblast cells indicating a context-dependent function. Our results indicate that GATA3 directly represses Gata2 in undifferentiated trophoblast cells, and a switch in chromatin occupancy between GATA3 and GATA2 (GATA3/GATA2 switch) induces transcription during trophoblast differentiation. We predict that this GATA3/GATA2 switch is an important mechanism for the transcriptional regulation of other trophoblast-specific genes.
Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.
The degree of urothelial differentiation in putative transitional (urothelial) proliferations in the female genital tract is still controversial. To further investigate the similarities (or dissimilarities) between female genital tract transitional proliferations and bladder urothelium, we evaluated the expression of S100P and GATA3, 2 proteins that we previously found to be strongly expressed in bladder urothelial tumors, in 25 benign ovarian Brenner tumors, 19 Walthard cell nests (17 tubal and 2 ovarian hilus), 1 mature teratoma with a benign urothelial proliferation, 2 proliferating (borderline) ovarian Brenner tumors, 1 malignant Brenner tumor, and 12 ovarian transitional cell carcinomas (TCC). Each lesion was also evaluated for p63 expression by immunohistochemistry. Immunostaining was performed on formalin-fixed, paraffin-embedded tissue sections using the avidin-biotin-peroxidase complex method. Eighty-eight percent of Brenner tumors were positive for S100P, whereas 96% and 100% were positive for GATA3 and p63, respectively. One of 2 proliferating Brenner tumors was positive for S100P, whereas both cases were positive for GATA3 and p63; the malignant Brenner tumor was positive for S100P and p63, but negative for GATA3. Only 17% of TCC were positive for S100p, whereas 33% and 50% of TCC were positive for GATA3 and p63, respectively. Tubal Walthard cell nests were either completely negative or showed only scattered positive staining for S100P; in contrast, 89.5% and 100% of Walthard nests, including the 2 ovarian cases were positive for GATA3 and p63. The teratoma-associated benign urothelial proliferation was also negative for S100P, but positive for GATA3 and p63. Although proliferating and malignant Brenner tumors may exhibit a more intermediate immunoprofile, expression of S100P, GATA3, and p63 by a majority of ovarian Brenner tumors underscores the similarity between these neoplasms and urothelial proliferations of bladder origin. The indeterminate phenotype seen in Walthard nests and ovarian TCC suggests that these proliferations may represent an incomplete or alternate form of differentiation.
Midbrain GABAergic neurons control several aspects of behavior, but regulation of their development and diversity is poorly understood. Here, we further refine the midbrain regions active in GABAergic neurogenesis and show their correlation with the expression of the transcription factor Gata2. Using tissue-specific inactivation and ectopic expression, we show that Gata2 regulates GABAergic neuron development in the mouse midbrain, but not in rhombomere 1, where it is needed in the serotonergic lineage. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. These studies identify Gata2 as an essential post-mitotic selector gene of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of GABAergic neurons in the midbrain.
GATA3 is a transcription factor closely associated with estrogen receptor alpha in breast carcinoma, with a potential prognostic utility. This study investigated the immunohistochemical expression of GATA3 in estrogen receptor alpha-positive and estrogen receptor alpha-negative breast carcinomas. One hundred sixty-six cases of invasive breast carcinomas with 10-year follow-up information were analyzed. Positive GATA3 and estrogen receptor alpha cases were defined as greater than 20% of cells staining. Time to cancer recurrence and time to death were analyzed with survival methods. Of 166 patients, 40 were estrogen receptor alpha negative and 121 estrogen receptor alpha positive. Thirty-eight (23%) recurrences and 51 (31%) deaths were observed. In final multivariable analyses, GATA3-positive tumors had about two thirds the recurrence risk of GATA3-negative tumors (hazard ratio = 0.65, P = .395) and comparable mortality risk (hazard ratio = 0.86, P = .730). In prespecified subgroup analyses, the protective effect of GATA3 expression was most pronounced among estrogen receptor alpha-positive patients who received tamoxifen (hazard ratio = 0.57 for recurrence and 0.68 for death). We found no statistically significant differences in recurrence or survival rates between GATA3-positive and GATA3-negative tumors. However, there was a suggestion of a modest-to-strong protective effect of GATA3 expression among estrogen receptor alpha-positive patients receiving hormone therapy.
GATA-binding protein 3 (GATA3) is a transcription factor that is crucial to mammary gland morphogenesis and differentiation of progenitor cells, and has been suggested to have a tumor suppressor function. The rs570613 single nucleotide polymorphism (SNP) in intron 4 of GATA3 was previously found to be associated with a reduction in breast cancer risk in the Cancer Genetic Markers of Susceptibility project and in pooled analysis of two case-control studies from Norway and Poland (P (trend) = 0.004), with some evidence for a stronger association with estrogen receptor (ER) negative tumours [Garcia-Closas M et al. (2007) Cancer Epidemiol Biomarkers Prev 16:2269-2275]. We genotyped GATA3 rs570613 in 6,388 cases and 4,995 controls from the Breast Cancer Association Consortium (BCAC) and 5,617 BRCA1 and BRCA2 carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). We found no association between this SNP and breast cancer risk in BCAC cases overall (OR(per-allele) = 1.00, 95% CI 0.94-1.05), in ER negative BCAC cases (OR(per-allele) = 1.02, 95% CI 0.91-1.13), in BRCA1 mutation carriers RR(per-allele) = 0.99, 95% CI 0.90-1.09) or BRCA2 mutation carriers (RR(per-allele) = 0.93, 95% CI 0.80-1.07). We conclude that there is no evidence that either GATA3 rs570613, or any variant in strong linkage disequilibrium with it, is associated with breast cancer risk in women.
To explore the effects of acute and chronic murine cytomegalovirus (MCMV) infections on the regulatory T cells (Treg) ratio and protein expression of the Th1/Th2 transcription factors T-bet/GATA-3.
120 BALB/c mice were randomly divided into 2 equal groups: MCMV-infected group undergoing infra-peritoneal injection of homogenate of salivary gland containing MCMV, and mock infection group undergoing infra-peritoneal injection of normal homogenate of salivary gland 1, 3, 7, 14, 28, 45, 60, 75, 90, and 120 days after infection 6 mice from each group were killed to examine the viral load of the heart, lung, liver, and kidney by plaque assay to access the status of MCMV infection. Suspension of splenocytes was prepared. The proportion of CD4+CD25+Foxp3+Treg in the splenocytes was measured by flow cytometry. Western blotting was used to detect the protein expression of T-bet/GATA-3.
The cutoff point between acute and chronic points was the 28th day. The CD4+CD25+Foxp3+Treg proportion in splenocytes significantly decreased during the acute infection stage and to the lowest level of (1.46+/-0.27)% at day 28, significantly lower than that of the mock infection group [(2.78+/-0.29)%, P<0.05]; then obviously increased in the chronic infection stage, increased to (4.51+/-0.24)% at day 60, significantly higher than that of the mock infection group [(2.69+/-0.12)%, P<0.05], and continued to increase still. The protein level (K value) of T-bet of the MCMV infection group peaked to the level of (0.618+/-0.053) on day 3, obviously higher than that of the mock infected group [(0.205+/-0.026)], then decreased to the level similar to that of the mock infection group on day 28, and was obviously lower than that of the mock infection group on day 75. Whereas the protein level of GATA-3 of the MCMV group increased to (0.836+/-0.061) on day 3, markedly higher than that of the mock infection group (0.398+/-0.022), peaked on day 7, then gradually decreased, and remained at the levels similar to those of the mock infection group from day 75 to day 120.
In the acute infection stage, MCMV up-regulates the T-bet and GATA-3 protein expression. But during the chronic infection stage, MCMV induces a marked proliferation and activation of Treg cells which further inhibit the Th1 and Th2 reactions, especially Th1 response. Treg proliferation may be an important mechanism of chronic and persistent CMV infection in the host.
To evaluate the balance of interleukin IL18 and its endogenous antagonist IL18 binding protein (IL18BP) in patients with idiopathic thrombocytopenic purpura (ITP), plasma IL18, IL18BP, interferon gamma (IFNG) and IL4 levels, as well as platelet counts were measured in patients with active ITP (n = 23), ITP in remission (n = 21) and in healthy subjects (n = 24) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction, the mRNA expression of IL18, IL18BP, IFNG, IL4, T-box (TBX21) and GATA-binding protein 3(GATA3) were studied in all subjects. The results showed that IL18 and IFNG protein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but that IL18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL18/IL18BP in patients with active disease. During remission stages, the levels of these cytokines were comparable to those of healthy controls. The elevated levels of IL18/IL18BP in plasma during active stages of disease suggest a possible role in the pathogenesis and course of ITP.
Systemic administration of cytosine-guanine (CpG) oligodeoxynucleotides provides neuroprotection against subsequent cerebral ischemic injury. We examined the genomic response of leukocytes and brain cells after ischemia in the context of CpG preconditioning.
RNA was isolated from circulating leukocytes and ischemic cortex 3 and 24 hours after middle cerebral artery occlusion after CpG or saline pretreatment and subjected to microarray analysis. Genes uniquely upregulated in CpG-pretreated mice were examined for overrepresented transcriptional regulatory elements.
CpG preconditioning induced a novel response to middle cerebral artery occlusion within circulating leukocytes that was dominated by natural killer cell-associated genes and the GATA-3 transcriptional regulatory element. Preconditioning also caused a novel brain response to stroke that was dominated by Type I interferon, interferon-associated genes, and transcriptional regulatory elements.
CpG preconditioning invokes novel leukocyte and brain responses to stroke. In this, CpG may be a unique preconditioning agent, coordinating peripheral and brain responses to protect against ischemic injury.
Quercetin is found to be the most active of the flavonoids in studies and many medicinal plants owe much of their activity to their high Quercetin content. Quercetin has demonstrated significant anti-inflammatory activity because of direct inhibition of several initial processes of inflammation. However, its anti-allergic effect in the Th1/Th2 immune response was poorly understood. Recently, it was shown that T-bet and GATA-3 were master Th1 and Th2 regulatory transcription factors. In this study, we have attempted to determine whether Quercetin regulates Th1/Th2 cytokine production, T-bet and GATA-3 gene expression in OVA-induced asthma model mice. Quercetin reduced the increased levels of IL-4, Th2 cytokine production in OVA-sensitized and -challenged mice. The other side, it increased IFN-gamma, Th1 cytokine production in Quercetin administrated mice. We also examined to ascertain whether Quercetin could influence Eosinophil peroxidase (EPO) activity. The administration of Quercetin before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that Quercetin plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of Quercetin in terms of its effects in a murine model of asthma, and also broaden current perspectives in our understanding of the immunopharmacological functions of Quercetin.
GATA-3 is a transcription factor that orchestrates gene expression profiles during embryogenesis of a variety of human tissues, including hematopoietic cells, skin, kidney, mammary gland, and the central nervous system. Among several other roles, GATA-3 has recently been identified as a key player of luminal cell differentiation in the mammary gland. The majority of breast cancers arise from luminal epithelial cells and hence GATA-3 appears to control a set of genes involved in the differentiation and proliferation of breast cancer. The expression of GATA-3 has a strong association with the expression of estrogen receptor-alpha (ER) in breast cancer, and there is mounting evidence that GATA-3 can be used as a clinical marker to determine response to hormonal therapy and to refine the prognosis of breast cancer patients. Here, we review the literature from the past 10 y on GATA-3 in normal and pathological states of the mammary gland. Conclusions from the literature are confirmed using meta-analyses performed by the Oncomine Research Platform.
Hypoparathyroidism is a disease characterized by hypocalcemia and hyperphosphatemia derived from deficient actions of parathyroid hormone (PTH). We report the case of 43-year-old Japanese man with PTH-deficient hypoparathyroidism introduced to an endocrinologist in our hospital. As he had complained of hearing disturbance since the age of 20, we decided to investigate the GATA3 gene. Direct sequencing of PCR products identified a novel heterozygous mutation, 432insG, in the GATA3 gene. The mutation introduces a premature stop codon at exon 4 (K302X), which results in a loss of both zinc finger domains of the GATA3 protein. However, because the mutation in the GATA3 gene found in this patient is highly likely to impair GATA3 function, we speculate that it is extremely unlikely that this patient has mutations in other genes that cause PTH-deficient hypoparathyroidism, in addition to the GATA3 mutation described here.
GATA3 is a critical transcription factor for many developmental processes. During T helper (Th) cell differentiation, GATA3 induces the Th2 and suppresses the Th1 pathway. Stimulation of the T cell receptor (TCR) of naive Th cells in the presence of interleukin 4 (IL-4) induces robust expression of GATA3; however, it is unclear where these signals integrate. Gata3 encodes two transcripts that differ in their alternative, untranslated first exons. We show here the involvement of the TCR-inducible transcription factor NFAT1 in the transcriptional regulation of both Gata3 transcripts following TCR stimulation of naive and differentiated Th2 cells. We also show that IL-4 is important for the initiation and establishment of Gata3 transcription in developing Th2 cells, especially from the distal promoter. The early function of IL-4 can be STAT6 dependent or independent. However, the establishment of the activity of the distal promoter is totally dependent on STAT6, whereas it is likely that the proximal promoter has additional activation mechanisms that are STAT6 independent. Our findings suggest that different combinations of transcription factors downstream of the IL-4 receptor (IL-4R) and TCR finely modulate Gata3 gene expression from its two promoters for optimal Th2 differentiation.
Th1 and Th17 cells are crucial in immune regulation and autoimmune disease development. By adding Stat6 deficiency to T-bet deficiency, and thus negating effects from elevated levels of IL-4/Stat6/GATA3 Th2 signals in T-bet-deficient cells, we investigated the signals important for Th1 and Th17 cell differentiation and their role in colitis development. The data reveal that Eomesodermin compensates T-bet deficiency for IFN-gamma and Th1 development. However, without T-bet, IFN-gamma production and Th1 differentiation are susceptible to inhibition by IL-6 and TGFbeta. As a result, Th17 development is strongly favored, the threshold for TGFbeta requirement is lowered, and IL-6 drives Th17 differentiation, elucidating a critical role for T-bet in directing T cell differentiation to Th1 vs Th17. In contrast to IL-6 plus TGFbeta-driven Th17, IL-6-driven Th17 cells do not express IL-10 and they induce a more intense colitis. Naive CD4 T cells deficient in Stat6 and T-bet also induce a Th17-dominant colitis development in vivo. Our data provide new insights into the choice between Th1 and Th17 development and their roles in autoimmunity.
Viperin (virus inhibitory protein, endoplasmic reticulum [ER]-associated, interferon-inducible) has been identified as a highly inducible ER protein that has antiviral activity. Here, we characterized the phenotype of mice deficient in viperin and examined the biological function of viperin in peripheral T-cell activation and differentiation. Splenic CD4(+) T cells deficient in viperin exhibited normal anti-T-cell receptor (TCR)-induced proliferation and IL-2 production, but produced significantly less T helper 2 (Th2) cytokines, including IL-4, IL-5, and IL-13, in association with impaired GATA3 activation, after stimulation with anti-CD3 antibody, which was not restored upon costimulation with anti-CD28. Th2 differentiation of viperin-deficient naive T cells was also impaired in the presence of strong TCR signaling and minimum IL-4, but not under optimal Th2-skewed conditions. In parallel, viperin-deficient T cells showed decreases in NF-kappaB1/p50 and AP-1/JunB DNA binding activities after TCR engagement. Thus, viperin facilitates TCR-mediated GATA-3 activation and optimal Th2 cytokine production by modulating NF-kappaB and AP-1 activities.
Naive CD4 T cells differentiate into functionally distinct T helper (Th) cells subsets or into regulatory T (Treg) cells in response to the cytokine milieu in which they encounter antigen. A recurring theme in post-thymic CD4 T cell differentiation is the cross-regulation of lineage choice by cytokines and transcription factors that are expressed in alternative lineages. For example, TGFbeta induces the de novo expression of the Treg cell signature transcription factor Foxp3, but iTreg differentiation is blocked by high concentrations of the Th2 cytokine IL4. However, whether IL4 can antagonise Foxp3 induction in more physiological settings remains to be addressed. Here we use a co-culture system to demonstrate that IL4 provided by Th2 cells in vitro is sufficient to block Foxp3 induction in naive CD4 T cells. In addition, we find that Foxp3 induction is efficiently blocked not only by the Th2 transcription factor Gata3, but also by PU.1, which is transiently induced during Th2 differentiation. These data suggest that iTreg differentiation may be affected by the polarity of immune responses.
Aim of this study was to evaluate the effect of zinc on the kinetic of development of CD34(+) cell progenitors towards NK cells in young and old ages. CD34(+) cells were purified from peripheral blood of healthy subjects and cultured in medium supplemented with interleukin-15, interleukin-7, Flt 3 ligand, and stem cell factor. The number of cells developed in culture was significantly lower in old than in young subjects. CD34(+) cells progressively lost CD34 antigen with a faster kinetics in old than in young donors. The percentage of primitive double positive CD34(+)CD133(+) cells inside the purified CD34(+) cells was greatly lower in old than in young subjects. These cells progressively decreased in cultures from young subjects whereas they remained at very low levels in old donors. Cells developed in culture acquired a NK phenotype mainly characterized by CD56(+)CD16(-) cells in young subjects and CD56(-)CD16(+) cells in old donors. These NK cells exerted a lower cytotoxic activity in old than in young subjects. The supplementation with zinc greatly increased the number of cells in culture and the percentage and the cytotoxic activity of NK cells both in young and old ages. In zinc supplemented cultures, a 3.6-fold and a 4.1-fold increased expression of GATA-3 transcription factor was observed in young and old donors, respectively. Our data demonstrate that zinc influences the proliferation and differentiation of CD34(+) progenitors both in young and old ages.
Major transcription factors controlling T(H)1 and T(H)2 development, such as T-box transcription factor and GATA3, might be centrally involved in asthma and atopic diseases. Only recently, the homeobox transcription factor H.20-like homeobox 1 (HLX1), interacting closely with T-box transcription factor, has been identified as an important regulator of T(H)1 differentiation and suppressor of T(H)2 commitment.
We investigated whether genetic variations in the HLX1 gene exist and whether these could affect the development of childhood asthma.
The HLX1 gene was resequenced in 80 chromosomes. Associations between identified polymorphisms, asthma, and atopic diseases were investigated in German children (total n = 3099) from the cross-sectional International Study of Asthma and Allergy in Childhood phase II. Functional properties of polymorphisms were studied by using luciferase reporter gene assays and electrophoretic mobility shift assays in T cells. All statistical analyses were performed with SAS/Genetics software (SAS Institute, Inc, Cary, NC).
Nineteen polymorphisms were identified in the HLX1 gene, and 2 tagging single nucleotide polymorphisms representing 7 polymorphisms were associated with childhood asthma in our study population. Two promoter polymorphisms, C-1407T and C-742G, contained in 1 tagging block were associated with asthma (odds ratio, 1.44; 95% CI, 1.11-1.86; P = .0061), significantly decrease promoter transactivation, and disrupt specificity protein-transcription factor binding in in vitro experiments.
Our data suggest that polymorphisms in the HLX1 gene increase the risk for childhood asthma. On the cellular level, altered binding of specificity protein-transcription factors to the HLX1 promoter and subsequent changes in HLX1 gene expression might contribute to these effects.
GATA3 gene encodes a transcription factor expressed during thymus, liver, kidney, adrenal gland, central and peripheral nervous systems, placenta and T lymphocytes embryonic development. Mutations of GATA3 cause Hypoparathyroidism, sensorineural Deafness and Renal dysplasia syndrome (HDR). We report the case of a girl with a terminal deletion of the short arm of chromosome 10 (10p12.1-pter), including both HDR locus and the DiGeorge critical region 2 (DGCR2), with HDR phenotype but not DiGeorge syndrome 2 features. The girl developed chronic renal failure during the first year of life, associated with sensorineural hearing loss, facial dysmorphic features and psychomotor development. She had hypodysplastic kidneys and bilateral grade 3-vesicoureteric reflux. Her karyotype was 46,XX,del(10)(p12.1-pter). Quantitative analysis by Real Time PCR on blood DNA confirmed the lack of one copy of GATA3 gene. She underwent renal transplantation at the age of 11. Our patient is the first case with a large deletion of the short arm of chromosome 10 - that certainly involves DGCR2 - with the HDR phenotype but without the clinical features of DGS2. This peculiarity suggests the hypothesis that the mechanisms underlying this syndrome may be more complex. It is therefore possible that DGS2 may be determined by locus heterogeneity.
The GATA3 transcription factor is expressed in many tissues such as the immune system, kidney, brain, endometrium, and mammary epithelial cells. As such it must co-ordinate a diverse transcriptional program to achieve specific outcomes in different tissues. One of the most interesting questions raised is whether GATA3 will be involved in the same pathways in every tissue or will be involved in distinct regulatory networks within different tissue types? While previous studies may imply the latter, with some known targets of GATA3 perhaps being specific to cell-type or tissue-type, the question has not been systematically addressed until now. With the advent of techniques such as co-expression meta-analysis a better understanding of the pathway partners of GATA3 can be obtained and specifically the partners within different tissue types can be found, yielding leads for future studies. Here, a recent technique of meta-analysis from the Oncomine database has been employed to probe this very question. Data obtained implies that GATA3 is involved in distinct pathways in different tissue types.
To investigate the effect of nebulized total ginkgo flavone glycosides (TFG) on Th1/Th2 imbalance in mice with athma.
Forty-eight BALB/C mice were randomly divided into four groups: group A (control group, n=12); group B (asthmatic model group, n=12); group C (TFG nebulized treated group, n=12); group D (dexamethasone intraperitoneal treated group, n=12). The asthmatic model was established by sensitivity and local activation with Ovalbumin(OVA) and aluminum hydroxide Al(OH)3. TFG (50 g x L(-1), per aerosol per six mice, 30 minutes) was nebulized 20 days after modeling, while dexamethasone (1 g x L(-1)) was intraperitoneal once daily for 10 days. Perfusate of bronchoalveolar lavage fluid(BALF) was collected on day 32. The level of IL-4, IFN-gamma in BALF, and the level of total IgE in serum was determined. The airway inflammation pathology change and the expression of GATA-3 protein in lungs was detected by immunohistochemical staining.
Compared with model group, the decreased content of IL-4(49.30 +/- 7.95) ng x L(-1) and increased level of IFN-gamma (49.08 x 5.46) ng x L(-1). were found in BALF, and the level of total IgE (9.47 +/- 1.52) microg x L(-1) in serum also decreased in TFG treated group. In model group, smooth muscle hypertrophing, mucous hyperemia, mucous layer thickening, and inflammatory cell in filtration were observed. Phlegmasia was appeared in the bronchi, which was filled with lots of mucus. In contrast, the inflammatory reaction in TFG and Dexamethasone treated group was less obvious. Expression of GATA-3 was markly increased in model group with decreased expression in TFG treated group.
Nebulized TFG showed a therapeutic effect for asthmatic mice, the mechanism may be explained by blockingnnnn GATA-3 expression and regulating the disorder Th1/Th2 imbalance.
Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1-infected individuals to a mean of 49.4 +/- SD 12.9% of CD8(+) T cells expressing Tim-3 in HIV-1-infected chronic progressors versus 28.5 +/- 6.8% in HIV-1-uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1-infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4(+) T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1-specific CD8(+) T cells. Tim-3-expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1-specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1-associated T cell dysfunction.
Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.
The rhesus monkey embryonic stem cell line 366.4 differentiates into serotonin neurons. We examined the genetic cascade during differentiation and compared ESC-derived serotonin neurons to adult monkey serotonin neurons. RNA was extracted from ESC colonies, embryoid bodies (EBs), neurospheres in selection (N1) and proliferation stages (N2), differentiated serotonin neurons (N3) and from laser captured (LC) serotonin neurons of spayed female macaques treated with placebo, estrogen (E), progesterone (P) or E+P. The RNA was labeled and hybridized to Rhesus Monkey Affymetrix Gene Chips (n=1 per stage and 2 per animal treatment). Gene expression was examined with GeneSifter software. 545 genes that were related to developmental processes showed a threefold or greater change between stages. TGFb, Wnt, VEGF and Hedgehog signaling pathways showed the highest percent of probe set changes during differentiation. Genes in the categories (a) homeobox binding and transcription factors, (b) growth factors and receptors, (c) brain and neural specific factors and (d) serotonin specific factors are reported. Pivotal genes were confirmed with quantitative RT-PCR. In the serotonin developmental cascade, FGFR2 was robustly expressed at each stage. GATA3 was robustly expressed in EBs. Sonic hedgehog (Shh), PTCH (Shh-R) and Fev1 transcription factor expression coincided with the induction of serotonin specific marker genes during N1-selection. A majority of the examined genes were expressed in adult serotonin neurons. However, in the ESC-derived neurons, there was significant over-representation of probe sets related to cell cycle, axon guidance & dorso-ventral axis formation. This analysis suggests that the 366.4 cell line possesses cues for serotonin differentiation at early stages of differentiation, but that ESC-derived serotonin neurons are still immature.
At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a (dr)) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a "cochlear-gravistatic" endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.
Allergic asthma is caused by aberrant helper T (T(H)) type 2 immune responses in susceptible individuals, characterized by airway hyperresponsiveness, chronic airway inflammation, and mucus hypersecretion. Its prevalence continues to increase, but optimal treatment remains a challenge. The transcription factor T-bet is a master regulator of T(H)1 lineage commitment and strongly promotes interferon gamma expression during T(H)1 cell differentiation.
The aim of this study was to explore the role of intranasal delivery of T-bet on the differentiation of T(H) cell subsets and airway inflammation in the ovalbumin (OVA)-induced mouse model of allergic airway inflammation.
BALB/c mice were sensitized by intraperitoneal injection of OVA and challenged with nebulized OVA. Four days before the inhalation challenge, the sensitized mice were subjected to intranasal delivery of a recombinant adeno-associated virus vector carrying murine T-bet gene (AAV-T-bet). Expression of the transcription factors T-bet, GATA3, and Foxp3 was then assayed in the lungs, and airway histology was analyzed along with other inflammatory parameters, such as eosinophils and cytokines in bronchoalveolar lavage (BAL) fluid, and total and OVA-specific immunoglobulin (Ig) E in serum.
Intranasal administration of AAV-T-bet efficiently balanced the T(H)1/T(H)2 transcription factor and cytokine profile and significantly decreased the number of eosinophils in BAL fluid. It also resulted in a reduction of peribronchial inflammation scores and serum IgE levels in OVA-sensitized and challenged mice during the effector phase.
Our data show that intranasal delivery of T-bet can promote a T(H)1 immune response, restore a balanced Th immune response, and inhibit airway inflammation during the challenge phase in a mouse model of allergic airway inflammation.
The transcription factor Gata3 is an important regulator of the development of thymus, the nervous system, ear, kidney, and adrenal glands. This study analyzes the role of Gata3 in the developing heart using a mouse strain containing an nlsLacZ reporter gene fused in frame to the Gata3 gene by homologous recombination. Using in situ hybridization, RT-PCR and Gata3-LacZ histochemistry, Gata3 expression was shown in various cardiac structures up to newborn stage. During looping stages (E9.5-E11.5) Gata3-LacZ activity recapitulated endogenous Gata3 and was abundantly expressed in the endocardial ridges and endothelium of distal outflow tract. Strong reporter gene expression was also noted in the mesenchyme of ventral branchial arches, and in the epithelium. In the atrioventricular canal expression was relatively lower. In the four-chambered heart stages (E13.5-E17.5) the LacZ-staining did not recapitulate the endogenous Gata3 transcript and showed rather lineage tracing of formerly Gata3-expressing cells in the hearts. beta-Galactosidase activity was detected in the cusps of semilunar valves, aorta, pulmonary trunk, innominate and common carotid arteries, and faintly in the atrioventricular valves. Gata3-null embryos die normally between E11 and E12. Pharmacological treatment with sympathomimetic beta-adrenergic receptor agonist lengthens the survival up to E18 when malformations of the heart such as ventricular septal defect (VSD), double-outlet of right ventricle (DORV), anomalies of the aortic arch (AAA) and persistent truncus arteriosus (PTA) were detected. The specified malformations correlate with the normal developmental pattern of Gata3-LacZ expression. The short outflow tract and insufficient rotation of truncus arteriosus during looping stages might be the main reasons underlying these malformations.
Both Ikaros and Notch are essential for normal T cell development. Collaborative mutations causing a reduction in Ikaros activity and an increase in Notch activation promote T cell leukemogenesis. Although the molecular mechanisms of this cooperation have been studied, its consequences in thymocyte development remain unexplored. In this study, we show that Ikaros regulates expression of a subset of Notch target genes, including Hes1, Deltex1, pTa, Gata3, and Runx1, in both Ikaros null T cell leukemia lines and Ikaros null primary thymocytes. In Ikaros null leukemia cells, Notch deregulation occurs at both the level of Notch receptor cleavage and expression of Notch target genes, because re-expression of Ikaros in these cells down-regulates Notch target gene expression without affecting levels of intracellular cleaved Notch. In addition, abnormal expression of Notch target genes is observed in Ikaros null double-positive thymocytes, in the absence of detectable intracellular cleaved Notch. Finally, we show that this role of Ikaros is specific to double-positive and single-positive thymocytes because derepression of Notch target gene expression is not observed in Ikaros null double-negative thymocytes or lineage-depleted bone marrow. Thus, in this study, we provide evidence that Ikaros and Notch play opposing roles in regulation of a subset of Notch target genes and that this role is restricted to developing thymocytes where Ikaros is required to appropriately regulate the Notch program as they progress through T cell development.
Cluster specific immunotherapy (SIT) is a modern form of allergen immunotherapy allowing safe administration of high allergen doses in a short time interval compared to classic SIT. In the current study, we investigated the safety profile and immunological effect of cluster SIT in children with allergic asthma due to house dust mite allergy.
A total of 34 children (6-18 years) with allergic asthma were assigned to cluster (n = 22) or classic SIT (n = 12). To achieve a maintenance dose of allergen extract, cluster patients received 14 injections of house dust mite allergen within 6 weeks, whereas the classic SIT group received 14 injections within 14 weeks. Safety was monitored by recording adverse events. Immunogenicity was measured by specific IgG(Mite) and IgG4(Mite), by antibody-blocking properties on basophil activation, and by the T cell subset transcription factors Foxp3, T-bet, and GATA-3.
There were no significant differences in local and systemic side effects between the two groups. In the cluster group, serum levels of specific IgG(Mite) (p < 0.001) and specific IgG4(Mite) (p < 0.001) significantly increased after 8 weeks, while it took 12 weeks in the classic SIT group. These data were confirmed by blocking CD63 expression as well as release of cysteinyl leukotrienes after in vitro basophil stimulation. No differences in transcription factor expression were found in the two groups.
Cluster SIT is safe in children. Additionally, our data demonstrated an even more rapid induction of specific immune tolerance. Cluster SIT is an attractive alternative to conventional up-dosing schedules with fewer consultations for the patients.
Sulfated glycans play critical roles in various cell recognition events among leukocytes. The 6-sulfated lactosamine glycans in particular have been widely noted for their importance because they are involved in cell recognition events mediated by cell-adhesion molecules such as selectins and sialic acid-recognizing molecules such as siglecs and also in the activation of CD44 in binding to extracellular matrix hyaluronate. A pro-inflammatory cytokine, tumor necrosis factor-alpha, induces expression of 6-sulfated glycans on human leukocytes. Here we report that the transcription of the GlcNAc6ST-1 gene, the gene encoding a sulfotransferase for 6-sulfated glycan synthesis, is induced in human T-lymphoid cells through tandem NF-kappaB and GATA motifs in its 5'-regulatory region. Results of our reporter assays, immunoprecipitation, and chromatin immunoprecipitation analyses indicated that GATA-3 and/or GATA-2, but not GATA-1, associates with NF-kappaB in a transcription factor complex on the 5'-regulatory region of the gene and acts synergistically with NF-kappaB in triggering GlcNAc6ST-1 transcription. Recently, a skin-homing subset of helper memory T cells exhibiting the Th2 marker CCR4 was shown to specifically express 6-sulfated glycans. The transactivation mechanism described here suggested that GlcNAc6ST-1 transcription is coordinated with the NF-kappaB/GATA-3 axis, which is known to figure heavily in Th2 cell differentiation. In line with this, in vitro differentiation of human T cells to Th2 cells was found to significantly induce GlcNAc6ST-1 transcription and 6-sulfated glycan expression.
To evaluate the association between single nucleotide polymorphisms (SNP) of GATA3 and Chinese with allergic rhinitis.
This study included 109 patients with allergic rhinitis and 112 healthy control people. SNP of two loci with restriction fragment length polymorphism was detected, rs1269486 at promoter region and rs2229360 located 92 bp downstream of STOP codon. Total IgE and specific IgE of Der p and Der f with different genotypes of the two loci were compared.
There was SNP at both loci of rs1269486 and rs2229360. The genotypes of rs1269486 were GG, GA and AA, and the genotypes of rs2229360 were CC, CT and TT. The frequency of genotype GG and allele G in patient group was significantly higher than that in control group (chi2 = 13.75, P <0.01; chi2 = 12.91, P <0.01), and the frequency of genotype GA and allele A in patient group was significantly lower than that in control group (chi2 = 11.90, P <0.01; chi2 =12.91, P <0.01). There were no significant differences between patient group and control group for the frequencies of genotypes and alleles at rs2229360 (P > 0.05). The haplotype frequency of GC in patient group was significantly higher than that in control group (chi2 = 114.59, P < 0.01), and the haplotype frequency of AC in patient group was significantly lower than that in control group (chi2 = 87.52, P <0.01). There was no significant difference of the levels of serum total IgE and specific IgE to Der p and Der f with the genotypes at the two loci (P >0.05).
The SNP at rs1269486 of GATA3 is associated with allergic rhinitis in Chinese.
Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats.
Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated.
We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment.
Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI.
Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.
The oestrogen receptor (ER) pathway is key for survival and progression in a significant proportion of breast cancers. The ER can be activated by oestrogen or activated due to "crosstalk" with growth factor receptor pathways. Activated ER signals through transcriptional and non-transcriptional mechanisms. Immunohistochemistry (IHC), in spite of the shortcomings, remains the method of choice as it provides for in situ assessment of ER expression within the tumour cells. This capability is lost in tissue grinding methods that assess oestrogen-binding activity or messenger RNAs in tumours. IHC is also not influenced by the presence of non-tumoural cells or low amounts of tumour cells within samples examined. It is clear that ER-positive tumours do not represent a single entity. Irrespective of the terminology used, low-grade ER-positive (also known as luminal A) tumours need to be differentiated from high-grade/highly proliferative ER-positive tumours. This can be done in a variety of ways including but not limited to analysis of FOXA1 and GATA-3 by IHC, and limited molecular profiling by Oncotype DX, MGH2-gene signature, intrinsic gene signature or MapQuant Dx. Several areas of ER biology are still poorly understood; these include: its function in the cytoplasm/plasma membrane, its role in the differentiation to proliferation switch, and pathways associated with resistance to hormonal therapy. A detailed understanding of these areas will permit better classification and a personalised approach to management of ER-positive breast cancers.
Legionella pneumophila infection of mice induces proinflammatory cytokines and Th1 immunity as well as rapid increases in serum levels of IL-12 and IFNgamma and splenic IL-12Rbeta2 expression. Delta-9-tetrahydrocannabinol (THC) treatment prior to infection causes a shift from Th1 to Th2 immunity and here we demonstrate that CB(1) and CB(2) cannabinoid receptors mediate different aspects of the shift. Using cannabinoid receptor antagonists and cannabinoid receptor gene deficient mice (CB(1) (-/-) and CB(2) (-/-)), we showed that both CB(1) and CB(2) receptors were involved in the THC-induced attenuation of serum IL-12 and IFNgamma. IFNgamma production is dependent upon signaling through IL-12Rbeta2 (beta2) and THC treatment suppressed splenic beta2 message; moreover, this effect was CB(1) but not CB(2)-dependent from studies with receptor antagonists and CB1(-/-) and CB2(-/-) mice. Furthermore, observed increases in IL-4 induced by THC, were not involved in the drug effect on beta2 from studies with IL-4 deficient mice. The GATA-3 transcription factor is necessary for IL-4 production and is selectively expressed in Th2 cells. GATA-3 message levels were elevated in spleens of THC-treated and L. pneumophila-infected mice and the effect was shown to be CB(2) but not CB(1)-dependent. Furthermore, GATA-3 regulatory factors were modulated in that Notch ligand Delta4 mRNA was decreased and Jagged1 increased by THC also in a CB2-dependent manner and splenic NFkappaB p65 was increased. Together, these results indicate that CB(1) and CB(2) mediate the THC-induced shift in T helper activity in L. pneumophila-infected mice, with CB(1) involved in suppressing IL-12Rbeta2 and CB(2) involved in enhancing GATA-3.
The zinc-finger transcription factor GATA3 serves as a master regulator of T-helper-2 (Th2) differentiation by inducing expression of the Th2 cytokines IL-4, IL-5 and IL-13 and by suppressing Th1 development. Here, we investigated how GATA3 affects Th17 differentiation, using transgenic mice with enforced GATA3 expression. We activated naïve primary T cells in vitro in the presence of transforming growth factor-beta and IL-6, and found that enforced GATA3 expression induced co-expression of Th2 cytokines in IL-17-producing T cells. Although the presence of IL-4 hampered Th17 differentiation, transforming growth factor-beta/IL-6 cultures from GATA3 transgenic mice contained substantial numbers of IL-17(+) cells, partially because GATA3 supported Th17 differentiation by limiting IL-2 and IFN-gamma production. GATA3 additionally constrained Th17 differentiation in vitro through IL-4-independent mechanisms, involving downregulating transcription of STAT3, STAT4, NFATc2 and the nuclear factor RORgammat, which is crucial for Th17 differentiation. Remarkably, upon myelin oligodendrocyte glycoprotein immunization in vivo, GATA3 transgenic mice contained similar numbers of IL-17-producing T cells in their lymph nodes as wild-type mice, but were not susceptible to autoimmune encephalomyelitis, possibly due to concomitant production of IL-4 and IL-10 induction. We therefore conclude that although GATA3 allows Th17 differentiation, it acts as an inhibitor of Th17-mediated pathology, through IL-4-dependent and IL-4-independent pathways.
To investigate the anti-inflammatory or immunomodulatory effect of shikonin on early stage and established murine collagen-induced arthritis (CIA).
Mouse were injected intraperitoneally with shikonin (5 mg/kg) for 10 days along before or after the onset of CIA. The arthritis response was monitored visually by macroscopic scoring. Reverse transcription-polymerase chain reaction and western blotting were employed to determine the mRNA and protein expression of cytokine in patella with adjacent synovium in CIA mouse. Histology of knee was used to assess the occurrence of cartilage destruction and bone erosion.
Shikonin (5 mg/kg) treatment along had no effect on macroscopic score and incidence of arthritis on early stage of CIA. However, a pronounced amelioration of macroscopic score and cartilage destruction was found in mouse treated with shikonin on established CIA for 10 days. Moreover, The mRNA levels of Th1 cytokines [tumor necrosis factor-alpha and interleukin (IL)-12] was significantly inhibited both in the synovial tissue and in the articular cartilage in treated groups compared with those in control groups, whereas the mRNA and protein levels of Th2 cytokines (IL-10 and IL-4) remained elevated throughout the treatment period. Moreover, the inflammatory cytokine, the mRNA and protein levels of IL-6 was down-regulated in mice with established CIA after treatment with shikonin. T-box expressed in T cells (T-bet) mRNA levels were decreased in shikonin compared with control group, and GATA-3 mRNA levels were higher than that in control group.
Shikonin treatment on established CIA can inhibit Th1 cytokines expression and induce Th2 cytokines expression in mice with established CIA. The inhibited effect of shikonin on Th1 cytokines expression may be mediated not only by inhibiting Th1 responses through T-bet mechanism, but also by inducing anti-inflammatory mediators such as IL-10 and IL-4 through a GATA-3 dependent mechanism.
The transcription factors GATA-3 and ThPOK are required for intrathymic differentiation of CD4(+) T cells, but their precise functions in this process remain unclear. Here we show that, contrary to previous findings, Gata3 disruption blocked differentiation into the CD4(+) T cell lineage before commitment to the CD4(+) lineage and in some contexts permitted the 'redirection' of major histocompatibility complex class II-restricted thymocytes into the CD8(+) lineage. GATA-3 promoted ThPOK expression and bound to a region of the locus encoding ThPOK established as being critical for ThPOK expression. Finally, ThPOK promoted differentiation into the CD4(+) lineage in a way dependent on GATA-3 but inhibited differentiation into the CD8(+) lineage independently of GATA-3. We propose that GATA-3 acts as a specification factor for the CD4(+) lineage 'upstream' of the ThPOK-controlled CD4(+) commitment checkpoint.
Histone deacetylase (HDAC) inhibitor treatments can augment the anti-tumor effects of retinoids in renal cancer cells. We studied the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and 13-cis retinoic acid (cRA) on two human renal cell carcinoma (RCC) lines. Cells were cultured in the presence of each drug for six days to determine the responses to monotherapy and to combination therapy. The proliferation of SKRC06 was inhibited with cRA treatment; the proliferation of SKRC39 was not. However, both RCC lines were sensitive to growth inhibition by dibutyryl cyclic AMP, with or without 13-cis RA. SAHA alone also reduced cell proliferation in both cell lines. To identify the alterations in gene expression that correlate with the responsiveness to treatment, gene microarray analyses were performed. Several retinoid-regulated genes exhibited much higher mRNA levels in SKRC06 than in SKRC39, even in the absence of drugs; these included crabp2, rargamma and cyp26A1. Combination treatment of cells with both SAHA and cRA induced several transcripts with known anti-cancer/immunomodulatory effects, including dhrs9, gata3, il1beta, phlda1, txk and vhl. Immunostaining confirmed the decreased expression of gata3 in human RCC specimens compared to normal kidney. Together, our results show that treatment of RCC with cRA and/or SAHA increases the expression of several genes and gene families that result in reduced cell proliferation.
T-cell precursors remain developmentally plastic for multiple cell generations after entering the thymus, preserving access to developmental alternatives of macrophage, dendritic-cell, and even mast-cell fates. The underlying regulatory basis of this plasticity is that early T-cell differentiation depends on transcription factors which can also promote alternative developmental programs. Interfactor competition, together with environmental signals, keep these diversions under control. Here the pathways leading to several lineage alternatives for early pro-T-cells are reviewed, with close focus on the mechanisms of action of three vital factors, GATA-3, PU.1, and Notch-Delta signals, whose counterbalance appears to be essential for T-cell specification.
The T(h)1/T(h)2 paradigm has been used for decades to characterize phenotypically different immune responses. Recent discoveries, e.g. T(h)17 cells are adding more dimensions to the helper T cell framework, and the T(h)1/T(h)2 paradigm is currently being extended to include these new phenotypes. Previous mathematical models cannot easily be extended to accommodate these new phenotypes, and therefore these discoveries call for a new type of models. We devised a new model of helper T cell differentiation that describes expression of, and interactions between, the master regulators determining the phenotypic polarization of helper T cells. The model is able to describe any number of master regulators and is grounded on transcription factors binding promoter sites and binding each other. The model allows for stable switches between several different phenotypes. Furthermore, the model accounts for the kinetics of FoxP3 and GATA3 mRNA expression measured after stimulating naive helper (CD4+CD45RA+) T cells under various circumstances. Due to its n-dimensional character, this model may easily be applied to other developmental processes that involve master regulators.
Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-peritoneally with antagonists of either B1 (R-954, 200 microg/kg) or B2 receptor (HOE140, 200 microg/kg) 30 minutes prior to ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript analyses by real-time PCR. Herein, B1R antagonism (R-954) was able to decrease serum creatinine levels, whereas B2R antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1beta transcription. Moreover, treatment with R-954 resulted in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal IRI.
CD154 (CD40-ligand) is a critical immune regulator. CD154 expression is tightly regulated and largely restricted to activated CD4 T cells. Using DNase I hypersensitivity site (HSS) mapping, we identified two novel HSS mapping to the human CD154 promoter element and just upstream. Both HSS were activation independent and CD4 T-cell specific. Approximately 350 bp of DNA sequence flanking the upstream HSS site was highly conserved between mouse and man, and was rich in binding sites for GATA and NFAT proteins. Gel shift and chromatin immunoprecipitation assays demonstrated both NFAT1 and the Th2 factor, GATA-3, bound this enhancer element in vitro and in vivo, respectively. A PstI/XbaI 345 bp fragment of this region acted as a transcriptional enhancer of the CD154 promoter in primary human CD4 T cells. Overexpression of repressor of GATA and a dominant negative GATA-3 protein independently inhibited transcription, whereas overexpression of wild-type GATA-3 enhanced transcriptional activity, by this element in primary CD4 T cells. Moreover, more interleukin-4-producing CD4 T cells expressed CD154 following activation than interferon-gamma-producing CD4 T cells. Thus, we identified a novel T-cell-specific, GATA-3 responsive, CD154 transcriptional enhancer, which may contribute to increased propensity of Th2 cells to express CD154.
The IL-18Ralpha-chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Ralpha) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report, we demonstrate the establishment of DH patterns that are distinct among undifferentiated CD4 T, Th1, and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation, and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Ralpha. These data provide further mechanistic insight into transcription factor-dependent establishment of Th subset-specific patterns of gene expression.
The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.
T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4(+) cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.
GATA-3 is a transcription factor that is specifically expressed in T helper 2 (Th2) cells and plays a critical role in the differentiation of Th2 cells from uncommitted CD4+ lymphocytes. In addition GATA-3 is essential for the gene expression of the cytokines IL-4, IL-5 and IL-13 that mediate allergic inflammation. In human T lymphocytes GATA-3 is normally localized to the cytoplasm, but on activation by antigen-presenting cells via the T cell receptor (CD3) and co-stimulatory receptor CD28 GATA-3 is phosphorylated by p38 MAP kinase and translocates to the nucleus via the nuclear import protein importin-alpha. Corticosteroids bound to glucocorticoid receptors inhibit GATA-3 function by competing for nuclear entry via importin-alpha and also by inhibiting p38 MAP kinase through the induction of MAP kinase phosphatase-1. GATA-3 is inhibited by the Th1 master regulatory transcription T-bet but in turn inhibits STAT-4 and thus T-bet so that Th2 polarization is maintained. Since GATA-3 appears to be a critical transcription factor for allergic inflammation it is an obvious target for inhibition. However, direct inhibition by inhaled specific oligonucleotides or interference RNA is not yet possible. Corticosteroids act as indirect inhibitors and in patients with corticosteroid resistance p38 MAP kinase inhibitors may also prove to be useful in the future.
Dlx homeobox transcription factors regulate epidermal, neural and osteogenic cellular differentiation. Here, we demonstrate the central role of Dlx3 as a crucial transcriptional regulator of hair formation and regeneration. The selective ablation of Dlx3 in the epidermis results in complete alopecia owing to failure of the hair shaft and inner root sheath to form, which is caused by the abnormal differentiation of the cortex. Significantly, we elucidate the regulatory cascade that positions Dlx3 downstream of Wnt signaling and as an upstream regulator of other transcription factors that regulate hair follicle differentiation, such as Hoxc13 and Gata3. Colocalization of phospho-Smad1/5/8 and Dlx3 is consistent with a regulatory role for BMP signaling to Dlx3 during hair morphogenesis. Importantly, mutant catagen follicles undergo delayed regression and display persistent proliferation. Moreover, ablation of Dlx3 expression in the telogen bulge stem cells is associated with a loss of BMP signaling, precluding re-initiation of the hair follicle growth cycle. Taken together with hair follicle abnormalities in humans with Tricho-Dento-Osseous (TDO) syndrome, an autosomal dominant ectodermal dysplasia linked to mutations in the DLX3 gene, our results establish that Dlx3 is essential for hair morphogenesis, differentiation and cycling programs.
The purpose of this review is to describe the development and function of the parathyroid gland from fish to mammals. We describe the molecular mechanisms regulating parathyroid gland embryogenesis and the clinical syndromes related to mutations in control genes.
Recent studies have shown that fish express parathyroid hormone. This is contrary to the long held view that the earliest animals to possess parathyroid hormone were amphibians. Two fish species have been demonstrated to express parathyroid hormone but the source and physiological function of this peptide in fish remains to be determined. There is strong recent evidence that regulation and development of the parathyroid gland in mammals is controlled by a cascade of genes. A number of these regulatory factors have been identified using genetically modified mouse models or as genes causing human disease. These include, Gcm2/GCMB, Pax1 and Pax9, Hox3a, Tbx1, GATA3, TBCE, Sox3, Eya1 and Six1/4. Expression of a number of these factors occurs in the gill in fish.
The function of parathyroid hormone and the parathyroid gland in humans is to regulate serum calcium levels to maintain homeostasis. Parathyroid hormone genes are present in fish but their function remains to be elucidated. Parathyroid development is regulated by a cascade of genes, which are now being rapidly defined in mouse models and in human mutations.
Two interferon gamma (IFN-gamma) genes are expressed in immune cells of teleost fish and are potentially implicated in B- and T-lymphocyte responses. IFN-gamma-2 shows structural and functional characteristics to other vertebrate IFN-gamma genes and is associated with T-lymphocyte function. Expression profiling shows IFN-gamma-2 upregulation in T-lymphocytes after phytohemagglutinin (PHA) stimulation in vitro. Unexpectedly, we found IFN-gamma-1, which is structurally different from IFN-gamma-2, to be expressed in lipopolysacharide (LPS)-stimulated IgM+ (B- lymphocyte enriched) fractions. Expression of T-box transcription factor T-bet, but not of GATA-binding protein 3 (GATA3), correlated with expression of both IFN-gamma genes. In-vivo parasite infection, but as predicted not zymosan-induced inflammation, resulted in concomitant upregulation of T-bet and IFN-gamma-2. This corroborates a genuine T-lymphocyte associated role for IFN-gamma-2.
Purified protein derivative (PPD) or tuberculin skin testing is used to identify infected individuals with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immunity to Mtb. In the present study, we compared PBMC cultures in the presence of tuberculin or Candida antigens using cytokine bead arrays and RNA microarrays. Measurements of different cytokines and chemokines in supernatants of PMBC cultures in the presence of PPD showed increased levels of interferon (IFN)-gamma in active tuberculosis infection (ATBI) and latent TB infected (LTBI) compared to controls, and increased levels of TNF-alpha in ATBI compared with LTBI. Also, we found increase of IL-6 in cultures of PPD positive and controls but not in the cultures with Candida. We also report the molecular signature of tuberculosis infection, in ATBI patients, the following genes were found to be up-regulated and absent in LTBI individuals: two kinases (JAK3 and p38MAPK), four interleukins (IL-7, IL-2, IL-6, and IFNbeta1), a chemokine (HCC-4) a chemokine receptor (CxCR5), two interleukin receptors (IL-1R2 and IL-18R1), and three additional ones (TRAF5, Smad2, CIITA, and NOS2A). By contrast, IL-17 and IGFBP3 were significantly up-regulated in LTBI. And, STAT4, GATA3, Fra-1, and ICOS were down-regulated in ATBI but absent in LTBI. Conversely, TLR-10, IL-15, DORA, and IKK-beta were down-regulated in LTBI but not in ATBI. Interestingly, the majority of the up-regulated genes found in ATBI were found in cultures stimulated with tuberculin (PPD) or Candida antigens, suggesting that these pathogens stimulate similar immunological pathways. We believe that the molecular signature distinguishing active from latent tuberculosis infection may require using cytokine bead arrays along with RNA microarrays testing cell cultures at different times following in vitro proliferation assays using several bacterial antigens and PPD.
Summary Analysis of T-helper cell differentiation to T-helper type 1 (Th1) and Th2 lineages has begun to reveal a complex mechanism whereby transcription factors, enzymes that either deposit or remove covalent modifications from histone tails and DNA methylating enzymes are recruited to cytokine genes. Each resultant cell lineage subsequently displays a programme of transcriptional restrictions that firstly, facilitates expression of a particular subset of signature cytokines and secondly, silences expression of the cytokines normally recognized as being markers of the opposite differentiation limb. Some essential proteins in this differentiative paradigm, such as the transcription factors GATA3 and T-bet, are well studied; however, the types of enzymatic activities that these proteins recruit in order to implement differentiation are more obscure. Recent genome-wide studies of histone modifications have begun to clarify how specific modifications of histones impact upon both transcriptional regulation and chromatin organization. Here we review how this information has enlightened our knowledge of how Th1/Th2 differentiation is orchestrated.
Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.
In this study, we attempt to determine whether lycopene regulates inflammatory mediators in the ovalbumin (OVA)-induced murine asthma model. To address this, mice were sensitized and challenged with OVA, and then treated with lycopene before the last OVA challenge. Administration of lycopene significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Administration of lycopene also resulted in a significant inhibition of the infiltration of inflammatory immunocytes into the bronchoalveolar lavage, and attenuated the gelatinolytic activity of matrix metalloproteinase-9 and the expression of eosinophil peroxidase. Additionally, lycopene reduced the increased levels of GATA-3 mRNA level and IL-4 expression in OVA-challenged mice. However, it increased T-bet mRNA level and IFN-gamma expression in lycopene-challenged mice. These findings provide new insight into the immunopharmacological role of lycopene in terms of its effects in a murine model of asthma.
The GATA family of transcription factors (GATA1-6) binds selected GATA sites in vertebrate genomes to regulate specific gene expression. Although vertebrate GATA factors have two highly conserved zinc finger motifs, how the two fingers act together to recognize functional DNA elements is not well understood. Here we determined the crystal structures of the C-terminal zinc finger of mouse GATA3 bound to DNA containing two variously arranged GATA binding sites. Our structures and accompanying biochemical analyses reveal two distinct modes of DNA binding by GATA to closely arranged sites. One mode involves cooperative binding by two GATA factors that interact with each other through protein-protein interactions. The other involves simultaneous binding of the N-terminal zinc finger (N-finger) and the C-terminal zinc finger of the same GATA factor. Our studies represent the first crystallographic analysis of GATA zinc fingers bound to DNA and provide new insights into the DNA recognition mechanism by the GATA zinc finger. Our crystal structure also reveals a dimerization interface in GATA that has previously been shown to be important for GATA self-association. These findings significantly advance our understanding of the structure and function of GATA and provide an important framework for further investigating the in vivo mechanisms of GATA-dependent gene regulation.
Studies of human asthma and of animal models of allergic inflammation/asthma highlight a crucial role for T(H)2 cells in the pathogenesis of allergic asthma. Repressor of GATA (ROG) is a POZ (BTB) domain-containing Kruppel-type zinc finger family (or POK family) repressor. A repressive function to GATA3, a master transcription factor for T(H)2 cell differentiation, is indicated.
The aim of this study was to clarify the regulatory roles of ROG in the pathogenesis of T(H)2-driven allergic diseases, such as allergic asthma.
We examined allergic airway inflammation and airway hyperresponsiveness (AHR) in 3 different mouse models, which use either ROG-deficient (ROG(-/-)) mice, ROG transgenic mice, or adoptive transfer of cells.
In ROG(-/-) mice T(H)2 cell differentiation, T(H)2 responses, eosinophilic airway inflammation, and AHR were enhanced. In ROG transgenic mice the levels of eosinophilic airway inflammation and AHR were dramatically reduced. Furthermore, adoptive transfer of T(H)2 cells with increased or decreased levels of ROG expression into the asthmatic mice resulted in reduced or enhanced airway inflammation, respectively.
These results indicate that ROG regulates allergic airway inflammation and AHR in a negative manner, and thus ROG might represent another potential therapeutic target for the treatment of asthmatic patients.
Aggressive hepatobiliary pancreatic surgery has been associated with high complication rates. Correlations of Th1/Th2 balance and toll-like receptor (TLR) 2/4 expression with postoperative infection following surgery were prospectively evaluated.
Plasma concentrations of interleukin (IL)-6, IL-10, soluble lymphocyte activation gene (sLAG)-3, and soluble CD30 were determined by enzyme-linked immunosorbent assay, and expression levels of T-bet, GATA-3, TLR2, and TLR4 mRNA in peripheral blood mononuclear cells were assayed by reverse transcription-polymerase chain reaction perioperatively in 56 consecutive patients who underwent hepatobiliary pancreatic surgery.
Of the 56 patients, 30 patients had postoperative infection. Postoperative plasma levels of IL-6 and IL-10 were significantly higher in patients with postoperative infection than in those without infection (P < 0.05). Plasma soluble CD30 level and GATA-3 mRNA expression level were significantly higher preoperatively, and remained higher by postoperative d 7 in patients with postoperative infection (P < 0.05). Soluble lymphocyte activation gene levels were not significantly different between the two groups. T-bet mRNA expression level was significantly higher on postoperative d 3, 7, and 14 in patients with postoperative infection (P < 0.05). Preoperative expression levels of GATA-3 mRNA correlated significantly with those of TLR2 and TLR4 mRNA (P < 0.05).
These results suggest that in patients with postoperative infection, Th1/Th2 balance shifts toward Th2 dominance preoperatively.
The results of our study demonstrated the association between the single nucleotide polymorphisms (SNPs) of GATA-3 and allergic rhinitis (AR).
AR is considered to be controlled by T-helper type 2 (Th2) cells. GATA-3 is the crucial transcription factor controlling the differentiation and function of Th2 cells. We aimed to evaluate the association between the SNPs of GATA-3 and AR.
GATA-3 SNPs in 109 AR patients and 112 healthy controls in China were detected with restriction fragment length polymorphism.
The genotypes at rs1269486 were GG, GA, and AA, and the genotypes at rs2229360 were CC, CT, and TT. The frequency of the G allele in the patient group was significantly higher than that in the control group (p<0.01), while the frequency of the A allele in the patient group was significantly lower than that in the control group (p<0.01). Meanwhile, the haplotype frequency of GC at the two loci in the patient group was significantly higher than that in the control group (p<0.01), and the haplotype frequency of AC in the patient group was significantly lower than that in the control group (p<0.01).
The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.
The transcription factor GATA3 has recently been found to be involved in the carcinogenesis for numerous cancers. We investigated this marker in relation to clinicopathologic characteristics, hormone receptors, other biomarkers, and survival in endometrial carcinoma.
A population-based study of 316 endometrial carcinomas with complete follow-up was studied for GATA3, estrogen receptor (ER)-alpha, ERbeta2, and progesterone receptor (PR) expression.
Positive GATA3 expression in hysterectomy specimens significantly correlated to high International Federation of Gynecology and Obstetrics stage, serous papillary/clear cell subtypes, high histologic grade, loss of PR expression, aneuploidy, high proliferation, pathologic p53 and p16 expression, and poor prognosis (P = .003). Loss of hormone receptors significantly correlated with aggressive phenotype and poor prognosis. Pathologic expression of GATA3/ERalpha in combination added independent prognostic information.
GATA3 expression is associated with an aggressive phenotype and adds independent prognostic information in addition to receptor status. Further studies of its value in tailored treatment protocols seem justified.
T helper (Th) 2 cells play a central role in the pathogenesis of allergic diseases such as allergic asthma, atopic dermatitis, and allergic rhinitis. We have found that YM-341619 hydrochloride, which suppressed IL-4-induced STAT6-dependent reporter gene expression, inhibited the differentiation of mouse spleen T cells into Th2 cells in vitro. YM-341619 suppressed the production of IL-4 and the expression of GATA-3 mRNA, a Th2 transcription factor, in T cells cultured with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-4. In contrast, the production of IFN-gamma and the expression of T-bet mRNA, a Th1 transcription factor, in T cells cultured with anti-CD3 antibody in the presence of IL-12, were not effected by YM-341619. Orally administered YM-341619 (0.003-0.03 mg/kg) reduced the plasma IgE level of DNP-Ascaris-sensitized rats, but not the IgG(2a) level. YM-341619 suppressed IL-4 and IL-13 production in the splenocytes of these DNP-Ascaris-sensitized rats without augmenting IFN-gamma production. YM-341619 also dose-dependently suppressed eosinophil accumulation in the lung (0.003-3 mg/kg, p.o.) and airway hyperresponsiveness (0.3-3 mg/kg, p.o.) induced by repeated exposure to ovalbumin in ovalbumin-sensitized rats. These results suggest that YM-341619 has the ability to suppress allergen-induced Th2 responses by selectively inhibiting the differentiation of CD4(+) T cells into the Th2 subset.
Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not to Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.
This study investigated interleukin-4 (IL-4), IL-4 delta 2, transforming growth factor beta (TGF-beta), TGF-beta RII, Foxp3, GATA-3, T-bet, and gamma interferon (IFN-gamma) transcription in peripheral blood samples of adult pulmonary tuberculosis patients prior to and after 1 week of therapy. Twenty patients with positive results for sputum culture for Mycobacterium tuberculosis were enrolled and treated with directly observed short-course antituberculosis chemotherapy. Early treatment response was assessed. At the end of the intensive phase of treatment (month 2), 12 patients remained sputum culture positive (slow responders) and 8 converted to a negative culture (fast responders). Only the expression levels of IL-4 (4-fold decrease) and IL-4 delta 2 (32-fold increase) changed significantly during the first week of therapy in the 20 patients. No baseline differences were present between the responder groups, but fast responders had significantly higher IL-4 transcripts than slow responders at week 1. Fast responders showed a 19-fold upregulation and slow responders a 47-fold upregulation of IL-4 delta 2 at week 1. Only slow responders also showed a significant decrease in IL-4 expression at week 1. There were no significant differences in expression of TGF-beta, TGF-beta RII, Foxp3, IFN-gamma, and GATA-3 between the groups. These data show that differential IL-4-related gene expression in the early stage of antituberculosis treatment accompanies differential treatment responses and may hold promise as a marker for treatment effect.
Inflammatory bowel diseases (IBD) are inflammatory diseases with a multifactorial component that involve the intestinal tract. The two relevant IBD syndromes are Crohn's disease (CD) and ulcerative colitis (UC). One factor involved in IBD development is a genetic predisposition, associated to NOD2/CARD15 and Toll-like receptor 4 (TLR4) polymorphisms that might favor infectious enterocolitis that is possibly associated to the development of IBD. The identification of specific immunologic alterations in IBD and their relationship to the etiology of the disease is a relevant research topic. The role of intra and extracellular molecules, such as transcription factors and cytokines that are involved in the inflammatory response, needs to be understood. The relevance of immunologic molecules that might drive the immune response to a T helper (Th) 1, Th 2 or the recently described Th 17 phenotype, has been demonstrated in animal models and clinical studies with IBD patients. CD and UC predominantly behave with a Th 1 and Th 2 immune phenotype, respectively. Recently, an association between CD and Th 17 has been reported. The knowledge acquired from immunologic and molecular research will help to develop accurate diagnostic methods and efficient therapies.
The IMiDs immunomodulatory drugs are an expanding family of compounds under investigation in a broad range of diseases because they exhibit immunomodulatory and anti-tumorigenic properties. Although the molecular targets remain unidentified, the broad activity of select IMiDs immunomodulatory drugs on cell signaling pathways and transcription regulation has been partly described. One characteristic of these compounds is their ability to act as a co-stimulus of TCR ligation leading to increased IL-2, TNF-alpha and IFN-gamma expression indicative of a Th1 phenotype. Because clinical evidence for this response has been observed in thalidomide and lenalidomide treated patients, we investigated the effect of CC-4047 on T cell activation and differentiation at the molecular level. We used primary human CD4(+) T cells as a model and found that CC-4047 enhances the expression of transcription factor T-bet in both naive and pre-polarized Th2 cells. This modulation leads to upregulation of Th1 markers and cytokine production. By increasing the expression of T-bet, CC-4047 promotes the differentiation of naive T-cells to Th1 as well as effectively reverting Th2 cells into Th1-like effector cells in vitro. These findings elucidate a novel mechanism of action of CC-4047 on T cell differentiation, suggesting that certain IMiDs immunomodulatory drugs may have expanded clinical application in treating both allergic diseases and certain T cell lymphomas where a predominant Th2 phenotype is displayed.
GATA binding protein 3 (GATA3) is an important regulator of central nervous system (CNS) development, but its expression pattern in the postnatal CNS has not been studied. In the present study, we examined the distribution of GATA3 mRNA in the mouse CNS at different postnatal stages by in situ hybridization. During the first 2 weeks of postnatal development, numerous GATA3-expressing cells were found in the intergeniculate leaf, ventral lateral geniculate nucleus, pretectal nucleus, nucleus of the posterior commissure, superior colliculus, inferior colliculus, periaqueductal grey, substantia nigra and raphe nuclei. Few notable changes in the profile of GATA3 expression occurred over this time period. As postnatal development progressed, however, GATA3 expression weakened, and was maintained in only a few regions of the adult CNS. Throughout the brain, we found that GATA3-expressing cells were NeuN-positive, and no colocalization with glial fibrillary acidic protein (GFAP) was observed. In the substantia nigra, GATA3 was exclusively expressed in cells of the reticulate part and some of which were found to be GABAergic. This study presents a comprehensive overview of GATA3 expression in the CNS throughout postnatal life, and the dynamics that we observed provide insights for further investigations of the roles of GATA3 in postnatal development and the maintenance of the mature CNS.
The immune response to allergens starts with stimulation of a naïve T helper (Th) cell and its differentiation into a Th2 cell, expressing the cytokines interleukin (IL)-4, IL-5 and IL-13 responsible for the allergic response. The initial pattern of cytokine expression is retained during restimulation and division of the Th2 cell to create a population of specific allergen-responsive memory Th2 cells. Both, the coordinate cytokine expression and the inherited cytokine memory are specified by epigenetic mechanisms. Th2-specific changes in chromatin configuration at the Th2 locus act locally to open DNA, allowing recruitment of transcriptional machinery and rapid induction of cytokine expression. Induction of the transcription factor GATA3 is critical to this process. Loss of DNA methylation at the Th2 locus during differentiation from a naïve Th cell correlates to increased histone acetylation, consistent with the expression of IL-4, IL-5 and IL-13. The silencing of the Th2 locus in Th1 cells was associated with repressive histone methylation. These data indicate the formation of a 'poised' chromatin configuration at the Th2 locus that in combination with specific transcription factors specifies the cytokine repertoire in daughter cells and allows the immediate, rapid induction of cytokines by those cells in response to allergen.
Chromosomal localization of nine porcine genes encoding transcription factors involved in adipogenesis was determined. BAC clones harboring sequences of selected genes CEBPA (SSC6q12), CEBPB (SSC17q23), CEBPD (SSC4q15), CEBPG (SSC6q12), PPARG (SSC13q24), SREBF1 (SSC10q17), DDIT3 (SSC12q15), GATA2 (SSC13q24 -->q31) and GATA3 (SSC5p12) were mapped by FISH. The positions of these genes in the human and pig genomes were compared. A potential role of the genes encoding adipogenesis factors as candidate genes for fatness traits as well as obesity-related phenotypes is discussed.
The transcription factor GATA3 has recently been shown to be necessary for mammary gland morphogenesis and luminal cell differentiation. There is also an increasing body of data linking GATA3 to the estrogen receptor alpha (ERalpha) pathway. Among these it was shown that GATA3 associates with the promoter of the ERalpha gene and ERalpha can reciprocally associate with the GATA3 gene. GATA3 has also been directly implicated in a differentiated phenotype in mouse models of mammary tumourigenesis. The purpose of our study was to compare coexpressed genes, by meta-analysis, of GATA3 and relate these to a similar analysis for ERalpha to determine the depth of overlap.
We have used a newly described method of meta-analysis of multiple cancer studies within the Oncomine database, focusing here predominantly upon breast cancer studies. We demonstrate that ERalpha and GATA3 reciprocally have the highest overlap with one another. Furthermore, we show that when both coexpression meta-analysis lists for ERalpha and GATA3 are compared there is a significant overlap between both and, like ERalpha, GATA3 coexpresses with ERalpha pathway partners such as pS2 (TFF1), TFF3, FOXA1, BCL2, ERBB4, XBP1, NRIP1, IL6ST, keratin 18(KRT18) and cyclin D1 (CCND1). Moreover, as these data are derived from human tumour samples this adds credence to previous cell-culture or murine based studies.
GATA3 is hypothesized to be integral to the ERalpha pathway given the following: (1) The large overlap of coexpressed genes as seen by meta-analysis, between GATA3 and ERalpha, (2) The highest coexpressing gene for GATA3 was ERalpha and vice-versa, (3) GATA3, like ERalpha, coexpresses with many well-known ERalpha pathway partners such as pS2.
Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity.
An exceptionally high B19V viral load in EMBs (115,091 viral copies/mug nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amino-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNgamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNgamma, IL2, IL27 and T-bet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low.
Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.
T helper type 2 (Th2) cells produce IL-4, IL-5, and IL-13 and play an important role in humoral immunity and allergic reactions. During Th2 cell differentiation, naïve CD4 T cells acquire 'Th2 cell identity', that is, the capability to produce selectively a large amount of Th2 cytokines. Th2 cell identity is maintained in memory Th2 cells. Significant advances in understanding of the molecular requirement for these processes have been made. The expression of GATA3, a master transcription factor for Th2 cell differentiation, is uniquely regulated by several distinct mechanisms. Molecular analyses of memory Th2 cells revealed that cell survival and the maintenance of Th2 cell function are epigenetically regulated by various nuclear factors, including Polycomb and Trithorax molecules.
Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.
Prostate cancer is the most frequently diagnosed cancer among men in the United States. In contrast, cancer of the seminal vesicle is exceedingly rare, despite that the prostate and seminal vesicle share similar histology, secretory function, androgen dependency, blood supply, and (in part) embryonic origin. We hypothesized that gene-expression differences between prostate and seminal vesicle might inform mechanisms underlying the higher incidence of prostate cancer.
Whole-genome DNA microarrays were used to profile gene expression of 11 normal prostate and 7 seminal vesicle specimens (including six matched pairs) obtained from radical prostatectomy. Supervised analysis was used to identify genes differentially expressed between normal prostate and seminal vesicle, and this list was then cross-referenced to genes differentially expressed between normal and cancerous prostate. Expression patterns of selected genes were confirmed by immunohistochemistry using a tissue microarray.
We identified 32 genes that displayed a highly statistically significant expression pattern with highest levels in seminal vesicle, lower levels in normal prostate, and lowest levels in prostate cancer. Among these genes was the known candidate prostate tumor suppressor GSTP1 (involved in xenobiotic detoxification). The expression pattern of GSTP1 and four other genes, ABCG2 (xenobiotic transport), CRABP2 (retinoic acid signaling), GATA3 (lineage-specific transcription), and SLPI (immune response), was confirmed by immunohistochemistry.
Our findings identify candidate prostate cancer genes whose reduced expression in prostate (compared to seminal vesicle) may be permissive to prostate cancer initiation. Such genes and their pathways may inform mechanisms of prostate carcinogenesis, and suggest new opportunities for prostate cancer prevention.
Mast cells are thought to participate in a wide variety of pathophysiological conditions. Mechanisms of regulation, however, of mast cell production and maturation are still to be elucidated. Mast cell developmental process is likely to be profoundly affected by cell-autonomous transcriptional regulators such as the GATA family and CCAAT/enhancer binding protein (C/EBP) family members. Extracellular regulators such as stem cell factor and IL-3 have essential roles in basal and inducible mast cell generation, respectively. The relationship, however, between the extracellular signaling and cellular transcriptional control is unclear, and the trigger of the mast cell development remains elusive. Notch signaling plays a fundamental role in the lymphopoietic compartment, but its role in myeloid differentiation is less clear. Here, we demonstrate that Notch signaling connects environmental cues and transcriptional control for mast cell fate decision. Delta1, an established Notch ligand, instructs bone marrow common myeloid progenitors and granulocyte-macrophage progenitors toward mast cell lineage at the expense of other granulocyte-macrophage lineages, depending on the function of the Notch2 gene. Notch2 signaling results in the up-regulation of Hes-1 and GATA3, whereas simultaneous overexpression of these transcription factors remarkably biases the progenitor fate toward the mast cell-containing colony-forming cells. C/EBPalpha mRNA was down-regulated in myeloid progenitors as a consequence of Hes-1 overexpression, in agreement with the recent proposal that the down-regulation of C/EBPalpha is necessary for mast cell fate determination. Taken together, signaling through Notch2 determines the fate of myeloid progenitors toward mast cell-producing progenitors, via coordinately up-regulating Hes-1 and GATA3.
NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.
Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.
In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.
CD43 is a highly glycosylated transmembrane protein that regulates T cell activation. CD43(-/-) T cells are hyperproliferative and the cytoplasmic tail of CD43 has been found to be sufficient to reconstitute wild-type proliferation levels, suggesting an intracellular mechanism. In this study, we report that upon TCR ligation CD43(-/-) T cells demonstrated no increase in tyrosine phosphorylation but a decreased calcium flux. Interestingly, CD43(-/-) T cells preferentially differentiated into Th2 cells in vitro, and CD43(-/-) T cells show increased GATA-3 translocation into the nucleus. In vivo, CD43(-/-) mice exhibited increased inflammation in two separate models of Th2-mediated allergic airway disease. In contrast, in Th1-mediated diabetes, nonobese diabetic CD43(-/-) mice did not significantly differ from wild-type mice in disease onset or progression. Th1-induced experimental autoimmune encephalomyelitis to MOG(35-55) was also normal in the CD43(-/-) mice. Nonetheless, the CD43(-/-) mice produced more IL-5 when restimulated with MOG(35-55) in vitro and demonstrated decreased delayed-type hypersensitivity responses. Together, these data demonstrate that although CD43(-/-) T cells preferentially differentiate into Th2 cells, this response is not sufficient to protect against Th1-mediated autoimmune responses.
Gata transcription factors are critical regulators of proliferation and differentiation implicated in various human cancers, but specific genes activated by Gata proteins remain to be identified. We previously reported that enforced expression of Gata3 during T cell development in CD2-Gata3 transgenic mice induced CD4(+)CD8(+) double-positive (DP) T cell lymphoma. Here, we show that the presence of the DO11.10 T-cell receptor transgene, which directs DP cells towards the CD4 lineage, resulted in enhanced lymphoma development and a dramatic increase in thymocyte cell size in CD2-Gata3 transgenic mice. CD2-Gata3 DP cells expressed high levels of the proto-oncogene c-Myc but the Notch1 signaling pathway, which is known to induce c-Myc, was not activated. Gene expression profiling showed that in CD2-Gata3 lymphoma cells transcription of c-Myc and its target genes was further increased. A substantial fraction of CD2-Gata3 lymphomas had trisomy of chromosome 15, leading to an increased c-Myc gene dose. Interestingly, most lymphomas showed high expression of the Notch targets Deltex1 and Hes1, often due to activating Notch1 PEST domain mutations. Therefore, we conclude that enforced Gata3 expression converts DP thymocytes into a pre-malignant state, characterized by high c-Myc expression, whereby subsequent induction of Notch1 signaling cooperates to establish malignant transformation. The finding that Gata3 regulates c-Myc expression levels, in a direct or indirect fashion, may explain the parallel phenotypes of mice with overexpression or deficiency of either of the two transcription factors.
GATA-3 is the master transcription factor for T helper 2 (Th2) cell differentiation and is critical for the expression of Th2 cytokines. Little is known, however, about the nature of the functional molecular complexes of GATA-3. We identified a high-mobility group (HMG)-box type transcription factor, lymphoid enhancer factor 1 (LEF-1), in the GATA-3 complex present in Th2 cells using a Flag-calmodulin-binding peptide (CBP)-tag based proteomics method. The interaction between GATA-3 and LEF-1 was confirmed by co-immunoprecipitation experiments using LEF-1-introduced T-cell lineage TG40 cells. The HMG-box domain of LEF-1 and two zinc finger domains of GATA-3 were found to be important for the physical association. The introduction of LEF-1 into developing Th2 cells resulted in the suppression of Th2 cytokine production. The suppression was significantly lower in the cells into which a HMG-box-deleted LEF-1 mutant was introduced. Moreover, LEF-1 inhibited the binding activity of GATA-3 to the interleukin (IL)-5 promoter. These results suggest that LEF-1 is involved in the GATA-3 complex, while also regulating the GATA-3 function, such as the induction of Th2 cytokine expression via the inhibition of the DNA-binding activity of GATA-3.
The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.
We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.
We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.
The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.
Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by a T(H)2-skewed eosinophilic inflammation, whereas chronic rhinosinusitis without nasal polyps (CRSsNP) represents a predominant T(H)1 milieu.
We aimed to study the direct tissue expression of transcription factors for T-cell subpopulations, including T regulatory cells, in relation to the cytokine expression patterns in the different disease subgroups.
The expression of forkhead box P3 (FOXP3), T-box transcription factor (T-bet), GATA-3, retinoid acid-related orphan receptor C (RORc), the suppressive cytokines TGF-beta1 and IL-10, and T(H)1/ T(H)2/ T(H)17 cytokines (IFN-gamma, IL-4, IL-5, IL-13, IL-17) were analyzed by means of RT-PCR in 13 CRSsNP, 16 CRSwNP, and 10 control samples. Additional protein measurements were performed for TGF-beta1 and IFN-gamma.
In CRSwNP, we observed a significantly lower FOXP3 mRNA and TGF-beta1 protein expression, but a significantly higher T-bet, GATA-3, IL-5, and IL-13 mRNA expression compared with controls, whereas RORc was not significantly different compared with controls. In CRSsNP, FOXP3, T-bet, GATA-3, and RORc expression was not significantly different from controls, whereas TGF-beta1 mRNA, IFN-gamma mRNA, and protein were significantly higher in CRSsNP compared with controls. For IL-17, no significant differences were noted among all groups.
We demonstrate for the first time a decreased FOXP3 expression accompanied by an upregulation of T-bet and GATA-3 and a downregulation of TGF-beta1 in CRSwNP versus controls and CRSsNP.
Atopic eczema is characterized by Th2-dominant immunity with the cytokine interleukin 13 and the transcription factor GATA binding protein 3 playing a critical role.
We assessed the association of polymorphisms in the IL13 and GATA3 genes with childhood eczema.
A birth cohort (n = 1456) was established on the Isle of Wight in 1989 and followed at the ages of 1 (n = 1167), 2 (n = 1174), 4 (n = 1218) and 10 years (n = 1373) to determine the prevalence of allergic disease including eczema. At 4 and 10 years, skin prick testing was performed. Whole blood samples (n = 923) were obtained at the 10-year assessment, stored frozen, and genotyped. Five polymorphisms from IL13 and seven from GATA3 were genotyped for this analysis. Repeated measurement analyses were conducted for the occurrence of eczema at ages 1, 2, 4 and 10 years. All analyses were adjusted for maternal and paternal eczema, low birth weight (< 2500 g), breastfeeding >or= 3 months and age.
IL13 was not associated with childhood eczema. For GATA3, the single nucleotide polymorphism (SNP) rs2275806 (promoter region) showed an increased odds ratio for atopic eczema independent of whether the comparison group had a positive skin prick test. The SNP rs444762 (intron 3 region) was associated with atopic eczema in comparison with children without eczema. The increased relative risks remained significant after adjustment for multiple testing only for rs2275806 (P < 0.05).
A SNP in GATA3 is associated with atopic eczema. This finding highlights the importance of GATA3 as an immune-modulating gene in atopic eczema.
Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-beta), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.
The inflammatory response observed in allergic disease involves multiple cell types but is orchestrated in part by the T(H)2 cytokines IL-4, IL-5, and IL-13. In recent years, the transcription factors that control the expression and function of these cytokines have been elucidated, including signal transducer and activator of transcription 6, GATA3, nuclear factor of activated T cells, and nuclear factor kappaB. These molecules are attractive targets for therapeutic intervention because they regulate the expression of numerous effector molecules and functions simultaneously. For instance, the immunosuppressive agents glucocorticoids and cyclosporin A both function by repressing the activity of transcription factors through a variety of mechanisms. In this review we examine the role of each transcription factor in allergic disease and discuss approaches that have been taken to therapeutically interfere with transcription factor function in allergic disease.
CBFbeta is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFbeta levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFbeta is also required at very early stages of natural killer (NK)-cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFbeta insufficient cells, nor can overexpression of Runx1 or CBFbeta overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFbeta, and both signals converge to activate the T-cell developmental program.
GATA3 and Notch1 are essential for T cell development at the earliest stage, but their mutual roles in this process remain to be clarified. In this study, we demonstrated that impairment of T lymphopoiesis in hematopoietic progenitor cells (HPC) of GATA3-deficient fetal liver (FL) on day 11.5 of gestation (E11.5) was rescued only by introduction of both GATA3 and the intracellular region of Notch1 but not by either alone. However, the introduction of GATA3 only was sufficient for T cell induction in GATA3-deficient FL cells at the advanced stage, where Notch signaling is well detectable. This indicates that Notch signaling is necessary for GATA3 to function for T cell fate specification but is not sufficient without GATA3. On the other hand, Notch signaling is sufficient for blockage of B cell development without GATA3, suggesting that T cell fate specification at the branching point does not result simply from the developmental arrest of B cell lineage by Notch signaling.
The Brn-3 family of transcription factors play a critical role in regulating expression of genes that control cell fate, including the small heat shock protein Hsp27. The aim of this study was to investigate the relationship between Brn-3a and Brn-3b and Hsp27 expression in the developing rodent heart. Brn-3a and Brn-3b were detected from embryonic days 9.5-10.5 (E9.5-E10.5) in the mouse heart, with significant increases seen later during development. Two isoforms (long and short) of each protein were detected during embryogenesis and postnatally. Brn-3a messenger RNA (mRNA) and protein were localized by E13.0 to the atrio-ventricular (AV) valve cushions and leaflets, outflow tract (OFT), epicardium and cardiac ganglia. By E14.5, Brn-3a was also localised to the septa and compact ventricular myocardium. An increase in expression of the long Brn-3a(l) isoform between E17 and adult coincided with a decrease in expression of Brn-3b(l) and a marked increase in expression of Hsp27. Hearts from Brn-3a-/- mice displayed a partially penetrant phenotype marked by thickening of the endocardial cushions and AV valve leaflets and hypoplastic ventricular myocardium. Loss of Brn-3a was correlated with a compensatory increase in Brn-3b and GATA3 mRNA but no change in Hsp27 mRNA. Reporter assays in isolated cardiomyocytes demonstrated that both Brn-3a and Brn-3b activate the hsp27 promoter via a consensus Brn-3-binding site. Therefore, Brn-3 POU factors may play an important role in the development and maintenance of critical cell types and structures within the heart, in part via developmental regulation of myocardial Hsp27 expression. Furthermore, Brn-3a may be necessary for correct valve and myocardial remodelling and maturation.
The GATA family of transcription factors plays essential roles in the specification and maintenance of differentiated cell types. GATA-3 was identified in a microarray screen of the mouse mammary gland as the most highly expressed transcription factor in the mammary epithelium and is expressed exclusively in the luminal epithelial cell population. Targeted deletion of GATA-3 in mammary glands leads to profound defects in mammary development and inability to specify and maintain the luminal cell fate in the adult mouse. In breast cancer, GATA-3 has emerged as a strong predictor of tumor differentiation, estrogen-receptor status, and clinical outcome. GATA-3 maintains tumor differentiation and suppresses tumor dissemination in a mouse model of breast cancer. This review explores our current understanding of GATA-3 signaling in luminal cell differentiation, both in mammary development and breast cancer.
GATA-3 is a zinc finger transcription factor that is expressed in T cell lineages as well as in the nervous system during development. In this study, we report that forced expression of GATA-3 resulted in an increased number of dopamine beta-hydroxylase (DBH)-expressing neurons in primary neural crest stem cell (NCSC) culture, suggesting that the DBH gene may be a downstream target gene of GATA-3. GATA-3 robustly transactivates the promoter function of the noradrenaline (NA)-synthesizing DBH gene, via two specific upstream promoter domains; one at -62 to -32 bp and the other at -891 to -853 bp. Surprisingly, none of these domains contain GATA-3 binding sites but encompass binding motifs for transcription factors Sp1 and AP4, respectively. Protein-protein interaction analyses both in vitro and in vivo and chromatin immunoprecipitation (ChIP) assays showed that GATA-3 effects its transcriptional regulatory function through physical interactions with these transcription factors.
To elucidate the structure of terminal inverted duplications and to investigate potential mechanisms of formation in two cases where there was mosaicism with cells of apparently normal karyotype.
A karyotype [46,XY,inv dup(4)(p16.3p15.1)/46,XY] performed on blood lymphocytes from a patient referred for developmental delay (case 1) demonstrated a normal karyotype in 60% of cells with a terminal inverted duplication 4p in the remainder. In case 2, referred for multiple fetal anomalies on an ultrasound scan, 33% of amniocyte colonies were karyotypically normal, with a terminal inv dup 10p in the remainder [46,XX,inv dup(10)(p15.3p11)/46,XX]. Duplicated FISH signals for GATA3 and NEBL loci (in case 2), and for the Wolf-Hirschhorn locus (case 1) confirmed the inverted structure of both duplications. In the GTL banded normal cells from both cases, there was a cryptic deletion detected by FISH of one copy of the subtelomere 4p (case 1, probe GS-36P21), and subtelomere 10p (case 2, probe GS-306F7). At pter on both inv dup chromosomes there was no FISH signal present for the specific subtelomere probe. However, a positive pantelomeric probe signal was detected at 4 pter and 10 pter in both the cryptically-deleted chromosomes and the inv dup chromosomes in the respective cell lines of both cases.
An inv dup structure was evident for both cases on GTL bands, and confirmed by the various FISH studies. The presence of telomere (TTAGGG repeat) sequences at pter on the inv dup chromosomes (where more proximal chromosome specific subtelomeric probes were negative) was indicated by the pantelomeric probe signals in both cases. We conclude the most likely mechanism of origin in both cases was by sub-telomeric breakage in the zygote at pter, and delayed repair/rearrangement until after one or more subsequent mitotic divisions. In these divisions, at least one breakage-fusion-bridge cycle occurred, to produce inverted duplications. It is proposed then that two differently "repaired" daughter cells proliferated in parallel. In one daughter cell line (with an overtly normal karyotype) there was deletion of the subtelomere and presumed repair through capping by a neo-telomere (i.e. "healing", as initially proposed by McClintock). This occurred in both cases presented. In the other daughter cell of each case, it is proposed that chromosome stabilization was achieved (after replication) by sister chromatid reunion to form a dicentric, which broke at a subsequent anaphase, to form an inverted duplication (with loss of the reciprocal product, and the other daughter cell line). One inv dup was repaired without an interstitial specific subtelomere (case 1) and one was repaired with a duplicated specific interstitial subtelomere (case 2). After repair TTAGGG repeats were detected by FISH at each respective new pter.
Allergic bronchial asthma is a chronic inflammatory disease of the airways. The transcription factor GATA-3 was shown to play an important role in TH2 cell activation, but also in the regulation of other cell types involved in bronchial asthma including mast cells, eosinophils, and epithelial cells. DNAzymes represent a new class of antisense molecules that combines the specificity of DNA base pairing with an inherent RNA-cleaving enzymatic activity.
To develop a GATA-3 mRNA-specific DNAzyme and analyze its allergy-preventing activity in murine models of experimental allergic asthma.
The most active DNAzyme (termed gd21) was selected by in vitro cleavage assays. Allergic airway inflammation was assessed by inflammatory cell and cytokine analysis within bronchoalveolar lavage. Lung histology, including goblet cell hyperplasia and lung function, was analyzed using head-out body-plethysmography.
Intranasal administration of gd21 prevented airway inflammation and mucus production and inhibited development of airway hyperresponsiveness to methacholine in models of acute allergic airway inflammation. Similar effects were also detected in a model of chronic experimental asthma. Interestingly, gd21 was at least as effective as other antisense molecules, and off-target effects were not detected. Further experiments indicated that pulmonary surfactant may facilitate the cellular uptake of gd21 by acting as an endogenous transfectant.
These results indicate that topical application of the GATA-3-specific DNAzyme is a promising novel approach for the treatment of allergic bronchial asthma.
The ectopic expression of the Notch receptor ligand delta-like 1 on stromal cells allows the induction of T cells from embryonic stem cells (ESCs). However, these in vitro-generated T cells are not transplantable because they are too immature to mount an immune response in an immunocompromised animal. We efficiently generated a subset of T cells called invariant natural killer T (iNKT) cells from ESCs derived from peripheral iNKT cells using somatic cell nuclear transfer (ntESCs). These iNKT cells matured autonomously in vivo and exhibited an adjuvant effect accompanying the production of interferon-gamma in an antigen-specific manner. This adjuvant effect culminated in the inhibition of inoculated tumor cell growth. Our results indicate that ntESC-derived iNKT cells are transplantable lymphocytes that will be beneficial for the induction of immune tolerance and the treatment of autoimmune diseases, tumors, and infections.
GATA3, a transcriptional activator, plays a critical role in the development of T-cells and differentiation to T helper type 2 cells. To date, no information is available on the role of GATA3 in the teleost immune system. We identified full-length cDNA and alternatively spliced variants of ginbuna crucian carp GATA3 (gbGATA3). The gbGATA3 gene is transcribed into multiple splice variants lacking either one or both zinc finger domains, although the sequences of both domains are fully conserved between ginbuna and other vertebrates. We found that alternative splice site and stop codon in gbGATA3 intron 3, located between exons that separately encode the two zinc finger domains, are conserved among teleosts, suggesting that teleost GATA3 gene can be translated into multiple isoforms. RT-PCR analysis revealed that the gbGATA3 is strongly expressed in the brain, thymus and gill of unstimulated fish. Moreover, gbGATA3 expression was detected in surface-IgM-negative lymphocytes among kidney cells sorted by FACS. Real-time PCR demonstrated that expression levels of full-length gbGATA3 and the splice variants differed with tissue type, but full length was always the predominantly expressed form. These results suggest that gbGATA3, including its splice variants, is involved in teleost T-cell function.
The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear.
Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs).
IL-4 and IFN-gamma production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs.
Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype.
CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components.
To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.
Inner ear stem cells can be isolated by neurosphere formation from the vestibular organs and the cochlea. The cells are pluripotent, with the potential to become hair cells and neurons, the cochlear cell types whose loss causes deafness. Here we describe the control of cell fate decisions that determine the phenotype adopted by these progenitors, and we determine whether differentiation to sensory neurons is preferred over other types of neurons. Differentiation of progenitor cells recapitulated developmental pathways of embryonic sensory neurons. Based on marker expression, retinoic acid increased the yield of neurons and the percentage of sensory neurons obtained and caused a sharp increase in Pax2, a key transcription factor of cranial placodes. Markers of embryonic auditory and other sensory neurons, GATA3, Brn3a, and islet1, could be detected after 3 days of differentiation of the cells, and markers of the sensory phenotype, peripherin, calretinin, TrkC, and TrkB were expressed after 10 days. The differentiated cells had tetrodotoxin-sensitive sodium currents and fired action potentials, and recordings revealed functional AMPA type-glutamate receptors, further indicating that these cells had developed neuronal features. Neurons differentiated from these stem cells grew processes to hair cells in vitro. Development of functional activity in cells with the markers of sensory neurons suggested that the inner ear stem cells might have the capacity to replace cells lost due to neural degeneration.
Pegylated granulocyte colony-stimulating factor (G-CSF) has recently been introduced as a new compound for mobilization of CD34(+) hematopoietic stem and progenitor cells. In this study, we compared the molecular and functional characteristics of CD34(+) cells mobilized by pegylated G-CSF with those mobilized by unconjugated G-CSF.
Gene expression of immunomagnetically enriched CD34(+) cells from leukapheresis products of patients who were given pegylated-G-CSF or unconjugated G-CSF was analyzed using Affymetrix HG Focus microarrays and quantitative reverse transcriptase polymerase chain reaction. Flow cytometry and fluorescence activated cell sorting was conducted to assess the CD34(+) subset composition and to obtain Lin(-), CD34(+), CD38(-) hematopoietic stem cells. Cell cycle assays and clonogenic assays were performed for functional corroboration.
Pegylated G-CSF and unconjugated G-CSF mobilized CD34(+) and hematopoietic stem cells with different molecular phenotypes and functional properties. The CD34(+) cells mobilized by pegylated G-CSF had higher expression levels of genes indicative of early hematopoiesis, including HOXA9, MEIS1 and GATA3. We found lower expression of genes characteristic of erythroid and later stages of myeloid differentiation and a lower functional burst-forming unit erythroid/colony-forming unit-granulocyte-macrophage ratio. Consistently, greater numbers of hematopoietic stem cells and common myeloid progenitors and fewer megakaryocyte-erthrocyte progenitors were found in the pegylated-G-CSF-mobilized CD34(+) cells. Additionally, sorted pegylated-G-CSF-mobilized hematopoietic stem cells displayed higher expression of HOXA9 in comparison to G-CSF-mobilized hematopoietic stem cells. In line with the gene expression data, CD34(+) cells mobilized by pegylated G-CSF, as well as sorted hematopoietic stem cells, showed a significantly greater cell cycle activity.
Stimulation with pegylated-G-CSF or G-CSF results in different expression of key regulatory genes and different functional properties of mobilized hematopoietic stem cells as well as their progeny, a finding that might be relevant for the application of these cells in blood stem cell transplantation.
GATA-3 is a transcription factor involved in human growth and differentiation. Gene expression profiling has shown that GATA-3 is highly expressed in the Luminal A subtype of breast cancer. A recent study found GATA-3 to be associated with favorable breast cancer pathologic features, including negative lymph node and positive estrogen receptor (ER) status. GATA-3 levels were also found to be an independent prognostic marker, with low expression predicting for breast cancer recurrence.
Our case series consists of 3,119 cases of invasive breast cancer in which GATA-3 expression was assessed by immunohistochemistry on tissue microarrays. We considered >5% nuclear staining to be a positive result for GATA-3.
Thirty-two percent of cases were GATA-3 positive. GATA-3 is almost exclusively expressed in ER+ patients and is also associated with lower tumor grade, older age at diagnosis, and the absence of Her2 overexpression. In univariate analysis, the presence of GATA-3 is a marker of good prognosis and predicted for superior breast cancer-specific survival, relapse-free survival, and overall survival. However, in multivariate models including patient age, tumor size, histologic grade, nodal status, ER status, and Her2 status, GATA-3 was not independently prognostic for these same outcomes. In the subgroups of ER+ patients treated with or without tamoxifen, GATA-3 was again nonprognostic for all outcomes.
GATA-3 is a molecular marker that is highly associated with ER expression, but it does not seem to have prognostic value independent of ER, nor does it predict for response to tamoxifen among ER-positive patients.
To explore the relationship between GATA-3 and IL-12 in patients with allergic rhinitis.
The expression of GATA-3 was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in 37 patients with allergic rhinitis and 12 control samples. IL-12 was detected by enzyme-linked immunosorbent assay.
The relative density ratio of GATA-3 to GAPDH in AR patients was 0.579 +/- 0.102, and in control group was 0.128 +/- 0.021. The concentrations of IL-12 were (53.7 +/- 12.3) ng/g and (121.9 +/- 20.4) ng/g in the two groups respectively. The expression of GATA-3 was negatively correlated to the expression of IL-12 in patients with allergic rhinitis.
The overexpression of GATA-3 in patients with allergic rhinitis was related to the decrease of IL-12, and to increasing the expression of IL-12 in topical tissue could suppress the expression of GATA-3 and may improve the therapeutic effectiveness of AR.
Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC). The etiology has not been clarified yet, but immune disorder is thought to be involved in the pathogenic physiology. Recently, general consensus has been reached that CD and UC are distinct, especially in respect of the immune response. Interestingly, smoking has diverse effects on CD, Th1-type enteritis, and on UC, Th2-type. However, the mechanisms remain obscure. Therefore, we hypothesized that nicotine altered the distinct immune responses in each form of IBD to affect their pathophysiology. In this study, we first demonstrated by RT-PCR analysis that human lamina propria T (LPT) cells had nicotinic acetylcholine receptor (nAChR), and express alpha7 nAChR subunit universally. In addition, the expression of T-bet mRNA in human LPT cells was significantly upregulated after the culture with 10(-7)M and 10(-5)M nicotine for 9 days, while chronic nicotine stimulation showed negligible effect on the expression of GATA-3 mRNA by real-time PCR. The effect of nicotine was inhibited by mecamylamine (MEC). These results suggested that nicotine could modulate the immune balance to Th1-dominant via nAChR in the intestine, to improve Th2-type enteritis. This may provide the experimental evidence for the fact that nicotine has a beneficial influence on UC, and exacerbates CD. Furthermore, it is of great interest that nicotine acts oppositely on CD and UC by modulation of the mucosal immune balance via the neurotransmitter receptor.
How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.
It has been suggested that T-cell immunoglobulin-and mucin-domain-containing molecule-1 (TIM-1) plays an important role in the development of allergic asthma, though its molecular mechanism remains unclear. Our aim was to examine the expression of TIM-1 and Th2-associated transcription factor GATA-3 in asthmatic mice, and to evaluate the correlation of TIM-1 and GATA-3 in the pathogenesis of allergic asthma. We examined TIM-1 expression in lung tissue and peripheral blood mononuclear cells (PBMCs) of asthmatic mice by real-time PCR and flow cytometry, respectively; we also investigated TIM-1 and GATA-3 expression in the spleen tissue of asthmatic mice by western blot. These results demonstrate that TIM-1 was significantly increased in pulmonary tissues and PBMCs in asthmatic mice after ovalbumin (OVA) challenge (P<0.05), and that the production of TIM-1 as well as GATA-3 was upregulated in the spleen of asthmatic mice. The production of TIM-1 correlated significantly with the production of GATA-3 in the spleen of asthmatic mice (r=0.753, P<0.05). The results of this study provide the first evidence that increased expression of TIM-1 in asthmatic mice is associated with the Th2-associated transcription factor GATA-3. The findings suggest a possible mechanism for how HAV infection and TIM-1 upregulation influence the development of allergic asthma.
Human invasive breast cancers (IBC) show enormous histologic and biological diversity. This study comprehensively evaluated diversity in ductal carcinoma in situ (DCIS), the immediate precursors of IBCs.
The extent of diversity for conventional histologic grade and standard prognostic biomarkers assessed by immunohistochemistry was evaluated in a series of pure DCIS (n = 200) compared with a contemporaneous series of IBCs (n = 200). A subset of the DCIS (n = 25) was evaluated by DNA microarrays for the presence of luminal, basal, and erbB2 intrinsic subtypes. The extent of diversity within individual cases of DCIS (n = 120) was determined by assessing multiple regions independently for histologic (nuclear) grade and several biomarkers by immunohistochemistry, which approximate microarrays in determining intrinsic subtypes.
DCIS showed a broad distribution of conventional histologic grades and standard biomarkers ranging from well to poorly differentiated, nearly identical to IBCs. Microarrays showed the same intrinsic subtypes in DCIS as in IBCs. However, higher resolution analysis showed that multiple histologic grades, biomarker phenotypes, and intrinsic subtypes often coexist within the same DCIS, and these diverse regions probably compete for dominance. Diversity within cases of DCIS was highly correlated with mutated p53 (P = 0.0007).
These results support the hypothesis that poorly differentiated DCIS gradually evolve from well-differentiated DCIS by randomly acquiring genetic defects resulting in increasingly abnormal cellular features. This diversity is amplified by defects resulting in genetic instability (e.g., p53 mutation), and the alterations are propagated to IBC in a manner independent of progression to invasion.
Glutathione S-transferase P1-1 is the main phase II xenobiotic metabolism enzyme in human placenta. Low level of its gene expression and corresponding ineffective protection of fetus from toxic compounds is associated with pregnancy disorders such as preeclampsia and abnormalities of fetus development. It was previously reported that environmental radioactive contamination caused down-regulation of GSTP1 transcription in human placenta, but mechanisms responsible for such changes were unclear. In the present study we have found that observed changes in transcription of this gene are not caused by promoter methylation because GSTP1 promoter was not methylated in any of analyzed 91 placental samples. Regulation of GSTP1 by methylation or transcription factors was not previously studied in human placenta. Using "Gene Expression Atlas" online software the placental expression profile of transcription factors known to interact with GSTP1 promoter in other cell types, was identified. According to computer analysis the genes coding for GATA2, GATA3, Fos-B, Nrf3 and MafK transcription factors are highly expressed in human placenta, while genes coding for c-Fos, Juns, Mafs, ERbeta, RARalpha and NF-kappaB factors have moderate level of expression. Competitive EMSA provided the evidence that ARE and NF-kappaB-like sites specifically interacted with placental nuclear proteins. Among these proteins transcription factors AP-1 and NF-kappaB were identified using corresponding consensus oligonucleotides as competitors in EMSA.
The objective of this study was to identify genes that are related to pathogenesis of carcinoma in situ (CIS) to invasive cervical cancer with the use of oligonucleotide microarray and reverse transcription-polymerase chain reaction (RT-PCR). Each two cases of normal cervix, CIS, and invasive cervical cancer were investigated with DNA microarray technology. Differential gene expression profiles among them were analyzed. Expression levels of selected genes from the microarray results were confirmed by RT-PCR. The expressions of 15,286 genes were compared and 458 genes were upregulated or downregulated by twofold or more compared with each other group. Among 458 genes, 22 genes were upregulated and 40 genes were downregulated by twofold or more in invasive cervical cancer group compared with CIS group. RT-PCR analysis confirmed upregulation of 18 genes and downregulation of 5 genes in invasive cervical cancer group. RBP1, TFRC, SPP1, SAA1, ARHGAP8, and NDRG1, which were upregulated, and GATA3, PLAGL1, APOD, DUSP1, and CYR61, which were downregulated, were considered as candidate genes associated with invasion of cervical cancer.
Endometrial cytokine expression is poorly understood. T-Bet and GATA-3 regulate cytokine expression in T-lymphocytes. Previous work has demonstrated expression of T-Bet in human endometrium. Changes in human endometrial T-Bet and GATA-3 mRNA and protein expression during the normal menstrual cycle were characterized. Human endometrium from each phase of the menstrual cycle underwent real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry to examine expression and localization. T-Bet and GATA-3 mRNA were increased in the late secretory phase. Progesterone receptor (PR) mRNA was increased during the proliferative and early secretory phases. T-Bet and GATA-3 proteins localized cytoplasmically in the late secretory phase. PR protein displayed nuclear localization and maximal immunostaining during the early secretory phase. T-Bet and GATA-3 are expressed in endometrial epithelium cyclically during the menstrual cycle. T-Bet and GATA-3 are both upregulated during the late secretory phase and in the same cell types. The expression patterns of T-Bet and GATA-3 oppose PR, suggesting antagonistic function and/or regulation between PR and T-Bet/GATA-3.
Inactivation of serotonin transporter (HTT) by pharmacologically in the neonate or genetically increases risk for depression in adulthood, whereas pharmacological inhibition of HTT ameliorates symptoms in depressed patients. The differing role of HTT function during early development and in adult brain plasticity in causing or reversing depression remains an unexplained paradox. To address this we profiled the gene expression of adult Htt knockout (Htt KO) mice and HTT inhibitor-treated mice. Inverted profile changes between the two experimental conditions were seen in 30 genes. Consistent results of the upstream regulatory element search and the co-localization search of these genes indicated that the regulation may be executed by Pax5, Pax7 and Gata3, known to be involved in the survival, proliferation, and migration of serotonergic neurons in the developing brain, and these factors are supposed to keep functioning to regulate downstream genes related to serotonin system in the adult brain.
In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.
Allergic asthma is an airway inflammatory disease characterized by chronically increased expression of multiple inflammatory proteins, including cytokines, chemokines, adhesion molecules, enzymes, and receptors. Several transcription factors are known to play a crucial role in the pathogenesis of chronic inflammatory diseases such as asthma, including signal transducer and activator of transcription factors, nuclear factor-kappaB, nuclear factor of activated T cells, activator protein-1 family proteins, and Th2 cellrelated transcription factors including GATA3, JunB, and c-Maf. Modulation of the activity of certain transcription factors has been shown to result in the inhibition of inflammation during asthma. Therefore, both agonists and inhibitors of transcription factors may be potential tools for the treatment of asthma. In this review, we summarize the current knowledge of the role of well-established transcription factors in asthma.
Ligustrazine is an alkaloid isolated from the rhizome of Chuanxiong (Ligusticum chuanxiong Hort), which is known to possess antioxidant, anti-inflammatory, anti-fibrosis and immunomodulative effects. It is used clinically to treat asthma as an assistant therapy of glucocorticoid. The purpose of this study was to explore the effects of intraperitoneal ligustrazine on Th1/Th2 cytokines in a rat asthma model and the underlying mechanism. SD rats were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. Within 24 hours after the last ovalbumin challenge, changes in airway histology were observed. The concentrations of IL-4 and IFN-gamma in bronchoalveolar lavage fluid (BALF) were measured by enzyme linked immunosorbent assay (ELISA). The protein expressions of GATA-3 and T-bet in lung were measured by Western blot. The results showed that an increase of Th2 cytokine and an inhibition of Th1 cytokine were accompanied by an increased expression of GATA-3 protein and a decreased expression of T-bet protein in rat asthmatic airways compared to those in normal control group. Intraperitoneal ligustrazine administration could significantly lower the level of IL-4 in BALF and the expression of GATA-3 protein in lung and also increase the level of IFN-gamma and T-bet in asthmatic rats, resulting in a decreased percentage of eosinophils (EOS) in BALF and ameliorated airway inflammatory cell infiltration. In conclusion, ligustrazine inhibits OVA induced airway inflammation by modulating key master switches GATA-3 and T-bet that result in reversing the Th2 cytokine patterns in asthma.
GATA-3, the only T cell-specific member of the GATA family of transcription factors, is essential for the intrathymic development of CD4+ T cells and for the differentiation of Th2 cells. However, whether distinct biochemical features, unique to GATA-3 compared with other GATA family members, are required to drive T cell transcriptional programs or whether the T cell-specific functions of GATA-3 can simply be ascribed to its expression pattern is unclear. Nor do we understand the protein structural requirements for each individual function of GATA-3. In this study, we report that a heterologous GATA factor, GATA-4, was competent in supporting the development of CD4+ T cells but could not fully compensate for GATA-3 in regulating the expression of Th cytokines. Specifically, GATA-3 was more potent than GATA-4 in driving the production of IL-13 due to a mechanism independent of DNA binding or chromatin remodeling of the IL-13 locus. The difference was mapped to a partially conserved region C-terminal to the second zinc finger. Converting a single proline residue located in this region of GATA-4 to its counterpart, a methionine of GATA-3, was sufficient to enhance the IL-13-promoting function of GATA-4 but had no effect on other cytokines. Taken together, our data demonstrate that the unique function of GATA-3 is conferred by both its cell type-specific expression and distinct protein structure.
Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNgamma, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+ CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFbeta, TGFbetaRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFbeta and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.
Transcription factor GATA-3 is the critical transcription factor for Th2 cell differentiation. In spite of its importance in Th2 cell differentiation, the molecular mechanism for its action in Th2 differentiation is poorly understood. Previous studies have suggested that GATA-3 may be involved in the chromatin remodeling in the Th2 cytokine locus. To determine whether GATA-3 exerts its effect on its target sites in the extrachromosomal status, cell transfection assay was performed. In this assay, 800 bp IL4 promoter-luciferase constructs linked with GATA-3 target sites were transfected into the M12 B cell line, D10 mouse Th2 cell lines, and human T lymphoma Jurkat cell lines with or without the GATA-3 expression vector. The GATA-3 effects on it target sites were minimal in the extrachromosomal status, supporting the previous propositions that GATA-3 functions at the chromatin level by remodeling chromatin structure.
Suppressors of cytokine signalling (SOCS) family members have been shown to play an important role in the balance of cytokines that determine the onset of T-helper type 1 (Th1)- and Th2-mediated immune responses. In particular, for cytokine-induced Src-homology 2 protein (CIS), SOCS1, SOCS3 and SOCS5, a role in the regulation of T cell differentiation has been discussed. However, only few data exist so far in the human system.
The aim of the present study was to analyse the relationship between these suppressors and Th1/Th2 regulation as well as allergic sensitizations within a population-based study.
Within the Lifestyle-Immune system-Allergy plus cohort study, mRNA was prepared from blood samples of 6-year-old children for the analysis of cytokines, transcription factors for T cell regulation and SOCS molecule expression by quantitative real-time polymerase chain reaction. In addition, total and specific IgE concentrations have been measured by the Pharmacia CAP System. A complete data set from 248 children was available. Results Among the SOCS molecules investigated, only SOCS1 showed a strong positive correlation to allergic sensitizations. In addition, an up-regulated SOCS1 expression correlated with down-regulated T-box expressed in T cells (Tbet) and higher expression levels of GATA-binding protein 3 (GATA-3) and IL-4. No association between SOCS1 and forkhead box P3 (FOXP3) was observed. For SOCS3, SOCS5 and CIS, a contradictory picture was found. The expression of these SOCS molecules was positively correlated with Tbet and FOXP3 and (with the exception of CIS) negatively with IL-4.
Our data suggest that SOCS3, SOCS5 and CIS, which correlate with an up-regulated Th1 and regulatory T cell activity, are without relevance for the allergic status. In contrast, SOCS1 might be involved in the development of a Th2-skewed immune response and subsequent allergic sensitizations.
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Cytokine-dependent T helper 1 (Th1) differentiation versus T helper 2 (Th2) differentiation is controlled by distinct transcription factors. Previously, we have demonstrated that immature human dendritic cells (DC) from blood donors with allergies show rapid phosphorylation of the Th2-associated signal transducer and activator of transcription 6 (STAT6) upon contact with protein allergens. In the present study we investigated whether this process is regulated by the downstream molecules suppressor of cytokine signalling (SOCS) and/or by the factors T-bet and GATA3. Therefore, immature DC of grass or birch pollen-allergic donors were treated with the respective Th2-promoting protein allergens, and, for comparison, with the Th1-promoting contact allergen 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI) or with the antigen tetanus toxoid. Changes in the mRNA levels of SOCS1, SOCS3, T-bet and GATA3 were analysed by quantitative real-time polymerase chain reaction. Exposure of DC to protein allergens led to the up-regulation of the Th2-associated genes SOCS3 and GATA3, whereas the contact allergen MCI/MI preferentially enhanced the expression of the Th1-associated gene T-bet. Treatment of immature DC with the antigen tetanus toxoid increased both Th1- and Th2-associated genes. Our data indicate that polarization of type 1 versus type 2 immune responses takes place already at the level of antigen-presenting cells, involving molecules similar to those used in T-cell polarization.
Recent studies have suggested that gene expression studies using urinary sediment might be a non-invasive approach to assessing activity and pathogenesis in glomerulonephritis. However, little information is available regarding the mRNA expression patterns of functional molecules, such as T-bet, GATA-3, FOXP3, and retinoic acid-inducible gene-I (RIG-I), in urinary sediment, from patients with immunocomplex-mediated glomerulonephritis. Fourteen lupus nephritis (LN) patients, 13 IgA nephropathy (IgAN) patients, and 12 healthy controls were enrolled in the study. The mRNA expressions of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment were measured using real time quantitative polymerase chain reaction. We also studied the expression of RIG-I in kidney tissue specimens obtained from LN and IgAN patients. Significant differences in the expression patterns of GATA-3, FOXP3 and RIG-I, and marginal differences in T-bet expression, were observed between the three study groups. Immunofluorescent staining for RIG-I was observed in the tissue specimens from the LN patients, but not in those from the IgAN patients. The mRNA expression patterns of T-bet, GATA-3, FOXP3 and RIG-I in urinary sediment differ according to diagnostic category. These results suggest that the measurement of these target gene expressions might be a useful, non-invasive method for clinical monitoring and studying of pathogenesis in glomerulonephritis.
GATA-3 plays a central role in the Th2-mediated immunoreaction. This study was aimed to construct and select plasmid vectors of siRNA which can effectively and specifically suppress the gene expression of GATA-3. Plasmid including PSi338, PSi717 and PSi1232 were designed and constructed for GATA-3 regarded as target gene and transfected into murine P815 cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detect the inhibition of GATA-3 mRNA as well as its protein in P815 cells. The results demonstrated that the expressions of mRNA and protein of GATA-3 in P815 cells were inhibited significantly by both of PSi338 and PSi717. It is concluded that PSi338 and PSi717 siRNA plasmid vectors have been successfully constructed, and both vectors are effective and specific siRNA plasmids for suppressing GATA-3 gene expression.
In T-cell acute lymphoblastic leukemia (T-ALL) the cardiac homeobox gene NKX2-5 (at 5q35) is variously deregulated by regulatory elements coordinating with BCL11B (at 14q32.2), or the T-cell receptor gene TRD (at 14q11.2), respectively. NKX2-5 is normally expressed in developing spleen and heart, regulating fundamental processes, including differentiation and survival. In this study we investigated whether NKX2-5 expression in T-ALL cell lines reactivates these embryonal pathways contributing to leukemogenesis. Among 18 known targets analyzed, we identified three genes regulated by NKX2-5 in T-ALL cells, including myocyte enhancer factor 2C (MEF2C). Knockdown and overexpression assays confirmed MEF2C activation by NKX2-5 at both the RNA and protein levels. Direct interactions between NKX2-5 and GATA3 as indicated by co-immunoprecipitation data may contribute to MEF2C regulation. In T-ALL cell lines LOUCY and RPMI-8402 MEF2C expression was correlated with a 5q14 deletion, encompassing noncoding proximal gene regions. Fusion constructs with green fluorescent protein permitted subcellular detection of MEF2C protein in nuclear speckles interpretable as repression complexes. MEF2C consistently inhibits expression of NR4A1/NUR77, which regulates apoptosis via BCL2 transformation. Taken together, our data identify distinct mechanisms underlying ectopic MEF2C expression in T-ALL, either as a downstream target of NKX2-5, or via chromosomal aberrations deleting proximal gene regions.
Inflammation plays a key role in the pathogenesis of an AAA (abdominal aortic aneurysm); however, the nature of the inflammatory factors and cellular response(s) involved in AAA growth is controversial. In the present study, we set out to determine the aortic levels of inflammatory cytokines in relation to downstream inflammatory transcription factors and cellular responses. A comparison of AAA wall samples with atherosclerotic wall samples taken from the same aortic region allowed AAA-specific inflammatory parameters to be identified that distinguish AAAs from ASD (aortic atherosclerotic disease). RT-PCR (real-time PCR), ELISA, Western blotting and immunohistochemistry were combined to assess cytokines and transcription factors at the mRNA and protein level, and their activation status. Compared with ASD, inflammatory parameters associated with Th1-type [T-bet, IL (interleukin)-2, IFN-gamma (interferon-gamma), TNF-alpha (tumour necrosis factor-alpha), IL-1alpha and cytotoxic T-cells] and Th2-type [GATA3, IL-4, IL-10, IL-13 and B-cells] responses were all increased in AAA samples. Evaluation of major downstream inflammatory transcription factors revealed higher baseline levels of C/EBP (CCAAT/enhancer-binding protein) alpha, beta and delta in the AAA samples. Baseline p65 NF-kappaB (nuclear factor kappaB) and c-Jun [AP-1 (activator protein-1)] levels were comparable, but their activated forms were strongly increased in the AAA samples. Downstream target genes of p65 NF-kappaB, c-Jun, IL-6 and IL-8 were hyperexpressed. Molecular and cellular processes associated with IL-6 and IL-8 hyperactivation were enhanced in the AAA samples, i.e. the expression of phospho-STAT-3 (signal transducer and activator of transcription-3) and perforin were elevated, and the content of plasma cells, neutrophils and vasa vasorum was increased. In conclusion, our findings demonstrate that an AAA is a general inflammatory condition which is characterized by enhanced expression and activation of pro-inflammatory transcription factors, accompanied by IL-6 and IL-8 hyperexpression and exaggerated downstream cellular responses, which together clearly distinguish an AAA from ASD.
Th2 cytokine expression is dependent on the transcription factor GATA-3. However, the molecular interactions of GATA-3 leading to Th2 cytokine gene activation have not been well characterized. Here, we reported a number of GATA-3 associated proteins in Th2 cells, and one of such proteins Pias1 functioned as a positive transcriptional coregulator for GATA-3. When overexpressed in Th2 cells, Pias1 enhanced the expression of IL-13, and to lesser degrees, IL-4 and -5. Conversely, Pias1 siRNA down-regulated the Th2 cytokine expression. In Leishmania major infection, manipulating Pias1 expression in parasite-reactive CD4 T cells altered severity of disease caused by Th2 responses. Mechanistically, Pias1 markedly potentiated GATA-3-mediated activation of the IL-13 promoter by facilitating the recruitment of GATA-3 to the promoter. In contrast, IL-5 promoter was modestly enhanced by Pias1 and no effect was observed on IL-4 promoter. Thus, both promoter activation and additional mechanisms are responsible for regulation by Pias1.
GATA3 activates transcription of the T(H)2 cytokines, including IL13, an important step in the allergic inflammatory pathway.
We sought to identify associations of single nucleotide polymorphisms of the genes GATA3 and IL13 and their interactions with rhinitis and allergic sensitization during childhood.
We performed genetic association studies in a cohort of children (n = 923) who have been evaluated for the development of rhinitis and allergic sensitization by means of skin prick tests (SPTs) at age 10 years. Pyrosequencing was used to genotype 7 polymorphisms from GATA3 and 5 from IL13. A novel model-selection procedure combining logistic regression models and classification was used to study the contributions of the polymorphisms and their interactions.
Combinations of polymorphisms and their interactions increase the risk for rhinitis and allergic sensitization at age 10 years. A model with rs1058240, rs379568, and rs4143094 (GATA3) and rs1800925 (IL13) and their interactions was selected to predict rhinitis and positive SPT responses. rs1058240 was associated with rhinitis and allergic rhinitis (P < .05), and the gene-gene interaction rs1058240:rs1800925 was associated with rhinitis (P = .043). The odds ratios for 4 genotype combinations were significant for rhinitis or SPTs (P < .044).
Gene-gene interaction between GATA3 and IL13 polymorphisms can influence the risk of childhood rhinitis. Our study suggests that set associations of polymorphisms are important in studying genetic associations for complex phenotypes, such as rhinitis and atopy.
Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR), using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open reading frame (ORF), which encodes a 444 amino acid protein. Semi-quantitative RT-PCR analysis of GATA-3 mRNA expression was done using the total RNAs from different normal tissues of a large white pig. The GATA-binding family of transcription factors comprised of a subgroup of DNA-binding proteins that both bound the consensus GATA motif and contained the class IV zinc finger motif. The molecular evolution tree was constructed based on the GATA-3 amino acid sequence and class IV zinc finger motif using mega 3.1. Phylogeny analysis of GATA factors isolated from vertebrates suggested that the six distinct vertebrate GATAs had descended from a common ancestral sequence, and the topology also suggested multiple modes of evolution including gene duplication and class IV zinc finger motif recombination. These data helped the authors in illuminating the pathways of divergent and convergent evolution of the GATA-binding family of transcription factors.
The activation and effector phenotype of T cells depend on the strength of the interaction of the TcR with its cognate antigen and additional signals provided by cytokines and by co-receptors. Lymphocytes sense both the presence of an antigen and also clues from antigen-presenting cells, which dictate the requisite response. CD43 is one of the most abundant molecules on the surface of T cells; it mediates its own signalling events and cooperates with those mediated by the T cell receptor in T cell priming. We have examined the role of CD43 signals on the effector phenotype of adult CD4+ and CD8+ human T cells, both alone and in the presence of signals from the TcR.
CD43 signals direct the expression of IFNgamma in human T cells. In freshly isolated CD4+ T cells, CD43 signals potentiated expression of the IFNgamma gene induced by TcR activation; this was not seen in CD8+ T cells. In effector cells, CD43 signals alone induced the expression of the IFNgamma gene in CD4+ T cells and to a lesser extent in CD8+ cells. The combined signals from CD43 and the TcR increased the transcription of the T-bet gene in CD4+ T cells and inhibited the transcription of the GATA-3 gene in both populations of T cells, thus predisposing CD4+ T cells to commitment to the T1 lineage. In support of this, CD43 signals induced a transient membrane expression of the high-affinity chains of the receptors for IL-12 and IFNgamma in CD4+ T cells. CD43 and TcR signals also cooperated with those of IL-12 in the induction of IFNgamma expression. Moreover, CD43 signals induced the co-clustering of IFNgammaR and the TcR and cooperated with TcR and IL-12 signals, triggering a co-capping of both receptors in CD4+ populations, a phenomenon that has been associated with a T1 commitment.
Our results suggest a key role for CD43 signals in the differentiation of human CD4+ T cells into a T1 pattern.
The ventral spinal cord consists of interneuron groups arising from distinct, genetically defined, progenitor domains along the dorsoventral axis. Many of these interneuron groups settle in the ventral spinal cord which, in mammals, contains the central pattern generator for locomotion. In order to better understand the locomotor networks, we have used different transgenic mice for anatomical characterization of one of these interneuron groups, called V2 interneurons. Neurons in this group are either V2a interneurons marked by the postmitotic expression of the transcription factor Chx10, or V2b interneurons which express the transcription factors Gata2 and Gata3. We found that all V2a and most V2b interneurons were ipsilaterally projecting in embryos as well as in newborns. V2a interneurons were for the most part glutamatergic while V2b interneurons were mainly GABAergic or glycinergic. Furthermore, we demonstrated that a large proportion of V2 interneurons expressed the axon guidance molecule EphA4, a molecule previously shown to be important for correct organization of locomotor networks. We also showed that V2 interneurons and motor neurons alone did not account for all EphA4-expressing neurons in the spinal cord. Together, these findings enable a better interpretation of neural networks underlying locomotion, and open up the search for as yet unknown components of the mammalian central pattern generator.
GATA-binding protein 3 (GATA3) is a transcription factor and a putative tumor suppressor that is highly expressed in normal breast luminal epithelium and estrogen receptor alpha (ER)-positive breast tumors. We hypothesized that common genetic variation in GATA3 could influence breast carcinogenesis. Four tag single-nucleotide polymorphisms (SNP) in GATA3 and its 3' flanking gene FLJ4598 were genotyped in two case control studies in Norway and Poland (2,726 cases and 3,420 controls). Analyses of pooled data suggested a reduced risk of breast cancer associated with two intronic variants in GATA3 in linkage disequilibrium (rs3802604 in intron 3 and rs570613 in intron 4). Odds ratio (95% confidence interval) for rs570613 heterozygous and rare homozygous versus common homozygous were 0.85 (0.75-1.95) and 0.82 (0.62-0.96), respectively (P(trend)=0.004). Stronger associations were observed for subjects with ER-negative, than ER-positive, tumors (P(heterogeneity)=0.01 for rs3802604; P(heterogeneity)=0.09 for rs570613). Although no individual SNPs were associated with ER-positive tumors, two haplotypes (GGTC in 2% of controls and AATT in 7% of controls) showed significant and consistent associations with increased risk for these tumors when compared with the common haplotype (GATT in 46% of controls): 1.71 (1.27-2.32) and 1.26 (1.03-1.54), respectively. In summary, data from two independent study populations showed two intronic variants in GATA3 associated with overall decreases in breast cancer risk and suggested heterogeneity of these associations by ER status. These differential associations are consistent with markedly different levels of GATA3 protein by ER status. Additional epidemiologic studies are needed to clarify these intriguing relationships.
Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance.
To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens.
C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses.
Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gammadelta+ and CD4+CD25+Foxp3+ cells in Peyer's patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut.
A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.
Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-gamma production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-beta mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen.
Inducible costimulator (ICOS) expression is critical for T cell-mediated immunity. We showed previously that T cell receptor and CD28 co-engagement up-regulate ICOS expression in activated T cells via the induction of NFATc2. Here, we examined the regulation of ICOS expression by Th-specific transcription factors T-bet and GATA-3. Overexpression of T-bet or GATA-3 alone could enhance, and NFATc2 could further synergize with either of them to increase, icos transcription. Although T-bet acted on the icos promoter, GATA-3 operated via an icos 3'-un-translated region element. Interestingly, NFATc2 was found to bind promiscuously the icos promoter in developing Th0, Th1, and Th2 cells but became selectively associated with T-bet at the promoter and with GATA-3 at the 3'-untranslated region in fully differentiated Th1 and Th2 cells, respectively. Collectively, our results reveal a temporally evolving circuit in which the non-selectively expressed NFATc2 cooperates with Th-restricted T-bet or GATA-3 to direct transcription of a costimulatory gene via distinct regulatory elements in different Th cells undergoing differentiation.
Signal transducer and activator of transcription 1 (STAT1), an intracellular signal transducer and activator of transcription centrally involved in many inflammatory pathways, was recently suggested to play an important role in allergy related immune responses.
Thus, we investigated the effect of polymorphisms in the STAT1 gene on the development of atopic sensitization and allergic diseases.
Haplotype tagging single nucleotide polymorphisms (SNPs) previously described in the STAT1 gene were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology in a cross-sectional study population of 3099 German children recruited and phenotyped by the International Study of Asthma and Allergy in Childhood, phase II (ISAAC II). Effects of single SNPs and haplotypes were studied using SAS/Genetics and Haploview.
The polymorphism C39134A (rs3771300), located in a potentially cis acting regulatory element in STAT1 intron 24, was inversely related to atopy measured by skin prick test, total and specific serum IgE levels while no effect on atopic disease risk was observed.
Our results indicate that STAT1 SNP C39134A may protect from atopic sensitization. Because of its location in a highly conserved noncoding sequence near a putative GATA3 binding site, this polymorphism represents an interesting target for further studies.
Secondary tumors and leukemias are major complications in Hodgkin lymphoma (HL). They likely arise from clonal selection of cells that have accumulated genomic lesions induced by chemo- and radiotherapy and may be further promoted by the loss of DNA repair and/or other pathways ensuring the fidelity of replicated DNA. To distinguish genomic imbalances associated with the development of acute myeloid leukemia (AML) in HL we used an array-based comparative genomic hybridization (aCGH) strategy on whole lymph node biopsies of HL patient. Genomic imbalances (amplifications and deletions) associated with AML outcome in 3 classic HL patients, at clinical diagnosis they exhibited a discrete individual variability. Three amplifications and 5 deletions were shared by all 3 patients. They involved AFM137XA11, a 9p11.2 pericentric region; FGFR1, the FGF receptor most frequently translocated in AML; PPARBP, a co-activator of nuclear receptors RARalpha, RXR and TRbeta1; AFM217YD10, a 17q25 telomeric region; FGR, an SRC2 kinase involved in cytokine production by NK and CD4+ NKT cells; GATA3, a Th2-specific transcription factor; TOP1, involved in DNA recombination and repair; WT1, a transcription factor involved in CD8+ T cell response against leukaemic blasts. Immunohistochemistry confirmed aCGH results and distinguished the distribution of either amplified or deleted gene products in neoplastic Reed Sternberg (RS) cells and non-neoplastic lymph node components.
Multiple biologic factors influence maintenance of immunologic responsiveness. Here, we studied whether epigenetics has a regulatory function in maintaining pre-established T(H)1-like and T(H)2-like immunity in human beings.
We focused on delineating the role of endogenous histone deacetylase (HDAC) activity in regulating cytokine recall responses.
Using RT-PCR and ELISA, the effect of increasing cellular acetylation on T(H)1/T(H)2 cytokine expression was systematically examined in 58 children by inhibiting HDAC activity with trichostatin A.
Phytohemagglutinin activation selectively stimulates antigen-experienced CD45RO+ T cells, eliciting recall cytokine responses. Trichostatin A reduced HDAC activity by approximately 1/3. The resulting cellular hyperacetylation led to increased T(H)2-associated (IL-13, 139%; IL-5, 168%; P < .0001) and reduced T(H)1-associated recall responses (IFN-gamma, 76%; CXCL10, 47%; P < .0001). IL-2 and IL-10 production were reduced 25% to 55% (P < .0001). These alterations in T(H)2-associated and T(H)1-associated recall responses were associated with increased expression of Gata-3 and sphingosine kinase 1, a T(H)1-negative regulator, independent of T-bet expression. Overall, inhibition of endogenous HDAC activity shifted T(H)1:T(H)2 ratios by 3-fold to 8-fold (P < or = .0001), skewing recall responses toward a more T(H)2-like phenotype, independent of the stimulus used.
Endogenous HDAC activity plays a crucial role in maintaining the balance of pre-established T(H)1-like and T(H)2-like responses, inhibiting excessive T(H)2 immunity.
To study the mechanism of imiquimod on asthma animals.
(1) 40 mice and 48 rats were divided into 4 groups: control, asthma, dexamethasone and imiquimod groups. The asthma model was established. The mice and rats in the imiquimod group were exposed to an aerosol of 0.15% imiquimod. Lung inflammation and airway responsiveness were measured 24 h after the last ovalbumin (OVA) challenge. The expression of Interleukin-4 (IL-4), interferon gamma (IFN-gamma), eotaxin, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), T-bet, GATA-3, STAT6 mRNA in the lung were determined by reverse transcription polymerase chain reaction (RT-PCR). The levels of eotaxin, MDC, and TARC in sera were tested by enzyme linked immunosorbent assay (ELISA). The expression of T-bet, GATA-3 and STAT6 proteins in the lung were measured by Western blot. (2) Parabronchial lymphnodes (PBLN) were isolated and cultured. The PBLN cells were divided into blank control, positive control, dexamethasone and drug groups (1 - 3 subgroups), cultured for different hours, and the expressions of IL-4 and IFN-gamma in supernatants were determined by ELISA, The mRNA expressions of the cytokines in cells weredetected by RT-PCR. (3) Flow cytometry was used to detect intracellular IL-4 and IFN-gamma production in spleen T lymphocytes. (4) CD(4)(+) T cell of spleen pellets were subject to assessment of T-bet and GATA-3 protein and mRNA expression respectively.
The expiration resistance was determined before and after injection of acetylcholine chloride (20 - 160 microg/ml), and expiration resistances of the asthmatic group (6.26 +/- 0.85), (11.55 +/- 3.09), (28.74 +/- 5.94), (3710.83 +/- 197.49) cm H(2)Oxml(-1)xs(-1), were significantly elevated compared with those of the control group (1.34 +/- 0.16), (3.47 +/- 0.49), (9.29 +/- 1.27), (25.22 +/- 5.44) cm H(2)Oxml(-1)xs(-1), D = 88.98, 56.00, 45.00, 108.00, all P < 0.01). The numbers of eosinophils and lymphocytes, the thicknesses of WA/Pi and ASM/Pi in the asthmatic group [(26.0 +/- 1.6)/mm(2), (45.2 +/- 3.2)/mm(2), 12.0 +/- 1.4, 6.7 +/- 0.6] were all significantly higher than those of the imiquimod group [(12.4 +/- 2.9)/mm(2), (24.2 +/- 3.7)/mm(2), 9.2 +/- 0.6, 4.0 +/- 0.5, D or q = 193.00, 16.92, 185.50, 7.66, all P < 0.01]. In the imiquimod group, the mRNA and protein expressions of T-bet (0.48 +/- 0.08, 0.48 +/- 0.17) were significantly increased compared with those of the asthmatic group (0.08 +/- 0.12, 0.18 +/- 0.06, D = 120.96, 177.98, all P < 0.01), the mRNA and protein expressions of GATA-3 in the imiquimod group were both significantly decreased compared with those of the asthmatic group (D = 166.96, 310.97, all P < 0.01). In the control group, only low concentrations of IFN-gamma [(22 +/- 5, 31 +/- 5) pg/ml] were detected in PBLN cell cultures. After 24 or 48 h stimulation, the concentrations of IFN-gamma in drug 2 subgroup [(149 +/- 31), (154 +/- 28) pg/ml] and drug 3 subgroup [(166 +/- 30), (158 +/- 31) pg/ml] were increased significantly; Levels of IL-4 [druug 2 subgroup: (23 +/- 5), (39 +/- 11) pg/ml, drug 3 subgroup: (43 +/- 13), (56 +/- 12) pg/ml] were increased slowly compared with those in the OVA group (drug 2 subgroup 24 h IL-4, D = 9.90; drug 3 subgroup 24 h IL-4, D = 8.79, drug 2 subgroup 48 h IL-4, D = 8.80, drug 3 subgroup 48 h IL-4, D = 8.10, drug 2 subgroup 24 h IFN-gamma, q = 4.80, drug 3 subgroup 24 h IFN-gamma, q = 6.40, drug 2 subgroup 48 h IFN-gamma, q = 3.95, drug 3 subgroup 48 h IFN-gamma, q = 4.31, all P < 0.05). After imiquimod treatment, the mRNA and protein levels of T-bet in imiquimod group CD(4)(+) T cells were increased significantly compared with those in OVA group, and the mRNA and protein levels of GATA-3 were decreased significantly in CD(4)(+) T cells of imiquimod group compared with those in OVA group. The eotaxin, MDC and TARC levels of serum in asthma group [(593 +/- 41) pg/ml, (170 +/- 20) pg/ml, (221 +/- 25) pg/ml] were significant different from those in control group [(288 +/- 66) pg/ml, (100 +/- 33) pg/ml, (84 +/- 49) pg/ml], (eotaxin: q = 12.20, MDC: q = 8.00, TARC: q = 10.50, all P < 0.01). MDC and TARC levels of serum in imiquimod group [(84 +/- 13) pg/ml, (163 +/- 35) pg/ml] decreased as compared with those in asthma group (MDC: q = 9.80, TARC: q = 4.50, all P < 0.01) and MDC levels in imiquimod group were no different with normal group (q = 1.80, P > 0.05). eotaxin levels of serum in imiquimod group [(501 +/- 76) pg/ml] increased as compared with those from normal group (q = 8.50, P < 0.01), and decreased as compared with those from asthma group (q = 3.70, P < 0.05). (4) The expression of eoaxin, MDC, TARC and STAT(6) on the bronchial epithelium in imiquimod group was decreased as compared with asthma group, but increased as compared with normal group. The eotaxin, MDC and TARC mRNA expression of the lung in asthma group (0.85 +/- 0.11, 0.96 +/- 0.10, 0.94 +/- 0.28) had significant differences from those in the control group (0.45 +/- 0.08, 0.39 +/- 0.09, 0.24 +/- 0.08, eotaxin: q = 3.00, MDC: q = 15.40, TARC: q = 5.90, all P < 0.01) and those in imiquimod group (0.65 +/- 0.17, 0.66 +/- 0.12, 0.66 +/- 0.34, eotaxin: q = 1.50, MDC: q = 8.10, TARC: q = 2.40, all P < 0.05).
These findings suggested that imiquimod can inhibit the airway inflammation of asthma animals by reducing GATA-3 mRNA and protein expression and increasing T-bet, STAT(6) mRNA and protein expression.
Hyperforin (Hyp) is an active compound contained in the extract of Hypericum perforatum, well known for its antidepressant activity. However, Hyp has been found to possess several other biological properties, including inhibitory effects on tumor invasion, angiogenesis, and inflammation. In this paper, we show that treatment with Hyp inhibited IFN-gamma production, with down-regulation of T-box (T-bet; marker of Th1 gene expression) and up-regulation of GATA-3 (marker gene of Th2) on IL-2/PHA-activated T cells. In parallel, we showed a strong down-regulation of the chemokine receptor CXCR3 expression on activated T cells. The latter effect and the down-modulation of matrix metalloproteinase 9 expression may eventually lead to the inhibition of migratory capability and matrix traversal toward the chemoattractant CXCL10 by activated lymphocytes that we observed in vitro. The effect of Hyp was thus evaluated on an animal model of experimental allergic encephalomyelitis (EAE), a classic, Th1-mediated autoimmune disease of the CNS, and we observed that Hyp attenuates the severity of the disease symptoms significantly. Together, these properties qualify Hyp as a putative, therapeutic molecule for the treatment of autoimmune inflammatory disease sustained by Th1 cells, including EAE.
A growing body of literature has examined and implicated DNA methylation as a critical epigenetic modification in T helper (Th) cell differentiation. The absence of DNA methyltransferases or methyl-binding proteins derepresses many cytokine loci, allowing their ectopic expression, while methylation of specific CpG residues is sufficient to prevent expression. Here, we characterize demethylation events of the Th2 cytokine locus control region (LCR). rad50 hypersensitive site 7 (RHS7), a hypersensitive site within this LCR, becomes demethylated in a STAT6-dependent manner and only in cells stimulated under type 2 conditions. Robust demethylation appears to require signaling contributions from both IL-4 receptor, via STAT6, and CD28, but it cannot be effected by GATA3. Finally, RHS7 is demethylated independently of cell division, consistent with an "active," rather than passive, mechanism. Taken together, these findings firmly connect RHS7 demethylation and Th2 LCR activation in the type 2 differentiation program.
A number of recent studies testify that calcitriol alone or in combination with corticosteroids exerts strong immune modulatory activity. As a new approach, we evaluated the protolerogenic potential of calcitriol and dexamethasone in acute T helper (Th)1-mediated colitis in mice. A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg) was applied to BALB/c mice. Calcitriol and/or dexamethasone were administered i.p. from days 0 to 3 or 3 to 5 following the instillation of the haptenating agent. Assessment of colitis severity was performed daily. Colon tissue was analyzed macroscopically and microscopically, and myeloperoxidase activity, as well as cytokine levels [tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-12p70, IL-1beta, IL-10, IL-4] were determined by enzyme-linked immunosorbent assay, T-bet, GATA family of transcription factors 3, a Th2 master regulator (GATA3), Foxp3, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), IL-23p19 and IL-17 expression by immunoblot analysis. The combination of the steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down-regulated, whereas Th2 markers like IL-4 and GATA3 were up-regulated. Apart from known steroid effects, calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFbeta, FoxP3, and CTLA4. Furthermore, analysis of dendritic cell mediators responsible for a proinflammatory differentiation of T cells revealed a significant reduction of IL-12p70 and IL23p19 as well as IL-6 and IL-17. Thus, our data support a rationale for a steroid-sparing, clinical application of calcitriol derivatives in inflammatory bowel disease. Furthermore they suggest that early markers of inflammatory dendritic cell and Th17 differentiation qualify as new target molecules for both calcitriol and highly selective immune-modulating vitamin D analogs.
Chemoattractant-receptor homologous molecule expressed on Th2 cells (CRTh2) is a receptor for prostaglandin (PG)D2, a lipid mediator involved in allergic inflammation. CRTh2 is expressed by Th2 cells, eosinophils and basophils and PDG(2)-CRTh2 signaling induces calcium mobilization, cell migration and expression of the Th2 cytokines IL-4, IL-5, and IL-13. Despite the role of CRTh2 in allergic inflammation, transcriptional regulation of this gene has not been studied. Here, we demonstrated that a reporter construct of the CRTh2 promoter was induced following T cell stimulation. This activity could be further enhanced by over-expression of GATA-3, but not NFAT2 or STAT6. Electromobility shift assay demonstrated GATA-3 binding to a probe from the CRTh2 promoter. This study provides the first detailed analysis of transcriptional regulation of the human CRTh2 promoter. These findings may help identify strategies to attenuate expression of this gene and influence the maintenance and proliferation of Th2 cells in allergic inflammation.
Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by in vitro studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis in vivo promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 post conception) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.
Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. HMGA2). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (GATA3, HMGA2, NRAP, SMC6L1, SPP1, RAB6IP2, TJP1 and two EST).
The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.
Alcohol consumption reduces peripheral NK cell numbers and compromises NK cell cytolytic activity; however, the underlying mechanism is not understood completely. It was found recently that the peripheral NK cell pool consists largely of bone marrow (BM)-derived and thymus-derived cells, which are phenotypically and functionally different. The effects of alcohol consumption on these subpopulations have not been studied previously. Using a well-established alcohol-feeding model, we found that chronic alcohol consumption decreases the percentage and number of peripheral NK cells, especially those expressing a mature phenotype. Alcohol consumption did not alter NK cells in the thymus. NK cells in the BM were increased significantly; however, proliferation rate was not altered by alcohol consumption, which increased CD127+ and decreased Ly49D+ NK cells in the spleen but not in the BM. Chronic alcohol consumption increased IFN-gamma-producing NK cells and GATA-3 expression in splenic NK cells. Collectively, these results indicate that chronic alcohol consumption perturbs the balance between thymus-derived and BM-derived NK cells. The increased proportion of thymus-derived NK cells in the spleen likely results from impaired NK cell release from the BM.
Following antigen challenge, adoptively transferred antigen-specific CD4(+) T cells induce allergic airway inflammation, comprised primarily of an increase in lymphocytes and eosinophils.
Our goal was to better understand the contribution of the GATA-3 transcription factor to the ability of adoptively transferred T cells to induce airway inflammation in the Brown Norway rat model of adoptively transferred asthma.
We transduced antigen-stimulated CD4+ T cells with recombinant retroviruses encoding enhanced green fluorescent protein (EGFP) only or EGFP and the GATA-3 transcription factor. Each population of transduced cells was adoptively transferred to naïve recipients that were then challenged with antigen. Airway inflammatory responses were then quantified.
Our data indicate that T cells transduced with retroviruses encoding GATA-3 expressed high levels of GATA-3 protein as well as T-helper type 2 cytokines. Following adoptive transfer and airway antigen challenge, these gene-modified T cells induced robust inflammatory responses in the lungs and draining lymph nodes. Increased numbers of total inflammatory cells and eosinophils were recovered in the bronchoalveolar lavage fluid (BALF). In addition, the number of antigen non-specific CD4+ T cells recovered in the BALF as well as the lung and draining lymph nodes was enhanced in recipients of GATA-3 overexpressing T cells following antigen challenge. Nevertheless, the transduced CD4+ T cells comprised only a small percentage of the population of CD4+ T cells infiltrating the lung and were not detectable at all in the draining lymph nodes.
These data provide evidence that GATA-3 plays a significant role in the ability of antigen-specific T cells to amplify allergic inflammatory responses in vivo by promoting the recruitment of endogenous antigen non-specific T cells to the lung.
GATA-3 is the key transcriptional factor for Th2 commitment in T cells and is strongly associated with asthma and allergic disease. We studied the silencing of the GATA-3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of GATA-3 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with OVA and instilled intratracheally (IT) with GATA-3 shRNAs lentiviral vector (Lenti-si-GATA-3) once, 48 hours before challenge. After three challenges with the OVA antigen, the mice were assessed for airway hyperresponsiveness (AHR) and inflammation. With infection of Lenti-si-GATA-3 in EL-4 cells, GATA-3 gene expression was abrogated and downstream Th2 cytokines, such as interleukin-4 (IL-4) and IL-5, were also significantly inhibited. IT delivery of Lenti-si-GATA-3 in OVA-immunized mice resulted in a strong inhibition of local GATA-3 gene expression. Treatment with Lenti-si-GATA-3 successfully alleviated OVA-induced airway eosinophilia and Th2 cytokine release. While evaluating AHR by means of enhanced pause (Penh) and pulmonary resistance (R(L)) using body plethysmography, it was found that the administration of Lenti-si-GATA-3 had significantly decreased AHR in OVA-immunized mice. These results suggest that inhibition of GATA-3 gene expression by shRNAs lentiviral vectors strongly attenuates antigen-induced airway inflammation and hyper-responsiveness in mice.
The principal aim of the immune system is to establish a balance between defense against pathogens and avoidance of autoimmune disease. This balance is achieved partly through regulatory T cells (Tregs). CD4(+)CD25(+) Tregs are either naturally occurring or induced by antigens and are characterized by the expression of the X-linked forkhead/winged helix transcription factor, Foxp3. Here we report a previously unrecognized subset of CD4(+)CD25(+) Tregs derived from CD4(+)CD25(-) T cells induced by nitric oxide (NO). The induction of Tregs (NO-Tregs) is independent of cGMP but depends on p53, IL-2, and OX40. NO-Tregs produced IL-4 and IL-10, but not IL-2, IFNgamma, or TGFbeta. The cells were GITR(+), CD27(+), T-bet(low), GATA3(high), and Foxp3(-). NO-Tregs suppressed the proliferation of CD4(+)CD25(-) T cells in vitro and attenuated colitis- and collagen-induced arthritis in vivo in an IL-10-dependent manner. NO-Tregs also were induced in vivo in SCID mice adoptively transferred with CD4(+)CD25(-) T cells in the presence of LPS and IFNgamma, and the induction was completely inhibited by N(G)-monomethyl-L-arginine, a pan NO synthase inhibitor. Therefore, our findings uncovered a previously unrecognized function of NO via the NO-p53-IL-2-OX40-survivin signaling pathway for T cell differentiation and development.
Interleukin (IL)-5 is a key regulator of eosinophilia in allergic inflammation and parasite infections but the mechanisms regulating IL-5 expression in activated human T lymphocytes are poorly understood. From studies on mouse cells, the activation protein (AP)-1 and GATA-3 sites in the proximal promoter region appear to be important in IL-5 regulation but the significance of an adjacent Ets/nuclear factor of activated T cell (NFAT) site has been less clear.
Interleukin-5 transcriptional activity was measured by transfection of reporter genes into the human HSB-2 cells and normal T lymphocytes. Expression vectors encoding transcription factors were used for transactivation studies and IL-5 expression measured using reporter genes and mRNA levels. Transcription factor binding was shown with chromatin immunoprecipitation (ChIP).
HSB-2 cells showed high inducible expression of IL-5 mRNA. Mutation of reporter gene plasmids showed the Ets/NFAT site was of equal importance to the AP-1 and GATA-3 sites in regulating IL-5 transcription. Transactivation by Ets1 increased luciferase expression 15-fold, in the absence of stimulation, and AP-1 (c-Fos/c-Jun) and GATA-3 gave transactivations of 85-fold, and 100-fold, respectively. Synergistic interactions were demonstrated between Ets1, GATA-3 and AP-1. Dominant-negative AP-1 inhibited IL-5 transcription. Transactivation by GATA-3 and synergy between GATA-3, Ets1 and AP-1 were verified measuring IL-5 mRNA levels. Chromatin immunoprecipitation showed increased binding of Ets1 and GATA-3 to the IL-5 promoter after stimulation. The importance of the Ets1 site and of synergistic interactions between the three transcription factors were verified with primary human T cells.
Ets1, GATA-3 and AP-1 synergize to regulate IL-5 transcription in human T cells.
The GATA family of transcription factors are implicated in early embryonic development. There are six factors in this family in vertebrates. GATA4 and GATA6 have been demonstrated to induce mouse embryonic stem (mES) cells differentiation toward extraembryonic endoderm (ExE). We investigated the effect of GATA3 on the differentiation of mES cells both in the ES cell and in the embryoid body (EB) states. The results demonstrate that GATA3 overexpression can initiate the ES cell differentiation program toward ExE. Furthermore, overexpression of GATA1 and GATA2 in ES cells and EBs resulted in similar effects. We believe this finding can augment our understanding of mouse ES cell differentiation.
Hypersensitivity pneumonitis (HP) is mediated by a Th1 immune response. Transcription factor GATA binding protein-3 (GATA-3) is believed to be a key regulator of Th2 differentiation and thus might play regulatory roles in the pathogenesis of hypersensitivity pneumonitis (HP).
We examined the effect of GATA-3 overexpression on the development of HP in mice.
Wild-type C57BL/6 mice and GATA-3-overexpressing mice of the same background were used in this study. HP was induced by repeated exposure to Saccharopolyspora rectivirgula, the causative antigen of farmer's lung.
Antigen exposure resulted in a marked inflammatory response with enhanced pulmonary expression of T-bet and the Th1 cytokine interferon (IFN)-gamma in wild-type mice. The degree of pulmonary inflammation was much less severe in GATA-3-overexpressing mice. The induction of T-bet and IFN-gamma genes was suppressed, but a significant induction of Th2 cytokines, including IL-5 and IL-13, was observed in the lungs of GATA-3-overexpressing mice after antigen exposure. Supplementation with recombinant IFN-gamma enhanced lung inflammatory responses in GATA-3-overexpressing mice to the level of wild-type mice. Because antigen-induced IFN-gamma production predominantly occurred in CD4+ T cells, nude mice were transferred with CD4+ T cells from either wild-type or GATA-3-overexpressing mice and subsequently exposed to antigen. Lung inflammatory responses were significantly lower in nude mice transferred with CD4+ T cells from GATA-3-overexpressing mice than in those with wild-type CD4+ T cells, with a reduction of lung IFN-gamma level.
These results indicate that overexpression of GATA-3 attenuates the development of HP by correcting the Th1-polarizing condition.
In contrast to CD(4)(+) helper T-lymphocytes (T(H)), little is known about the transcriptional regulation of CD(8)(+) cytotoxic T-lymphocytes (Tc) and its role in the pathogenesis of asthma is unclear. This study was conducted to investigate the effect of T-bet and GATA-3 mRNA expression on profiles of type 1 and type 2 cytotoxic T lymphocytes in asthmatic children.
Totally 38 asthmatic children, including acute attack group composed of 20 cases (age 3 - 13 years, mean 6.2 +/- 2.9), remission group with 18 cases (age 3 - 12 years, mean 6.1 +/- 2.5) and 20 healthy control children (age 3 - 12, 6.9 +/- 2.7) were recruited in this study from Sep. 2005 to Mar. 2006. The mRNA expression of T-bet and GATA-3 in the peripheral blood mononuclear cells were detected by using semi-quantitative PCR and Tc1, Tc2 cell numbers by flow cytometry analysis system.
T-bet mRNA in asthmatic children was lower than that in control group and lower in attack stage than in remission stage (0.14 +/- 0.04, 0.21 +/- 0.03, 0.28 +/- 0.03, P < 0.05). In contrast, GATA-3 mRNA was higher in asthmatic children than in control group and higher in attack stage than in remission stage (0.49 +/- 0.09, 0.44 +/- 0.08, 0.37 +/- 0.04, P < 0.05). It was shown that Tc1 percentage was lower in asthmatic children than those of control group and lower in attack stage than those of remission stage (6.6 +/- 2.4, 14.2 +/- 4.3, 31.2 +/- 3.8, P < 0.05). Tc2 percentage in asthmatic children was higher than that of control group and higher in attack stage than that of remission stage (10.0 +/- 4.2, 5.4 +/- 2.2, 3.5 +/- 1.1, P < 0.05). Spearman correlation analysis revealed that T-bet mRNA was positively correlated with Tc1 percentage (r = 0.704) and negatively correlated with Tc2 percentage (r = -0.629). GATA3 mRNA was negatively correlated with Tc1 percentage (r = -0.612) and positively correlated with Tc2 percentage (r = 0.673). The T-bet/GATA-3 mRNA ratio was positively correlated with Tc1 percentage (r = 0.731) and Tc1/Tc2 (r = 0.773), while negatively correlated with Tc2 percentage (r = -0.642).
The imbalance of T-bet/GATA-3 mRNA expression is closely correlated with skewed Tc2 dominance in asthmatic children.
Recent advance in mouse genomics has provided us lots of information concerning genes playing critical roles during organogenesis of parathyroid gland. And human counterparts of murine hypoparathyroidism produced by gene manipulation have been identified. Large part of genes which is responsible for hypoparathyroidism consisted from transcription factors such as TBX1, GATA3, SOX3. This may contribute to the abnormalities in the other organogenesis. Systematic survey of the anomalies in face and the other organ is important to confirm the diagnosis. And people should keep in mind that PTH could not be produced only in parathyroid in parathyroid agenesis.
Hypoparathyroidism is characterized by decreased actions of parathyroid hormone (PTH) either by reduced secretion of or resistance to PTH. The understanding in the pathogenesis of hypoparathyroidism has been greatly increased by recent findings in genetic abnormalities of patients with hypoparathyroidism. A new classification of hypoparathyroidism based upon these genetic abnormalities is proposed, and a diagnostic approach to patients with hypoparathyroidism is summarized.
CD4(+) T helper cells differentiate into T helper 1 (Th1) or Th2 effector lineages, which orchestrate immunity to different types of microbes. Both Th1 and Th2 differentiation can be induced by Notch, but what dictates which of these programs is activated in response to Notch is not known. By using T cell-specific gene ablation of the Notch effector RBP-J or the Notch1 and 2 receptors, we showed here that Notch was required on CD4(+) T cells for physiological Th2 responses to parasite antigens. GATA-3 was necessary for Notch-induced Th2 differentiation, and we identified an upstream Gata3 promoter as a direct target for Notch signaling. Moreover, absence of GATA-3 turned Notch from a Th2 inducer into a powerful inducer of Th1 differentiation. Therefore, Gata3 is a critical element determining inductive Th2 differentiation and limiting Th1 differentiation by Notch.
Notch signaling plays multiple roles to direct diverse decisions regarding cell fate during T cell development. During helper T (Th) cell differentiation, Notch is involved in generating optimal Th2 cell responses. Here, we present data investigating how Notch mediates Th2 cell differentiation. Notch showed a CD4(+) T cell intrinsic role in promoting IL-4 expression that required GATA-3. In the absence of Notch signals, Gata3 expression was markedly diminished. Introduction of an activated allele of Notch1 into CD4(+) T cells led to the specific and direct upregulation of a developmentally regulated Gata3 transcript that included the exon 1a sequences. Furthermore, Notch acted in parallel with GATA-3 to synergistically activate IL-4 expression. Together, these data implicate Gata3 as a direct transcriptional Notch target that acts in concert with Notch signaling to generate optimal Th2 cell responses.
Both bone marrow stromal cells (BMSCs) and transcription factors (GATA-1, GATA-2 and GATA-3) are important in the normal hematopoiesis and the pathogenesis of hematopoietic disease. The purpose of this study was to investigate the expression of GATA-1, GATA-2 and GATA-3 genes in the bone marrow (BM) microenvironment from patients with chronic aplastic anemia (cAA) and normal individuals. Mononuclear cells (MNCs) were isolated from BM of patients with cAA (8 cases) and normal controls (9 cases). Adherent cells (i.e. BMSCs) were collected after long-term culture in vitro. The semi-quantitative expression levels of GATA genes were analyzed by using RT-PCR-enzyme linked immunosorbent assay (RT-PCR-ELISA). The BMSCs with cAA grew slowly compared with the normal BMSCs. In BMSCs, only the expression ratio of GATA-3 gene from cAA group (50.0%) was significant lower than the normal controls (P < 0.05), the expression ratios of other GATA genes from cAA group were similar to the normal controls. There was no difference in the expression level of GATA-1 gene in the BMSCs between normal controls and cAA group. The expression level of GATA-2 gene in BMSCs from cAA was significantly lower than that from normal controls (P < 0.05). The expression level of GATA-3 gene in BMSCs from cAA was significantly higher than that from normal controls (P < 0.05). The dominant expression of GATA-3 gene was found in the BMSCs from cAA and normal controls. GATA genes can be expressed in the BMSCs and may play a role in the regulation of hematopoiesis in normal individuals, as well as in patients with cAA. The change of expression levels of GATA genes may influence the hematopoiesis in BM microenvironment and relate to the pathogenesis and development of aplastic anemia.
Risk of allergic diseases has been linked to abnormal patterns of fetal immune development, suggesting that priming of the immune system may occur in utero. The aim of the study was to investigate whether the pattern of immune response in cord blood mononuclear cells (CBMC) shows association with allergic diseases and IgE sensitization at 2 yr of age, and to study the effect of maternal probiotic supplementation on CBMC immune responses. CBMC were isolated from 98 neonates in a randomized double-blinded intervention study. CBMC were stimulated with beta-lactoglobulin, and phytohemaglutinin (PHA). Secretion of interferon-gamma (IFN-gamma), interleukin-5 (IL-5), and IL-13 was measured by an ELISA; IL-2, IL-4, and IL-10 by a cytokine bead assay. T-cell polarization-associated IL-4 receptor and IL-12R expressions, and the respective transcription factors GATA-3 and T-bet were analyzed with RT-PCR. The above responses were compared with the development of allergic diseases and IgE sensitization at 2 yr of age, and with the maternal probiotic or placebo supplementation. PHA-stimulated GATA-3 expression and IL-2 secretion in CBMC were higher in IgE-sensitized children at an age of 2 yr than in the non-sensitized, non-allergic children (p = 0.03 and 0.026). PHA-induced expression of GATA-3 correlated with IL-5 (p = 0.003, r = 0.300) and IL-13 (p = 0.007, r = 0.278) secretion of CBMC, and IL-5 secretion of beta-lactoglobulin-stimulated CBMC was higher in IgE-sensitized children at 2 yr of age than in the non-sensitized, non-allergic children (p = 0.013). Probiotic bacteria had no effect on CBMC immune responses. In CBMC-enhanced induction of GATA-3, which activates several Th2 cytokines genes, was a risk factor for IgE sensitization. The immune deviation towards Th2-type immunity developed already in utero and seemed to modulate the pattern of immune response favoring an IgE response to environmental antigens.
To observe the effect of moxibustion on Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway of synovial cells in rheumatoid arthritis (RA) rabbits for revealing the underlying mechanism of moxibustion in the treatment of RA.
A total of 30 rabbits (Japanese Big-ear White species) were randomized (stratified random) into control, model and moxibustion groups with 10 cases in each. RA model was established by injection of Freund's Complete Adjuvant (FCA, 0.5 mL/kg) into the animal's bilateral joint cavities. Moxibustion was applied to bilateral "Shenshu" (BL 23), 5 cones every time, once daily (except Sundays), 3 weeks altogether. The synovial tissue of joint was sampled for analyzing the expression of signal molecules associated with JAK-STAT pathway with gene chip and bioinformation analytical techniques.
Compared with normal control group, the perimeters of both knee joints in model group increased significantly from the 3rd day to 21st day after injection of FCA (P < 0.01), while compared with model group, those in moxibustion group decreased considerably from the 6th day on (P < 0.05, 0.01). In comparison with control group, JAK-STAT pathway-associated genes with up-regulated expression in model group were C/EBP beta, CBP, CRP, GATA3, IFNAR1, IFNAR2, IFNGR2, IL-10Rb, INDO, SH2B, STAT3, STAT6, JAK3 and GP130, and those with down-regulated expression were A2M, MIG and IL-2Rr, suggesting an abnormal activation of JAK-STAT pathway; while in comparison with model group, the related gene up-regulated in moxibustion group in the expression was IL22R and those down-regulated were Cyclin D1, C/EBP beta, CRP, GATA3, IFNAR2, INDO, JAK2, JAK3, V-JUN, STAT3, STAT5, SH2B and OSM, showing that moxibustion had an apparent inhibitory effect on AR-induced abnormal activation of some genes as C/EBP beta, GATA3, IFNAR2, INDO, etc.
Moxibustion can resist inflammation and eliminate swelling in RA rabbits, which may be closely with its effect in inhibiting abnormal activation of JAK-STAT pathway in synovial cells.
The development of new stem cell-based technologies is creating new hopes in regenerative medicine. Hearing-impaired individuals should benefit greatly from the development of a cell-based regenerative strategy to treat deafness. An important achievement would be to develop a human-based system that could bring the advances made in animal models closer to clinical application. In this work, we have explored the suitability of the developing fetal cochlea to be used as a source for the extraction of auditory progenitor/stem cells. We have established cultures that express critical markers such as NESTIN, SOX2, GATA3 and PAX2. These cultures can be expanded in vitro for several months and differentiating markers such as ATOH1/HATH1 and POU4F3/BRN3C can be induced by manipulating the culture conditions using specific growth factors such as bFGF, EGF and retinoic acid.
During T-cell development, thymocytes with intermediate avidity for antigen-MHC complexes are positively selected and then differentiate into functional cytotoxic and helper T cells. This process is controlled by signalling from the T-cell receptor (TCR). Here, we show that the c-Myb transcription factor is a critical downstream regulator of positive selection, promoting the development of helper T cells and blocking the development of cytotoxic T cells. A gain-of-function c-Myb transgene stops development of cytotoxic T cells, instead causing accumulation of a precursor population. Conversely, loss of c-Myb in selecting cells results in significantly fewer helper T cells. In c-Myb-null thymocytes, Gata3, a critical inducer of T-helper cell fate, is not upregulated in response to T-cell receptor signaling, following selection. We show that Gata3 is a direct target of c-Myb, and propose that c-Myb is an important regulator of Gata3, required for transduction of the T-cell receptor signal for subsequent helper cell lineage differentiation.
A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the DeltadblGATA mouse. We demonstrate here that bone marrow progenitors isolated from DeltadblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured DeltadblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured DeltadblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1E(B)), located approximately 3700 bp downstream to the previously described first exon found in hemopoietic cells (1E(A)) and approximately 42 bp upstream to another variant first exon, 1E(C). These data suggest that cultured progenitors are able to circumvent the effects of the DeltadblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.
Interleukin (IL) 25 (IL-17E), a distinct member of the IL-17 cytokine family, plays important roles in evoking T helper type 2 (Th2) cell-mediated inflammation that features the infiltrations of eosinophils and Th2 memory cells. However, the cellular sources, target cells, and underlying mechanisms remain elusive in humans. We demonstrate that human Th2 memory cells expressing distinctive levels of IL-25 receptor (R) are one of the responding cell types. IL-25 promotes cell expansion and Th2 cytokine production when Th2 central memory cells are stimulated with thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs), homeostatic cytokines, or T cell receptor for antigen triggering. The enhanced functions of Th2 memory cells induced by IL-25 are associated with sustained expression of GATA-3, c-MAF, and JunB in an IL-4-independent manner. Although keratinocytes, mast cells, eosinophils, and basophils express IL-25 transcripts, activated eosinophils and basophils from normal and atopic subjects were found to secrete bioactive IL-25 protein, which augments the functions of Th2 memory cells. Elevated expression of IL-25 and IL-25R transcripts was observed in asthmatic lung tissues and atopic dermatitis skin lesions, linking their possible roles with exacerbated allergic disorders. Our results provide a plausible explanation that IL-25 produced by innate effector eosinophils and basophils may augment the allergic inflammation by enhancing the maintenance and functions of adaptive Th2 memory cells.
The epidermis is a multi-layered stratified epithelium continuously renewed by differentiating keratinocytes that develops by the action of p63, a member of the p53 family. The TP63 contains two promoters, resulting in the expression of different proteins, containing (TAp63) or not (DeltaNp63) an amino-terminal transactivation domain, which contribution in skin formation is not fully understood. We found that p63 binds and transactivate GATA-3 promoter, which in turn transactivate IKKalpha, two pivotal regulators of epithelial development. Indeed, GATA-3 is a regulator of cell lineage in skin and hair follicles formation. To further study the relationship between GATA-3 and p63 isoforms here we investigated their expression during keratinocyte differentiation, in human epidermis and hair follicle.
To explore the correlation between the expression of GATA-3 and the level of local cytokines (IL-5, IL-6 and IL-8).
The levels of IL-5, IL-6 and IL-8 in ethmoid sinus mucosa were titrated in 45 patients with chronic rhinosinusitis and 11 normal subjects by ELISA. Patients were divided into AR group (with allergic rhinitis) and NAR group (without allergic rhinitis) . Semi-quantitative RT-PCR and immunohistochemical staining were used to examine the GATA-3 expression in nasal mucosa. The correlation between the expression of GATA-3 and the levels of cytokines was evaluated.
IL-5, IL-6 and IL-8 levels in both AR and NAR groups were significantly elevated compared with normal group (all P < 0.01 for AR group; P < 0.05, 0.05, 0.01 for NAR group, respectively), and they were much higher in AR group in comparison with NAR group (P < 0.01, 0.05, 0.01, respectively). Semi-quantitative RT-PCR showed that AR and NAR groups had markedly greater level of GATA-3 mRNA than that in control group (P < 0.01, respectively), and the level of GATA-3 mRNA in AR group was further higher than that in NAR group (P < 0.01). Immunohistochemical staining illustrated that GATA-3 was primarily presented in cytoplasma and the GATA-3 positive cells were mainly infiltrating inflammatory cells in submucosa. The mean GATA-3 positive-staining rate was (27. 90 +/- 16.75)% and (10.22 +/- 0.05)% in AR and NAR group, which were markedly higher than (1.30 +/- 1.78)% in control group (P < 0.01, respectively). Pearson correlation analysis demonstrated that GATA-3 positive-staining rate was closely correlated with IL-5 level, but not IL-6 and IL-8. The correlation coefficient was 0. 712 for GATA-3 and IL-5 (P < 0.01), 0.200 for GATA-3 and IL-6 (P > 0.05), 0.089 for GATA-3 and IL-8 (P > 0.05).
Activation of GATA-3 might be one of the mechanisms for induction of IL-5 expression in chronic rhinosinusitis . Concomitance of allergic rhinitis with chronic rhinosinusitis further increased expression of GATA-3, and subsequently enhanced IL-5 expression. Chronic sinusitis may be related to allergy, and GATA-3 may play a key role in the pathogenesis of chronic sinusitis.
The present study underlines the importance of gemfibrozil, a lipid-lowering drug and an activator of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), in inhibiting the disease process of adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of relapsing-remitting multiple sclerosis. Clinical symptoms of EAE, infiltration of mononuclear cells, and demyelination were significantly lower in SJL/J female mice receiving gemfibrozil through food chow than those without gemfibrozil. It is noteworthy that the drug was equally effective in treating EAE in PPAR-alpha wild-type as well as knockout mice. Gemfibrozil also inhibited the encephalitogenicity of MBP-primed T cells and switched the immune response from a Th1 to a Th2 profile independent of PPAR-alpha. Gemfibrozil consistently inhibited the expression and DNA-binding activity of T-bet, a key regulator of interferon-gamma (IFN-gamma) expression and stimulated the expression and DNA-binding activity of GATA3, a key regulator of IL-4. Gemfibrozil treatment decreased the number of T-bet-positive T cells and increased the number of GATA3-positive T cells in spleen of donor mice. The histological and immunohistochemical analyses also demonstrate the inhibitory effect of gemfibrozil on the invasion of T-bet-positive T cells into the spinal cord of EAE mice. Furthermore, we demonstrate that the differential effect of gemfibrozil on the expression of T-bet and GATA3 was due to its inhibitory effect on NO production. Although excess NO favored the expression of T-bet, scavenging of NO stimulated the expression of GATA-3. Taken together, our results suggest gemfibrozil, an approved drug for hyperlipidemia in humans, may find further therapeutic use in multiple sclerosis.
Human natural killer (NK) cells are the key contributors of innate immune response and the effector functions of these cells are enhanced by cytokines such as interleukine 2 (IL2). We utilized genome-wide transcriptional profiling to identify gene expression signatures and pathways in resting and IL2 activated NK cell isolated from peripheral blood of healthy donors.
Gene expression profiling of resting NK cells showed high expression of a number of cytotoxic factors, cytokines, chemokines and inhibitory and activating surface NK receptors. Resting NK cells expressed many genes associated with cellular quiescence and also appeared to have an active TGFbeta (TGFB1) signaling pathway. IL2 stimulation induced rapid downregulation of quiescence associated genes and upregulation of genes associated with cell cycle progression and proliferation. Numerous genes that may enhance immune function and responsiveness including activating receptors (DNAM1, KLRC1 and KLRC3), death receptor ligand (TNFSF6 (FASL) and TRAIL), chemokine receptors (CX3CR1, CCR5 and CCR7), interleukin receptors (IL2RG, IL18RAB and IL27RA) and members of secretory pathways (DEGS1, FKBP11, SSR3, SEC61G and SLC3A2) were upregulated. The expression profile suggested PI3K/AKT activation and NF-kappaB activation through multiple pathways (TLR/IL1R, TNF receptor induced and TCR-like possibly involving BCL10). Activation of NFAT signaling was supported by increased expression of many pathway members and downstream target genes. The transcription factor GATA3 was expressed in resting cells while T-BET was upregulated on activation concurrent with the change in cytokine expression profile. The importance of NK cells in innate immune response was also reflected by late increased expression of inflammatory chemotactic factors and receptors and molecules involved in adhesion and lymphocyte trafficking or migration.
This analysis allowed us to identify genes implicated in cellular quiescence and the cytokines and cytotoxic factors ready for immediate immune response. It also allowed us to observe the sequential immunostimulatory effects of IL2 on NK cells improving our understanding of the biology and molecular mediators behind NK cell activation.
Signalling lymphocytic activation molecule (SLAM) and interleukin (IL)-18 induce interferon (IFN)-gamma production from Th1 cells. The allergen-induced SLAM and IL-18 mRNA expressions are increased during subcutaneous immunotherapy (SCIT), but nothing is known about their role during sublingual immunotherapy (SLIT). Transcription factor GATA-3 is associated with Th2 cells but its role in SCIT and SLIT is yet unexplored. This study was undertaken to analyse the allergen induced in vitro mRNA expression of IL-18, SLAM and GATA-3 in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR) during SLIT.
Ten patients with AR undergoing pollen SLIT with a weekly dose of 200,000 SQ-U, 10 with 24,000 SQ-U of mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included. Peripheral blood mononuclear cell were stimulated with birch extract prior to, after 1 and 2 years of the treatment. The mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan); Applied Biosystems, Foster City, CA, USA).
The expression of IL-18 mRNA was increased in the high-dose group in comparison to the placebo group after 1 year of therapy (P = 0.028) and had an inverse correlation with the late phase skin reaction after the second study year (r = -0.41, P = 0.041). SLAM mRNA expression increased in the high-dose group from baseline to 1 year (P = 0.028) and correlated with IL-10 (r = 0.96, P < 0.0001) and transforming growth factor-beta (r = 0.80, P = 0.0037) mRNA expression. No significant changes were seen in GATA-3 mRNA expression.
During SLIT, IL-18 and SLAM are upregulated, suggesting that the Th2 type inflammatory response is downregulated during SLIT by increased Th1 type response.
The transcription factor GATA-3 is required for normal mammary gland development, and its expression is highly correlated with estrogen receptor alpha (ER alpha) in human breast tumors. However, the functional role of GATA-3 in ER alpha-positive breast cancers is yet to be established. Here, we show that GATA-3 is required for estradiol stimulation of cell cycle progression in breast cancer cells. The role of GATA-3 in estradiol signaling requires the direct positive regulation of the expression of the ER alpha gene itself by GATA-3. GATA-3 binds to two cis-regulatory elements located within the ER alpha gene, and this is required for RNA polymerase II recruitment to ER alpha promoters. Reciprocally, ER alpha directly stimulates the transcription of the GATA-3 gene, indicating that these two factors are involved in a positive cross-regulatory loop. Moreover, GATA-3 and ER alpha regulate their own expression in breast cancer cells. Hence, this transcriptional coregulatory mechanism accounts for the robust coexpression of GATA-3 and ER alpha in human breast cancers. In addition, these results highlight the crucial role of GATA-3 for the response of ER alpha-positive breast cancers to estradiol. Moreover, they identify GATA-3 as a critical component of the master cell-type-specific transcriptional network including ER alpha and FoxA1 that dictates the phenotype of hormone-dependent breast cancer.
GATA-3 is essential for T cell development from the earliest stages. However, abundant GATA-3 can drive T lineage precursors to a non-T cell fate, depending on Notch signaling and developmental stage. Here, overexpression of GATA-3 blocked the survival of pro-T cells when Notch-Delta signals were present but enhanced viability in their absence. In fetal thymocytes at the double-negative 1 (DN1) stage and DN2 stage but not those at the DN3 stage, overexpression of GATA-3 rapidly induced respecification to the mast cell lineage with high frequency by direct transcriptional 'reprogramming'. Normal DN2 thymocytes also showed mast cell potential when interleukin 3 and stem cell factor were added in the absence of Notch signaling. Our results suggest a close relationship between the pro-T cell and mast cell programs and a previously unknown function for Notch in T lineage fidelity.
Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-1 (TCF-1) (gene name Tcf7). To identify additional regulators of T cell specification, a cDNA library from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin(-)Sca-1(+)Kit(+)CD27(-)) and multipotent progenitors (Lin(-)Sca-1(+)Kit(+)CD27(+)), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bcl11b, TCF-1 (Tcf7), and HEBalt, Notch target Deltex1, Deltex3L, Fkbp5, Eva1, and Tmem131. Like GATA3 and Deltex1, Bcl11b, Fkbp5, and Eva1 were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only Bcl11b and HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative 1 and double negative 2) corresponding to T lineage specification. Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway.
Human CD34+ cord blood (CB) cells are hematopoietic progenitors useful for stem cell transplantation, even after ex vivo expansion. We investigated the effect of tumor necrosis factor (TNF) on lymphoid development from cultured CD34+ CB cells.
Human CD34+ CB cells were cultured in cytokine mixes with or without TNF. Preculture during 60 hours was followed by in vitro differentiation assays, including fetal thymus organ culture and coculture on murine stromal MS-5 cells. In a next step, experiments were extended to CD34+CD38- and CD34+CD38+ CB cells and prolonged preculture.
Preculture in the presence of TNF improved differentiation into T cells and diminished the ability to generate B cells, while NK potential and myeloid development were unaffected. Sorted CD34+CD38- CB cells were more potent T-cell precursors after preculture in TNF, compared to CD34+CD38+ CB cells. In precultured CD34+CD38- CB cells, TNF increased GATA3 but decreased EBF1 expression, in line with the skewed lymphoid differentiation induced by TNF. However, when preculture in the presence of TNF was extended to 1 week, T-cell precursors were lost.
After short-term culture of CD34+ CB cells in the presence of TNF, T-cell generation is stimulated at the expense of B-cell generation. T-cell progenitors are enriched in the CD34+CD38- fraction. These results have implications on the culture conditions to be used for CB CD34+ cells prior to transplantation.
Regulatory T cells (T-reg cells) have been described as an important cell population in the UV treatment of inflammatory skin diseases.
We have treated five patients with generalized atopic eczema (AE) using medium-dose (15 cycles of 50 J/cm(2), total dose of 750 J/cm(2)) UVA1 therapy and have analyzed the skin-infiltrating T-cellular subsets before and after therapy. Skin biopsies were split for immunohistochemistry and Real-time PCR and analyzed for CD4, Fox-P3, GATA-3, and IL-10 transcription as well as for CD3, CD4, CD152, Fox-P3, and GITR staining.
In all the investigated patients, we observed a good clinical response to UVA1. As described previously, the number of epidermal T cells slightly declined after irradiation. However, we did not observe a general decrease in T cell numbers. Within the population of T cells, no specific difference in the kinetics of Fox-P3-positive cells and Fox-P3-specific mRNA was noted as compared with GATA-3 positive T cells. These results were paralleled by RT-PCR for IL-10 and staining for CD152, a surface molecule that has been described for T-reg cells.
In our hands, the clinical benefit of UVA1 treatment in AE patients does not seem to be due to a preferential survival/proliferation of T-reg cells.
Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.
Some polyacetylenes from the plant Bidens pilosa have been reported to treat diabetes. In this study, we report that the cytopiloyne from B. pilosa, which is structurally different from the above-mentioned polyacetylenes and inhibits CD4(+) T cell proliferation, effectively prevents the development of diabetes in nonobese diabetic mice as evidenced by a normal level of blood glucose and insulin and normal pancreatic islet architecture. Cytopiloyne also suppresses the differentiation of type 1 Th cells but promotes that of type 2 Th cells, which is consistent with it enhancing GATA-3 transcription. Also, long-term application of cytopiloyne significantly decreases the level of CD4(+) T cells inside pancreatic lymph nodes and spleens but does not compromise total Ab responses mediated by T cells. Coculture assays imply that this decrease in CD4(+) T cells involves the Fas ligand/Fas pathway. Overall, our results suggest that cytopiloyne prevents type 1 diabetes mainly via T cell regulation.
Cross-talk between TGF-beta and IL-6 has been shown to direct the differentiation of CD4(+) cells into special IL-17-secreting cells, which are termed Th17 cells. In this study, we demonstrated that TGF-beta and IL-6 could stimulate CD8(+) cells to differentiate into noncytotoxic, IL-17-producing cells in MLC. These IL-17-producing CD8(+) cells exhibit a unique granzyme B(-)IFN-gamma(-)IL-10(-) phenotype. The mRNA level of Th2/T cytotoxic 2 (Tc2) transcription factors GATA3 and Th1/Tc1 transcription factors T-box expressed in T cell (T-bet) as well as its target H2.O-like homeobox (Hlx) is decreased in CD8(+) cells from TGF-beta- and IL-6-treated MLC. In addition, these CD8(+) cells display a marked up-regulation of retinoic acid-related orphan receptor-gammat, a key IL-17 transcription factor. These results demonstrate that the existence of an IL-17-producing CD8(+) subset belongs to neither the Tc1 nor the Tc2 subset and can be categorized as a T noncytotoxic 17 (Tnc17) subset.
The sphingosine-1-phosphate analogue FTY720 is known to alter migration and homing of lymphocytes via sphingosine-1-phosphate receptors. However, several studies indicate that its mode of action is more complex and that FTY720 may also directly influence cytokine effector functions. Therefore, we studied the effect of FTY720 in T helper type (Th2)-mediated oxazolone-induced colitis in mice. Following rectal oxazolone instillation, Th2 cells producing IL-13 induce a progressive colitis resembling human ulcerative colitis. A rectal enema of oxazolone [90 mg/kg body weight] was applied to BALB/c mice. FTY720 was administered i.p. from day 0 to 3 or from day 3 to 5 following the instillation of the haptenating agent. Assessment of severity of colitis was performed daily. FTY720 plasma levels were detected using LC-MS/MS-analysis. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity as well as cytokine levels of lamina propria CD4(+) T-cells and T1/ST2 expression were determined. Treatment with FTY720 prominently reduced the clinical and histopathologic severity of oxazolone-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of FTY720 were associated with a prominent reduction of the key effector Th2 cytokines IL-13, IL-4 and IL-5. Strikingly, FTY720 inhibited GATA3 and T1/ST2 expression which represent highly relevant markers for Th2 differentiation and Th2 effector function, respectively. Our data provide the first evidence that FTY720 exhibits beneficial prophylactic as well as therapeutic effects in Th2-mediated experimental colitis by directly affecting Th2 cytokine profiles probably by reducing T1/ST2, thus offering a new auspicious therapeutic instrument for the treatment of human ulcerative colitis.
Breast cancer is a complex and heterogeneous disease at the molecular level. Evolution is difficult to predict according to classical histoclinical prognostic factors. Different studies highlight the importance of large-scale molecular expression analyses to improve taxonomy of breast cancer and prognostic classification. Identification of new molecular markers that refine this taxonomy and improve patient management is a priority in the field of breast cancer research.Nectins are cell adhesion molecules involved in the regulation of epithelial physiology. We present here Nectin-4/PVRL4 as a new histological and serological tumor associated marker for breast carcinoma.
Expression of Nectin-4 protein was measured on a panel of 78 primary cells and cell lines from different origins and 57 breast tumors by FACS analysis and immunohistochemistry (IHC), respectively. mRNA expression was measured by quantitative PCR. Serum Nectin-4 was detected by ELISA and compared with CEA and CA15.3 markers, on panels of 45 sera from healthy donors, 53 sera from patients with non-metastatic breast carcinoma (MBC) at diagnosis, and 182 sera from patients with MBC. Distribution of histological/serological molecular markers and histoclinical parameters were compared using the standard Chi-2 test.
Nectin-4 was not detected in normal breast epithelium. By contrast, Nectin-4 was expressed in 61% of ductal breast carcinoma vs 6% in lobular type. Expression of Nectin-4 strongly correlated with the basal-like markers EGFR, P53, and P-cadherin, and negatively correlated with the luminal-like markers ER, PR and GATA3. All but one ER/PR-negative tumors expressed Nectin-4. The detection of Nectin-4 in serum improves the follow-up of patients with MBC: the association CEA/CA15.3/Nectin-4 allowed to monitor 74% of these patients compared to 67% with the association CEA/CA15.3. Serum Nectin-4 is a marker of disease progression, and levels correlate with the number of metastases (P = 0.038). Serum Nectin-4 is also a marker of therapeutic efficiency and correlates, in 90% of cases, with clinical evolution.
Nectin-4 is a new tumor-associated antigen for breast carcinoma. Nectin-4 is a new bio-marker whose use could help refine breast cancer taxonomy and improve patients' follow-up. Nectin-4 emerges as a potential target for breast cancer immunotherapy.
Decisions by uncommitted cells to differentiate down one lineage pathway or another is fundamental to developmental biology. In the immune system, lymphocyte precursors commit to T- or B-cell lineages and T-cell precursors to CD4 or CD8 independently of foreign antigen. T and B cells must also decide whether or not to respond to antigen and when a response is initiated, what sort of response to make such as the type of antibody, CD4 or CD8, and CD4 Th1 or Th2. The two basic mechanisms for these decision-making processes are selection and instruction. Selection depends on prior stochastic production of precommitted cells, which are then selected to respond by an appropriate signal; for example, CD8 and CD4 responses selected by peptide presented in association with major histocompatibility complex class I or II. In contrast, instruction occurs when an uncommitted precursor embarks upon a differentiation pathway in response to a particular set of signals; for example, Th1 and Th2 lineage commitment. In this paper, the signals that determine Th1 and Th2 differentiation are examined with a mathematical model and shown to act as a bistable switch permitting either Tbet or Gata3 to be expressed in an individual cell but not both. The model is used to show how the Tbet Gata3 network within an individual cell interacts with cytokine signals between cells and suggests how Th1 and Th2 lineage commitment can become irreversible. These considerations provide an example of how mathematical models can be used to gain a better understanding of lymphocyte differentiation in an immune response.
Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1a were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.
The morphologic distinction between prostate and urothelial carcinoma can be difficult. To identify novel diagnostic markers that may aid in the differential diagnosis of prostate versus urothelial carcinoma, we analyzed expression patterns in prostate and bladder cancer tissues using complementary DNA microarrays. Together with our prior studies on renal neoplasms and normal kidney, these studies suggested that the gene for placental S100 (S100P) is specifically expressed in benign and malignant urothelial cells. Using tissue microarrays, a polyclonal antiserum against S100P protein stained 86% of 295 urothelial carcinomas while only 3% of 260 prostatic adenocarcinomas and 1% of 133 renal cell carcinomas stained. A commercially available monoclonal antibody against S100P stained 78% of 300 urothelial carcinomas while only 2% of 256 prostatic adenocarcinomas and none of 137 renal cell carcinomas stained. A second gene, GATA3, also showed high level expression in urothelial tumors by cDNA array. A commercially available monoclonal antibody against GATA3 stained 67% of 308 urothelial carcinomas, but none of the prostate or renal carcinomas. For comparison, staining was also performed for p63 and cytokeratin 5/6. p63 stained 87% of urothelial carcinomas whereas CK5/6 stained 54%. Importantly, when S100P and p63 were combined 95% of urothelial carcinomas were labeled by one or both markers. We conclude that the detection of S100P and GATA3 protein expression may help distinguish urothelial carcinomas from other genitourinary neoplasms that enter into the differential diagnosis.
To investigate the effect of rosiglitazone on the expression of T-bet/GATA-3 in peripheral blood T lymphocytes and cytokine derived from Th1/Th2 in patients with acute asthma and to compare its mechanism with that of dexamethasone (DXM).
Peripheral blood T lymphocytes from 10 patients with acute asthma (A group, A) and 10 healthy volunteers (B group, B) were isolated, purificated and cultured, and were divided into 2 groups (3 h and 24 h) based on the treatment time, and each group was further divided, on the basis of the treatment given, into 3 sub-groups respectively: control group (A(1) and B(1)), rosiglitazone treated group (A(2) and B(2)) and DXM treated group (A(3) and B(3)). The levels of IFN-gamma and IL-4 in the culture supernatant were detected by Enzyme-linked immunoadsorbent assay (ELISA) and the expression levels of T-bet mRNA/GATA-3 mRNA in T lymphocytes was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
The levels of INF-gamma and INF-gamma/IL-4 in the culture supernatant of T lymphocytes and T-bet mRNA and T-bet mRNA/GATA-3 mRNA in T lymphocytes from the A at 3 h and 24 h were 357 +/- 31, 783 +/- 47, 5.5 +/- 1.0, 8.4 +/- 1.5, 18.7 +/- 3.7, 11.9 +/- 2.9, 1.20 +/- 0.11, 0.290 +/- 0.020, and those from the B at 3 h and 24 h were 659 +/- 41, 1394 +/- 120, 11.5 +/- 3.0, 17.4 +/- 4.0, 29.0 +/- 4.0, 18.9 +/- 4.0, 3.82 +/- 0.81, 0.870 +/- 0.090, the differences were significant between these groups (t = 18.59, 5.95, 14.87, 6.25, 6.06, 4.63, 10.23, 18.67, all P < 0.05, respectively). The levels of IL-4 in the culture supernatant of T lymphocytes and GATA-3 mRNA in T lymphocytes from the A at 3 h and 24 h were 65 +/- 6, 96 +/- 9, 16.4 +/- 4.2, 41 +/- 6, and those from the B at 3 h and 24 h were 57 +/- 5, 83 +/- 7, 7.8 +/- 2.2, 23 +/- 4, the differences were significant between these groups (t = 3.19, 3.90, 5.77, 7.76, all P < 0.05, respectively). The levels of IFN-gamma, IL-4 and IFN-gamma/IL-4 in the culture supernatant of T lymphocytes from the A and B at 3 h were 357 +/- 31, 65 +/- 6, 5.5 +/- 1.0, 659 +/- 41, 57 +/- 5, 11.5 +/- 3.0, and those from the A and B at 24 h were 783 +/- 47, 96 +/- 9, 8.4 +/- 1.5, 1394 +/- 120, 83 +/- 7, 17.4 +/- 4.0, The differences were significant between these groups (t = 23.8, 9.48, 5.03, 18.21, 9.01, 3.53, all P < 0.05, respectively). The levels of T-bet mRNA and T-bet mRNA/GATA-3 mRNA in T lymphocytes from the A and B at 3 h were 18.7 +/- 3.7, 1.20 +/- 0.11, 29.0 +/- 4.0, 3.82 +/- 0.81, and those from the A and B at 24 h were 11.9 +/- 2.9, 0.290 +/- 0.020, 18.9 +/- 4.0, 0.870 +/- 0.090, the differences were significant between these groups (t = 4.62, 5.66, 29.67, 11.5, all P < 0.05, respectively). The levels of GATA-3 mRNA in T lymphocytes from the A and B at 3 h were 16.4 +/- 4.2, 7.8 +/- 2.2, it was lower than those from the A and B at 24 h (41 +/- 6, 23 +/- 4, t = 10.70, 10.11, all P < 0.05, respectively). The levels of T-bet mRNA from the A showed a positive correlation with the level of IFN-gamma (r = 0.581, P < 0.05), and a negative correlation with the level of IL-4 (r = -0.493, P < 0.05); The levels of GATA-3 mRNA from the A showed a negative correlation with the level of IFN-gamma (r = -0.501, P < 0.05), and a positive correlation with the level of IL-4 (r = 0.579, P < 0.05); The ratio of T-bet mRNA to GATA-3 mRNA from the A showed a positive correlation with the ratio of IFN-gamma to IL-4 (r = 0.696, P < 0.05).
Rosiglitazone could regulate the balance of IFN-gamma and IL-4 through effect on the expression of T-bet mRNA and GATA-3 mRNA.
To study the expression of T-bet, GATA-3 and FOXP3 mRNA in asthmatic peripheral blood mononuclear cells (PBMC) before and after stimulation by Dermatophagoides farinae antigen (Df), and the relationship among the three transcription factors.
Twenty-five patients of asthma and 15 healthy controls were included. The mRNA levels of T-bet, GATA-3, and FOXP3 in PBMC before and after Df stimulation were detected by semiquantitative reverse transcription-polymerase chair reaction (RT-PCR).
There was no statistical difference (t = 0.78, 0.38 respectively, all P > 0.05) in the levels of T-bet and FOXP3 mRNA between the patients (0.3 +/- 0.4, 0.42 +/- 0.24 respectively) and the healthy controls (0.4 +/- 0.3, 0.39 +/- 0.37 respectively). However, GATA-3 mRNA was statistically different (t = 2.27, P < 0.05) between the patients (1.0 +/- 0.3) and the healthy controls (0.8 +/- 0.3). After the stimulation of PBMC by Df, the levels of T-bet, GATA-3 and FOXP3 mRNA showed statistical difference (t = 2.30, 3.79, 2.08 respectively, P < 0.05, < 0.01, < 0.05 respectively) between the patients (0.33 +/- 0.39, 1.58 +/- 1.44, 0.11 +/- 0.32 respectively) and the healthy controls (0.03 +/- 0.40, 0.11 +/- 0.53, 0.43 +/- 0.66 respectively). The FOXP3 mRNA level in the asthmatics was negatively correlated with T-bet and GATA-3 mRNA (r = -0.46, P < 0.05, r = -0.62, P < 0.01 respectively).
There are a predominant expression of GATA-3 and T-bet, but a decreased expression of FOXP3 in Df-stimulated PBMCs from patients with asthma.
To investigate the roles of transcription factor GATA-3 and T-bet at the fetal-maternal interface in the pathogenesis of unexplained recurrent spontaneous abortion (URSA).
The expression of GATA-3 and T-bet mRNA was examined by in situ hybridization. Decidua was obtained from 20 women with URSA and 20 normal pregnant (NP) women.
(1) The number of GATA-3 positive cells per high power field in women with URSA (25 +/- 16) was significantly lower than those in NP women (38 +/- 16) (P < 0.05). (2) The number of T-bet positive cells per high power field in women with URSA (59 +/- 17) was significantly higher than those in NP women (46 +/- 18) (P < 0.05). (3) Numbers of cells expressing GATA-3 mRNA correlated significantly with T-bet mRNA positive cell numbers in decidua (r = -0.55, P < 0.01).
The results demonstrate that upregulated expression of T-bet and downregulated expression of GATA-3 are found in the decidua of women with URSA. Imbalance of GATA-3/T-bet transcription factors might interfere with type 1 helper T cell (Th1)/type 2 helper T cell (Th2) differentiation at the fetal-maternal interface and cause URSA.
Transcription factors play a crucial role during hematopoiesis by orchestrating lineage commitment and determining cellular fate. Although tight regulation of transcription factor expression appears to be essential, little is known about the epigenetic mechanisms involved in transcription factor gene regulation. We have analyzed DNA methylation profiles of 13 key transcription factor genes in primary cells of the hematopoietic cascade, lymphoma cell lines and lymph node biopsies of diffuse large B-cell- and T-cell-non-Hodgkin lymphoma patients. Several of the transcription factor genes (SPI1, GATA3, TCF-7, Etv5, c-maf and TBX21) are differentially methylated in specific cell lineages and stages of the hematopoietic cascade. For some genes, such as SPI1, Etv5 and Eomes, we found an inverse correlation between the methylation of the 5' untranslated region and expression of the associated gene suggesting that these genes are regulated by DNA methylation. Differential methylation is not limited to cells of the healthy hematopoietic cascade, as we observed aberrant methylation of c-maf, TCF7, Eomes and SPI1 in diffuse large B-cell lymphomas. Our results suggest that epigenetic remodelling of transcription factor genes is a frequent mechanism during hematopoietic development. Aberrant methylation of transcription factor genes is frequently observed in diffuse large B-cell lymphomas and might have a functional role during tumorigenesis.
Sphingosine 1-phosphate (S1P) is a natural lipid mediator that regulates immune cell traffic, Ab production, and T cell cytokine generation by mechanisms that enhance Th2 activities. Responses to S1P are controlled principally by the diverse expression patterns of its receptors in different cells. In T cells, the type 1 (S1P(1)) and type 4 (S1P(4)) G protein-coupled receptors are predominant. S1P(1) mainly transduces effects on T cell migration and trafficking, whereas S1P(4) transduces immunosuppression via its effects on T cell proliferation and cytokine production. Using T cell-specific S1P(1) transgenic (TG) mice, we investigated the regulatory effects of the S1P-S1P(1) axis on T cell cytokine production. The production of IL-4, but not IL-2 or IFN-gamma, was significantly up-regulated >10-fold in activated CD4 T cells from S1P(1) TG mice compared with those from wild-type mice. Quantitative real-time PCR analysis revealed that IL-4 up-regulation was initiated at the mRNA level as early as 4 h after T cell activation. The up-regulation of IL-4 mRNA was mediated by c-Maf, Jun B, and Gata3 as demonstrated by increases in their protein expression and DNA-binding activities. In contrast, the expression and DNA-binding activities of T-bet, FosB, C-Fos, Jun D, Fra-1, Fra-2, and c-Jun all were identical in wild-type and TG CD4 T cells. Immunological assays showed that increased IL-4 levels induced greater production of IgE. Thus, the S1P-S1P(1) axis specifically up-regulates c-Maf, Jun B, and Gata3, which consequently enhance IL-4 production that may lead to a Th2 phenotype.
We believe that immunotherapy with HKL as an adjuvant induces Thl-like TReg cells that can inhibit AHR and airway inflammation. These antigen-specific TReg cells are induced with CD8alpha+ DCs producing IL-12 and IL-10, produce IFN-gamma and IL-10, and also express T-bet and Foxp3. These Th1Reg cells are distinct from antigen-specific TReg cells induced with respiratory tolerance, which can also inhibit AHR and airway inflammation. These Th2-like TReg cells are induced with CD8alpha- DCs producing IL-10, they express IL-10, GATA3 and Foxp3. So allergen immunotherapy is effective in large part because it induces regulatory T cells. With HKL you get Th1Reg cells, and with other forms of immunotherapy you may get Th2Reg cells. We believe that with further refinements, allergen immunotherapy that rapidly induces allergen specific TReg cells will indeed be the magic bullets for allergy and asthma.
WNT family members are secreted-type glycoproteins regulating cell fate, planar cell polarity, cell adhesion, and cell movement. WNT signals are context-dependently transduced to the canonical pathway for the transcriptional up-regulation of MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, and also to the non-canonical pathway for the activation of RHOA, JNK, PKC, NFAT and NLK signaling cascades. We cloned and characterized the wild-type human WNT8B, while another group the aberrant human WNT8B with Gly230Ala and Arg284Leu amino-acid substitutions. Although WNT8B is undetectable in normal adult tissues by using Northern blot analyses, WNT8B is expressed in gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, and embryonal tumors. Here, comparative integromics on WNT8B orthologs were investigated by using bioinformatics (Techint) and human intelligence (Humint). Cow Wnt8b gene was identified within NW_001494361.1 genome sequence. Predicted sequence XM_582222.3 was an artificial cow Wnt8b with aberrant prediction for the first exon. Cow Wnt8b complete coding sequence was found to encode a 350-amino-acid protein, which showed 96.9% total-amino-acid identity with human WNT8B. Comparative proteomics revealed that N-terminal signal peptide, 22 Cys residues, two Asn-linked glycosylation sites, Gly230, and Arg284 of human WNT8B were conserved among mammalian WNT8B orthologs. Comparative genomics revealed that POU/OCT- and GATA-binding sites in the 5'-flanking promoter region were conserved among human, chimpanzee, cow, mouse, and rat WNT8B orthologs. In silico expression analyses revealed that human WNT8B was expressed in embryoid body derived from embryonic stem (ES) cells, hepatocyte progenitors derived from ES cells, fetal brain, diffuse-type gastric cancer, colorectal cancer, prostate cancer, and ovarian fibrotheoma. Based on the expression profiles of POU and GATA family transcription factors, it was revealed that WNT8B expression in hepatocyte progenitors derived from human ES cells is due to POU5F1 (OCT3/OCT4) and GATA3, and also that WNT8B expression in diffuse-type gastric cancer is due to POU5F1 and GATA6.
Genomic regulatory networks specify how cellular gene expression responds to external temporal and spatial stimuli, ensuring that correct cell fate decisions are made and the appropriate cell phenotypes are adopted. In mammary epithelial cells, the hierarchy of stem and progenitor cells and the genetically specified program of transcriptional activity are beginning to be elucidated and integrated. A novel role for Gata-3 in specifying and maintaining mammary cell fate has recently been identified. These reports offer an understanding of how mammary cells assume and maintain a variety of cell behaviours and functions, and how a mammary cell may potentially subvert these constraints during carcinogenesis.
The transcription factor GATA3 is essential at multiple stages of T cell development, including the earliest double-negative stages, beta-selection and CD4 single-positive thymocytes. Here, we show that in CD2-GATA3 transgenic mice, with enforced GATA3 expression driven by the CD2 promoter, thymocytes have reduced levels of CD5, which is a negative regulator of TCR signaling participating in TCR repertoire fine-tuning. Reduction of CD5 expression was most prominent in CD4(+)CD8(+) double-positive (DP) cells and was associated with increased levels of the transcription factor E2A. Conversely, GATA3-deficient DP thymocytes showed consistently higher CD5 levels and defective TCR up-regulation during their development towards the CD4(lo)CD8(lo) subpopulation. CD2-GATA3 transgenic mice carrying the MHC class II-restricted TCR DO11.10 also manifested decreased CD5 levels. As in these TCR-transgenic mice reduced CD5 expression cannot result from an effect of GATA3 on repertoire selection, we conclude that enforced GATA3 interferes with the developmentally regulated increase of CD5 levels. Enforced GATA3 expression in DO11.10 transgenic mice was also accompanied by enhanced TCR expression during CD4 positive selection. Because GATA3 is induced by TCR signaling in DP thymocytes, our findings indicate that GATA3 establishes a positive feedback loop that increases TCR surface expression in developing CD4 lineage cells.
GATA-3 was first cloned as a T cell specific transcription factor in 1991 and its importance in the transcriptional control of T helper type 2 cell (Th2) differentiation was established in the mid to late 90's. A role for GATA-3 during thymic development has long implied by its continuous and regulated expression through out T lineage development, but the absolute requirement for GATA-3 during early T lymphoid commitment/survival previously precluded definitive answers to this question. Several technical breakthroughs have fueled fruitful investigation in recent years and uncovered unexpected and critical roles for GATA-3 in CD4 thymocyte survival, invariant natural killer T cell generation and function, and also in beta selection. Not only does GATA-3 participate in nearly every stage of T cell development from common lymphoid progenitor to Th2, conditional knockout studies have indicated that the influence of GATA-3 also extends beyond the immune system.
To explore the molecular mechanism of Chinese herbs for tonifying Shen on asthma by adjusting the imbalance of Th1/Th2.
Peripheral blood mononuclear cells (PBMC) separated from anticoagulated venous blood of 20 children with asthma in remission stage (3 ml from each) were equally divided into three groups, the blank group and the Chuankezhi (CKZ) groups cultured with media without or with CKZ of different concentrations respectively, for 48 h in vitro. The mRNA expressions of T-bet, GATA-3, IFN-gamma and IL-4 in the sediment collected from the cultures were measured by real-time fluorescence quantitative polymerase chain reaction technology.
As compared with those in the blank group, in the CKZ groups, T-bet mRNA expression and IFN-gamma mRNA expression were significantly higher, especially in CKZ II group (P < 0.01 and P < 0.05 resepectively); GATA-3 mRNA expression was insignificantly different (P > 0.05); IL-4 mRNA expression was significantly lower (P < 0.01). Moreover, the ratios of T-bet/GATA-3 and IFN-gamma/IL-4 were higher in the CKZ groups than those in the blank group, respectively, though showing insignificant difference in the former (P > 0.05), the difference in the latter was certainly significant (P < 0.05).
Chinese herbs for tonifying Shen can adjust the imbalance of Th1/Th2 in multiple layers by enhancing the function of Th1 cells and attenuating the function of Th2 cells, which may be realized through various links as regulating the expression of transcription factors and cytokines.
Three Gata transcription factors (Gata1, -2, and -3) are essential for hematopoiesis. These factors are thought to play distinct roles because they do not functionally replace each other. For instance, Gata2 messenger RNA (mRNA) expression is highly elevated in Gata1-null erythroid cells, yet this does not rescue the defect. Here, we test whether Gata2 and -3 transgenes rescue the erythroid defect of Gata1-null mice, if expressed in the appropriate spatiotemporal pattern. Gata1, -2, and -3 transgenes driven by beta-globin regulatory elements, directing expression to late stages of differentiation, fail to rescue erythropoiesis in Gata1-null mutants. In contrast, when controlled by Gata1 regulatory elements, directing expression to the early stages of differentiation, Gata1, -2, and -3 do rescue the Gata1-null phenotype. The dramatic increase of endogenous Gata2 mRNA in Gata1-null progenitors is not reflected in Gata2 protein levels, invoking translational regulation. Our data show that the dynamic spatiotemporal regulation of Gata factor levels is more important than their identity and provide a paradigm for developmental control mechanisms that are hard-wired in cis-regulatory elements.
Lupus nephritis is characterised by intrarenal inflammation and lymphocyte activation.
To examine the profile of cytokine gene expression in glomerulus and tubulointerstitium in patients with lupus nephritis.
36 consecutive patients with systemic lupus erythematosus having active renal disease were recruited, and they were required to undergo kidney biopsy. Glomerular and tubulointestitial cytokine expression of interleukin (IL)2, 4, 10, 12, 18, interferon gamma (IFN)gamma, T-bet (the Th1 transcription factor), GATA-3 (the Th2 transcription factor), transforming growth factor beta and monocyte chemoattractant protein (MCP)1 were studied by laser microdissection of the renal biopsy specimen, followed by real-time quantitative PCR.
There were 13 patients with World Health Organization class III nephritis, 14 patients with class IV nephritis and 9 patients with class V nephritis. There was a significant correlation between serum C3, C4 and anti-double strand DNA antibody level with glomerular expression of T-bet, IFNgamma and IL2. There was a significant correlation between histological activity index and glomerular expression of IL12, IL18, IL10 and MCP1. In addition, the degree of glomerular leucocyte infiltration significantly correlated with glomerular expression of IFNgamma, IL10, IL12 and IL18. By contrast, histological chronicity index correlated with the tubulointerstitial expression of IL2, MCP1 and GATA-3.
Intraglomerular expression of certain target genes correlate with the severity of systemic as well as histological activity, whereas the tubulointerstitial expression of other target genes correlate with the degree of chronic kidney scarring. This result may shed light on the immunopathogenesis of lupus nephritis.
T helper type 2 (Th2) cell differentiation requires the expression of GATA-3, a transcription factor that allows transcriptional activation of Th2 cytokine genes through chromatin remodelling. We investigated the role of the negative costimulatory receptor cytotoxic T-lymphocyte antigen 4 (CTLA-4) in the regulation of GATA-3 expression, Th2 differentiation and immunoglobulin production during the immune response to allergens. BALB/c mice were immunized with a recombinant major allergenic component of Parietaria judaica pollen, rPar j I, and treated with blocking anti-CTLA-4 or control antibodies. Results showed that in vivo CTLA-4 blockade enhanced the Par j I-specific immunoglobulin E (IgE) serum level. In contrast, Par j I-specific IgG2a serum level was reduced, suggesting that CTLA-4 blockade skewed immunoglobulin production towards interleukin-4 (IL-4) -dependent immunoglobulin isotypes. Consistently, CTLA-4 blockade increased the frequency of Par j I-specific Th2 cells but not Th1 cells, as well as IL-4 and IL-5 but not interferon-gamma production. Our data also showed that CTLA-4 blockade enhanced the GATA-3 : T-bet messenger RNA ratio. Interestingly, in vivo CTLA-4 blockade did not increase the frequency of GATA-3 protein-expressing cells. In contrast, it enhances GATA-3 protein level per cell. Further, in vitro results show that the anti-CTLA-4 monoclonal antibody, by competing with CD80 for CTLA-4 binding, induced an enhancement in the frequency of IL-4-producing cells that correlates with the increase in GATA-3 protein level per cell. In conclusion, CTLA-4, by affecting the level of GATA-3 per cell, contributes to keeping this factor under the threshold required to become a Th2 effector cell. Consequently, it affects IgE/IgG2a production and contributes to the outcome of allergen-specific immune responses.
The critical role for GATA family proteins in maintaining the normal (non-transformed) cell state is corroborated by the recent findings of mutations or methylation in GATA genes both in primary cancers and tumor lines including breast. Previously, microarray profiling studies determined that the highest expression of both GATA3 and ESR1 (estrogen receptor alpha) is seen in tumors associated with the most favorable survival outcomes, whereas the lowest expression of GATA3 is detected in tumor subtypes showing the worst outcomes. At this time, genes and pathways that are regulated by GATA3 in the mammary gland are not well defined. We have previously established a requirement for FOG (Friend Of GATA) cofactors during mouse development. Here we report that in the murine mammary gland Fog2 gene expression is upregulated upon pregnancy and lactation with prominent expression in the epithelial cells of the gland during post-lactational regression. Mammary-specific deletion of Fog2 identified a role for this gene during gland involution; excision of the Fog2 gene leads to the accelerated involution of the gland despite diminished levels of the remodeling enzymes. Importantly, the levels of several genes linked to the control of cancerous transformation in the breast (Esr1, Prg and Foxa1) are significantly reduced upon Fog2 excision. This implicates FOG2 in the maintenance of epithelial cell differentiation in the mammary gland and in performing a protective role in breast cancer.
The Natural Killer cell maturation from CD34(+) hematopoietic cell precursors is a complex process that requires the synergistic effect of different cytokines and growth factors. Although there have been a number of important advances in our understanding of the Natural Killer differentiation, the developmental step leading to mature Natural Killer cells is still poorly defined. We evaluated the effect of two zinc concentrations (10 and 20microM) on the kinetic of development of CD34(+) cell progenitors towards Natural Killer cells. CD34(+) cells were purified from peripheral blood and cultured in medium supplemented with interleukin-15, interleukin-7, Flt 3 ligand, and stem cell factor. CD34(+) cells underwent proliferation and progressively lost CD34 antigen and acquired a CD56(+) phenotype. These CD56(+) cells exerted cytotoxic activity and expressed the CD94 inhibitory receptor. The supplementation with zinc greatly increased both the number of cells in culture and the absolute number of CD56(+) cells. Zinc induced higher levels of cytotoxic activity and a higher number of perforin-producing and of CD94-bearing CD56(+) cells in comparison with zinc unsupplemented cultures in early stages of Natural Killer cell development. The zinc-induced changes in CD34-derived CD56(+) cells were associated with an increased expression of GATA-3, a zinc-finger transcription factor providing for maturation and activity of T and Natural Killer cells. The increase was related to a higher CD56(+) cell number (10microM zinc), or to an increased GATA-3 mRNA transcription in CD56(+) cells (20microM zinc). Our data demonstrate that zinc influences the proliferation and differentiation of CD34(+) progenitors.
In Th1 and Th2 memory lymphocytes, the genes for the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) are imprinted for expression upon restimulation. This cytokine memory is based on expression of the transcription factors T-bet for IFN-gamma, and GATA-3 for IL-4, and epigenetic modification of the cytokine genes. In Th2 cells, expression of the cytokine IL-10 is also induced by GATA-3. Here, we show that this induction is initially not accompanied by epigenetic modification of the IL-10 gene. Only after repeated restimulation of a memory Th2 cell in the presence of IL-4, extensive histone acetylation of the IL-10 gene is detectable. This epigenetic imprinting correlates with the development of a memory for IL-10 in repeatedly restimulated Th2 cells. In Th1 cells, IL-10 expression is induced by IL-12, but the IL-10 gene lacks detectable histone acetylation. Accordingly, IL-10 expression in restimulated memory Th1 cells remains conditional on the presence of IL-12. This finding defines a potential anti-inflammatory role for IL-12 in Th1 recall responses. While in primary Th1 responses IL-12 is required to induce expression of the pro-inflammatory cytokine IFN-gamma, in secondary Th1 responses IFN-gamma re-expression is independent of IL-12, which still is able to induce expression of the anti-inflammatory cytokine IL-10.
Interleukin-12 (IL-12) is a key cytokine for the development of T helper type 1 (Th1) responses; however, naïve CD4(+) T cells do not express IL-12Rbeta2, and are therefore unresponsive to IL-12. We have examined the mechanisms that control Th1-specific expression of the human IL-12Rbeta2 gene at early time points after T-cell stimulation. We have identified a Th1-specific enhancer element that binds signal transducer and activator of transcription 4 (STAT4) in vivo in developing Th1 but not Th2 cells. T-cell receptor (TCR) signaling induced histone hyperacetylation and recruitment of BRG1, the ATPase subunit of the SWI/SNF-like BAF chromatin remodeling complex, to the IL-12Rbeta2 regulatory regions and was associated with low-level gene transcription at the IL-12Rbeta2 locus. However, high-level IL-12Rbeta2 expression required TCR triggering in the presence of IL-12. Our results indicate a synergistic role of TCR-induced chromatin remodeling and cytokine-induced STAT4 activation to direct IL-12Rbeta2 expression during Th1 cell development.
GATA-3 plays a critical role in allergic diseases by regulating the release of cytokines from Th2 lymphocytes. However, the molecular mechanisms involved in the regulation of GATA-3 in human T lymphocytes are not yet understood. Using small interfering RNA to knock down GATA-3, we have demonstrated its critical role in regulating IL-4, IL-5, and IL-13 release from a human T cell line. Specific stimulation of T lymphocytes by costimulation of CD3 and CD28 to mimic activation by APCs induces translocation of GATA-3 from the cytoplasm to the nucleus, with binding to the promoter region of Th2 cytokine genes, as determined by chromatin immunoprecipitation. GATA-3 nuclear translocation is dependent on its phosphorylation on serine residues by p38 MAPK, which facilitates interaction with the nuclear transporter protein importin-alpha. This provides a means whereby allergen exposure leads to the expression of Th2 cytokines, and this novel mechanism may provide new approaches to treating allergic diseases.
The E2A transcription factors are required for normal T lymphopoiesis and to prevent T-lymphocyte progenitor transformation. Ectopic expression of E2A proteins in E2A-deficient lymphomas results in growth arrest and apoptosis, indicating that these cells remain responsive to the targets of E2A. Here we identify the transcriptional repressor growth factor independent 1B (Gfi1b) as a target of E2A that promotes growth arrest and apoptosis in lymphomas. Gfi1b expression in primary T-lymphocyte progenitors is dependent on E2A and excess Gfi1b prevents the outgrowth of T lymphocyte progenitors in vitro. Gfi1b represses expression of Gata3, a transcription factor whose appropriate regulation is required for survival of lymphomas and T-lymphocyte progenitors. We also show that ectopic expression of Gata3 in lymphomas promotes expression of Gfi1b, indicating that these proteins may function in an autoregulatory loop that maintains appropriate levels of Gata3. Therefore, we propose that E2A proteins prevent lymphoma cell expansion, at least in part through regulation of Gfi1b and modulation of Gata3 expression.
The expression of IL-5 correlated tightly with the maturation and differentiation of eosinophils, and is considered as a cytokine responsible for allergic inflammation. We report here that inhibition of HDAC activity by Trichostatin A (TSA) and sodium butyrate (NaBu), the two specific HDAC inhibitors, resulted in the elevation of both endogenous and exogenous activity of IL-5 promoter. We demonstrated that both the mRNA expression and protein production of IL-5 were stimulated by TSA and NaBu treatments. ChIP assays showed that treatments of TSA and NaBu caused hyperacetylation of histones H3 and H4 on IL-5 promoter in Jurkat cells, which consequently promoted the exogenous luciferase activity driven by this promoter. Moreover, site-directed mutagenesis studies showed that the binding sites for transcription factors NFAT, GATA3 and YY1 on IL-5 promoter were critical for the effects of TSA and NaBu, suggesting that the transcriptional activation of IL-5 gene by these inhibitors was achieved by affecting HDAC function on IL-5 promoter via transcription factors. These data will contribute to elucidating the unique mechanism of IL-5 transcriptional control and to the therapy of allergic disorders related to IL-5.
Naive CD4(+) T cells were reported to produce small amounts of IL-4 in vitro, which are implicated to be sufficient to initiate T(h)2 response in vivo. However, IL-4-producing naive CD4(+) T cells are difficult to study in vivo because they are present in low numbers shortly after the first antigen exposure. Here, we used IL-4/green fluorescence protein (GFP) reporter mice (G4 mice) to track the initial response of CD4(+) IL-4-producing cells. We first established a flow cytometry method to estimate the number of GFP(+) cells. We demonstrated the effectiveness of this method by showing that the responding CD4(+)GFP(+) cells exhibited an activated phenotype, possessed the capacity to express IL-5 and IL-13, but not IFN-gamma mRNA, and showed enhanced levels of GATA3 and c-maf mRNA expression. More importantly, we showed that the cell lines derived from FACS-sorted CD4(+)GFP(+) cells were antigen specific. By using this newly established method, we showed that the majority of responding GFP(+) cells were CD4(+) T cells. Our study provides direct ex vivo evidence to show that a small percent of CD4(+) T cells that have no previous experience of antigenic stimulation might produce IL-4 to initiate T(h)2 response.
Aberrancies in T-cell polarization including expression of chemokine receptors have been reported in human leucocyte antigen (HLA) class II associated autoimmune diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis. We asked whether these aberrancies are present at birth in newborn infants carrying the HLA risk haplotypes for T1D. Sixty-seven cord blood (CB) samples from infants were screened for T1D-associated HLA risk genotypes (HLA-DR4-DQ8 and/or DR3-DQ2 without protective alleles). CB lymphocytes were stimulated with phytohaemagglutinin in type 1 (interleukin (IL)-12, anti-IL4) or type 2 (IL-4, anti-IL12) cytokine environment for 6 days. The expression of chemokine and cytokine receptors on T cells was determined by flow cytometry, secretion of cytokines was analysed with enzyme-linked immunosorbent assay, and transcription factors were analysed using real-time reverse transcriptase-polymerase chain reaction. After culture of CB lymphocytes in type 2 cytokine environment newborn infants carrying DR4-DQ8 haplotype (n=18) showed reduced percentage of CD4 T cells expressing CCR4 (P=0 x 009) and the level of CCR4 mRNA was decreased (P=0 x 008). In addition, lower secretion of IL-13 and expression of GATA-3 in CB lymphocytes cultured in type 2 cytokine environment were found in the infants with DR4-DQ8 haplotype (P=0 x 020 and P=0 x 004, respectively) in comparison to newborn infants without DR4-DQ8 and DR3-DQ2 haplotypes (n=37). Poor in vitro induction of type 2 immune responses in newborn infants with DR4-DQ8 haplotype suggests that the HLA genotype associated with risk of autoimmunity may affect the T cell polarization already at birth, which in turn may contribute to the risk for autoimmunity later in life.
Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.
GATA-3 and T-box expressed in T cells (T-bet) play central roles in Th-cell development and function. Consistently, studies in mice document their selective expression in Th1 and Th2 cells, respectively. In contrast, it is not clear whether these genes are regulated in human Th cells. Here we show that T-bet expression is polarized to a comparable degree in human and mouse Th-cell cultures, while only mouse GATA3 is subject to substantial regulation. This did not reflect differential skewing efficiency in human versus mouse cultures, as these contained similar frequencies of IFN-gamma- and IL-4-producing cells. However, GATA-3 was expressed at significantly higher levels in human IL-4-producing cells enriched via capture with monoclonal antibodies (mAbs) against the PGD(2) receptor, CRTH2, the best selective Th2-cell surface marker to date. Along with increased IL-4 and GATA-3, CRTH2(+) Th cells isolated from Th2-skewed cultures or the circulating memory pool exhibited markedly decreased IFN-gamma and T-bet expression. Thus, the human GATA-3 gene is not regulated in response to polarizing signals that are sufficient to direct Th2-specific expression in mouse cells. This postulates the involvement of an additional level of complexity in the regulation of human GATA-3 expression and stresses the existence of nontrivial differences in the regulation of human versus mouse T-cell function.
The hypoparathyroidism-deafness-renal (HDR) dysplasia syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. We investigated 21 HDR probands and 14 patients with isolated hypoparathyroidism for GATA3 abnormalities. Thirteen different heterozygous germline mutations were identified in patients with HDR. These consisted of three nonsense mutations, six frameshifting deletions, two frameshifting insertions, one missense (Leu348Arg) mutation and one acceptor splice site mutation. The splice site mutation was demonstrated to cause a pre-mRNA processing abnormality leading to the use of an alternative acceptor site 8 bp downstream of the normal site, resulting in a frameshift and prematurely terminated protein. Electrophoretic mobility shift assays (EMSAs) revealed three classes of GATA3 mutations: those that lead to a loss of DNA binding which represent over 90% of all mutations, and involved a loss of the carboxy-terminal zinc finger; those that resulted in a reduced DNA-binding affinity; and those (e.g. Leu348Arg) that did not alter DNA binding or the affinity but likely altered the conformational change that occurs during binding in the DNA major groove as predicted by a three-dimensional modeling. These results elucidate further the molecular mechanisms underlying the altered functions of mutants of this zinc finger transcription factor and their role in causing this developmental anomaly. No mutations were identified in patients with isolated hypoparathyroidism, thereby indicating that GATA3 abnormalities are more likely to result in two or more of the phenotypic features of the HDR syndrome and not in one, such as isolated hypoparathyroidism.
The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer. To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (beta3-integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.
Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery.
We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4- NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed.
Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4- NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines.
D-pinitol has been demonstrated to exert insulin-like and anti-inflammatory activities. However, its anti-allergic effect in the Th1/Th2 immune response is poorly understood. Recently, it was shown that T-bet and GATA-3 are master Th1 and Th2 regulatory transcription factors. In this study, we have attempted to determine whether D-pinitol regulates Th1/Th2 cytokine production, T-bet and GATA-3 gene expression in OVA-induced asthma model mice. We also examined to ascertain whether D-pinitol could influence eosinophil peroxidase (EPO) activity. After being sensitized and challenged with ovalbumin (OVA) showed typical asthmatic reactions. These reactions included an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid, an increase in inflammatory cell infiltration into the lung tissue around blood vessels and airways, airway luminal narrowing, and the development of airway hyper-responsiveness (AHR). The administration of D-pinitol before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that D-pinitol plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of D-pinitol in terms of its effects in a murine model of asthma, and also broaden current perspectives in our understanding of the immunopharmacological functions of D-pinitol.
The differentiation of mouse naïve CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling at the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. However, little is known about how chromatin remodeling of these Th2 cytokine gene loci occurs in human Th2 cells. We herein established an in vitro culture system in which both Th1 and Th2 cells are efficiently differentiated from human peripheral blood naïve CD4 T cells. This system allowed us to investigate the chromatin status at the Th2 cytokine gene loci and the IFNgamma locus in human Th2 and Th1 cells, respectively. In typical individuals, the chromatin remodeling indicated by the induction of hyper-acetylation of histone H3 lysine 9 and hyper-methylation of histone H3 lysine 4 was induced at the whole Th2 cytokine gene loci in developing Th2 cells. We more precisely assessed the methylation status of histone H3 lysine 4 at the Th2 cytokine gene loci (IL-5 exon 3, IL-5 promoter, IL-5/RAD50 intergenic region, RAD50 promoter, CGRE, CNS1, IL-13 promoter, IL-4 promoter, and VA enhancer regions) and the IFNgamma locus in developing Th1 and Th2 cells prepared from 20 healthy volunteers. Th2-cell specific chromatin remodeling was induced at most of the Th2 cytokine gene loci. In parallel with the induction of chromatin remodeling, GATA3 mRNA was preferentially expressed in developing Th2 cells, whereas T-bet, HLX and ROG mRNA was selectively expressed in developing Th1 cells.
The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.
Hodgkin's lymphoma (HL) is characterised by an ineffective immune response that is predominantly mediated by CD4+ T-cells.
To analyse the expression of the key regulatory T-cell transcription factors (TFs) in the T-cells of HL involved tissues in order to assess the nature of the T(H) immune response in HL.
By immunohistochemistry, GATA3 was strongly and T-bet exclusively expressed in a subset of interfollicular lymphocytes in the reactive lymphoid tissues. In classical HL (CHL), which is generally located in the interfollicular zones, a predominance of T-bet+ T-cells and lesser amounts of GATA3+ and c-Maf+ T-cells was found, concordant with the pattern of the normal interfollicular compartment. In reactive lymphoid tissues, c-Maf was observed mostly in T-lymphocytes within the germinal centres (GCs). Nodular lymphocyte predominance type of Hodgkin's lymphoma (NLPHL) and progressively transformed germinal centres cases, showed a majority of c-Maf+ T-cells, consistent with the pattern in normal GCs. NLPHL cases uniformly showed c-Maf+/CD57+ T-cell rosettes around the neoplastic cells; these rosettes were absent in "paragranuloma-type" T-cell/histiocyte rich B-cell lymphoma.
T-cell TF expression profiles of the reactive T-cells in both subtypes of HL are in accordance with the expression profile observed in the distinct lymphoid compartments.
Sensory receptors in the vestibular organs of birds can regenerate after ototoxic injury. Notably, this regenerative process leads to the restoration of the correct patterning of hair cell phenotype and afferent innervation within the repaired sensory epithelium. The molecular signals that specify cell phenotype and regulate neuronal guidance during sensory regeneration are not known, but they are likely to be similar to the signals that direct these processes during embryonic development. The present study examined the recovery of hair cell phenotype during regeneration in the avian utricle, a vestibular organ that detects linear acceleration and head orientation. First, we show that Type I hair cells in the avian vestibular maculae are immunoreactive for the extracellular matrix molecule tenascin and that treatment with the ototoxic antibiotic streptomycin results in a nearly complete elimination of tenascin immunoreactivity. Cells that express tenascin begin to recover after about 2 weeks and are then contacted by calyx terminals of vestibular neurons. In addition, our previous work had shown that the zinc finger transcription factor GATA3 is uniquely expressed within the striolar reversal zone of the utricle (Hawkins et al. [2003] Hum Mol Genet 12:1261-1272), and we show here that this regionalized expression of GATA3 is maintained after severe hair cell lesions and after transplantation of the sensory epithelium onto a chemically defined substrate. In contrast, the expression of three other supporting cell markers--alpha- and beta-tectorin and SCA--is reduced following ototoxic injury. These observations suggest that GATA3 expression may maintain positional information in the maculae during sensory regeneration.
The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. Here, we describe a conditional Gata3-/- mouse (K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates.
Serotonin is an important neurotransmitter with multiple functions in the whole central nervous system. Its synthesis, however, is restricted to a very limited number of cells in the brainstem raphe nuclei with a vast axonal network. These cells express markers of the serotonin lineage such as the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 2, the serotonin transporter, and the transcription factor Pet1. Pet1 together with Lmx1b, Nkx2.2, Mash1, Gata2, Gata3, and Phox2b form a transcriptional network, which specifies the differentiation of serotonergic neurons around embryonic day 11 in the mouse. These cells are generated in rhombomeres r1-r3 and r5-r7 caudal to the midbrain- hindbrain organizer under the control of the fibroblast growth factors 4 and 8 and sonic hedgehog (SHH) from precursors, which have produced motoneurons before. Because serotonin is a relevant pathophysiological factor in several neurological diseases such as bipolar disorder and depression tools to generate or maintain serotonergic neurons might be of therapeutic value. Such tools can be assessed in embryonic stem cells, which can be differentiated in vitro to produce serotonergic neurons. Culture systems for these cells including embryoid bodies based and monolayer differentiation have been established, which allows the generation of up to 50% serotonergic neurons in all neurons developed.
Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined.
We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E-deficient (ApoE-/-) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE-/- Ccr5-/- mice, ApoE-/- Ccr1-/- mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE-/- Ccr5-/- mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-gamma content were increased in ApoE-/- Ccr1-/- mice.
Genetic deletion of Ccr5 but not Ccr1 in ApoE-/- mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.
The GATA family of transcription factors plays fundamental roles in cell-fate specification. However, it is unclear if these genes are necessary for the maintenance of cellular differentiation after development. We identified GATA-3 as the most highly enriched transcription factor in the mammary epithelium of pubertal mice. GATA-3 was found in the luminal cells of mammary ducts and the body cells of terminal end buds (TEBs). Upon conditional deletion of GATA-3, mice exhibited severe defects in mammary development due to failure in TEB formation during puberty. After acute GATA-3 loss, adult mice exhibited undifferentiated luminal cell expansion with basement-membrane detachment, which led to caspase-mediated cell death in the long term. Further, FOXA1 was identified as a downstream target of GATA-3 in the mammary gland. This suggests that GATA-3 actively maintains luminal epithelial differentiation in the adult mammary gland, which raises important implications for the pathogenesis of breast cancer.
T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) are transcriptional factors that play a crucial role in Th1 and Th2 development. We investigated the immunomodulatory roles of T-bet and GATA-3 and Th1/Th2 related cytokines in the pathogenesis of systemic lupus erythematosus (SLE) and their association with disease activity.
Gene expressions of T-bet, GATA-3, interferon-gamma (IFN-gamma), and interleukin 4 (IL-4) in peripheral blood mononuclear cells, and plasma concentrations of the Th1/Th2 cytokines IFN-gamma, IL-18, and IL-4, were assayed in 80 patients with SLE and 40 sex and age matched healthy subjects by real-time quantitative polymerase chain reaction and ELISA.
The mRNA levels of T-bet and IFN-gamma and the relative expression levels of T-bet/GATA-3 and IFN-gamma/IL-4 were significantly higher, in contrast to the lower expressions of GATA-3 and IL-4, in SLE patients than controls (all p < 0.05). In all SLE patients, there were significant correlations in mRNA expression of T-bet with IFN-gamma (r = 0.590, p < 0.0001), and of GATA-3 with IL-4 (r = 0.245, p = 0.029). The relative expressions of T-bet/GATA-3 and IFN-gamma/IL-4 correlated with lupus disease activity (r = 0.229, p = 0.042; r = 0.231, p = 0.040, respectively). Plasma IL-18 concentration was increased significantly in all SLE patients (p < 0.05). The elevated plasma Th1/Th2 cytokine ratio IL-18/IL-4 correlated positively with disease activity in all SLE patients (r = 0.250, p = 0.025).
There is an association between expression of Th1/Th2 transcription factors and cytokines in SLE. The elevated gene expressions of Th1/Th2 transcription factors and cytokines should provide a useful tool for assessing the functional status of T-helper lymphocytes in SLE disease development.
Skin lies at the interface between the complex physiology of the body and the external environment. This essential epidermal barrier, composed of cornified proteins encased in lipids, prevents both water loss and entry of infectious or toxic substances. We uncover that the transcription factor GATA-3 is required to establish the epidermal barrier and survive in the ex utero environment. Analysis of Gata-3 mutant transcriptional profiles at three critical developmental stages identifies a specific defect in lipid biosynthesis and a delay in differentiation. Genomic analysis identifies highly conserved GATA-3 binding sites bound in vivo by GATA-3 in the first intron of the lipid acyltransferase gene AGPAT5. Skin from both Gata-3-/- and previously characterized barrier-deficient Kruppel-like factor 4-/- newborns up-regulate antimicrobial peptides, effectors of innate immunity. Comparison of these animal models illustrates how impairment of the skin barrier by two genetically distinct mechanisms leads to innate immune responses, as observed in the common human skin disorders psoriasis and atopic dermatitis.
The Polycomb group (PcG) gene products regulate the maintenance of the homeobox gene expression in Drosophila and vertebrates and also the cell cycle progression in thymocytes and Th2 cell differentiation in mature T cells. We herein studied the role of PcG gene bmi-1 product in Th1/Th2 cell differentiation and found that Bmi-1 facilitates Th2 cell differentiation in a Ring finger-dependent manner. Biochemical studies indicate that Bmi-1 interacts with GATA3 in T cells, which is dependent on the Ring finger of Bmi-1. The overexpression of Bmi-1 resulted in a decreased ubiquitination and an increased protein stability of GATA3. In bmi-1-deficient Th cells, the levels of Th2 cell differentiation decreased as the degradation and ubiquitination on GATA3 increased. Therefore, Bmi-1 plays a crucial role in the control of Th2 cell differentiation in a Ring finger-dependent manner by regulating GATA3 protein stability.
Protein kinase C theta (PKCtheta) is essential for T cell activation, as it is required for the activation of NF-kappaB and expression of IL-2. PKCtheta has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCtheta in the regulation of T helper cells, we used PKCtheta-deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCtheta produced significantly less TNF-alpha and IL-2. The expression of Th2 cytokines, including IL-4, IL-5, IL-10, IL-13 and IL-24 was significantly reduced in PKCtheta-deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCtheta-deficient T cells. Overexpression of GATA3 by retroviral infection in PKCtheta-deficient T cells resulted in increased expansion of IL-4-producing T cells and higher IL-4 production than that of wild type Th2 cells. IL-5, IL-10, IL-13 and IL-24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCtheta regulates Th2 cytokine expression via GATA3.
The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.
The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKalpha, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKalpha and Ets-1, respectively. We also found that DeltaNp63 upregulates IKKalpha indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKalpha in epithelial development.
A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome.
We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section.
The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149).
With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.
Among neurogenic factors, the neuropeptides have an important regulatory influence on immune system activity and may lead to allergic sensitization.
The aim of our study was to investigate the relationship of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) on modulation of Th1/Th2 balance and allergic sensitization in children.
Within the LISAplus (Life style-Immune system-Allergy) study, blood samples of 321 six-year-old children were analysed for concentration of neuropeptides, Th1 and Th2 cytokines, transcription factors for T cell regulation and suppressors of cytokine signalling. In addition, samples were screened for specific IgE against inhalant and food allergens.
Children with high SOM values showed a Th2 polarization and a reduced expression of FOXP3, the marker for regulatory T cells. High (VIP) levels correlated inversely with the expression of T cell transcription factors (Tbet and SOCS3). In contrast, elevated levels of SP were associated with reduced GATA3 and SOCS3 expression and with increased IFN-gamma concentrations. Allergic sensitization was more prevalent in children with higher SOM and VIP concentrations but not associated with SP levels.
Our data reveal an association between neuropeptides and modulatory effects on immune cells in vivo, especially on Th1/Th2 balance with a correlation to allergic sensitization in children. We suggest that elevated SOM and VIP concentrations and the inducing factors should be considered as allergy risk factors.
Although invariant NKT (iNKT) cells participate in many aspects of immune responses, the molecular mechanisms regulating their development, maturation, and activation are still poorly understood. GATA-3 is a T cell-specific transcription factor that is also expressed in iNKT cells. The critical role of GATA-3 in conventional alphabeta T cells has been well documented, but whether GATA-3 also regulates the development and function of iNKT cells is unknown. In the present study, we report that deficiency of GATA-3 results in cell-intrinsic defects in the thymic development and peripheral maturation of murine iNKT cells. In addition, GATA-3 is also required for survival, activation, and effector functions of this unique population of T cells. Our data also reveal a previously unidentified peripheral maturation step that is GATA-3 dependent.
Recent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells.
Promoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays.
We confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas.
Our study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.
Selectin-dependent cell adhesion mediates inflammatory extravasation and routine homing of lymphocytes. Most resting peripheral T lymphocytes lack expression of sialyl Lewis X, the carbohydrate ligand for selectins, and are induced to strongly express it upon activation. T helper 1 (Th1) cells are known to more preferentially express sialyl Lewis X as compared with T helper 2 (Th2) cells upon activation. The molecular basis for this preferential expression, however, has not been elucidated to date. Here we show that the gene for fucosyltransferase VII (FUT7), the rate-limiting enzyme for sialyl Lewis X synthesis, is a unique example of the human genes with binding sites for both GATA-3 and T-bet, two opposing factors for Th1 and Th2 development, and is regulated transcriptionally by a balance of the two interacting transcription factors. T-bet promotes and GATA-3 represses FUT7 transcription. Our results indicated that T-bet interferes with the binding of GATA-3 to its target DNA, and also that GATA-3 significantly interferes with the binding of T-bet to the FUT7 promoter. T-bet has a binding ability to GATA-3, CBP/P300, and Sp1 to form a transcription factor complex, and GATA-3 regulates FUT7 transcription by phosphorylation-dependently recruiting histone deacetylase (HDAC)-3/HDAC-5 and by competing with CBP/P300 in binding to the N terminus of T-bet. Suppression of GATA-3 activity by dominant-negative GATA-3 or repressor of GATA (ROG) was necessary to attain a maximum expression of FUT7 and sialyl Lewis X in human T lymphoid cells. These results indicate that the GATA-3/T-bet transcription factor complex regulates the cell-lineage-specific expression of the lymphocyte homing receptors.
To investigate the expression of the T cell-specific transcription factors T-bet/GATA-3 in recurrent aphthous ulcerations (RAU).
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from RAU patients and healthy controls. Expressions of T-bet and GATA-3 mRNA and protein in the PBMCs were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. The concentrations of gamma-interferon (gamma-IFN) and interleukin-4 (IL-4) in the serum were determined by enzyme-linked immunosorbent assay (ELISA).
The expressions of T-bet mRNA were 0.73 +/- 0.14 in the control group and 0.12 +/- 0.04 in the recurrent aphthous ulcerations group, the levels of T-bet protein expressions were 0.73 +/- 0.18 in the control group and 0.24 +/- 0.09 in the recurrent aphthous ulcerations group, there was a significant difference between the two groups (P < 0.01). The levels of GATA-3 mRNA expressions were 0.89 +/- 0.11 in the control group and 1.30 +/- 0.13 in the recurrent aphthous ulcerations group; GATA-3 protein expressions were 1.09 +/- 0.16 in the control group and 1.80 +/- 0.16 in the recurrent aphthous ulcerations group, there was a significant difference between the two groups (P < 0.01). In the control group, the concentration of gamma-interferon in the serum was (22.15 +/- 1.52) ng/L, which was significantly elevated compared with that in the recurrent aphthous ulcerations group (16.32 +/- 0.22) ng/L (P < 0.01), in the control group, the concentration of IL-4 in the serum was (25.58 +/- 2.59) ng/L, which was significantly lower than that of the recurrent aphthous ulcerations group (43.60 +/- 3.15) ng/L (P < 0.01). The ratio of gamma-IFN and IL-4 was positively correlated with the ratio of T-bet and GATA-3 mRNA and protein expression (r = 0.96, 0.94, P < 0.01).
Imbalance of transcription factors T-bet and GATA-3 may be one of the key factors in immune dysregulation of recurrent aphthous ulcerations.
SATB1 (special AT-rich sequence binding protein 1) organizes cell type-specific nuclear architecture by anchoring specialized DNA sequences and recruiting chromatin remodeling factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4 and Il13, located in the 200-kb T-helper 2 (T(H)2) cytokine locus on mouse chromosome 11. We show that on T(H)2 cell activation, SATB1 expression is rapidly induced to form a unique transcriptionally active chromatin structure at the cytokine locus. In this structure, chromatin is folded into numerous small loops, all anchored to SATB1 at their base. In addition, histone H3 is acetylated at Lys9 and Lys14, and the T(H)2-specific factors GATA3, STAT6 and c-Maf, the chromatin-remodeling enzyme Brg1 and RNA polymerase II are all bound across the 200-kb region. Before activation, the T(H)2 cytokine locus is already associated with GATA3 and STAT6, showing some looping, but these are insufficient to induce cytokine gene expression. Using RNA interference, we show that on cell activation, SATB1 is required not only for compacting chromatin into dense loops at the 200-kb cytokine locus but also for inducing Il4, Il5, Il13 and c-Maf expression. Thus, SATB1 is a necessary determinant for the hitherto unidentified higher-order, transcriptionally active chromatin structure that forms on T(H)2 cell activation.
A zinc finger transcription factor, GATA3, plays an essential role in the development of T cells and the functional differentiation into type 2 Th cells. Two transactivation domains and two zinc finger regions are known to be important for the GATA3 function, whereas the role for other regions remains unclear. In this study we demonstrated that a conserved YxKxHxxxRP motif (aa 345-354) adjacent to the C-terminal zinc finger domain of GATA3 plays a critical in its DNA binding and functions, including transcriptional activity, the ability to induce chromatin remodeling of the Th2 cytokine gene loci, and Th2 cell differentiation. A single point mutation of the key amino acid (Y, K, H, R, and P) in the motif abrogated GATA3 functions. A computer simulation analysis based on the solution structure of the chicken GATA1/DNA complex supported the importance of this motif in GATA3 DNA binding. Thus, we identified a novel conserved YxKxHxxxRP motif adjacent to the C-terminal zinc finger domain of GATA3 that is indispensable for GATA3 DNA binding and functions.
Human GATA3 haploinsufficiency leads to HDR (hypoparathyroidism, deafness and renal dysplasia) syndrome, demonstrating that the development of a specific subset of organs in which this transcription factor is expressed is exquisitely sensitive to gene dosage. We previously showed that murine GATA-3 is essential for definitive kidney development, and that a large YAC transgene faithfully recapitulated GATA-3 expression in the urogenital system. Here we describe the localization and activity of a kidney enhancer (KE) located 113 kbp 5' to the Gata3 structural gene. When the KE was employed to direct renal system-specific GATA-3 transcription, the extent of cell autonomous kidney rescue in Gata3-deficient mice correlated with graded allelic expression of transgenic GATA-3. These data demonstrate that a single distant, tissue-specific enhancer can direct GATA-3 gene expression to confer all embryonic patterning information that is required for successful execution of metanephrogenesis, and that the dosage of GATA-3 required has a threshold between 50% and 70% of diploid activity.
Effects of cannabinoids (CBs) are mediated by two types of receptors, CB1 and CB2. In this report, we investigated whether CBs regulate gene expression of their cognate receptors in T cells and studied underlying mechanisms in CD4+ Jurkat T cells. Transcription of the CB1 gene was strongly induced in response to Delta9-tetrahydrocannabinol (THC), whereas the CB2 gene was not regulated. The induction of CB1 gene expression is mediated by CB2 receptors only, as demonstrated by using the CB1 and CB2 agonists R(+)-methanandamide and JWH 015, respectively, and combinations of THC plus CB1- and CB2-specific antagonists. After activation of CB2 receptors, the transcription factor STAT5 is phosphorylated. STAT5 then transactivates IL-4. Induction of IL-4 mRNA as well as IL-4 protein release from the cells are necessary for the following induction of the CB1 gene. This was demonstrated by using decoy oligonucleotides against STAT5, which blocked IL-4 and CB1 mRNA induction, and by using the IL-4 receptor antagonist IL-4 [R121D,Y124D], which blocked the up-regulation of CB1 gene transcription. Transactivation of the CB1 gene in response to IL-4 is then mediated by the transcription factor STAT6, as shown by using decoy oligonucleotides against STAT6. An increase in CB1-mediated phosphorylation of MAPK in cells prestimulated with CB2-specific agonists suggests up-regulation of functional CB1 receptor proteins. In summary, up-regulation of CB1 in T lymphocytes in response to CBs themselves may facilitate or enhance the various immunomodulatory effects related to CBs.
Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.
We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPdelta, GATA domain-binding protein 3 (GATA3), C/EBPbeta, peroxisome proliferator-activated receptor (PPAR) gamma2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor alpha, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARgamma coactivator 1alpha, PPARgamma coactivator 1beta, and PPARdelta] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.
Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 +/- 1.9 vs. 7.5 +/- 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 +/- 0.26 vs. 0.78 +/- 0.17 microm; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARgamma2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPbeta only tended to be lower (p = 0.07).
This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.
In breast cancer, the determination of estrogen receptor (ER) expression is crucial for the decision on therapeutic strategies. Current ER expression analysis is based on immunohistochemical (IHC) staining of ER on formalin fixed tissue sections. However, low levels of ER expression frequently escape detection because of varying sensitivities of routine histopathological laboratories. Moreover, in estimating ER by IHC the receptor protein only is tested instead of the complex underlying ER pathway, which reflects its biological activity. To overcome this limitation, we have used the microarray technology to study 56 samples of invasive ductal carcinoma. We infer a robust and reliable signature of 10 genes, which is associated with ER expression and presumably therapeutically relevant biological processes. In a meta-analysis, the signature was tested on 3 further independent microarray gene expression data sets, covering different laboratories, array platforms, and clinics. The classification based on the signature showed a very low misclassification rate. In summary, the expression of few genes is sufficient to determine ER status. Future decisions on antiestrogen based therapy in breast cancer could be based on this signature rather than on immunostaining alone.
Multiple sclerosis (MS) is an autoimmune disease, showing a great degree of variance in temporal disease activity. We have recently demonstrated that peripheral blood NK cells biased for secreting IL-5 (NK2 bias) are associated with the remission state of MS. In this study, we report that MS patients in remission differentially express CD11c on NK cell surface (operationally defined as CD11chigh or CD11clow). When we compared CD11chigh or CD11clow patients, the expression of IL-5 and GATA-3 in NK cells supposed to endow a disease-protective NK2 phenotype was observed in CD11clow but not in CD11chigh patients. In contrast, the CD11chigh group showed a higher expression of HLA-DR on NK cells. In vitro studies demonstrated that NK cell stimulatory cytokines such as IL-15 would up-regulate CD11c expression on NK cells. Given previous evidence showing an association between an increased level of proinflammatory cytokines and temporal disease activity in MS, we postulate that inflammatory signals may play a role in inducing the CD11chigh NK cell phenotype. Follow-up of a new cohort of patients showed that 6 of 10 CD11chigh MS patients developed a clinical relapse within 120 days after evaluation, whereas only 2 of 13 CD11clow developed exacerbated disease (p = 0.003). As such, a higher expression of CD11c on NK cells may reflect the temporal activity of MS as well as a loss of regulatory NK2 phenotype, which may allow us to use it as a potential biomarker to monitor the immunological status of MS patients.
Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.
The basic helix-loop-helix transcription factor Hand2, together with Ascl1, Phox2a, Phox2b and Gata2/Gata3, is induced by bone morphogenetic proteins in neural crest-derived precursor cells during sympathetic neuron generation. Hand2 overexpression experiments and the analysis of its function at the Dbh promotor implicated Hand2 in the control of noradrenergic gene expression. Using the zebrafish hand2 deletion mutant hands off, we have now investigated the physiological role of hand2 in the development of sympathetic ganglia. In hands off mutant embryos, sympathetic precursor cells aggregate to form normal sympathetic ganglion primordia characterized by the expression of phox2b, phox2a and the achaete-scute family member zash1a/ascl1. The expression of the noradrenergic marker genes th and dbh is strongly reduced, as well as the transcription factors gata2 and tfap2a (Ap-2alpha). By contrast, generic neuronal differentiation seems to be unaffected, as the expression of elavl3 (HuC) is not reduced in hands off sympathetic ganglia. These results demonstrate in vivo an essential and selective function of hand2 for the noradrenergic differentiation of sympathetic neurons, and implicates tfap2a and gata2 as downstream effectors.
The ecotropic viral integration site 1 (EVI1) gene was identified as a common locus of retroviral integration in myeloid tumors found in mice. EVI1 gene is highly conserved through evolution and human gene EVI1 on chromosome 3q26 encodes zinc fingers-containing transcription factor. EVI1 is expressed in nonhematopoietic tissues but not in normal blood or bone marrow. EVI1 was detected in hematopoietic cells in retrovirus-induced myeloid leukemias in mice and several reports documented EVI1 expression in human myelodysplastic syndromes and other hematologic malignancies without 3q26 translocations. EVI1 is abnormally expressed in human myeloid leukemias that are associated with the t(3;3)(q21;q26), t(3;21)(q26;q22), inv(3)(q21q26) and other chromosomal rearrangements. EVI1 is overexpressed in some ovarian cancers and human colon cancer cell lines and may play a role in the initiation and/or progression of solid tumors, as well as hematopoietic malignancies. EVI1 is a transcriptional repressor which inhibits transforming growth factor beta (TGFbeta) family signalling by binding signal transducers (Smad proteins) and recruiting transcriptional corepressors. TGFbeta is an important regulator of proliferation, differentiation, apoptosis and migration of cells. EVI1 inhibits TGFbeta-mediated apoptosis. Knockdown of EVI1 function by small interference RNA increases the sensitivity of malignant cells to TGFbeta-mediated or other inducer-mediated apoptosis. Overexpressed EVI-1 blocks granulocyte and erythroid differentiation and possess the ability of growth promotion in some types of cells. EVI1 functions in some cases as a transcriptional activator which stimulates for example GATA2 and GATA3 promoters. The study of EVI1 target genes will help to clear the mechanism by which EVI1 upregulates cell proliferation, impairs cell differentiation, and induces cell transformation.
Haploinsufficiency of the zinc finger transcription factor GATA3 causes the triad of hypoparathyroidism, deafness and renal dysplasia, known by its acronym HDR syndrome. The purpose of the current study was to describe in detail the auditory phenotype in human HDR patients and compare these to audiometrical and histological data previously described in a mouse model of this disease. Pure tone audiometry, speech audiometry, speech in noise, auditory brainstem responses and transiently evoked otoacoustic emissions were measured in 2 patients affected by HDR syndrome. Both patients were affected by a moderate-to-severe sensorineural hearing loss. Speech reception thresholds were shifted and speech recognition in noise was disturbed. No otoacoustic emissions could be generated in either patient. Auditory brainstem response interpeak intervals were normal. The human and murine audiological phenotypes seem to correspond well. Hearing loss in HDR syndrome is moderate to severe, seems to be slightly worse at the higher end of the frequency spectrum and may be progressive with age. The absence of otoacoustic emissions and the loss of frequency selectivity suggest an important role for outer hair cells in causing the hearing loss.
Gata3 is a positional candidate gene for allergic asthma. We determined allergen-induced GATA-3 mRNA and protein expression in asthma susceptible and resistant mice and generated Gata3 sequence data. Our data indicate that the Gata3 gene in isolation is not a causative agent of asthma susceptibility in our model.
Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice raised in nonair-controlled conventional circumstances spontaneously develop atopic dermatitis-like skin lesions; however, the underlying mechanisms remain unclear.
We wanted to identify the critical intracellular signaling molecules in T cells that induce atopic dermatitis-like skin legions in NC/Nga mice.
We examined the levels of signal transduction and cytokine production in T cells, particularly those in atopic dermatitis-like lesions induced by the topical injection of mite antigens in NC/Nga mice under specific pathogen-free conditions.
In NC/Nga mice maintained under specific pathogen-free conditions, the capability of T(H)1/T(H)2 and T cytotoxic 1/T cytotoxic 2 (Tc1/Tc2) cell differentiation increased significantly. T-cell antigen receptor-mediated activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase cascade and nuclear factor-kappaB (NF-kappaB) signaling were enhanced in NC/Nga T cells. The expression of T(H)2 cytokines (IL-4, IL-13, and IL-5) and that of GATA-binding protein 3 (GATA3), avian musculoaponeurotic fibrosarcoma (c-Maf), NF-kappaB, and activator protein 1 (AP1) selectively increased in draining lymph node T cells of NC/Nga mice. Moreover, the cell transfer of inhibitory NF-kappaB mutant-infected T(H)2 cells reduced ear thickness in the mite antigen-induced skin lesion of NC/Nga mice.
Hyperresponsive T(H)2 cells with an enhanced activity of NF-kappaB and AP1 play a crucial role in the pathogenesis of atopic dermatitis-like skin lesions in NC/Nga mice.
These results indicate potential therapeutic usefulness of developing selective inhibitors for NF-kappaB in the treatment of human atopic dermatitis.
Gata3 mutant mice expire of noradrenergic deficiency by embryonic day (E) 11 and can be rescued pharmacologically or, as shown here, by restoring Gata3 function specifically in sympathoadrenal (SA) lineages using the human DBH promoter to direct Gata3 transgenic expression. In Gata3-null embryos, there was significant impairment of SA differentiation and increased apoptosis in adrenal chromaffin cells and sympathetic neurons. Additionally, mRNA analyses of purified chromaffin cells from Gata3 mutants show that levels of Mash1, Hand2 and Phox2b (postulated upstream regulators of Gata3) as well as terminally differentiated SA lineage products (tyrosine hydroxylase, Th, and dopamine beta-hydroxylase, Dbh) are markedly altered. However, SA lineage-specific restoration of Gata3 function in the Gata3 mutant background rescues the expression phenotypes of the downstream, as well as the putative upstream genes. These data not only underscore the hypothesis that Gata3 is essential for the differentiation and survival of SA cells, but also suggest that their differentiation is controlled by mutually reinforcing feedback transcriptional interactions between Gata3, Mash1, Hand2 and Phox2b in the SA lineage.
The critical role of IL-5 (interleukin-5) in eosinophilic inflammation implicates it as a therapeutic target for allergic diseases. The aim of the present study was to elucidate the molecular basis for the involvement of reversible histone acetylation in IL-5 transcriptional regulation. We provide evidence that HDAC4 (histone deacetylase 4) and p300, a known HAT (histone acetyltransferase), reversibly controlled the activity of the IL-5 promoter in vivo and in vitro, with a concurrent alteration of histone H3 acetylation status at the promoter regions. The nucleo-cytoplasmic shuttling of HDAC4 was shown to play an important role in the suppressive function of HDAC4 in IL-5 gene expression. Point mutation and reporter ChIP (chromatin immunoprecipitation) studies determined that the four transcription factors binding on the IL-5 promoter, i.e. C/EBPbeta (CAAT/enhancer-binding protein beta), GATA3 (GATA binding protein 3), NFAT (nuclear factor of activated T cells) and YY1 (Yin and Yang 1), were essential for the recruitment of HDAC4. Consistent with these observations, HDAC4 was found to form protein complexes with GATA3 and YY1, and to co-exist in the nuclei with GATA3. We propose that the unique regulatory mechanism of IL-5 gene transcription involves the reversible histone modification catalysed by HDAC4 and p300, which are recruited by the transcription factors. The dynamic balance in IL-5 transcriptional regulation is achieved through interactions among HATs/HDACs, histones and transcription factors. These data contribute to understanding the molecular mechanisms of IL-5 regulation, which is crucial to the development of new therapeutic strategies for IL-5-related allergic diseases.
Familial hypoparathyroidism may be caused by mutations of several genes. The CaSR and GATA3 genes are the two candidates most commonly responsible for this condition.
We collected five unrelated families with familial hypoparathyroidism and examined their CaSR and GATA3 genes.
Blood samples from these five families and 50 ethnically matched unrelated controls were acquired. Biochemistry screening and formal audiogram were performed to evaluate the affected individuals. All the exons and exon-intron boundaries of the GATA3 and CaSR genes were sequenced.
We identified three novel mutations in the GATA3 gene responsible for familial hypoparathyroidism and deafness: 1) a frameshift deletion occurring in codon 160 (478delG) was hypothesized to disrupt dual zinc fingers as well as one transactivating domain; 2) a donor splice site mutation at exon 4/intron 4 boundary (IVS4 + 2 T to GCTTACTTCCC) was predicted to lead to truncated GATA3 proteins that lack both N- and C-terminal zinc-containing fingers; and 3) a missense mutation R353S was predicted to disrupt the helical turn and thus changed the angle between the C-terminal zinc finger and the adjacent C-terminal tail. Except for a previously described polymorphism, G990R, we did not find any genetic variants in the CaSR gene.
This is the first article presenting mutations of the GATA3 gene responsible for familial hypoparathyroidism and deafness in the Chinese population. Our results expand the spectrum of mutations and report the first splice donor site mutation of the GATA3 gene. In contrast, we do not find causal sequence variants of the CaSR gene from our collection of familial hypoparathyroidism.
Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.
The head mesoderm is the mesodermal tissue on either side of the brain, from forebrain to hindbrain levels, and gives rise to the genuine head muscles. Its relatedness to the more posterior paraxial mesoderm, the somites, which generate the muscles of the trunk, is conversely debated. To gain insight into the molecular setup of the head mesoderm, its similarity or dissimilarity to the somitic mesoderm, and the implications of its setup for the progress of muscle formation, we investigated the expression of markers (1) for mesoderm segmentation and boundary formation, (2) for regional specification and somitogenesis and (3) for the positive and negative control of myogenic differentiation. We show that the head mesoderm is molecularly distinct from somites. It is not segmented; even the boundary to the first somite is ill-defined. Importantly, the head mesoderm lacks the transcription factors driving muscle differentiation while genes suppressing differentiation and promoting cell proliferation are expressed. These factors show anteroposteriorly and dorsoventrally regionalised but overlapping expression. Notably, expression extends into the areas that actively contribute to the heart, overlapping with the expression of cardiac markers.
The zinc finger transcription factor GATA-3 is a master regulator of type 2 T-helper cell development. Interestingly, in GATA-3-/- mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA-3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA-3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA-3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at -61 to -39 bp. Surprisingly, this domain does not contain GATA-3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site-directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA-3. Furthermore, protein-protein interaction assays showed that GATA-3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA-3 may regulate TH gene transcription via a novel and distinct protein-protein interaction, and directly contributes to NA phenotype specification.
All 3 hematopoietic GATA transcription factors, GATA-1, GATA-2, and GATA-3, are acetylated, although the in vivo role of this modification remains unclear. We examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 does not impair its nuclear localization, steady-state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to all examined cellular target sites in vivo, including genes that are normally activated and repressed by GATA-1. Together, these results suggest that acetylation regulates chromatin occupancy of GATA-1. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo.
Naïve T helper (Th) cells differentiate in response to antigen stimulation into either Th1 or Th2 effector cells, which are characterized by the secretion of different set of cytokines. Th2 differentiation, which is critical for allergic airway disease, is triggered by signals of the T-cell receptor (TCR) and the cytokines generated during polarization, particularly IL-4. We determine here the potential role of the signaling adapter p62 in T-cell polarization. We report using p62-/- mice and cells that p62 acts downstream TCR activation, and is important for Th2 polarization and asthma, playing a significant role in the control of the sustained activation of NF-kappaB and late synthesis of GATA3 and IL-4 by participating in the activation of the IKK complex.
To study the mechanisms of hyporesponsiveness of HBV-specific CD4+ T cells by testing TH1 and TH2 commitment and regulatory T cells.
Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4+CD25+ cells.
Level of mRNAs for T-bet, IL-12R beta2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4+CD25highCTLA-4+ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4+ cells and CD4+CD25+ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAg-specific TH1 antibody compared with control antibody (P < 0.01, 0.34% +/- 0.12% vs 0.15% +/- 0.04%). Deletion of CD4+CD25+ T cells increased the amount of HBcAg-specific TH1 antibody when compared with lymphocytes reconstituted using regulatory T cells (P < 0.01, 0.03% +/- 0.02% vs 0.18% +/- 0.05%).
The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.
To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction.
Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR).
Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation.
bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.
Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.
The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte-derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate-labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon- gamma or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4(+) T cells and in autologous CD4(+) T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells.
The engagement of CD137 (4-1BB), an inducible T cell costimulatory receptor and member of the TNF receptor superfamily, by agonistic Abs can promote strong tumor and viral immunity mediated by CD8(+) T cells and stimulate IFN-gamma production. However, its role in Th2-mediated immune responses has not been well defined. To address this issue, we studied the function of CD137 engagement using an allergic airway disease model in which the mice were sensitized with inactivated Schistosoma mansoni eggs followed by S. mansoni egg Ag challenge directly in the airways and Th1/2 cytokine production was monitored. Interestingly, treatment of C57BL/6 mice with agonistic anti-CD137 (2A) during sensitization completely prevents allergic airway inflammation, as shown by a clear inhibition of T cell and eosinophil infiltration into the lung tissue and airways, accompanied by diminished Th2 cytokine production and reduced serum IgE levels, as well as a reduction of airway hyperresponsiveness. At various time points after immunization, restimulated splenocytes from 2A-treated mice displayed reduced proliferation and Th2 cytokine production. In accordance with this, agonistic Ab to CD137 can directly coinhibit Th2 responses in vitro although it costimulates Th1 responses. CD137-mediated suppression of Th2 response is independent of IFN-gamma and T regulatory cells. Our study has identified a novel pathway to inhibit Th2 responses in a CD137-dependent fashion.
Recent studies have demonstrated that Th2 cytokines, such as interleukin-4 and interleukin-13, enhance fibrotic processes by activating fibroblast proliferation and collagen production, whereas interferon-gamma, a Th1 cytokine, inhibits these processes. Th1 and Th2 cells both differentiate from common T precursor cells, with transcription factor GATA-3 a key regulator of Th2 differentiation. In the present study, therefore, we examined the effects of GATA-3 overexpression on the development of pulmonary fibrosis in a mouse model. Wild-type C57BL/6 mice and GATA-3-overexpressing (GATA-3-tg) mice of the same background were intratracheally treated with bleomycin. The survival rate after bleomycin was significantly decreased in GATA-3-tg mice compared with wild-type mice. The degree of pulmonary fibrosis was much greater in GATA-3-tg mice than in wild-type mice 28 days after bleomycin treatment. Lung interferon-gamma concentration was significantly decreased in GATA-3-tg mice compared with wild-type mice by 7 days after either saline or bleomycin treatment. The concentration of transforming growth factor-beta, a fibrogenic cytokine, was significantly higher in GATA-3-tg mice than in wild-type mice. Exogenous administration of interferon-gamma to GATA-3-tg mice improved the degree of pulmonary fibrosis and thus increased survival. These results indicate that overexpression of GATA-3 enhances the development of pulmonary fibrosis, possibly by reducing interferon-gamma levels in the lung.
Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.
The generalized T-cell activation characterizing HIV-1 and SIVmac infections in humans and macaques (MACs) is not found in the non-pathogenic SIVagm infection in African green monkeys (AGMs). We have previously shown that TGF-beta1, Foxp3 and IL-10 are induced very early after SIVagm infection. In SIVmac-infected MACs, plasma TGF-beta1 induction persists during primary infection 1. We raised the hypothesis that MACs are unable to respond to TGF-beta1 and thus cannot resorb virus-driven inflammation. We therefore compared the very early expression dynamics of pro- and anti-inflammatory markers as well as of factors involved in the TGF-beta1 signaling pathway in SIV-infected AGMs and MACs.
Levels of transcripts encoding for pro- and anti-inflammatory markers (tnf-alpha, ifn-gamma, il-10, t-bet, gata-3) as well as for TGF-beta1 signaling mediators (smad3, smad4, smad7) were followed by real time PCR in a prospective study enrolling 6 AGMs and 6 MACs.
During primary SIVmac infection, up-regulations of tnf-alpha, ifn-gamma and t-bet responses (days 1-16 p.i.) were stronger whereas il-10 response was delayed (4th week p.i.) compared to SIVagm infection. Up-regulation of smad7 (days 3-8 p.i.), a cellular mediator inhibiting the TGF-beta1 signaling cascade, characterized SIV-infected MACs. In AGMs, we found increases of gata-3 but not t-bet, a longer lasting up-regulation of smad4 (days 1-21 p.i), a mediator enhancing TGF-beta1 signaling, and no smad7 up-regulations.
Our data suggest that the inability to resorb virus-driven inflammation and activation during the pathogenic HIV-1/SIVmac infections is associated with an unresponsiveness to TGF-beta1.
Steatohepatitis enhances the severity of liver injury caused by acute inflammation. The purpose of this study was to test the hypothesis that fatty liver due to chronic choline-deficient diet exacerbates concanavalin A (ConA)-induced liver hepatitis, which is predominantly facilitated by T cells. Male C57BL/6 mice were fed either control choline-sufficient diet (CSD) or choline-deficient diet (CDD) for 6 weeks before ConA administration. Mice were sacrificed 3, 9, and 24 hours after ConA injection. Liver injury measured by aspartate aminotransferase (AST), alanine aminotransferase (ALT), pathology, and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was minimal in mice fed either diet before ConA exposure. However, ConA-induced liver injury was significantly greater in CDD-fed mice compared with control-fed mice. Liver cytokines were assessed by quantitative real-time polymerase chain reaction (PCR). The expression of T helper (Th) 1 cytokines tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and interferon gamma (IFN-gamma) were dramatically elevated after ConA in CDD-fed mice compared with control-fed mice. CDD also enhanced ConA-induced STAT4 activation, but not STAT6. Notably, regulators of T-cell differentiation were strongly shifted toward a predominant Th1 profile. T-bet, regulator of the Th1 response, was up-regulated in CDD-fed mice, whereas Th2 regulator GATA-3 was significantly suppressed in CDD-fed mice after ConA. Moreover, the expression of suppressor of cytokine signaling (SOCS)-1, SOCS-3, and repressor of GATA-3 (ROG) favored a predominant Th1 cytokine response in CDD-fed mice. In conclusion, these data support the hypothesis that hepatosteatosis caused by CDD is associated with more severe ConA-induced hepatitis due to a predominant shift toward Th1 response.
During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile.
Horses with recurrent airway obstruction (RAO) present many similarities with human asthmatics including airway inflammation, hyperresponsiveness, reversible obstruction, and increased NF-kappaB expression. Studies in experimental asthma models have shown that transcriptions factors such as activator protein-1 (AP-1), GATA-3, cyclic AMP response element binding protein (CREB) and CAAT/enhancer binding protein (C/EBP) may also play an important role in airway inflammation. The purpose of this study was to measure DNA binding activity of these transcription factors in the airways of horses with RAO and to compare it to pulmonary function and bronchoalveolar lavage fluid (BALF) cytology. Seven horses with RAO and six control animals were studied during a moldy hay challenge and after 2 months at pasture. Pulmonary function, BALF cytology and transcription factors' activities in bronchial brushings were measured during hay and pasture exposures. During moldy hay challenge, RAO-affected horses developed severe airway obstruction and inflammation and a significantly higher airway AP-1 binding activity than in controls. After 2 months on pasture, pulmonary function and airway AP-1 binding activity were not different between RAO and control horses. The DNA binding activity of CREB in airways of RAO-affected horses increased significantly after 2 months at pasture and became higher than in controls. A significant positive correlation was detected between AP-1 binding activity and indicators of airway obstruction and inflammation. Airway GATA-3, CEBP and CREB binding activities were negatively correlated with indices of airway obstruction. However, contrarily to CREB binding activity, GATA-3 and CEBP binding activities were not different between RAO and control horses and were unaffected by changes in environment. These data support the view that AP-1 and CREB play a role in modulating airway inflammation in horses with RAO.
Oestrogen (E) is essential for normal and cancer development in the breast, while anti-oestrogens have been shown to reduce the risk of the disease. However, little is known about the effect of E on gene expression in the normal human breast, particularly when the epithelium and stroma are intact. Previous expression profiles of the response to E have been performed on tumour cell lines, in the absence of stroma. We investigated gene expression in normal human breast tissue transplanted into 9-10-week-old female athymic nude (Balb/c nu/nu) mice. After 2 weeks, when epithelial proliferation is minimal, one-third of the mice were treated with 17beta-oestradiol (E2) to give human luteal-phase levels in the mouse, which we have previously shown to induce maximal epithelial cell proliferation. RNA was isolated from treated and untreated mice, labelled and hybridized to Affymetrix HG-U133A (human) GeneChips. Gene expression levels were generated using BioConductor implementations of the RMA and MAS5 algorithms. E2 treatment was found to represent the largest source of variation in gene expression and cross-species hybridization of mouse RNA from xenograft samples was demonstrated to be negligible. Known E2-responsive genes (such as TFF1 and AREG), and genes thought to be involved in breast cancer metastasis (including mammoglobin, KRT19 and AGR2), were upregulated in response to E treatment. Genes known to be co-expressed with E receptor alpha in breast cancer cell lines and tumours were both upregulated (XBP-1 and GREB1) and downregulated (RARRES1 and GATA3). In addition, genes that are normally expressed in the myoepithelium and extracellular matrix that maintain the tissue microenvironment were also differentially expressed. This suggests that the response to oestrogen in normal breast is highly dependent upon epithelial-stromal/myoepithelial interactions which maintain the tissue microenvironment during epithelial cell proliferation.
The essential features of persistent severe asthma include structural changes in the airway wall (remodelling). It is not known whether these are the sequelae of chronic inflammation or indeed its initiators. Several transcription factors have been implicated in the inflammatory process in asthma, including the glucocorticoid receptor (GR), NFkappaB, Activator Protein-1 (AP-1), Nuclear Factor of Activated T-cells (NF-AT), cyclic AMP Response Element Binding Protein and more recently, the CCAAT/Enhancer Binding Protein (C/EBP), Peroxisome Proliferator-activated Receptor (PPAR) and the bZIP transcription factor, Nrf2. Could a pathological de-regulation of one of these transcription factors explain the broad spectrum of asthma pathology and can their modulation lead to better symptom control? Although some of the transcription factors seem to be valid targets (NFkappaB, Nrf2 or STAT6) or tools (PPARgamma, -alpha and C/EBP-alpha) for new therapeutic approaches, since many transcription factors play a central role in tissue and organ homeostasis, a longterm general suppression or overexpression, would cause severe side effects in other organs. Cell type specific application of decoy or antisense oligonucleotides for NFkappaB, Nrf2 or STAT6, or specific agonists for PPARgamma and -alpha may help to control the inflammatory response in lung epithelial cells and infiltrated immune cells, but additional, unwanted, effects on other resident cells of the lung cannot be excluded and a beneficial effect over known anti-asthma drugs has first to be proven. In order to progress with such novel therapeutic strategies, the only option seems to be to link transcription factor inhibitors/activators to a cell type specific delivery system.
The Mixed-Lineage Leukemia (MLL) gene, a mammalian homolog of the Drosophila trithorax, is implicated in regulating the maintenance of Hox gene expression and hematopoiesis. The physiological functions of MLL in the immune system remain largely unknown. Although MLL(+/-) CD4 T cells differentiate normally into antigen-specific effector Th1/Th2 cells in vitro, the ability of memory Th2 cells to produce Th2 cytokines was selectively reduced. Furthermore, histone modifications at the Th2 cytokine gene loci were not properly maintained in MLL(+/-) memory Th2 cells. The reduced expression of MLL in memory Th2 cells resulted in decreased GATA3 expression accompanied with impaired GATA3 locus histone modifications. The direct association of MLL with the GATA3 locus and the Th2 cytokine gene loci was demonstrated. Memory Th2 cell-dependent allergic airway inflammation was decreased in MLL(+/-) Th2 cell-transferred mice. Thus, a crucial role for MLL in the maintenance of memory Th2 cell function is indicated.
Statins, best known for their lipid-lowering actions, also possess immunomodulatory properties. Recent studies have shown a Th2-biasing effect of statins, although the underlying mechanism has not been identified. In this study, we investigated whether simvastatin can exercise a Th2-promoting effect through modulation of function of dendritic cells (DCs) without direct interaction with CD4+ T cells. Exposure of DCs to simvastatin induced the differentiation of a distinct subset of DCs characterized by a high expression of B220. These simvastatin-conditioned DCs up-regulated GATA-3 expression and down-regulated T-bet expression in cocultured CD4+ T cells in the absence of additional simvastatin added to the coculture. The Th2-biased transcription factor profile induced by simvastatin-treated DCs also was accompanied by increased Th2 (IL-4, IL-5, and IL-13) and decreased Th1 (IFN-gamma) cytokine secretion from the T cells. The Th2-promoting effect of simvastatin was found to depend on the chitinase family member Ym1, known to be a lectin. Anti-Ym1 antibody abolished the Th2-promoting effect of simvastatin-treated DCs. Also, simvastatin was unable to augment Ym1 expression in DCs developed from STAT6-/- or IL-4R alpha-/- mice. Thus, modulation of Ym1 production by DCs identifies a previously undescribed mechanism of Th2 polarization by statin.
To explore the profiles of type 1 and type 2 T cells in chronic idiopathic thrombocytopenic purpura (ITP) patients.
Samples of peripheral blood were collected from 30 chronic ITP patients, 8 males and 22 females, aged 34, 20 being in the active stage, and 10 in the remission stage, and 20 healthy persons, 7 males and 13 females, aged 31. Peripheral blood mononuclear cells (PBMCs) were isolated, cultured, and activated with PMA/ionomycin. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-gamma and interleukin (IL)-4 in the PBMCs so as to identify the Th1 cells (IFN-gamma(+) IL-4(+) CD4(+) cells), Th2 cells (IFN-gamma(-) IL-4(+) CD4(+) cells), Tc1 cells (IFN-gamma+ IL-4(-) CD8(+) cells), and Tc2 cells (IFN-gamma(-) IL-4(+) CD8(+) cells). The ratios of Th2/Tc2, Th1/Th2, and Tc1/Tc2 were calculated. Samples of spleen tissue were collected from 8 patients with chronic ITP, 3 males and 5 females, aged 30, and 6 patients with hereditary spherocytosis (HS), 3 males and 3 females, aged 35, who underwent splenectomy. Splenocytes were isolated, cultured, and activated with PMA/ionomycin. Real-time PCR was used to detect the mRNA expression of T-bet and GATA-3 in the PBMCs and splenocytes.
The Th1/Th2 ratio of the patients in active stage was 25.62, significantly higher than those of the patients in remission stage (9.86) and the control (8.29, both P < 0.01), and the Tc1/Tc2 ratio of the patients in active stage was 30.23, significantly higher than those of the patients in remission stage (10.10) and the controls (12.58, both P < 0.01). The Th1/Th2 ratio of the splenocytes of the patients in active stage was 41.46, significantly higher than that of the controls (16.30, P < 0.01), and the Tc1/Tc2 ratio of the splenocytes of the active ITP patients was 35.80, not significantly higher than that of the controls (16.30, P = 0.082). The GATA-3 mRNA expression level of the PBMCs of the active ITP patients was one-fifth of that of the controls (P < 0.05) and the GATA-3 mRNA expression level of the splenocytes of the ITP patients was 0.34 of that of the HS patients (P < 0.05), however, there was no difference in the T-bet expression among the 3 groups. The T-bet/GATA-3 ratio of the PBMCs of the active ITP patients was 5.85 times that of the controls and the T-bet/GATA-3 ratio of the splenocytes of the active ITP patients was 2.68 times that of the controls (both P < 0.01).
ITP is a T1 cells (Th1 and Tc1) predominant disease. The T-bet/GATA-3 ratio may provide a surrogate marker of T1/T2 cytokine balance. Shifting the cytokine patterns from T1 to T2 may be a potential immunotherapy for ITP.
To identify the change of the mRNA and protein expression of T-bet and GATA-3 in lung tissues, and to investigate the association between the imbalanced T cell-specific transcription factors T-bet/GATA-3 and the airway inflammation in asthmatic rats.
Twenty-four male SD rats were randomly divided into a control group and an asthmatic group. Airway responsiveness was measured and the change of airway histology was observed. The concentrations of interleukin-4 (IL-4), IL-5, and interferon-gamma (IFN-gamma) in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of IL-4, IL-5, IFN-gamma, T-bet and GATA-3 in the lungs were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively.
The expiration resistance after injection of acetylcholine chloride (20, 40, 80, 160 microg/ml) in the asthmatic group was (6.26 +/- 0.85), (11.55 +/- 3.09), (28.74 +/- 5.94), (3,710.83 +/- 197.49) cm H2O.ml(-1).s(-1) respectively; and that in the control group was (1.51 +/- 0.18), (2.15 +/- 0.36), (6.08 +/- 1.06), (37.17 +/- 6.12) cm H2O.ml(-1).s(-1) respectively; the difference being significant between the two groups (all P < 0.01). In the asthmatic group, the numbers of eosinophils and lymphocytes, the thicknesses of WA/Pi and ASM/Pi were (26.0 +/- 1.6)/mm(2), (45.2 +/- 3.2)/mm(2), 12.0 +/- 1.4, 6.7 +/- 0.6, respectively; and those of the control group were (2.9 +/- 1.2)/mm(2), (8.8 +/- 1.8)/mm(2), 6.4 +/- 0.8, 2.7 +/- 0.5, respectively; all were significantly different between the two groups (all P < 0.01). In the asthmatic group, the concentrations of IL-4, IL-5, and IFN-gamma in BALF were (23.4 +/- 0.7) pg/ml, (24.8 +/- 0.5) pg/ml, (21.7 +/- 1.1) pg/ml, respectively, and those of the control group were (9.3 +/- 0.3) pg/ml, (12.5 +/- 0.3) pg/ml, (65.8 +/- 2.1) pg/ml, respectively; all were significantly different between the two groups (all P < 0.01). In the control group, the mRNA ratio of T-bet to GATA-3 (0.73 +/- 0.32) was significantly increased compared with the asthmatic group (0.06 +/- 0.09, P < 0.01). There was also a significant difference in the ratio of protein expression of T-bet to GATA-3 between the control group (0.75 +/- 0.25) and the asthmatic group (0.09 +/- 0.04, P < 0.01). The ratio of protein expression of T-bet and GATA-3 was correlated negatively with expiration resistance (r = -0.959, -0.919, -0.949, all P < 0.01), the numbers of eosinophils and lymphocytes in lung tissues (r = -0.832, -0.831, all P < 0.01), the thicknesses of WA/Pi and ASM/Pi (r = -0.837, -0.863, all P < 0.01) and the concentrations of IL-4, IL-5 in BALF (r = 0.921, 0.920, all P < 0.01), the mRNA of IL-4, IL-5 in lung tissues (r = -0.964, -0.931, all P < 0.01), but positively with the concentrations of IFN-gamma in BALF and the mRNA of IFN-gamma in lung tissues (r = -0.934, 0.983, all P < 0.01).
Imbalance of transcription factors T-bet and GATA-3, a reflection of the immune imbalance in asthma, may play a key role in the formation of airway inflammation in the disease.
Mast cells are able to produce a huge panel of mediators including the Th2-type cytokine IL-9, which is considered to be a key mediator for the pathogenesis of allergic asthma, but detailed information on the regulation of IL-9 transcription in mast cells has been scarce. Herein we provide evidence that the erythroid/myeloid transcription factor GATA-1, which is not expressed in Th2 cells, is a potent activator of IL-9 expression in murine bone marrow-derived mast cells (BMMC). Furthermore, in mast cells, but not in Th2 cells, production of IL-9 is sensitive to inhibition of p38 MAP kinase. As transactivation mediated by GATA-1 is also sensitive to inhibition of p38 MAP kinase, and GATA-1 is a target for p38 MAP kinase-mediated phosphorylation in vitro, we conclude that both signaling molecules represent a part of a mast cell-specific signaling network that regulates the expression of IL-9.
Imiquimod is an imidazoquinoline, which class of compounds are known to have antiviral and antitumoural properties. In recent studies, it was shown that imiquimod modulates the T helper cell type Th1/Th2 response by inducing the production of Th1 cytokines like IFN-gamma, and by inhibiting the Th2 cytokines like interleukin (IL)-4. Several investigators have shown that T-bet and GATA-3 are master Th1 and Th2 regulatory transcription factors. This study investigated whether imiquimod treatment inhibited airway inflammation by modulating transcription factors T-bet and GATA-3.
Thirty-six male SD rats were randomly divided into a control group, an asthmatic group, and an imiquimod group, which was exposed to an aerosol of 0.15% imiquimod. Twenty-four hours after the last ovalbumin (OVA) challenge, airway responsiveness was measured and changes in airway histology were observed. The concentrations of IL-4, IL-5 and IFN-gamma in bronchoalveolar lavage fluid (BALF) and serum were measured by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of IL-4, IL-5, IFN-gamma, T-bet and GATA-3 in lung and in CD4(+) T cells were determined by reverse transcription polymerase chain reaction (RT-PCR). The protein expressions of T-bet and GATA-3 were measured by Western blot.
It was demonstrated that imiquimod 1) attenuated OVA induced airway inflammation; 2) diminished the degree of airway hyperresponsiveness (AHR); 3) decreased the Th2 type cytokines and increased Th1 type cytokines mRNA and protein levels; 4) modulated the Th1/Th2 reaction by inhibiting GATA-3 production and increasing T-bet production.
Imiquimod treatment inhibits OVA induced airway inflammation by modulating key master switches GATA-3 and T-bet that result in committing T helper cells to a Th1 phenotype.
To explore whether local blockade of T-box expressed in T cells (T-bet) expression in the lungs could lead to airway inflammation.
Twenty-four rats were randomly divided into 4 groups: saline group, ovalbumin (OVA)-sensitized group, nonsense group, and the antisense group. The OVA-sensitized rats were sensitized and challenged with OVA, and the rats in the nonsense and antisense groups were subjected to an aerosol delivery of the nonsense and antisense oligonucleotides (AS-ODN) of T-bet (0.1%, w/v). The levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and IL-5 in the bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the mRNA and the protein expression of T-bet and GATA-3 genes were examined by in situ hybridization and Western blot analysis, respectively.
The expression of T-bet mRNA and protein in the lungs of the rats in the antisense group were inhibited effectively. The lungs of the rats in the antisense and OVA-sensitized groups showed eosinophil and lymphocyte inflammatory infiltration, and eosinophilia located predominantly around the airways. The number of GATA-3 mRNA-positive cells and the level of GATA-3 protein in the lungs of the rats in the antisense and the OVA-sensitized groups significantly increased. The level of IL-4 and IL-5 in the BALF in the antisense and OVA-sensitized groups were elevated, but the level of IFN-gamma decreased markedly.
Antisense ODN-induced local blockade of T-bet expression leads to airway inflammation with a selective alteration in patterns of cytokine expression and recruitment of eosinophil cells similar to that in the OVA-sensitized animals.
High-altitude climate therapy is a well-established therapeutic option, which improves clinical symptoms in asthma. However, little is known about the underlying immunological mechanisms. The study investigates the influence of high-altitude climate therapy on airway inflammation and cellular components of specific and unspecific immune response. Exhaled NO significantly decreased within 3 weeks of therapy in patients with allergic and intrinsic, moderate and severe asthma. Interleukin-10 (IL-10)-secreting peripheral blood mononuclear cells (PBMC) increased within 3 weeks of therapy in six of 11 patients, whereas transforming growth factor-beta(1)-secreting PBMC remained stable. Furthermore, monocyte activation, assessed by CD80 expression significantly decreased during therapy. The frequency of CRTH2-expressing T cells decreased, while regulatory T cells (T(reg)) remained stable. FOXP3 and GATA-3 mRNA expression in CD4(+) T cells did not change, while interferon-gamma and IL-13 mRNA expression decreased in eight of 10 patients. The current data demonstrate that high-altitude climate therapy reduces local airway inflammation. Furthermore, monocytes switch towards a tolerogenic phenotype under high-altitude climate therapy. The T(reg)/Th2 ratio increases; however, because of the absence of antigens/allergens, no de novo differentiation of Th2 nor T(reg) cells is observed. The high-altitude climate therapy therefore may form the immunological basis for the endogenous control of allergen-driven diseases.
Genetic analyses identified Ses1 as a significant quantitative trait locus influencing the carrier state of 129S6 mice following a sublethal challenge with Salmonella enterica serovar Enteritidis. Previous studies have determined that Slc11a1 was an excellent candidate gene for Ses1. Kinetics of infection in 129S6 mice and Slc11a1-deficient (129S6-Slc11a1(tm1Mcg)) mice demonstrated that the wild-type allele of Slc11a1 contributed to the S. enterica serovar Enteritidis carrier state as early as 7 days postinfection. Gene expression profiling demonstrated that 129S6 mice had a significant up-regulation of proinflammatory genes associated with macrophage activation at day 10 postinfection, followed by a gradual increase in immunoglobulin transcripts, whereas 129S6-Slc11a1(tm1Mcg) mice had higher levels of immunoglobulins earlier in the infection. Quantitative reverse transcription-PCR revealed an increase in Th1 cytokine (Ifng and Il12) and Th1-specific transcription factor Tbx21 expression during infection in both the 129S6 and 129S6-Slc11a1(tm1Mcg) strains. However, the expression of Gata3, a transcription factor involved in Th2 polarization, Cd28, and Il4 was markedly increased in Slc11a1-deficient mice during infection, suggesting a predominant Th2 phenotype in 129S6-Slc11a1(tm1Mcg) animals following S. enterica serovar Enteritidis infection. A strong immunoglobulin G2a response, reflecting Th1 activity, was observed only in 129S6 mice. All together, these results are consistent with an impact of Slc11a1 on Th cell differentiation during chronic S. enterica serovar Enteritidis infection. The presence of a Th2 bias in Slc11a1-deficient mice is associated with improved bacterial clearance.
Airway remodeling is an important feature of chronic asthma that causes irreversible airflow obstruction. Although asthma is considered to be a Th2 disease, the role of T-bet and GATA-3, the key transcription factors for differentiation toward Th1 and Th2 cells, in the pathogenesis of airway remodeling is poorly understood.
We therefore examined the effects of GATA-3 or T-bet induction of Th1/Th2 bias on the development of airway remodeling in mice.
The development of airway remodeling after repeated allergen challenges was analyzed using transgenic mice overexpressing either GATA-3 or T-bet.
The degrees of subepithelial fibrosis and airway smooth muscle hyperplasia after repeated allergen exposure were significantly enhanced in mice overexpressing GATA-3, compared with wild-type mice. Allergen-induced goblet cell hyperplasia and mucus hypersecretion were significantly lower in mice overexpressing T-bet than in wild-type mice. Eosinophilic airway inflammation increased in mice overexpressing GATA-3, but decreased in mice overexpressing T-bet after repeated allergen exposure. Cytokine analysis revealed that the Th1/Th2 cytokine balance shifted to Th2 in lung homogenates and lung T cells of mice overexpressing GATA-3, whereas this balance shifted to Th1 in those of mice overexpressing T-bet after allergen exposure. Lung transforming growth factor-beta and eotaxin levels were associated with the degree of subepithelial fibrosis and eosinophilic airway inflammation, respectively.
Overall, the results indicate that development of airway remodeling is regulated by the lung Th1/Th2 bias induced by GATA-3 and T-bet.
Frequent users of smokeless tobacco (ST) have an increased risk for developing oral cancer. Nicotine and its derivatives may contribute to tumorigenesis through stimulation of nicotinic acetylcholine receptors (nAChRs) in target cells. Emerging evidence indicates that nAChRs can be stimulated by the nicotine-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) that can induce oral cavity tumors in laboratory animals. This study was designed to elucidate the receptor-mediated mechanisms of the initiation and progression of NNK-, and NNN-induced oral cancers. We used Het-1A cells that were found to express alpha3, alpha5, alpha7, alpha9, beta2 and beta4 nAChR subunits. Both NNK and NNN competed with nicotinic radioligands for binding to Het-1A cells. NNK showed a higher than NNN affinity to the [3H]nicotine-labeled binding sites, and NNN-to the [3H]epibatidine-sensitive nAChRs. NNK and NNN increased proliferative potential of Het-1A cells and produced an anti-apoptotic effect, which was alleviated by antagonists. alpha-Bungarotoxin was most effective against NNK and mecamylamine against NNN. Treatment of Het-1A cells with either NNK or NNN led to acquisition of capability of anchorage independent growth and ability to produce tumors in nude mice, both of which can be by inhibited by antagonists. To elucidate the signaling mechanisms, we studied transcription of the genes encoding the cell cycle, apoptosis and signal transduction regulators at both the mRNA and protein levels. The Het-1A cells stimulated with nitrosamines showed multifold increases of the mRNA transcripts encoding PCNA and Bcl-2, and upregulated expression of the transcription factors GATA3, nuclear factor-kappaB, and STAT-1. The STAT-1 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results establish the role of specific nAChR subtypes in tobacco-related carcinogenesis and open a novel avenue for oral cancer chemoprevention.
A receptive endometrial environment requires adequate immunological tolerance to protect the implanting embryo from maternal immune rejection. Studies in mice implicate CD4+CD25+ T-regulatory (Treg) cells as essential mediators of immune tolerance in pregnancy. The aim of this study was to evaluate the link between Treg cells and fertility in women. Expression of Foxp3, a master regulator of Treg cell differentiation, was quantified in endometrial tissue from women experiencing primary unexplained infertility and normal fertile women. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women meeting rigorously defined criteria for unexplained infertility after experiencing repeated failed cycles of IVF treatment (infertile, n = 10), or women classified as proven fertile (control, n = 12). Expression of Foxp3 mRNA was reduced approximately two-fold in the tissue of infertile women. In contrast, mRNAs encoding T cell transcription factors T-bet and GATA3, associated with differentiation of Th1 and Th2 CD4+ T cells respectively, were unchanged. Treg cell differentiation is controlled by TGFbeta, but the relative abundance in endometrial tissue of TGFbeta1, TGFbeta2, TGFbeta3 mRNAs was not changed in infertile women. Cytokines influencing Th1 and Th2 cell differentiation, including IFNgamma, IL-2, IL-4, IL-5, IL-10 and IL-12p40, as well as dendritic cell-regulating cytokines IL-1alpha, IL-1beta, IL-6, LIF, GM-CSF and TNFalpha were also expressed similarly regardless of fertility status. The finding of reduced endometrial Foxp3 implicates impaired differentiation of uterine T cells into the Treg phenotype as a key determinant of fertility in women. The factors underpinning this aberration in the immune response remain to be identified.
Murine neonatal immunity is typically Th2 biased. This is characterized by high-level IL-4 production at all phases of the immune response and poor IFN-gamma memory responses. The differential expression of Th1/Th2 cytokines by neonates and adults could arise if the critical regulators of Th differentiation and function, STAT6 and T-bet, operate differently during the neonatal period. To test this idea, the Th cell responses of wild-type, T-bet-deficient, or STAT6-deficient mice were compared in vitro and in vivo. The absence of these factors had similar qualitative effects on the development of effector function in neonates and adults, i.e., if a Th lineage was inhibited or enhanced in adult animals, a similar phenomenon was observed in neonates. However, there was a striking difference observed in the in vivo Th1 memory responses of STAT6-deficient mice initially immunized as neonates. Antigen-specific IFN-gamma production was increased 50-100-fold in STAT6-deficient neonates, achieving levels similar to those of STAT6-deficient adults. These findings demonstrate that STAT6 and T-bet signals are central in shaping Th responses in wild-type neonates, as in adult mice, and that the master regulators of Th cell development and function are already firmly established in early life.
The TCR signals for the release of CD4 effector function are poorly understood. Itk plays an essential role in Th2, but not Th1, responses. However, when Itk is required during Th2 development is unclear. We followed the fate of Itk-deficient T cells during Th2 development in vitro and in vivo using an IL-4/GFP reporter. Surprisingly, a similar frequency of itk(-/-) CD4(+) cells differentiated and committed to the Th2 lineage as wild-type cells. However, Itk-deficient Th2 cells failed to exert effector function upon TCR triggering. Loss of function was marked by defective transcriptional enhancement of Th2 cytokines and GATA3. IL-4 production in itk(-/-) Th2s could be rescued by the expression of kinase-active Itk. Thus, Itk is necessary for the release, but not gain, of Th2 function. We suggest that the liberation of effector function is tightly controlled through qualitative changes in TCR signals, facilitating postdifferentiation regulation of cytokine responses.
GATA2 is expressed in the pituitary during development and in adult gonadotropes and thyrotropes. It is proposed to be important for gonadotrope and thyrotrope cell fate choice and for TSH production. To test this idea, we produced a pituitary-specific knockout of Gata2, designed so that the DNA-binding zinc-finger region is deleted in the presence of a pituitary-specific recombinase transgene. These mice have reduced secretion of gonadotropins basally and in response to castration challenge, although the mice are fertile. GATA2 deficiency also compromises thyrotrope function. Mutants have fewer thyrotrope cells at birth, male Gata2-deficient mice exhibit growth delay from 3-9 wk of age, and adult mutants produce less TSH in response to severe hypothyroidism after radiothyroidectomy. Therefore, Gata2 appears to be dispensable for gonadotrope and thyrotrope cell fate and maintenance, but important for optimal gonadotrope and thyrotrope function. Gata2-deficient mice exhibit elevated levels of Gata3 transcripts in the pituitary gland, suggesting that GATA3 can compensate for GATA2.
T helper type 1 (Th1) development is facilitated by interrelated changes in key intracellular factors, particularly signal transducer and activator of transcription (STAT)4, T-bet, and GATA-3. Here we show that CD4+ cells from T-bet-/- mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels but exhibit virtually normal Th1 differentiation provided that GATA-3 levels are regulated at an early stage by anti-interleukin (IL)-4 blockade of IL-4 receptor (R) signaling. In addition, under these conditions, Th1 cells from T-bet-/- mice manifest IFNG promotor accessibility as detected by histone acetylation and deoxyribonuclease I hypersensitivity. In related studies, we show that the negative effect of GATA-3 on Th1 differentiation in T-bet-/- cells arises from its ability to suppress STAT4 levels, because if this is prevented by a STAT4-expressing retrovirus, normal Th1 differentiation is observed. Finally, we show that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. These findings lead to a model of T cell differentiation that holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of STAT4/IL-12Rbeta2 chain unless GATA-3 levels or function is regulated by T-bet. Thus, the principal function of T-bet in developing Th1 cells is to negatively regulate GATA-3 rather than to positively regulate the IFNG gene.
IL-10 is a major regulator in inflammatory responses. Although various transcription factors were defined to enhance IL-10, the molecular mechanism for the initiation of Il-10 transcription, remains unknown. mRNA profiling of six distinct primary CD4+ T cell populations showed differential expression of the transcription factor GATA-3 correlated with levels of IL-10 expression. We showed that ectopic expression of GATA-3 in naive primary CD4+ T cells enhanced expression of IL-10 by these cells and uncovered a possible mechanism for this effect. We found that GATA-3 induced changes of the chromatin structure at the Il-10 locus and that these changes occur even in the absence of IL-4. Furthermore we found that in the presence of GATA-3 the histones at the Il-10 locus become acetylated. Despite being recruited in vivo to two locations on the Il-10 locus, GATA-3 did not transactivate the IL-10 promoter. We therefore suggest a key role of GATA-3 in instructing Il-10 gene expression in primary CD4+ T cells, possibly by switching and stabilizing the Il-10 locus into a transcriptionally competent status.
We report here on a girl and her father with HDR syndrome (Hypoparathyroidism, sensorineural Deafness and Renal anomaly syndrome). The proband, an 11 year-old girl, complained of periodic tetany lasting for 6 years, and also used a hearing aid because of sensorineural hearing impairment. Furthermore, she had hemimegalencephaly, and had been taking an anti-epileptic agent to treat psychomotor seizures for 6 years. Endocrine assessment showed modest hypocalcemia, hyperphosphatemia and hypophosphaturia with lower normal parathyroid hormone concentration, and she had no renal abnormalities. Her father, who was 40 years old at the time of the investigation, had sensorineural hearing impairment, a lower than normal calcium level and normal renal function. Direct sequencing after PCR amplification of genomic DNA revealed a novel insertional mutation (405insC) in the GATA3 gene of both patients. This mutation was hypothesized to disrupt dual zinc fingers as well as one transactivating domain. The present findings lend additional support to the notion that the phenotype cannot be precisely estimated from the genotype in HDR syndrome.
The prognosis of a patient with estrogen receptor (ER) and/or progesterone receptor (PR) -positive breast cancer can be highly variable. Therefore, we developed a gene expression-based outcome predictor for ER+ and/or PR+ (ie, luminal) breast cancer patients using biologic differences among these tumors.
The ER+ MCF-7 breast cancer cell line was treated with 17beta-estradiol to identify estrogen-regulated genes. These genes were used to develop an outcome predictor on a training set of 65 luminal epithelial primary breast carcinomas. The outcome predictor was then validated on three independent published data sets. Results The estrogen-induced gene set identified in MCF-7 cells was used to hierarchically cluster a 65 tumor training set into two groups, which showed significant differences in survival (P = .0004). Supervised analyses identified 822 genes that optimally defined these two groups, with the poor-prognosis group IIE showing high expression of cell proliferation and antiapoptosis genes. The good prognosis group IE showed high expression of estrogen- and GATA3-regulated genes. Mean expression profiles (ie, centroids) created for each group were applied to ER+ and/or PR+ tumors from three published data sets. For all data sets, Kaplan-Meier survival analyses showed significant differences in relapse-free and overall survival between group IE and IIE tumors. Multivariate Cox analysis of the largest test data set showed that this predictor added significant prognostic information independent of standard clinical predictors and other gene expression-based predictors.
This study provides new biologic information concerning differences within hormone receptor-positive breast cancers and a means of predicting long-term outcomes in tamoxifen-treated patients.
GATA-3 (GATA binding protein 3) and the proto-oncogene c-maf are Th2-regulating transcription factors that control the expression of interleukin (IL)-4 and IL-5, while T-bet (T-box expressed in T cells) is a Th1-specific transcription factor that controls the expression of interferon (IFN-gamma). Allergen provocation in asthmatics induces a Th2-dominated cytokine profile, but so far it is unknown whether the skewed cytokine expression is reflected by the expression of the respective transcriptional regulators.
The aim of this study was to determine the regulation of Th1- and Th2-specific transcription factors and cytokines in 10 atopic subjects with mild asthma and 5 nonatopic healthy controls at baseline and after segmental sham and allergen challenge.
The mRNA expression of GATA-3, c-maf and T-bet was determined by real-time polymerase chain reaction in bronchoalveolar lavage (BAL) cells and bronchial biopsies. The percentage of IL-4+, IL-5+ and IFN-gamma+ BAL T cells was determined by flow cytometry, and BAL levels of these cytokines were measured by ELISA.
In BAL cells of asthmatics, the mRNA expression of all transcription factors was increased after allergen challenge. In bronchial biopsies, the basal expression of GATA-3 was increased in asthmatics compared to healthy controls but decreased after allergen challenge. Compared to sham challenge, the percentage of IL-5+/CD4+ BAL T cells was increased after allergen challenge in asthmatics while the percentage of IFN-gamma+/CD4+ and IFN-gamma+/CD8+ T cells was decreased. Expression of c-maf mRNA in BAL cells correlated with IL-4+/CD4+ BAL cells and BAL IL-5 levels.
Segmental allergen challenge in asthmatics leads to increased GATA-3, c-maf and T-bet expression in BAL cells but not in bronchial biopsies.
Patent lymphatic filariasis is characterized by a profound down-regulation of immune responses with both parasite Ag-specific tolerance and bystander suppression. Although this down-regulation is confined to the Th1 arm of the immune system in response to parasite Ag, we hypothesized a more generalized suppression in response to live parasites. Indeed, when we examined the cytokine profile of a cohort of filaria-infected (n = 10) and uninfected (n = 10) individuals in response to live infective-stage larvae or microfilariae of Brugia malayi, we found significant impairment of both Th1 and Th2 cytokines characterized by diminished production of IFN-gamma, TNF-alpha, IL-4, IL-5, and IL-10 in infected patients. The molecular basis of this impaired Th1/Th2 response was examined, and we identified three major networks of immunoregulation and tolerance. First, impaired induction of T-bet and GATA-3 mRNA underlies the Th1/Th2 deficiency in infected individuals. Second, regulatory networks, as evidenced by significantly increased expression of Foxp3 (natural regulatory T cell marker) and regulatory effectors such as TGF-beta, CTLA-4, PD-1, ICOS, and indoleamine 2,3-dioxygenase play an important role in immunosuppression. Third, the compromise of effector T cell function is mediated by the enhanced induction of anergy-inducing factors cbl-b, c-cbl (cbl is abbreviation for Casitas B lymphoma), Itch, and Nedd4. Indeed, blocking CTLA-4 or neutralizing TGF-beta restored the ability to mount Th1/Th2 responses to live parasites and reversed the induction of anergy-inducing factors. Hence, we conclude that a profound impairment of live parasite-specific Th1 and Th2 immune responses occurs in lymphatic filariasis that is governed at the transcriptional level by a complex interplay of inhibitory mediators.
The pathophysiological characteristics of bronchial asthma consist of chronic inflammation of airways, airway hyperresponsiveness, and bronchoconstriction. Studies have shown that T helper type 2 (Th2) cytokines produced by both T cells and mast cells in the airway contribute substantially to the initiation of inflammation in both experimental and human bronchial asthma. GATA-3 is a transcription factor essential to the production of Th2 cytokines by T lymphocytes. To clarify the role of GATA-3-expressing T cells in the pathophysiology of bronchial asthma, we utilized transgenic (Tg) mice carrying the GATA-3 gene and the ovalbumin (OVA)-specific T cell receptor gene (GATA-3-Tg/OVA-Tg). Mice were intranasally administrated OVA without systemic immunization. Airway responses were analyzed with noninvasive and invasive whole body plethysmographs. GATA-3-Tg/OVA-Tg mice exhibited significantly higher IL-13 and IL-4 protein expression in the airway. Although there were no differences in the types of infiltrating cells between GATA-3-Tg/OVA-Tg and GATA-3-non-Tg/OVA-Tg mice and no significant increase in IgE level in either group compared with nontreated mice, the response after ACh inhalation was significantly elevated in GATA-3-Tg/OVA-Tg on the seventh day of intranasal treatment with OVA. This hyperresponsiveness was inhibited by 5-lipoxygenase inhibitor and IL-13 neutralization, suggesting that airway responses were induced through IL-13 and leukotriene pathway. In conclusion, airway hyperresponsiveness, a characteristic of bronchial asthma, is regulated at the level of GATA-3 transcription by T lymphocytes in vivo.
Naive CD4+ T-cell differentiation to T helper 1 (Th1) and Th2 cells is dependent on T-bet and GATA-3 factors, respectively. T-bet and GATA-3, indeed, through chromatin remodelling allow transcriptional activation of Ifngamma and Th2 cytokine (Il4, Il5, Il13) genes, respectively. We investigated the effects of the negative costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) on GATA-3 and T-bet mRNA expression and Th cell differentiation in mouse naive CD4+ T cells. Our results show that CTLA-4 inhibits GATA-3 mRNA expression and Th2 cell differentiation. At variance, CTLA-4 does not affect T-bet mRNA expression and Th1 cell differentiation. GATA-3 mRNA expression is inhibited when CD4+ cells are stimulated under both neutral (i.e. absence of cytokines) and Th2-polarizing (i.e. presence of interleukin (IL)-4) conditions, the effect being larger under the latter condition. Hence CTLA-4 might affect the IL-4/signal transducer and activator of transcription-6 (STAT6) pathway leading to GATA-3 mRNA up-regulation. We found, indeed, that CTLA-4 engagement inhibits STAT6 activation leaving unaffected the STAT6 protein level. Moreover, CTLA-4 engagement drastically inhibits IL-4Ralpha mRNA and protein up-regulation under Th2-polarizing conditions. Thus, CTLA-4 exerts a tight control on Th2 cell differentiation by negatively regulating both the CD3/CD28 and the IL-4/STAT6 pathways.
Naïve CD4 T cells can differentiate into at least two different types of T helpers, Th1 and Th2 cells. Th2 cells, capable of producing IL-4, IL-5 and IL-13, are involved in humoral immunity against extracellular pathogens and in the induction of asthma and other allergic diseases. In this review, we summarize recent reports regarding the transcription factors involved in Th2 differentiation and cell expansion, including Stat5, Gfi-1 and GATA-3. Stat5 activation is necessary and sufficient for IL-2-mediated function in Th2 differentiation. Enhanced Stat5 signaling induces Th2 differentiation independent of IL-4 signaling; although it does not up-regulate GATA-3 expression, it does require the presence of GATA-3 for its action. Gfi-1, induced by IL-4, promotes the expansion of GATA-3-expressing cells. Analysis of conditional Gata3 knockout mice confirmed the critical role of GATA-3 in Th2 cell differentiation (both IL-4 dependent and IL-4 independent) and in Th2 cell proliferation and also showed the importance of basal GATA-3 expression in inhibiting Th1 differentiation.
Systemic lupus erythematosus (SLE) is characterized by the aberrant activation of T lymphocytes. Since T-bet and GATA-3 are the principal transcription factors for the differentiation of type-1 and type-2 helper T lymphocytes, respectively, we studied their mRNA expression in the urinary sediment of SLE patients and compared this with their urinary and intra-renal protein expression.
We studied 100 SLE patients and 10 healthy subjects. Urinary mRNA expression of T-bet and GATA-3 were studied by the real-time quantitative polymerase chain reaction. Intra-renal and urinary expressions of T-bet and GATA-3 were studied by immunohistochemistry and western blotting, respectively.
The urinary mRNA and protein expressions of T-bet were significantly higher in SLE patients with active nephritis than those with inactive disease (mRNA: P < 0.001; protein: P = 0.004). The urinary mRNA expression of T-bet correlated with the SLE disease activity index (SLEDAI) score (r = 0.55, P < 0.001) and the histological activity index (r = 0.48, P = 0.03). On the other hand, the urinary mRNA and protein expressions of GATA-3 were significantly lower in SLE patients with active nephritis (mRNA: P < 0.001; protein: P = 0.006), and GATA-3 mRNA expression inversely correlated with the SLEDAI score (r = 0.38, P < 0.001). For the 22 SLE patients with kidney biopsy, tubular expressions of T-bet and GATA-3 significantly correlated with the histological activity index (T-bet: r = 0.57, P = 0.006; GATA-3: r = -0.79, P < 0.001).
Patients with active lupus nephritis have increased T-bet and depressed GATA-3 expression in the urinary sediment and kidney tissue, indicating a predominant Th1 type of T-lymphocyte activation.
Age-related changes in lymphocytes are most prominent in the T cell compartment. There have been substantial numbers of reports on T cell function in aged mice and humans, such as on the production of Th1 and Th2 cytokines, but the results show considerable variation and contradictions. In the present study, we used 8- to 12-mo-old aging mice and a well-established in vitro Th1/Th2 cell differentiation culture system to identify molecular defects in Th1/Th2 cell differentiation that can be detected in the relatively early stages of aging. The capability to differentiate into Th2 cells is reduced in aging mouse CD4(+) T cells. Decreased activation of the ERK MAPK cascade upon TCR stimulation, but normal intracellular-free calcium ion concentration mobilization and normal IL-4-induced STAT6 activation were observed in aging mouse CD4(+) T cells. In addition, reduced expression of GATA3 was detected in developing Th2 cells. Chromatin remodeling of the Th2 cytokine gene locus was found to be impaired. Th2-dependent allergic airway inflammation was milder in aging mice compared with in young adult mice. These results suggest that the levels of Th2 cell differentiation and resulting Th2-dependent immune responses, including allergic airway inflammation, decline during aging through defects in the activation of the ERK MAPK cascade, expression of GATA3 protein and GATA3-dependent chromatin remodeling of the Th2 cytokine gene locus. In the present study, we provide the first evidence indicating that a chromatin-remodeling event in T cells is impaired by aging.
Previously, a modest association was observed between the progesterone receptor +331G/A gene variant and breast cancer risk. Here, in a larger sample of breast cancer cases and controls (n = 1,322/n = 1,953) nested in the Nurses' Health Study cohort, we confirm a significant association (odds ratio, 1.41; 95% confidence interval, 1.10-1.79) and suggest a molecular model. The association of the +331G/A variant with breast cancer was particularly strong among obese women (body mass index > 30; odds ratio, 2.87; 95% confidence interval, 1.40-5.90). To help understand the molecular mechanism by which this variant may predispose women to breast cancer, we identified nearby transcription factor binding sites. This search predicted a binding site for the GATA family of transcriptional regulators adjacent to this hPR polymorphism. Importantly, we found GATA3, GATA4, and GATA6 are expressed in normal breast tissue and two breast cancer cell lines, whereas GATA5 is minimally expressed in normal mammary tissue and more strongly expressed in two breast cancer cell lines. This finding was relevant because GATA5 bound the site adjacent to the +331G/A polymorphism, and activated the hPR (-711 to +822)-luciferase reporter plasmid in breast cancer cells. Overexpression of GATA5 increased expression of the endogenous hPR transcript, and GATA5 more strongly activated an hPR promoter construct encoding the PR-B isoform. Finally, hPR promoter constructs including the +331A were more strongly activated by GATA5 than constructs including +331G. Our findings suggest that GATA5 interacts with the +331G/A polymorphism to stimulate hPR-B expression in mammary cells, which may contribute to breast cancer susceptibility.
Opioids and cannabinoids are both associated with analgetic, psychotropic, and immunomodulatory effects. It has been suggested that both systems interact on multiple levels. We hypothesized that cannabinoids induce opioid receptors and investigated cannabinoid-dependent expression of the mu-opioid receptor subtype in a human T cell model. We report that activation of the peripheral cannabinoid receptor type 2 leads to a de novo induction of mu-opioid receptor transcription in Jurkat E6.1 cells. We show that interleukin-4 is transcriptionally induced in response to cannabinoids and that an interleukin-4 receptor antagonist blocks cannabinoid-dependent induction of mu-opioid receptors, indicating that induced expression of interleukin-4 is required in this process. Induction of interleukin-4 is blocked by decoy oligonucleotides directed against STAT5, indicating the requirement of this transcription factor. In addition, we show cannabinoid-dependent phosphorylation of STAT5. Further experiments demonstrate that interleukin-4 then induces phosphorylation of STAT6, which directly transactivates the mu-opioid receptor gene. In addition, STAT6 induces expression of the transcription factor GATA3, which also contributes to mu-opioid receptor gene transcription. The responsive promoter region of the human mu-opioid receptor gene with the binding sites for both factors was mapped to nt -1001 to -950. To demonstrate functional mu-opioid receptor proteins, morphine-mediated phosphorylation of mitogen-activated protein kinase was investigated. We show that phosphorylation of mitogen-activated protein kinase occurs only in cannabinoid-prestimulated Jurkat E6.1 cells and that it is blocked by the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. In summary, these findings provide a first example for cannabinoid-opioid-interactions in cells of the immune system.
An inbred A/J mouse respiratory challenge model was validated for vaccine testing against Chlamydia (C.) pneumoniae and used to screen the C. pneumoniae genome for vaccine candidates by expression library immunization (ELI). Biolistic delivery of genetic vaccine constructs elicited Th2-like immunity that was associated with inefficient elimination of C. pneumoniae. Delivery by injection elicited protective Th1-like responses. Since biolistic delivery of pools of ORFs was used in first round screening, the screen presumably selected against potent immunogens. Nevertheless, it was sufficiently accurate to identify three weakly protective antigens among all putative C. pneumoniae ORFs. The results suggest ELI discovery of highly protective C. pneumoniae vaccine candidates requires tight control of the Th1 immunity elicited by the genetically delivered library of test antigens.
To obtain a global view of the immunological alterations occurring in early systemic sclerosis (SSc) by transcriptional profiling of peripheral blood cells (PBCs).
Oligonucleotide microarrays were used to compare PBC gene expression profiles in 18 SSc cases (<2 yr duration) and 18 controls matched for race, gender and ethnicity. SSc cases had no prior or current exposure to cytotoxic drugs. PAXgene tubes were used to stabilize RNA during phlebotomy. Changes in gene expression were independently validated by real-time polymerase chain reaction.
SSc PBCs demonstrated differential expression of 18 interferon-inducible genes. Six of these genes were identical to the interferon signature genes in lupus peripheral blood mononuclear cells. Notably, SSc PBCs also had increased expression of allograft inflammatory factor (AIF1) and several selectins and integrins involved in cellular adhesion to the endothelium. Global analysis of 284 known biological pathways revealed that 13 were differentially regulated in SSc PBCs, including two pathways (IL2RB and GATA3) that lead to T(H)2 polarization.
Transcriptional profiling reliably discriminates between PBCs from SSc and normal donors despite the fact that they represent a heterogeneous cell population. Multiple biological pathways were differentially regulated in SSc PBCs, but a common thread across these pathways was alterations in protein tyrosine kinase 2beta and mitogen-activated protein kinase signalling. Although the SSc PBC gene expression profile demonstrated some parallels with the lupus interferon gene signature, there was also increased expression of transcripts encoding proteins that target PBCs to the endothelium, which might be relevant to the vasculopathy of SSc.
NKT cells are unique in that they can produce high levels of both Th1 and Th2 cytokines, yet little is known about how NKT cells control the transcription of Th2 cytokines. The expression of IL-4 by NKT cells is independent of the Th2-associated transcription factor Stat6. We have found that Stat6 is critical for the expression of IL-5, IL-10, and IL-13 by NKT cells. However, the Th2 cell-associated transcription factor GATA-3, normally induced by Stat6 activation, is expressed at low levels in NKT cells. CD4+ NKT cells are highly enriched for Th2 cytokine expression compared with CD4- NKT cells, and we searched for transcription factors that are up-regulated in CD4+ NKT cells that could control Th2 cytokine expression. We found that the NFAT family member NFAT2 is selectively increased in CD4+ NKT cells. We tested the roles of NFAT2 and also GATA-3 in Th2 cytokine expression by retrovirus-mediated gene transduction into NKT cells and nonpolarized conventional T cells. Expression of NFAT2 increased the expression of IL-4 in both NKT cells and conventional T cells, and NFAT2 activated IL-10 in conventional T cells but not in NKT cells. GATA-3 strongly activated IL-4, IL-5, and IL-13 expression in conventional T cells but had comparatively weak effects on these cytokines in NKT cells. Thus, NFAT2, GATA-3, and Stat6 have surprisingly different roles in NKT cells than in conventional T cells. We propose that one mechanism by which CD4+ NKT cells express IL-4 independent of Stat6 is via increased NFAT2 activity.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly malignant tumors affecting adolescents and adults. There have been a few reports on chromosomal aberrations of MPNSTs; however, the tumor-specific alteration remains unknown. We characterized the genomic alterations in 8 MPNSTs and 8 schwannomas by metaphase comparative genomic hybridization (CGH). In 5 of 8 MPNSTs, microarray CGH was added for more detailed analyses. Frequent gains were identified on 3q13-26, 5p13-14, and 12q11-23 and frequent losses were at 1p31, 10p, 11q24-qter, 16, and 17. Microarray CGH revealed frequent gains of EGFR, DAB2, MSH2, KCNK12, DDX15, CDK6, and LAMA3, and losses of CDH1, GLTSCR2, EGR1, CTSB, GATA3, and SULT2A1. These genes seem to be responsible for developing MPNSTs. The concordance rate between metaphase CGH and microarray CGH was 66%. Metaphase CGH was useful for identifying chromosomal alterations before applying microarray CGH.
Despite the radical surgical resection performed in patients with colorectal carcinoma, there is a high rate of tumor recurrence. Over an observation period of 3 years, 18% of the patients in our collective suffered a tumor relapse with local or distinct metastases after initial R0-resection. Some evidence suggests that this may be due to suppression of anti-tumor responses, a phenomenon that might be attributed to regulatory T cells. The aim of our study was to investigate the tumor-specific immune response depending on the UICC stage of patients with colorectal cancer. The cellular immune responses against defined antigens that are overexpressed in most of the patients with colorectal cancer were characterized. For this purpose, the tumor suppressor gene, p53, was chosen as the tumor-associated antigen that exhibits mutations and overexpression in up to 60% of colorectal carcinoma. We observed that p53 induced both IFN-gamma and IL-10 secretion. The predominance of IL-10 production indicated that regulatory T cells directly participate in modulating the anti-tumor immune response. IL-10 levels in the blood as well as the expression of regulatory T-cell specific genes at the tumor site correlate with the UICC stage of the disease. These results may provide an explanation for the poor prognosis and increased recurrence rate in patients with advanced carcinoma.
IL-5 is a key regulator of eosinophilic inflammation and is selectively expressed by antigen-activated Th2 lymphocytes. An important role for the proximal AP-1 and GATA sites in regulating IL-5 transcription is generally accepted but the significance of an adjacent Ets/NFAT site has remained unclear. We have investigated its role using the mouse Th2 clone D10.G4.1. Transcription of IL-5 reporter gene plasmids could be induced in D10 cells by phorbol myristate acetate/cyclic adenosine monophosphate (PMA/cAMP) stimulation and significantly further enhanced by activation of the mitogen-activated protein (MAP) kinase pathways. Strong induction of IL-5 mRNA was also induced by PMA/cAMP. Mutagenesis showed that the Ets/NFAT site is of critical importance along with the AP-1 and GATA sites in regulating IL-5 transcription stimulated by PMA/cAMP and MAP kinase activation. Transactivation was used to investigate the transcription factors which could function at the three sites and possible synergistic interactions. AP-1 (c-Fos/c-Jun) strongly induced IL-5 transcription and dominant negative AP-1 constructs confirmed that AP-1 plays an important role in regulating IL-5 expression. Ets1, unlike other members of the Ets/NFAT family, synergized strongly with AP-1 suggesting that Ets1 is the family member which functions at the Ets/NFAT site. AP-1/Ets1 transactivation also stimulated IL-5 mRNA expression. Ets1 binding to the proximal promoter region, demonstrated by chromatin immunoprecipitation, was stimulated by PMA/cAMP. The absolute dependence on the binding sites for Ets1, AP-1 and GATA-3 together with the strong synergy between Ets1 and AP-1 suggest close cooperative interactions between the three transcription factors in the regulation of IL-5 expression in mouse T cells.
GATA binding protein 3 (GATA3) is a transcriptional activator highly expressed by the luminal epithelial cells in the breast. Here we did a meta-analysis of the available breast cancer cDNA data sets on a cohort of 305 patients and found that GATA3 was one of the top genes with low expression in invasive carcinomas with poor clinical outcome. To validate its prognostic utility, we did a tissue microarray analysis on a cohort of 139 consecutive invasive carcinomas (n = 417 tissue samples) immunostained with a monoclonal antibody against GATA3. Low GATA3 expression was associated with higher histologic grade (P < 0.001), positive nodes (P = 0.002), larger tumor size (P = 0.03), negative estrogen receptor and progesterone receptor (P < 0.001 for both), and HER2-neu overexpression (P = 0.03). Patients whose tumors expressed low GATA3 had significantly shorter overall and disease-free survival when compared with those whose tumors had high GATA3 levels. The hazard ratio of metastasis or recurrence according to the GATA3 status was 0.31 (95% confidence interval, 0.13-0.74; P = 0.009). Cox multivariate analysis showed that GATA3 had independent prognostic significance above and beyond conventional variables. Our data suggest that immunohistochemical analysis of GATA3 may be the basis for a new clinically applicable test to predict tumor recurrence early in the progression of breast cancer.
There is now overwhelming evidence to support a major role for T cells in asthma, in particular the involvement of T helper type 2 (Th2) cells in atopic allergic asthma as well as nonatopic and occupational asthma. There may also be a minor contribution from T cytotoxic type 2 CD8+T cells. Several Th2 cytokines have potential to modulate airway inflammation, in particular interleukin-13 which induces airway hyperresponsiveness independently of IgE and eosinophilia in animal models. The identification of transcription factors controlling Th1, Th2 and T-regulatory cell (T(Reg)) development further support the Th2 hypothesis since GATA3 is overexpressed and T-bet underexpressed in the asthmatic airway and Foxp3 is induced in asthma by corticosteroid treatment. Specific T-cell-directed immunotherapy may allow induction/modulation of T-cell responses, and elucidation of the mechanisms of T(Regs) may allow further optimization of immunotherapy. Recent advances in the understanding of dendritic cell function in directing T-cell responses may uncover further therapeutic targets. Efficacy of cyclosporin and anti-CD4 treatment in chronic severe asthma argues for continued T-cell involvement, but whether remodeling contributes to pathology inaccessible to antiinflammatory treatment or T-cell immunotherapy remains an important question.
Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.
Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells.
We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines.
The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA.
CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters.
CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.
Anterior chamber associated immune deviation (ACAID) is characterized by a Th2 cell response. GATA-3 has been shown to be necessary for the activation of Th2 cells. This study was designed to examine the expression of GATA-3 in the development of ACAID.
ACAID was induced by injection of 50 microg interphotoreceptor retinoid binding protein (IRBP) into the anterior chamber (AC) of Wistar rats. Delayed-type hypersensitivity (DTH) was evaluated on day 3, 7, 14, 21, 28 after IRBP inoculation. GATA-3 expression was detected using immunohistochemical staining. The expression of GATA-3 mRNA at different time points after AC injection of IRBP was assayed by reverse transcriptase polymerase chain reaction (RT-PCR).
A significant DTH reaction was observed in Wistar rats on day 3 and 5 after IRBP inoculation. The DTH reaction was decreased 7 days after IRBP inoculation. GATA-3 expression was weak at both mRNA and protein levels in the normal spleen, but was significantly increased on day 5, 7, 14, and 21 after AC injection of IRBP.
The expression of GATA-3 is increased during ACAID, suggesting that GATA-3 may be involved in the development of ACAID.
The mammalian pro- and mesonephros are transient embryonic kidneys essential for urogenital system development. The nephric (Wolffian) duct, which is a central constituent of both structures, elongates caudally along a stereotypical path to reach the hindlimb level where it induces metanephros (adult kidney) formation, while the remaining duct gives rise to the male genital tract (epidydimis, vas deferens). The transcription factors Pax2 and Pax8 are essential for the initiation of pro- and mesonephros development. In a cDNA microarray screen for genes specifically expressed in the pro/mesonephros and regulated by Pax proteins, we identified Gata3, a transcription factor gene associated with hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. Gata3 is already expressed in the pronephric anlage, together with Pax2 and Pax8, suggesting that it may be a direct Pax2/8 target gene. Inactivation of Gata3 by insertion of an Ires-GFP reporter gene resulted in a massive increase in nephric duct cellularity, which was accompanied by enhanced cell proliferation and aberrant elongation of the nephric duct. Interestingly, however, the nephrogenic cord extended, with delayed kinetics, along the entire caudal path up to the level of the hindlimb bud, indicating that extension of the nephric duct and cord is controlled by different guidance cues. At the molecular level, the nephric duct of Gata3(-/-) embryos is characterized by the loss of Ret expression and signaling, which may contribute to the guidance defect of the nephric duct. Together, these results define Gata3 as a key regulator of nephric duct morphogenesis and guidance in the pro/mesonephric kidney.
Human early thymic precursors have the potential to differentiate into multiple cell lineages, including T cells and plasmacytoid dendritic cells (pDCs). This decision is guided by the induction or silencing of lineage-specific transcription factors. The ETS family member Spi-B is a key regulator of pDC development, whereas T-cell development is critically dependent on GATA-3. Here we show that triggering of the Notch1 signaling pathway by Delta-like1 controls the T/pDC lineage decision by regulating the balance between these factors. CD34+ CD1a- thymic progenitor cells express Notch1, but down-regulate this receptor when differentiating into pDCs. On coculture with stromal cell lines expressing either human Delta-like1 (DL1) or Jagged1 (Jag1) Notch ligands, thymic precursors express GATA-3 and develop into CD4+ CD8+ TCRalphabeta+ T cells. On the other hand, DL1, but not Jag1, down-regulates Spi-B expression, resulting in impaired development of pDCs. The Notch1-induced block in pDC development can be relieved through the ectopic expression of Spi-B. These data indicate that DL1-induced activation of the Notch1 pathway controls the lineage commitment of early thymic precursors by altering the levels between Spi-B and GATA-3.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
Glucocorticoid-induced TNFR family-related protein (GITR) is expressed at low levels on resting T cells, B cells and macrophages but at high levels on regulatory T cells (Treg). Although GITR expression is up-regulated on CD4+ effector cells upon activation, the role of GITR in Th1 and Th2 cell development is unclear. We report here that activation of GITR signalling by anti-GITR antibody markedly enhanced the induction of both Th1 and Th2 cytokine production by naive CD4+CD25- T cells. Consistent with this observation, anti-GITR antibody significantly enhanced the expression of the key Th1 (T-bet) and Th2 (GATA3) transcription factors in vitro. Administration of anti-GITR mAb in a murine model of arthritis significantly exacerbated the severity and onset of joint inflammation with elevated production of TNF-alpha, IFN-gamma, IL-5, and collagen-specific IgG1. Administration of anti-GITR mAb also significantly exacerbated murine allergic airways inflammation with elevated production of OVA-specific IFN-gamma, IL-2, IL-4, IL-5, and IgE. Finally, we demonstrated that adoptive transfer of CD4+GITR+ T cells effectively abolished airway inflammation induced in SCID mice reconstituted with CD4+GITR- T cells. Our results therefore provide direct evidence that GITR can modulate both Th1- and Th2-mediated inflammatory diseases, and may be a potential target for therapeutic intervention.
The noradrenergic cell type is characterized by the expression of proteins involved in the biosynthesis, transport, and secretion of noradrenaline and is dependent on the sequential and combinatorial expression of numerous transcription factors, including Phox2a, Phox2b, dHAND, GATA2, GATA3, and MASH1. Phox2a and Phox2b transactivate the promoter of the gene encoding the noradrenergic biosynthetic enzyme, dopamine beta-hydroxylase (DBH), and dHAND potentiates the activity of Phox2a. In this study, we use chromatin immunoprecipitation assays to identify target genes of the Phox2 proteins and dHAND. All three proteins are bound to the DBH and PHOX2B promoter regions in SH-SY5Y neuroblastoma cells. The interaction between Phox2a and dHAND is analyzed by fluorescent anisotropy, which demonstrates that dHAND causes an eightfold increase in the affinity of Phox2a for its recognition sites on the DBH promoter region. The Phox2 proteins are not found on the genes encoding other noradrenergic enzymatic or transport proteins but are reciprocally bound to each other's promoters in SH-SY5Y cells. Together with Phox2a and Phox2b, dHAND is bound to the PHOX2B promoter and is also associated with the GATA2 and eHAND genes in the absence of the Phox2 proteins. These results demonstrate the direct interactions of the Phox2 and dHAND transcription factors within a noradrenergic cell type. The Phox2 proteins were found to share all target genes, whereas dHAND binds to genes independently of Phox2a.
We recently showed that dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) prime naive CD4(+) T cells to differentiate into T helper type 2 (Th2) cells that produced high amounts of tumor necrosis factor-alpha (TNF-alpha), but no interleukin (IL)-10. Here we report that TSLP induced human DCs to express OX40 ligand (OX40L) but not IL-12. TSLP-induced OX40L on DCs was required for triggering naive CD4(+) T cells to produce IL-4, -5, and -13. We further revealed the following three novel functional properties of OX40L: (a) OX40L selectively promoted TNF-alpha, but inhibited IL-10 production in developing Th2 cells; (b) OX40L lost the ability to polarize Th2 cells in the presence of IL-12; and (c) OX40L exacerbated IL-12-induced Th1 cell inflammation by promoting TNF-alpha, while inhibiting IL-10. We conclude that OX40L on TSLP-activated DCs triggers Th2 cell polarization in the absence of IL-12, and propose that OX40L can switch IL-10-producing regulatory Th cell responses into TNF-alpha-producing inflammatory Th cell responses.
GATA-3 was shown to bind to two sites of the IL-4 gene promoter in human T-cell lines PER-117 and Jurkat. A motif located in the region of position -860 and responsible for GATA-3 binding was detected for the first time. Mutation or deletion of this site increased the promoter activity. The findings suggest a direct involvement of GATA-3 in regulation of the human IL-4 gene transcription as a repressor of the promoter activity.
The stability of helper T cell fates is not well understood. Using conditional introduction of dominant-negative factors, we now show that T-bet and GATA-3 are far more critical in establishment than maintenance of IFN-gamma and IL-4 activity during Th1 and Th2 maturation, respectively. We also show that a genetic interaction between T-bet and its target Hlx seems to be required for Th1 maturation, but that Hlx may also be dispensable for maintenance of a transcriptionally permissive ifng gene. In parallel to progressive activator independence in the permissive lineage, the ifng gene becomes more recalcitrant to switching as the forbidden lineage matures. T-bet plus Hlx can disrupt ifng silencing when introduced into developing Th2 cells, but they fail to perturb ifng silencing in mature Th2 cells. In contrast, a hypermorphic allele of T-bet can reverse silencing of the ifng gene in mature Th2 cells. These results suggest that signature gene activity of helper T cells is initially plastic but later becomes epigenetically fixed and offer an initial strategy for inducing mature cells to switch their fate.
The effects of 17beta-estradiol (E2) on immune function have been extensively reported. The effects are dependent on concentration and duration of exposure and potential differences in signaling between the known E2 receptors, estrogen receptors (ER) alpha and ERbeta. Through the use of ER-deficient mice, we and others have begun to demonstrate the role of the two known receptors in modulating immune functional activities. Previous studies have shown that cells of the innate immune system have altered function (bactericidal capacity) and patterns of cytokine expression (increased proinflammatory cytokine expression) through amelioration of ERalpha signaling. In this study, we extend these studies to analysis of T cell differentiation and proliferation in APC-dependent and APC-independent in vitro assay systems. Our results demonstrate that ERalpha deficiency in splenic macrophages, but not CD11c+ splenic dendritic cells pulsed with OVA significantly enhances proliferative responses and IFN-gamma production by transgenic OVA peptide-specific (OT-II) CD4+ T cells when compared with Ag-pulsed APC from wild-type littermates. The addition of E2 in this culture system did not significantly affect the production of IFN-gamma. In addition, when purified CD4+ T cells from ERalpha-deficient and wild-type littermates were stimulated with anti-CD3/CD28 Ab in the absence of E2, there were no significant differences in IFN-gamma or IL-4 production. However, the addition of E2 significantly increased IL-4 secretion, as well as increased GATA-3 mRNA levels from ERalpha-replete CD4+ T cells, while this effect was abrogated in ERalpha-deficient CD4+ T cells.
Listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, is a potent inducer of interleukin (IL)-12, IL-18 and interferon (IFN)-gamma. We have shown that LLO facilitates development of T cells mediating protective immunity against L. monocytogenes through the induction of IFN-gamma production at an early stage. Based on this finding, it is postulated that LLO inhibits differentiation of Th2 cells and the Th2 immune response. By using a murine model of ovalbumin (OVA)-induced allergic rhinitis, we investigated whether LLO has an ability to modulate the Th2-type immune disorder. In mice sensitized intraperitoneally with ovalbumin (OVA)/alum and challenged intranasally with OVA, a large number of eosinophils migrated into the nasal tissue, and high titres of anti-OVA IgE and IgG(1) antibodies were detected in sera. However, LLO treatment during sensitization markedly inhibited the eosinophil infiltration and production of these anti-OVA antibodies. A large number of T cells from mice sensitized and challenged with OVA produced high level of IL-4 and IL-5 but not IFN-gamma after stimulation with OVA. In contrast, OVA-specific IFN-gamma-producing T cells were preferentially induced in mice treated with LLO at the time of sensitization. In the absence of LLO administration, the expression level of GATA-3 and SOCS-3 in CD4(+) T cells was enhanced after sensitization with OVA. LLO treatment resulted in a reduction of GATA-3 and SOCS-3 expressions but induced the transcription of T-bet instead. Taken together, these data show clearly that LLO is capable of inhibiting Th2 immune response by skewing differentiation of antigen-specific T cells into Th1 cells.
T-bet is an important Th1 driving transcription factor regulated by IFN-gamma/STAT1 pathway. T-bet turns on IFN-gamma transcription in CD4+ T cells and T-bet-deficient cells fail to differentiate to Th1 direction. Previous reports have characterized function of T-bet mainly in murine cells and very little is known about its functions in human cells. Here, we studied T-bet expression kinetics in parallel with GATA3 during Th1/Th2 polarization. We demonstrate that in addition to CD3/CD28 activation, cytokines IL-12 and IFN-alpha in the presence of neutralizing anti-IFN-gamma enhanced T-bet mRNA and protein expression in human CD4+ cells. T-bet is known to be a potent inducer of IFN-gamma. Even though IFN-gamma and IL-12 stimulation induced similar levels of T-bet protein in human CD4+ cells, IFN-gamma-treated cells produced considerably less IFN-gamma than cells treated with IL-12. Therefore, high T-bet protein expression does not necessarily correlate with IFN-gamma production. In addition, we show that the immunosuppressive cytokine TGF-beta inhibits T-bet and GATA3 protein expression only if it is present prior to primary T cell activation and is maintained in the cultures during the early polarization of Th1/Th2 cells. In conclusion, we report new insights into the cytokine regulation of T-bet in human CD4+ T cells.
The differentiation of naïve T helper (Th) cells is induced by TCR activation and IL-12/STAT4 or IL-4/STAT6 signaling pathways, forming Th1 and Th2 cells, respectively. In this study, oligonucleotide arrays were used to identify genes regulated during the initiation of human Th1 and Th2 cell differentiation at 2 and 6 h in presence or absence of immunosuppressive TGF-beta. As a result the immediate targets of IL-12, IL-4 and TGF-beta were identified. The effects of IL-12 at this early stage were minimal and consistent with the known kinetics of IL-12Rbeta2 expression. IL-4, however, was observed to rapidly regulate 63 genes, 26 of which were differentially expressed at both the 2- and 6-h time points. Of these IL-4 regulated genes, one-third have previously been observed to display expression changes in the later phases of the polarization process. Similarly to the key regulators, TBX21 and GATA3, the transcription factors SATB1, TCF7 and BCL6 were differentially regulated at the protein level during early Th1 and Th2 cell polarization. Moreover, the developing Th1 and Th2 cells were demonstrated to be responsive to the immunosuppressive TGF-beta and IL-10. In this study, a panel of novel factors that may be important regulators of the differentiation process was identified.
Type 2 cytokines are usually predominant in tumor patients and associated with tumor progression. To explore whether reversing of type 2 predominance could be a promising strategy in tumor immunotherapy, PBMC of 35 lung cancer patients and 19 healthy subjects were prepared and subjected to be examined for cytokine secretion and gene expression. Tetra-Methylpyrazine (TTMP), extracted from a traditional Chinese medicinal herb which has been used in clinic to reverse the Th2 status of cancer patients in China, was added to PBMC culture. Determined by RT-PCR, the positive percentages of mRNA expression of type 1 cytokines (8.6% for IFN-gamma and 11.4% for IL-2) were lower than those of type 2 cytokines (71.4% for IL-4, 60% for IL-6 and 80% for IL-10) in patients' PBMCs. The potential of gene expressing (measured as relative intensity to the ratio of beta-actin) in the patients for type 1 cytokines was also in a low level (0.111 for IFN-gamma, 0.119 for IL-2) in comparison with a relative high level for type 2 cytokines (0.319 for IL-4, 0.303 for IL-6 and 0.377 for IL-10). Meanwhile, both positive percentage and relative intensity of gene expression were lower for a type 1 cytokine-related transcription factor T-bet (31.4% and 0.142, respectively) than those for type 2 cytokine-related GATA3 (85.7% and 0.378, respectively). The blood serum levels of IFN-gamma and IL-2 in the patients were slightly lower but not significantly when compared with healthy control. In contrast, the levels IL-4 and IL-6 in patients were significantly higher than those in healthy subjects by ELISA analysis. TTMP could enhance supernatant concentration and gene expression levels of IFN-gamma, IL-2 and T-bet, but reduce those of type 2 cytokines. These results demonstrate that the lung cancer patients had a predominant expression of type 2 cytokines and TTMP could reverse the type 2 dominant status, which might offer an alternative therapeutic regime for lung cancer patients.
Previous studies have shown that tumor cells predominantly express Th2 type cytokines and transcription factors. GATA-3, as a Th2-specific transcription factor, plays a central role in positive-regulating Th2 development. So whether the expression of GATA-3 in tumor cells has any effect on tumor development is a question of interest. In the present study, we inhibited the expression of GATA-3 in tumor cells through antisense RNA blockade technique, and observed its effects on tumor in vitro and in vivo. Our results showed that antisense GATA-3 treatment could inhibit the expression of TNF-alpha and Th2 cytokines in tumor cells, and antisense-induced blockade of GATA-3 could also depress tumor growth in tumor-bearing mice. We suggest that the ratio of T-bet/GATA-3 can be evaluated as a more important marker of the status of Th1/Th2 type. And our results might provide some evidence about the molecular regulatory mechanisms in tumor cell development.
Th1 and Th2 cytokines secreted by polarized effector T cells play a pivotal role in the development of autoimmune and allergic diseases. However, the genetic basis of cytokine production by T lymphocytes in humans is poorly understood. In this study, we investigated the genetic contribution to cytokine production and regulation of T cell-specific transcription factors in a prospective twin study. We found a substantial genetic contribution to the production of Th1 cytokines such as IFN-gamma and TNF-alpha with heritabilities of 0.85 (95% confidence intervals, 0.74-0.95) and 0.72 (0.50-0.93), respectively, whereas no genetic influence on production of the Th2 signature cytokine IL-4 was observed. Furthermore, the intrapair variability in IFN-gamma production by isolated T cells was lower in monozygotic than in dizygotic twins. In contrast to GATA-3, NFAT, and NF-kappaB, intrapair variability of T-bet, the master transcription factor of Th1 cells, was very low among monozygotic and high among dizygotic twins, indicative of a strong genetic influence on T-bet (heritability 0.93, 95% confidence interval, 0.84-1.0). Our data provide novel insights into the genetic regulation of human Th cell polarization. These data suggest that signature cytokines and cytokine signaling events of Th1 rather than Th2 cells are genetically determined and implicate that Th2-associated diseases in humans might be due to genetic variations in Th1 cytokine regulation via T-bet. This concept is highlighted by the recent finding that inactivation of the T-bet gene in mice results in development of clinical hallmark features of asthma.
GILZ (glucocorticoid-induced leucine zipper), a gene induced by dexamethasone, is involved in control of T lymphocyte activation and apoptosis. In the present study, using Gilz transgenic mice (TG), which overexpress GILZ in the T-cell lineage, we demonstrate that Gilz is implicated in T helper-2 (Th-2) response development. After in vitro stimulation by CD3/CD28 antibodies, peripheral naive CD4+ T cells from TG mice secrete more Th-2 cytokines such as interleukin-4 (IL-4), IL-5, IL-13, and IL-10, and produce less Th-1 cytokines such as interferon-gamma (IFN-gamma) than wild-type mice (WT). CD4+ TG lymphocytes up-regulated Th-2 cytokine expression in the specific response to ovalbumin chicken egg (OVA) antigen immunization. Up-regulation correlated with increased expression of GATA-3 and signal transducer and activator of transcription 6 (Stat6), Th-2-specific transcription factors and decreased expression of T-bet, a transcription factor involved in Th-1 differentiation. Finally, in TG mice delayed-type hypersensitivity, a Th-1 response, was inhibited and bleomycin-induced pulmonary fibrosis, a Th-2 mediated disease, was more severe. These results indicate that Gilz contributes to CD4+ commitment toward a Th-2 phenotype and suggest this contribution may be another mechanism accounting for glucocorticoid immunomodulation.
This study was designed to investigate the effects of allitridin on the expression of transcription factors T-bet and GATA-3 in mice infected by murine cytomegalovirus (MCMV). A BALB/c mouse model system of MCMV infection was established. Twenty mice were allocated randomly into an allitridin-treated group (n = 10) and a placebo control group (n = 10). The same dose (25 mg/kg/day) and regimen of allitridin were used in the treated group in the 24 hours after virus infection; the same volume of saline solution was injected in placebo control mice. In an additional blank control group (n = 10), the same volume of saline solution was injected. The expression levels of the transcription factors T-bet and GATA-3 were measured by reverse transcription-polymerase chain reaction. The expression levels of the T helper (Th) 1 cytokine interferon-gamma (IFN-gamma) and the Th2 cytokine interleukin (IL)-10 in supernatant of spleen cell culture were measured by enzyme-linked immunosorbent assay. MCMV infection markedly down-modulated the expression of IFN-gamma and T-bet and significantly up-modulated the expression of IL-10 and GATA-3. Allitridin induced significantly (P < .01) increased expression of the transcription factor T-bet and the Th1 cytokine IFN-gamma and markedly (P < .01) decreased expression of the transcription factor GATA-3 and the Th2 cytokine IL-10. Thus MCMV infection could lead to disequilibrium of Th1/Th2 cytokine expression: The level of the Th1 cytokine IFN-gamma was decreased significantly, and Th2 cytokine IL-10 was overexpressed markedly. Allitridin could up-regulate the expression of T-bet and IFN-gamma and inhibit the expression of GATA-3 and IL-10 in MCMV-infected mice, indicating a Th1 dominant state, which should enhance the specific cellular immune reactions against cytomegalovirus (CMV) and be helpful for clearance of CMV from the host.
T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.
To investigate effects of CpG oligodeoxynucleotide (CpG ODN) on the mRNA expression of transcription factors GATA binding protein 3 (GATA-3) and T-box expressed in T cells (T-bet) in asthmatic mice.
An asthmatic mouse model was established and treated with CpG ODN. Total inflammatory cells and eosinophils in bronchoalveolar lavage fluid (BALF) were counted and inflammatory cell infiltration in lung tissue was evaluated. Interferon-gamma and interleukin-4 concentrations in BALF and splenocyte culture supernatants were detected using an enzyme-linked immunosorbent assay. Transcription factor GATA-3 and T-bet mRNA expression in splenocytes and lung tissue were detected by reverse transcription-polymerase chain reaction.
Total inflammatory cells and eosinophils in BALF were reduced in the CpG ODN-treated group compared with the asthma group, and inflammatory cell infiltration in lung tissue was also significantly alleviated. CpG ODN treatment increased the interferon-gamma concentration but decreased the interleukin-4 concentration in both BALF and splenocyte culture supernatants. GATA-3 mRNA expression was reduced in both lung tissue and splenocytes in the CpG ODN-treated group, while the mRNA ratio of T-bet to GATA-3 in splenocytes was increased.
CpG ODN treatment inhibits airway inflammatory cell infiltration and regulates interferon-gamma/interleukin-4 synthesis in asthmatic mice, possibly through a mechanism of downregulation of GATA-3 mRNA expression in both lung tissue and splenocytes.
We previously showed that a positive impact of peritoneal defense response on the outcome of peritoneal dialysis (PD)-related peritonitis is characterized by an increased pattern of peritoneal CD4/CD8 T-cell ratio with a predominant CD4(+)-T helper subtype 1 phenotype. To further explore longitudinal changes in peritoneal immunity during PD-related peritonitis, we examined the production of interleukin 12 (IL-12), IL-18, and interferon gamma (IFN-gamma) in peritoneal dialysate effluent (PDE) and kinetic expression of the transcription factors T box expressed in T cells (T-bet) and guanine adenine thymine adenine (GATA) binding protein 3 (GATA-3) in peritoneal T cells during peritonitis. Correlations between these observations and responses to antibiotics were analyzed.
IL-12, IL-18, and IFN-gamma protein and IFN-gamma, T-bet, and GATA-3 messenger RNA (mRNA) were measured in PDE during various phases of peritonitis in 40 patients undergoing PD. Patients were divided into 2 groups according to whether they had a rapid versus delayed response to antibiotic treatment.
In the early phase of peritonitis, IL-12, IL-18, and IFN-gamma levels in PDE were significantly greater in the rapid-response group (P < 0.05). Changes in peritoneal IL-12 and IL-18 levels preceded changes in IFN-gamma levels. The kinetics of IFN-gamma, T-bet, and GATA-3 mRNA expression in peritoneal T cells, measured by means of real-time polymerase chain reaction, differed between the 2 groups. In the rapid-response group, IFN-gamma and T-bet mRNA expression increased, whereas that of GATA-3 decreased over time. Results were opposite in the delayed-response group, with IFN-gamma and T-bet levels decreasing and GATA-3 levels increasing over time.
These data suggest that local IL-12 and IL-18 production is part of a protective early immune response to PD-related peritonitis. High IL-12 and IL-18 levels in PDE during the early phase of peritonitis correlated with a predominant type 1 immune response and favorable outcome.
Cancers, including that of the breast, are the result of multiple contributing factors including aberrant gene expression. Indeed, the CYP19 gene encoding P450 aromatase, the key enzyme for estrogen biosynthesis, is up-regulated in breast tumors predominantly via the cAMP-responsive gonad-type PII promoter, ultimately leading to increased intratumoral estrogen production and tumor growth. Thus, identifying the molecular factors involved in aromatase PII promoter regulation is essential for our understanding and treatment of the disease. Because we have previously shown activity of the murine aromatase PII promoter to be markedly up-regulated by GATA factors with respect to the gonads, we hypothesized that GATA factors are also key determinants of human PII promoter-driven aromatase transcription in breast tumors. We now show that GATA3 and GATA4 are indeed expressed in several breast cancer cells lines. Consistent with the cAMP dependence of the PII promoter, activation elicited by GATA3 or GATA4 alone and the striking synergism between GATA3 or GATA4 and the nuclear receptor liver receptor homolog (LRH)-1 was intimately linked to forskolin treatment or overexpression of protein kinase A (PKA) catalytic subunit. PKA-mediated phosphorylation increases the interaction between GATA3 and LRH-1 and the requirement for PKA in aromatase PII promoter stimulation involves at least three specific amino acid residues: GATA3 Ser308, GATA4 Ser261, and LRH-1 Ser469. Finally, we show that the human LRH-1 promoter is itself a target for GATA factors. Thus, taken together, our results suggest that GATA factors likely contribute to aberrant aromatase expression in breast tumors through two distinct, yet complementary mechanisms.
Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.
Development can occur by either instructive or stochastic processes. My colleagues and I have studied the contributions of these processes to differentiation of naïve CD4+ T-cells to either a Th1 or Th2 phenotype. Our initial discovery that pathogens in our in vitro priming system led to the development of Th1 cells through the action of interleukin-12 was important evidence of a link between innate and adaptive immunity. Subsequent studies in our laboratory revealed an important role for GATA-3 autoactivation in Th2 development. Other interesting projects that have emerged as a result of our Th cell differentiation studies include understanding the role of the inhibitory immunoreceptor B- and T-lymphocyte attenuator in the immune response, as well as the role of the transcription factor ERM in both T-cells and spermatogenesis. We currently maintain our interests in the Th differentiation field by trying to understand the role of type 1 interferons in Th1 development and the role of alternate promoters for the GATA-3 gene, among other things, but are also actively embarking on studies related to the choice between divergent cell types during embryonic stem cell differentiation.
CC chemokine receptor 5 (CCR5) is the major HIV-1 coreceptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA elements, we show that the transcription factors GATA binding protein 1 (GATA-1) and GATA-3 differentially suppress the expression of CCR5 in stem-cell-derived dendritic cells and primary human T-cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing T helper 1 (Th1)-associated genes, interferon-gamma (IFNgamma), and CXC chemokine receptor-3 (CXCR3). GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 could also serve as a surrogate for GATA-3 in its canonic role of programming Th2 gene expression. These findings provide insight into GATA-3-mediated gene regulation during T-cell differentiation. Importantly, decoding the mechanisms of GATA-1-mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells.
Gata-3 has been shown to specifically alter its expression patterns in different types of cancers. Recent evidence suggests that an interference of Gata-3 exists in the TGF-beta signaling pathway. To determine the role of Gata-3 in pancreatic cancer, pancreatic cancer samples were analyzed in comparison to normal pancreatic tissues. Furthermore, four different pancreatic cancer cell lines with different alterations of the TGF-beta pathway were studied. To evaluate if a potential relationship with TGF-beta signaling pathway exists, we correlated mRNA expression levels with the expression of TGF-betas, TGF-beta receptors, and Smad-3. Finally, we analyzed the influence of TGF-beta on Gata-3 expression in vitro. All pancreatic cancer samples demonstrated a marked overexpression of Gata-3 mRNA and protein. Immunohistochemical staining revealed strong and persistent cytoplasmic Gata-3 immunoreactivity in cancer cells. In an electrophoretic mobility shift assay, a disturbed nuclear translocation was confirmed. The expression of Gata-3 showed a significant correlation with the expression of TGF-betas, TGF-beta receptors, and Smad-3. TGF-beta responsive cell lines showed a downregulation of Gata-3 mRNA upon TGF-beta exposure, whereas in TGF-beta-unresponsive cell lines, Gata-3 mRNA expression persisted at high levels. Furthermore, strong specific upregulation of Gata-3 impaired nuclear translocation and its cooperative action with the TGF-beta pathway, suggesting that Gata-3 plays a central role in human pancreatic cancer.
The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. Here, we describe an unexpected role of the HMG-box-containing gene Sox9 in hair development.
Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, we demonstrate that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, we show that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrate overexpression in skin tumors in mouse and man.
We conclude that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Our genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma.
The consequences that drug withdrawal has on immune functioning has only recently been appreciated; however, given the wide variety of use and abuse of opiate analgesics, understanding the decrements to immune function that withdrawal from these drugs causes is of crucial importance. In previous work, we have demonstrated that morphine treatment contributes to immunosuppression by polarizing Th cells toward the Th2 lineage. In the current study, it was hypothesized that morphine withdrawal would result in Th2 differentiation and subsequent immune dysfunction. To address this hypothesis, mice were chronically treated with morphine for 72 h followed by a 24-h withdrawal period. It was determined that 24-h morphine withdrawal resulted in a decrease in IFN-gamma, the Th1 signature cytokine, whereas the Th2 cytokine, IL-4, was increased. In addition, Western blot and EMSA experiments revealed that morphine withdrawal-induced Th2 differentiation was mediated through the classical Th2 transcription factors Stat-6 and GATA-3. In addition, the consequence of morphine withdrawal in the presence of an immune stimulation was also examined by treating mice in vivo with LPS before morphine withdrawal. Following withdrawal, it was found that the Th1-polarizing cytokine IL-12 was significantly decreased, providing further support for the observation that withdrawal results in Th2 differentiation by possibly impacting the generation of an appropriate innate immune response which directs subsequent adaptive Th1/Th2 responses.
CD4+ T cells with a block in the NF-kappaB signaling pathway exhibit decreases in Th1 responses and diminished nuclear levels of multiple transactivating NF-kappaB/Rel/IkappaB proteins. To determine the lineage-intrinsic contributions of these transactivators to Th differentiation, T cells from mice deficient in specific subunits were cultured in exogenous cytokines promoting either Th1 or Th2 differentiation. RelB-deficient cells exhibited dramatic defects in Th1 differentiation and IFN-gamma production, whereas no consistent defect in either Th1 or Th2 responses was observed with c-Rel-deficient cells. In sharp contrast, Bcl-3-null T cells displayed no defect in IFN-gamma production, but their Th2 differentiation and IL-4, IL-5, and IL-13 production were significantly impaired. The absence of RelB led to a dramatic decrease in the expression of T-box expressed in T cells and Stat4. In contrast, Bcl-3-deficient cells exhibited decreased GATA-3, consistent with evidence that Bcl-3 can transactivate a gata3 promoter. These data indicate that Bcl-3 and RelB exert distinct and opposing effects on the expression of subset-determining transcription factors, suggesting that the characteristics of Th cell responses may be regulated by titrating the stoichiometry of transactivating NF-kappaB/Rel/IkappaB complexes in the nuclei of developing helper effector cells.
The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.
GATA-3 appears to be key to the Th2 response. However, few in vivo experiments have examined the function of GATA-3 in Th1 and Th2 immune responses. We developed two lines of GATA-3-transgenic (Tg) mice harboring the SRalpha or lck promoters and examined the Th2 immune responses of mice infected with the intestinal nematode Nippostrongylus brasiliensis and the Th1 responses with purified derivative of tuberculin (PPD) immunization. Numbers of peripheral blood eosinophils in all GATA-3-Tg mice increased 10- to 20-fold after primary infection with N. brasiliensis and 25-100-fold after secondary infection. The number of eosinophils in infected GATA-3-Tg mice was significantly higher than that in infected control littermates. Total IgE levels after primary infection in GATA-3-Tg mice were 8-450-fold increased, which was significantly higher than those of control mice. Mesenteric lymph node cells of infected GATA-3-Tg mice upon stimulation with N. brasiliensis antigen secreted more IL-5 and IL-13 than those of control mice. However, production of IL-4 and IFN-gamma were comparable between GATA-3-Tg and controls. Mice immunized with PPD were intradermally challenged with PPD to induce delayed type hypersensitivity (DTH). The amount of footpad swelling caused by the DTH reaction in GATA-3-Tg mice was significantly smaller than that of control littermates. Inguinal lymph node cells from GATA-3-Tg mice stimulated with PPD in vitro secreted more IL-5, IL-10 and less IFN-gamma than those of control littermates. These results suggested that Th1 and Th2 driven conditions enhance IL-5 production in GATA-3-Tg mice through the direct binding of GATA-3 to the IL-5 promoter region. The influence of GATA-3 on IL-13, IFN-gamma and IL-10 production varied according to the stimulating conditions. However, IL-4 production was not significantly elevated in GATA-3-Tg mice, indicating that IL-4 and IL-5 production was differentially regulated in these mice.
Type 1 diabetes is the result of an inflammatory T helper 1 (Th1) lymphocyte-mediated beta cell destructive process. The majority of diabetes-prone BioBreeding (BBdp) rats fed wheat protein-based diets, such as NTP-2000, develop type 1 diabetes and display a mild coeliac-like enteropathy. Mesenteric lymph nodes (MLNs), which drain the gut, are the major inductive site where dietary antigens are recognised in the gut-associated lymphoid tissue (GALT). We hypothesised that this compartment could be a site of abnormal wheat protein-induced Th1 cell activation.
MLN cells were isolated from BBdp and BB control (BBc) rats that were fed NTP-2000 or a hydrolysed casein (HC)-based diet at ages that pre-date classic insulitis. The inflammatory status, phenotype and proliferation of these cells in response to wheat protein were determined.
The expression ratio of T-bet : Gata3, master transcription factors for Th1 and Th2 cytokines, was increased in the MLN from NTP-2000-fed BBdp rats compared with that from BBc rats, mainly due to decreased Gata3 expression. CD3(+)CD4(+)IFN-gamma(+) T cells were more prevalent in the MLN of wheat-fed BBdp rats, but remained at control levels in BBdp rats fed a diabetes-retardant HC diet. BBdp MLN cells proliferated in response to wheat protein antigens in a specific, dose-dependent manner, and >93% of cells were CD3(+)CD4(+) T cells. This proliferation was associated with a low proportion of CD4(+)CD25(+) T cells and a high proportion of dendritic cells in the MLN of BBdp rats.
Before insulitis is established, the MLNs of wheat-fed BBdp rats contain an unusually high proportion of Th1 cells that proliferate specifically in response to wheat protein antigens.
Adult idiopathic thrombocytopenic purpura (ITP) is a chronic acquired organ-specific autoimmune hemorrhagic disease characterized by the production of antibodies against antigens on the membranes of platelet, resulting in enhanced Fc-mediated destruction of the platelets by macrophages in the reticuloendothelial system. Dysfunctional cellular immunity is considered important in the pathophysiology of ITP. The aim of this study was to explore the profile of type1 and type2 T cells in chronic ITP patients.
The balance of Th1/Th2 and Tc1/Tc2 was studied by simultaneous analysis of intracellular cytokines of peripheral blood mononuclear cells and splenocytes in short-term cultures activated with PMA/ionomycin as well as mRNA expression of T-bet and GATA-3 in peripheral blood mononuclear cells and splenocytes using real-time polymerase chain reaction.
Patients with active disease but not patients in remission had significant higher Th1/Th2 (p<0.01) and Tc1/Tc2 (p<0.01) ratios in peripheral blood (PB) and significant higher Th1/Th2 ratio in splenocytes (p<0.01) than those in the control group. The Tc1/Tc2 ratio in splenocytes in ITP patients was higher than that in control, but did not reach significant difference (p=0.082). GATA-3 mRNA expression in ITP patients was significantly lower both in PB (p<0.01) and in splenocytes (p<0.01) than in corresponding samples from controls while there was no difference in T-bet expression.
Our data indicate that ITP is a T1 cell (Th1 and Tc1) predominant disease although the precise mechanisms await further functional assay. The T-bet/GATA-3 ratio may provide a surrogate marker of T1/T2 cytokine balance. Shifting the cytokine patterns from T1 to T2 might be a potential immunotherapy for ITP.
Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome.
A T helper (Th)1 cytokine profile is predominant in the inflamed synovium of rheumatoid arthritis (RA). Since the situation in the blood is more controversial, we studied the Th1/Th2 balance in the peripheral blood of RA patients using mRNA markers. Total RNA was isolated directly from whole blood from 20 RA patients and 14 healthy controls. T-bet and GATA-3 transcription factors associated with Th1 and Th2 responses respectively, and IFNgamma and IL-4 mRNA levels were measured by real-time PCR. In RA but not in control samples, T-bet mRNA levels correlated positively with IFNgamma mRNA levels, and negatively with CRP levels. Accordingly, RA patients were divided into two groups according to CRP levels. In comparison to RA patients with a low CRP (CRP < 40 mg/l), patients with a high CRP (CRP>or=40 mg/l) had lower IFNgamma/beta-actin, T-bet/beta-actin mRNA levels and T-bet/GATA-3 expression ratios. In conclusion, RA blood cells showed a decreased Th1 situation as indicated by low IFNgamma and T-bet mRNA expression. This pattern was found only in patients with the most active disease.
Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.
Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is a Th1-mediated inflammatory demyelinating disease of the CNS. AMP-activated protein kinase was reported recently to have anti-inflammatory activities by negatively regulating NF-kappaB signaling. In this study, we investigated the prophylactic and therapeutic efficacy of an AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), in active and passive EAE induced by active immunization with PLP(139-151) or MOG(35-55) and in adoptive transfer of PLP(139-151)-sensitized T cells, respectively. In vivo treatment with AICAR exerted both prophylactic and therapeutic effects on EAE, attenuating the severity of clinical disease. The anti-inflammatory effects of AICAR were associated with the inhibition of the Ag-specific recall responses and inhibition of the Th1-type cytokines IFN-gamma and TNF-alpha, whereas it induced the production of Th2 cytokines IL-4 and IL-10. Treatment of PLP(139-151)-specific T cells in vitro with AICAR decreased their expression of T-bet in response to IL-12, a Th1 transcription factor, whereas in response to IL-4, it induced the expression and phosphorylation of Th2 transcription factors GATA3 and STAT6, respectively. Moreover, treatment of APCs in vitro with AICAR inhibited their capability to present the proteolipid protein peptide to PLP(139-151)-specific T cells. In an irrelevant Th1-mediated, OT-2 TCR transgenic mouse model, AICAR impaired in vivo Ag-specific expansion of CD4(+) T cells. Together, these findings show for the first time that AICAR is a novel immunomodulator with promising beneficial effects for the treatment of multiple sclerosis and other Th1-mediated inflammatory diseases.
Primary T helper 2 cells are heterogeneous, expressing subsets of cytokines at varying levels. Mechanisms controlling this spectrum of phenotypes are still unclear. The ETS family transcription factor PU.1 is expressed in Th2 but not Th1 cells. Th2 cytokine production is decreased in cultures transduced with a PU.1-expressing retrovirus and increased in Th2 cells following RNAi that decreases PU.1 expression. In primary cultures, PU.1 expression is restricted to a subpopulation of Th2 cells that express CCL22 and a subset of Th2 cytokines. PU.1 regulates the Th2 phenotype by interfering with GATA-3 DNA binding without altering GATA-3 protein levels. Thus, the expression of PU.1 in subsets of Th2 cells establishes a defined cytokine profile and contributes towards establishing the spectrum of cytokine production observed in Th2 populations.
Administration of bee venom (BV) elicits anti-inflammatory, anti-nociceptive and anti-allergic effects in various animal models. This study was designed to evaluate the direct effects of BV on helper T cell activities and on Th1/Th2 lineage development using both in vitro and in vivo conditions. In the Th1 skewed condition, BV increased the expression of IFN-gamma mRNA and enhanced the expression of T-bet on purified CD4(+) T cells from splenocytes of BALB/c mice. On the other hand, BV treatment did not alter the expression of IL-4 or GATA-3 in a Th2 driven environment. To elucidate the effects of BV on Th1/Th2 lineage development under in vivo conditions, BV was given by intraperitonial injection to BALB/c mice. It significantly increased the CD4(+) T cell population and enhanced IFN-gamma expression, while IL-4 transcripts were not altered upon in vivo activation using an anti-CD3 antibody injection. Taken together, these results imply that BV induces Th1 lineage development from CD4(+) T cells by increasing the expression of a Th1-specific cytokine, IFN-gamma. In addition, this result may be mediated by inducing a Th1-specific transcription factor, T-bet.
Helper T cells and T cell cytokines play central roles in allergic disorders including bronchial asthma. We reported enhanced IL-5 production by peripheral blood T cells of asthmatic patients. A transcription factor, GATA-3, has been implicated in IL-5 gene expression. This study was undertaken to clarify the role of GATA-3 in the upregulation of IL-5 synthesis in asthmatic patients.
Peripheral CD4+ T cells were transfected with an IL-5 promoter reporter construct as well as its mutants in the presence or absence of a GATA-3 expression vector. Messenger RNA expression level of GATA-3 in CD4+ T cells of asthmatic subjects was compared to that of healthy donors.
IL-5 promoter activity in CD4+ T cells was enhanced by overexpression of GATA-3, whereas it was diminished by the introduction of mutations in the putative GATA-3 binding sites. The GATA-3 expression level in CD4+ T cells of asthmatic patients was equivalent to that of healthy controls.
The expression level of GATA-3 may not be an essential factor to cause IL-5 hyperproduction in bronchial asthma, though GATA-3 is crucially involved in IL-5 gene transcription in human peripheral CD4+ T cells.
Naive T helper (Th) lymphocytes are induced to express the il4 (interleukin-4) gene by simultaneous signaling through the T cell receptor and the interleukin (IL)-4 receptor. Upon restimulation with antigen, such preactivated Th lymphocytes can reexpress the il4 gene independent of IL-4 receptor signaling. This memory for expression of the il4 gene depends on epigenetic modification of the il4 gene locus and an increased expression of GATA-3, the key transcription factor for Th2 differentiation. Here, we have identified a phylogenetically conserved sequence, the conserved intronic regulatory element, in the first intron of the il4 gene containing a tandem GATA-3 binding site. We show that GATA-3 binds to this sequence in a position- and orientation-dependent manner, in vitro and in vivo. DNA demethylation and histone acetylation of this region occurs early and selectively in differentiating, IL-4-secreting Th2 lymphocytes. Deletion of the conserved element by replacement of the first exon and part of the first intron of the il4 gene with gfp leads to a defect in the establishment of memory for expression of IL-4, in that reexpression of IL-4 still requires costimulation by exogenous IL-4. The conserved intronic regulatory element thus links the initial epigenetic modification of the il4 gene to GATA-3 and serves as a genetic control element for memory expression of IL-4.
T-bet (T-box expressed in T cells) and GATA-3 are transcription factors that play a critical role in the development of Th1 and Th2 cells, as do genes of the SOCS (suppressor of cytokine signaling) family, albeit indirectly. Another transcription factor, Foxp3, is a master regulator of natural regulatory T cells (Tregs). To identify the role of these factors in impaired Th1 responses of patent filarial infection, analysis of cytokine, SOCS, and transcription factor mRNA expression was performed on purified T cells of filaria-infected individuals (n = 6) and uninfected controls (n = 6). As expected (and in contrast to cells of uninfected individuals), there was a significant depression of gamma interferon (IFN-gamma) and a concomitant increase in interleukin-4 (IL-4), IL-5, and IL-10 mRNA expression following stimulation with parasite antigen (BmA) but not with a polyclonal T-cell (anti-CD3) stimulus. T-bet (but not GATA-3) was expressed at significantly lower levels in cells of filaria-infected individuals in response to BmA compared with those from the uninfected group, accounting, at least partially, for the diminished IFN-gamma expression. Second, we found no significant differences in expression of Foxp3 between the two groups, although induction of Foxp3 expression correlated with induced expression levels of IL-10, implicating Tregs in the IL-10 expression seen. Finally, parasite-specific T-cell expression of SOCS-1, SOCS-5, and SOCS-7 was significantly diminished among infected patients; in contrast, expression of SOCS-3 increased. Our data therefore indicate that the impaired Th1 responses observed in patent lymphatic filariasis are associated with decreased expression of T-bet, SOCS-1, SOCS-5, and SOCS-7 and increased expression of SOCS-3 in T cells.
The pathogenesis of allergic asthma involves the interplay of inflammatory cells and resident airway cells, and of their secreted mediators including cytokines, chemokines, growth factors and inflammatory mediators. Tyrosine kinase signaling cascades play a critical role in the pathogenesis of allergic airway inflammation. Receptor tyrosine kinases (e.g. epidermal growth factor receptor [EGFR] and platelet-derived growth factor receptor) are important for the pathogenesis of airway remodeling. Stimulation of non-receptor tyrosine kinases (e.g. Lyn, Lck, Syk, ZAP-70, Btk, Itk and JAK) is the earliest detectable signaling response upon activation of immune receptors (T cell receptor, B cell receptor and FCepsilonR1), cytokine receptors and chemokine receptors in inflammatory cells. Activation of tyrosine kinases invokes multiple downstream signaling pathways, including phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB), leading to cell differentiation, survival, proliferation, degranulation and chemotaxis. Inhibitors targeted at different enzyme molecules of the tyrosine kinase signaling cascade might afford therapeutic potential for asthma. Anti-inflammatory effects of pharmacological agents targeted at tyrosine kinases, Syk, Itk, signal transducer and activator of transcription-1, NF-kappaB, GATA3, EGFR, PI3K, MEK1/2, p38 MAPK and JNK have been reported in animal models of allergic airway inflammation. Therefore, development of inhibitors targeted at the tyrosine kinase signaling cascade is an attractive strategy for the treatment of asthma.
The type 2 T-helper (Th2) lymphocyte can be regarded as an important target cell for the treatment of allergic asthma as it plays a crucial role in the initiation, progression and persistence of disease. Several strategies to target Th2 cells can be envisioned. Drugs that prevent Th2-cells from migrating into the lung tissue, such as antibodies to the chemokine receptor CCR4 and inhibitors of the adhesion molecule VLA-4, are promising for the treatment of asthma. To inhibit Th2-cell activation, novel asthma drugs that act on Th2-selective transciption factors such as GATA3 are being developed. Although initial strategies aimed to block the action of Th2-derived cytokines, the generation of counter-regulatory Th1 lymphocytes and regulatory T cells is currently being explored.
Peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a role in adipocyte differentiation and insulin sensitization. We identified and characterized a new C/T substitution at position -689 (-689C>T) in the P2 promoter of PPARgamma in a putative GATA binding site. By electrophoretic mobility shift assay, both GATA2 and GATA3 proteins could bind weakly to the wild-type P2 -689 GATA binding site but not to the mutated site. Neither GATA2 nor GATA3 was able to regulate significantly the P2 promoter activity in a reporter-luciferase assay, whatever the allele at position -689 was, suggesting that the -689 putative GATA site was probably not a functional target for GATAs. However, the presence of the -689T allele rendered the P2 promoter less active at the basal state. We genotyped a population of 1155 men and women for the -689C>T polymorphism and looked for possible associations with anthropometric and lipid variables. The carriers of the -689T allele had elevated body weight and LDL-cholesterol concentrations compared with the homozygous for the common allele. Haplotype analyses including the -681C>G (P3 promoter), -689C>T (P2 promoter), and Pro12Ala (exon B) polymorphisms were performed. Carriers of the G-T-Ala haplotype (corresponding to the P3 -681C>G, P2 -689C>T and Pro12Ala polymorphisms in this order) had elevated LDL-cholesterol concentrations and body weight compared with C-C-Pro individuals. In conclusion, we identified a new polymorphism in the P2 promoter of PPARgamma. The P3 -681C>G, P2 -689C>T, and Pro12Ala polymorphisms and related haplotypes were associated with higher body weight and plasma LDL-cholesterol concentrations.
The cis- and trans-acting factors that are critical for placenta-specific expression of the human syncytin gene are unknown. We identified a 146-base pair (bp) region of the 5'-flanking region of the human syncytin gene from nt-294 to -148 that is essential for basal gene expression in human BeWo and JEG3 choriocarcinoma cell lines but not in hepatoblastoma and kidney cell lines. Ligation of the 146-bp fragment to a SV40 promoter or a human beta-globin minimal promoter markedly enhanced promoter activity in the placenta cells but not in the liver and kidney cells. DNase I footprint assays indicated that nuclear extracts from BeWo cells but not HepG2 cells protected four regions (FP1-FP4) of the 146-bp fragment. Site-directed mutagenesis of an SP1-binding site in FP3 and a GATA-binding site in FP4 significantly repressed promoter activity in the placenta cells. Overexpression of SP1 (Sp1 transcription factor) and GATA2 (GATA binding protein 2) and GATA3 induced syncytin promoter activity but had little or no effect on the activities of syncytin promoter fragments containing mutations in the SP1- and GATA-binding sites. GATA2 and -3 mRNA levels increased markedly during spontaneous in vitro differentiation of human cytotrophoblast cells when the cytotrophoblast cells fused to form a syncytium. These findings strongly suggest that the 146-bp region of the 5'-flanking region (nt-294/-148) of the human syncytin gene acts as a placenta-specific enhancer. Binding of SP1 and GATA family members to this enhancer is critical for cell-specific expression of the syncytin gene.
The transcription factor GATA-3 is one regulator of Th1/Th2 differentiation. In sheep, we recently discovered a putative GATA-binding site (WGATAR) in the second intron of the Th1-cytokine gene interleukin 2 (IL2), showing a single nucleotide polymorphism (G/C). As genetic variations in cytokine genes are thought to regulate cytokine production, we studied the significance of this polymorphism for IL2 transcription. Sheep with different IL2 genotypes were identified by single-strand conformation polymorphism (SSCP)-analysis and IL2 transcription levels in peripheral blood mononuclear cells (PBMC) isolated from these animals were compared. For this purpose, transcription of IL2 mRNA was quantified by real-time polymerase chain reaction in unstimulated PBMC and in PBMC incubated for 4h in the presence of concanavalin A (ConA) or phorbol 12-myristate 13-acetate plus ionomycin (PMA/I). Compared to unstimulated cells, stimulation with ConA and PMA/I increased the IL2 mRNA transcription in average by 300- and 20-fold, respectively. Nevertheless, no significant differences in IL2 transcription between the genotypes could be detected. These findings were confirmed by band shift studies using different oligonucleotides containing variations of the potential binding motif, which showed no differences in the gel mobility after incubation with nuclear extract containing GATA-3. The obtained results argue against an impact of this polymorphism on the IL2 transcription and the genetic disease resistance in sheep.
Expression of estrogen receptor-alpha (ERalpha) as determined by immunohistochemistry of tumor tissue is currently the most clinically useful test to predict hormone responsiveness of breast cancer. Thirty percent of ERalpha-positive breast cancers do not respond to hormonal therapy. GATA-3 is a transcription factor that is expressed in association with ERalpha and there is evidence that GATA factors influence response to estrogen. In this pilot study, we investigated whether GATA-3 expression is associated with hormone response in breast cancer.
Breast cancer tissue was stained for GATA-3 expression by immunohistochemistry in ERalpha-positive cancers from 28 patients, 14 of whom were defined as hormone unresponsive (cases) and 14 of whom were age-matched controls with hormone-responsive, ERalpha-positive cancers (controls).
Comparing cases and controls, there were no differences in expression of ERalpha; progesterone receptor, ErbB2; or tumor grade. Using 20% nuclear staining to characterize tumors as GATA-3 positive or GATA-3 negative, 6 of 14 (43%) cancers in the hormone-unresponsive group and none of the controls were classified as GATA-3 negative (odds ratio, 8.2; 95% confidence interval, 1.2-infinity; p = 0.031). Using different cut points to characterize GATA-3 positivity yielded very similar results, indicating a positive association between lack of GATA-3 expression and lack of response to hormonal therapy.
The study suggests that analyzing ERalpha-positive breast tumors for GATA-3 using immunohistochemistry might improve prediction of hormone responsiveness. The association between GATA-3 expression and hormone response suggests that GATA-3 may play a role in mechanisms controlling response to estrogen.
Almost every neonate receives Bacillus Calmette-Guerin (BCG) vaccination in China. The authors' previous study showed that BCG promoted cord blood monocyte-derived dendritic cells maturation and induced high level of interleukin (IL)-10, medium level of interferon (IFN)-gamma, but low level of IL-4 production by cord naive T cells. The experiments in the present study were designed to explore the effects of neonatal BCG vaccination on immune functional development of splenic T cells in mice in vivo.
Neonatal BALB/c mice were inoculated with BCG intraperiotoneally. Four weeks later, spleen cells of mice were isolated and surface molecular markers of CD4, CD25 and CD44 and intracellular IFN-gamma, IL-10, and IL-4 in CD3(+) T cells were detected by flow cytometry. Furthermore, mRNA expression of transcription factor T-bet, Foxp3 and GATA-3 were analyzed by RT-PCR.
The percentage of total CD4(+) T cells decreased [(23.50 +/- 2.59)% vs. (47.38 +/- 10.41)%, P < 0.01] but the percentage of CD25(+) [(24.92 +/- 2.74)% vs. (20.27 +/- 2.85)%, P < 0.05] and CD44(+) [(89.29 +/- 2.56)% vs. (82.98 +/- 5.51)%, P < 0.05] T cells in CD4(+) T cells was higher in BCG-vaccinated mice than that in controls. Meanwhile, the percentage of IFN-gamma positive [(6.52 +/- 2.40)% vs. (3.13 +/- 2.03)%, P < 0.05] and IL-10 positive [(14.81 +/- 3.65)% vs. (10.90 +/- 1.61)%, P < 0.05] but not IL-4 positive [(1.17 +/- 0.46)% vs (1.51 +/- 0.75)%, P > 0.05] cells in CD3(+) T cells of BCG-vaccinated mice was significantly higher than that of non-BCG-vaccinated mice. In comparison with BCG-naive mice, T-bet was significantly high in BCG-vaccinated mice [T-bet/beta-actin 0.44 +/- 0.11 vs. 0.28 +/- 0.06, P < 0.05], but there was no significant difference in GATA-3 [GATA-3/beta-actin 0.46 +/- 0.08 vs. 0.50 +/- 0.10,P > 0.05] and Foxp3 [Foxp3/beta-actin vs. 0.27 +/- 0.11 and 0.30 +/- 0.16, P > 0.05] mRNA expression between the two groups.
Neonatal BCG vaccination could induce strong Th1 but weak Th2 response as reported previously. Though neonatal BCG vaccination was not capable of inducing CD4(+)CD25(+) regulatory T cell response with Foxp3 expression, it caused increase of IL-10(+) CD3(+) cells which might represent some regulatory T cells producing IL-10.
CD8(+) T cells play an important role in the induction of the autoimmune response in non-obese diabetic (NOD) mice. Here we describe abnormalities in the control of cytokine production by NOD CD8(+) T cells. NOD CD8(+) T cells had an increased propensity to produce IFN-gamma upon TCR activation, in both adult and 2-week-old mice. NOD CD8(+) T cells had a reduced capacity to produce IL-4 in type 2 conditions compared to CD8(+) T cells from the diabetes-resistant strains BALB/c and C57BL/6. Both GATA-3 and c-Maf, two positive transactivators for IL-4 gene expression, were expressed in type 2 conditions at comparable levels in NOD CD8(+) T cells. The GATA-3 was functional since normal levels of IL-5 were produced and the IL-4 promoter was hyperacetylated in NOD CD8(+) T cells. In contrast, c-Maf failed to bind to its responsive element as determined by chromatin immunoprecipitation (ChIP) assay. These results suggest that NOD CD8(+) T cells possess an increased propensity to produce IFN-gamma and impaired c-Maf-dependent DNA binding activities in vivo that lead to reduced IL-4 production following TCR activation. These defects may facilitate the development of the autoimmune response by inducing an overall type 1-biased immune response in NOD mice.
Recently, a member of the GATA-binding family of transcription factors was shown to be involved in human hypoparathyroidism, sensorineural deafness, and renal abnormality (HDR) syndrome. We report here a Japanese family in which two of the members are affected with HDR syndrome. Sequence analysis of GATA3 showed a heterozygous novel mutation in this family: an unusual mutation at exon 3 (709insC) resulting in a premature stop at codon 302 with a loss of both of the zinc finger domains.
Glucocorticoids are the mainstay of asthma therapy and mediate the repression of a number of cytokine genes, such as Interleukin (IL)-4, -5, -13, and granulocyte macrophage colony-stimulating factor (GM-CSF), which are central to the pathogenesis of asthmatic airway inflammation. The glucocorticoid receptor (GR) mediates repression by a number of diverse mechanisms. We have previously suggested that one such repressive activity is by direct binding of GR to elements within the GM-CSF enhancer that are recognized by the nuclear factor of activated T cells.activator protein 1 (NF-AT.AP-1) complex. We reasoned that, because many cytokine genes activated in asthma are transcriptionally regulated by the recruitment of this complex to DNA, their binding sites might provide a target for GR to mediate its repressive effects. Here, we show that transcriptional repression of the Interleukin-5 gene involves recruitment of GR to a DNA region located within the IL-5 proximal promoter, which is bound by NF-AT and AP-1 proteins. GR recruitment had a profound effect upon the activation capacity of GATA3, which has a binding site close to the NF-AT.AP-1 domain in both IL-5 and IL-13 promoters. Repression by GR involves co-repressor recruitment, because treatment of transfected cells with the deacetylase inhibitor trichostatin A caused a partial relief of repression. Additionally, repression could be augmented by co-transfection of cells with a histone deacetylase (HDAC1). These data suggest that the local recruitment of GR causes repression by inhibiting transcriptional activation by GATA3, a key tissue-specific determinant of expression of Th2 cytokines.
15-Deoxy-delta(12, 14)-prostaglandin J2 (15d-PG J2) is a regulator of a nuclear transcriptional factor, peroxisome proliferator-activated receptor (PPAR)-gamma. A previous study has demonstrated that 15d-PG J2 enhanced acute lung injury induced by lipopolysaccharide (LPS) in mice. 15d-PG J2 induced mucin-producing cells in the bronchial epithelium, especially in the presence of LPS. The present study investigated the effects of 15d-PG J2 on the activation of GATA-3 and Signal Transducer and Activator of Transcription (STAT) 6, important transcriptional factors in mucus secretion, in the lung in the presence or absence of LPS. ICR mice were divided into 4 experimental groups that intratracheally received vehicle, lipopolysaccharide (LPS: 125 microg/kg), 15d-PG J2 (1 mg/kg), or 15d-PG J2 + LPS. The nuclear localization of GATA-3 and phosphorylated STAT 6 was evaluated 2 h after the intratracheal administration. 15d-PG J2 enhanced the nuclear localization of GATA-3 in the presence of LPS, whereas the nuclear localization of phosphorylated STAT 6 was not altered in the groups. These results suggest that the enhancing effects of 15d-PG J2 on the production of mucin-producing cells might be related, at least in part, to the activation of GATA-3.
Infection of ruminants with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) leads to a chronic and often fatal granulomatous enteritis known as Johne's disease. Most infections with M. paratuberculosis occur during the first 6 months of life, and there is some evidence for transmission in utero. Once established, infections typically exist in a subclinical state for several years. Recent gene-expression profiling studies suggested the hypothesis that inherent gene-expression profiles in peripheral blood mononuclear cells (PBMCs) from M. paratuberculosis-infected cattle may be different than expression profiles in PBMCs from uninfected controls. If true, this would suggest that it is possible to identify an M. paratuberculosis infection "signature" through transcriptional profiling of peripheral immune cells. In addition, identification of groups or classes of genes showing inherently different expression in PBMCs from M. paratuberculosis-infected cattle relative to PBMCs from uninfected controls might highlight important interactions between this pathogen and the host immune system. In this report, we describe studies aimed at testing this hypothesis. Our novel results indicate that, indeed expression profiles of at least 42 genes are inherently different in freshly isolated PBMCs from M. paratuberculosis-infected cattle when compared to similar cells from uninfected controls. Gene-expression differences observed following microarray analysis were verified and expanded upon by quantitative real-time PCR (Q-RT-PCR). Our results indicate that T cells within PBMCs from M. paratuberculosis-infected cows have adopted a predominant Th 2-like phenotype (enhanced expression of IL-5, GATA 3, and possibly IL-4 mRNA), that cells within infected cow PBMCs may exhibit tissue remodeling deficiencies through higher expression of tissue inhibitor of matrix metalloproteinase (TIMP) 1 and TIMP2 RNA and lower expression of matrix metalloproteinase (MMP) 14 RNA than similar cells from healthy controls, and that cells within the PBMC population of M. paratuberculosis-infected cows are likely poised for rapid apoptosis (upregulation of CIDE-A, Bad, TNFRI, and Fas).
Transforming growth factor (TGF)-beta, a pleiotropic cytokine that has multiple effects on immune responses, has been shown to inhibit interleukin (IL)-4/GATA-3 expression as well as T helper 2 (Th2) differentiation. Consistent with these reports, we found that priming T cells from DO11.10 transgenic mice with antigen in the presence of TGF-beta inhibited GATA-3 expression and the development of IL-4-producing T cells. Unexpectedly, the inhibition of Th2 development was accompanied by a substantial increase in the number of interferon-gamma (IFN-gamma)-producing cells. T cells primed with TGF-beta secreted IFN-gamma in response to both T-cell receptor ligation and IL-12/IL-18 stimulation, and expressed high levels of T-bet and low levels of GATA-3. The TGF-beta-mediated enhancement of T helper 1 (Th1) priming was independent of IL-12 and signal transducer and activator of transcription (STAT)-4, but required endogenous IFN-gamma. TGF-beta-mediated enhancement of the IFN-gamma-dependent, IL-12-independent pathway of Th1 priming was mediated primarily by the inhibition of IL-4 produced by memory/activated T cells in the unfractionated CD4+ responder population. Nevertheless, TGF-beta did not inhibit this pathway of Th1 differentiation when purified naive CD4+ T cells were used as responders. These data have important implications for strategies being considered for the use of TGF-beta-producing T cells for the treatment of autoimmune disorders.
The TEC-family protein tyrosine kinases ITK, RLK and TEC have been identified as key components of T-cell-receptor signalling that contribute to the regulation of phospholipase C-gamma, the mobilization of Ca(2+) and the activation of mitogen-activated protein kinases. Recent data also show that TEC kinases contribute to T-cell-receptor-driven actin reorganization and cell polarization, which are required for productive T-cell activation. Functional studies have implicated TEC kinases as important mediators of pathways that control the differentiation of CD4(+) T helper cells. Here, we review studies of signalling pathways that involve TEC kinases and how these pathways might contribute to the regulation of T-helper-cell differentiation and function.
To investigate whether dengue-2 patients with and without dengue hemorrhagic fever had different virus load, immune mediators, or T helper (Th) reaction, we simultaneously measured virus load, immune mediators and the Th1/Th2 transcription factors T-bet/GATA-3 mRNA expression in a large outbreak of dengue-2 infections in Southern Taiwan. Results showed that virus load was not significantly different between patients with and without dengue hemorrhagic fever. Patients with dengue fever had higher IFN-gamma levels, but patients with dengue hemorrhagic fever had significantly higher IL-10 levels. Further studies showed that patients with dengue hemorrhagic fever had a significantly lower T-bet than those with dengue fever, but GATA-3 mRNA expression in peripheral blood leukocytes was not significant difference between both groups. In conclusion, altered Th1 reaction as reflected by lower T-bet mRNA expression associated with higher IL-10 levels might be involved in the pathogenesis of dengue hemorrhagic fever.
Seven protopanaxatriol-type ginsenosides and their aglycones including PPT, PT, -Re, -Rg (1), -F (1), -Rh (1), 20(R)-Rh (1) which are closely related in structure were studied for their effects on type 1 and type 2 cytokines production from murine splenocytes and their related mechanisms were examined. The results indicate that PPT, PT and ginsenoside-Re show hardly any or weak effects on concanavalin A (Con A)-induced production of IFN-gamma and IL-4. Ginsenoside-Rh (1) and 20(R)-Rh (1) induce a Con A-induced type 1 cytokine pattern by increasing the production of interleukin-12 (IL-12), the expression of IFN-gamma, T-bet and enhancing NF-kappaB DNA binding activity. In contrast ginsenosides-Rg (1) and -F (1) cause a Con A-induced type 2 cytokines response by increasing the expression of IL-4, GATA-3 and enhancing NF-kappaB DNA binding activity. Thus, these protopanaxatriol-type ginsenosides have different immunomodulatory effects, which might explain the complex immunomodulatory effect of Panax ginseng.
Transcription factor Gata3 is implicated in the formation of autosomal dominant hypoparathyroidism, sensorineural deafness, and renal anomaly (HDR) syndrome. We pursued to identify the potential Gata3 target genes by profiling the gene expression pattern in E9.5 Gata3-/- mouse embryos. Altogether four independent microarray hybridizations were carried out on NIA Mouse15K cDNA arrays. We discovered two hundred and sixty one genes that are downregulated in Gata3 mutant embryos at E9.5 (with a minimal 2.0-fold change). The majority of the differentially expressed genes belong to two functional groups--genes involved in transcription regulation and cellular signaling. One of the genes discovered to be downregulated in Gata3 mutant embryos was tumor suppressor gene Disabled 2. The validity of this finding was checked by using the whole mount in situ hybridization technology. This study revealed that the sites, where Dab2 is downregulated in the mutant embryos partly overlap with the Gata3 expression domains, including the mid-embryo region, branchial arches and facio-acoustic (VII-VIII) neural crest complex. This is the first time when tumor supressor gene Dab2 is shown to be implicated in the defective phenotype of Gata3 mutant mice.
Interleukin-7 (IL-7) promotes survival and expansion of lymphoid precursors. We show here that, in addition, IL-7 has a fundamental role, as early as the stage of the multipotent (B/T/NK) common lymphoid progenitor (CLP), in maintaining the B cell differentiation program open. CLPs generated in the absence of IL-7 have normal T/NK differentiation potential, but severely impaired B potential. Accordingly, CLPs from IL-7-deficient mice express lower amounts of early B cell factor (EBF) and Pax5 than wild-type CLPs, but similar amounts of GATA-3. Importantly, induced overexpression of EBF is sufficient to restore the B potential in these cells. These results indicate that IL-7 directs commitment of CLPs by modulating EBF expression. This is the first example of a cytokine influencing lymphoid lineage commitment in multipotent progenitors and highlights the relevance of the expression of a functional IL-7 receptor at the CLP stage.
Although caspase activation is critical for T cell proliferation following activation, the role of caspases in T cell differentiation is unclear. In this study, we have examined the effect of inhibition of caspases on the process of Th1/Th2 differentiation. Naive CD4+ T cells activated under neutral differentiation conditions in the presence of the pan caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (Z-VAD) fluoromethylketone showed increased Th2 cell differentiation concomitant with an up-regulation of GATA-3. Z-VAD induced optimal Th2 differentiation when T cells were stimulated under strong primary activation conditions. Treatment of naive CD4+ T cells with Z-VAD under strong activation conditions led to a 6-fold increase in IL-4 mRNA compared with control-treated T cells. The Z-VAD-induced increase in IL-4 transcription occurred within 24 h of activation and was independent of Stat6. IFN-gamma mRNA expression was not affected by Z-VAD at the 24-h time point. Z-VAD did not augment IL-4 expression from a committed Th2 cell, suggesting that caspases regulate IL-4 expression specifically during primary T cell activation. Z-VAD did not augment IL-12-driven Th1 differentiation. Activation of T cells in the presence of Z-VAD led to a specific increase in the expression of the transcription factor c-fos. Lastly, retrovirus-mediated expression of the antiapoptotic protein Bcl-2 resulted in an enhancement of Th2 cytokine expression, suggesting that inhibition of caspase activation by Bcl-2 can also modulate IL-4 expression. These findings reveal a novel regulatory mechanism of cytokine expression by caspases, and may explain how signaling pathways that inhibit apoptosis tend to promote Th2 differentiation.
Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-kappaB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-alpha and IFN-gamma and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.
ST2 is a member of the interleukin-1 receptor family and is expressed in type-2 T helper (Th2) cells. Here, we have studied the molecular mechanism responsible for the transcriptional regulation of the ST2 gene in Th2 cells using a mouse thymoma cell line, EL-4. The ST2 gene has distal and proximal promoters. ST2 mRNA was produced from the distal promoter in EL-4 cells stimulated with both phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP). The region of approximately 100 bp upstream of transcription start site, containing two GATA consensus sites, was indispensable for the activation of the distal promoter in reporter gene analysis. An electrophoretic mobility shift assay showed that transcription factor GATA-3 bound one of the GATA consensus sites (from -84 to -79) with nuclear extracts from PMA plus Bt2cAMP-stimulated EL-4 cells. The overexpression of GATA-3 enhanced the activity of the distal promoter. On the other hand, mutations of the GATA consensus site canceled out the enhancement by GATA-3. These data suggest that GATA-3 is an important transcription factor for the expression of the ST2 gene in Th2 cells.
The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of a member of the GATA-binding family of transcription factors, GATA3. This dual zinc finger transcription factor binds DNA with its C-terminal zinc finger (ZnF2) and stabilizes this binding with its N-terminal zinc finger (ZnF1). ZnF1 also interacts with other zinc finger proteins, notably Friend of GATA (FOG). The HDR syndrome has been described in patients with mutations affecting both ZnF1 and ZnF2 domains; the former result in inefficient interaction with FOG, and the latter result in disruption of DNA binding. We report a patient with renal failure, hypoparathyroidism, and bilateral hearing loss. Assessment of family members indicated that the disease arose as a de novo mutation in her mother. Analysis of GATA3 in the family revealed a heterozygous missense mutation resulting in a nonconservative change of a single amino acid (R276P) in the ZnF1 domain. Functional analysis using dissociation electrophoretic mobility shift and yeast two-hybrid assays showed reduced binding affinity to the GATA motifs but normal interaction with FOG in vitro. These results are consistent with the predicted functions of human GATA3-ZnF1 from three-dimensional molecular modeling and with HDR being a result of GATA3 haploinsufficiency.
CD94/NKG2A is an inhibitory receptor expressed by most human NK cells and a subset of T cells that recognizes HLA-E on potential target cells. To study the transcriptional regulation of the human NKG2A gene, we cloned a 3.9-kb genomic fragment that contains a 1.65-kb region upstream of the exon 1, as well as exon 1 (untranslated), intron 1 and exon 2. Using deletion mutants, we identified a region immediately upstream from the most upstream transcriptional initiation site that led to increased transcriptional activity from a luciferase reporter construct in YT-Indy (NKG2A positive) cells relative to Jurkat and K562 (both NKG2A negative) cells. We also localized a DNase I hypersensitivity site to this region. Within this 80-bp segment, we identified two GATA binding sites. Mutation of GATA binding site II (-2302 bp) but not GATA binding site I (-2332 bp) led to decreased transcriptional activity. Pull-down assays revealed that GATA-3 could bind oligonucleotide probes containing the wild type but not a mutated GATA site II. Using chromatin immunoprecipitation assays, we showed that GATA-3 specifically binds to the NKG2A promoter in situ in NKL and primary NK cells, but not in Jurkat T cells. Moreover, coexpression of human GATA-3 with an NKG2A promoter construct in K562 cells led to enhanced promoter activity, and transfection of NKL cells with small interfering RNA specific for GATA-3 reduced NKG2A cell surface expression. Taken together, our data indicate that GATA-3 is an important transcription factor for regulating NKG2A gene expression.
ICOS is the third member of the CD28 family molecules and plays a critical role in many T cell-dependent immune responses. Although accumulated data suggest that ICOS costimulatory signals play an important role in Th2-mediated immune responses, the molecular basis for this selective differentiation mechanism is largely unknown. To clarify this mechanism, we used DO11.10 TCR transgenic ICOS-/- mice and evaluated the nature of ICOS costimulatory signals during the process of Ag-specific activation and differentiation of naive CD4+ T cells. Results obtained from these experiments demonstrated that Ag stimulation of naive CD4+ T cells in the absence of an ICOS signal resulted in impaired Th2 development. Unlike previous reports, we found that primary IL-4 production by these T cells was intact and that IL-4R sensitivity of these T cells was reduced as evidenced by a profound defect in IL-4-induced Stat6 phosphorylation and the early induction of GATA-3. The fact that ICOS ligation of wild-type T cells significantly enhanced IL-4-induced Stat6 phosphorylation and primary GATA-3 induction, but not IL-4 transcription, of naive CD4+ T cells was consistent with the results obtained from ICOS-/- T cell experiments. These observations led us to propose that the predominant effect of ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4R-mediated signaling.
Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.
The hematopoietic stem cell (HSC) compartment is composed of long-term reconstituting (LTR) and short-term reconstituting (STR) stem cells. LTR HSC can reconstitute the hematopoietic system for life, whereas STR HSC can sustain hematopoiesis for only a few weeks in the mouse. Several excellent gene expression profiles have been obtained of the total hematopoietic stem cell population. We have used five-color FACS sorting to isolate separate populations of LTR and STR stem cell subsets. The LTR HSC has the phenotype defined as Lin- Sca+ Kit+ 38+ 34-; two subsets of STR HSC were obtained with phenotypes of Lin- Sca+ Kit+ 38+ 34+ and Lin- Sca+ Kit+ 38- 34+. The microarray profiling study reported here was able to identify genes specific for LTR functions. In the interrogated genes (approximately 12,000 probe sets corresponding to 8,000 genes), 210 genes are differentially expressed, and 72 genes are associated with LTR activity, including membrane proteins, signal transduction molecules, and transcription factors. Hierarchical clustering of the 210 differentially expressed genes suggested that they are not bone marrow-specific but rather appear to be stem cell-specific. Transcription factor-binding site analysis suggested that GATA3 might play an important role in the biology of LTR HSC.
Cell lineage specification depends on both gene activation and gene silencing, and in the differentiation of T helper progenitors to Th1 or Th2 effector cells, this requires the action of two opposing transcription factors, T-bet and GATA-3. T-bet is essential for the development of Th1 cells, and GATA-3 performs an equivalent role in Th2 development. We report that T-bet represses Th2 lineage commitment through tyrosine kinase-mediated interaction between the two transcription factors that interferes with the binding of GATA-3 to its target DNA. These results provide a novel function for tyrosine phosphorylation of a transcription factor in specifying alternate fates of a common progenitor cell.
The zebrafish is an established animal model system that profits from the availability of strong experimental approaches in both genetics and embryology. As a vertebrate, zebrafish can be used to model many aspects of human development and disease. GATA transcription factors play important roles in the development of many organ systems, including those for hematopoietic, cardiovascular, reproductive, and gut-endoderm derived tissues. The six vertebrate GATA factors are highly conserved in zebrafish at the level of sequence, expression pattern, and function. The identification of mutants, establishment of transgenic GFP reporter fish, and the ease of performing loss- and gain-of-function experiments have all contributed new insight into our understanding of the regulation and function of GATA factors. We review recent advances toward this goal using the zebrafish system with a focus on hematopoiesis and cardiogenesis, and suggest how comparative genetics using the zebrafish genes might reveal core conserved properties, as well as changes in gene function that reflect different morphogenetic programs utilized by various vertebrate embryos.
GATA factors regulate critical events in hematopoietic lineages (GATA-1/2/3), the heart and gut (GATA-4/5/6) and various other tissues. Transgenic approaches have revealed that GATA genes are regulated in a modular fashion by sets of enhancers that govern distinct temporal and/or spatial facets of the overall expression patterns. Efforts are underway to resolve how these GATA gene enhancers are themselves regulated in order to elucidate the genetic and molecular hierarchies that govern GATA expression in particular developmental contexts. These enhancers also afford a raft of tools that can be used to selectively perturb and probe various developmental events in transgenic animals.
Immune responses characterized by T H 2 type cells and IgE are important for the development of asthma and atopy. The transcription factors STAT6, GATA3, and STAT4 mediate the cytokine-induced development of naive CD4 + T cells into either T H 1 or T H 2 type.
We studied genetic variation of the STAT6, GATA3, and STAT4 genes and examined whether single nucleotide polymorphisms (SNPs) in these loci were associated with asthma or serum high IgE levels in the Finnish asthmatic families.
With denaturing high-performance liquid chromatography we screened all exons and exon-intron boundaries of the genes in 14 to 22 patients. All identified SNPs were genotyped in 120 nuclear families, and the haplotypes were analyzed by Haplotype Pattern Mining based statistical analysis. When potential association was observed, the analysis was replicated among 245 asthmatic patients and 405 population-based control subjects.
A total of 23 SNPs were identified, of which 8 were not previously listed in the SNP database. Interestingly, a haplotype analysis of GATA3 showed 3 related haplotypes that associated with different asthma and atopy related phenotypes among both the family and case-control data sets. For STAT6 and STAT4, no significant association to asthma or serum total IgE levels was observed.
We identified a panel of novel SNPs in genes coding for proteins important in the T H 1/T H 2 cell differentiation. SNPs of the GATA3 gene showed an initial association to asthma-related phenotypes. Elucidation of the importance of the identified panel of SNPs in other T H 1/T H 2 mediated diseases will be of great interest.
We have previously demonstrated that GATA-2 and GATA-3 are expressed in adipocyte precursors and control the preadipocyte-to-adipocyte transition. Constitutive expression of both GATA-2 and GATA-3 suppressed adipocyte differentiation, partially through direct binding to the peroxisome proliferator-activated receptor gamma (PPARgamma) promoter and suppression of its basal activity. In the present study, we demonstrate that both GATA-2 and GATA-3 form protein complexes with CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta, members of a family of transcription factors that are integral to adipogenesis. We mapped this interaction to the basic leucine zipper domain of C/EBPalpha and a region adjacent to the carboxyl zinc finger of GATA-2. The interaction between GATA and C/EBP factors is critical for the ability of GATA to suppress adipocyte differentiation. Thus, these results show that in addition to its previously recognized function in suppressing PPARgamma transcriptional activity, interaction of GATA factors with C/EBP is necessary for their ability to negatively regulate adipogenesis.
Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.
The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified-one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left-right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFbeta3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFbetaR2, and TGFbetaR3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development.
Success in treating severe sepsis will require relevant tools to monitor the patient immunoinflammatory status. This study aimed to investigate the feasibility of measuring a panel of immunological mediator mRNAs in whole blood and to study their prognostic values in septic shock patients. At the onset of shock, compared to healthy volunteers, mRNA levels in septic shock patients were increased for IL-10, IL-1beta, and high mobility group B1 (HMGB1) and decreased for transforming growth factor beta 1, the Th1, and Th2 transcription factors, T-bet and GATA-3, respectively. Single parameter analysis highlighted an increased expression of IL-10 and HMGB1 mRNA in nonsurvivors and a significant rise over time of GATA3 in survivors. Combining the expression levels of four genes, hierarchical cluster analysis showed that up to 95% of the patients with a similar outcome displayed transcriptional similarities. These results illustrate both the potential of whole blood mRNA quantification assays and the interest of a multiparametric strategy to better stratify septic patients.
Multiple-sequence alignment analysis is a powerful approach for understanding phylogenetic relationships, annotating genes, and detecting functional regulatory elements. With a growing number of partly or fully sequenced vertebrate genomes, effective tools for performing multiple comparisons are required to accurately and efficiently assist biological discoveries. Here we introduce Mulan (http://mulan.dcode.org/), a novel method and a network server for comparing multiple draft and finished-quality sequences to identify functional elements conserved over evolutionary time. Mulan brings together several novel algorithms: the TBA multi-aligner program for rapid identification of local sequence conservation, and the multiTF program for detecting evolutionarily conserved transcription factor binding sites in multiple alignments. In addition, Mulan supports two-way communication with the GALA database; alignments of multiple species dynamically generated in GALA can be viewed in Mulan, and conserved transcription factor binding sites identified with Mulan/multiTF can be integrated and overlaid with extensive genome annotation data using GALA. Local multiple alignments computed by Mulan ensure reliable representation of short- and large-scale genomic rearrangements in distant organisms. Mulan allows for interactive modification of critical conservation parameters to differentially predict conserved regions in comparisons of both closely and distantly related species. We illustrate the uses and applications of the Mulan tool through multispecies comparisons of the GATA3 gene locus and the identification of elements that are conserved in a different way in avians than in other genomes, allowing speculation on the evolution of birds. Source code for the aligners and the aligner-evaluation software can be freely downloaded from http://www.bx.psu.edu/miller_lab/.
The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of naïve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Naïve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for naïve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when naïve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.
To test the efficacy of combined high-throughput analyses (HTA) in target gene identification, screening criteria were set using >fivefold difference by microarray and statistically significant changes (p<0.01) in SAGE and EST. Microarray analysis of two normal and seven breast cancer samples found 129 genes with >fivefold changes. Further SAGE and EST analyses of these genes identified four qualified genes, ERBB2, GATA3, AGR2, and ANXA1. Their expression pattern was validated by RT-PCR in both breast cell lines and tissue samples. Loss of ANXA1 in breast cancer was further confirmed at mRNA level by Human Breast Cancer Tissue Profiling Array and at protein level by immunohistochemical staining. This study demonstrated that combined HTA effectively narrowed the number of genes for further study, while retaining the sensitivity in identifying biologically important genes such as ERBB2 and ANXA1. A distinctive loss of ANXA1 in breast cancer suggests its involvement in maintaining normal breast biology.
To evaluate the relationship between GATA-3 and IL-4, IL-5 in patients with allergic rhinitis.
The expression of GATA-3 was detected by immunochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) in 23 patients with allergic rhinitis. IL-4, IL-5 were detected by enzyme-linked immunosorbent assay (ELISA).
The positive rate of GATA-3 was 86.9% (20/23) and the relative density ratio of GATA-3 and glyceraldehyde phosphate dehydrogenase (GAPDH) were 0.602 +/- 0.11 in patients with allergic rhinitis. The concentrations of IL-4, IL-5 were (135.5 +/- 66.4) pg/mg, (77.5 +/- 29.4) pg/mg, respectively. The expressions of GATA-3 and IL-4, IL-5 had positive relationship (r = 0.45 and 0.62, P < 0.05).
GATA-3 contributed to the production of IL-4, IL-5 in patients with allergic rhinitis.
BALB/c mice immunized with cruzipain, a major Trypanosoma cruzi antigen, produce specific and autoreactive immune responses against heart myosin, associated with cardiac functional and structural abnormalities. Preferential activation of the Th2 phenotype and an increase in cell populations expressing CD19+, Mac-1+ and Gr-1+ markers were found in the spleens of these mice. The aim of the present study was to investigate whether cardiac autoimmunity could be induced by cruzipain immunization of C57BL/6 mice and to compare the immune response elicited with that of BALB/c mice. We demonstrate that immune C57BL/6 splenocytes, re-stimulated in vitro with cruzipain, produced high levels of IFNgamma and low levels of IL-4 compatible with a Th1 profile. In contrast to BALB/c mice, spleens from cruzipain immune C57BL/6 mice revealed no significant changes in the number of cells presenting CD19+, Mac-1+ and Gr-1+ markers. An increased secretion of TGFbeta and a greater number of CD4+ TGFbeta+ cells were found in immune C57BL/6 but not in BALB/c mice. These findings were associated with the lack of autoreactive response against heart myosin and a myosin- or cruzipain-derived peptide. Thus, the differential immune response elicited in C57BL/6 and BALB/c mice upon cruzipain immunization is implicated in the resistance or pathogenesis of experimental Chagas' disease.
SAP is an adaptor mutated in X-linked lymphoproliferative disease. It plays a critical role in T helper 2 (T(H)2) cytokine production. This function was suggested to reflect the capacity of SAP to associate with SLAM family receptors and enable tyrosine phosphorylation signaling by these receptors through SAP-mediated recruitment of Src-related kinase FynT. Here, we addressed by genetic means the importance of the SAP-FynT interaction in normal T cell functions. By creating a mouse in which the FynT binding site of SAP was inactivated in the germ line (sap(R78A) mouse) and by analyzing mice lacking SAP, FynT or SLAM, evidence was obtained that the SAP-FynT cascade is indeed crucial for normal T(H)2 functions in vitro and in vivo. These data imply that SAP is necessary for T(H)2 cytokine regulation primarily as a result of its capacity to recruit FynT. They also establish a previously unappreciated role for FynT in SAP-dependent T(H)2 cytokine regulation.
XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.
The linked IL-4 and IL-13 cytokine genes, which are activated and silenced in T helper (Th) 2 and Th1 cells, respectively, are flanked by the equivalently expressed RAD50 and KIF3A genes. A scan of DNase I hypersensitivity and DNA methylation across approximately 100 kb of the KIF3A/IL-4/IL-13/RAD50 cluster revealed differences in chromatin structure between Th1 and Th2 cells at the 3' end of the RAD50 gene, a region previously shown to contain a locus control region (LCR) regulating Th2-specific expression of IL-4 and IL-13. Naive CD4 T cells did not exhibit any DNase I hypersensitivity in this region, but stimulation under either Th1 or Th2 conditions caused rapid development of three hypersensitive sites. An additional hypersensitive site developed rapidly only under Th2 conditions, through a mechanism dependent on signal transducers and activators of transcription 6 (STAT6) but not GATA3. Our data point to a physical separation in the actions of STAT6 and its downstream effector GATA3 during Th2 differentiation: STAT6 directly remodels the RAD50 LCR, whereas GATA3 acts only in the vicinity of the IL-4 gene. We suggest that the RAD50 LCR has a complex and dual role in Th1 and Th2 differentiation, communicating early T cell antigen receptor and cytokine signals to the IL-4/IL-13 locus in both differentiating cell types.
To determine the effect of anterior chamber associated immune deviation (ACAID) on endotoxin-induced uveitis (EIU) and the possible mechanism.
ACAID animal model was induced by injection of 5 microl IRBP (10 microg/microl) into the anterior chamber (AC) of Spar-Dawley (SD) rats. Then 200 microg LPS was injected into hind footpads at different time points after AC inoculation. The animals were randomly divided into 3 groups: positive control (LPS injection only), 3 d group (LPS injection 3 days after IRBP inoculation), 7 d group (LPS injection 7 days after IRBP inoculation). Delayed type hypersensitivity (DTH) was examined to determine the development of ACAID. Then the serum level of IL-10 was evaluated by ELISA, and GATA-3 expression at the different time points after IRBP injection was assayed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot on mRNA and protein level respectively. The ocular inflammation was observed clinically; at the same time, the eye was extirpated and histological examination was performed.
In control and 3 d groups, significant DTH reaction was induced, but the serum level of IL-10 could not be detected and GATA-3 expression was not increased. While in 7 d group, the DTH reaction could not be induced, and IL-10 and GATA-3 expression increased significantly at both the mRNA and protein levels. The clinical manifestation was significantly alleviated in the 7 d group; Histological examination displayed that the inflammatory cells were significantly reduced in iris/ciliary body, anterior and posterior chambers, vitreous body and retina of the rats in 7 d group.
The development of ACAID can reduce the ocular inflammation induced by LPS, that is related to the increase of GATA-3 and IL-10 expression.
Gata2 and Gata3 belong to the Gata family of transcription factors in vertebrates that bind to a consensus "GATA" DNA sequence. The Gata3 gene is one of the earliest markers for the developing mouse inner ear. Ear morphogenesis is blocked in Gata3-deficient embryos, whereas nothing was known of the role of Gata2 in mouse inner ear. Here, we have compared the expression patterns of Gata2 and Gata3 during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 has been inactivated. The expression of the two Gata genes is highly overlapping at embryonic day (E)10.5 but becomes increasingly distinct later. Whereas Gata2 is predominantly expressed in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. No phenotypic abnormalities were observed in the inner ear of Gata2-/- embryos before lethality at E10.5 and Gata3 expression was unchanged. In contrast, a delay and strong reduction of Gata2 expression was detected in Gata3-/- otic epithelium.
Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.
Originally defined by their patterns of cytokine production, Th1 and Th2 cells have been described more recently to express other genes differentially as well, at least in vitro. In this study we compared the expression of Th1- and Th2-associated genes directly during in vivo sensitization to ovalbumin (OVA) in Th1- and Th2-polarized models of airways inflammation. Th1-polarized airway inflammation was achieved by the intranasal instillation of adenoviral vectors (Ad) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-12, followed by daily aerosolizations of OVA; instillation of Ad/GM-CSF alone with OVA aerosolization led to Th2-polarized responses. Lymph nodes were obtained at various time-points, RNA extracted, and analysed by real-time quantitative polymerase chain reaction (PCR). Consistent with reports from in vitro and human studies, mice undergoing Th1-polarized inflammation showed preferential expression of the transcription factor t-bet, the chemokines IFN-gamma inducible protein (IP)-10 and macrophage inflammatory protein 1 alpha (MIP-1-alpha), and the chemokine receptor CCR5. In contrast, the transcription factor GATA-3, the chemokines I-309 and thymus and activation regulated chemokine (TARC), and the chemokine receptors CCR3 and CCR4 were preferentially expressed in the Th2 model. Importantly, we also show that Ad/transgene expression remains compartmentalized to the lung after intranasal instillation. Flow cytometric analysis of lung myeloid dendritic cells indicated that B7.1 was expressed more strongly in the Th1 model than in the Th2 model. These studies provide a direct comparison of gene expression in in vivo Th1- and Th2-polarized models, and demonstrate that molecular events in the lymph nodes can be altered fundamentally by cytokine expression at distant mucosal sites.
IL-13 is considered to be a key modulator in the pathogenesis of Th2-induced allergic inflammation, although little is known about the regulation of IL-13 transcription in mast cells. In T cells, involvement of GATA-3 in cell type-specific expression of the IL-13 gene has been reported. However, the mechanisms that induce rapid transactivation of the IL-13 gene in response to various types of stimulation have hitherto remained unknown. In this report, we describe our investigation of the promoter region necessary for IL-13 transcription; we have found that both AP-1 and GATA proteins are indispensable for IL-13 transcription in mouse mast cells. In our investigation, we focused on the functional interaction between GATA and AP-1 in the IL-13 promoter context. Transfection experiments have revealed that GATA-1 and GATA-2 proteins are able to associate with AP-1 proteins. We have also shown that overexpression of GATA-1 induced excess AP-1 binding to the IL-13 promoter as well as a significant increase in IL-13 production in mast cells. The results of the present study have shown that direct interaction between AP-1 and GATA proteins plays an important role in IL-13 transcription in mast cells.
The potency of TCR signaling during primary CD4+ T cell activation influences initial cytokine expression patterns and subsequent polarization toward either Th1 or Th2 subsets. In this study, we demonstrate that the T cell inhibitor placental protein 14 (PP14; glycodelin) preferentially inhibits Th1 cytokine responses and chemokine expression when present during ex vivo priming of CD4+ T cells. PP14 synergizes with exogenously added IL-4 in skewing T cell responses. Significantly, PP14 impairs the down-regulation of GATA-3 transcriptional regulator expression that normally accompanies T cell activation, which is a prerequisite for Th1 development. Taken together, these data document for the first time the ability of PP14 to skew Th responses.
CD4 T cell differentiation is a complex process affected by many transcription factors interacting in a tightly regulated manner. We have previously shown that CIITA-deficient mouse Th1 cells expressed Th2-type cytokines, while IFN-gamma expression was normal. In this study, we show that CIITA-deficient Th1 cells contain three distinct populations: cells secreting IL-4 alone, IFN-gamma alone, and both IL-4 and IFN-gamma together. This novel phenotype is stable over multiple rounds of stimulation in the presence of Th1-inducing factors. CIITA-deficient Th1 cells require TCR-mediated signaling to express Th2 cytokines, and this occurs with similar kinetics as wild-type Th2 cells. Both GATA-3 and IL-4 appear to be required for CIITA-deficient Th1 cells to express Th2-type cytokines. Interestingly, however, CIITA-deficient Th1 cells can produce IL-4 in the absence of exogenous IL-4. Introducing either CIITA or antisense GATA-3 during Th1 differentiation partially reduces Th2-type cytokine expression. With the exception of Th2-type cytokine expression, Th1 differentiation occurs normally in the absence of CIITA, as measured by expression of T-bet, IL-12Rbeta2, IL-18Ralpha, and IFN-gamma. Therefore, CIITA plays a key role to repress Th2-type cytokine expression as naive CD4 T cells differentiate toward the Th1 lineage.
The Human T-cell leukemia virus type I (HTLV-I) non-structural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T-cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, T helper2 (Th2) differentiation, and a role of GATA3 during the immune response have been reported. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and the GATA3 promoter and repressor regions. Results demonstrated an interaction between Tax and the GATA3 promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).
To address how heritable patterns of gene expression are acquired during the differentiation of Th1 and Th2 cells, we analyzed the nuclear position of lineage-restricted cytokine genes and their upstream regulators by 3-dimensional fluorescence in situ hybridization. During Th1 differentiation, GATA-3 and c-maf loci, which encode upstream regulators of Th2 cytokines, were progressively repositioned to centromeric heterochromatin as defined by a gamma-satellite repeat probe and/or the nuclear periphery, compartments that have been associated with transcriptional repression. A third transcription factor locus, T-bet, which controls Th1-specific programs, was subject to de novo CpG methylation in a Th2 cell clone. In contrast, we did not find repositioning of the cytokine gene loci IL-2, IL-3, IL-4 or IFN-gamma during T helper cell differentiation. Instead, IFN-gamma was constitutively associated with the nuclear periphery, even when primed for expression in Th1 cells. Our results suggest that Th1/Th2 lineage commitment and differentiation involve repositioning of the regulators of cytokine expression, rather than the cytokine genes themselves.
To review potential or current therapies that decrease IgE synthesis or effects.
Relevant literature in peer-reviewed journals and abstracts from national meetings.
Key articles were selected by the authors.
Modulation of IgE-mediated diseases can occur at several levels. Transcription factors may be altered to differentiate lymphocytes into a TH1 phenotype, thus decreasing TH2-driven IgE production. This may be accomplished by inhibiting GATA-3 with peroxisome proliferator-activated receptor agonists or promoting T-bet expression with CpG motifs. Inhibiting IgE-promoting cytokines may be accomplished by blocking the effects or synthesis of interleukin 4 (IL-4) or IL-13 by suplatast tosilate. Cytokine therapy with anti-IL-4 or anti-IL-13 has the potential to directly influence IgE-mediated diseases, but strategies aimed at IL-4 alone have been disappointing. Clinical trials with interferon-gamma or IL-12, 2 cytokines important in promoting TH1 and inhibiting TH2 responses, have been fraught with adverse effects that make their use limited. The use of plasmids encoding interferon-gamma or IL-12 has shown promise in animal models. Inhibition of IgE synthesis has been demonstrated with anti-CD23 antibodies. Early human studies have been very encouraging, and larger studies are under way. The only IgE immunomodulator currently available for use is omalizumab. Omalizumab is effective for allergic asthma in children and adults.
Newer therapies hold great promise for the future treatment of allergic respiratory diseases, but clinical trials are necessary to accurately evaluate risk-benefit ratios of IgE immunomodulators.
Expression of the transcription factor GATA-3 is strongly associated with T helper type 2 (T(H)2) differentiation, but genetic evidence for its involvement in this process has been lacking. Here, we generated a conditional GATA-3-deficient mouse line. In vitro deletion of Gata3 diminished both interleukin 4 (IL-4)-dependent and IL-4-independent T(H)2 cell differentiation; without GATA-3, T(H)1 differentiation occurred in the absence of IL-12 and interferon-gamma. Gata3 deletion limited the growth of T(H)2 cells but not T(H)1 cells. Deletion of Gata3 from established T(H)2 cells abolished IL-5 and IL-13 but not IL-4 production. In vivo deletion of Gata3 using OX40-Cre eliminated T(H)2 responses and allowed the development of interferon-gamma-producing cells in mice infected with Nippostrongylus brasiliensis. Thus, GATA-3 serves as a principal switch in determining T(H)1-T(H)2 responses.
NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-gamma) and Th2 (IL-4) responses following stimulation of their invariant Valpha14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Valpha14 ligand alpha-galactosylceramide (alpha-GalCer) remains unclear. To address this issue, LFA-1-/- (CD11a-/-) mice were used to investigate IL-4 and IFN-gamma production by NKT cells following alpha-GalCer stimulation. Intriguingly, LFA-1-/- mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to alpha-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in alpha-GalCer-activated NKT cells from LFA-1-/- mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.
NK cells differentiate into either NK1 or NK2 cells that produce IFN-gamma or IL-5 and IL-13, respectively. Little is known, however, about the molecular mechanisms that control NK1 and NK2 cell differentiation. To address these questions, we established an in vitro mouse NK1/NK2 cell differentiation culture system. For NK1/NK2 cell differentiation, initial stimulation with PMA and ionomycin was required. The in vitro differentiated NK2 cells produced IL-5 and IL-13, but the levels were 20 times lower than those of Th2 or T cytotoxic (Tc)2 cells. No detectable IL-4 was produced. Freshly prepared NK cells express IL-2Rbeta, IL-2RgammaC, and IL-4Ralpha. After stimulation with PMA and ionomycin, NK cells expressed IL-2Ralpha. NK1 cells displayed higher cytotoxic activity against Yac-1 target cells. The levels of GATA3 protein in developing NK2 cells were approximately one-sixth of those in Th2 cells. Both NK1 and NK2 cells expressed large amounts of repressor of GATA, the levels of which were equivalent to CD8 Tc1 and Tc2 cells and significantly higher than those in Th2 cells. The levels of histone hyperacetylation of the IL-4 and IL-13 gene loci in NK2 cells were very low and equivalent to those in naive CD4 T cells. The production of IL-5 and IL-13 in NK2 cells was found to be STAT6 dependent. Thus, similar to Th2 cells, NK2 cell development is dependent on STAT6, and the low level expression of GATA3 and the high level expression of repressor of GATA may influence the unique type 2 cytokine production profiles of NK2 cells.
Differentiation of uncommitted T cells into Th1 and Th2 subpopulations depends on both intracellular events controlling expression of transcription factors T-bet and GATA-3 and interactions between cells mediated by cytokines, particularly IL4 and IFNgamma. A great deal is known about the intracellular and extracellular events involved in Th1 and Th2 (Th) differentiation, but how these are integrated in T-cell populations or indeed why extracellular cytokine control is required after a decision has been made at a transcriptional level is not at all understood. We present a mathematical model of CD4+ T-cell differentiation that describes both intracellular and extracellular processes and the interactions between them. It shows how antigen stimulation in conjunction with cytokines and other extracellular signals gives rise to rapid, reversible and mutually exclusive expression of T-bet or GATA-3 due to feedback between the transcription factors and their signalling pathways. After transient signalling by APC, continued Th1 and Th2 differentiation is shown to require cytokine production by the proliferating T cells. Moreover, intercellular communication by T-cell-derived cytokines lowers the threshold of APC signals required for Th differentiation. This provides an explanation for enhanced Th differentiation by pre-existing memory T cells. The model also predicts that Th differentiation can be reversed at the single cell level before commitment by manipulating the cytokine environment. It suggests a mechanism for switching between Th1 and Th2 in the so-called irreversible state that may be developed as a novel therapeutic means of manipulating Th1 and Th2 responses.
Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.
The T helper type 2 (T(H)2) locus control region is important in the regulation of the genes encoding the cytokines interleukins 4, 5 and 13. Using the chromosome conformation capture technique, we found that in T cells, natural killer cells, B cells and fibroblasts, the promoters for the genes encoding T(H)2 cytokines are located in close spatial proximity, forming an initial chromatin core configuration. In CD4(+) T cells and natural killer cells, but not B cells and fibroblasts, the T(H)2 locus control region participates in this configuration. The transcription factors GATA3 and STAT6 are essential for the establishment and/or maintenance of these interactions. Intrachromosomal interactions in the T(H)2 cytokine locus may form the basis for the coordinated transcriptional regulation of cytokine-encoding genes by the T(H)2 locus control region.
Although a number of transcription factors (TFs) have been identified that play a pivotal role in the development of hematopoietic lineages, only little is known about factors that may influence development and lineage commitment of natural killer (NK) or NK-like T (NKT)-cells. Obviously to fully appreciate the NK- and NKT-cell differentiation process, it is important to identify and characterize the TFs effecting the NK- and NKT-cell lineage. Furthermore, these TFs may play a role in NK- or NKT-cell leukemias, in which the normal differentiation program is presumably disturbed. The present study analyzed the expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3, IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK-cell lines together with 5 malignant NKT-cell lines, 5 T-cell acute lymphoblastic leukemia (ALL) cell lines including 3 gamma/delta T-cell receptor (TCR) type and 2 alpha/beta TCR type, and 3 B-cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1, IKAROS and IRF1 were found to be positive for all cell lines tested whereas GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative for all cell lines tested except in the three positive BCP-cell lines. GATA2 was positive for 3/5 T-cell lines but negative for the other cell lines. GATA3 was positive for 7/7 NK-, 4/5 NKT-, 5/5 T- and 2/3 BCP-cell lines. TBET was positive for all NK- and NKT-cell lines and negative for all T- and BCP-cell lines except one BCP-cell line. In contrast to the expression of TBET, TCF1 was negative for all NK- and NKT-cell lines, being positive for 4/5 T- and 1/3 BCP-cell lines. Expression analysis of TFs revealed that NK- and NKT-cell lines showed identical profiles, clearly distinct from those of the other T-ALL or BCP-ALL leukemia-derived cell lines..
GATA3 is an essential transcription factor that was first identified as a regulator of immune cell function. In recent microarray analyses of human breast tumors, both normal breast luminal epithelium and estrogen receptor (ESR1)-positive tumors showed high expression of GATA3. We sequenced genomic DNA from 111 breast tumors and three breast-tumor-derived cell lines and identified somatic mutations of GATA3 in five tumors and the MCF-7 cell line. These mutations cluster in the vicinity of the highly conserved second zinc-finger that is required for DNA binding. In addition to these five, we identified using cDNA sequencing a unique mis-splicing variant that caused a frameshift mutation. One of the somatic mutations we identified was identical to a germline GATA3 mutation reported in two kindreds with HDR syndrome/OMIM #146255, which is an autosomal dominant syndrome caused by the haplo-insufficiency of GATA3. The ectopic expression of GATA3 in human 293T cells caused the induction of 73 genes including six cytokeratins, and inhibited cell line doubling times. These data suggest that GATA3 is involved in growth control and the maintenance of the differentiated state in epithelial cells, and that GATA3 variants may contribute to tumorigenesis in ESR1-positive breast tumors.
Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5-/- pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5-/- progenitors efficiently differentiate in vitro into CD4+CD8+ alphabeta and gammadelta T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5-/- progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRbeta, gamma, and delta loci in the correct temporal sequence. Notch-stimulated Pax5-/- progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same time, Notch signaling rapidly induces cell surface expression of the c-Kit receptor and transcription of the target genes Deltex1 and pre-Talpha concomitant with the activation of TCR Vbeta germline transcription and the regulatory genes GATA3 and Tcf1. These data suggest that Notch1 acts upstream of GATA3 and Tcf1 in early T cell development and regulates Vbeta-DJbeta rearrangements by controlling the chromatin accessibility of Vbeta genes at the TCRbeta locus.
Chromosome 10p terminal deletions have been associated with a DiGeorge like phenotype. Haploinsufficiency of the region 10p14-pter, results in hypoparathyroidism, sensorineural deafness, renal anomaly, that is the triad that features the HDR syndrome. Van Esch (2000) identified in a HDR patient, within a 200 kb critical region, the GATA3 gene, a transcription factor involved in the embryonic development of the parathyroids, auditory system and kidneys. We describe a new male patient, 33-year-old, with 10p partial deletion affected by hypocalcemia, basal ganglia calcifications and a severe autistic syndrome associated with mental retardation. Neurologically he presented severe impairment of language, hypotonia, clumsiness and a postural dystonic attitude. A peripheral involvement of auditory pathways was documented by auditory evoked potentials alterations. CT scan documented basal ganglia calcifications. Hyperintensity of the lentiform nuclei was evident at the MRI examination. Renal ultrasound scan was normal. Haploinsufficiency for GATA3 gene was documented with FISH analysis using cosmid clone 1.2. Phenotypic spectrum observed in del (10p) is more severe than the classical DGS spectrum. GATA3 has been found to regulate the development of serotoninergic neurons. A serotoninergic dysfunction may be linked with autism in this patient.
Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 and Phox2a. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and Phox2a expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, Phox2a, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development.
Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1beta or TNF-alpha treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1beta or TNF-alpha in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1beta or TNF-alpha. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.
The active metabolite of vitamin D (1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))) is known to modulate the immune response in Th1 cell-directed diseases. To investigate the role of vitamin D in Th2 cell-directed diseases, experimental allergic asthma was induced in vitamin D receptor (VDR) knockout and in wild-type (WT) mice. As expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils, elevated Th2 cytokine levels, mucous production, and airway hyperresponsiveness. The administration of 1,25(OH)(2)D(3) had no effect on asthma severity. The only discernable effect of 1,25(OH)(2)D(3) on experimental allergic asthma in WT mice was an increased expression of two Th2-related genes (soluble CD23 and GATA-3) in lungs of BALB/c mice exposed to Ag through the nasal route only. By contrast, asthma-induced VDR knockout mice failed to develop airway inflammation, eosinophilia, or airway hyperresponsiveness, despite high IgE concentrations and elevated Th2 cytokines. The data suggest that although 1,25(OH)(2)D(3) induced these Th2-type genes, the treatment failed to have any affect on experimental asthma severity. However, VDR-deficient mice failed to develop experimental allergic asthma, suggesting an important role for the vitamin D endocrine system in the generation of Th2-driven inflammation in the lung.
The retina is a neural tissue especially differentiated for vision and, thus, the inner blood-retinal barrier (inner BRB) specific molecules may play an essential role in maintaining neural functions in the retina. The purpose of the present study was to identify selectively expressed genes at the inner blood-retinal barrier compared with the blood-brain barrier (BBB).
A comparison of expressed genes between conditionally immortalized rat retinal (TR-iBRB) cell lines and brain capillary endothelial (TR-BBB) cell lines was performed using mRNA differential display analysis and quantitative real time PCR analysis. The rat M-cadherin gene was cloned by performing 5' RACE, and its protein expression was detected by immunoblot analysis.
Eight clones were identified as highly expressed genes in TR-iBRB cells including GATA-binding protein-3 (GATA-3), cytosolic branched chain amino transferase (BCATc), and M-cadherin (cadherin-15). The rat M-cadherin gene was cloned from TR-iBRB cells, for the first time, and has >86% amino acid sequence identity to the previously cloned mammalian M-cadherins. Rat M-cadherin expression in TR-iBRB cells was much greater than that in TR-BBB cells as far as mRNA and protein levels were concerned.
M-cadherin, GATA-3, and BCATc are highly expressed in TR-iBRB cells compared with TR-BBB cells and may indeed be involved in unique functions at the inner BRB.
The lower jaw skeleton is derived from cephalic neural crest (CNC) cells that reside in the mandibular region of the first pharyngeal arch. Endothelin-A receptor (Ednra) signaling in crest cells is crucial for their development, as Ednra(-/-) mice are born with severe craniofacial defects resulting in neonatal lethality. In this study, we undertook a more detailed analysis of mandibular arch development in Ednra(-/-) embryos to better understand the cellular and molecular basis for these defects. We show that most lower jaw structures in Ednra(-/-) embryos undergo a homeotic transformation into maxillary-like structures similar to those observed in Dlx5/Dlx6(-/-) embryos, though lower incisors are still present in both mutant embryos. These structural changes are preceded by aberrant expansion of proximal first arch gene expression into the distal arch, in addition to the previously described loss of a Dlx6/Hand2 expression network. However, a small distal Hand2 expression domain remains. Although this distal expression is not dependent on either Ednra or Dlx5/Dlx6 function, it may require one or more GATA factors. Using fate analysis, we show that these distal Hand2-positive cells probably contribute to lower incisor formation. Together, our results suggest that the establishment of a 'mandibular identity' during lower jaw development requires both Ednra-dependent and -independent signaling pathways.
HDR syndrome is a newly recognized very rare syndrome defined by the combination of hypoparathyroidism, renal dysplasia and sensorineural deafness. Haplo-insufficiency of the GATA3 gene is the underlying mechanism of the HDR syndrome. Here we report a 16 year-old girl with HDR syndrome who presented with generalized psoriasis, which is an unusual presentation. Skin lesions rapidly disappeared after replacement therapy with calcium. It is suggested that generalized psoriasis might have been induced by hypocalcemia due to hypoparathyroidism associated with HDR syndrome.
To study interferon gamma (IFN gamma) production and the expression of T-bet and GATA-3, the transcription factors associated with Th1 and Th2, in peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis before and during infliximab treatment, so as to distinguish between a disease specific and a disease activity dependent defect.
Rheumatoid PBMC were obtained at weeks 0 and 6 of infliximab treatment and cultured for seven days with or without interleukin (IL)12 or the combination of IL12 and IL18. IFN gamma concentrations in supernatants were determined by ELISA. mRNA expression of IFN gamma, IL4, T-bet, and GATA-3 was determined by real time RT-PCR in whole blood at weeks 0 and 22.
A reduction in spontaneous IFN gamma production and in the response to Th1 inducing cytokines occurred in rheumatoid PBMC. Reduction of systemic inflammation with infliximab treatment increased IFN gamma production in response to IL12 or IL12+IL18. The IFN gamma/IL4 expression ratio of rheumatoid blood before treatment was lower than in healthy controls but was increased by infliximab treatment. T-bet expression or T-bet/GATA-3 ratio of rheumatoid blood was less than in controls. The T-bet/GATA-3 ratio was not influenced by infliximab treatment.
Regulation of T-bet and GATA-3 or IFN gamma and IL4 expression appeared different. The IFN gamma/IL4 ratio might express the blood Th1/Th2 balance better than the T-bet/GATA-3 ratio. Reduced IFN gamma production by rheumatoid PBMC and levels of IFN gamma and IL4 mRNA expression in blood were linked to disease improvement, indicating an association between this systemic Th1 feature and disease activity.
Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.
To clarify the functional state of T cells in Kawasaki disease, we analysed mRNA expression levels of Th1/Th2 cytokines (IFN-gamma and IL-4) along with Th1/Th2-inducing transcription factors, T-bet and GATA-3, which play pivotal roles in the development of Th1 and Th2 cells, respectively. By real-time PCR, IFN-gamma mRNA levels in peripheral blood mononuclear cells (PBMNC) were significantly decreased in Kawasaki disease patients compared with those with measles, and tended to be lower than those in healthy controls. T-bet mRNA levels were significantly decreased in patients with Kawasaki disease compared with healthy controls. In addition, IL-4 and GATA-3 mRNA levels were significantly decreased in Kawasaki disease compared with healthy controls. Regulatory cytokine mRNA levels (TGF-beta and IL-10) were also decreased in Kawasaki disease. The mRNA levels of IFN-gamma showed a significant positive correlation with those of T-bet in Kawasaki disease. These results suggest that the suppressed function of Th1 and Th2, associated with the suppression of both T-bet and GATA-3 gene expression, may be one of the immunological characteristics of Kawasaki disease.
The role of Stat6 (signal transducers and activators of transcription) in the recruitment and activation of eosinophils has been studied in detail in asthma and other allergic diseases. In this study, we demonstrated that eosinophil responses occur in a Stat6-independent manner in mice infected with the intestinal nematode, Nippostrongylus brasiliensis. Stat6-deficient (Stat6(-/-)) mice cannot expel N. brasiliensis and establish chronic infections. Prominent blood and intestinal eosinophilia were induced after day 14 postinfection (p.i.) and maintained at this level in Stat6(-/-) mice, whereas in wild-type mice eosinophil responses reached a peak on day 10 p.i. and declined thereafter. The introduction of a secondary infection of N. brasiliensis into wild-type mice induced rapid and exaggerated eosinophilia, whereas secondary infection in Stat6(-/-) mice resulted in almost the same eosinophil responses as those of the primary infection, suggesting a lack of memory responses. Blood eosinophilia was also induced in Stat6(-/-) mice implanted with N. brasiliensis in the small intestine, suggesting that intestinal exposure to parasitic antigen is sufficient to induce eosinophil responses. Furthermore, this prominent eosinophil response of Stat6(-/-) mice after day 14 was closely associated with an increase of interleukin (IL)-5 production in serum and intestine. Neither IL-4 nor eotaxin were significantly induced in Stat6(-/-) mice after infection with N. brasiliensis. We also found that mRNA for IL-5, GATA-3 and eosinophil peroxidase (EPO) are induced in the intestine of Stat6(-/-) mice on day 14 p.i. Taken together, these results provide evidence for Stat6-independent IL-5 production and subsequent eosinophil responses after chronic infection with N. brasiliensis.
Coordinated programs of gene expression during cell differentiation can be controlled by master transcription factors. The differentiation of helper T (Th) lymphocytes during the immune response has been shown to occur along alternative pathways designated as Th1 and Th2. Induction of the Th1 and Th2 pathways is associated with the conversely regulated expression of the master factors T-bet and GATA-3, respectively. Both autoactivation and inhibition of GATA-3 play a crucial role in this process. We develop mathematical models of the underlying regulatory networks to provide a framework for the analysis of experimental data. Modeling concepts for gene expression dynamics are introduced, and paradigms for the behavior of gene-regulatory networks are reviewed. A mechanistic model for the regulation of GATA-3 in Th cells is developed that accounts for autoactivation and regulation by external differentiation signals. This system works as a bistable switch that enables the triggering of a differentiation program by transient inductive signals. GATA-3 inhibitors (such as FOG-1 and ROG) modulate GATA-3 expression by yet unidentified mechanisms. Three potential modes of inhibition, sequestration by a binding protein, repression of basal transcription, and repression of autoactivation, are predicted to have distinct, and strongly concentration-dependent, regulatory effects on GATA-3 dynamics. Based on these results, we develop a model for the cross-regulation of the alternative Th1 and Th2 differentiation programs which are governed by the dynamics of T-bet and GATA-3, respectively. The steady states of this model correlate with naïve, Th1-polarized, and Th2-polarized phenotypes. Our analysis makes predictions on the stability of the Th1 and Th2 programs and raises questions on the relation between transcription factor regulation and epigenetic determination in cell differentiation.
Interleukin (IL)-4-induced STAT6 activation and the subsequent up-regulation of GATA3 are crucial for the induction of chromatin remodeling of the Th2 cytokine gene loci as Th2 cells undergo development. This study probes the role of these molecules in the maintenance of memory Th2 cells. IL-4 was not required to maintain the capability for Th2 cytokine production in in vivo generated antigen-specific memory Th2 cells. Histone H3-K9/14 hyperacetylation and intergenic transcripts associated with the IL-4 gene locus were preserved in the absence of IL-4, but those associated with the IL-13 gene were partially IL-4-dependent. Histone H3-K4 methylation of the IL-13 and IL-4 gene loci was fully preserved in memory Th2 cells and accompanied by memory cell-specific accumulation of Pol II complex to highly restricted sites. Thus, memory Th2 cells maintain a unique Th2-specific remodeled chromatin in the IL-4 and IL-13 gene loci by active molecular events that are IL-4-independent.
To identify the T helper cell predominant differentiation in asthmatic patients and to explore the modulation of T cell-specific transcription factors T-bet/GATA-3.
Thirty-two asthmatic patients were enrolled, among whom 18 were atopic defined by positive antigen skin test and 12 were children. Lymphocytes were isolated from peripheral blood and incubated with PHA (100 microg/ml) at 37 degrees C for 48 hours. INF-gamma and IL-4 concentrations in the supernatant were detected by ELISA. The T-bet and GATA-3 mRNA expression levels in lymphocytes were assayed by reverse transcription-polymerase chain reaction (RT-PCR) while the ratio of CCR3+ and CCR5+ cells in lymphocytes was counted by flow cytometry (FCM) after direct immunofluorescence staining.
IL-4 concentration in the lymphocyte supernatant of the asthmatic group was (118 +/- 25) microg/L, which was significantly elevated compared to that of the healthy control group (75 +/- 12) microg/L (P < 0.01). When subgroups of asthmatic patients were compared, the results showed that atopic subjects had a higher IL-4 level than non-atopic subjects [(126 +/- 23) microg/L vs (107 +/- 26) microg/L, P < 0.01], but no significant difference was demonstrated between adults and children [(118 +/- 25) micro g/L vs (121 +/- 25) microg/L, P > 0.05]. Significantly lower concentration of INF-gamma in the asthmatic group was detected as compared to the control [(651 +/- 85) microg/L vs (1 179 +/- 332) microg/L, P < 0.001]. The concentration of INF-gamma was higher in atopic subjects than in non-atopic subjects [(618 +/- 89) micro g/L vs (680 +/- 83) microg/L, P < 0.01], but no difference was found between adults and children. The percentage of CCR3+ cells in lymphocytes was (9.4 +/- 5.8)% in the asthmatic group and (4.9 +/- 2.3)% in the control (P < 0.05), while the percentages of CCR5+ cells was (6 +/- 7)% and (13 +/- 7)%, respectively (P < 0.05). RT-PCR revealed that T-bet mRNA expression levels were as follows: 0.13 +/- 0.03 in the asthmatic group and 0.18 +/- 0.04 in the control (P < 0.01); 0.120 +/- 0.030 in atopic subjects and 0.140 +/- 0.010 in the non-atopic subjects (P < 0.05); 0.120 +/- 0.020 in children and 0.130 +/- 0.020 in adults (P > 0.05). The levels of GATA-3 mRNA expression were 0.43 +/- 0.07 in asthma and 0.29 +/- 0.09 in the control (P < 0.01), however, no differences were found between atopic and non-atopic, children and adults (0.50 +/- 0.12 vs 0.40 +/- 0.10, 0.44 +/- 0.09 vs 0.43 +/- 0.07, respectively, P > 0.05). A positive correlation was found between concentration of INF-gamma and T-bet mRNA level (r=0.663, P < 0.01), while no correlation with GATA-3 mRNA expression was found. The concentration of IL-4 was negatively correlated with T-bet mRNA level (r=-0.250, P < 0.05) and positively with GATA-3 mRNA level (r=0.72, P < 0.01). It was interesting that a closer relationship existed between the ratio of T-bet to GATA-3 and the ratio of INF-gamma to IL-4 (r=0.873, P < 0.01).
In asthma there is a tendency of Th2 polarization with over-production of Th2-like cytokines in which T-bet deficiency may be a key factor. T-bet might direct T cells to Th1 differentiation while GATA-3 orientated Th2 maturation. Considering the fact that committed Th2 cells underwent re-differentiation induced by T-bet, this novel Th1-specific transcription factor is a fascinating target gene for modifying to restore the Th1 and Th2 balance.
The regulatory mechanism by which GATA-3 suppresses IFN-gamma gene expression was investigated. A reduction of GATA-3 using RNA interference technology enhanced, whereas overexpression of GATA-3 suppressed IFN-gamma mRNA expression. IL-4 expression was reciprocally affected by GATA-3. GATA-3-mediated down-regulation of IFN-gamma was achieved through the inhibition of its promoter/enhancer activity. Two GATA elements located in the cis-regulatory elements did not contribute to the suppression of IFN-gamma promoter activity, even though they behaved as binding sites for GATA-3. The effect of GATA-3 on IFN-gamma promoter was lost upon removal of the region encompassing -257 to -172. Among several transcription factors putatively interacting with this region, Stat4, which enhanced IFN-gamma promoter activity, was down-regulated by GATA-3 at gene transcription level. Although GATA-3 has the capacity to interact with the cis-regulatory elements, it suppresses IFN-gamma gene transcription via down-regulation of Stat4.
The paired box transcription factor, Pax2, is important for cochlear development in the mouse inner ear. Two mutant alleles of Pax2, a knockout and a frameshift mutation (Pax21Neu), show either agenesis or severe malformation of the cochlea, respectively. In humans, mutations in the PAX2 gene cause renal coloboma syndrome that is characterized by kidney abnormalities, optic nerve colobomas and mild sensorineural deafness. To better understand the role of Pax2 in inner ear development, we examined the inner ear phenotype in the Pax2 knockout mice using paint-fill and gene expression analyses. We show that Pax2-/- ears often lack a distinct saccule, and the endolymphatic duct and common crus are invariably fused. However, a rudimentary cochlea is always present in all Pax2 knockout inner ears. Cochlear outgrowth in the mutants is arrested at an early stage due to apoptosis of cells that normally express Pax2 in the cochlear anlage. Lack of Pax2 affects tissue specification within the cochlear duct, particularly regions between the sensory tissue and the stria vascularis. Because the cochlear phenotypes observed in Pax2 mutants are more severe than those observed in mice lacking Otx1 and Otx2, we postulate that Pax2 plays a key role in regulating the differential growth within the cochlear duct and thus, its proper outgrowth and coiling.
Helicobacter pylori infection leads to chronic gastric inflammation. The current study determined the response of human APCs, NK cells, and T cells toward the bacteria in vitro. Human monocyte-derived dendritic cells (DC) were incubated with bacteria for 48 h. Intact H. pylori at a multitude of infection 5 stimulated the expression of MHC class II (4- to 7-fold), CD80, and CD86 B7 molecules (10- to 12-fold) and the CD83 costimulatory molecule (>30-fold) as well as IL-12 secretion (>50-fold) in DCs, and thereby, strongly induced their maturation and activation. CD56(+)/CD4(-) NK cells, as well as CD4(+)/CD45RA(+) naive T cells, were isolated and incubated with DCs pulsed with intact bacteria or different cellular fractions. Coculture of H. pylori-pulsed DCs with NK cells strongly potentiated the secretion of TNF-alpha and IFN-gamma. Coculture of naive T cells with H. pylori-pulsed DCs significantly enhanced TNF-alpha, IFN-gamma, and IL-2 secretion as well as T-bet mRNA levels, while GATA-3 mRNA was lowered. However, the effect appeared attenuated compared with coculture with Escherichia coli. A greater stimulation was seen with naive T cells and DCs pulsed with H. pylori membrane preparations. Intact H. pylori potently induced the maturation and activation of human monocyte-derived DC and thereby promote NK and Th1 effector responses. The strong activation of NK cells may be important for the innate immune response. Th1-polarized T cells were induced especially by incubation with membrane preparations of H. pylori, suggesting that membrane proteins may account for the specific adaptive immune response.
Jawless fishes occupy a critical phylogenetic position in understanding the origin of the adaptive immune system. Here, we performed large-scale expressed sequence tag analysis of leukocytes isolated from the inshore hagfish Eptatretus burgeri. Although we found many immunity-related genes such as those involved in lymphocyte or hematopoietic cell signaling and development as well as cytokine and cytokine receptor genes, MHC molecules or antigen receptors were not identified. We characterized two hagfish cDNAs that closely resembled mammalian proteins with essential roles in adaptive immunity, one encoding a GATA3-like molecule and another encoding a Bruton's tyrosine kinase (Btk)-like molecule. The GATA3-like gene of hagfish was equidistant from GATA3 and GATA2 in jawed vertebrates. Similarly, the hagfish Btk-like molecule was not Btk itself, but qualified as a pre-duplicated form of Btk and Bmx in jawed vertebrates. In total, our work provides circumstantial evidence that adaptive immunity is unique to jawed vertebrates.
Patients with HDR syndrome suffer from hypoparathyroidism, deafness, and renal dysplasia due to a heterozygous deletion of the transcription factor GATA3. Since GATA3 is prominently expressed in both the inner ear and different parts of the auditory nervous system, it is not clear whether the deafness in HDR patients is caused by peripheral and/or central deficits. Therefore, we have created and examined heterozygous Gata3 knockout mice. Auditory brainstem response (ABR) thresholds of alert heterozygous Gata3 mice, analyzed from 1 to 19 months of age, showed a hearing loss of 30 dB compared to wild-type littermates. Neither physiological nor morphological abnormalities were found in the brainstem, cerebral cortex, the outer or the middle ear. In contrast, cochleae of heterozygous Gata3 mice showed significant progressive morphological degeneration starting with the outer hair cells (OHCs) at the apex and ultimately affecting all hair cells and supporting cells in the entire cochlea. Together, these findings indicate that hearing loss following Gata3 haploinsufficiency is peripheral in origin and that this defect is detectable from early postnatal development and maintains through adulthood.
Vasoactive intestinal peptide and its G protein-coupled receptors, VPAC(1) and VPAC(2), regulate critical aspects of innate and adaptive immunity. T cell VPAC(2)Rs mediate changes in cytokine generation, which potently increase the Th2/Th1 ratio and consequently shift the effector responses toward allergy and inflammation. To examine mechanisms of VPAC(2) promotion of the Th2 phenotype, we analyzed controls of IL-4 transcription in CD4 T cells from T cell-targeted VPAC(2) transgenic (Tg), VPAC(2) knockout, and wild-type (WT) mice. c-maf and junB mRNA, protein, and activity were significantly up-regulated to a higher level in TCR-stimulated CD4 T cells from Tg mice compared with those from knockout and WT C57BL/6 mice. In contrast, GATA3, T-bet, and NFATc levels were identical in WT and Tg CD4 T cells. Vasoactive intestinal peptide binding to VPAC(2) on CD4 T cells specifically induces an up-regulation of the Th2-type transcription factors c-Maf and JunB, which consequently enhances IL-4 and IL-5 production, leading to a Th2-type phenotype.
In breast tumours and breast cancer cell (BCC) lines, microarray analyses have revealed that a series of genes are expressed in close association with the oestrogen receptor-alpha (ER-alpha) gene, ESR1. Three of them, GATA3, HNF3A (also known as FOXA1), and XBP1 encode transcription factors. Here, we present these factors and we discuss their potential involvement in the ER-alpha-mediated actions in BCC. We notably show the relations that exist, or that might exist, between these factors and the oestrogen-inducible trefoil factor TFF1.
Interleukin-12 (IL-12) is a potent inducer of interferon-gamma production by T cells and is a major factor for the development of T-helper 1 (Th1) cells. It exerts its biological effects through binding to the IL-12 receptor (IL-12R), a heterodimer composed of a 1 and a beta2 subunits. The signaling beta2 chain is expressed on Th1 cells and to a lesser extent on Th0 cells, but not on Th2 cells, rendering these latter cells unresponsive to IL-12. Polymorphisms in the coding region of the IL-12Rbeta2 gene were shown to be associated with atopic disease. Here, we analyzed the 5'-regulatory region of the human IL-12Rbeta2 gene by denaturing high-performance liquid chromatography (Transgenomic WAVE system, San Jose, CA). We found five novel single-nucleotide polymorphisms (SNPs) in the proximal 1.2 kb IL-12Rbeta2 promoter region, i.e. -237C/T, -465A/G, -1023A/G, -1033T/C, and -1035A/G. SNP -465A/G is of particular interest as it determines the integrity of a GATA consensus site. By functional comparison of both -465 alleles in transient transfection assays, we show that promoter activity is increased in case of the -465G allele, disrupting the intact GATA site. Comparison of the prevalence of -465A/G SNP alleles in small cohorts of allergic asthmatic and healthy control individuals provided no evidence for an altered distribution in the asthmatic population. In conclusion, we have identified a novel polymorphic GATA site that may affect transciptional activity of the human IL-12Rbeta2 gene under GATA3-mediated, Th2-polarizing conditions.
Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer.
The transcriptional control of the differentiation of central serotonergic (5-HT) neurons in vertebrates has recently come under scrutiny and has been shown to involve the homeobox genes Nkx2-2 and Lmx1b, the Ets-domain gene Pet1 (also known as Fev) and the zinc-finger gene Gata3. The basic helix-loop-helix (bHLH) gene Ascl1 (also known as Mash1) is coexpressed with Nkx2-2 in the neuroepithelial domain of the hindbrain, which gives rise to 5-HT neurons. Here we show in the mouse that Ascl1 is essential for the birth of 5-HT neurons, both as a proneural gene for the production of postmitotic neuronal precursors and as a determinant of the serotonergic phenotype for the parallel activation of Gata3, Lmx1b and Pet1. Thus Ascl1, which is essential for noradrenergic differentiation, is also a determinant of the serotonergic phenotype.
An initial activation signal via the TCR in a restricted cytokine environment is critical for the onset of Th cell development. Cytokines regulate the expression of key transcriptional factors, T-bet and GATA-3, which instruct the direction of Th1 and Th2 differentiation, through changes in chromatin conformation. In this study, we investigated the kinetics of IL-4-mediated signaling in a transgenic mouse, expressing human IL-4R on a mouse IL-4alphaR-deficient background. These experiments, allowing induction with human IL-4 at defined times, demonstrated that an IL-4 signal was required at the early stage of TCR-mediated T cell activation for lineage commitment to Th2, along with structural changes in chromatin, which take place in the conserved noncoding sequence-1 and -2 within the IL-4 locus. At later times, however, IL-4 failed to promote efficient Th2 differentiation and decondensation of chromatin, even though GATA-3 was clearly induced in the nuclei by IL-4 stimulation. Moreover, IL-4-mediated Th2 instruction was independent from cell division mediated by initial TCR stimulation. The role of IL-4 signaling may have a time restriction during Th2 differentiation. In late stages of initial T cell activation, the chromatin structure of the IL-4 locus retains condensation state. These results demonstrate that IL-4-induced GATA-3 expression is time-restriction switch for Th2 differentiation.
The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.
Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1, Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclear beta-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.
GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division.
We report a novel approach to gene expression profiling using the Nottingham Prognostic Index (NPI) to stratify 26 patients with invasive breast carcinoma. As an aggregate index of parameters reflecting metastatic potential, growth rate, and genetic instability the NPI has distinct advantages over other clinicopathologic features used to segregate breast cancer patients. As a continuous variable it offers a responsive and sensitive means of modeling a continuum of clinical aggressiveness. Using RNA extracted from 26 tumors and cDNA microarrays with 23 343 unique genetic elements, 84 genes and expressed sequence tags were identified whose expression patterns correlated with NPI. Differential expression by immunohistochemistry (IHC) was also observed for two of three genes evaluated by this method. Correlation was determined by the Spearman rank correlation method with null distribution analysis. Among the 84 genetic elements were seven previously implicated in neoplastic progression (including the two demonstrating differential expression by IHC), 11 without specific cancer association but localized to chromosomal sites whose loss or gain has been identified in cytogenetic studies of breast carcinoma, and 73 not previously associated with breast carcinoma. Collectively, the expression patterns of these 84 elements have potential to distinguish high and low NPI patient samples. These data add support to the assertion that prognostic groups of breast carcinoma are reflected in distinguishable expression profiles of a limited set of genes.
Cellular FLIP long form (c-FLIP(L)) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIP(L) in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIP(L) with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIP(L)-transgenic mice. In this study we show that activated CD4(+) T cells from c-FLIP(L)-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIP(L)-transgenic CD4(+) T cells parallels impaired NF-kappa B activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-gamma and increased Th2 cytokines. The Th2 bias of c-FLIP(L)-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIP(L) can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.
Interleukin 5 (IL-5) plays a unique role in allergic inflammatory responses, and the understanding of molecular mechanisms underlying the generation of IL-5-producing cells is crucial for the regulation of allergic disorders. Differentiation of naive CD4 T cells into type-2 helper (Th2) cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13, and IL-5 genes. Histone hyperacetylation of the IL-5 gene displayed a delayed kinetics compared with that of the IL-4 and IL-13 genes, suggesting a distinct remodeling mechanism for the IL-5-gene locus. Here we studied the role of CD28 costimulation in the generation of IL-5-producing cells and the histone hyperacetylation of the IL-5 gene locus. CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus that appeared to be mediated through NF-kappaB activation and subsequent up-regulation of GATA3. The CD28 costimulation-sensitive histone hyperacetylation spanned almost the entire intergenic region between the IL-5 and RAD50 accompanied with intergenic transcript. Thus, this is the first demonstration that CD28 costimulation controls a chromatin-remodeling process during Th2 cell differentiation.
Atopic dermatitis is characterized by Th2-dominant immunity. Recently many intracellular molecules have been reported to regulate cytokine expression and T cell differentiation. GATA-3 and T-box expressed in T cells (T-bet) are transcription factors that play a critical role in the development of Th2 and Th1 immunity, respectively. Suppressor of cytokine signalling (SOCS)-3 and SOCS-5, are negative regulators of the cytokine signalling induced by IL-12 and IL-4, respectively. Txk is a transcription factor that activates IFN-gamma gene directly. The present study was designed to identify intracellular molecules that are responsible for the pathogenesis and the imbalance of cytokines in atopic dermatitis. Semi-quantitative RT-PCR revealed that in peripheral blood mononuclear cells without any stimulation the levels of mRNA for GATA-3 and SOCS-3 were elevated, the levels of mRNA for Txk were depressed and the levels of mRNA for T-bet and SOCS-5 were comparable in patients with atopic dermatitis as compared with healthy controls. In addition, successful therapy normalized levels of mRNA for GATA-3 and Txk, although those for the others including IL-4, IL-5, IL-10, IL-13 and IFN-gamma did not change. Levels of Txk mRNA correlated with those of IFN-gamma, while the mRNA levels of the other regulators did not correlate with those of any of the cytokines. These results suggest GATA-3 and Txk might be involved in skin lesions, while SOCS-3 might be associated with an imbalance of cytokines that is difficult to normalize in atopic dermatitis.
Differentiation of naïve CD4 T cells into T helper (Th) 2 cells requires signaling through the T cell receptor and an appropriate cytokine environment. IL-4 is critical for such Th2 differentiation. We show that IL-2 plays a central role in this process. The effect of IL-2 on Th2 generation does not depend on its cell growth or survival effects. Stat5a(-/-) cells show diminished differentiation to IL-4 production, and forced expression of a constitutively active form of Stat5a replaces the need for IL-2. In vivo IL-2 neutralization inhibits IL-4 production in two models. Studies of restriction enzyme accessibility and binding of Stat5 to chromatin indicate that IL-2 mediates its effect by stabilizing the accessibility of the Il4 gene. Thus, IL-2 plays a critical role in the polarization of naive CD4 T cells to the Th2 phenotype.
The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.
The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 --> Stop and Arg-367 --> Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 --> Arg and Asn-320 --> Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast two-hybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.
The mechanisms by which Th1 and Th2 cells inter-regulate in vivo are still poorly understood. In this study we examined the plasticity of Th1 cell differentiation and how Th2 cells may down-regulate these responses. We show here that IL-4 affects Th1 cell responses by two developmentally regulated mechanisms. During the commitment phase of naive CD4+ T cells, IL-4 inhibits Th1 cell differentiation and induces a reversion of developing Th1 cells to the Th2 lineage. In contrast, for effector Th1 cells IL-4 does not affect the developmental process, but only the transcription of the IFN-gamma gene. We further show that the difference in IL-4 responsiveness correlates with a loss, in effector Th1 cells, of IL-4-dependent up-regulation of GATA-3 expression despite normal activation of STAT6. Transient inhibition of IFN-gamma production by differentiated effector cells may explain why Th1 and Th2 responses can co-exist in vivo although Th2 effector cells dominate functionally, as observed in some infectious or autoimmune mice models.
IL-4 secreting and nonsecreting cells from Th2 cultures have a similar probability of producing IL-4 upon subsequent stimulation, implying that there is stochastic element in IL-4 production by stimulated Th2 cells. Purified IL-4 producers and nonproducers have similar Gata3 and c-maf mRNA expression. Il4 gene accessibility, analyzed by restriction enzyme accessibility (REA) at sites in the promoter, in the second intron (DNase I hypersensitivity sites HSII and HSIII) and in CNS-1 in the two populations was also similar. However, upon TCR stimulation, site VA, which is 5 kB 3' of exon 4, displayed a striking increase in accessibility but REA was 2- to 3-fold greater in producers than nonproducers. Cyclosporin A treatment inhibited VA opening, implying the involvement of NFAT in increased VA accessibility. Induction of VA accessibility is sensitive to cycloheximide, suggesting an additional factor(s) is needed. Thus, opening of VA is a probabilistic event determining which Th2 cells transcribe Il4.
Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. Here, we identify a Sonic hedgehog (Shh)-regulated cascade of transcription factors that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1), which project to the forebrain, but not for the induction of caudal 5-HT neurons, which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. Our findings reveal that the identity of closely related 5-HT subclasses occurs through distinct responses of adjacent rostrocaudal progenitor domains to broad ventral inducers.
Expansion of adipose tissue mass results from increased number and size of adipocyte cells. We hypothesized that subcutaneous abdominal preadipocytes in obese individuals might have an intrinsically higher propensity to differentiate into adipocytes. Thus we investigated the relationship between obesity and the level of in vitro preadipocyte differentiation in Pima Indians. Subcutaneous abdominal stromal vascular fractions containing preadipocytes were cultured from 58 nondiabetic subjects [31 M/27 F, 30 +/- 6 yr, body fat 34 +/- 8% by dual-energy X-ray absorptiometry (means +/- SD)]. The average percentage of preadipocyte differentiation (PDIFF; cell count by microscopy) was 11 +/- 11% (range 0.2-51%). PDIFF correlated negatively with percent body fat (r = -0.35, P = 0.006) and waist circumference (r = -0.45, P = 0.0004). Multiple regression analysis indicated that waist circumference (P = 0.01), sex (P = 0.01), and percent body fat (P = 0.05) were significant determinants of PDIFF. Molecular characterization of predifferentiated cultured cells was performed by real-time PCR measurements of glucocorticoid receptor-alpha (GRalpha), insulin-like growth factor I receptor (IGF-IR), peroxisome proliferator-activated receptor-gamma (PPARgamma), enhancer-binding protein GATA-3, CCAAT/enhancer-binding protein-alpha undifferentiated protein (CUP/AP-2alpha), and endothelial cell-specific marker 2 (ECSM2). The mRNA concentrations of GRalpha correlated with PDIFF (r = 0.29, P = 0.03), but the others did not (IGF-IR, r = 0.003, P = 1.0; PPARgamma, r = -0.1, P = 0.5; GATA-3, r = 0.02, P = 0.9; CUP/AP-2alpha, r = -0.2, P = 0.1; ECSM2, r = 0.04, P = 0.7). Contrary to our hypothesis, the results may indicate a blunted in vitro differentiation potential of preadipocytes in centrally obese individuals. The lower differentiation potential of preadipocytes in the obese subjects might be due, at least partly, to decreased glucocorticoid receptor expression.
T helper type 2 (Th2) cells secrete IL-4, IL-5, IL-10, and IL-13 and mediate allergic and asthmatic disease. GATA-3 is a Th2-specific transcription factor that appears in overexpression studies and transgenic systems to function as a Th2 lineage determinant. Because GATA-3 is also crucial for development of the T lineage and throughout thymic development, direct demonstration that GATA-3 is required for Th2 development by targeted deletion has been lacking. Using a conditional knockout approach, we found that GATA-3 is required for optimal Th2 cytokine production in vitro and in vivo. Our data also show that GATA-3 expression must be sustained to maintain the Th2 phenotype.
T(H)2 cytokines play a central role in the pathogenesis of allergic asthma. We previously showed that the "antiasthma" Chinese herbal formula MSSM-002 exhibited therapeutic effects on established allergic airway responses in a murine model of allergic asthma. However, the mechanisms underlying these effects are largely unknown.
The objective of this study was to determine whether and how MSSM-002 modulates an established T(H)2 response and whether the actions of MSSM-002 on T(H)2 cell differs from corticosteroids.
T(H)2 polarized splenocytes (T(H)2-SPCs) from mice with antigen-induced airway hyperresponsiveness and T(H)2 cloned cells, D10 G4.1 (D10), were cultured in the presence or absence of antigen with or without MSSM-002 and dexamethasone, and the proliferative responses and cytokine profiles were determined. Apoptosis and T(H)2 transcription factor GATA-3 expression and binding to IL-4 gene promoter and V(A) enhancer in MSSM-002-treated D10 cells were also determined.
MSSM-002 significantly decreased antigen-induced proliferation and IL-4 and IL-5 production but increased IFN-gamma production by T(H)2-SPCs, whereas dexamethasone suppressed IFN-gamma as well as IL-4 and IL-5. Anti-IL-12 antibody, although abrogating MSSM-002 induction of IFN-gamma, had no significant effect on MSSM-002 suppression of IL-4 and IL-5 secretion. MSSM-002 also suppressed T(H)2 cytokine secretion by D10 cells, and in contrast to dexamethasone, MSSM-002 did not induce apoptosis of D10 cells. MSSM-002 markedly suppressed GATA-3 mRNA and protein expression and the binding to IL-4 gene promoter and V(A) enhancer in D10 cells.
MSSM-002, in contrast to the overall suppression of T cells by dexamethasone, exhibits immunomodulatory actions on T(H)2 cells caused, at least partially, by downregulation of GATA-3.
The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell-mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/- individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell-mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo.
We have reported that injection of marijuana cannabinoids, such as Delta(9)-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) gamma production and enhancement of IL-4 production suggesting THC-induced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL-12Rbeta2 or GATA3 gene products. The results showed that THC suppressed IL-12Rbeta2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rbeta2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rbeta2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells.
Chronic morphine treatment in animal models has been shown to alter a number of immune parameters including suppression of cellular immunity. T helper cell differentiation into Th2 effector cell may be a major contributing factor to impaired cellular immunity following chronic drug abuse. We had previously shown that chronic morphine treatment in vivo and in vitro decreases IL-2 and IFNgamma (Th1) protein levels and increases IL-4 and IL-5 (Th2) protein levels in a time-dependent manner. In addition in this paper, we show that chronic morphine treatment resulted in a decrease in IFNgamma and IL-2 mRNA and an increase in IL-4 and IL-5 mRNA accumulation in murine splenocytes. Furthermore, chronic morphine treatment inhibited IFNgamma promoter activity and increased IL-4 promoter activity in respective promoter transfected primary T cells. In addition, we also demonstrate that chronic morphine treatment resulted in an increase in GATA 3 binding to DNA consensus elements in electromobility shift assays and an increase in GATA 3 protein and mRNA levels. In contrast, chronic morphine treatment resulted in a decrease in T-bet mRNA levels. From these data, we conclude that chronic morphine treatment differentiates T helper cell to Th2 effector cells by modulating key master switches that results in committing T helper cell to a Th2 phenotype.
To determine the levels of maturation and differentiation of murine CD4 single-positive (SP) T cells, we compared the secondary responses of staphylococcal enterotoxin A (SEA)-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts prepared from whole or heat-stable antigen(low) CD4 SP T cells. Proliferative responses upon re-stimulation with SEA were strong in adult splenic CD4 SP T cell blasts, but quite weak in neonatal thymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2 production was weaker in neonatal thymic blasts than in the adult splenic CD4 SP T cell blasts. In contrast, SEA-induced IL-4 production was high in neonatal thymic CD4 SP T cell blasts, and low in adult splenic and thymic CD4 SP T cell blasts. Expression of GATA-3, that directs production of IL-4 in T cells, examined at protein and mRNA levels, was higher in neonatal thymic cells than in adult thymic and splenic cells. These results suggest that neonatal and adult thymic CD4 SP T cells in the final stage of maturation are relatively immature compared with adult splenic CD4 SP T cells. The cytokine production profile of neonatal thymic CD4 SP T cells suggests that they are inclined towards a T(h)2 response.
The expression of mRNA for pre-Talpha is specific for human plasmacytoid dendritic cells (PDC), a population ontogenically close to T cells. The latter need Gata-3 transcription factor to develop. PU1 and RelB are two transcription factors involved in the development of murine myeloid DC (MDC). To determine the lineage origin of human thymic DC, the expression of these genes was investigated.
Fresh thymic DC, CD34(+)CD1a(-) progenitors, and progenitor-derived DC populations were sorted, analyzed, and compared to blood DC.
Three DC populations were found in the thymus. 1) CD123(-/lo)HLA-DR(hi) DC expressing PU1 and RelB; 2) CD123(hi)HLA-DR(+) DC expressing only pre-Talpha, the expression of which was similar to that of MDC and PDC from peripheral blood; and 3) a new mature CD123(hi)HLA-DR(hi) PDC population with pre-Talpha, PU1 and RelB mRNAs. In culture, most CD34(+)CD1a(-) progenitors remained CD1a(-)CD123(-); had a T and natural killer cell differentiation potential; and expressed Gata-3 mRNA contrary to DC precursors. A few cells (10%) became CD1a(+)CD123(+) expressing pre-Talpha, PU1, and RelB mRNAs and were able to differentiate into typical Langerhans cells with transforming growth factor-beta. Coculture of thymic progenitors on a murine cell line generated CD123(hi)CD1a(-) cells with typical PDC morphology, expressing pre-Talpha but not PU1 and RelB transcripts. Activated PDC acquired myeloid antigens, and up-regulated PU1 and RelB mRNAs while down-regulating pre-Talpha mRNA expression.
Both DC maturation pathways may arise from distinct precursors but are interconnected. DC differentiation seems to occur from Gata-3(-) precursors upstream of T and natural killer precursors.
The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins are newly identified immunomodulators. In vivo treatment of SJL/J mice with lovastatin reduced the duration and clinical severity of active and passive experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Lovastatin induced the expression of GATA3 and the phosphorylation of STAT6, whereas it inhibited tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT4. Inhibition of the Janus kinase-STAT4 pathway by lovastatin modulated T0 to Th1 differentiation and reduced cytokine (IFN-gamma and TNF-alpha) production, thus inducing Th2 cytokines (IL-4, IL-5, and IL-10). It inhibited T-bet (T box transcription factor) and NF-kappaB in activated T cells and significantly reduced infiltration of CD4- and MHC class II-positive cells to CNS. Further, it stabilized IL-4 production and GATA-3 expression in differentiated Th2 cells, whereas in differentiated Th1 cells it inhibited the expression of T-bet and reduced the production of IFN-gamma. Moreover, lovastatin-exposed macrophage and BV2 (microglia) in allogeneic MLRs induced the production of the anti-inflammatory cytokine IL-10. These observations indicate that the anti-inflammatory effects of lovastatin are mediated via T cells as well as APCs, because it modulates the polarization patterns of naive T cell activation in an APC-independent system. Together, these findings reveal that lovastatin may have possible therapeutic value involving new targets (in both APCs and T cells) for the treatment of multiple sclerosis and other inflammatory diseases.
GATA transcription factors play important roles in a variety of developmental processes. Recently, we discovered that GATA factors also play a key role in adipogenesis. Two isoforms, GATA-2 and GATA-3, are specifically expressed in murine preadipocytes but not mature adipocytes. Continuous expression of GATA factors in preadipocyte cell lines inhibits terminal differentiation into mature adipocytes. In contrast, GATA-3-deficient mouse embryonic stem cells possess a higher capacity to convert to adipocytes. The inhibitory effect of GATA on adipogenesis is mediated in part by suppression of promoters of adipogenic factors, including peroxisome proliferator-activated receptor gamma, but additional mechanisms are also likely to be in effect. These findings indicate that GATA factors function as molecular gatekeepers at the onset of terminal adipocyte differentiation. Whether GATA factors are also involved in the commitment of multipotent mesenchymal stem cells to progenitors of the adipogenic lineage is under investigation.
In this study, we investigated the role of hepatocyte growth factor (HGF) in blood formation during Xenopus development. First, we examined the gene expression of HGF and its receptor, c-met, by whole-mount in situ hybridization during development. Strong signals of HGF as well as c-met were detected early in the developing ventral mesoderm, which later gives rise to the ventral blood island. Furthermore, to study the role of HGF, we blocked the HGF signaling pathway in Xenopus embryos by using truncated c-met lacking the tyrosine kinase domain. Injection of truncated c-met mRNA resulted in a marked decrease in the number of circulating blood cells. Similar results were obtained using morpholino antisense HGF oligonucleotides. Moreover, we also analyzed the expression of several early primitive blood markers in the blood island of these embryos. RNA in situ analysis revealed a significant reduction (or absence) of stem cell leukemia (SCL), alpha-globin, and GATA-1 expression, but not GATA-2 expression. In contrast, no significant difference was observed in the levels of expression of early definitive blood markers, SCL, GATA-2, and GATA-3 in the dorsolateral plate, as analyzed by in situ hybridization. Overall, the present study demonstrated that HGF is necessary for primitive hematopoiesis by regulating the expression of SCL.
The NF-AT family is a group of potent transcription factors that are essential for T cell activation in vitro. However, NF-ATc2-deficient Th cells display hyperproliferation in response to stimulation, suggesting that NF-ATc2 functions as a negative regulator of Th cell activation/proliferation. In this study we show that the transcriptional repressor of GATA (ROG) is a direct target gene of NF-ATc2 and that NF-ATc2-deficient Th cells are unable to fully up-regulate ROG upon stimulation. Restoration of ROG expression in vivo partly corrects the hyperproliferation of NF-ATc2-deficient Th cells by attenuating TCR signals. Our data, therefore, depict a ROG-mediated negative feedback mechanism of T cell activation.
The transcription factor GATA-3 has been shown to play an important role for the in vitro induction of T(h)2 cells. To clarify how the in vivo immune response is governed under GATA-3 function, we generated double-transgenic mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice. After immunization with OVA, the double-transgenic mice showed increased expression of GATA-3 in antigen-reactive fresh CD4(+) T cells, and higher production of IL-5 and IL-13 in cultured spleen cells in the presence of cognate antigen without any polarizing conditions for T(h)2 cells. Moreover, the immunized double-transgenic mice showed a higher increase of in vivo secretion of IL-5 and IL-13 in bronchoalveolar lavage fluid after OVA aerosol challenging. The serum levels of OVA-specific IgG1, IgE and IgA antibodies were much higher in the immunized double-transgenic mice than TCR transgenic mice. These findings provide direct evidence that antigen-stimulated CD4(+) T cells in the immunized mice have already been committed into T(h)2 cells producing IL-5 and IL-13 selectively through enhanced GATA-3 expression in vivo, thereby inducing higher production of antigen-specific antibody for three isotypes other than IgM.
To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found that while remained unmethylated the ABL, CAV, EPO, GATA3, LKB1, NEP, NFL, NIS and p27KIP1 genes, varying extents of the HCC specific hypermethylation were found associated with the ABO, AR, CSPG2, cyclin a1, DBCCR1, GALR2, IRF7, MGMT, MT1A, MYOD1, OCT6, p57KIP2, p73, WT1 genes, and demethylation with the MAGEA1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more frequently than in their neighboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for the association with, the hypermethylation of the cyclin a1 gene was more prevalent in the non-cirrhosis group (P=0.021) while the hypermethylated p16INK4a gene was more common in the cirrhosis group (P=0.017). The concordant methylation behaviors of nineteen genes, including the four previously studied and their association with cirrhosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us to shape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, as well as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.
The transcription factor GATA-3 is expressed at every stage of thymic development, but its role in thymocyte differentiation is unknown. The fact that RAG chimeric animals lacking GATA-3 cannot generate early thymocytes from common lymphoid progenitors has thus far precluded investigation of the function of GATA-3 in the thymus. To address this, we generated mice deficient in GATA-3 at early and late stages of thymic differentiation. Our studies revealed that GATA-3 is involved in beta selection and is indispensable for single-positive CD4 thymocyte development. Thus, our data demonstrate that the coordinated and regulated expression of GATA-3 at each stage of thymic development is critical for the generation of mature T cells.
IL-27, a novel heterodimeric cytokine produced by antigen-presenting cells, signals through the T cell cytokine receptor (TCCR)/WSX-1 expressed on naïve CD4+ T cells and natural killer cells. TCCR/WSX-1 deficiency results in delayed T helper type 1 (TH1) development through an unresolved mechanism. We report here that IL-27 stimulation in developing murine T helper cells potently induces the expression of the major TH1-specific transcription factor T-bet and its downstream target IL-12R beta2, independently of IFN gamma. In addition, IL-27 suppresses basal expression of GATA-3, the critical TH2-specific transcription factor that inhibits TH1 development by down-regulating signal transducer and activator of transcription (Stat) 4. IL-27 signaling through TCCR/WSX-1 induces phosphorylation of Stat1, Stat3, Stat4, and Stat5. Stat1 is required for suppression of GATA-3, but T-bet induction by IL-27 can also be mediated through a Stat1-independent pathway. Despite its TH1-like signaling profile, IL-27 is not sufficient to drive the differentiation of CD4+ T cells into IFN gamma-producing cells. Similarly, IL-27 induces T-bet expression in primary natural killer cells, but this does not result in an increase of IFN gamma production or cytotoxic activity. Therefore, although IL-27 is unable to drive IFN gamma production on its own, it plays an important role in the early steps of TH1 commitment by contributing in a paracrine manner to the control of IL-12 responsiveness.
To investigate the changes of the GATA-3 expression and development of anterior chamber associated immune deviation (ACAID) after anterior chamber (AC) injection of interphotoreceptor retinoid binding protein (IRBP) under ocular inflammation.
ACAID was induced by injection of IRBP into the AC of 30 Spar-Dawley (SD) rats. Then the animals were divided into -4 days group, -24 hours group, 0 hour group, 3 days group, and 7 days group according to the lipopolysaccharide (LPS) injection 4 days and 24 hours before, or 0 hour, 3 days and 7 days after IRBP inoculation respectively. 6 rats were used as controls 8 days after IRBP injection, Serum interleukin-4 (IL-4) was evaluated to count the development of ACAID; Western blot and RT-PCR were used to determine the protein and mRNA levels of GATA-3 expression.
In -24 hours group and 0 hour group, the ocular inflammation reached a maximum 24 hours after LPS injection; on 8 days after IRBP inoculation, serum IL-4 couldn't be detected and GATA-3 expression were not changed both on mRNA and protein levels compared with control group. In -4 days group, the ocular inflammation was subsided gradually 24 hours and disappeared 96 hours after LPS injection; serum IL-4 and GATA-3 expression were significantly elevated 8 days after IRBP injection. In 7 days group, the serum IL-4 and GATA-3 expression in spleen increased 8 days after IRBP inoculation.
In ocular inflammation, the up-regulation of GATA-3 expression is inhibited and ACAID development is blocked after antigen was injected into anterior chamber. Once GATA-3 is up-regulated, LPS injection cannot affect ACAID development.
Upon TCR engagement, naive CD4 T cells differentiate toward the Th1 or Th2 phenotype. IL-4, acting through Stat6, plays a major role in Th2 differentiation; IL-2 has also been reported to be essential. Here, we report that retroviral (RV)-mediated expression of a constitutively active Stat5A mutant (STAT5A1*6) can fully restore IL-4 production when naive CD4 T cells are primed in the absence of IL-2. Furthermore, STAT5A1*6 expression causes Th2 differentiation in the absence of IL-4 or in Stat6- or IL-4Ralpha-deficient cells. Infection with STAT5A1*6-NGFR-RV does not enhance GATA-3 expression. STAT5A1*6-NGFR-RV and GATA-3-GFP-RV each render the Il4 gene accessible, but the sites of restriction enzyme accessibility are different. Stat5A binds to HSII and HSIII sites of the Il4 gene. Coinfection with STAT5A1*6-NGFR-RV and GATA-3-GFP-RV results in optimal Th2 priming.
The GATA-3 transcription factor has a determinant role in T cell specification and is an essential mediator of T helper 2-type polarized immune responses. While both committed NK precursors and mature NK cells express GATA-3, a role of this transcription factor in murine NK cell differentiation is not known. We found that NK cells, in contrast to T cells, can be generated in the absence of GATA-3. However, while GATA-3 antagonizes IFN-gamma production in differentiating T cells, GATA-3-deficient NK cells paradoxically produced less IFN-gamma compared to control NK cells and failed to provide early protection in vivo against infection with Listeria monocytogenes. Surprisingly, GATA-3 was essential for NK cell homing to the liver. Our results suggest that GATA-3 promotes NK cell maturation and acts in this lineage to specify distinct effector phenotypes.
Using conditional gene targeting in mice, we show that BMP receptor IA is essential for the differentiation of progenitor cells of the inner root sheath and hair shaft. Without BMPRIA activation, GATA-3 is down-regulated and its regulated control of IRS differentiation is compromised. In contrast, Lef1 is up-regulated, but its regulated control of hair differentiation is still blocked, and BMPRIA-null follicles fail to activate Lef1/beta-catenin-regulated genes, including keratin genes. Wnt-mediated transcriptional activation can be restored by transfecting BMPRIA-null keratinocytes with a constitutively activated beta-catenin. This places the block downstream from Lef1 expression but upstream from beta-catenin stabilization. Because mice lacking the BMP inhibitor Noggin fail to express Lef1, our findings support a model, whereby a sequential inhibition and then activation of BMPRIA is necessary to define a band of hair progenitor cells, which possess enough Lef1 and stabilized beta-catenin to activate the hair specific keratin genes and generate the hair shaft.
Th1 and Th2 cells arise from a common precursor cell in response to triggering through the TCR and cytokine receptors for IL-12 or IL-4. This leads to activation of complex signaling pathways, which are not known in detail. Disturbances in the balance between type 1 and type 2 responses can lead to certain immune-mediated diseases. Thus, it is important to understand how Th1 and Th2 cells are generated. To clarify the mechanisms as to how IL-12 and IL-4 induce Th1 and Th2 differentiation and how TGF-beta can inhibit this process, we have used oligonucleotide arrays to examine the early polarization of Th1 and Th2 cells in the presence and absence of TGF-beta. In addition to genes previously implicated in the process, we have identified 20 genes with various known and unknown functions not previously associated with Th1/2 polarization. We have also further determined which genes are targets of IL-12, IL-4, and TGF-beta regulation in the cells induced to polarize to Th1 and Th2 directions. Interestingly, a subset of the genes was coregulated by IL-12 or IL-4 and TGF-beta. Among these genes are candidates that may modulate the balance between Th1 and Th2 responses.
How T cells regulate interleukin 4 (IL-4) expression is not completely understood. We show here that single-positive thymocytes express IL-4, but attenuate GATA-3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. Type 2 polarization blocks Dnmt1 recruitment, enhances histone H3 Lys4 methylation (indicative of accessible chromatin) and initiates DNA demethylation of the locus. Dnmt1-/- CD4 and CD8 T cells derepress IL-4 expression considerably, demethylate DNA and increase H3 Lys4 methylation without affecting GATA-3 expression, demonstrating that Dnmt1 and DNA methylation are essential for proper Il4 regulation. These results indicate that Dnmts, DNA and histone methylation, and transcription factors 'collaborate' to determine appropriate Il4 expression patterns.
Erythropoietin production switches from fetal liver to adult kidney during development. GATA transcription factors 2 and 3 could be involved in modulating this switch, because they were shown to negatively regulate erythropoietin gene transcription through a promoter proximal GATA site. Herein, we analyzed the role of several GATA factors in the regulation of the erythropoietin gene in human liver and in hepatoma cells. Although GATA-3 expression in hepatocytes increases during human development, erythropoietin mRNA accumulation is unaltered in mutant mice lacking GATA-3. We found that GATA-2, -3, -4, and -6 are all expressed in human hepatocytes and that GATA-4 exhibits the most prominent Epo promoter binding activity in vitro and in vivo. Inhibition of GATA-4 expression by RNA interference leads to a dramatic reduction in Epo gene transcription in Hep3B cells. Moreover, GATA-4 expression is high and limited to hepatocytes in the fetal liver, whereas GATA-4 expression in the adult liver is low and restricted to epithelial cells surrounding the biliary ducts. Thus, GATA-4 is critical for transcription of the Epo gene in hepatocytes and may contribute to the switch in the site of Epo gene expression from the fetal liver to the adult kidney.
A T helper 1 (Th1)/Th2 imbalance is thought to contribute to the pathogenesis of autoimmune diseases. The differentiation of T cells into Th1 or Th2 subtypes is under the regulation of several transcription factors. Among these, transcription factor GATA-3 is thought to play an indispensable role in the development of T cells and the differentiation of Th2 cells. To examine how a Th1/Th2 imbalance affects the development of autoimmune disease, GATA-3 was overexpressed in the T lymphocytes of C57BL/6 x BXSB/MpJ-Yaa F(1) (Yaa) mice. Yaa mice developed autoimmune nephritis similarly to BXSB/MpJ-Yaa mice, which are commonly used as a model for Th1-dominant murine lupus. GATA-3 overexpression in T cells improved the 50% mortality incidence time for GATA-3-transgenic Yaa mice (41.6 wk), compared with Yaa mice (30.9 wk), and reduced proteinuria, serum creatinine levels, and the severity of glomerulonephritis in GATA-3-transgenic Yaa mice. GATA-3 overexpression in Yaa mice led to simultaneously elevated Th2 Ig (IgG1) and decreased Th1 Ig (IgG2a and IgG3) production and serum IFN-gamma levels. Although IL-4 production remained unchanged, intracellular cytokine analyses demonstrated that IL-5 was induced and IFN-gamma was suppressed in stimulated T cells from the GATA-3-transgenic Yaa mice. These results indicated that abundant GATA-3 was unable to stimulate complete differentiation of Th2 cells but did counteract the dominance of Th1 cells and alleviated the disease severity in Yaa mice. These data suggest that transcriptional regulation therapy may have potential as an effective strategy for treating autoimmune glomerulonephritis.
Activation of naive T cells through the TCR and cytokine signals directs their differentiation into effector or memory subsets with different cytokine profiles. Here, we tested the flexibility of human Th1 or Th2 differentiation by forced expression of transcription factors T-bet and GATA-3. Ectopic expression of T-bet and GATA-3 in freshly isolated human T(N) cells resulted in their differentiation to a Th1 and Th2 phenotype, respectively, in the absence of polarizing cytokines. Introduction of GATA-3 into lineage-committed Th1 cells induced the expression of Th2-specific cytokines (IL-4 and IL-5) and chemotactic receptors (CCR4, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, these cells partially maintained their Th1-specific profile (IFN-gamma and IL-12Rbeta2 expression). Conversely, expression of T-bet in lineage-committed Th2 cells caused a more profound switch to the Th1 phenotype, including the up-regulation of CXCR3 and down-regulation of CCR4 and CRTH2. Interestingly, similar to the naive T cell subset, central memory T cells were also largely programmed toward Th1 or Th2 effector cells upon expression of T-bet and GATA-3, respectively. However, expression of these transcription factors in effector memory T cells was much less influential on cytokine and chemokine receptor expression profiles. Our results reveal remarkable plasticity in the differentiation programs of human memory T cells. This flexibility is progressively diminished as cells mature from naive to effector T cells. These findings have important implications in understanding the molecular mechanisms of human T cell differentiation and for devising novel therapeutic strategies aimed at immunomodulation of skewed effector T cell responses.
We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.
Neonates are known to have poor cellular immunity, especially poor Th1 response. We investigated how neonatal mononuclear cells raised different Th1/Th2 reactions in response to different antigens. Employing Dermatophagoides pteronyssinus (Der p) extract and varicella zoster virus (VZV) as antigens, we assessed Th1/Th2 reactions as demonstrated by IL-4/IFNgamma production and mRNA expression, and transcriptional factors T-bet/GATA-3 mRNA expression in mononuclear cells from human umbilical cord blood (CBMC). Results showed that VZV induced a dramatic increase of IFNgamma production by adult peripheral blood mononuclear cells (PBMC), whereas VZV did not drive CBMC to release significant IFNgamma production (1614.7+/-362.0 vs. 49.0+/-29.3,p<0.005). However, Der p induced higher IFNgamma production by CBMC than VZV (298.1+/-171.8 vs. 49.0+/-29.3, P=0.047). In contrast, VZV did not induce significant IL-4 production either by CBMC or by PBMC. Der p induced a comparative IL-4 production by CBMC and PBMC (2.58+/-0.84 vs. 2.04+/-0.37, p>0.05). A real-time RT-PCR analysis of IL-4 and IFNgamma mRNA expression showed that VZV induced a significantly higher IFNgamma, but not IL-4, mRNA expression in PBMC than CBMC. Der p did not induce significant difference of IFNgamma or IL-4 mRNA expression in PBMC and CBMC. VZV enhanced Th1-related transcription factor T-bet mRNA expression, in association with later down-regulation of Th2-related GATA-3 mRNA expression in PBMC. However, VZV did not up-regulate T-bet or down-regulate GATA-3 expression significantly in CBMC. In contrast, Der p induced an early GATA-3 expression and later T-bet expression in CBMC. These results suggest that different antigens trigger various Th1/Th2 reactions in PBMC and CBMC resulting from kinetic changes of T-bet/GATA-3 expression.
The differentiation of naive T-helper (Th) cells towards Th1 or Th2 cells is regulated by the transcription factors T-box expressed in T-cells (T-bet) and GATA-binding protein-3 (GATA-3). In the present study, the gene expression of T-bet and GATA-3 was measured by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in Th1 and Th2 cells derived from purified splenic CD4+ T cells from DO11.10/Rag2(-/-) transgenic mice and control BioBreeding (BBc) Wistar rat splenic T cells stimulated under Th1 or Th2 conditions. In both sets of experiments, changes in the ratio of expression of T-bet and GATA-3 reflected changes in the Th1-specific cytokine interferon-gamma (IFN-gamma) and Th2-specific cytokine interleukin (IL)-4. T-bet gene expression was not maintained in fully polarized rat Th1 cells whereas GATA-3 gene expression was maintained in long-term polarized rat Th2 cells, indicating that maintenance of Th1/Th2 status occurred more as a result of altered GATA-3 mRNA expression than T-bet. These transcription factors are up-regulated in several cells that produce type 1 and type 2 cytokines and can be analyzed readily by RT-PCR using total RNA isolated from mixed cell populations or cultured splenocytes thereby providing a surrogate marker of Th1/Th2 cytokine balance under a variety of conditions.
GATA-3 plays a central role in regulating Th1 and Th2 cell differentiation. Upon interleukin (IL)-4 binding to its receptor, GATA-3 is induced through the action of Stat6. GATA-3 regulates Th2 cytokine expression not only at the transcription level, such as directly binding to the promoters of the IL-5 and IL-13 gene, but also by the involvement in the remodeling of the chromatin structure and opening the IL-4 locus. As a master control, GATA-3 stabilizes the Th2 phenotype by two methods. First, GATA-3 shuts down Th1 development through the repression the IL-12 receptor beta2-chain expression. Second, GATA-3 augments its own expression by a positive feedback autoregulation. In this article, we review the recent study of the function of GATA-3 in Th1 and Th2 differentiation.
Steroidogenesis is a tightly regulated process that is dependent on pituitary hormones. In steroidogenic tissues, hormonal stimulation triggers activation of an intracellular signalling pathway that typically involves cAMP production, activation of PKA, and phosphorylation of target transcription factors. In the classic cAMP signalling pathway, phosphorylation of CREB (cAMP response element (CRE)-binding protein) and its subsequent binding to cAMP-response elements (CREs) in the regulatory regions of target genes play a key role in mediating cAMP responsiveness. However, the cAMP responsive regions of several genes expressed in steroidogenic tissues do not contain consensus CREs indicating that other transcription factors are also involved. We have been studying the role played by the GATA family of transcription factors. GATA factors are expressed in a variety of tissues including the adrenals and gonads. Since the regulatory regions of several steroidogenic genes contain GATA elements, we have proposed that GATA factors, particularly GATA-4 and GATA-6, might represent novel downstream effectors of hormonal signalling in steroidogenic tissues. In vitro experiments have revealed that GATA-4 is indeed phosphorylated in steroidogenic cells and that phosphorylation levels are rapidly induced by cAMP. GATA-4 phosphorylation is mediated by PKA. Phosphorylation increases GATA-4 DNA-binding activity and enhances its transcriptional properties on multiple steroidogenic promoters. We now define a new molecular mechanism whereby phospho-GATA factors contribute to increased transcription of steroidogenic genes in response to hormonal stimulation.
Chromatin remodeling of type 2 cytokine gene loci occurs during differentiation of naive CD4 and CD8 T cells into type 2 helper (Th2) and cytotoxic (Tc2) T cells. IL-4 production and histone hyperacetylation in IL-4-associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells; however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1, and HDAC2 and exhibited repression of IL-4 gene activation. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus.
To evaluate the role of transcription factor GATA-3 in the pathogenesis of nasal polyps.
The expression of GATA-3 was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) in 28 cases of nasal polyps and 17 specimens of normal nasal epithelium. The expression of interleukin-5 (IL-5) in these specimens was detected by enzyme-linked immunosorbent assay (ELISA) meanwhile.
Immunohistochemistry showed, as confirmed by RT-PCR, a positive rate of GATA-3 of 89.3% in the nasal polyp (25/28) and 29.4% in the normal nasal mucosa (5/17), P < 0.05. GATA-3 was mainly distributed in submucous inflammatory cells. The relative density ratio of GATA-3 to GAPDH was 0.618 +/- 0.137 in nasal polyp and 0.21 +/- 0.11 in normal nasal mucosa (P < 0.05) as indicated by RT-PCR and agarose electrophoresis. The concentrations of IL-5 were 69.4 +/- 15.1 pg/ml and 25.7 +/- 13.0 pg/mg in the two groups respectively. The expression of GATA-3 was positively correlated to the expression of IL-5 in the nasal polyp.
Contributing to the overexpression of such cytokines as IL-5, GATA-3 may play an important role in the pathogenesis of nasal polyps.
Multipotent skin stem cells give rise to epidermis and its appendages, including the hair follicle. The Lef-1/Tcf family of Wnt-regulated transcription factors plays a major role in specification of the hair shaft, but little is known about how the equally important hair channel, the inner root sheath (IRS), develops in concert to shape and guide the hair. In a microarray screen to search for transcriptional regulators of hair follicle morphogenesis, we identified GATA-3, a key regulator of T-cell lineage determination. Surprisingly, this transcription factor is essential for stem cell lineage determination in skin, where it is expressed at the onset of epidermal stratification and IRS specification in follicles. GATA-3-null/lacZ knock-in embryos can survive up to embryonic day 18.5 (E18.5), when they fail to form the IRS. Skin grafting unveiled additional defects in GATA-3-null hairs and follicles. IRS progenitors failed to differentiate, whereas cortical progenitors differentiated, but produced an aberrant hair structure. Curiously, some GATA-3-null progenitor cells expressed mixed IRS and hair shaft markers. Taken together, these findings place GATA-3 with Lef-1/Wnts at the crossroads of the IRS versus hair shaft cell fate decision in hair follicle morphogenesis. This newfound function for GATA-3 in skin development strengthens the parallels between the differentiation programs governing hair follicle and lymphocyte differentiation.
Allergic asthma is characterized by airway hyperresponsiveness, eosinophilia, and mucus accumulation and is associated with increased IgE concentrations. We demonstrate here that peroxisome proliferator-activated receptors (PPARs), PPAR-alpha and PPAR-gamma, which have been shown recently to be involved in the regulation of various cell types within the immune system, decrease antigen-induced airway hyperresponsiveness, lung inflammation, eosinophilia, cytokine production, and GATA-3 expression as well as serum levels of antigen-specific IgE in a murine model of human asthma. In addition, we demonstrate that PPAR-alpha and -gamma are expressed in eosinophils and their activation inhibits in vitro chemotaxis and antibody-dependent cellular cytotoxicity. Thus, PPAR-alpha and -gamma (co)agonists might be of therapeutic interest for the regulation of allergic or inflammatory reactions by targeting both regulatory and effector cells involved in the immune response.
Th2 cytokine is predominant in tumor patients and was found to be associated with tumor progression. Reversing of Th2 dominant status is thought to be a promising strategy. In the present study, peripheral blood mononuclear cells (PBMNC) of 37 lung cancer patients and 19 healthy subjects were prepared and used for examination of cytokine secretion and gene expression. The positive percentage of mRNA transcripts of Th1 cytokines (8.1% for IFNgamma and 13.5% for IL-2) in patients' PBMNC were lower than those of Th2 cytokines (70.3% for IL-4, 64.9% for IL-6 and 83.8% for IL-10). The gene expression capacity (measured as relative intensity to ratio of beta-actin) of patients for Th1 cytokines was low, but constitutively relatively high for Th2 cytokines. Both positive percentage and relative intensity were lower in transcript factor for Th1 cytokine, T-bet (40.5% and 0.139, respectively) than those for Th2 cytokine, GATA3 (89.2% and 0.364, respectively). Traditional Chinese medicine, Astragalus (AG) was observed to reverse Th2 status of lung cancer. AG enhanced culture supernatant and gene expression levels of Th1 cytokine (IFNgamma and IL-2) and its transcript factor (T-bet), and reduced those of Th2 cytokines in cultured PBMNC of lung cancer patients. These results demonstrated that traditional Chinese medicine AG might reverse the Th2 predominant status in lung cancer patients, which is a probable alternative therapeutic regime in future.
The vitamin A metabolite, retinoic acid (RA), affects Th1 and Th2 development. The effect is partly exerted through the modulation of antigen-presenting cell functions, but it remains unclear whether RA directly exerts its effect on T cells to influence Th1/Th2 development. To clarify this problem, we used two experimental systems with isolated T cells in vitro. In one system, isolated CD4+CD8+ thymocytes differentiated into Th1 and Th2 by two transient stimulations with defined combinations of ionomycin and phorbol myristate acetate followed by treatment with IL-2 and IL-4 and/or IL-12. In the second system, functional differentiation was induced in purified naive CD4 T cells from DO-11.10 TCR-transgenic and RAG-2-deficient mice with cytokines and antibodies to CD3 and CD28. In both systems, all-trans-RA at > or = 1 nM concentrations suppressed Th1 development, but enhanced Th2 development. 9-cis-RA elicited similar effects. The optimal enhancement of Th2 development in the second system, however, was achieved with a delayed addition of RA. The presence of RA during the initial stimulation period often suppressed Th2 development. The RA receptor (RAR) antagonists, LE540 and LE135, but not the retinoic X receptor (RXR) antagonist, PA452, inhibited the effect of RA on Th1/Th2 development. Accordingly, the RAR agonists, Am80 and Tp80, but not the RXR agonists, HX600 and TZ335, mimicked the effect of RA. The RXR agonists enhanced the effect of the RAR agonists only slightly, if at all. These results indicate that, via RAR, RA directly suppresses Th1 development and directly enhances Th2 development with its timely addition.
The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation, although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha, GATA1, GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells, indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes.
Interleukin-4 (IL-4) is crucial for the differentiation of naive T helper (T(H)) cells into the T(H)2 effector cells that promote humoral (antibody) immunity and provide protection against intestinal helminths. IL-4 also has a central role in the pathogenesis of allergic inflammation. Many transcription factors are involved in the regulation of expression of the gene encoding IL-4. Initiation of transcription of the gene encoding IL-4 in naive T(H) cells is regulated by the T(H)2-specific transcription factor GATA3, whereas acute expression of the gene encoding IL-4 in T(H)2 cells is mediated by inducible, ubiquitous transcription factors after antigen encounter. This review focuses on acute activation of the gene encoding IL-4 in T cells and discusses therapeutic perspectives at the transcriptional level.
The homeobox Six genes, homologues to Drosophila sine oculis (so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1(+/-) mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1(-/-) mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1(-/-) embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1 dependent, we first clarified that Eya1 expression was unaffected in Six1(-/-) otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1(-/-) otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional specification of the otic vesicle. Finally, our data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle.
The Th2 cytokine genes IL4, IL5, and IL13 are clustered and expressed in a cell lineage-specific manner. We investigated the global locus-specific regulation of these genes using BAC transgenic mice containing the murine Th2 cytokine cluster carrying an IL4 promoter-luciferase reporter. IL4 promoter activity in effector CD4 T cells from these transgenic mice was strong, Th2 specific, and copy number dependent, suggesting the presence of an LCR in the locus. The production of IL4 and IL13, but not IL5, by these cells was also copy number dependent. Deletion analysis defined a 25 kb fragment in the RAD50 gene as the region containing the LCR activity. Expression of the IL4 promoter-luciferase reporter was transactivated by GATA-3 irrespective of position in the locus, suggesting the global nature of this regulation. The LCR itself, however, does not respond directly to GATA-3.
GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.
Because of the different functions of IL-4, IL-13 and IL-5, it would perhaps be surprising if common transcriptional mechanisms occur. However, because of the physical proximity of their genes within the human 5q locus, chromatin remodelling during T-cell differentiation may make transcription of all the genes permissible. If co-ordinate regulation were to take place, it might be argued that similar factors might be involved in expression of all of the cytokines. Emerging data on c-Maf and GATA3 regulation of IL-4 and IL-5 respectively argues that diverse proteins may be required for transcriptional activation. Alternatively, these factors may be responsible for regulating transcriptional competence, allowing fine control over generation of particular cytokines depending upon recognition of physiological cues. If competent for transcription, common factors, such as members of the NFAT and/or AP-1 families, may operate to regulate cytokine levels. To support this, we have recently identified a conserved palindrome located within the promoters of the different Th2-type cytokines, which acts as an enhancer of transcription. Central to the capacity to express Th2 cytokines is likely to be the ability to remodel chromatin at the locus. It remains to be determined whether a single factor, or combination of factors acts to regulate this event. It is also unclear what the boundaries of remodelling within the locus are, i.e. whether IL-4 and IL-13 may be within open chromatin and IL-5 in a closed environment, and whether there is a hierarchy which determines whether particular cytokines are preferentially expressed irrespective of competence.
We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed approximately 10-fold more H4/ICOS on their surfaces and produced approximately 10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.
Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.
Antigen and cytokine receptor signals act in synergy to direct the differentiation of CD4+ T cells. These signals initiate reciprocal activation and silencing of the interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) cytokine gene loci, changes that are heritably maintained in the resulting T helper type 1 (T(H)1) or T(H)2 cells and their progeny. Early, unpolarized transcription and chromatin remodeling of the poised cytokine genes of naive T cells is followed by consolidation and spreading of epigenetic changes and the establishment of self-reinforcing transcription factor networks. Recent studies have begun to elucidate the molecular mechanisms that establish and maintain polarized cytokine gene expression, and thus the cellular identity of differentiated helper T cells.
The interleukin 13 alpha 2 receptor (IL-13Ra2) has been shown to be expressed in most malignant glioblastoma cells. Recent studies suggest that IL-13Ra2 serves as a dominant negative inhibitor or a decoy receptor for IL-13. To investigate the transcriptional regulation of this receptor, we cloned and characterized the promoter for the human IL-13Ra2 gene. Our results demonstrate that this promoter contains three TATA boxes and one CCAAT site. Several putative transcriptional factor binding sites for nuclear factor of activated T cells 1, AP1 (c-JUN and c-FOS), AP2, GABP, OCT1, GATA3, PRE, and C-ETS1 were predicted in the promoter region. Using the secreted alkaline phosphate reporter gene assay, we investigated the functional activity of the human IL-13Ra2 promoter by transient transfection in glioma cell lines U118, U87, and T98, which differ in their expression of the human IL-13Ra2 protein. The different secreted alkaline phosphate activities among these 3 cell lines suggest that the expression of human IL-13Ra2 is regulated at the transcriptional level. Methylation analysis showed that expression of IL-13Ra2 may not be the result of methylation of the CpG dinucleotides in the promoter region of the gene. Deletion analysis identified a 64 base pair (bp) region that is necessary for human IL-13Ra2 promoter activity. This 64-bp sequence contains cis-elements for AP1, nuclear factor of activated T cells, and AP2. The possible role of AP1 in the regulation of human IL-13Ra2 promoter activity was suggested by in vitro mutagenesis and c-JUN N-terminal kinase inhibition analysis.
It is well-known that the expression of T helper (Th) type 2 cytokines such as interleukin (IL)-4 and IL-5, and their receptors, is up-regulated within the airways of allergic asthmatic patients. Furthermore, higher numbers of cells producing GATA-3, c-MAF, and signal transducer and activator of transcription factor (STAT)-6, which are transcription factors (TFs) that are implicated in the regulation and signaling of the Th2 cytokines, have been observed in bronchial biopsy specimens from asthmatic patients but not in those of healthy control subjects.
We examined whether these mediators also can be detected in induced sputum. Immunoreactivity for IL-4Ralpha, IL-5Ralpha, GATA-3, c-MAF, and STAT-6 was investigated in samples of induced sputum from asthmatic patients (n = 8) and healthy control subjects (n = 8).
Our results showed that the numbers of cells expressing IL-4 receptor alpha (Ralpha) and IL-5Ralpha were higher in samples from asthmatic patients compared to those of control subjects (p < 0.01). More cells exhibiting GATA-3, c-MAF, and STAT-6 immunoreactivity also were found in asthmatic patients vs those in control subjects (p < 0.005). Furthermore, the expression of STAT-6 and IL-4Ralpha, GATA-3 and IL-5Ralpha, and c-MAF with both IL-4Ralpha and IL-5Ralpha was correlated (p < 0.05).
This study demonstrated that induced sputum provides sufficient sensitivity for examining the pathways of cytokine signaling, cytokine receptor signaling, and intracellular signaling. Furthermore, these data show correlations between the expression of Th2 cytokine receptors and associated TFs in the human lung, which has not been documented previously.
To examine the participation of transcription factor GATA-3 in Th2 immune responses in vivo, we generated transgenic mice having several copies of GATA-3 with LCK promotor. Mice were infected with the intestinal nematode Heligmosomoides polygyrus to induce Th2 immune responses. Upon antigen stimulation, IL-5 and IL-13 production of mesenteric lymph node cells from H. polygyrus-infected mice, was significantly enhanced in GATA-3-transgenic mice compared with nontransgenic control mice. However, IL-4 production was the same in GATA-3-transgenic and control mice. H. polygyrus-infected GATA-3-transgenic mice exhibited significantly more peripheral blood eosinophils and total IgE levels compared with control mice. These results suggest that GATA-3 promotes IL-5 and IL-13 production, and that the function of these cytokines results in eosinophilia and hyper-IgE, respectively.
The sensory receptors for hearing and balance are the hair cells of the cochlea and vestibular organs of the inner ear. Permanent hearing and balance deficits can be triggered by genetic susceptibilities or environmental factors such as infection. Unlike mammalian hair cells that have a limited capacity for regeneration, the vestibular organ of the avian ear is constantly undergoing hair cell regeneration, whereas the avian cochlea undergoes regeneration only when hair cells are damaged. In order to gain insights into the genetic programs that govern the regenerative capacity of hair cells, we interrogated custom human cDNA microarrays with sensory epithelial cell targets from avian inner ears. The arrays contained probes from conserved regions of approximately 400 genes expressed primarily in the inner ear and approximately 1500 transcription factors (TF). Highly significant differences were observed for 20 inner-ear genes and more than 80 TFs. Genes up-regulated in the cochlea included BMP4, GATA3, GSN, FOXF1 and PRDM7. Genes up-regulated in the utricle included SMAD2, KIT, beta-AMYLOID, LOC51637, HMG20B and CRIP2. Many of the highly significant changes were validated by Q-PCR and in situ methods. Some of the observed changes implicated a number of known biochemical pathways including the c-kit pathway previously observed in melanogenesis. Twenty differentially expressed TFs map to chromosomal regions harboring uncloned human deafness loci, and represent novel candidates for hearing loss. The approach described here also illustrates the power of utilizing conserved human cDNA probes for cross-species comparisons.
The normal immunoregulatory mechanisms that maintain homeostasis in the intestinal mucosa, despite continuous provocation by environmental antigens, are jeopardized in inflammatory bowel diseases. Although previous studies have suggested that intestinal intraepithelial lymphocytes prevent spontaneous intestinal inflammation, there is limited knowledge about the characteristics of regulatory cells in the intestinal intraepithelial lymphocytes population. Here we show that CD4(+)CD8 alpha alpha(+) double-positive cells present in the intestinal intraepithelial lymphocytes population can suppress T helper 1-induced intestinal inflammation in an IL-10-dependent fashion. CD4(+) T cells stimulated along the Th2 but not the Th1 lineage, when transferred to RAG-1-/- mice, acquire CD8 alpha alpha expression on reaching the intestinal epithelium, and on arrival there, augment their production of IL-10. We show that a precursor CD4(+) T cell after limited, but not repeated, stimulation by IL-4 is able to become a double-positive-regulatory cell on exposure to the intestinal microenvironment in mice. Both CD8 alpha alpha acquisition and IL-10 production depend critically on the NF-kappa B-GATA-3-axis that we have previously shown is essential for differentiation to the Th2 phenotype and for the induction of airway inflammation. Our studies identify a mechanism for the generation of regulatory T cells in the intestine that may play an important role in controlling inflammatory bowel disease.
Results from experimental models, in vitro studies, and clinical data indicate that granulocyte colony-stimulating factor (G-CSF) stimulation alters T-cell function and induces Th2 immune responses. The immune modulatory effect of G-CSF on T cells results in an unexpected low incidence of acute graft-versus-host disease in peripheral stem cell transplantation. However, the underlying mechanism for the reduced reactivity and/or alloreactivity of T cells upon G-CSF treatment is still unknown. In contrast to the general belief that G-CSF acts exclusively on T cells via monocytes and dendritic cells, our results clearly show the expression of the G-CSF receptor in class I- and II- restricted T cells at the single-cell level both in vivo and in vitro. Kinetic studies demonstrate the induction and functional activity of the G-CSF receptor in T cells upon G-CSF exposure. Expression profiling of T cells from G-CSF-treated stem cell donors allowed identification of several immune modulatory genes, which are regulated upon G-CSF administration in vivo (eg, LFA1-alpha, ISGF3-gamma) and that are likely responsible for the reduced reactivity and/or alloreactivity. Most importantly, the induction of GATA-3, the master transcription factor for a Th2 immune response, could be demonstrated in T cells upon G-CSF treatment in vivo accompanied by an increase of spontaneous interleukin-4 secretion. Hence, G-CSF is a strong immune regulator of T cells and a promising therapeutic tool in acute graft-versus-host disease as well as in conditions associated with Th1/Th2 imbalance, such as bone marrow failure syndromes and autoimmune diseases.
Immune response messenger RNAs (mRNA) were compared in liver during self-limited (resolved) and chronic neonatal woodchuck hepatitis virus (WHV) infection. At week 14 postinfection (mid-acute phase), mRNAs for leukocyte markers (CD3, CD4, CD8), type 1 cytokines and related transcription factors (IFN-gamma, TNF-alpha, STAT4, T-bet), and IL-10 were increased in livers from resolving infections, but mRNAs of other type 1 (IL-2) and type 2 (IL-4, STAT6, and GATA3 markers remained at baseline levels. Increased coexpression of IFN-gamma and TNF-alpha mRNAs correlated in most cases with lower levels of intrahepatic WHV covalently closed circular DNA (cccDNA). At the same time point postinfection, livers from woodchucks that eventually progressed to chronic infection had baseline or slightly elevated levels of CD and type 1 mRNAs, which were significantly lower (or elevated less frequently) compared with resolving woodchucks. Earlier, at week 8, there were no differences between the two outcome settings. During these early time points and at a later stage in chronic infection (15 months), type 2 mRNAs in carrier liver remained at baseline levels or, when elevated, were never in excess of those in resolving woodchucks. In conclusion, the onset and maintenance of neonatal chronic WHV infection are not associated with antagonistic type 2 immunoregulation of type 1 responses in liver. Accordingly, chronicity develops in association with a primary deficiency in the intrahepatic CD responses, especially involving CD8(+) T lymphocytes, and in both extracellular (cytokine) and intracellular (transcriptional) type 1 response mediators. This has relevant implications for future treatment of chronic hepatitis B virus (HBV) infection in humans.
To further understand the interaction among GATA-3, Stat4, and T-bet in helper T cell development, we first showed that retroviral expression of GATA-3 in developing Th1 cells suppresses Th1 development through downregulation of Stat4 rather through downregulation of the IL-12Rbeta2 chain. Correspondingly, Stat4 levels are greatly suppressed during physiological Th2 development. Then, using cells doubly infected with GFP- and YFP-expressing retroviruses, we showed that retroviral GATA-3 expression in developing Th1 cells does not block Th1 development in cells coexpressing Stat4 but does so in cells coexpressing T-bet. Finally, we showed that retroviral Stat4 expression could facilitate Th2-->Th1 conversion in cells bearing an IL-12Rbeta2 transgene, even in cells lacking T-bet. These findings reassert that Stat4 signaling is a central element of Th1/Th2 development.
There is considerable evidence to support a role for T cells in asthma, particularly the involvement of T(H)2 cells both in atopic allergic asthma and in nonatopic and occupational asthma. There might also be a minor contribution from T(C)2 CD8+ T cells. Several T(H)2 cytokines have the potential to modulate airway inflammation, particularly IL-13, which induces airway hyperresponsiveness independently of IgE and eosinophilia in animal models. The identification of transcription factors controlling T(H)1 and T(H)2 development further support the T(H)2 hypothesis because GATA3 is overexpressed and T-bet is underexpressed in the asthmatic airway. Specific T cell directed immunotherapy might allow induction, modulation, or both of T-cell responses, and elucidation of the mechanisms of regulatory T cells might allow further optimization of immunotherapy. Recent advances in our understanding of dendritic cell function in directing T-cell responses might uncover further therapeutic targets. The efficacy of cyclosporin A and anti-CD4 treatment in patients with chronic severe asthma argues for continued T-cell involvement, but whether remodeling contributes to pathology inaccessible to anti-inflammatory treatment or T-cell immunotherapy will be an important future question.
Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4(+) T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit alpha enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.
The B cell lymphoma (BCL)-6 transcriptional repressor protein is an important regulator of Th2 responses. Mice deficient in BCL-6 develop severe Th2-type inflammation that can develop even in the absence of IL-4 signaling. We have investigated the mechanism for how BCL-6 regulates Th2 cell differentiation and have found that IL-6 signaling can promote dramatically increased levels of Th2 differentiation in BCL-6(-/-) CD4 T cells compared with wild-type CD4 T cells. IL-6 can induce a low level of Th2 cytokine expression in BCL-6(-/-)STAT6(-/-) cells but not in STAT6(-/-) cells. Since the promoters for Th2 cytokines such as IL-4, IL-5, IL-10, and IL-13 do not contain consensus BCL-6 DNA binding sites, we investigated whether BCL-6 might regulate the GATA-3 transcription factor that activates the expression of multiple Th2 cytokines. Consistent with the idea that BCL-6 represses GATA-3 expression, we found that GATA-3 levels are up-regulated in BCL-6(-/-)STAT6(-/-) CD4 T cells compared with STAT6(-/-) CD4 T cells. Retrovirus-mediated expression of BCL-6 in BCL-6(-/-)STAT6(-/-) T cells as well as developing wild-type Th2 cells leads to a potent repression of IL-4 and IL-10 secretion. Retrovirus-mediated expression of BCL-6 in both BCL-6(-/-)STAT6(-/-) and wild-type T cells also leads to a significant decrease in GATA-3 protein levels. Surprisingly, BCL-6 does not appear to regulate GATA-3 mRNA levels and thus BCL-6 appears to regulate GATA-3 expression at a posttranscriptional level. Regulation of GATA-3 protein levels is likely a key mechanism for how BCL-6 regulates Th2 cytokine expression and Th2 differentiation independently of STAT6. These data also point to a novel regulatory mechanism for BCL-6 separate from transcriptional repression.
SPACRCAN is a novel proteoglycan present in the interphotoreceptor matrix (IPM) of the rat and human retina that resists aqueous extraction through its binding to hyaluronan. The purpose of this study was: to clone mouse Spacrcan; to characterize the promoter elements; to define the deduced amino acid sequence; to establish the time of Spacrcan expression during retinal development; and to determine the time of appearance and distribution of SPACRCAN protein. Spacrcan cDNA clone was obtained through PCR amplification of a mouse retina cDNA library, and RT-PCR amplification and 5'RACE of mouse retina RNA. The deduced polypeptide sequence of mouse SPACRCAN contains a signal peptide at the N-terminal, seven N-link glycosylation sites, numerous potential O-linked glycosylation sites in a central mucin-like domain, two glycosaminoglycan attachment sites, five potential hyaluronan-binding motifs, two epidermal growth factor-like domains, and a hydrophobic stretch of 23 amino acids near the C-terminal. Comparison of the genomic structure of mouse and human SPACRCAN showed significant structure conservation. Analysis of the promoter region revealed several important putative regulatory elements including a Ret-1/PCE-1 element, an 11 base motif for Crx binding, six copies of PIRE, a Ret-4 element, three copies of AP-1, a CRE element, and five copies of GATA3. Northern blot analysis and immunohistochemistry were used to determine the tissue specificity of Spacrcan mRNA and to localize SPACRCAN in developing retina. Spacrcan mRNA is expressed in both retina and pineal gland and was detectable as early as embryonic day 15. The protein is first detectable in the IPM at postnatal day 8 where it increases in concert with the extension of photoreceptor inner and outer segments from the outer retinal surface. The presence of several unique regulatory elements in the promoter region and characteristic molecular features shared with the orthologue in human and rat suggest an important functional role of SPACRCAN in the IPM. The time of appearance of the SPACRCAN protein during retinal development suggests that this matrix protein may establish the extracellular microenvironment into which photoreceptor outer segments are elaborated.
Atopy has been linked to chromosome 11q22, a region that harbors the IL18 gene. IL-18 enhances IL-4/IL-13 production and induces IgE production that is directly associated with the pathogenesis of atopic disorders.
We sought to investigate whether genetic abnormalities in the regulatory regions of the IL18 gene predispose, in part, to susceptibility to atopy.
Among a white population of 105 families, the oldest child was examined with regard to atopic phenotypes and single-nucleotide polymorphisms (SNPs) within the IL18 gene.
We have identified 5 novel SNPs in the IL18 gene (-920[t/c], -133[c/g], and -132[a/g] in promoter 2 [upstream of exon 2]; +179[c/a; Ser35Ser] in exon 4; and +486[c/t; Phe137Phe] in exon 6). Three SNPs are located in promoter 2, and one (-133[c/g]; nuclear factor 1 site) was significantly associated with high serum IgE levels (P =.001; odds ratio, 3.96) and specific sensitization to common allergens (P =.005; OR, 4.12). In addition, previously identified SNPs in exon 1 (+113[t/g] and +127[c/t]) and in promoter 1 (-137[g/c], GATA3 site) of the IL18 gene were significantly associated with high IgE levels (P < or =.005; OR, 3.27-3.90) and specific sensitization (P =.02 to.008; OR, 3.27-3.83). The SNP +127(g/t) in exon 1 was also a susceptibility locus for seasonal allergic rhinitis (P =.008; OR, 3.22).
IL18 might be responsible for the linkage effects seen in the chromosomal region 11q22, which has been found previously with the phenotype "sensitization to mite allergen." Thus a suspected direct role of IL18 in the pathogenesis of atopy has been strengthened by the presence of 8 common SNPs in the promoter regions of IL18.
The relative importance of the cytokine milieu versus cytolytic T lymphocyte-associated antigen 4 (CTLA-4) and T cell receptor signal strength on T cell differentiation remains unclear. Here we have generated mice deficient for signal transducer and activator of transcription 6 (STAT6) and CTLA-4 to determine the role of CTLA-4 in cytokine-driven T cell differentiation. CTLA-4-deficient T cells bypass the need for STAT6 in the differentiation of T helper type 2 (T(H)2) cells. T(H)2 differentiation of cells deficient for both STAT6 and CTLA-4 is accompanied by induction of GATA-3 and the migration of T(H)2 cells to peripheral tissues. CTLA-4 deficiency also affects the balance of the nuclear factors NFATc1 and NFATc2, and enhances activation of NF-kappaB. These results suggest that CTLA-4 has a critical role in T cell differentiation and that STAT6-dependent T(H)2 lineage commitment and stabilization can be bypassed by increasing the strength of signaling through the T cell receptor.
Pax-5 gene is important transcription factor in B-lymphopoiesis and B-cell development. To understand the regulatory mechanism of pax-5 expression, the immediate 5'-flanking region (6 671 bp) of human pax-gene exon 1B was isolated and characterized. Analysis of the total sequence showed that the proximal promoter includes 3 CAT boxes, 1 SP1 and 1 E box. However, there was no consensus sequence for a TATA box in the 5'-flanking region. Putative regulatory sites of further upstream in the sequence revealed 6 LMO(2)COM, 5 NFAT, 2 LPOLYA-B, 3 GATA1, 2 AP4, 10 MZF1, 1 ETS1-B, 1 GATA3, 1 NKX25, 2 RORA1, 1 LYF1, 2 Ikaros2, 2 TCF11, 1 GATA-C and 1 FREAC7. Therefore, the 5'-flanking region of human pax-5 exon 1B could be involved in regulating the expression of human pax-5 and B-cell differentiation and development.
The signal transducer and activator of transcription (STAT)1 is a cytoplasmic-transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon gamma(IFN-gamma). The phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFN-gamma-inducible genes, such as the interferon regulatory factor (IRF)-1. We show here that gamma irradiation reduces the IFN-gamma mRNA expression. The inhibition of the STAT1 phosphorylation and the IRF-1 expression by gamma irradiation was also observed. In contrast, the mRNA levels of IL-5 and transcription factor GATA-3 were slightly induced by gamma irradiation when compared to the non-irradiated sample. Furthermore, we detected the inhibition of cell-mediated immunity by gamma irradiation in the allogenic-mixed lymphocytes' reaction (MLR). These results postulate that gamma irradiation induces the polarized-Th2 response and interferes with STAT1 signals, thereby causing the immunosuppression of the Th1 response.
After encountering antigen, helper T (T(H)) cells undergo differentiation to effector cells, which can secrete high levels of interferon-gamma, interleukin-4 (IL-4), IL-10 and other immunomodulators. How T(H) cells acquire, and remember, new patterns of gene expression is an area of intensive investigation. The process is remarkably plastic, with cytokines being key regulators. Extrinsic signals seem to be integrated into cell-intrinsic programming, in what is becoming an intriguing story of regulated development. We summarize the latest insights into mechanisms that govern the lineage choices that are made during T(H)-cell responses to foreign pathogens.
CD4+ T cell priming under T helper type I (T(H)1) or T(H)2 conditions gives rise to polarized cytokine gene expression. We found that in these conditions human naive T cells acquired stable histone hyperacetylation at either the Ifng or Il4 promoter. Effector memory T cells showed polarized cytokine gene acetylation patterns in vivo, whereas central memory T cells had hypoacetylated cytokine genes but acquired polarized acetylation and expression after appropriate stimulation. However, hypoacetylation of the nonexpressed cytokine gene did not lead to irreversible silencing because most T(H)1 and T(H)2 cells acetylated and expressed the alternative gene when stimulated under opposite T(H) conditions. Such cytokine flexibility was absent in a subset of T(H)2 cells that failed to up-regulate T-bet and to express interferon-gamma when stimulated under T(H)1 conditions. Thus, most human CD4+ T cells retain both memory and flexibility of cytokine gene expression.
Regulation of gene expression during the ontogeny of haematopoiesis in the human fetal bone marrow is poorly understood. Studies in mice demonstrated that GATA-1, -2 and -3 play pivotal roles in haematopoiesis. In this study, we identified GATA-1-, GATA-2- and GATA-3-expressing cells in bone marrow sections and analysed the expression of GATA-transcription factors during the development of human fetal bone marrow haematopoiesis using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). We showed that GATA-1, -2 and -3 were expressed only in haematopoietic cells in the bone marrow. RT-PCR analysis demonstrated that (1) GATA-1 expression significantly increased during gestation; (2) GATA-2 expression peaked at the onset of medullary haematopoiesis, declined thereafter, and remained at a constant level after 30 weeks post conception; and (3) GATA-3 expression revealed no changes during development. The results indicated that the onset of medullary haematopoiesis in humans is accompanied by high expression of GATA-2, reflecting high proliferation rates of early haematopoietic progenitor cells, whereas expression of GATA-1 mirrors haematopoietic activity.
The development of Th subset is accompanied by subset-specific chromatin remodeling of cytokine gene loci. In this study, we show that the C-terminal, but not the N-terminal zinc finger (N-finger) of GATA-3 mediates the association with the IL-4/IL-13 intergenic DNase I hypersensitive site and the induction of an extended DNase I hypersensitivity on the IL-4/IL-13 locus. Consistently, deletion of the transactivation domains or the C-finger, but not the N-finger, abrogated the induction of IL-4 and IL-13 as well as the down-regulation of IFN-gamma. In contrast, the N-finger of GATA-3 was indispensable for the binding to the IL-5 promoter and the induction of IL-5. The selective use of the N-finger may underlie the differential roles of GATA-3 in the induction of IL-4, IL-13, and IL-5.
The development of T helper (Th)2 responses is a key step in the pathogenesis of asthma. Interleukin (IL)-4 is thought to be important, although not strictly necessary, for Th2 differentiation, although triggers of IL-4-independent Th2 polarization have not been identified. We examined whether IL-4 is necessary for Th2-polarized responses during granulocyte macrophage colony-stimulating factor (GM-CSF)-driven respiratory mucosal sensitization. Balb/c wild type (WT) or IL-4 knockout (4KO) mice were exposed to aerosolized ovalbumin (OVA) in the context of airway GM-CSF expression. We examined the extent of Th2 polarization using real-time quantitative polymerase chain reaction on lymph node mRNA, flow cytometric analysis of lung Th cells, and measurement of cells, cytokines, and immunoglobulins in bronchoalveolar lavage (BAL) and serum. GATA-3 and CCR3, -4, and -8 were expressed in the lymph nodes of WT and 4KO mice at similar levels, as were IL-5 and IL-13 levels in the BAL, T1/ST2 on lung Th cells, and BAL eosinophils after recall challenge. With the exception of immunoglobulin production, expression of GATA-3, CCR-3, -4, -8, IL-5, and T1/ST2, and the generation of blood eosinophilia, were intact in mice doubly deficient in both IL-4 and IL-13. We conclude that IL-4 is not required for the generation of Th2-polarized responses in the presence of GM-CSF.
Previously, we have shown in a model of hypersensitivity pneumonitis that Th1-biased C57BL/6 mice are susceptible and Th2-biased DBA/2 mice are resistant to disease. We also showed that this was explained in part by differential regulation of IL-12 by IL-4. For these reasons, we postulated that C57BL/6 and DBA/2 mice differentially express IL-4. In this study, we show that C57BL/6 immune cells express Th2 but not Th1 cytokines at lower levels than DBA/2 cells. We also found that C57BL/6 splenocytes exhibit decreased mRNA stability of Th2 cytokines, relative to DBA/2 splenocytes. Stability of IL-2 and IFN-gamma were similar in the two strains of mice. Differences in Th2 cytokine mRNA stability between C57BL/6 and DBA/2 cells were not due to sequence polymorphism at specific regions of the IL-4/IL-13 locus. Furthermore, expression of Th1- and Th2-specific transcription factors T-bet and GATA-3, as well as the nuclear factor of activated T cells transcription factor, NFATc, was not significantly different between the two mice. Our data suggest that decreased mRNA stability of Th2 cytokines in C57BL/6 splenocytes may underlie the differential susceptibility to hypersensitivity pneumonitis between C57BL/6 and DBA/2 mice. Moreover, our results indicate that regulation of mRNA stability may serve as an important mechanism underlying Th1/Th2 immune polarization.
It is widely accepted that a strong Th2 response is responsible for nonhealing Leishmania major infections in BALB/c mice. This Th2 response has been thoroughly documented by measuring the levels of Th2 cytokines produced by CD4(+) T cells present in the lymphoid organs by enzyme-linked immunosorbent assay and PCR. However, the cytokine profile of L. major-specific Th2 cells has never been determined. In this study, we used the recently described Th2 marker T1/ST2 to characterize Th2 cells during the course of nonhealing L. major infection. We analyzed the intracellular cytokine profile of CD4(+) T1/ST2(+) T cells and showed that they clearly displayed a Th2 phenotype, as they expressed interleukin 4 (IL-4), IL-10, and IL-5. In addition, we detected another population of Th2 cells among the CD4(+) T1/ST2(-) T cells that expressed IL-4 and IL-10 but excluded IL-5. In summary, we show here that two type 2 subpopulations are present in the lymphoid organs of L. major-infected BALB/c mice; Th2 cells from both subsets expressed IL-4 and IL-10, but they could be distinguished by their expression of IL-5 and T1/ST2.
Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including that of the vertebrate central nervous system. However, the role of GATA proteins in spinal cord development remains unresolved. In this study, we investigated the expression and function of two GATA proteins, GATA2 and GATA3, in the developing chick spinal cord. We show that both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, we demonstrate that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, we further show that Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, we also demonstrate that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, our findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord.
Differentiation of naive CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling of Th2 cytokine gene loci. Hyperacetylation of histone H3 on nucleosomes associated with the interleukin (IL)-4, IL-13 and IL-5 genes was observed in developing Th2 cells but not in Th1 cells. Histone hyperacetylation on IL-5 gene-associated nucleosomes was Th2-specific but occurred with delayed kinetics, and hyperacetylation on RAD50 gene-associated nucleosomes was T cell antigen receptor stimulation-dependent but not Th2-specific. The induction of the Th2-specific histone hyperacetylation was STAT6- and GATA3-dependent, and interestingly, it was accompanied by the expression of intergenic transcripts within the IL-13 and IL-4 gene loci. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream from the IL-13 gene, corresponding with the 5'-border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyltransferase complexes including CBP/p300, and RNA polymerase II. Also, the CGRE showed a significant enhancing effect on the Th2 cytokine gene promoters. Thus, the CGRE may play a crucial role for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci.
We have developed an in vitro differentiation assay in which human naive CD4(+) cells are driven toward either the Th1 or Th2 phenotype. We have examined the interrelationships among the expression of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-gamma in individual cells using intracellular cytokine staining at various times during the differentiation process. We provide direct evidence that the Th2 cytokines IL-4, IL-5, and IL-13, unlike the other cytokines, are regulated by a coordinated mechanism. We also show that IL-10 is expressed by a different subset of cells that is prevalent at early stages of Th2 differentiation, but then diminishes. Additionally we demonstrate that while naive cells can express IL-2 upon activation, they cannot express GM-CSF. Commitment to GM-CSF expression occurs during differentiation in a Th1/Th2 subset-independent manner. Furthermore, we have examined the levels of GATA3, c-Maf, T-bet, and Ets-related molecule during human Th1/Th2 differentiation and suggest that differences in the levels of these critical transcription factors are responsible for commitment toward the Th1 or Th2 lineage.
Certain IFN regulatory factor (IRF) transcription factors indirectly influence T helper (Th) cell differentiation by regulating the production of IL-12. Here, we show that IRF4 directly regulates Th cell differentiation in vitro and in vivo during murine leishmaniasis. In the absence of IRF4, IL-12-induced Th1 cell differentiation was compromised, while IL-4 failed to induce Th2 cell differentiation. Instead, IL-4 tended to induce Th1 cells, defined by production of IFN-gamma and TNF. Although early IL-4 signaling was normal in IRF4(-/-) Th cells, the protein GATA-3, a transcription factor critical for Th2 development, was not up-regulated following IL-4 treatment. Retroviral overexpression of GATA-3 rescued Th2 differentiation. Therefore, IRF4 deficiency manifests itself as severely dysregulated Th cell differentiation.
Based on previous animal experiments that suggest the plasticity of peripheral blood-derived, CD34(-) stem cell lines, the aim of this study was to isolate CD34(-) stem cell lines from human peripheral blood cells and obtain evidence of their multipotency and plasticity.
Adherent growing cells were isolated from peripheral blood mononuclear cells from a healthy volunteer donor and different cell clones were established after SV40 large-T-antigen-mediated immortalization. The immunophenotype of the cell lines was investigated by flow cytometry. One particular cell clone, V54/2, was stained with rhodamine 123, and the Rh123(low) and Rh123(high) subpopulations were sorted for a reverse transcriptase polymerase chain reaction gene expression survey and distinct differences in morphology and biologic behavior.
The peripheral blood-derived and fibroblast-like cell line V54/2 expressed high levels of CD10 and CD105 and showed only a very low level expression of CD34 (<1.0%) and CD117 (c-kit). Among the entire CD34(-)CD105(+) cell population that transcribed factors such as Myb, Tie-1, and VEGF, there was a small Rh123(low)CD34(+) subpopulation that transcribed significant levels of several members of the GATA family of transcription factors. The morphology of the Rh123(low)CD34(+) (also expressing the P-glycoprotein) was different compared to the Rh123(high)CD34(-) population. Mesenchymal differentiation into glial fibrillary acidic protein (GFAP)(+) glial cells could be shown from the entire CD34(-)CD105(+) cell population.
The findings provide evidence that it is possible to isolate CD34(-)CD105(+) mesenchymal stem cell lines from human peripheral blood cells that contain a small subpopulation of CD34(+) and GATA-transcribing cells. Those cells are potential hematopoietic progenitors and can be recruited from the CD34(-) stem cell pool. The plasticity of stem cells seems to require essential molecular tools, such as a panel of transcription factors, to respond to the environmental demand within a biologic system.
How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.
Many light-responsive promoters contain GATA motifs and a number of nuclear proteins have been defined that interact with these elements. Type-IV zinc-finger proteins have been extensively characterised in animals and fungi and are referred to as GATA factors by virtue of their affinity for promoter elements containing this sequence. We previously identified cDNA sequences representing four Arabidopsis thaliana type-TV zinc-finger proteins. Here we define the organisation and expression of GATA-1, GATA-2, GATA-3 and GATA-4 as well as DNA-binding characteristics of their encoded proteins. Transcripts from all four genes can be detected in all tissues examined suggesting that they are not developmentally regulated at the level of transcription. In vitro binding experiments with Escherichia coli-derived recombinant proteins were performed using motifs previously defined as targets for nuclear GATA-binding proteins. These studies reveal differences in DNA binding specificity of GATA-1 as compared to the other three proteins. In vivo protein-DNA interactions monitored by yeast one-hybrid assays reveal different binding characteristics as compared to those defined with E. coli-derived recombinant protein. Trans-activation of gene expression by the four Arabidopsis proteins via some, but not all, DNA elements tested indicates that the Arabidopsis proteins can form functional interactions with previously defined promoter elements containing GATA motifs. We conclude that the Arabidopsis type-IV zinc-finger proteins may represent the previously defined family of nuclear GATA-binding proteins implicated in light-responsive transcription.
To determine the role of GATA-3 (a transcript factor that has influences on Th2 cell differentiation) in the development of anterior chamber associated immune deviation (ACAID).
Immunohistochemistry and Western blot were used to determine the localization and relative protein levels of GATA-3 respectively at different time points after anterior chamber injection of 50 microgram interphotoreceptor retinoid binding protein (IRBP).
GATA-3 was detected weakly in normal spleen and increased significantly at 5, 7, 14 and 21 days after anterior chamber inoculation. It was detected mainly in CD(4)(+) T cells in spleen.
The expression of GATA-3 increases earlier than development of ACAID. It suggests that GATA-3 play an important role in Th2 commitment during ACAID development.
The differentiation of neural progenitors into the many classes of neurons that exist in the mature spinal cord is a process that relies heavily on the activation of precise combinations of transcription factors. Defining these transcription factor combinations is an important aspect of research in developmental neurobiology that promises to provide incredible insights into the structure, function, and pathology of the central nervous system. The present study aimed to investigate a possible role for the Stem Cell Leukemia (SCL) gene, a basic helix-loop-helix (bHLH) transcription factor gene, in the specification of a population of neural cells in the ventral neural tube. Section RNA in situ hybridisation revealed that SCL is transiently expressed within the V2 postmitotic domain of the developing mouse spinal cord between 10.5 and 13.5 days post coitum. Double-immunofluorescence experiments were subsequently carried out to directly compare the expression of SCL with other V2-specific markers at the cellular level. These experiments revealed that SCL is expressed in a medially restricted subpopulation of GATA-3 producing cells, suggesting a possible role for this factor in the differentiation of the GATA population of V2 interneurons.
Partial monosomy 10p is a rare chromosomal aberration. Patients often show symptoms of the DiGeorge/velocardiofacial syndrome spectrum. The phenotype is the result of haploinsufficiency of at least two regions on 10p, the HDR1 region associated with hypoparathyroidism, sensorineural deafness, and renal defects (HDR syndrome) and the more proximal region DGCR2 responsible for heart defects and thymus hypoplasia/aplasia. While GATA3 was identified as the disease causing gene for HDR syndrome, no genes have been identified thus far for the symptoms associated with DGCR2 haploinsufficiency. We constructed a deletion map of partial monosomy 10p patients and narrowed the critical region DGCR2 to about 300 kb. The genomic draft sequence of this region contains only one known gene, BRUNOL3 ( NAPOR, CUGBP2, ETR3). In situ hybridization of human embryos and fetuses revealed as well as in other tissues a strong expression of BRUNOL3 in thymus during different developmental stages. BRUNOL3 appears to be an important factor for thymus development and is therefore a candidate gene for the thymus hypoplasia/aplasia seen in partial monosomy 10p patients. We did not find BRUNOL3 mutations in 92 DiGeorge syndrome-like patients without chromosomal deletions and in 8 parents with congenital heart defect children.
Over the past year, vertebrate GATA factors have been found to participate directly in several signal-transduction pathways. Smad3, phosphorylated by TGF-beta signalling, interacts with GATA3 to induce differentiation of T helper cells. Hypertrophic stimuli act through RhoA GTPase and ROCK kinase to activate GATA4 in cardiac myocytes. In the liver, GATA4 is elevated by BMP and FGF signalling, and is able to bind to chromatin targets. Invertebrate GATA factors play a central role in specifying the mesendoderm.
Peripheral T cell differentiation is accompanied by chromatin changes at the signature cytokine loci. Using chromatin immunoprecipitation we demonstrate that profound increases in histone acetylation occur at the IFN-gamma and IL-4 loci during Th1/Th2 differentiation. These changes in histone acetylation status are locus and lineage specific, and are maintained by the transcription factors Tbet and GATA3 in a STAT-dependent manner. Our results suggest a model of cytokine locus activation in which TCR signals initiate chromatin remodeling and locus opening in a cytokine-independent fashion. Subsequently, cytokine signaling reinforces polarization by expanding and maintaining the accessible state at the relevant cytokine locus (IL-4 or IFN-gamma). In this model, GATA3 and Tbet serve as transcriptional maintenance factors, which keep the locus accessible to the transcriptional machinery.
We have used Affymetrix high-density gene arrays to generate a temporal profile of gene expression during differentiation of UB/OC-1, a conditionally immortal cell line derived from the mouse cochlea. Gene expression was assessed daily for 14 days under differentiating conditions. The experiment was replicated in two separate populations of cells. Profiles for selected genes were correlated with those obtained by RT-PCR, TaqMan analysis, immunoblotting, and immunofluorescence. The results suggest that UB/OC-1 is derived from a population of nonsensory epithelial cells in the greater epithelial ridge that have the potential to differentiate into a hair-cell-like phenotype, without the intervention of Math1. Elements of the Notch signaling cascade were identified, including the receptor Notch3, with a transient up-regulation that suggests a role in hair cell differentiation. Several genes showed a profile similar to Notch3, including the transcriptional co-repressor Groucho1. UB/OC-1 also expressed Me1, a putative partner of Math1 that may confer competence to differentiate into hair cells. Cluster analysis revealed expression profiles for neural guidance genes associated with Gata3. The temporal dimension of this analysis provides a powerful tool to study genetic mechanisms that underlie the conversion of nonsensory epithelial cells into hair cells.
Immunological memory involves the fast recall of cytokine expression by T helper (Th) lymphocytes. Two distinct profiles of cytokine expression, Th1 and Th2, can be induced by antigen and polarizing signals during activation of naive Th cells and can subsequently be reexpressed on stimulation by antigen alone. The transcription factor GATA-3 induces Th2 development. GATA-3 is activated by the Th2-polarizing stimulus, IL-4, and has recently been observed to autoactivate its transcription. Based on these experimental data, we developed a mathematical model of GATA-3 expression that assumes independent activation of GATA-3 transcription by IL-4 and by GATA-3. Cooperativity of GATA-3 transcriptional activation is shown to create a threshold for autoactivation, resulting in the coexistence of two distinct GATA-3 expression states: a state of basal expression and a state of high expression sustained by autoactivation. Suprathreshold IL-4 signals induce a transition from basal to high GATA-3 expression. Thus, GATA-3 autoactivation creates a bistable system that can memorize a transient inductive signal. The model further predicts conditions under which the state of high GATA-3 expression can be abolished, which may extinguish the Th2 cytokine memory.
Memory is one of the key features of the adaptive immune system. Specific T and B lymphocytes are primed for a particular antigen and upon challenge with it will react faster than naive lymphocytes. They also memorize the expression of key effector molecules, in particular cytokines, which determine the type and scale of an immune reaction. While in primary activations differential expression of cytokine genes is dependent on antigen-receptor signaling and differentiation signals, in later activations the expression is triggered by antigen-receptor signaling and dependent on the cytokine memory. The molecular basis of the cytokine memory implies differential expression of transcription factors and epigenetic modifications of cytokine genes and gene loci. GATA-3 for Th2 and T-bet for Th1 cells expressing interleukin-4 or interferon-gamma, respectively, are prime candidates for key transcription factors of cytokine memory. The essential role of epigenetic modifications is suggested by the requirement of DNA synthesis for the establishment of a cytokine memory in Th lymphocytes. At present the molecular link between transcription factors and epigenetic modifications of cytokine genes in the establishment and maintenance of cytokine memory is not clear. The initial cytokine memory is not stable against adverse differentiation signals, while in repeatedly stimulated lymphocytes it is stabilized by a variety of mechanisms.
GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. The transcriptional regulation of GATA-3, however, remains elusive. In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene. The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro. By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes. More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro. Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.
To identify cellular genes that may be involved in human papillomavirus (HPV)-mediated immortalization mRNA differential display analysis was performed on preimmortal and subsequent immortal stages of four human keratinocyte cell lines transformed by HPV type 16 or 18 DNA. This yielded a cDNA fragment encoding the transcription factor GATA-3 that was strongly reduced in intensity in all immortal stages of the four cell lines. A marked reduction in both GATA-3 mRNA and protein expression in HPV-immortalized cell lines was confirmed by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry and was also shown to be apparent in cervical carcinoma cell lines. Immunohistochemical analysis of cervical tissue specimens showed a clear nuclear staining for GATA-3 in normal cervical squamous epithelium (n = 14) and all cervical intraepithelial neoplasia (CIN) I (n = 6) and CIN II lesions (n = 2). In contrast, 11% (1 of 9) of CIN III lesions and 67% (8 of 12) of cervical squamous cell carcinomas revealed a complete absence of GATA-3 immunostaining. Hence, complete down-regulation of GATA-3 expression represents a rather late event during cervical carcinogenesis. Whether GATA-3 down-regulation is etiologically involved in HPV-mediated immortalization and cervical carcinogenesis remains to be examined.
Naïve T cells differentiate into effector cells upon stimulation with antigen, a process that is accompanied by changes in the chromatin structure of effector cytokine genes. Using histone acetylation to evaluate these changes, we showed that T cell receptor (TCR) stimulation results in early activation of the genes encoding both interleukin 4 and interferon-gamma. We found that continued culture in the presence of polarizing cytokines established a selective pattern of histone acetylation on both cytokine genes; this correlated with restricted access of the transcription factor NFAT1 to these gene regulatory regions as well as mutually exclusive gene expression by the differentiated T cells. Our data point to a biphasic process in which cytokine-driven signaling pathways maintain and reinforce chromatin structural changes initiated by the TCR. This process ensures that cytokine genes remain accessible to the relevant transcription factors and promotes functional cooperation of the inducible transcription factor NFAT with lineage-specific transcription factors such as GATA-3 and T-bet.
IL-4 is important in Th2 differentiation and in cell expansion. Stat6 is necessary and sufficient for both functions. Although Gata3 is critical for Th2 polarization, it is not sufficient to mediate IL-4-driven cell expansion. We report that growth factor independent-1 (Gfi-1), a Stat6-dependent transcriptional repressor, strikingly increases Th2 cell expansion by promoting proliferation and preventing apoptosis. Cells infected with a Gfi-1 retrovirus show striking enhancement of IL-2-induced Stat5 phosphorylation and repression of p27(Kip-1) expression, suggesting a potential mechanism for the Gfi-1 growth effect. The synergy of Gfi-1 and Gata3 provides a mechanism through which IL-4 could selectively promote Th2 cell expansion.
The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused with luciferase. The construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoterluciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferase constructs. N(G)-monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed.
Pathogenic infections lead to activation of innate immunity followed by induction of a type 1 T cell subset and, therefore, provide a good model to evaluate when T cells commit to type 1 T cells. Here we show a two-step mechanism of T cell subset commitment during pathogenic infection. The first step is mediated by the basal function of macrophage/dendritic cells and is antigen independent. This step modulates the committed precursor frequency of T cell subsets and influences the expression of T-box expressed in T cells (T-bet) and GATA-3 genes. IL-12 and NK cells are not required for this step. The second step requires antigenic stimulation of T cells together with IL-12 or IL-4, and influences on the expression of T-bet and GATA-3. We propose a two-step T cell subset commitment pathway based on these observations. Therefore, pathogenic infections influence functional T cell commitment before T cells encounter nominal antigen.
PU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Ralpha expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects.
The transcription factors T-bet and GATA3 are important reciprocal determinants of Th1 and Th2 T helper cell differentiation. Recent evidence suggests that these factors may affect airway immunopathology in asthma.
T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes.
To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization.
PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation.
These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.
The T helper lymphocyte is responsible for orchestrating an appropriate immune response to pathogens. To do so, it has evolved into two specialized subsets that direct type 1 and type 2 immunity. Here, we discuss the genetic programs that control lineage commitment of progenitor T helper cells along each of these pathways.
We previously demonstrated that CD81-/- mice fail to develop Th2-biased immune responses and allergen-induced airway hyper-reactivity. Because CD81 is expressed on both activated T and on B cells, we examined the role of CD81 expression by each cell type. We established an in vitro system by backcrossing the CD81 deletion to TCR transgenic (Tg) mice and to BCR Tg mice. Here we demonstrate that CD81 expression by T cells is critical for their induction of IL-4 synthesis by B cells. CD81-/- TCR Tg T cells were impaired in IL-4 production compared to CD81+/+ TCR Tg T cells, whereas CD81-/- and CD81+/+ BCR Tg B cells induced equivalent amounts of IL-4 in CD81+/+ TCR Tg T cells. CD81-/- TCR Tg T cells expressed reduced levels of ICOS, GATA-3, STAT6 and phosphorylated STAT6 when activated by antigen-presenting B cells. Taken together, these results indicate that CD81 expression by T cells greatly enhances cognate T-B cell interactions and greatly augments intracellular activation pathways leading to Th2 polarization.
To study the relation between the expression of transcription factor T-bet/GATA3 and Th1/Th2 type cytokines in peripheral blood mononuclear cells (PBMC) from lung cancer patients and their interference by the traditional Chinese herbal medicine.
The gene expression of Th1/Th2 type cytokine IFN gamma, IL-2, IL-4, IL-6, IL-10, transcription factor T-bet/GATA3 and tumor tissue specific mRNA CEA, CK19 in PBMC from lung cancer patients were detected by reverse transcription-polymerase chain reaction RT-PCR. Meanwhile, the change of IFN gamma, IL-4, T-bet and GATA3 in PBMC before and after being cultured with the traditional Chinese herbal medicine-Astragulus and Tetramethylpyrazine was also observed.
Predominant expression of Th2 type cytokines was detected in 42 lung cancer patients. The positive rates of IL-4, IL-6, IL-10, IFN gamma and IL-2 were 27/42, 24/42, 31/42, 4/42 and 5/42, respectively. But, the positive rates of transcription factor T-bet and GATA3 were 16/42 and 34/42. Moreover, the expression intensity of T-bet was lower in the CEA and CK19 positive patients than the negative ones. On the contrary, the expression intensity of GATA3 was significantly higher in the same patients.
Predominant expression of Th2 type cytokines may be related to lower expression of T-bet or higher expression of GATA3. This condition can be interfered by the traditional Chinese herbal medicine-Astragulus and Tetramethylpyrazine.
Recent studies raise the possibility that T helper (Th) polarization may be attributable to generalized activation and regulated silencing rather than regulated activation of target cytokine genes. The binding of transcription factors GATA-3 or T-bet to specific enhancers does recruit transcription factors such as NFAT-1 to IL-4 or IFNgamma promoters, respectively; however, GATA-3 also intrinsically suppresses T-bet and vice versa. Silencing of GATA-3/T-bet, which is influenced by factors such as cytokines, is associated with irreversible Th polarization. For the first few divisions (perhaps reflecting the situation in lymph nodes), naive Th cells retain pluripotency; after further cell divisions (perhaps under the influence of an inflammatory cytokine milieu) they may become polarized appropriately to respond to the specific environment.
The commitment of naive T cells to polarized Th cells requires specific changes in their transcription factors. Retrovirally overexpressed GATA-3 has been reported to induce the Th2 cytokine profile in developing Th1 cells. In this study, we examined the role of the N-terminal finger (Nf) of GATA-3 in Th2 cell development. The Nf, as well as the C-terminal finger and the transactivation domain, is critical for the induction of the Th2 phenotype. Using the GATA-3-Nf as a bait, our yeast two-hybrid screening identified friend of GATA (FOG) in the Th2 cell-specific library. Naive T cells express significant levels of FOG mRNA, which was rapidly down-regulated upon commitment to both Th1 and Th2 lineages. In reporter assays, FOG blocked the GATA-3-mediated activation of several cytokine promoters. Finally, retroviral expression of FOG in developing Th2 cells suppressed both IL-4 and IL-5 and allowed for IFN-gamma production, which was accompanied by a significant level of T-bet mRNA expression. Serial deletion mutation analysis indicated that the N-terminal region, but not the consensus C-terminal binding protein-binding motif, of FOG is critical for the effects. Our results clearly indicate that 1) FOG is a repressor of GATA-3 in naive T cells and 2) the down-regulation of FOG induces Th2 cell differentiation by releasing GATA-3 from its repression.
The GATA family of transcription factors regulates development of multiple tissues. Several GATA factors have two promoters directing distinct tissue-specific expression. Although GATA-3 acts in both neuronal and thymocyte development, no alternative promoter usage has been reported. We examined various cell types and tissues for potential alternative GATA-3 transcripts and identified an alternative transcript directed by a promoter located 10 kb upstream of the recognized promoter. Sequences within this promoter and alternative first exon are highly conserved between mouse and human genomes. This new promoter is expressed selectively in the brain but is essentially undetectable in the thymus. In contrast, the recognized promoter is selectively expressed in the thymus but not in the brain. We also observed a gradual increase in expression from this new promoter during Th2 development. These results indicate that similar to other GATA factors, the GATA-3 gene can be controlled by two promoters that may direct lineage- and tissue-specific expression.
Asthma therapy, like other therapies, has been moving towards a molecular basis for several years. This year, there have been several preclinical studies published which utilize attributes or facets of DNA to address asthma therapeutics. These include antisense oligonucleotides (against the nuclear transcription factor GATA-3 and the mast cell chemotactic agent, stem cell factor), gene transfer (of interleukin-18, both by plasmid and viral vectors), and CpG oligodeoxynucleotides (which suppress Th2 and stimulate Th1 responses). No clinical experience has yet been reported for any of these areas of research in asthma, but clinical trials are ongoing utilizing CpG oligonucleotides.
To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.
After activation, CD4 helper T (Th) cells differentiate into Th1 or Th2 effector cells. These two subsets produce distinct profiles of cytokines and regulate different immune responses. Here we discuss transcription factors and signaling pathways that are selectively expressed or activated in Th1 and Th2 cells to regulate cytokine gene expression, cell proliferation and apoptosis.
CD30 ligand (CD30L), but not its cognate receptor CD30, is frequently expressed on acute myeloid leukaemia (AML) blasts. In the present study, we found that leukaemic blasts presenting surface CD30L displayed a characteristic cytokine-receptor pattern that makes them ideal targets for those cytokines usually produced by Th2-type cell subsets. In particular, even though a broad distribution of Th2 cytokine receptors by AML blasts was shown, we demonstrated the almost exclusive expression of interleukin 4 (IL-4) receptor (R), in the absence of its cognate cytokine, by CD30L+ AML. Furthermore, a number of Th2-associated markers, including CD30, IL-4 and GATA-3, were expressed by residual T cells derived from CD30L+ AML but not from CD30L- AML, in which the presence of the Th1-associated marker LAG-3 was documented in some cases. The production of IL-4 in the absence of interferon gamma (IFN-gamma) was also detected in CD3+/CD30+ T cells from CD30L+ AML. These results, along with the shift toward IL-4-producing specific T-cell clones observed in CD30L+ AML samples by enzyme-linked Immunospot (ELISpot) assay, were consistent with the hypothesis of a Th2 polarization taking place in T cells from CD30L+ AML. The notion that IL-4 was able to enhance in vitro proliferation of CD30L+/IL-4R+ purified leukaemic blasts suggests that the selective interaction of IL-4-producing CD30+ T cells with CD30L+ leukaemic progenitors may have a role in the progression of this particular AML subset.
Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.
Using a transgenic approach, we studied the role of GATA-3 in T cells. As previously shown, enforced GATA-3 expression in transgenic mice inhibits Th1 differentiation of CD4 T cells, but unexpectedly, both type 1 (interferon gamma) and type 2 (interleukin (IL)-4 and IL-13) cytokine genes were activated in the transgenic CD8 T cells. Because IL-13 gene expression was highly enhanced in vivo by GATA-3 expression, we studied the human and the mouse IL-13 gene promoters and found an evolutionary-conserved association of a consensus GATA binding site and two GATG motifs. We showed that efficient GATA-3 binding to this regulatory sequence required these three motifs and that the affinity of the GATA zinc fingers for this association was five times higher than for the consensus GATA binding site alone. Transfections in a T cell line or transactivation by GATA-3 showed that the combination of the three sites was required for full transcriptional activity of the IL-13 gene promoter. Finally we showed that this association of binding sites causes a very high sensitivity of the IL-13 gene promoter to small variations in the level of GATA-3 protein. Altogether, these results indicate an important role of GATA-3 in CD8 cytokine gene expression and demonstrate that a critical network of GATA binding sites highly modulates GATA-3 activity.
The development of the immune system and the host response to microbial infection rely on the activation and silencing of numerous, differentially expressed genes. Since the mid-1980s, a primary goal has been to identify transcription factors that regulate specific genes and specific immunological processes. More recently, there has been a growing appreciation of the role of chromatin structure in gene regulation. Before most activators of a gene access their binding sites, a transition from a condensed to a decondensed chromatin structure appears to take place. The activation of transcription is then accompanied by the remodeling of specific nucleosomes. Conversely, the acquisition of a more condensed chromatin structure is often associated with gene silencing. Chromatin structure is a particularly significant contributor to gene regulation because it is likely to be a major determinant of cell identity and cell memory. That is, the propagation of decondensed chromatin at specific loci through DNA replication and cell division helps a cell remember which genes are expressed constitutively in that cell type or are poised for expression upon exposure to a stimulus. Here we review recent progress toward understanding the role of chromatin in the immune system. The interleukin-4 gene serves as a primary model for exploring the events involved in the acquisition and heritable maintenance of a decondensed chromatin structure. Studies of the interleukin-12 p40 and interferon-beta genes are then reviewed for insight into the mechanisms by which the remodeling of specific nucleosomes in the vicinity of a promoter can contribute to rapid activation following cell stimulation. Finally, basic principles of gene silencing are discussed.
gammadelta T cells predominantly produce IFN-gamma upon activation. To determine the basis for default production of IFN-gamma by gammadelta T cells, we analyzed the transcription factors T-box expressed in T cells (T-bet) and GATA-3. T-bet, absent in naive cells, was induced upon TCR signaling, with IFN-gamma production. T-bet also regulated IL-4 synthesis, as gammadelta cells isolated from T-bet-deficient mice displayed enhanced IL-4 levels with reduced IFN-gamma production. Notably, T-bet expression after TCR signaling in gammadelta cells was not down-regulated by IL-4, in conjunction with a higher ratio of T-bet:GATA-3 expression than that found in CD4(+) T cells. Indeed, overexpression of GATA-3 failed to inhibit IFN-gamma secretion in gammadelta cells to the degree seen in CD4(+) T cells. These results indicate that T-bet enhances IFN-gamma secretion and suppresses IL-4 secretion in gammadelta cells, and that GATA-3 fails to counterbalance T-bet-mediated IFN-gamma production, accounting for the default synthesis of IFN-gamma by these T lymphocytes.
The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8-E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5-10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5-13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling.
Allergen-induced recruitment of T lymphocytes and eosinophils to the airways is associated with increased expression of the transcription factor GATA-3. In this study, the relationship between airway inflammation and GATA-3 expression in the lungs was investigated using ragweed-sensitized C57BL/6J mice. Intratracheal ragweed challenge increased both the number of GATA-3-expressing cells in the perivascular and peribronchial regions and the amount of expression per cell. Interleukin (IL)-4 and IL-5 levels in bronchoalveolar lavage fluid were upregulated in parallel with GATA-3 expression. GATA-3 mRNA and protein colocalized to eosinophils. Eosinophils isolated from the lungs and stimulated with phorbol 12-myristate 13-acetate and/or A-23187 released IL-5. The release was inhibited by actinomycin D, which indicates that de novo synthesis of the cytokine was involved. Western blot analysis of proteins from isolated eosinophils demonstrated expression of the p50 subunit of nuclear factor-kappaB, a transcription factor that is implicated in control of GATA-3 expression. These data provide evidence that allergen challenge increases GATA-3 and proinflammatory cytokine expression by pulmonary eosinophils, which could provide positive feedback for the inflammatory response.
Members of the GATA family of zinc finger transcription factors are genetically controlled "master" regulators of development in the hematopoietic and nervous systems. Whether GATA factors also serve to integrate epigenetic signals on target promoters is, however, unknown. The transforming growth factor-beta (TGF-beta) superfamily is a large group of phylogenetically conserved secreted factors controlling cell proliferation, differentiation, migration, and survival in multiple tissues.
GATA-3, a key regulator of T helper cell development, was found to directly interact with Smad3, an intracellular signal transducer of TGF-beta. Complex formation required a central region in GATA-3 and the N-terminal domain of Smad3. GATA-3 mediated recruitment of Smad3 to GATA binding sites independently of Smad3 binding to DNA, and the two factors cooperated synergistically to regulate transcription from the IL-5 promoter in a TGF-beta-dependent manner. Treatment of T helper cells with TGF-beta promoted the formation of an endogenous Smad3/GATA-3 nuclear complex and stimulated production of the Th2 cytokine IL-10 in a Smad3- and GATA-3-dependent manner.
Although Smad proteins are known to interact with a number of general transcription factors, these are insufficient to explain the tissue-specific biology of TGF-beta proteins. Through its interaction with Smad3, GATA-3 is able to integrate a genetic program of cell differentiation with an extracellular signal, providing a molecular framework for the effects of TGF-beta on the development and function of specific subsets of immune cells and possibly other cell types.
We recently identified the allantois as a site producing hemopoietic and endothelial cells capable of colonizing the bone marrow of an engrafted host. Here, we report a detailed investigation of some early cytological and molecular processes occurring in the allantoic bud, which are probably involved in the production of angioblasts and hemopoietic cells. We show that the allantois undergoes a program characterized by the prominent expression of several "hemangioblastic" genes in the mesoderm accompanied by other gene patterns in the associated endoderm. VEGF-R2, at least from stage HH17 onward, is expressed and is shortly followed by transcription factors GATA-2, SCL/tal-1, and GATA-1. Blood island-like structures differentiate that contain both CD45(+) cells and cells accumulating hemoglobin; these structures look exactly like blood islands in the yolk sac. This hemopoietic process takes place before the establishment of a vascular network connecting the allantois to the embryo. As far as the endoderm is concerned, GATA-3 mRNA is found in the region where allantois will differentiate before the posterior instestinal portal becomes anatomically distinct. Shortly before the bud grows out, GATA-2 was expressed in the endoderm and, at the same time, the hemangioblastic program became initiated in the mesoderm. GATA-3 is detected at least until E8 and GATA-2 until E3 the latest stage examined for this factor. Using in vitro cultures, we show that allantoic buds, dissected out before the establishment of circulation between the bud and the rest of the embryo, produced erythrocytes of the definitive lineage. Moreover, using heterospecific grafts between chick and quail embryos, we demonstrate that the allantoic vascular network develops from intrinsic progenitors. Taken together, these results extend our earlier findings about the commitment of mesoderm to the endothelial and hemopoietic lineages in the allantois. The detection of a prominent GATA-3 expression restricted to the endoderm of the preallantoic region and allantoic bud, followed by that of GATA-2, is an interesting and novel information, in the context of organ formation and endoderm specification in the emergence of hemopoietic cells.
Peroxisome proliferator activator receptor (PPAR)-binding protein (PBP) is an important coactivator for PPARgamma and other nuclear receptors. It has been identified as an integral component of a multiprotein thyroid hormone receptor-associated protein/vitamin D(3) receptor-interacting protein/activator-recruited cofactor complexes required for transcriptional activity. Here, we show that PBP is critical for the development of placenta and for the normal embryonic development of the heart, eye, vascular, and hematopoietic systems. The primary functional cause of embryonic lethality at embryonic day11.5 observed with PBP null mutation was cardiac failure because of noncompaction of the ventricular myocardium and resultant ventricular dilatation. There was a paucity of retinal pigment, defective lens formation, excessive systemic angiogenesis, a deficiency in the number of megakaryocytes, and an arrest in erythrocytic differentiation. Some of these defects involve PPARgamma and retinoid-sensitive sites, whereas others have not been recognized in the PPAR-signaling pathway. Phenotypic changes in four organ systems observed in PBP null mice overlapped with those in mice deficient in members of GATA, a family of transcription factors known to regulate differentiation of megakaryocytes, erythrocytes, and adipocytes. We demonstrate that PBP interacts with all five GATA factors analyzed, GATA-1, GATA-2, GATA-3, GATA-4, and GATA-6, and show that the binding of GATA-1, GATA-4, and GATA-6 to PBP is not dependent on the nuclear receptor recognition sequence motif LXXLL (where L is leucine and X is any amino acid) in PBP. Coexpression of PBP with GATA-3 markedly enhanced transcriptional activity of GATA-3 in nonhematopoietic cells. These observations identify the GATA family of transcription factors as a new interacting partner of PBP and demonstrate that PBP is essential for normal development of vital organ systems.
The development of naive CD4+ T cells into a T helper (Th) 2 subset capable of producing interleukin (IL)-4, IL-5, and IL-13 involves a signal transducer and activator of transcription (Stat)6-dependent induction of GATA-3 expression, followed by Stat6-independent GATA-3 autoactivation. The friend of GATA (FOG)-1 protein regulates GATA transcription factor activity in several stages of hematopoietic development including erythrocyte and megakaryocyte differentiation, but whether FOG-1 regulates GATA-3 in T cells is uncertain. We show that FOG-1 can repress GATA-3-dependent activation of the IL-5 promoter in T cells. Also, FOG-1 overexpression during primary activation of naive T cells inhibited Th2 development in CD4+ T cells. FOG-1 fully repressed GATA-3-dependent Th2 development and GATA-3 autoactivation, but not Stat6-dependent induction of GATA-3. FOG-1 overexpression repressed development of Th2 cells from naive T cells, but did not reverse the phenotype of fully committed Th2 cells. Thus, FOG-1 may be one factor capable of regulating the Th2 development.
The Tec kinases Rlk and Itk are critical for full T cell receptor (TCR)-induced activation of phospholipase C-gamma and mitogen-activated protein kinase. We show here that the mutation of Rlk and Itk impaired activation of the transcription factors NFAT and AP-1 and production of both T helper type 1 (TH1) and TH2 cytokines. Consistent with these biochemical defects, Itk-/- mice did not generate effective TH2 responses when challenged with Schistosoma mansoni eggs. Paradoxically, the more severely impaired Rlk-/-Itk-/- mice were able to mount a TH2 response and produced TH2 cytokines in response to this challenge. In addition, Rlk-/-Itk-/- cells showed impaired TCR-induced repression of the TH2-inducing transcription factor GATA-3, suggesting a potential mechanism for TH2 development in these hyporesponsive cells. Thus, mutations that affect Tec kinases lead to complex alterations in CD4+ TH cell differentiation.
Naïve CD4(+) helper T (T(H)) cells respond to stimulation by terminally differentiating into two mature classes, T(H)1 cells, which express interferon gamma (IFN-gamma), and T(H)2 cells, which express interleukin 4 (IL-4). The transcriptional activators T-bet and Gata-3 mediate commitment to the T(H)1 and T(H)2 fates, respectively, including chromatin remodeling of signature genes. The cytokine IL-12 fosters growth of committed T(H)1 cells, while IL-4 fosters growth of committed T(H)2 cells. IL-12 and IL-4 also play critical roles in commitment by promoting transcriptional silencing of Gata-3 and T-bet, respectively. We now show that both T-bet and Gata-3 are induced in a cell cycle-independent manner in bipotent progenitor cells. In contrast, both lineage-restricted gene induction by the activator proteins and heritable silencing of the transcription of each activator, the hallmarks of terminal differentiation, are cell cycle dependent. We found that cells that cannot cycle remain uncommitted and bipotent in response to the most polarizing signals for maturation. These results provide mechanistic insight into a mammalian model of terminal differentiation by illustrating that cell cycle-coupled epigenetic effects, as originally described in yeast, may represent an evolutionarily conserved strategy for organizing signaling and cell fate.
1alpha,25-Dihydroxyvitamin D3 (vitD3) is an immunoregulatory hormone with beneficial effects on Th1 mediated autoimmune diseases. Although the inhibitory effects of vitD3 on macrophages and dendritic cells are well documented, any direct effects of vitD3 on Th cell development are not clearly defined. Using CD4(+)Mel14(+) T cells derived from mice on a BALB/c and a C57BL/6 genetic background we examined the effect of vitD3 on Th cell development. We demonstrated that vitD3 affects Th cell polarization by inhibiting Th1 (IFN-gamma production) and augmenting Th2 cell development (IL-4, IL-5, and IL-10 production). These effects were observed in cultures driven with splenic APC and Ag, as well as with anti-CD3 and anti-CD28 alone, indicating that CD4(+) cells can also be direct targets for vitD3. The enhanced Th2 development by vitD3 was found in both BALB/c and C57BL/6 mice. An increased expression of the Th2-specific transcription factors GATA-3 and c-maf correlated with the increased production of Th2 cytokines after vitD3 treatment. The vitD3-induced effects were largely mediated via IL-4, because neutralization of IL-4 almost completely abrogated the augmented Th2 cell development after vitD3 treatment. These findings suggest that vitD3 acts directly on Th cells and can, in the absence of APC, enhance the development of a Th2 phenotype and augment the expression of the transcription factors c-maf and GATA-3. Our findings suggest that the beneficial effects of vitD3 in autoimmune diseases and transplantation operate through prevention of strong Th1 responses via the action on the APC, while simultaneously directly acting on the T cell to enhance Th2 cell development.
Following bone marrow transplantation, patients often suffer from immune incompetence by reduced or late T cell development. Moreover, adult bone marrow stem cells have a lower capacity to generate T cells compared with fetal liver- and umbilical cord blood-derived progenitors. Therefore, enhancing thymic-dependent T cell generation might hold great therapeutic potential. GATA-3 is a transcription factor that is essential in T cell development. In this study we examined the therapeutic potential of GATA-3 to enhance T cell generation by overexpressing GATA-3 in T cell progenitors followed by fetal thymic organ culture (FTOC). We observed that early during FTOC, there was an enhanced differentiation toward the double positive stage of T cell development. From day 10 of FTOC, however, overexpression of GATA-3 induced a severe reduction in thymic cellularity, which probably correlates with the absence of a functional TCR-beta chain. We further show that the frequency of apoptosis was increased in GATA-3-transduced thymocytes. Despite the absence of a functional TCR-beta chain, GATA-3 transduced progenitors were able to differentiate into CD8beta(+) double positive thymocytes. This study shows that a strictly regulated expression of GATA-3 is essential for normal T cell development and this puts severe restrictions on the potential therapeutic use of continuously overexpressed GATA-3.
IL-13, a Th2 cell-specific cytokine, is a major effector molecule mediating several pathological features of allergic asthma. However, the transcriptional regulation of the IL-13 gene remains unclear. Here we demonstrate, by using intracellular cytokine staining, that IL-13 is not always coexpressed with other Th2 cytokines in normal Th cells on a single cell basis. In addition, we identified and cloned a minimal inducible and cell type-specific promoter of the murine IL-13 gene. The cell type specificity of the minimal IL-13 promoter is mediated by a functionally critical GATA-3 site that binds endogenous GATA-3 proteins, whereas the induction by PMA/ionomycin is mediated by distinct cis-acting elements. Furthermore, by expressing GATA-3 in wild-type and c-maf transgenic Th1 cells, we demonstrate that the expression of IL-13 is regulated by a mechanism distinct from that regulating the expression of IL-4, and that the expression of Th1 and Th2 cytokine genes does not have to be mutually exclusive in effector Th cells.
GATA-binding proteins are a subfamily of zinc finger transcription factors with six members (GATA-1-6) that interact with the GATA deoxyribonucleic acid (DNA) sequence. This sequence is found in the regulatory regions of many genes including those encoding T-helper 2 (Th2)-like cytokines, receptors, adhesion molecules and enzymes, which may be important in the pathogenesis of bronchial asthma. The expression of GATA-3, 4 and -6 was investigated in peripheral blood T-lymphocytes and monocytes and bronchial biopsies from 11 normal subjects and 10 steroid-naive asthmatic patients. Using Western blot analysis, T-cells from asthmatic subjects expressed 5 times the level of GATA-3 compared to that in normals. Confocal microscopy indicated that GATA-3 expression was both nuclear and cytoplasmic. GATA DNA binding complex containing GATA-3 was elevated in Th2 cells as determined by electrophorectic mobility shift assay. In contrast, monocytes from normal and asthmatic subjects expressed GATA-4 and -6 in equal amounts, but no GATA-3 was found. Using immunohistochemistry in bronchial biopsies, epithelial cells expressed high levels of GATA-3, GATA-4 and GATA-6 proteins. Comparison of Western blots of bronchial biopsies showed no significant differences between normal and asthmatic subjects. In conclusion, the increased expression of GATA-3 in asthmatic T-cells may underlie augmented T-helper 2-like cytokines in this disease. However, the unaltered GATA-3 expression in epithelial cells suggests a distinct role for GATA-3 in these cells unrelated to T-helper 2-like cytokine release. Finally, no evidence was found for an increased expression of GATA-4 and GATA-6 in asthma.
The objective of this study was to define phenotypic changes of antigen-presenting cells (APCs) and T cells in a murine model of antigen-induced airways inflammation that involves intraperitoneal sensitization with ovalbumin (OVA)/adjuvant followed by antigen aerosolization. We investigated the APC and T-cell compartments both after sensitization (primary immune response) and after challenge (secondary immune response) at the thoracic lymph nodes (initiation site) and the lung (effector site). Our findings document a major cellular expansion in the lymph nodes after both sensitization and challenge. After sensitization, this expansion was comprised mainly of B cells, a considerable proportion of which expressed B7.2. At this time, T cells were markedly expanded and activated as assessed by CD69 expression; further, although GATA-3 and signal transducer and activator of transcription-6 were expressed at this time point, expression of interleukin (IL)-4, IL-5, and IL-13 messenger RNA (mRNA) levels were marginal. However, in vitro stimulation of lymph-node cells with OVA led to cytokine production. In contrast, 24 h after challenge, but not after sensitization, there was a major expansion of dendritic cells and macrophages in the lungs. This expansion was associated with enhanced expression of both B7.1 and B7.2. We also observed expansion of activated CD3(+)/CD4(+) T cells expressing the T helper-2-associated marker T1/ST2 in the lung, most notably 5 d after challenge. Further, IL-4, IL-5, and IL-13, but not interferon-gamma mRNA were expressed at high levels 3 h after challenge. This study helps to elucidate the "geography" of immune responses generated in a conventional murine model of allergic airways inflammation.
Interleukin-5 (IL-5) is a T-cell cytokine involved in Type 2 diseases and is commonly described as being coordinately regulated with other Type 2 cytokines, such as IL-4 and IL-13. Considering the unique control of eosinophilia by IL-5, such coordinate regulation would be surprising. In fact, the biological specificity of eosinophilia and its control by IL-5 suggests a unique and independent control of IL-5 regulation. In this report we show the binding of GATA-3 to three sites in the human IL-5 promoter in the human T-cell line PER117. The previously identified -70 site and another site at position -152 are shown to positively regulate IL-5 transcription. More importantly, the site located at -400 acts as a powerful repressor of IL-5 transcription with mutagenesis of this site allowing a high level expression of IL-5 without the activation of other factors normally required for IL-5 expression. Whereas GATA-3 has been proposed to be involved in the regulation of the IL-4/IL-5/IL-13 locus, we show here that it has another function in controlling IL-5 transcription that supports the observed unique biological function of this cytokine.
Recently, a member of the GATA-binding family of transcription factors was shown to be involved in the human hypoparathyroidism, sensorineural deafness and renal anomalies (HDR) syndrome. Deletion-mapping studies and subsequent mutation analysis revealed that haploinsufficiency for GATA3 is the underlying mechanism of the HDR syndrome. Here we discuss the clinical characteristics of the HDR syndrome and present an overview of the role of GATA3 and related GATA-binding transcription factors during vertebrate embryonic development and their involvement in human disease.
IL-5 is produced by the T(H)2 subset of CD4(+) T lymphocytes and is necessary for the eosinophilia typical of allergic conditions. Glucocorticoids such as dexamethasone are highly effective inhibitors of eosinophilic inflammation, and one of their effects is inhibition of IL-5 gene expression.
We wished to examine the effect of dexamethasone on the binding of nuclear factors from primary human CD4(+) T lymphocytes to the RE-I and RE-II positively acting regulatory elements of the IL-5 promoter.
CD4(+) T cells, purified from PBMCs by magnetic bead separation, were activated with anti-CD3 antibody and phorbol myristate acetate. Nuclear extracts were tested in electrophoretic mobility shift assays with probes based on RE-I and RE-II.
In extracts from activated cells, the RE-II region of the promoter formed a complex that was shown by supershift assay to contain NFATc. This complex was abolished by treatment of the cells with dexamethasone before activation and was weak or absent in unactivated cells. By contrast, binding to the RE-I region and to the GATA-3 site within RE-I was observed in resting cells and was not affected by activation or treatment with dexamethasone.
Dexamethasone inhibits the inducible binding of factors to the RE-II region but does not affect the constitutive binding to the RE-I region. Characterization of such molecular effects of glucocorticoids could enable the development of specific inhibitors of IL-5 expression that lack the side effects of glucocorticoids.
Polycomb group (PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNgamma was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.
Three GATA family transcription factors are involved in various aspects of hematopoiesis. Their lineage-restricted expression correlates well with their function in selective lineage commitment and differentiation. We focused on the role of GATA-3 to determine whether an intrinsic variation among different GATA proteins, in addition to the distinct expression pattern, determines lineage specification.
Using a retroviral vector, we introduced the GATA-3 gene into primary murine hematopoietic stem cells (HSC) and examined their development in in vitro suspension culture and colony-forming assays as well as in vivo competitive repopulation studies.
Although GATA-3 expression normally is restricted to lymphoid precursor and committed T cells, overexpression of GATA-3 in HSC results in cessation of cell expansion followed by selective induction of megakaryocytic and erythroid differentiation and inhibition of myeloid and lymphoid precursor development in liquid suspension culture and in vitro colony-forming assays. Competitive repopulation studies show that transplanted GATA-3-expressing HSC/progenitor cells give one wave of erythrocyte development but fail to expand in the bone marrow or to reconstitute other lineages.
The selective megakaryocytic/erythroid differentiation in HSC with enforced GATA-3 expression suggests a functional redundancy among GATA proteins and indicates that the specific lineage fate determination by individual GATA proteins is largely regulated at the level of expression in a lineage and developmental-stage restricted fashion, whereas the identity of the GATA factor may not be as important.
The transcription factor GATA-3 is essential for early T cell development and differentiation of naive CD4(+) T cells into Th2 effector cells. To study the function of GATA-3 during T cell-mediated immune responses in vivo, we investigated CD2-GATA3-transgenic mice in which GATA-3 expression is driven by the CD2 locus control region. Both in the CD4(+) and the CD8(+) T cell population the proportion of cells exhibiting a CD44(high)CD45RB(low)CD62L(low) Ag-experienced phenotype was increased. In CD2-GATA3-transgenic mice, large fractions of peripheral CD4(+) T cells expressed the IL-1 receptor family member T1/ST2, indicative of advanced Th2 commitment. Upon in vitro T cell stimulation, the ability to produce IL-2 and IFN-gamma was decreased. Moreover, CD4(+) T cells manifested rapid secretion of the Th2 cytokines IL-4, IL-5, and IL-10, reminiscent of Th2 memory cells. In contrast to wild-type CD4(+) cells, which lost GATA-3 expression when cultured under Th1-polarizing conditions, CD2-GATA3-transgenic CD4(+) cells maintained expression of GATA-3 protein. Under Th1 conditions, cellular proliferation of CD2-GATA3-transgenic CD4(+) cells was severely hampered, IFN-gamma production was decreased and Th2 cytokine production was increased. Enforced GATA-3 expression inhibited Th1-mediated in vivo responses, such as Ag-specific IgG2a production or a delayed-type hypersensitivity response to keyhole limpet hemocyanin. Collectively, these observations indicate that enforced GATA-3 expression selectively inhibits Th1 differentiation and induces Th2 differentiation. The increased functional capacity to secrete Th2 cytokines, along with the increased expression of surface markers for Ag-experienced Th2-committed cells, would argue for a role of GATA-3 in Th2 memory formation.
The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.
A current paradigm has ICOS participating in TH2 costimulation. New data indicates ICOS regulates not only TH2 cells, but also TH1s.
Endothelial vascular cell adhesion molecule-1 (VCAM-1) is expressed in response to cytokine stimulation and plays a critical role in inflammatory reactions. Previously, we developed a novel VCAM-1 inhibitor that acts through a mechanism independent of nuclear factor-kappaB activity. It suppresses the binding activity of GATA proteins in cytokine-stimulated endothelial cells, which may be related to the anti-VCAM-1 induction effect of this drug. In this study, we investigated the role of GATA proteins in the induction of VCAM-1 by tumor necrosis factor-alpha (TNF-alpha) in human endothelial cells. The mRNA expression of GATA-6 was increased, whereas GATA-3 mRNA was decreased by TNF-alpha stimulation. Electrophoretic mobility shift assay showed that TNF-alpha stimulation increased the DNA binding of GATA-6 but decreased that of GATA-3. Experiments using protein overexpression or antisense oligonucleotides revealed that GATA-6 potently acts as a positive regulator of VCAM-1 gene transcription. In contrast, overexpression of GATA-3 was able to suppress TNF-alpha-induced VCAM-1 expression. Our results provide evidence of the importance of GATA proteins in the induction of VCAM-1 by TNF-alpha in vascular endothelial cells. The switch from GATA-3 to GATA-6 is taken to be an important transcriptional control event in TNF-alpha induction of VCAM-1.
IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R alpha-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Ralpha, as well as the wild-type (WT) hIL-4Ralpha, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.
Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4-producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate-labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine.
We report on GATA3 analysis and the phenotypic spectrum in nine Japanese families with the HDR syndrome (hypoparathyroidism, sensorineural deafness, and renal dysplasia) (MIM 146255). Fluorescence in situ hybridisation and microsatellite analyses showed heterozygous gross deletions including GATA3 in four families. Sequence analysis showed heterozygous novel mutations in three families: a missense mutation within the first zinc finger domain at exon 4 (T823A, W275R), an unusual mutation at exon 4 (900insAA plus 901insCCT or C901AACCCT) resulting in a premature stop at codon 357 with loss of the second zinc finger domain, and a nonsense mutation at exon 6 (C1099T, R367X). No GATA3 abnormalities were identified in the remaining two families. The triad of HDR syndrome was variably manifested by patients with GATA3 abnormalities. The results suggest that HDR syndrome is primarily caused by GATA3 haploinsufficiency and is associated with a wide phenotypic spectrum.
A highly conserved sequence block (CSB) located in the mouse and human T cell receptor (TCR) Jalpha loci is recognized by tissue-specific factors and up-regulates TCR alpha enhancer activity. In this study, the properties of CSB-interacting factors were further explored to decipher the function of this cis-acting element. Thymocytes corresponding to different developmental stages were found capable of forming differential CSB-nucleoprotein complexes. Pronounced changes in the CSB-complex-forming activity were observed during the transition from double-negative to double-positive thymocytes. Furthermore, we showed that transcription factors Oct-1, Oct-2 and GATA-3 interacted with CSB both in vitro, as evidenced by electrophoretic mobility shift assays, and in vivo, as demonstrated by chromatin immunoprecipitation assays in mouse thymus. Importantly, we also demonstrated that GATA-3 associated in vivo with TCR alpha enhancer, the activity of which is known to be required in regulating chromatin accessibility to the V(D)J recombinase. Thus, CSB may temporally regulate local chromatin structure and help to spread TCR alpha enhancer activity over the entire 70-kb Jalpha locus by forming developmentally regulated CSB-nucleoprotein complexes and by interacting with other cis-regulatory element-nucleoprotein complexes present within the TCR alpha / delta locus.
Differentiated T helper 1 (Th1) and T helper 2 (Th2) T-cells show striking differences in their patterns of cytokine expression. This process is initiated by stimulation with antigen and the cytokines IL-12 and IL-4, respectively, and requires antigen-induced transcription factors such as NFAT and cytokine-induced transcription factors such as STAT4, induced by IL-12, and STAT6, induced by IL-4. This results in induction and maintained expression of subset-specific transcription factors including T-bet in Th1 cells and GATA3 in Th2 cells, which are involved in ensuring the commitment of T-cells to Th1 or Th2 lineages. Here we review the signalling pathways and transcription factors that mediate T-cell differentiation, and describe the epigenetic changes in chromatin structure, locus accessibility and DNA methylation that are known to accompany this process.
Using a transgenic approach, we examined distal regulatory elements located in the IL-4 locus and the role of GATA-3 at these elements. The intergenic DNase I hypersensitive sites (HSS) showed strong enhancement, and the intronic enhancer (IE) and HS5/HS5a sites showed weaker enhancement of the IL-4 promoter. Elements in the 3' region of the IL-4 gene contributed to Th2 specificity. All individual enhancers were T cell activation dependent but not Th2 specific, with the exception of IE. However, when these distal elements were combined into a "minilocus," expression was strongly enhanced and Th2 specific. GATA-3 mediated strong enhancement of IL-4 promoter activity in Th1 cells when the promoter was embedded in the minilocus or linked to HSS and IE, demonstrating that GATA-3 acts through these elements to regulate IL-4 gene expression.
The precise nature of allergen-specific cytokine responses in atopics versus non-atopics, in particular the 'Th1 polarity' of responses in non-atopics, remains controversial. This is due in part to the relative insensitivity of cytokine detection systems, and associated variations in kinetics of cytokine production and catabolism in in vitro culture systems. As an alternative to cytokine measurement, this study focuses on expression of the transcription factor GATA-3 for analysis of allergen-specific Th cell responses.
Cord blood mononuclear cells were Th1- or Th2-polarized by culture in IL-12- or IL-4-employing established methods; PBMC from house dust mite (HDM)-sensitive atopics and controls were stimulated overnight with HDM; cytokine production was measured by ELISA and GATA-3 mRNA expression by PCR.
Cytokine-driven Th2 polarization of naive T cells is associated with marked upregulation of GATA-3 expression, whereas a reciprocal expression pattern accompanies differentiation towards the Th1 cytokine phenotype. In T cells from HDM skin prick test-positive (HDM-SPT+/HDM-IgE+) volunteers, overnight stimulation results in marked upregulation of GATA-3 expression, compared to an equally marked downregulation of expression in T cells from SPT-/IgE- subjects. In subjects who are HDM-SPT+ but IgE-, GATA-3 expression levels remained relatively stable during culture with HDM.
Upregulation of GATA-3 expression in PBMC is a hallmark of the early phase of Th2 recall responses to specific allergen in atopics. The reciprocal expression pattern observed in HDM-specific recall responses of non-atopics provides independent confirmation of the presence of underlying Th1-like immunity in these subjects. The parallel findings in neonatal T cells suggest that the same approach may be utilized for monitoring the progress of allergen-specific Th1/Th2 memory development during early childhood, and hence in assessment of risk for future allergic disease.
IL-12 is a potent inducer of IFN-gamma production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling beta2-subunit of the IL-12R and, therefore, do not signal in response to IL-12. The question was raised as to what extent the loss of the IL-12Rbeta2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL-12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA-3 expression and induction of T-bet expression. These reversed cells are further characterized by a marked IL-12Rbeta2 chain expression and fully restored IL-12-inducible STAT4 activation. The IL-12-induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL-4 and in the absence of IL-12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen-specific Th2 cell clones. These findings suggest the possibility of restoring IL-12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL-12, and that IL-12-promoting immunotherapy can be beneficial for Th2-mediated immune disorders, targeting both naive and memory effector T cells.
The expression of IL-4 and IL-5 is increased in patients with atopic asthma compared with control subjects and correlates with indices of pulmonary function. In nonatopic asthma the expression of IL-4, unlike IL-5, fails to correlate with pulmonary function, and compared with their atopic counterparts, these patients have fewer cells expressing IL-4 receptor (IL-4R). As such, a deficiency in the IL-4 signaling pathway may be implicated in nonatopic asthma. The transcription factors GATA-3 and cMAF mediate IL-4 and IL-5 synthesis, whereas signal transducer and activator of transcription 6 (STAT-6) is critical for IL-4R signaling.
This study examines the expression profile of these transcription factors in asthma, according to atopic status.
With immunocytochemistry, the expression of GATA-3, cMAF, and STAT-6 protein was determined in sections of bronchial biopsy specimens from patients with atopic asthma (n = 7), patients with nonatopic asthma (n = 8), and control subjects (n = 8).
Higher numbers of cells expressing GATA-3 and cMAF were observed in patients with atopic and those with nonatopic asthma than in control subjects and patients with tuberculosis (P <.001). There were also more STAT-6-immunoreactive cells in patients with atopic and those with nonatopic asthma than in control subjects (P <.0001, P <.05). Notably, however, fewer cells expressing STAT-6 protein were observed in nonatopic versus atopic asthma (P <.0001).
These results demonstrate the upregulation of GATA-3 and cMAF in both variants of asthma and indicate that reduced IL-4R signaling, because of lower STAT-6 expression, may be a feature of nonatopic asthma.
Naive CD4+ T cells activated through TCR/CD28 under Th1 or Th2 conditions expressed canonical cytokine patterns irrespective of cell division. Only cells that had divided fewer than four times were capable of reexpressing alternative cytokines when restimulated under opposing conditions. Although T cells transcribed both IFN-gamma and IL-4 within hours in a Stat4-/Stat6-independent manner, neither T-bet nor GATA-3 was induced optimally without Stat signals, and polarized cytokine expression was not sustained. Cytokine genes were positioned apart from heterochromatin in resting T cell nuclei, consistent with rapid expression. After polarization, the majority of silenced cytokine alleles were repositioned to heterochromatin. Naive T cells transit through sequential stages of cytokine activation, commitment, silencing, and physical stabilization during polarization into differentiated effector subsets.
Development of Th2 subset of CD4+ T cells involves the interleukin-4 (IL-4)- and Stat6-dependent increase in GATA-3 expression during primary activation. Recently we reported that the phenotypic stability and factor independence of Th2 cells involves acquisition of an intracellular pathway that maintains GATA-3 expression. Evidence from retroviral expression studies implied that this pathway involved an autoactivation of GATA-3 expression, since Stat6-deficient T cells induced endogenous GATA-3 when infected with GATA-3-expressing retroviruses. That study left unresolved the issue of whether GATA-3 autoactivation was direct or indirect. Several other Th2-specific transcription factors have been described, including c-Maf and JunB. We therefore examined the ability of these other transcription factors to induce GATA-3 expression and promote Th2 development. Neither c-Maf nor JunB induced Th2 development in Stat6-deficient CD4+ T cells, in contrast to GATA-3. Consistent with this indication of a possible direct autoactivation pathway, we also observed that heterologous GATA family proteins GATA-1, GATA-2, and GATA-4 were also capable of inducing GATA-3 expression in developing Stat6-deficient T cells and promote Th2 development. Mutational analysis revealed evidence for two distinct mechanisms of GATA-3 action. IL-4 induction by GATA-3 required each of the functional domains to be present, whereas repression of gamma interferon could occur even when mutants of GATA-3 lacking the second transactivation domain, TA2, were expressed. The GATA-dependent induction of the GATA-3 but not the other GATA genes in T cells suggests that T-cell-specific cis elements within the GATA-3 locus likely cooperate with a general GATA recognition motif to allow GATA-3-dependent autoactivation.
The transcription factor GATA-3 is expressed in T helper 2 (TH2) but not TH1 cells and plays a critical role in TH2 differentiation and allergic airway inflammation in vivo. Mice that lack the p50 subunit of nuclear factor kappa B (NF-kappa B) are unable to mount airway eosinophilic inflammation. We show here that this is not due to defects in TH2 cell recruitment but due to the inability of the p50-/- mice to produce interleukin 4 (IL-4), IL-5 and IL-13: cytokines that play distinct roles in asthma pathogenesis. CD4+ T cells from p50-/- mice failed to induce Gata3 expression under TH2-differentiating conditions but showed unimpaired T-bet expression and interferon gamma (IFN-gamma) production under TH1-differentiating conditions. Inhibition of NF-kappa B activity prevented GATA-3 expression and TH2 cytokine production in developing, but not committed, TH2 cells. Our studies provide a molecular basis for the need for both T cell receptor and cytokine signaling for GATA-3 expression and, in turn, TH2 differentiation.
Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T-cell-specific transcription factor GATA-3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2-specific cytokine production. Investigating GATA-3 gene regulation in human T cells we have found that naive T cells highly express GATA-3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down-regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA-3, interleukin (IL)-5 and IL-4 gene expression kinetics, supporting the positive action of GATA-3 on Th2-specific cytokine production. A possible relationship between GATA-3 gene expression and the down-regulation of the IL-12 receptor (beta2-chain; IL-12Rbeta2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T-cell commitment the presence of IL-4 in the culture was essential to maintain or enhance GATA-3 transcription, while IL-12 was not necessary for full repression of GATA-3. Finally, we showed selective GATA-3 up-regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA-3 expression in Th1 cells upon activation.
Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.
In summary, a multitude of regulatory systems are employed to cause the selective activation of target cytokine genes in Th1 and Th2 effector cells. These mechanisms involve both positive and negative regulation and employ at least three kinds of mechanisms. In the first, selective expression of transcription factors such as GATA3 in Th2 cells and the homeobox gene HLX in Th1 cells occurs, and appears in both cases to play a causal role. Another example of this would be c-maf, discovered by the Glimcher laboratory. A second mechanism is by the selective accumulation of protein through posttranscriptional mechanisms. Thus, junB accumulates in Th2 cells despite the fact that the junB mRNA levels are not different between Th1 and Th2 cells. Finally, the selective use of signaling pathways, in the case studied here MAP kinase pathways, leads to the selective activation of target genes. We believe that transcriptional up-regulation of rac2 leads to the coupling of both the p38 and JNK MAP kinase pathways to the T-cell receptor and/or costimulatory receptors, thereby providing a lineage-specific signal.
CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.
Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus ("Hb switching" model), whereas the transition involves replacement of red-blood-cell (RBC) populations ("RBC replacement" model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180-189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to "primitive" erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to "definitive" hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.
GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. Recently, it was shown that the expression of GATA-3 is further induced in CD4+ helper T cells upon differentiation into type 2 but not type 1 effector cells. Here, we report the molecular cloning of a GATA-3 interacting protein, repressor of GATA (ROG). ROG is a lymphoid-specific gene and is rapidly induced in Th cells upon stimulation with anti-CD3. In in vitro assays, ROG represses the GATA-3-induced transactivation. Furthermore, overexpression of ROG in Th clones inhibits the production of Th cytokines. Taken together, our results suggest that ROG might play a critical role in regulating the differentiation and activation of Th cells.
Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.
The initiation of primary immune responses is the key function of specialized antigen presenting cells, the dendritic cells (DC). DC of myeloid origin capture antigens in tissues, migrate to lymphoid organs and stimulate T cell responses. A subset of DC has been described which expresses lymphoid determinants and has potential regulatory functions. Conditional transformation of chicken bone marrow progenitors with v-relER, a v-rel estrogen receptor (ER) fusion gene, allows expansion of progenitors that can be induced to differentiate into DC in vitro. In this paper we describe that v-relER cells exhibit both myeloid and lymphoid surface markers, while B cell, T cell and NK (natural killer)-specific surface markers are absent. v-relER DC express, however, cytoplasmic CD3 protein and mRNA for CD8alpha and the lymphoid transcription factor GATA-3. These data suggest that v-relER DC might be related to the lymphoid subset of DC described in mammals.
During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.
Haematopoietic precursors first colonizing the avian embryonic thymus are derived from the intraembryonic sites located around the dorsal aortae. These intraembryonic precursors have previously been demonstrated to include cells that harbour T-cell progenitor capacity and express the Ikaros transcription factor, known to be a prerequisite for lymphocyte development. In this study, we further evaluated the properties of these prethymic cells. We show that early intraembryonic cells and prethymic progenitors already express the GATA-3 transcription factor. The chicken homologue of T-cell factor-1, chTcf, is also detected in cells isolated from the avian para-aortic region. However, these intraembryonic cells retain their T-cell receptor gamma loci in germline configuration. Interestingly, chTcf was found to express different alternatively spliced isoforms during early ontogeny and thymic T-cell development, which indicates developmentally regulated expression of chTcf variants. Taken together, these results demonstrate that, although the avian prethymic progenitor cells express T-lineage-associated transcription factors, they have not yet undergone TCR rearrangements. It is therefore suggested that activation of lineage-associated genes is an early event in the generation of haematopoietic progenitor cells during ontogeny.
Breast cancer is characterized by an important histoclinical heterogeneity that currently hampers the selection of the most appropriate treatment for each case. This problem could be solved by the identification of new parameters that better predict the natural history of the disease and its sensitivity to treatment. A large-scale molecular characterization of breast cancer could help in this context. Using cDNA arrays, we studied the quantitative mRNA expression levels of 176 candidate genes in 34 primary breast carcinomas along three directions: comparison of tumor samples, correlations of molecular data with conventional histoclinical prognostic features and gene correlations. The study evidenced extensive heterogeneity of breast tumors at the transcriptional level. A hierarchical clustering algorithm identified two molecularly distinct subgroups of tumors characterized by a different clinical outcome after chemotherapy. This outcome could not have been predicted by the commonly used histoclinical parameters. No correlation was found with the age of patients, tumor size, histological type and grade. However, expression of genes was differential in tumors with lymph node metastasis and according to the estrogen receptor status; ERBB2 expression was strongly correlated with the lymph node status (P < 0.0001) and that of GATA3 with the presence of estrogen receptors (P < 0.001). Thus, our results identified new ways to group tumors according to outcome and new potential targets of carcinogenesis. They show that the systematic use of cDNA array testing holds great promise to improve the classification of breast cancer in terms of prognosis and chemosensitivity and to provide new potential therapeutic targets.
We have identified a novel Kruppel-type zinc finger (ZF) gene, SKAT-2, which is selectively expressed by murine Th2 cells. The protein encoded by this gene has 14 C2H2-type ZF tandemly arrayed at its C terminus and N-terminal SCAN box and KRAB domains. SKAT-2 is tissue restricted in expression at the RNA level, detectable only in brain and at low levels in kidney and spleen and few hematopoietic cell lines. By in situ hybridization, SKAT-2 expression was found to peak in antigen-stimulated CD4(+) T cells after 2-3 days of culture under Th2 but not Th1 biasing conditions. This pattern of expression closely mirrored that of GATA-3 in the same cells. In transient transfection experiments in phorbol 12-myristate 13-acetate/ionomycin-stimulated EL4 cells, SKAT-2 was found to up-regulate the activity of the IL-4 but not the IL-5 promoter, contrasting with the ability of GATA-3 to activate both promoters. This result was confirmed using clones of EL4 cells stably expressing an inducible form of SKAT-2, thus SKAT-2 is a novel Th2-specific gene that may play a role in selective regulation of cytokine genes in T cells.
The genomic sequence of the human GCMa/GCM1 gene, a mammalian homologue of Drosophila melanogaster GCM, was determined. Drosophila GCM is a neural transcription factor that regulates glial cell fate. The mammalian homolog however, is a placenta-specific transcription factor that is necessary for placental development. The 22 kb DNA sequence spanning the GCMa gene contains six exons and five introns, encoding a 2.8 kb cDNA. Overall genomic organization is similar for the human and mouse. Several potential binding sites for transcription factors like GATA, Oct-1, and bHLH proteins were found in the 5'-flanking region of the human gene. A DNA motif for GCM protein binding exists in the 5'-flanking region that is highly homologous with that of the mouse gene. The location of this gene was mapped to chromosome 6 using fluorescence in situ hybridization.
cAMP is an important second messenger with immunomodulatory properties. Elevation of intracellular cAMP in T cells, induced by agents such as IL-1alpha or PGs, inhibits T cell activation. In effector T cells, an increase in the level of intracellular cAMP inhibits cytokine production in Th1 cells but stimulates cytokine production in Th2 cells. Here we report that cAMP-induced effects in Th2 cells occur independently of the protein kinase A pathway, which is the major mediator of cAMP-induced signaling events in most cell types. Instead, cAMP stimulates activation of p38 mitogen-activated protein kinase in Th2 cells. This appears to be a Th2-selective event because cAMP barely increased p38 phosphorylation in Th1 cells. We show that in Th2 cells, cAMP promotes the production of both IL-5 and IL-13, which play distinct but critical roles in asthma pathogenesis. Our data also show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which we have shown is a critical regulator of Th2 cytokine gene expression and, in turn, of airway inflammation in mice. Thus, Th2-specific GATA-3 expression and p38 mitogen-activated protein kinase activation together provide a molecular basis for the differential effects of cAMP in the two T helper cell subsets.
TGF-beta is an important immunomodulatory cytokine that can inhibit differentiation of effector T cells. In this report, we address the molecular mechanisms through which TGF-beta inhibits differentiation of CD4(+) cells into Th type 2 cells. We demonstrate that TGF-beta inhibits GATA-3 expression in developing Th cells. We also show that inhibition of GATA-3 expression by TGF-beta is a major mechanism of inhibition of Th2 differentiation by TGF-beta as ectopic expression of GATA-3 in developing T cells overcomes the ability of TGF-beta to inhibit Th2 differentiation. TGF-beta likely inhibits GATA-3 expression at the transcriptional level and does so without interfering with IL-4 signaling.
The T helper subsets Th1 and Th2 regulate specific types of immune responses by producing distinct sets of cytokines. Differentiation of the T helper subsets from their common precursors, naive CD4+ T cells, is induced by antigen stimulation and controlled by various other conditions. Of these conditions, the contributions of the cytokine environment have been the best characterized. The presence of interleukin-4 (IL-4) directs the differentiation towards Th2 cells, whereas IL-12 induces Thl differentiation. The Th2 signature cytokine genes encoding IL-4, IL-13, and IL-5 are clustered, and noncoding regions such as the intergenic region of the IL-4 and IL-13 genes are highly conserved from mice to humans. Alteration of the chromatin structure of this Th2 cytokine cluster region is detected as nuclease-accessible regions specific to Th2 cells. Activation of STAT6 promotes Th2 differentiation by inducing Th2-specific transcription factors, including GATA3. Expression of GATA3 induces Th2 differentiation and enhances the Th2 cell-specific chromatin accessibility, indicating that GATA3 is a key protein that regulates differentiation through chromatin remodeling. T helper subset differentiation provides a good system to study gene expression regulation at the chromatin level.
Genes that control the early stages of adipogenesis remain largely unknown. Here, we show that murine GATA-2 and GATA-3 are specifically expressed in white adipocyte precursors and that their down-regulation sets the stage for terminal differentiation. Constitutive GATA-2 and GATA-3 expression suppressed adipocyte differentiation and trapped cells at the preadipocyte stage. This effect is mediated, at least in part, through the direct suppression of peroxisome proliferator-activated receptor gamma. GATA-3-deficient embryonic stem cells exhibit an enhanced capacity to differentiate into adipocytes, and defective GATA-2 and GATA-3 expression is associated with obesity. Thus, GATA-2 and GATA-3 regulate adipocyte differentiation through molecular control of the preadipocyte-adipocyte transition.
TGF-beta plays an important role in immune regulation in vivo and affects T cell differentiation in vitro. Here we describe how TGF-beta modulates Th2 development in vitro and investigate its mechanisms of action. TGF-beta down-regulated Th2 development of naive CD4+ Mel-14high T cells derived from the DO11.10 ovalbumin-specific TCR-transgenic mouse, and this was observed both in cultures driven with anti-CD3 and anti-CD28 and with splenic APC and antigen. TGF-beta down-regulated GATA-3 expression in developing Th2 and these cells showed a diminished IL-4-induced STAT6 activation. We found, however, that naive cells driven in Th2 conditions with TGF-beta did not show a significantly decreased STAT6 activation, suggesting that TGF-beta inhibits Th2 development via a STAT6-independent mechanism.
The transgenic mouse line Ggamma/T-15 containing the fetal globin promoter linked to SV40 T antigen unexpectedly results in androgen-independent prostate carcinomas. Given the key role of GATA-1 transcription factor in fetal globin gene promoter activity, we investigated whether specific GATA family members are expressed in the prostate and whether they can regulate prostate-specific genes. We found that GATA-2 and -3 are the predominant GATA family members expressed in human and mouse prostate and that GATA mRNA levels are not regulated by androgen. We identified six GATA sites flanking an androgen-response element located in the far-upstream enhancer of the prostate-specific antigen (PSA) gene. These GATA sites are targets for GATA factors and are essential for optimal androgen induction of transfected PSA enhancer/promoter plasmids in LNCaP, a PSA and androgen receptor expressing human prostate cancer cell line. Our results suggest that prostatic GATA-2 and -3 are involved in the androgen regulation of the PSA gene.
Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.
Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA-3 is acetylated in T cells and that a mutation introduced into amino acids 305-307 (KRR-GATA3) creates local hypoacetylation in GATA-3. Remarkably, KRR-GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T-cell homing to systemic lymphnodes, and prolonged T-cell survival after activation. These findings have significant implications in that the acetylation state of GATA-3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA-3 in T-cell survival and homing to secondary lymphoid organs.
Terminal deletions of chromosome 10p result in a DiGeorge-like phenotype that includes hypoparathyroidism, heart defects, immune deficiency, deafness and renal malformations. Studies in patients with 10p deletions have defined two non-overlapping regions that contribute to this complex phenotype. These are the DiGeorge critical region II (refs 1, 2), which is located on 10p13-14, and the region for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome (Mendelian Inheritance in Man number 146255), which is located more telomeric (10p14-10pter). We have performed deletion-mapping studies in two HDR patients, and here we define a critical 200-kilobase region which contains the GATA3 gene. This gene belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development. Investigation for GATA3 mutations in three other HDR probands identified one nonsense mutation and two intragenic deletions that predicted a loss of function, as confirmed by absence of DNA binding by the mutant GATA3 protein. These results show that GATA3 is essential in the embryonic development of the parathyroids, auditory system and kidneys, and indicate that other GATA family members may be involved in the aetiology of human malformations.
GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1 gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1-deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.
By DNase I hypersensitivity analysis, we have identified an inducible, cyclosporin A-sensitive enhancer located 3' of the interleukin-4 (IL-4) gene. The enhancer binds the Th2-specific transcription factor GATA3 in vivo but is not perceptibly influenced by the absence of a second Th2-specific factor, cMaf. The antigen-inducible transcription factor NFAT1 binds the IL-4 enhancer and the IL-4 promoter only in stimulated Th2 cells; conversely, NFAT1 binds to the interferon (IFN)-gamma promoter only in stimulated Th1 cells. Our results support a model whereby transcription factors such as NFAT1, which are nonselectively induced in antigen-stimulated T cells, gain access to cytokine regulatory regions only in the appropriate subset of differentiated T cells in vivo. This restricted access enables antigen-dependent and subset-specific transcription of cytokine genes.
Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.
Given the critical role of cytokines in the regulation of an inflammatory response, we investigated whether certain cytokines are expressed in the brains of normal mice during maturation that could contribute to the immune-privileged nature of the CNS or potentially influence an immune-mediated illness such as experimental allergic encephalomyelitis. The gene expression of IFN gamma (Th1 cytokine) and IL-4 (Th2 cytokine) was analyzed in the brain of several strains of mice. IFN gamma was not detectable. However, IL-4 was present in the brains of neonatal mice, but not adult mice. Resident CNS cells are believed to be the source of the IL-4, because mice deficient in T cells (SCID and RAG2-/-) expressed the IL-4 gene in the CNS. Further analysis indicated that the gene expression of the Th2 cytokine transcription factor, GATA-3, correlated with IL-4 and IL-10 expression in the brain. Since GATA-3-deficient mice have an abnormal CNS, brain-derived Th2 cytokines may play an important role in CNS development, as well as potentially contribute to the immune-privileged nature of the brain.
Allergic rhinitis is a complex upper airways disorder characterized by the infiltration of eosinophils and T(H2)-type T lymphocytes. GATA-3 is a novel transcription factor recently shown to regulate IL-5 and, possibly, IL-4 gene expression. We previously reported that GATA-3 is increased within the bronchial mucosa of allergic asthmatic subjects compared with control subjects.
In the present study we set out to determine whether there is also an increased number of cells expressing GATA-3 messenger (m)RNA within the nasal mucosa of patients with allergic rhinitis.
Inferior turbinate biopsy specimens were obtained from patients with allergic rhinitis and nonatopic control subjects before and after local allergen provocation in vivo. To assess the contribution of resident cells expressing GATA-3 mRNA, we also performed isolated explant studies in which nasal mucosal tissue from subjects with allergic rhinitis and nonatopic control subjects was cultured in allergen-treated medium. The presence of mRNA coding for GATA-3, IL-5, IL-4, IL-13, and GM-CSF was assessed by using in situ hybridization.
The number of GATA-3 mRNA(+) cells was increased after local allergen provocation in vivo (increase in GATA-3 mRNA(+) cells [mean +/- SEM]: subjects with allergic rhinitis, 11.3 +/- 8.7; control subjects, 1.2 +/- 4.1; P <.05) and in explanted nasal mucosa in vitro (subjects with allergic rhinitis, 10. 2 +/- 3.8; control subjects, 2.7 +/- 4.4; P <.05). The gene expression of GATA-3 was significantly correlated to the numbers of IL-5 (r = 0.87) and GM-CSF (r = 0.79) mRNA(+) cells but not with IL-4 or IL-13 mRNA(+) cells.
In summary, the expression of the transcription factor GATA-3 was increased after allergen challenge, and this was evident in the absence of de novo inflammatory cell recruitment. GATA-3 may be a potential target in the treatment of allergic diseases, such as rhinitis.
Recombination activating gene-1 (RAG-1) and RAG-2 are expressed in lymphoid cells undergoing the antigen receptor gene rearrangement. A study of the regulation of the mouse RAG-2 promoter showed that the lymphocyte-specific promoter activity is conferred 80 nucleotide (nt) upstream of RAG-2. Using an electrophoretic mobility shift assay, it was shown that a B-cell-specific transcription protein, Pax-5, and a T-cell-specific transcription protein, GATA-3, bind to the -80 to -17 nt region in B cells and T cells, respectively. Mutation of the RAG-2 promoter for Pax-5- and GATA-3-binding sites results in the reduction of promoter activity in B cells and T cells. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively. (Blood. 2000;95:3845-3852)
Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3-/- mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine beta-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.
GATA3 is a specific T-cell transcription factor involved in the expression of T-cell receptor (TCR). In order to characterize the relationship between HTLV-1 infection, which has been reported to be associated with down-regulation of genes belonging to the TCR/CD3 complex, and the transcription factor GATA3, we evaluated, by semi-quantitative RT-PCR, the expression of GATA3 gene in HTLV-1 carriers and individuals with related diseases. The study included 4 asymptomatic carriers, 2 patients with adult T-cell leukaemia/lymphoma (ATLL), 1 patient with HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis (TSP) and 7 healthy blood donors. A considerable decrease in the expression of the GATA3 mRNA was observed in all subjects infected by HTLV-1 and no expression of GATA3 mRNA was observed in 1 subject with ATLL and in 1 with HAM/TSP.
Gamma delta T cells secrete Th1- and Th2-like cytokines that help mediate innate and acquired immunity. We have addressed the mechanism whereby murine gamma delta T cells acquire the capacity to differentially produce such cytokines. Splenic gamma delta T cells could be polarized into IFN-gamma- or IL-4-secreting cells in vitro; however, in contrast to CD4+ alpha beta T cells, gamma delta T cells predominantly produced IFN-gamma, even in the presence of IL-4, a finding independent of genetic background. Like CD4+ Th1 cells, IFN-gamma-producing cells expressed the IL-12 receptor beta 2-chain after activation in the presence of IL-12; however, unlike Th2 cells, IL-4-primed gamma delta T cells also expressed this receptor, even in the absence of IFN-gamma and despite the presence of the transcription factor GATA-3. IL-12 also induced IL-4-primed gamma delta T cells to proliferate and to translocate Stat3/Stat4, indicating signaling through the IL-12 receptor. These molecular events can account for the predominant production of IFN-gamma by gamma delta T cells in the presence of IL-12, despite the availability of IL-4. Early and predominant production of IFN-gamma by gamma delta T cells likely is critical for the roles that these cells play in protection against intracellular pathogens and in tumor immunity.
The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.
It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, we analysed the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc), with a particular emphasis on intraembryonic hemogenic sites. We show here that both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. (1) Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase of intraembryonic precursors determination. (2) A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. (3) Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonisation. Furthermore, a strong GATA-3 signal allowed us to uncover previously unreported mesodermal aggregates beneath the aorta. A combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation.
Stat6 is critical for IL-4-mediated Th2 cell development, but its molecular mechanism remains unclear. Here we constructed Stat6:ER, a Stat6-estrogen receptor fusion protein that can be activated by 4-hydroxy-tamoxifen, independently of IL-4 and endogenous Stat6. Retrovirus-mediated introduction of Stat6:ER into developing Th1 cells induced Th2-specific cytokines and suppressed IFNgamma production in a 4-HT-dependent manner and in the absence of IL-4. It also induced GATA-3 and c-maf expression and downregulated IL-12Rbeta2 chain expression. Its decreased ability to induce the Th2 phenotype with progressing Th1 cell commitment correlated with a decreased induction of GATA-3 and c-maf. This study indicates that Stat6 functions upstream of GATA-3 and c-Maf to induce Th2 development.
The relative contribution of T cell receptor-versus CD28-mediated signals in co-stimulation of resting CD4 T cells is thought to influence their functional differentiation towards T helper (Th) 1 versus Th2 subsets. We have used a conventional and a mitogenic CD28-specific monoclonal antibody to assess the effect of polyclonal T cell activation through CD28 alone on CD4 subset differentiation. In vivo, mitogenic but not conventional anti-CD28 induces massive lymphocytosis, the Th2 cytokines interleukin (IL)-4 and IL-10, and Th2-dependent immunoglobulin isotypes, most notably IgE. In vitro, it is shown that mitogenic anti-CD28 primes for IL-4-dependent induction of IL-4 expression much more efficiently than conventional co-stimulation. At the molecular level, we show for the first time that the activation of the "Th2 promoting" transcription factor GATA-3 requires co-stimulation by CD28 and is also induced by mitogenic anti-CD28 alone. We suggest that CD28-dependent induction of GATA-3 in concert with other transcription factors, which are preferentially induced by strong CD28-signals, primes CD4 T cells for IL-4-dependent Th2 differentiaton.
In this paper, we show that the transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons.
The cytokines IL-4, IL-5, and IL-13, secreted by Th2 cells, have distinct functions in the pathogenesis of asthma. We have previously shown that the transcription factor GATA-3 is expressed in Th2 but not Th1 cells. However, it was unclear whether GATA-3 controls the expression of all Th2 cytokines. Expression of a dominant-negative mutant of GATA-3 in mice in a T cell-specific fashion led to a reduction in the levels of all the Th2 cytokines IL-4, IL-5, and IL-13. Airway eosinophilia, mucus production, and IgE synthesis, all key features of asthma, were severely attenuated in the transgenic mice. Thus, targeting GATA-3 activity alone is sufficient to blunt Th2 responses in vivo, thereby establishing GATA-3 as a potential therapeutic target in the treatment of asthma and allergic diseases.
Here we describe the first real-time study of nuclear protein interaction with a composite DNA regulatory region. We studied the interplay between the three target sites of the negative regulatory element (NRE) of HIV-1 LTR, comprising a noncanonical GATA site overlapping two negative regulatory regions, USF and NFIL-6, and their corresponding transcription factors in nuclear extracts. By bandshift analysis, no GATA binding activity could be detected between LTR NRE and different nuclear extracts, although evidenced by in vitro footprinting. Additionally, the LTR NRE and a USF oligonucleotide showed identical retarded complexes. BIAcore study of these interactions revealed the binding of huGATA-3, as well as USF, to the immobilized LTR NRE oligonucleotide. Competition analyses, performed with GATA, USF, and NFIL-6 oligonucleotides, clearly showed that this regulatory region could bind both huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 proteins in the complexes formed with LTR NRE was ascertained using specific anti-huGATA-3 and anti-USF2 polyclonal antibodies.
We have investigated in detail the expression patterns of two Gata genes, cGata2 and cGata3, during early chick development. In addition to confirming previously described expression of these two genes in developing brain, kidney and blood islands, this study reveals several important novel expression domains during very early stages of development. cGata2 is expressed in the area opaca in pre-primitive streak stages, forming a gradient along the A-P axis (strongest anteriorly). Both genes are expressed strongly in the entire non-neural ectoderm from stage 4+, and neither is expressed in prospective neural plate at any stage. Unlike other previously described non-neural markers, neither gene is expressed in the dorsal neural tube. We also describe dynamic expression of cGata2 and cGata3 during eye, ear and gut development.
GATA3 is a transcription factor expressed in the inner ear during the early stages of development. A monoclonal antibody revealed that it is expressed in spiral ganglion cells and in all cells of the developing auditory sensory epithelium in the mouse before the hair cells differentiate at embryonic days 14-16. Expression decreases selectively in the hair cells as they differentiate progressively from the base to the apex of the developing organ of Corti. GATA3 subsequently decreases in the supporting cells and cannot be detected by immunofluorescence in any cell of the adult sensory epithelium. It is not expressed in the vestibular sensory epithelia or surrounding tissues from embryonic day 14. We suggest that GATA3 could act as a repressor of critical genes involved in cell differentiation in the organ of Corti, enabling a progressive formation of the adult cellular pattern.
Analysis of gene expression on a medium- or large-scale is an increasingly recognized method for functional and clinical investigations based on the now extensive catalog of known or partially sequenced genes. The accessibility of this approach can be enhanced by using readily available technology (macroarrays on Nylon, radioactive detection) and the IMAGE resource to assemble sets of targets. We have set up such a medium-scale, flexible system and validated it by the study of quantitative expression levels for 120 genes in six cell lines, including three mammary carcinoma cell lines. A number of important parameters are identified as necessary for the assembly of a valid set and the obtention of good-quality quantitative data. The extensive data assembled in this survey identified potential targets of carcinogenesis, for example the CRABP2 and GATA3 transcription factor genes. We also demonstrate the feasibility of this procedure for relatively small tumor samples, without recourse to probe amplification methods.
The spatial and temporal analysis of GATA-3 expression pattern in the human embryo revealed its expression in new anatomical sites. These include the endoderm of the primitive foregut, pharynx and allantois, the branchial arches and the mesenchymal cells surrounding the stomach and dorsal aorta. On the other hand, human (h) GATA-3 expression in the central nervous system, somites and embryonic kidney confirms the tissue specificity of this gene throughout vertebrate evolution.
GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3+ cells throughout T cell development in the thymus, the proportions of GATA-3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR alpha and beta gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3+ cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25- double-negative (CD4-CD8-) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.
The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.
The development and function of T lymphocytes are regulated tightly by signal transduction pathways that include specific cell-surface receptors, intracellular signaling molecules, and nuclear transcription factors. Since 1988, several families of functionally important T cell transcription factors have been identified. These include the Ikaros, LKLF, and GATA3 zinc-finger proteins; the Ets, CREB/ATF, and NF-kappa B/Rel/NFAT transcription factors; the Stat proteins; and HMG box transcription factors such as LEF1, TCF1, and Sox4. In this review, we summarize our current understanding of the transcriptional regulation of T cell development and function with particular emphasis on the results of recent gene targeting and transgenic experiments. In addition to increasing our understanding of the molecular pathways that regulate T cell development and function, these results have suggested novel targets for genetic and pharmacological manipulation of T cell immunity.
Here we examine the expression of transcription factors GATA-2 and GATA-3 during early stages of embryonic development in the central nervous system (CNS) of the mouse. GATA-2 is expressed as early as 9 dpc in the hindbrain, in ventral rhombomere 4, and transiently in ventral rhombomere 2 (r2). From 9.5 to 11.5 dpc, activation of the gene spreads to many sites of early neuronal differentiation, such as the olfactory bulbs, the pretectum, and the oculomotor nucleus in the midbrain, a thin stripe of cells lining the floor plate from the mesencephalon to the cervical spinal cord and a ventral column of cells spanning the neural tube from rostral hindbrain and including motor neuron as well as ventral interneuron precursors. GATA-3 is expressed in a pattern very similar to that of GATA-2. Distinguishing features are the lack of expression in r2 at 9 dpc and a slight delay in its activation. In addition, GATA-2 is activated in both the ventricular and the subventricular zones of the neural tube, whereas GATA-3 is restricted mainly to the subventricular zone. Expression analyses performed on GATA-2 -/- mouse embryos between E9.5 and 10.5 dpc established that: (i) the expression of GATA-3 in the developing CNS of the mouse embryo is dependent on the presence of GATA-2 and (ii) loss of GATA-2 leads to severe defects in neurogenesis, which strongly suggests that GATA-2 is involved, as in hematopoiesis, in the maintenance of the pool of ventral neuronal progenitors.
IL-12 and IL-4 are dominant factors driving the development of Th1 and Th2 cells, respectively, by their activation of Stat-4 and Stat-6 signaling molecules. Activation of Stat factors, although specific, is a rapid event; however, differentiation of Th cells takes place over several days. Thus, it is unlikely that the expression of effector cytokines is mediated solely by Stat factors. Recently there have been indications that link other molecular factors to Th subset development. The transcription factor GATA-3 is selectively expressed in Th2 cells and has been shown to induce the expression of Th2 cytokines in developing Th1 cells. Using retroviral infection of naive T cells to introduce GATA-3 cDNA, we measured its direct effects on the development of Th1 cytokine production. We now show that ectopic expression of GATA-3 in developing Th1 cells significantly inhibits IFN-gamma, as well as enhancing IL-4 and IL-5 production. Furthermore, GATA-3 inhibits production of IFN-gamma by developing Th1 cells in the complete absence of IL-4. Thus, antagonism of Th1 development by GATA-3 may facilitate rapid divergence of Th subsets toward a Th2 phenotype in concert with other factors.
Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.
To better understand the molecular basis for the hormone-responsive phenotype in breast cancer, we have used a human cDNA array to compare patterns of gene expression between breast carcinoma cell lines discordant for estrogen receptor (ER) expression. These experiments indicated abundant expression of the transcription factor GATA-3 in the ER-positive cell lines MCF7 and T-47D, with minimal or no expression in the ER-negative cells lines MDA-MB-231 and HBL-100. Northern blot analysis of a panel of human breast carcinoma cell lines demonstrated a correlation between ER and GATA-3 expression. Studies of MCF7 cells grown in the absence or presence beta-estradiol indicated that GATA-3 expression was not responsive to estradiol. Protein immunoprecipitation and gel shift analysis confirmed the presence of functional GATA-3 protein in MCF7 but not in HBL-100 nuclear extracts. A panel of 47 primary breast cancers was characterized for expression of ER and GATA-3 using immunoperoxidase assay. In primary tumors, a statistically significant correlation between ER and GATA-3 expression was established (p < 0.0001, chi2). Our results indicate that GATA-3, in association with ER, is likely to regulate genes critical to the hormone-responsive breast cancer phenotype.
Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.
The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity.
High expression of IL-5 by T cells in the airways of asthmatic individuals is believed to play a fundamental role in the eosinophilia associated with this disease. Recently, the transcription factor GATA-3 was shown to be critical for IL-5 gene expression in TH2 cells in vitro.
Our aim was to examine the expression of GATA-3 mRNA and its colocalization within the airways of asthmatic and nonasthmatic individuals.
We investigated the association between GATA-3 gene expression, airway inflammatory cells, and IL-5 gene expression in bronchoalveolar lavage fluid and bronchial biopsy specimens from atopic asthmatic subjects (n = 10) and normal control subjects (n = 10).
We report that GATA-3 mRNA expression is significantly increased in the airways of asthmatic subjects compared with those of normal control subjects (P <.001). Numbers of cells expressing GATA-3 transcripts correlated significantly with reduced airway caliber (P <.05) and airways hyperresponsiveness (P <.05) in asthmatic subjects. Colocalization studies showed that the majority (approximately 60% to 90%) of GATA-3 mRNA+ cells in asthmatic airways were CD3(+) T cells, with smaller contributions from major basic protein+ eosinophils and tryptase+ mast cells. The density of GATA-3 mRNA+ cells correlated significantly with the numbers of cells expressing IL-5 mRNA (P <.001, r = 0.879 for bronchoalveolar lavage fluid; P <. 05, r = 0.721 for biopsy specimens). Furthermore, double in situ hybridization demonstrated that approximately 76% of GATA-3 mRNA+ cells coexpressed IL-5 mRNA and that 91% of IL-5 mRNA+ cells coexpressed GATA-3 mRNA.
The results of this study provide the first evidence of increased GATA-3 gene expression in association with IL-5 mRNA+ cells in asthmatic airways. These findings support a causal association between augmented GATA-3 expression and dysregulated IL-5 expression in atopic asthma.
We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice. In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals. A lacZ reporter gene was first inserted into both large GATA-3 YACs. The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZ insertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ "knock-ins"). We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers). Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5' and 3' flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.
GATA-3 is essential for murine embryonic development, but elucidating the genetic controls over the complex temporal and tissue-specific transcriptional regulatory pattern of this transcription factor gene has been problematic. Here we report the isolation and characterization of two yeast artificial chromosomes (YACs) bearing the murine GATA-3 gene. Ordered deletions of both YACs show that they define a 1-megabase pair contig spanning the GATA-3 locus. We found that a 120-kb YAC transgene, including 35 kb of 5' as well as 60 kb of 3' flanking sequence, confers normal GATA-3 expression at sites not revealed previously through analysis of plasmid transgenic lines. However, even this 120-kb YAC does not contain sufficient information to recapitulate the complete GATA-3 expression program during embryogenesis. While not complete in its regulatory capacity, the YAC transgene is nonetheless able to complement several homozygous GATA-3 mutant phenotypes and thereby prolong embryonic life.
Lung Kruppel-like factor (LKLF) is a member of the Kruppel-like family of zinc finger transcription factors and is closely related to erythroid kruppel-like factor (EKLF), which is necessary for beta-globin gene expression. While EKLF is expressed exclusively in erythroid cells, LKLF is expressed temporally during early embryonic development and predominantly in the adult mouse lung. To understand the role this novel transcription factor plays in development as well as tissue differentiation and function, animals lacking LKLF were produced using gene targeting technology. Mice lacking LKLF die in utero between day 11.5 and 13.5 of embryonic life and exhibit retarded growth, craniofacial abnormalities, abdominal bleeding and signs of anaemia. Although the yolk sac erythropoiesis is normal in mutant embryos, in vitro fetal liver cultures of these embryos fail to give rise to erythroid cells. Expression of other erythroid specific genes such as EKLF, GATA1 and GATA3 is unaltered in these animals. These findings demonstrate the LKLF function is indispensable during normal embryonic development, and although both LKLF and EKLF recognize common DNA motifs, they do not substitute for each other.
Recently, the transcription factor GATA-3 was shown to be selectively expressed in Th2 but not Th1 cells and to augment Th2-specific cytokines. Here, we show that loss of GATA-3 expression by developing Th1 cells requires IL-12 signaling through Stat4 and does not simply result from an absence of IL-4. Moreover, we demonstrate a novel role for GATA-3 in directly repressing Th1 development distinct from its positive actions on Th2-specific cytokines. GATA-3 inhibits Th1 cytokines by a cell-intrinsic mechanism that is not dependent on IL-4 and that may involve repression of IL-12 signaling. Thus, GATA-3 expression and IL-12 signaling are mutually antagonistic, which facilitates rapid dominance of one pathway during early Th development, producing a stable divergence in cytokine profiles.
We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.
Previously, we have shown that TAL1 and the LIM-only protein gene (LMO) are regularly coactivated in T-cell acute lymphoblastic leukemia (T-ALL). This observation is likely to relate to the findings that TAL1 and LMO are highly synergistic in T-cell tumorigenesis in double-transgenic mice. To understand the molecular mechanisms of functional synergy between TAL1 and LMO in tumorigenesis and transcriptional regulation, we tried to identify downstream target genes regulated by TAL1 and LMO by a subtractive PCR method. One of the isolated genes, that for retinaldehyde dehydrogenase 2 (RALDH2), was regularly expressed in most of the T-ALL cell lines that coexpressed TAL1 and LMO. Exogenously transfected TAL1 and LMO, but not either alone, induced RALDH2 expression in a T-ALL cell line, HPB-ALL, not expressing endogeneous TAL1 or LMO. The RALDH2 transcripts in T-ALL were, however, mostly initiated within the second intron. Promoter analysis revealed that a GATA site in a cryptic promoter in the second intron was essential and sufficient for the TAL1- and LMO-dependent transcriptional activation, and GATA3 binds to this site. In addition, forced expression of GATA3 potentiated the induction of RALDH2 by TAL1 and LMO, and these three factors formed a complex in vivo. Furthermore, a TAL1 mutant not binding to DNA also activated the transcription of RALDH2 in the presence of LMO and GATA3. Collectively, we have identified the RALDH2 gene as a first example of direct transcriptional target genes regulated by TAL1 and LMO in T-ALL. In this case, TAL1 and LMO act as cofactors for GATA3 to activate the transcription of RALDH2.
To study the regulatory mechanism of gp91phox gene expression in eosinophils, we transiently transfected eosinophil-committed HL-60-C15 cells with gp91phox promoter constructs, and identified a negative element from bp -267 to -246 of the gp91phox gene, the deletion of which caused an 83% increase in promoter activity. Electrophoresis mobility shift assays demonstrated GATA-3 binds to the GATA consensus site from bp -256 to -250. An 81% increment in promoter activity was obtained when a mutation was introduced in the GATA-3 binding site of the bp -267 to +12 construct, which is comparable to that of the bp -245 to +12 construct. We therefore conclude that GATA-3 specifically binding to the GATA site negatively regulates the expression of the gene in HL-60-C15 cells.
Transgenic mice with human CD3epsilon gene have been shown to exhibit early arrest of T cell development in the thymus. The present study shows that, instead of T cells, B cells are generated in the thymus of a line, tg epsilon26, of the human CD3epsilon transgenic mice. The accumulation of mature B cells in the thymus was found only in tg epsilon26 mice, not in other human CD3epsilon transgenic mouse lines or other T cell-deficient mice, including CD3-epsilon knockout mice and TCR-beta/TCR-delta double knockout mice. Hanging drop-mediated transfer into 2-deoxyguanosine-treated thymus lobes showed that lymphoid progenitor cells rather than thymus stromal cells were responsible for abnormal B cell development in tg epsilon26 thymus, and that tg epsilon26 fetal liver cells were destined to become B cells in normal thymus even in the presence of normal progenitor cells undergoing T cell development. These results indicate that lymphoid progenitor cells in tg epsilon26 mice are genetically defective in thymic choice between T cells and B cells, generating B cells even in normal thymus environment. Interestingly, tg epsilon26 thymocytes expressed GATA-3 and TCF-1, but not LEF-1 and PEBP-2alpha, among T cell-specific transcription factors that are involved in early T cell development, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cell lineage. Thus, tg epsilon26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.
Previously, we analyzed the proximal IL-4 promoter in directing Th2-specific activity. An 800-base pair proximal promoter conferred some Th2-selective expression in transgenic mice. However, this region directed extremely low reporter mRNA levels relative to endogenous IL-4 mRNA, suggesting that full gene activity requires additional enhancer elements. Here, we analyzed large genomic IL-4 regions for enhancer activity and interaction with transcription factors. The proximal IL-4 promoter is only moderately augmented by GATA-3, but certain genomic regions significantly enhanced GATA-3 promoter transactivation. Some enhancing regions contained consensus, GATA sites that bound Th2-specific complexes. However, retroviral transduction of GATA-3 into developing T cells induced IL-5 to full Th2 levels, but only partially restored IL-4 production. Thus, we propose that GATA-3 is permissive, but not sufficient, for full IL-4 enhancement and may act through GATA elements surrounding the IL-13/IL-4 gene locus.
The cytokines IL-4 and IL-5 are often coordinately produced by Th2 cells as in asthma. However, it is unclear whether similar molecular mechanisms underlie transcription of the two genes. We have previously shown that the transcription factor GATA-3 is expressed in Th2 but not Th1 cells and is crucial for activation of the IL-5 promoter by different stimuli. In a different study, GATA-3 was shown to be sufficient for the expression of IL-4 and other Th2 cytokine genes. Here, we show that ectopic expression of GATA-3 is sufficient to drive IL-5 but not IL-4 gene expression. Also, in Th2 cells, antisense GATA-3 RNA inhibits IL-5 but not IL-4 promoter activation. The induction of IL-5 gene expression by GATA-3 involves high affinity binding of GATA-3 to an inverted GATA repeat in the IL-5 promoter.
Recent studies in early Xenopus and zebrafish embryos have demonstrated that posteriorizing, non-axial signals arising from outside the organizer (or shield) contribute to A/P patterning of the neural axis, in contradiction to the classical Spemann model in which such signals were proposed to be solely organizer derived. Our studies on the early expression of the transcription factors GATA-2 and 3 in both Xenopus and zebrafish nonneural ectoderm lend support to the existence of such non-axial signaling in the A/P axis. Thus we find that the earliest expression of GATA-2 and 3 is located in nonneural ectoderm and is strongly patterned in a graded manner along the A/P axis, being high anteriorly and absent from the most posterior regions. This results by early neurula stages in three broad zones: an anterior region which is positive for both GATA-2 and 3, a middle region which is positive for GATA-2 alone and a posterior region in which neither gene is expressed. These regions correspond to head, trunk and tail ectoderm and may represent the beginnings of functional segmentation of nonneural ectoderm, as suggested in the concept of the 'ectomere'. We find that A/P patterning of GATA expression in nonneural ectoderm may occur as early as late blastula/early gastrula stages. We investigate which posteriorizing signals might contribute to such distinct non axial ectodermal patterning in the A/P axis and provide evidence that both FGF and a Wnt family member contribute towards the final A/P pattern of GATA expression in nonneural ectoderm.
The tissue- and stage-specific assembly of Ag receptor genes is regulated by transcriptional control elements positioned within Ig and TCR loci. To further understand the role of cis-acting elements in these regulatory mechanisms, we have characterized a transcriptional promoter that drives germline expression of TCR beta gene segments in vivo. The activity of this promoter, termed PD beta, is restricted to a highly conserved 400-bp region located directly upstream from D beta 1-coding sequences. Maximal PD beta activity requires a TATA element situated within the D beta 1 recombination signal sequences and consensus binding sites for the ubiquitous SP1 and the T cell-specific GATA-3 transcription factors. When linked to active enhancer elements, PD beta directs transcription in most cell types; however, the TCR beta enhancer (E beta) stimulates PD beta function specifically in precursor T lymphocytes. These findings suggest that PD beta/E beta interactions may contribute to differential regulation of regions within the TCR beta locus during thymocyte development.
TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human gammadelta lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.
Allergens and infections with parasitic helminths preferentially induced Th2 immune responses associated with elevated levels of serum immunoglobulin E (IgE) and expansion of eosinophils and mast cells. Interleukin-4 (IL-4) is a key cytokine in the differentiation of naive CD4+ T cells into Th2 cells, which produce a panel of cytokines including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [1] and have been shown to trigger recovery from gastrointestinal nematodes [2]. Nonetheless, mice deficient for IL-4 have been shown to develop residual Th2 responses [3-5] and can expel the nematode Nippostrongylus brasiliensis [6], suggesting that there is a functional equivalent of IL-4 in these processes. IL-13 is a cytokine that shares some, but not all, biological activities with IL-4 [7,8]. There is now compelling evidence that IL-4 and IL-13 share receptor components, including IL-4R alpha and IL-13R alpha 1 [9]. In order to dissect the roles of IL-4 and IL-13 in the regulation of Th2 cells and in the response to nematode infections, we looked for differences between mice deficient for either the IL-4 gene or the IL-4R alpha gene. Unlike IL-4, IL-4R alpha was required for control of N. brasiliensis, and Th2 development during infection--as characterized by cytokine production, GATA-3 and surface CD30 expression--was more severely affected in IL-4R alpha-/- mice than in IL-4-/- mice. Injection of recombinant IL-13 induced worm expulsion in otherwise incompetent RAG2-/- mice. Our results suggest that IL-13 regulates Th2 responses to nematode infection and requires IL-4R alpha.
Cas-Br-E and Graffi are two murine viruses that induce myeloid leukemia in mice: while Cas-Br-E induces mostly non-T, non-B leukemia composed of very immature cells, Graffi causes exclusively a granulocytic leukemia (E. Rassart, J. Houde, C. Denicourt, M. Ru, C. Barat, E. Edouard, L. Poliquin, and D. Bergeron, Curr. Top. Microbiol. Immunol. 211:201-210, 1995). In an attempt to understand the basis of the myeloid specificity of these two retroviruses, we used DNase I footprinting analysis and gel mobility shift assays to identify a number of protein binding sites within the Cas-Br-E and Graffi U3 regions. Two protected regions include potential GATA binding sites. Methylation interference analysis with different hematopoietic nuclear extracts showed the importance of the G residues in these GATA sites, and supershift assays clearly identified the binding factors as GATA-1, GATA-2, and GATA-3. Transient assays with long terminal repeat (LTR)-chloramphenicol acetyltransferase constructs showed that these three GATA family members are indeed able to transactivate Cas-Br-E and Graffi LTRs. Thus, the availability and relative abundance of the various members of the GATA family of transcription factors in a given cell type could influence the transcriptional tissue specificity of murine leukemia viruses and hence their disease specificity.
Transcription factors of the GATA-family are essential for proper development of diverse tissues or cell types. GATA-1 is required for differentiation of two hematopoietic lineages (red blood cells and megakaryocytes), whereas GATA-3 is essential for T-cell development. Functional studies suggest that many properties of the GATA-family of proteins are shared and largely interchangeable. To test whether the function of GATA-1 in erythroid differentiation can be replaced by another GATA-factor, we generated a knock-in mutation of the GATA-1 locus in which GATA-3 cDNA was introduced by gene targeting. Mutant embryos (designated G1G3ki), though embryonic lethal, exhibit partial rescue, characterized by increased survival of erythroid precursor cells and improved hemoglobin production. The basis for the incomplete extent of rescue is likely to be complex, but may be accounted for, in part, by insufficient accumulation of GATA-3 protein (compared with the normal level of GATA-1). Our findings suggest that GATA-3 protein is functional when expressed in an erythroid environment and is competent to act on at least a subset of erythroid-expressed target genes in vivo.
Until now the allantois has not been considered as a hematopoietic organ. Here we report experimental evidence demonstrating the in situ emergence of both hematopoietic and endothelial precursors in the avian allantoic bud. When the prevascularized allantoic bud from a quail embryo was grafted in the coelom of a chicken host, hematopoietic and endothelial cells later were found in the bone marrow of the host. Because the graft was located at a distance from the limb bud, these cells could reach the bone marrow only by the circulatory pathway. This blood-borne seeding may be accomplished by distinct hematopoietic and endothelial precursors, or by hemangioblasts, the postulated common precursors of these two lineages; we consider the latter interpretation more likely. We also show by reverse transcription-PCR that the allantois region expresses very early the GATA genes involved in hematopoiesis and some beta-globin chain genes.
Expression of the IL-5 gene is restricted to the Th2 subset of helper T cells. We have previously defined four cis-regulatory elements of the IL-5 promoter responding to PMA and cAMP in EL-4 cells. We now report that the 1.2-kb region of the IL-5 promoter directs expression of the IL-5 gene in a Th2 clone but not a Th1 clone, indicating that transcription from the IL-5 promoter is Th2 specific. For the functioning of the IL-5 promoter in a Th2 clone, IL-5C and IL-5CLE0 were critical. IL-5CLE0 interacted with both constitutive and inducible nuclear factors (designated NFIL-5CLE0), which existed in both Th1 and Th2 clones, whereas IL-5C interacted with a constitutive nuclear factor (designated NFIL-5C), which was found only in Th2 but not in Th1 clones. Th2 specificity of NFIL-5C was also confirmed using in vitro-differentiated Th1 and Th2 cells derived from TCR-transgenic mice. The sequence for NFIL-5C binding bears homology with GATA-binding sites. The NFIL-5C complex was supershifted by an anti-GATA-3 Ab and inhibited by an oligonucleotide containing GATA-binding sites. We showed preferential expression of GATA-3 in Th2 cells. Finally, we demonstrated that in vitro-translated GATA-3 bound to IL-5C and overexpression of GATA-3 augmented stimulation-dependent IL-5 promoter activity in EL-4 cells. Taken together, our results provide evidence that GATA-related factors may be involved in Th2-specific expression of the IL-5 gene.
Relative affinities of transcriptional regulatory elements for their respective factor have been essentially studied by bandshift analysis. Here we report a real-time study of factor/DNA interactions using a surface plasmon resonance approach and further characterization of recovered proteins involved in this interaction. For this purpose, human GATA-3, either recombinant or in nuclear extracts, and three natural GATA elements of the HIV-1 long terminal repeat (sites 1, 2, and 3) were chosen, in which only site 2 is a noncanonical GATA site. Direct analysis of sensorgrams, with recombinant huGATA-3, allowed the comparison of association and dissociation profiles of the three DNA regions and their ranking according to their relative affinities. This result, confirmed by competitions with each GATA site, demonstrated the higher relative affinity (at least sevenfold) of site 3. Interactions between the canonical and unique GATA site 3 and nuclear extracts were also studied in real time and provided information on its association and dissociation rates for native huGATA-3. Finally, recovered protein was identified as genuine huGATA-3 by SDS-PAGE, Western blotting, and bandshift assays.
Interleukin-5 (IL-5), which is produced by CD4(+) T helper 2 (Th2) cells, but not by Th1 cells, plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4(+) T helper cells, Th1 and Th2, the molecular basis for Th1- and Th2-specific gene expression remains to be elucidated. We previously established a critical role for the transcription factor GATA-3 in IL-5 promoter activation in EL-4 cells, which express both Th1- and Th2-type cytokines. Our studies reported here demonstrate that GATA-3 is critical for expression of the IL-5 gene in bona fide Th2 cells. Whereas mutations in the GATA-3 site abolished antigen- or cAMP-stimulated IL-5 promoter activation in Th2 cells, ectopic expression of GATA-3 in Th1 cells or in a non-lymphoid, non-IL-5-producing cell line activated the IL-5 promoter. During the differentiation of naive CD4(+) T cells isolated from T cell receptor transgenic mice, GATA-3 gene expression was up-regulated in developing Th2 cells, but was down-regulated in Th1 cells, and antigen- or cAMP-activated Th2 cells (but not Th1 cells) expressed the GATA-3 protein. Thus, GATA-3 may play an important role in the balance between Th1 and Th2 subsets in immune responses. Inhibition of GATA-3 activity has therapeutic potential in the treatment of asthma and other hypereosinophilic diseases.
GATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of its contribution to the development of specific cell fates in the many tissues where it is expressed during embryogenesis. We show here that the elements controlling GATA-3 regulation can be precisely refined, using transgenic mice, to discrete cis-acting domains: within 6 kb surrounding the transcriptional initiation site, separate sequences were found to control the expression of mGATA-3 in early muscle masses, in a subset of PNS neurons, in the genital tubercle, and in the branchial arches. The branchial arch regulatory element is particularly robust and was refined to a discrete enhancer sequence lying between nt -2832 and -2462 from the transcription initiation site. The enhancer contains potential binding sites for many well-characterized transcription factors, suggesting that mGATA-3 transcriptional activity may be regulated by these proteins (or related family members) in the mesenchyme of the arches that contribute to formation of the jaw. These studies show that discrete regulatory elements required for the elaboration of complex developmental programs can be individually localized, suggesting that the developmentally transient expression of individual transcription factors collaboratively contributes to the temporal and spatial pattern of cellular differentiation leading to the formation of adult anatomy.
The 5'-flanking region from -78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position -113 to -78. In the present studies, we have characterized three regulatory elements in the proximal 5'-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from -52 to -43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from -62 to -53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.
CD4 T cells potentiate the inflammatory or humoral immune response through the action of Th1 and Th2 cells, respectively. The molecular basis of the differentiation of these cells from naive T cell precursors is, however, unclear. We found that GATA-3 was selectively expressed in Th2 cells. GATA-3 is expressed at a high level in naive, freshly activated T cells and Th2 lineage cells, but subsides to a minimal level in Th1 lineage cells as naive cells commit to their Th subset. Antisense GATA-3 inhibited the expression of all Th2 cytokine genes in the Th2 clone D10. GATA-3 directly activated an IL-4 promoter-luciferase reporter gene in M12 cells. In transgenic mice, elevated GATA-3 in CD4 T cells caused Th2 cytokine gene expression in developing Th1 cells. Thus, GATA-3 is necessary and sufficient for Th2 cytokine gene expression.
The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.
Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region, within the human IL-5 gene promoter, that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of these family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and PMA/A23187 stimulation are necessary for the IL-5 promoter activation. The requirement of another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNA of three GATA-binding proteins, hGATA-2, hGATA-3 and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/ A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms specific DNA-protein complex with the -70 GATA site. The electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity to the -70 GATA site among the three GATA-binding proteins. When the transactivation ability was compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.
In the hematopoietic lineage, the transcription factors GATA-1 and GATA-2 show restricted and largely overlapping expression profiles, but GATA-2 is uniquely expressed in early hematopoietic progenitors. GATA-3 is found exclusively in T cells of hematopoietic lineage. To clarify whether these expression profiles are preserved or changed during the development of malignancies, we analyzed the expression of GATA factors in the blasts from leukemic children. A total of 18 myelogenous leukemia and 24 lymphoblastic leukemia (ALL) cases were investigated. In the majority of the former cases, GATA-2 mRNA expression and the expression of CD34 and c-kit antigens on leukemic cells were demonstrated. In contrast, GATA-2 mRNA and c-kit antigen could not be detected in CD34-positive cells from ALL patients. GATA-3 mRNA was expressed in all T-ALL cases, but not in any precursor B-ALL. These findings suggest that down-regulation of GATA-2 and expression of GATA-3 are important events for the commitment of cells to lymphoid and T cell lineage, respectively. The expression profiles of GATA factors in leukemic cells are generally consistent with those in their normal counterparts, and thus provide a useful tool to determine the lineage commitment of unclassified leukemia.
To evaluate the functional role of GATA motifs in the 5'-flanking region of a kidney-specific AQP-2 water channel gene, we sought to isolate a GATA factor(s) expressed in collecting ducts and determined the role on the AQP-2 promoter. Two cDNAs encoding GATA factors were isolated from rat kidney, whose sequences were highly homologous with human GATA-2 and -3. Reverse-transcription PCR using dissected nephron segments revealed that rat GATA-3 but not GATA-2 was expressed in collecting ducts, thus indicating that GATA-3 could interact with GATA motifs in the AQP-2 promoter. Transactivation experiments utilizing the rat GATA-3 expression vector indicated that rat GATA-3 increased the AQP-2 promoter activity about fourfold. These results indicated that GATA motifs in the 5'-flanking region of the hAQP-2 gene were functional cis-elements and that GATA-3 in collecting ducts may be one of the important regulators of AQP-2 expression in vivo.
We examined regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter and showed that Hep3B and HepG2 cells express human GATA-2 (hGATA-2) mRNA and protein. Nuclear extracts of QT6 cells transfected with hGATA-1, 2, or 3 transcription factors showed specific binding to the GATA element in the human Epo gene promoter by gel mobility shift assay. Transient transfection of Hep3B cells with hGATA-1, 2, or 3 showed that each of these transcription factors significantly decreased the level of expression of Epo mRNA as assessed by a competitive polymerase chain reaction. Transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (growth hormone [GH]) construct showed significant inhibition of the Epo promoter. Antisense oligonucleotide for hGATA-2 transcription factor significantly increased the Epo protein in Hep3B cells under 1% O2 for 24 hours incubation. Furthermore, transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (luciferase) construct also showed significant inhibition of the Epo promoter. However, transfection of the mutated GATA sequence of the Epo-luciferase gene with hGATA-1, 2, and 3 interfere with the inhibition of the Epo promoter. We conclude that the hGATA-1, 2, and 3 transcription factors specifically bind to the GATA element in the human Epo gene promoter and negatively regulate Epo gene expression.
TAL1, which is frequently activated in T cell acute lymphoblastic leukemia (T-ALL), encodes lineage-specific basic helix-loop-helix (bHLH) proteins that bind specifically to E-box DNA motif upon dimerization with ubiquitous basic helix-loop-helix proteins E47 or E12. RBTN1 and RBTN2, also frequently activated in T-ALL, encode proteins only with tandem cysteine-rich LIM domains. We found that aberrant expression of TAL1 detected in 11 out of 14 T-ALL cell lines was invariably accompanied by that of either RBTN1 or RBTN2. Forced expression of TAL1 together with RBTN1 or RBTN2, but not TAL1 alone, strongly induced artificial reporter genes in a TAL1/RBTN-negative T-ALL cell line, HPB-ALL. Such collaborative transcriptional activity of TAL1 and RBTN was not, however, observed in non-T cell lines, suggesting further involvement of some T cell-specific cofactors. In this context, we carried out preliminary evaluation of a potential role of the T cell-specific GATA-binding protein, GATA3, in the transcriptional activity of TAL1 and RBTN. We also showed that coexpression of TAL1 and RBTN1 in HPB-ALL strongly induced TALLA1, a highly specific T-ALL marker whose positivity correlated 100% with ectopic expression of TAL1 among various T-ALL cell lines. Collectively, ectopic TAL1 and RBTN1 or -2, together with some endogenous T cell-specific cofactors like GATA3, constitute a highly collaborative set of transcription factors whose aberrant activity in T cells may lead to leukemogenesis by modulating expression of downstream genes such as TALLA1.
We previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that both of these factors regulate transcription of the placental lactogen I gene, as well as the related proliferin gene, in trophoblast giant cells in vivo. Placentas lacking GATA-3 accumulate placental lactogen I and proliferin mRNAs to a level 50% below that reached in the wild-type placenta. Mutation of the GATA-2 gene had a similar effect on placental lactogen I expression, but led to a markedly greater reduction (5- to 6-fold) in proliferin gene expression. Placentas lacking GATA-2 secrete significantly less angiogenic activity than wild-type placentas as measured in an endothelial cell migration assay, consistent with a reduction in expression of the angiogenic hormone proliferin. Furthermore, within the same uterus the decidual tissue adjacent to mutant placentas displays markedly reduced neovascularization compared to the decidual tissue next to wild-type placentas. These results indicate that GATA-2 and GATA-3 are important in vivo regulators of trophoblast-specific gene expression and placental function, and reveal a difference in the effect of these two factors in regulating the synthesis of related placental hormones.
Commitment and differentiation of hematopoietic stem cells are associated with the progressive restriction of cellular proliferation and the progressive expression of a subset of genes encoding the markers of mature cells. These two processes are genetically regulated and, in this paper, I review the expression and function of the GATA family of transcription factors as an example of this genetic regulation. GATA cis-acting elements are found in most of the regulatory regions of T-lymphoid, erythrocytic and megakaryocytic restricted genes. These GATA motifs are recognized by the members of a family of transcriptional regulators: the GATA family. Three members of this family, GATA-1, 2 and 3 are expressed in hematopoietic cells. They are necessary for the erythrocytic and megakaryocytic lineages (GATA-1), for the T-lymphoid lineage (GATA-3), and for the proliferation of uncommitted hematopoietic precursors (GATA-2). GATA-1 displays at least four functions: activation of the erythrocytic and megakaryocytic specific genes, regulation of the epsilon-->gamma globin switch and control of the cell cycle. These two last functions will be discussed to show the multiple facets of GATA-1 in the genetic regulation of hematopoiesis.
THE zinc-finger transcription factor GATA-3 is expressed in haematopoietic cells and in the developing kidney and nervous system. Within the haematopoietic lineages, expression of GATA-3 is restricted to thymocytes and T cells. Functionally important GATA-3 binding sites have been identified in multiple T-cell-specific genes. Mice containing homozygous null mutations of the GATA-3 gene die on embryonic day 12, precluding a detailed assessment of the role of GATA-3 in haematopoietic development. Here we have used murine embryonic stem (ES) cells containing homozygous mutations in the GATA-3 gene (GATA-3(-/-)) in conjunction with the RAG-2(-/-) (ref. 10) and C57BL/6 complementation systems to study the role of GATA-3 in mammalian haematopoiesis. Our results show that GATA-3(-/-) ES cells can contribute to the development of the mature erythroid, myelomonocytic and B-cell lineages, but fail to give rise to thymocytes or mature peripheral T cells. The differentiation of GATA-3(-/-) T cells is blocked at or before the earliest double-negative (CD4-/CD8-) stage of thymocyte development, such that the GATA-3(-/-) ES cells are unable to contribute measurably to the double-negative thymocyte population. These findings suggest that GATA-3 is an essential and specific regulator of early thymocyte development.
Flow cytometric and immunocytochemical analyses of murine fetal thymus (FT) cells with antibodies to various surface markers and transcription factors reveal that the synthesis of TCF-1 and GATA-3 protein begins simultaneously in a fraction of the most immature population of FT cells, which have the phenotype of CD4-CD8-CD44+CD25-. No TCF-1-producing cells is found in the fetal liver (FL). In CD44+CD25- FT cells, the production of TCF-1 is immediately followed by intracellular expression of CD3 epsilon. It is also found that the T cell development from FL, but not FT, progenitors in the FT organ culture system is severely inhibited by the addition of antisense oligonucleotides for either TCF-1 or GATA-3. These results strongly suggest that TCF-1 and GATA-3 play essential roles in the initiation of the earliest steps of T cell development in the thymus.
Based on the presence of T cell receptor-beta (TcR-beta) gene rearrangements in L428 and HDLM-1 cells, the expression of CD2 in HDLM-1 cells, and the presence of immunoglobulin heavy-chain (IgH) gene rearrangement in KM-H2 cells, some researchers have concluded that these long-term cell lines derived from patients with Hodgkin's disease are lymphoid in nature. The information obtained from these cell lines has also been used in arguments for a lymphoid origin of H-RS cells in tissue despite the frequent absence of lymphoid markers and Ig/TcR gene rearrangements in these cells. We questioned whether one can use the limited expression of lymphoid markers or the limited gene rearrangement to conclude that H-RS cells have a lymphoid origin, because these markers may be aberrant in tumor cells. In this study, we examined the expression of two T-cell-specific transcription factors (TCF-1 and GATA-3) and one B-cell-specific transcription factor (BSAP) in cultured H-RS cells by using a gel mobility shift assay. The sensitivity and specificity of this assay for determination of cell lineage have been established in a large number of cultured human and murine cell lines. All three types of H-RS cell lines were consistently negative for BSAP, TCF-1, and GATA-3. The absence of GATA-3 was confirmed in H-RS cells in tissues by an in situ hybridization technique. Virtually all B-cell lines, with the exception of some myeloma cell lines, are positive for BSAP, which is the transcription factor for promoters for several B-cell markers, including VpreB1, lambda 5, CD19, and CD20. All T-cell lines tested were positive for TCF-1 and GATA-3, which are the transcription factors for promoters for several T-cell-restricted markers, including CD2, CD3, TcR, and lck. The absence of BSAP, TCF-1, and GATA-3 clearly indicates an underlying difference between H-RS cells and lymphoid cells.
The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.
Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs.
Interleukin-5 (IL-5) plays a central role in the growth and differentiation of eosinophils and contributes to several disease states including asthma. Accumulating evidence suggests a role for cAMP as an immunomodulator; agents that increase intracellular cAMP levels have been shown to inhibit production of cytokines predominantly produced by T helper (Th) 1 cells such as IL-2 and interferon gamma (IFN-gamma). In contrast, the production of IL-5, predominantly produced by Th2 cells, is actually enhanced by these agents. In this report, we have performed transient transfection experiments with IL-5 promoter-reporter gene constructs, DNase I footprinting assays, and electrophoretic mobility shift assays to investigate the key regulatory regions necessary for activation of the IL-5 promoter by dibutyryl cAMP and phorbol esters in the mouse thymoma line EL-4. Taken together, our data demonstrate the critical importance of two sequences within the IL-5 5'-flanking region for activation by these agents in EL-4 cells: one, a highly conserved 15-base pair element present in genes expressed by Th2 cells, called the conserved lymphokine element 0 (CLE0; located between -53 and -39 in the IL-5 promoter), and the other, two overlapping binding sites for the transcription factor GATA-3 (but not GATA-4) between -70 and -59. Taken together, our data suggest that activation via the unique sequence combination GATA/CLE0 results in selective expression of the IL-5 gene in response to elevated levels of intracellular cAMP.
Restriction mapping and sequence analysis of the gene encoding the rat beta 2 adrenergic receptor (beta 2AR) revealed an error affecting most of the 5'-flanking domain, located between -192 and -2245 bp in the only published sequence of this gene [Buckland et al., Nucleic Acids Res. 18 (1990) 682]. The correct sequence lacks a TATA box, contains 67% G + C residues in the first 500 nucleotides upstream from the start codon, and contains putative glucocorticoid, thyroid hormone and cAMP-response elements.
The transcription factor GATA-3 contributes to the expression of several genes critical for T-cell function and development, including the T-cell receptor alpha and beta chains. We report here the isolation of a human cDNA clone which encodes a 3' segment of a novel protein with three zinc fingers. This protein, which we termed G3B, can interact with GATA-3 in vitro. Its mRNA is expressed in T and B cells. Thus, G3B may represent a potential regulator of GATA-3 function.
We have designed a new strategy to isolate unknown DNA regions interacting with one or several related regulatory proteins. It involves trapping such DNAs by their cognate binding proteins followed by PCR amplification, as described previously [Kinzler, K. & Vogelstein, B. (1989) Nucleic Acids Res. 17, 3645-3653]. To overcome the inability of such a procedure to discriminate between functional and non-functional binding sites as well as to specifically trap short DNA motifs from the whole higher eukaryotic genome, we have used as starting material DNA isolated from transcriptionally competent chromatin fractions, instead of total genomic DNA. To test our strategy, we selected human DNA sequences that bind members of the GATA family, known to recognize similar WGATAR motifs. These proteins are expressed in different cell types in which they regulate the transcription of different sets of genes; thus, transcriptionally active chromatin containing GATA motifs should differ according to the cell type. We have trapped and analyzed DNA fragments isolated from an active chromatin fraction, from erythroid cells and lymphoid cells, using GATA-1 and GATA-3 proteins, respectively. We show that regulatory GATA sequences known to be in open chromatin in erythroid cells (typified by the HSIII fragment of the beta-globin locus control region) or in lymphoid cells (typified by a fragment of the CD2 locus control region) are dramatically enriched in a cell-specific manner, demonstrating the potency of the method. The sequences of the erythroid or lymphoid DNA fragments isolated through the procedure described here were determined and display subset-site preference for GATA-1 and GATA-3.
GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.
Endothelin-1 (ET-1) is a 21-amino-acid vasoactive peptide initially characterized as a product of endothelial cells. Reporter gene transfection experiments have indicated that a GATA site and an AP1 site are essential for ET-1 promoter function in endothelial cells, and GATA-2 appears to be the active GATA factor which regulates ET-1 expression. To look for interactions between AP1 and GATA-2, transactivation experiments were performed with expression vectors encoding c-Jun, c-Fos, and GATA-2. Cooperativity between the AP1 complex and GATA-2 was observed as a synergistic increase in transcriptional activity of the ET-1 reporter plasmid. In addition, AP1 was able to potentiate the action of GATA-2 on reporter constructs lacking a functional AP1 site. In a similar fashion, GATA-2 was able to potentiate the action of AP1 despite deletion of the GATA site. Experiments with GATA-1 and GATA-3 expression vectors provided evidence that this capacity to interact with AP1 may be a characteristic of all GATA family members. Biochemical evidence for AP1-GATA interaction was provided by immunoprecipitation experiments. A GATA-2-specific antiserum was shown to immunoprecipitate in vitro-synthesized Jun and Fos protein from reticulocyte lysate. Also, antisera directed against Jun and Fos were able to immunoprecipitate from nuclear extracts a GATA-binding protein, indicating the association of AP1 and GATA proteins in vivo.
Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.
GATA-3 is a transcription factor involved in the differentiation of T lymphocytes and additionally expressed in several chicken and mouse embryonic tissues. Using in situ hybridization, we found that the human GATA-3 gene is selectively expressed in the developing kidney. GATA-3 mRNA is first detected in the Wolffian duct from the time of its emergence in the embryonic intermediate mesoderm and further expressed in the collecting ducts of the mesonephros until its involution. In the metanephros, GATA-3 is expressed in the ureteric bud where it is constitutively transcribed, throughout development, along the branching process that gives rise to the whole collecting system of the definitive kidney. Besides the Wolffian duct and derivatives, we also report the expression of GATA-3 gene in the glomerular mesangium and adjacent endocapillary cells, in both meso- and metanephros. This early and specific expression of the GATA-3 gene suggests a role for this transcription factor in the differentiation of the human kidney.
The first lymphoid cell line derived from an amphibian (Xenopus) thymic tumor shows an extreme form of lineage infidelity. Although it has rearranged in-frame the two alleles of the heavy chain, deleted one light chain locus, and rearranged abortively the two alleles of the second light chain locus, the cell line does not produce immunoglobulin molecules or message. It expresses a variety of T-cell characteristic markers such as Xenopus pan T-cell markers, CD8 equivalent and GATA3 transcription factor. It does not express any major histocompatibility complex class I or class II molecules. It resembles some rare types of mammalian leukemias.
In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.
SCL, GATA-1, GATA-2 and GATA-3 encode lineage restricted haemopoietic transcription factors. We have previously shown that SCL, GATA-1 and GATA-2 are expressed in multipotent progenitors prior to lineage commitment, but are down-regulated during granulocyte/monocyte differentiation. The phenomenon of gene extinction in cell hybrids may reveal negative regulatory mechanisms operating during normal differentiation. We have therefore analysed the regulation of SCL, GATA-1, GATA-2 and GATA-3 in cell hybrids formed by the fusion of cell lines representing different haemopoietic lineages. Expression of GATA-3 was extinguished in both human and murine erythroid x T cell hybrids, an observation which suggests that erythroid cells contain factors capable of repressing GATA-3 expression. By contrast expression of SCL, GATA-1 and GATA-2 was not extinguished in erythroid x T or in erythroid x B cell hybrids. These data suggest that T cells and B cells do not contain trans-acting factors capable of down-regulating expression of SCL, GATA-1 or GATA-2, and therefore raise the possibility that a 'hit and run' mechanism may repress these genes during normal haemopoiesis. HpaII sites within the SCL promoter were unmethylated in erythroid cells but methylated in T cells. Erythroid x T and erythroid x B cell hybrids contained both methylated and unmethylated SCL promoters, thus implicating a heritable cis-acting mechanism in the regulation of the SCL gene in lymphoid cell lines. These results provide the first analysis of SCL and GATA gene regulation in stable cell hybrids.
Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.
The GATA family of transcription factors regulates a wide variety of genes, including those involved in differentiation of erythrocytes and T lymphocytes. We report here the creation of a dominant negative mutant of GATA-3, KRR, which effectively blocks wild-type GATA-1, GATA-2, and GATA-3 transactivation when co-expressed in transient assays. KRR was generated by site-directed mutagenesis while investigating a putative activation domain of GATA-3, located between its two zinc fingers. The GATA-3 KRR mutation does not affect expression, nuclear translocation, or the ability to bind to a consensus GATA sequence. KRR can suppress the activity of the minimal T cell receptor (TCR) alpha and beta enhancers by 12- and 3.4-fold, respectively. However, KRR did not have a significant effect on the activity of larger TCR-alpha and -beta enhancer fragments. Thus, functional redundancy in the TCR-alpha and -beta enhancers can compensate for the loss of GATA-3 activity.
The molecular determinants governing tissue-specific gene expression in the placenta are at present only poorly defined, particularly with respect to the regulation of specific hormone genes whose products are vital to embryonic development and the maintenance of a nurturing maternal environment. In continuing our analysis of the trophoblast-specific expression of the mouse placental lactogen I gene, we now demonstrate that the transcription factors GATA-2 and GATA-3 regulate the activity of this gene promoter. These factors are expressed in placental trophoblast cells, with peak levels of the GATA-2, GATA-3 and placental lactogen I mRNAs each accumulating at midgestation. Analysis of a region of the placental lactogen I gene promoter, previously shown to be sufficient for directing trophoblast-specific transcription, revealed the presence of three consensus binding sites for GATA-2 or GATA-3. Both GATA-2 and GATA-3 bind to these sites in vitro and mutation of these sites results in a significant decrease in promoter activity as assayed by transient transfection into the choriocarcinoma-derived cell line Rcho-1, which expresses endogenous GATA-2 and GATA-3. Furthermore, overexpression of GATA factors in Rcho-1 cells stimulates transcription from a co-transfected placental lactogen I gene promoter. Most significantly, expression of GATA-2 or GATA-3 was found to induce transcription from this promoter in transfected non-trophoblast (fibroblast) cells. These data indicate that GATA factors are both limiting and required transcriptional regulatory molecules in placental trophoblasts, and that the tissue specificity of the placental lactogen I gene is determined, at least in part, by GATA-2 and/or GATA-3.
We describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephalon, pons and inner ear) later in gestation. GATA-3 also shows a restricted expression pattern in the peripheral nervous system, including terminally differentiating cells in the cranial and sympathetic ganglia. In addition to this distinct pattern in the nervous system, mGATA-3 is also expressed in the embryonic kidney and the thymic rudiment, and further analysis showed that it is expressed throughout T lymphocyte differentiation. To begin to investigate how this complex gene expression pattern is elicited, cloning and transcriptional regulatory analyses of the mGATA-3 gene were initiated. At least two regulatory elements (one positive and one negative) appear to be required for appropriate tissue-restricted regulation after transfection of mGATA-3-directed reporter genes into cells that naturally express GATA-3 (T lymphocytes and neuroblastoma cells). Furthermore, this same region of the locus confers developmentally appropriate expression in transgenic mice, but only in a subset of the tissues that naturally express the gene.
The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.
Three high-affinity binding sites for the GATA family of transcriptional regulators have been identified within the T-cell receptor beta-chain (TCR beta) transcriptional enhancer, and their functional significance has been determined in an effort to understand the T-cell specificity of the enhancer more fully. One site, TE4, is important for activity of the enhancer in T cells. Neither site TE1 nor site TE2 can functionally replace a mutated TE4 site in T cells; however, the same protein, probably GATA-3, binds all three sites, as judged by electrophoretic mobility shift, oligonucleotide competition, and proteolytic clipping assays. These data suggest that additional proteins are critical for the ability of GATA-3 to activate the TCR beta enhancer. In fibroblasts, the GATA sequence at site TE1 appears to bind a negative regulator. Since this is not true in B cells, B cells and fibroblasts appear to have different mechanisms for negative regulation of the TCR beta enhancer.
Mouse embryonic stem (ES) cells lacking the transcription factor GATA-1 do not produce mature red blood cells either in vivo or in vitro. To define the consequences of GATA-1 loss more precisely, we used an in vitro ES cell differentiation assay that permits enumeration of primitive (EryP) and definitive (EryD) erythroid precursors and recovery of pure erythroid colonies. In contrast to normal ES cells, GATA-1- ES cells fail to generate EryP precursors. EryD precursors, however, are normal in number but undergo developmental arrest and death at the proerythroblast stage. Contrary to initial expectations, arrested GATA-1(-)-definitive proerythroblasts express GATA target genes at normal levels. Transcripts of the related factor GATA-2 are remarkably elevated in GATA-1- proerythroblasts. These findings imply substantial interchangeability of GATA factors in vivo and suggest that GATA-1 normally serves to repress GATA-2 during erythropoiesis. The approach used here is a paradigm for the phenotypic analysis of targeted mutations affecting hematopoietic development.
GATA3, a member of the GATA family that is abundantly expressed in the T-lymphocyte lineage, is thought to participate in T-cell receptor gene activation through binding to enhancers. To understand GATA3 gene regulation, we cloned the human gene and the 5' end of the mouse GATA3 gene. We show that the human GATA3 gene contains six exons distributed over 17 kb of DNA. The two human GATA3 zinc fingers are encoded by two separate exons highly conserved with those of GATA1, but no other structural homologies between these two genes can be found. The human and mouse GATA3 transcription units start at a major initiation site. The promoter sequence analysis of these two genes revealed that they are embedded within a CpG island and share structural features often found in the promoters of housekeeping genes. Finally, we show that a DNA fragment containing the human GATA3 transcription unit, 3 kb upstream from the initiation site and 4 kb downstream from the polyadenylation site, displays T-cell specificity.
The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.
GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.
The DNA motif WGATAR has been identified within transcriptional regulatory domains of globin and other erythroid-specific genes and the activator proteins that bind to this regulatory element, the GATA factors, belong to a multi-gene family that is expressed in chicken erythroid cells. Here we show that, as in chickens, multiple members of the GATA factor family are expressed in human and murine erythroid cells. During the early stages of chicken embryogenesis (well before blood island formation), each of the GATA family members is transcribed with a unique temporal and spatial pattern. In the primitive erythroid lineage, transcription of the embryonic epsilon-globin gene parallels GATA-1 expression while the switch to beta-globin transcription in definitive erythroid cells is directly preceded by a pronounced increase in GATA-3 accumulation. The timing and pattern of expression of these different mRNAs during avian erythroid development and differentiation suggests that temporally regulated changes in GATA factor expression are required for vertebrate hematopoiesis.
Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.
The GATA 'zinc-finger' transcription factors are thought to have important roles in the control of hematopoiesis. GATA-1 and GATA-2 are found in the erythroid, mast cell, and megakaryocytic lineages, and GATA-3 in T lymphocytes. GATA-1 is required for erythroid development and has recently been shown by gene transfer to direct megakaryocytic differentiation of the primitive myeloid cell line 416B. Here we show that enforced expression in 416B cells of either the GATA-2 or GATA-3 gene also induces megakaryocytic differentiation, as assessed by cellular morphology, acetylcholinesterase activity, polyploid DNA content, and loss of Mac-1 expression. No erythroid or mast cell differentiation was found. Unexpectedly, the level of endogenous GATA-1 mRNA had increased 20- to 30-fold among the transfectants, whereas that of GATA-2 mRNA was unaltered and endogenous GATA-3 transcripts remained undetectable. This finding suggests that GATA-2 and GATA-3 lie upstream of GATA-1 in a regulatory hierarchy and that, in 416B cells, GATA-1 may mediate the phenotypic changes induced by GATA-2 or GATA-3. Furthermore, 416B cells treated with the DNA demethylating agent 5-azacytidine underwent megakaryocytic differentiation accompanied by a marked increase in the level of GATA-1 mRNA but not that of GATA-2 or GATA-3. These results strongly implicate GATA factors in megakaryocytic differentiation and suggest that, at least for 416B cells, GATA-1 is a dominant regulator of maturation along this lineage.
Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.
GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.
A family of transcriptional activating proteins, the GATA factors, has been shown to bind to a consensus motif through a highly conserved C4 zinc finger DNA binding domain. One member of this multigene family, GATA-3, is most abundantly expressed in T lymphocytes, a cellular target for human immunodeficiency virus type 1 (HIV-1) infection and replication. In vitro DNase I footprinting analysis revealed six hGATA-3 binding sites in the U3 region (the transcriptional regulatory domain) of the HIV-1 LTR. Cotransfection of an hGATA-3 expression plasmid with a reporter plasmid whose transcription is directed by the HIV-1 LTR resulted in 6- to 10-fold stimulation of LTR-mediated transcription, whereas site specific mutation of these GATA sites resulted in virtual abrogation of the activation by hGATA-3. Further, deletion of the hGATA-3 transcriptional activation domain abolished GATA-dependent HIV-1 trans-activation, showing that the stimulation of viral transcription observed is a direct effect of cotransfected hGATA-3. Introduction of the HIV-1 plasmids in which the GATA sites have been mutated into human T lymphocytes also caused a significant reduction in LTR-mediated transcription at both the basal level and in (PHA- plus PMA-) stimulated T cells. These observations suggest that in addition to its normal role in T lymphocyte gene regulation, hGATA-3 may also play a significant role in HIV-1 transcriptional activation.
The expression of the hematopoietic transcription factors GATA-1, GATA-2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins.
The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.
In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed.
The GATA family of C4 zinc-finger transcription factors has been implicated in tissue-specific gene regulation in birds and mammals. One of the members of this family, GATA-3, is reportedly expressed specifically in the T-cell lineage, where it interacts with GATA motifs in the TCR-alpha, TCR-beta, and TCR-delta enhancers, thereby controlling the T-cell phenotype. To evaluate the differentiation control properties of GATA-3, we have now documented its expression pattern during lymphoid differentiation and murine embryogenesis. The onset of GATA-3 expression in the lymphoid lineage was studied in a panel of lymphoid (precursor) cell lines by Northern blot analysis. GATA-3 was uniquely expressed in T-lineage lymphocytes expressing TCR and CD3 genes; it was absent from TCR/CD3 mRNA-negative prothymocytes and from all B-lineage cells. In order to obtain information on the expression of GATA-3 outside the immune system, in situ hybridization was performed on mouse embryos on day 11.5-14.5 of gestation. GATA-3 mRNA was detected in fetal thymus and in erythroid cells. Outside the haemopoietic system, we detected GATA-3 mRNA throughout the central nervous system, in kidney, in the epidermis, lens fibers, the inner ear, whisker follicles, and in the primary palate. These data provide new clues about the potential role of GATA-3 during mouse development, and will aid the interpretation of currently ongoing gene knockout experiments.
The transcription factors GATA-1, GATA-2, and GATA-3 were found to be expressed in several mouse and rat mast cell lines that contain mast cell carboxypeptidase A (MC-CPA) and other proteases in their cytoplasmic granules. GATA-1 mRNA was not detected in P815 cells, an immature mouse mastocytoma-derived cell line that lacks electron-dense granules and has low levels of secretory granule proteases. Because the 5'-flanking regions of the mouse and human MC-CPA genes contained a conserved GATA-binding motif 51 base pairs upstream of their translation initiation sites, the ability of GATA-binding proteins to regulate the promoter activity of the MC-CPA gene was examined in rat basophilic leukemia cells, mouse P815 cells, and transfected mouse P815 cells that expressed GATA-1. In all three mast cell lines, the promoter activity of the MC-CPA gene depended on the GATA binding site. GATA-1, GATA-2, and GATA-3 are thus the first DNA-binding proteins identified in mast cells which regulate the promoter activity of a gene that encodes a secretory granule protease.
Proteins that recognize the core sequence GATA are important regulators of hematopoietic-specific gene transcription. We have characterized cDNAs encoding the Xenopus laevis homologues of three related transcription factors, designated GATA-1, -2, and -3. Comparative sequence analysis reveals strong conservation of the zinc-finger DNA-binding domain among all vertebrate GATA-binding proteins. GATA-2 and GATA-3 polypeptides are homologous throughout their entire sequences, whereas GATA-1 sequence is conserved only in the region responsible for DNA binding. In Xenopus, RNAs encoding GATA-binding proteins are expressed in both larval and adult erythroid cells. GATA-1, -2, and -3 RNAs are first detectable in early gastrula (Nieuwkoop developmental stage 11). This is earlier than the appearance of the early larval alpha T1 globin RNA (stage 15), beta T1 globin RNA (stage 26), or blood island formation (stage 30). The expression of GATA-1, -2, and -3 in early development may signal an early commitment of mesoderm to form hematopoietic tissue.
The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein containing a zinc-finger domain. We have isolated a full-length human Gata3 cDNA and characterized its role in TCR gene regulation. Northern blot analysis showed that the Gata3 mRNA is present in T-cell, but not B-cell, macrophage or HeLa cell lines. The in vitro transcribed and translated Gata3 cDNA product bound specifically to the enhancer elements of at least three TCR genes containing the above consensus sequence. This binding specificity was consistent with the in vivo activity of the TCR enhancer-binding element in transient transfection assays. Hence, the Gata3 protein, together with other T-cell-specific enhancer-binding proteins, may form the basis of tissue-specific expression of the TCR genes.
A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.
Nuclear cap binding protein complex (CBC) is a heterodimer of a small subunit (Cbc2 in yeast) that binds the m(7)G cap and a large subunit (Sto1 in yeast) that interacts with karyopherins. In order to probe the role of cap recognition in yeast CBC function, we introduced alanine mutations (Y24A, F91A, D120A, D122A, R129A, and R133A) and N-terminal deletions (NΔ21 and NΔ42) in the cap-binding pocket of Cbc2. These lesions had no effect on vegetative growth, but they ameliorated the cold-sensitivity of tgs1Δ cells that lack trimethylguanosine caps (a phenotype attributed to ectopic association of CBC with the m(7)G cap of the normally TMG-capped U1 snRNA), thereby attesting to their impact on cap binding in vivo. Further studies of the Cbc2-Y24A variant revealed synthetic lethality or sickness with null mutations of proteins involved in early steps of spliceosome assembly (Nam8, Mud1, Swt21, Mud2, Ist3, and Brr1) and with otherwise benign mutations of Msl5, the essential branchpoint binding protein. Whereas the effects of weakening CBC-cap interactions are buffered by other actors in the splicing pathway during mitotic growth, the NΔ42 allele causes a severe impediment to yeast sporulation and meiosis. RNA analysis revealed a selective defect in the splicing of MER3 and SAE3 transcripts in cbc2-NΔ42 diploids during attempted sporulation. An intronless MER3 cDNA fully restored sporulation and spore viability in the cbc2-NΔ42 strain, signifying that MER3 splicing is a limiting transaction. These studies reveal a new level of splicing control during meiosis that is governed by nuclear CBC.
The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.
One of the major abiotic stress conditions limiting healthy growth of trees is salinity stress. The use of gene manipulation for increased tolerance to abiotic stress has been successful in many plant species. Overexpression of the Arabidopsis SALT TOLERANT1 (STO1) gene leads to increased concentrations of 9-cis-epoxycarotenoid dioxygenase3, a vital enzyme in Arabidopsis abscisic acid biosynthesis. In the present work, the Arabidopsis STO1 gene (AtSTO1) was overexpressed in poplar to determine if the transgene would confer enhanced salt tolerance to the generated transgenics. The results of multiple greenhouse trials indicated that the transgenic poplar lines had greater levels of resistance to NaCl than wild-type plants. Analysis using RT-PCR indicated a variation in the relative abundance of the STO1 transcript in the transgenics that coincided with tolerance to salt. Several physiological and morphological changes such as greater overall biomass, greater root biomass, improved photosynthesis, and greater pith size were observed in the transgenics when compared to controls undergoing salt stress. These results indicated overexpression of AtSTO1 improved salt tolerance in poplar.
Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.
We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.
We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure).
Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.
An important objective of plant-pathogen interactions research is to determine where resistance proteins detect pathogen effectors to mount an immune response. Many nucleotide binding-Leucine-rich repeat (NB-LRR) resistance proteins accumulate in the plant nucleus following effector recognition, where they initiate the hypersensitive response (HR). Here, we show that potato (Solanum tuberosum) resistance protein R3a relocates from the cytoplasm to endosomal compartments only when coexpressed with recognized Phytophthora infestans effector form AVR3a(KI) and not unrecognized form AVR3a(EM). Moreover, AVR3a(KI), but not AVR3a(EM), is also relocalized to endosomes in the presence of R3a. Both R3a and AVR3a(KI) colocalized in close physical proximity at endosomes in planta. Treatment with brefeldin A (BFA) or wortmannin, inhibitors of the endocytic cycle, attenuated both the relocalization of R3a to endosomes and the R3a-mediated HR. No such effect of these inhibitors was observed on HRs triggered by the gene-for-gene pairs Rx1/PVX-CP and Sto1/IpiO1. An R3a(D501V) autoactive MHD mutant, which triggered HR in the absence of AVR3a(KI), failed to localize to endosomes. Moreover, BFA and wortmannin did not alter cell death triggered by this mutant. We conclude that effector recognition and consequent HR signaling by NB-LRR resistance protein R3a require its relocalization to vesicles in the endocytic pathway.
Shortening eukaryotic poly(A) tails represses mRNA translation and induces mRNA turnover. The major cytoplasmic deadenylase, the Ccr4-Not complex, is a conserved multisubunit assembly. Ccr4-Not is organized around Not1, a large scaffold protein that recruits two 3'-5' exoribonucleases, Caf1 and Ccr4. We report structural studies showing that the N-terminal arm of yeast Not1 has a HEAT-repeat structure with domains related to the MIF4G fold. A MIF4G domain positioned centrally within the Not1 protein recognizes Caf1, which in turn binds the LRR domain of Ccr4 and tethers the Ccr4 nuclease domain. The interactions that form the nuclease core of the Ccr4-Not complex are evolutionarily conserved. Their specific disruption affects cell growth and mRNA deadenylation and decay in vivo in yeast. Thus, the N-terminal arm of Not1 forms an extended platform reminiscent of scaffolding proteins like eIF4G and CBP80, and places the two nucleases in a pivotal position within the Ccr4-Not complex.
In response to osmotic stress, global translation is inhibited, but the mRNAs encoding stress-protective proteins are selectively translated to allow cell survival. To date, the mechanisms and factors involved in the specific translation of osmostress-responsive genes in Saccharomyces cerevisiae are unknown. We find that the mRNA cap-binding protein Cbc1 is important for yeast survival under osmotic stress. Our results provide new evidence supporting a role of Cbc1 in translation initiation. Cbc1 associates with polysomes, while the deletion of the CBC1 gene causes hypersensitivity to the translation inhibitor cycloheximide and yields synthetic "sickness" in cells with limiting amounts of translation initiator factor eIF4E. In cbc1Δ mutants, translation drops sharply under osmotic stress, the subsequent reinitiation of translation is retarded, and "processing bodies" containing untranslating mRNAs remain for long periods. Furthermore, osmostress-responsive mRNAs are transcriptionally induced after osmotic stress in cbc1Δ cells, but their rapid association with polysomes is delayed. However, in cells containing a thermosensitive eIF4E allele, their inability to grow at 37ºC is suppressed by hyperosmosis, and Cbc1 relocalizes from nucleus to cytoplasm. These data support a model in which eIF4E-translation could be stress-sensitive, while Cbc1-mediated translation is necessary for the rapid translation of osmostress-protective proteins under osmotic stress.
Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5'-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m(7)G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.
Functional stacking of broad spectrum resistance (R) genes could potentially be an effective strategy for more durable disease resistance, for example, to potato late blight caused by Phytophthora infestans (Pi). For this reason, three broad spectrum potato R genes (Rpi), Rpi-sto1 (Solanum stoloniferum), Rpi-vnt1.1 (S. venturii) and Rpi-blb3 (S. bulbocastanum) were selected, combined into a single binary vector pBINPLUS and transformed into the susceptible cultivar Desiree. Among the 550 kanamycin resistant regenerants, 28 were further investigated by gene specific PCRs. All regenerants were positive for the nptII gene and 23 of them contained the three Rpi genes, referred to as triple Rpi gene transformants. Detached leaf assay and agro-infiltration of avirulence (Avr) genes showed that the 23 triple Rpi gene transformants were resistant to the selected isolates and showed HR with the three Avr effectors indicating functional stacking of all the three Rpi genes. It is concluded that Avr genes, corresponding to the R genes to be stacked, must be available in order to assay for functionality of each stack component. No indications were found for silencing or any other negative effects affecting the function of the inserted Rpi genes. The resistance spectrum of these 23 triple Rpi gene transformants was, as expected, a sum of the spectra from the three individual Rpi genes. This is the first example of a one-step approach for the simultaneous domestication of three natural R genes against a single disease by genetic transformation.
Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.
Pre-messenger RNA splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Despite the striking evolutionary conservation of the spliceosomal components and their functions, controversy persists about the relative importance of splicing in Saccharomyces cerevisiae-particularly given the paucity of intron-containing genes in yeast. Here we show that splicing of one pre-messenger RNA, SUS1, a component of the histone H2B ubiquitin protease machinery, is essential for establishing the proper modification state of chromatin. One protein complex that is intimately involved in pre-mRNA splicing, the yeast cap-binding complex, appears to be particularly important, as evidenced by its extensive and unique genetic interactions with enzymes that catalyze histone H2B ubiquitination. Microarray studies show that cap binding complex (CBC) deletion has a global effect on gene expression, and for approximately 20% of these genes, this effect is suppressed when ubiquitination of histone H2B is eliminated. Consistent with this finding of histone H2B dependent effects on gene expression, deletion of the yeast cap binding complex leads to overubiquitination of histone H2B. A key component of the ubiquitin-protease module of the SAGA complex, Sus1, is encoded by a gene that contains two introns and is misspliced when the CBC is deleted, leading to destabilization of the ubiquitin protease complex and defective modulation of cellular H2B levels. These data demonstrate that pre-mRNA splicing plays a critical role in histone H2B ubiquitination and that the CBC in particular helps to establish the proper state of chromatin and proper expression of genes that are regulated at the level of histone H2B ubiquitination.
To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5' splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5' splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5' splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.
The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways.
A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis) mutants, both of which are also involved in abscisic acid (ABA) biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination.
Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in different abi3 mutants. Expression profiling revealed a potentially novel regulation of auxin metabolism and transport in an ABA deficient mutant, sis7-1/nced3-4/sto1-4.
Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.
Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.
The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.
We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Delta and rrp6-Delta. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this "retention signal" causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.
We previously demonstrated an increased degradation of mRNAs in mutants of Saccharomyces cerevisiae having blocks in nuclear export. The degradation activity, designated DRN (degradation of mRNA in the nucleus), requires Cbc1p, a nuclear cap-binding protein, and Rrp6p, a nuclear exosome component. Microarray procedures were used to determine the half-lives of mRNAs from normal and mutant strains, leading to the tentative identification of hundreds of normal mRNAs that were notably stabilized when either CBC1 or RRP6 were deleted. Northern blot analysis of representative mRNAs confirmed the diminished degradation. One representative of this group, SKS1 mRNA, was also shown by a cytological procedure to be preferentially retained in the nucleus compared with typical mRNAs. We suggest that all normal mRNAs are subjected to degradation by DRN, but the degree of degradation is determined by the degree of nuclear retention. Furthermore, these mRNAs particularly susceptible to DRN were also diminished by overproduction of Cbc1p, demonstrating a regulatory role for CBC1. This conclusion was corroborated by finding an inverse relationship of the CBC1 and SKS1 mRNA levels in normal strains grown under different conditions.
We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media. sto1 plants were more tolerant in vitro than wild type to Na(+) and K(+) both for germination and subsequent growth but were hypersensitive to Li(+). Postgermination growth of the sto1 plants on sorbitol was not improved. Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 [Phaseolus vulgaris] and to NCED3 GB:AB020817 [Arabidopsis]), a key enzyme in the abscisic acid (ABA) biosynthetic pathway. STO1 transcript abundance was substantially reduced in mutant plants. Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered. Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype. Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation. Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster. The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses.
SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis.
The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin. The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.
To study the function of GCR3, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Candida albicans gene which complements the growth defect of the (delta)gcr3 mutant was isolated. Transformants of this gene also recovered the glycolytic enzyme activities. Its DNA sequencing predicted an 886 amino acid protein with 30.4% identity to the Gcr3p of S. cerevisiae.
To study the functions of DNA topoisomerase I and Hpr1 protein, a suppressor mutant of the temperature-sensitive growth of an hpr1 top1-5ts double mutant was isolated. The isolated triple mutant showed cold-sensitive growth. By complementation of this phenotype, the suppressor gene was cloned. DNA sequencing showed it to be GCR3, a gene involved in the expression of glycolytic genes. Further analysis showed that gcr3 mutations also suppressed the temperature-sensitive growth of hpr1 single mutants. Experiments with gcr3 truncation mutants also suggested a genetic interaction between GCR3 and HPR1. The fact that top1 suppressed the growth defect of gcr3 suggested an interaction between those two genes also. Plasmid DNA isolated from gcr3 mutants was significantly more negatively supercoiled than normal, suggesting that Gcr3 protein, like topoisomerase I and Hpr1p, affects chromatin structure, perhaps during transcription.
G4 nucleic acids are quadruplex structures involving guanine-rich sequences that form in vitro under moderate conditions. Experimental evidence exists supporting biological functions for these elements; however, direct demonstration of G4 nucleic acids in vivo has not yet been achieved. Here we purify and characterize a yeast protein, G4p2, which has a specific affinity for G4 nucleic acids. G4p2 binds equivalently to RNA and DNA in G4 form. The Keq for G4p2 binding to a G4 DNA oligomer is 2.2 x 10(8) M-1 under near physiological conditions. We have cloned and sequenced the gene encoding G4p2 and have shown it to be identical to MPT4 and STO1. MPT4 was isolated in a screen for multicopy suppressors of staurosporine sensitivity in POP2 cells. Pop2 is a complex regulatory factor that participates, in part, in the repression of certain genes in the absence of glucose (Sakai, A., Chibazakura, T., Shimizu, Y., and Hishinuma, F. (1992) Nucleic Acids Res. 20, 6227-6233). STO1 was isolated as a multicopy suppressor of TOM1, an uncharacterized mutation that leads to temperature-sensitive cell cycle arrest at the G2/M boundary. Suppression of these mutations by G4p2 indicate this G4 nucleic acid binding protein may function in signal transduction pathways regulated by protein kinases, which control carbon source utilization, and in cell cycle progression.
Based on the requirements of test protocols developed to evaluate clinical MRS single slice and volume localisation sequences, two clinical test objects, STO1 and STO2 have been developed. The properties of a range of potential construction materials have been assessed, demonstrating that the water/Perspex interface produced minimum susceptibility effects. The design of the objects has been evaluated in trials on different magnetic resonance instruments, with size and loading being adjusted to allow use on currently available equipment. Appropriate test solutions for 31P and 1H measurements have been developed and their properties evaluated.
Adenoviruses recovered from the northern Stockholm area during 1987-1992 have been subjected to restriction endonuclease analysis. Adenoviruses of all subgenera (A-F) were represented and a considerable degree of serotype variation was seen, e.g. the rarely encountered subgenus A viruses were frequently isolated in the present study. Of 16 subgenus A isolates, Ad31 predominated with 12 strains which were equally distributed into the DNA-variants D2 and D7. Analysis of 38 Ad3 isolates revealed four DNA-variants: D1, D3, D10, and "Sto1". The ten Ad7 isolates belonged all to the DNA-variant D5 of Ad7. Of 27 Ad1 strains, 11 belonged to D10, followed by the DNA-variants D4 and D7 with four strains each. Among Ad2 isolates, D2 or D2-like strains prevailed (23/28). Of six Ad5 strains, three belonged to the DNA-variant D2. The most notable subgenus D event was a nosocomial outbreak of keratokonjunctivitis due ot Ad19a. In addition, a collection of heterogenous subgenus D strains was detected, most of which untypable by RE-analysis. Among the six Ad4 isolates of subgenus E, both genomic clusters (p and a, respectively) of Ad4 were recognized. Concerning the clinical important subgenus F adenoviruses, only two strains of Ad40 were detected as compared to 12 strains of Ad41, all of which ascribed to the DNA-variant D12 of Ad41.
Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting several glycolytic enzyme activities. The recessive single nuclear gene mutation, named gcr3, caused an extremely defective growth phenotype on fermentable carbon sources such as glucose, while growth on respiratory media was almost normal. The GCR3 gene was obtained by growth complementation from a genomic DNA library, and the complemented strains had normal enzyme levels. GCR3 gene was sequenced, and a 99,537-Da protein was predicted. The predicted GCR3 protein was fairly acidic (net charge, -34). The C-terminal region was highly charged, and an acidic stretch was found in it.
The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5'-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.
The influenza virus RNA polymerase synthesizes three types of RNA: genomic vRNA, anti-genomic cRNA and mRNA. Both vRNA and cRNA are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mRNAs are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mRNA, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mRNA. We found that viral mRNA associates with the viral non-structural protein 1 (NS1), cellular poly(A)-binding protein 1 (PABP1), the 20 kDa subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eIF4E. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mRNP) complex. We propose a model in which viral mRNAs, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mRNAs for splicing, nuclear export and translation.
Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure.
5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G2(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C).
Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies.
Preconditioning of the myocardium rapidly induces a number of transcription factors, which are likely to be responsible for a cascade of transcriptional changes underlying the development of delayed adaptation. Identifying these changes provides insight into the molecular pathways elicited by sub-lethal ischaemia and the mechanism leading to delayed adaptation. Genes up-regulated in rabbit myocardium in vivo by ischaemic preconditioning following reperfusion for 2 h, 4 h and 6 h post-treatment were identified by representational difference analysis of cDNA (cDNA. RDA). The area of the left ventricle rendered ischaemic by preconditioning or the equivalent area of sham-treated animals was isolated and cDNA.RDA performed. Three novel genes and six genes with known function where identified, including the TGFbeta receptor interacting protein 1, the alpha isoform of the A subunit of PP2 and the cap binding protein NCBP1. To determine whether expression of these genes correlated with preconditioning per se, expression was measured in myocardium after both ischaemic as well as heat shock induced preconditioning following 2 h, 4 h, and 6 h reperfusion. These genes were induced in rabbit myocardium in vivo by both ischaemia and heat shock, consistent with a fundamental role in the development of delayed adaptation. The well described role of PP2 in modulating the mitogen-activated protein kinase pathway and promoting cell survival is consistent with our previous work, which identified the reperfusion injury salvage kinase pathway in mediating the protective effects of ischaemic preconditioning. Expression of Trip1 and Ncbp1 also implicates TGFbeta signalling pathways and RNA processing and transport in delayed adaptation to stress in the myocardium.
We report the case of a 40-year-old woman who recovered from a diffuse metastatic renal cell carcinoma that developed from a kidney allograft. She was successfully treated by the induction of tumor rejection. Immunosuppression was discontinued, and transplant nephrectomy was deliberately delayed based on the expectation that the tumor mass would trigger the alloimmune response, which was stimulated with pegylated interferon-α-2a. Three years later, the patient remained in complete remission. Despite this severe context, the present case shows that the poor prognosis of allograft metastatic renal cell carcinoma could be dramatically reversed by taking advantage of the donor tumor origin to actively induce a specific alloimmune rejection of the tumor. This article is protected by copyright. All rights reserved.
Neonatal infection can sensitize the adult substantia nigra (SN) to secondary insults, causing a decrease in antioxidant capacity which may lead to Parkinson's disease in adults. We studied the prolonged effect of systemic infection by (i.p.) administration of lipopolysaccharide (LPS) on interleukin (IL)-1β, the antioxidant regulator nuclear factor-erythroid 2-related factor 2 (Nrf2), and the peroxisome proliferator-activated receptor γ coactivator (PGC)-1α in rat SN.
Five-day-old rat pups were treated with LPS (i.p. 2 mg/kg). After 65 days, the mRNA level of IL-1β was significantly increased, in parallel with a decrease in that of the rate-limiting enzyme in glutathione synthesis, the γ-glutamylcysteine ligase catalytic subunit (γGCLc), Nrf2, and brain-derived neurotrophic factor (BDNF). Protein levels of γGCLc and Nrf2 were decreased while IL-1β protein was significantly increased. These LPS-induced long-term changes correlated with a decrease in phosphorylated (active) AKT (pAKT) and phosphorylated (inactive) GSK-3β (pGSK-3β). In another set of experiments, a 0.1% Spirulina-containing diet was given to lactating mothers 24 h before the LPS treatment of the pups. The Spirulina-supplemented diet decreased IL-1β protein expression in SN and elevated the mRNA level of γGCLc, Nrf2 protein, PGC-1α protein, and pAKT.
Early-life infection can negatively affect Nrf2, pAKT, and pGSK-3β for a long time in SN. A diet enriched with antioxidant and anti-inflammatory phytochemicals can partly restore some, but not all, of the effects on the antioxidant defense, possibly via normalizing effects on pAKT.
Thorium is natural actinide metal with potential use in nuclear energetics. Contamination by thorium, originated from mining activities or spills, represents environmental risk due to its radioactivity and chemical toxicity. A promising approach for cleaning of contaminated areas is phytoremediation, which need to be based, however, on detail understanding of the thorium effects on plants. In this study we investigated transcriptomic response of tobacco roots exposed to 200μM thorium for one week. Thorium application resulted in up-regulation of 152 and down-regulation of 100 genes (p-value <0.01, fold change ≥2). The stimulated genes were involved in components of jasmonic acid and salicylic acid signaling pathways and various abiotic (e.g. oxidative stress) and biotic stress (e.g. pathogens, wounding) responsive genes. Further, up-regulation of phosphate starvation genes and down-regulation of genes involved in phytic acid biosynthesis indicated that thorium disturbed phosphate uptake or signaling. Also expression of iron responsive genes was influenced. Negative regulation of several aquaporins indicated disturbance of water homeostasis. Genes potentially involved in thorium transport could be zinc-induced facilitator ZIF2, plant cadmium resistance PCR2, and ABC transporter ABCG40. This study provides the first insight at the processes in plants exposed to thorium.
Colorectal cancer constitutes 33% of all cancer morbidity, so the research of the new methods for colorectal cancer diagnosis and chemotherapy monitoring is gaining its momentum. Diagnostic instruments are being sought, which enable the detection of single malignant cells based on the analysis of tissue material potentially reusable at further stages of diagnostic management. The most common approach to tissue specimen processing is paraffin-embedding. Yet, paraffin may cause background noise in spectroscopic measurements with the wavenumber ranging between 900cm(-1) and 3500cm(-1). However, the study by Depciuch et al. (2016) proved that appropriate specimen processing and paraffin-embedding technique as well as a strict measurement methodology may eliminate paraffin vibrations. As a result, spectroscopic measurements may become a reliable and precise method for the diagnosis and treatment monitoring in patients with colorectal cancer as long as the high standards of specimen processing are maintained. Chemotherapy is the main medical treatment in colorectal cancer. Unfortunately, the absence of tools which enable monitoring its efficacy leads to the partial response or non-response frequently seen in affected patients. Hence, diagnostic instruments are also being sought capable of monitoring treatment efficacy so as to enable early changes of chemotherapy regimen thus increasing the chance of cure. The paper aims at comparing the results of FTIR (Fourier Transform Infrared) spectroscopy in several types of colon tissue: healthy colon, cancerous colon, post-chemotherapy colon and healthy surgical margin of colon cancer sample. The obtained FTIR spectra along with the Principal Component Analysis-Linear Discriminant Analysis (PCA-LDC) as well as bandwidth analysis of the primary amide region revealed some differences between the spectra of healthy tissues as compared to cancerous tissues (pre- or post-chemotherapy). Apart from confirming that FTIR spectroscopy is a good source of information on the composition of analysed samples, this fact supports its application as a tool to facilitate understanding the pathophysiology of various conditions and to monitor efficacy of chemotherapy in cancer patients.
High percentage of human cervical malignancy is related to human papillomavirus (HPV) infections. Thus, it is important to find novel non-invasive treatment strategies among various therapeutic HPV vaccines. In current study, we investigated the protective and therapeutic effects of DNA- and protein-based vaccines using HPV16 E7 as a model antigen in tumor mice model. In this line, the full length of high-mobility group box 1 (HMGB1) protein as well as an HMGB1-derived short peptide (Hp91) was used as an adjuvant for stimulating adaptive immunity and developing the potency of these vaccines.
DNA vaccination of HPV16 E7 with HMGB1 was performed as the complexed and conjugated forms. The immunostimulatory properties of Hp91 peptide along with Hp121 control peptide were compared to Montanide 720 in protein vaccination.
Our data showed that co-immunization of HPV16 E7 protein with Hp91 peptide or Hp91+Hp121 peptides significantly increased the secretion of IFN-γ, IgG2a antibody response, and protected 100% of mice against a TC-1 tumor challenge. Furthermore, the linkage of HMGB1 with E7 antigen led to enhance the immunogenicity of DNA vaccine especially in combination with Hp91 and Hp121 peptides.
These findings suggest that Hp91 peptide, and the full length of HMGB1 gene could be an efficient adjuvant for improvement of therapeutic HPV protein- and DNA-based vaccines, respectively.
DFT/B3LYP calculations were employed to study the effects of tautomerism and explicit/implicit water molecules on Nuclear Quadrupole Resonance (NQR) and Nuclear Magnetic Resonance (NMR) tensors of nitrogen nuclei in tetrazole-5-thione structure. The obtained results revealed that nuclear quadrupole coupling constant (χ) and isotropic chemical shielding (σiso ) values of nitrogen nuclei in tetrazole ring of five possible tautomeric forms of tetrazole-5-thione, i.e. two thione forms called tautomers A and E and three thiol forms called tautomers B, C, and D, were functions of resonance energy(E2 ) values of nitrogen lone pairs. Furthermore, it was observed that by increasing participation of lone pairs of nitrogen atoms in the ring resonance interactions, the σiso values around them were increased; while their χ and qzz values were decreased. However, the results indicated that with exception of tautomer B, the order of qzz and χ values of nitrogen nuclei in tetrazole ring was exactly opposite of the order of resonance energy values for the same nitrogen nuclei in all tautomers and their mono-hydrated complexes. In addition, a significant decrease was noticed in χ and qzz values when a water molecule was put in different positions near the tetrazole ring in tautomers A-E. The mentioned result can be attributed to hydrogen bond formation between nitrogen nuclei and the oxygen of water. In mono-hydrated complexes, the σiso values around nitrogen atoms acting as hydrogen donors in hydrogen bond formation (N-H….OH2 ) were decreased, while its values were increased for nitrogen atoms acting as hydrogen acceptors in hydrogen bond formation(N….H-OH).
Although multiple serum antiretinal autoantibodies (ARAs) have been reported in patients with paraneoplastic and non-paraneoplastic autoimmune retinopathy ((n)pAIR), not all retinal antigens involved in (n)pAIR are specified. This study aims to serologically identify patients with presumed (n)pAIR through determination of both known and unknown ARAs by autoantibody profiling.
An antigen suspension bead array using 188 different antigens representing 97 ocular proteins was performed to detect ARAs in serum samples of patients with presumed (n)pAIR (n = 24), uveitis (n = 151) and cataract (n = 21). Logistic regressions were used to estimate the associations between ocular antigens and diagnosis. Validation of interphotoreceptor matrix proteoglycan 2 (IMPG2) and recoverin antigens was performed by immunohistochemistry and immunoblot, respectively.
Samples of patients with presumed (n)pAIR exhibited a broad spectrum of ARAs. We identified retinal antigens that have already been described previously (e.g. recoverin), but also identified novel ARA targets. Most ARAs were not specific for (n)pAIR since their presence was also observed in patients with cataract or uveitis. High titers of autoantibodies directed against photoreceptor-specific nuclear receptor and retinol-binding protein 3 were more common in patients with presumed (n)pAIR compared to uveitis (p = 0.015 and p = 0.018, respectively). The presence of all other ARAs did not significantly differ between groups. In patients with presumed (n)pAIR, anti-recoverin autoantibodies were the most prevalent ARAs. Validation of bead array results by immunohistochemistry (anti-IMPG2) and immunoblot (anti-recoverin) showed concordant results in (n)pAIR patients.
Patients with (n)pAIR are characterized by the presence of a broad spectrum of ARAs. The diagnosis of (n)pAIR cannot be based on the mere presence of serum ARAs, as these are also commonly present in uveitis as well as in age-related cataract patients.
All obese women are categorised as being of equally high risk of gestational diabetes (GDM) whereas the majority do not develop the disorder. Lifestyle and pharmacological interventions in unselected obese pregnant women have been unsuccessful in preventing GDM. Our aim was to develop a prediction tool for early identification of obese women at high risk of GDM to facilitate targeted interventions in those most likely to benefit. Clinical and anthropometric data and non-fasting blood samples were obtained at 15+0-18+6 weeks' gestation in 1303 obese pregnant women from UPBEAT, a randomised controlled trial of a behavioural intervention. Twenty one candidate biomarkers associated with insulin resistance, and a targeted nuclear magnetic resonance (NMR) metabolome were measured. Prediction models were constructed using stepwise logistic regression. Twenty six percent of women (n = 337) developed GDM (International Association of Diabetes and Pregnancy Study Groups criteria). A model based on clinical and anthropometric variables (age, previous GDM, family history of type 2 diabetes, systolic blood pressure, sum of skinfold thicknesses, waist:height and neck:thigh ratios) provided an area under the curve of 0.71 (95%CI 0.68-0.74). This increased to 0.77 (95%CI 0.73-0.80) with addition of candidate biomarkers (random glucose, haemoglobin A1c (HbA1c), fructosamine, adiponectin, sex hormone binding globulin, triglycerides), but was not improved by addition of NMR metabolites (0.77; 95%CI 0.74-0.81). Clinically translatable models for GDM prediction including readily measurable variables e.g. mid-arm circumference, age, systolic blood pressure, HbA1c and adiponectin are described. Using a ≥35% risk threshold, all models identified a group of high risk obese women of whom approximately 50% (positive predictive value) later developed GDM, with a negative predictive value of 80%. Tools for early pregnancy identification of obese women at risk of GDM are described which could enable targeted interventions for GDM prevention in women who will benefit the most.
Allogeneic transplantation (Tx) of induced pluripotent stem cells (iPSCs) is a promising tissue regeneration therapy. However, this inevitably induces macrophage-mediated immune response against the graft, limiting its therapeutic efficacy. Monitoring the magnitude of the immune response using imaging tools would be useful for prolonging graft survival and increasing the therapy longevity. Minimally invasive quantitative detection of activated macrophages by medical imaging technologies such as positron emission tomography (PET) imaging targets translocator protein (TSPO), which is highly expressed on mitochondrial membrane, especially in activated macrophage. N,N-diethyl-2-[4-(2-fluoroethoxy) phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide (DPA-714) is known as a TSPO ligand used in clinical settings. We herein hypothesized that immune rejection of the transplanted iPSC-derived cardiomyocytes (iPSC-CMs) of allogeneic origin may be quantitated using 18F-DPA-714-PET imaging study. iPSC-CM cell-sheets of C57BL/6 mice origin were transplanted on the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (syngeneic Tx group), or Balb/c mice as an allogeneic model (allogeneic Tx group). 18F-DPA-714-PET was used to determine the uptake ratio, calculated as the maximum standardized uptake value in the anterior and septal wall of the LV. The uptake ratio was significantly higher in the allogeneic Tx group than in the syngeneic group or the sham group at days 7 and day 10 after the cell transplantation. In addition, the immunochemistry showed significant presence of CD68 and CD3-positive cells at day 7 and 10 in the transplanted graft of the allogeneic Tx group. The expression of TSPO, CD68, IL-1 beta, and MCP-1 was significantly higher in the allogeneic Tx group than in the syngeneic Tx and the sham groups at day 7. The 18F-DPA-714-PET imaging study enabled quantitative visualization of the macrophages-mediated immune rejection of the allogeneic iPSC-cardiac. This imaging tool may enable the understanding and monitoring host-immune response of the host, allogeneic cell transplantation therapy.
BACKGROUND Traumatic brain injury (TBI) induces edema on the uninjured side (i.e., contralateral brain tissue; CBT). We evaluated the role of AQP4 in CBT edema formation following TBI. MATERIAL AND METHODS Mild or severe TBI was induced using a controlled cortical impact model in rats, immediately followed by intraventricular siRNA infusions. The effects of AQP4 siRNA on CBT edema were assessed at up to 168 h. RESULTS Mild or severe TBI induced different patterns of CBT edema. Furthermore, following mild TBI, brain water content (BWC) was increased at 72 h thereafter and AQP4 expression was increased after 168 h, relative to non-injured rats (i.e., sham). AQP4 interference reduced AQP4 expression 48 h thereafter and BWC 72 h thereafter, relative to control siRNA. In contrast, following severe TBI, BWC was increased 1 h thereafter and AQP4 expression was transiently enhanced after 1 h, relative to sham. However, AQP4 interference reduced AQP4 expression after 1 h and BWC 24 h thereafter, relative to control siRNA. Finally, apparent diffusion coefficient (ADC) value in CBT was positively correlated with AQP4 expression level following severe, but not mild, TBI. AQP4 interference disrupted this correlation. CONCLUSIONS AQP4 interference reduces CBT edema formation, and ADC value may predict TBI severity.
To compare diagnostic performance and confidence of a standard visual reading and combined 3-dimensional stereotactic surface projection (3D-SSP) results to discriminate between Alzheimer disease (AD)/mild cognitive impairment (MCI), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD).[F]fluorodeoxyglucose (FDG) PET brain images were obtained from 120 patients (64 AD/MCI, 38 DLB, and 18 FTD) who were clinically confirmed over 2 years follow-up. Three nuclear medicine physicians performed the diagnosis and rated diagnostic confidence twice; once by standard visual methods, and once by adding of 3D-SSP. Diagnostic performance and confidence were compared between the 2 methods.3D-SSP showed higher sensitivity, specificity, accuracy, positive, and negative predictive values to discriminate different types of dementia compared with the visual method alone, except for AD/MCI specificity and FTD sensitivity. Correction of misdiagnosis after adding 3D-SSP images was greatest for AD/MCI (56%), followed by DLB (13%) and FTD (11%). Diagnostic confidence also increased in DLB (visual: 3.2; 3D-SSP: 4.1; P < 0.001), followed by AD/MCI (visual: 3.1; 3D-SSP: 3.8; P = 0.002) and FTD (visual: 3.5; 3D-SSP: 4.2; P = 0.022). Overall, 154/360 (43%) cases had a corrected misdiagnosis or improved diagnostic confidence for the correct diagnosis.The addition of 3D-SSP images to visual analysis helped to discriminate different types of dementia in FDG PET scans, by correcting misdiagnoses and enhancing diagnostic confidence in the correct diagnosis. Improvement of diagnostic accuracy and confidence by 3D-SSP images might help to determine the cause of dementia and appropriate treatment.
Granulocytic sarcoma (GS) is defined as leukemia infiltration in any organ other than the bone marrow. GS rarely occurs in the pancreas. Here, we present the first report of GS in the pancreas on F-fluorodexyglucose positron emission tomography/computed tomography (F-FDG PET/CT).
A 19-year-old male patient with acute myeloid leukemia received a human leukocyte antigen-haploidentical stem cell transplant as a second transplant while in second complete remission.
After a second stem cell transplant, obstructive pancreatitis accompanied by a mass in the pancreatic head was observed. FDG-PET/CT revealed abnormal activity in the head of the pancreas and the skin in the patient's left breast area.
Pathological examination demonstrated relapsed acute myeloid leukemia in both the lesions.
This is the first report showing the F-FDG PET/CT findings of GS in the pancreas.
F-FDG PET/CT may help determine the stage of GS.
The differential subcellular localizations of β-catenin (including membrane, cytoplasm, and nucleus) play different roles in the progression of colorectal cancer (CRC). However, the correlation between each subcellular localization of β-catenin and the prognosis of CRC patients remains undetermined.
Systematic strategies were applied to search for eligible published studies in the PubMed, Embase, and Web of Science databases. The correlation between each subcellular localizations of β-catenin expression and patients' clinicopathological features or prognosis was analyzed.
Finally, this meta-analysis, including 6238 cases from 34 studies, revealed that β-catenin overexpression in the nucleus (HR: 1.50[95% CI: 1.08-2.10]) or reduced expression of β-catenin in the membrane (HR: 1.33[95% CI: 1.15-1.54]) significantly correlated with lower 5-year overall survival (OS). Conversely, overexpression of β-catenin in the cytoplasm (HR: 1.00[95% CI: 0.85-1.18]) did not show significant association with 5-year OS.
This study suggested that β-catenin overexpression in the nucleus or reduced expression in the membrane, but not its overexpression in cytoplasm, could serve as a valuable prognostic predictor for CRC. However, additional large and well-designed prospective studies are required to verify our results.
Gastric cancer (GC) ranks as the fourth most frequent in incidence and second in mortality among all cancers worldwide. The development of effective treatment approaches is an urgent requirement. Growth hormone-releasing hormone (GHRH) and GHRH receptor (GHRH-R) have been found to be present in a variety of tumoral tissues and cell lines. Therefore the inhibition of GHRH-R was proposed as a promising approach for the treatment of these cancers. However, little is known about GHRH-R and the relevant therapy in human GC. By survival analyses of multiple cohorts of GC patients, we identified that increased GHRH-R in tumor specimens correlates with poor survival and is an independent predictor of patient prognosis. We next showed that MIA-602, a highly potent GHRH-R antagonist, effectively inhibited GC growth in cultured cells. Further, this inhibitory effect was verified in multiple models of human GC cell lines xenografted into nude mice. Mechanistically, GHRH-R antagonists target GHRH-R and down-regulate the p21-activated kinase 1 (PAK1)-mediated signal transducer and activator of transcription 3 (STAT3)/nuclear factor-κB (NF-κB) inflammatory pathway. Overall, our studies establish GHRH-R as a potential molecular target in human GC and suggest treatment with GHRH-R antagonist as a promising therapeutic intervention for this cancer.
Quantum tunneling is a ubiquitous phenomenon in nature and crucial for many technological applications. It allows quantum particles to reach regions in space which are energetically not accessible according to classical mechanics. In this "tunneling region," the particle density is known to decay exponentially. This behavior is universal across all energy scales from nuclear physics to chemistry and solid state systems. Although typically only a small fraction of a particle wavefunction extends into the tunneling region, we present here an extreme quantum system: a gigantic molecule consisting of two helium atoms, with an 80% probability that its two nuclei will be found in this classical forbidden region. This circumstance allows us to directly image the exponentially decaying density of a tunneling particle, which we achieved for over two orders of magnitude. Imaging a tunneling particle shows one of the few features of our world that is truly universal: the probability to find one of the constituents of bound matter far away is never zero but decreases exponentially. The results were obtained by Coulomb explosion imaging using a free electron laser and furthermore yielded He2's binding energy of [Formula: see text] neV, which is in agreement with most recent calculations.
Sex determination is a fundamental developmental pathway governing male and female differentiation, with profound implications for morphology, reproductive strategies, and behavior. In animals, sex differences between males and females are generally determined by genetic factors carried by sex chromosomes. Sex chromosomes are remarkably variable in origin and can differ even between closely related species, indicating that transitions occur frequently and independently in different groups of organisms. The evolutionary causes underlying sex chromosome turnover are poorly understood, however. Here we provide evidence indicating that Wolbachia bacterial endosymbionts triggered the evolution of new sex chromosomes in the common pillbug Armadillidium vulgare We identified a 3-Mb insert of a feminizing Wolbachia genome that was recently transferred into the pillbug nuclear genome. The Wolbachia insert shows perfect linkage to the female sex, occurs in a male genetic background (i.e., lacking the ancestral W female sex chromosome), and is hemizygous. Our results support the conclusion that the Wolbachia insert is now acting as a female sex-determining region in pillbugs, and that the chromosome carrying the insert is a new W sex chromosome. Thus, bacteria-to-animal horizontal genome transfer represents a remarkable mechanism underpinning the birth of sex chromosomes. We conclude that sex ratio distorters, such as Wolbachia endosymbionts, can be powerful agents of evolutionary transitions in sex determination systems in animals.
Nuclear lamins are the major components of the nuclear lamina at the periphery of the nucleus, supporting the nuclear envelope and participating in many nuclear processes, including DNA replication, transcription and chromatin organization. A group of diseases, the laminopathies, is associated with mutations in lamin genes. One of the most striking cases is Hutchinson-Gilford progeria syndrome (HGPS) which is the consequence of a lamin A dominant negative mutant named progerin. Due to the abnormal presence of a permanent C-terminal farnesyl tail, progerin gradually accumulates on the nuclear membrane, perturbing a diversity of signalings and transcriptional events. The accumulation of progerin has led to the speculation that progerin possesses higher stability than the wild type lamin A protein. However, the low solubility of lamin proteins renders traditional immunoprecipitation-dependent methods such as pulse-chase analysis ineffective for comparing the relative stabilities of mutant and wild type lamins. Here, we employ a novel platform for inferring differences in lamin stability, which is based on normalization to a co-translated reporter protein following porcine teschovirus-1 2A peptide-mediated co-translational cleavage. The results obtained using this method support the notion that progerin is more stable than lamin A. Moreover, treatment of FTI reduces progerin relative stability to the level of wild type lamin A.
Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the micro-symbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Ys were shown previously to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism(s) by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH (SHI/STY) transcription factor gene family, called STY1, STY2 and STY3, are demonstrated to be among likely direct targets of LjNF-YA1 and our results point to their involvement in nodule formation.
This study evaluates the implementation of volumetric-modulated arc therapy (VMAT) using multicriteria optimization (MCO) in the RayStation treatment planning system (TPS) for complex sites, namely extremity and body sarcoma. The VMAT-MCO algorithm implemented in RayStation is newly developed and requires an integrated, comprehensive analysis of plan generation, delivery, and treatment efficiency. Ten patients previously treated by intensity-modulated radiation therapy (IMRT) with MCO were randomly selected and replanned using VMAT-MCO. The plan quality was compared using homogeneity index (HI) and conformity index (CI) of the planning target volume (PTV) and dose sparing of organs at risk (OARs). Given the diversity of the tumor location, the 10 plans did not have a common OAR except for skin. The skin D50 and Dmean was directly compared between VMAT-MCO and IMRT-MCO. Additional OAR dose points were compared on a plan-by-plan basis. The treatment efficiency was compared using plan monitor units (MU) and net beam-on time. Plan quality assurance was performed using the Sun Nuclear ArcCHECK phantom and a gamma criteria of 3%/3 mm. No statistically significant differences were found between VMAT- and IMRT-MCO for HI and CI of the PTV or D50 and Dmean to the skin. The VMAT-MCO plans showed general improvements in sparing to OARs. The VMAT-MCO plan set showed statistically significant improvements over the IMRT-MCO set in treatment efficiency per plan MU (p &lt; 0.05) and net beam-on time (p &lt; 0.01). The VMAT-MCO plan deliverability was validated. Similar gamma passing rates were observed for the two modalities. This study verifies the suitability of VMAT-MCO for sarcoma cancer and highlighted the comparability in plan quality and improve-ment in treatment efficiency offered by VMAT-MCO as compared to IMRT-MCO.
Skeletal uptake of radiolabeled-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetramethylene phosphoric acid (e.g., 177Lu-DOTMP) complex, is used for bone pain palliation. The moderate energy of β-emitting 177Lu (T½ = 6.7 d, Eβmax = 497keV) has been considered as a potential radionuclide for development of the bone-seeking radiopharmaceutical. Since the specific activity of the radiolabeled carrier molecules should be high, the "no-carrier-added radionuclides" have sig-nificant roles in nuclear medicine. Many researchers illustrated no-carrier-added 177Lu production; among these separation techniques such as ion exchange chromatography, reversed phase ion-pair, and electrochemical method, extraction chromatography has been considered more capable than other methods. In order to optimize the conditions, some effective factors on separation of Lu/Yb were investigated by EXC. The NCA 177Lu, produced by this method, was mixed with 300 μl of DOTMP solution (20 mg in 1 mL of 0.5 M NaHCO3, pH = 8) and incu-bated under stirring at room temperature for 45 min. Radiochemical purity of the 177Lu-DOTMP complex was determined using radio-thin-layer chromatography (RTLC) method. The complex was injected to wild-type rats and biodistribution was then studied for seven days. The NCA 177Lu was produced with specific activ-ity of 48 Ci/mg and with a radinuclidic purity of 99.99% through irradiation of enriched 176Yb target (1 mg) in a thermal neutron flux of 4 × 1013 n.cm-2.s-1 for 14 days. 177Lu-DOTMP was obtained with high radiochemical purities (&gt; 98%) under optimized reaction conditions. The radiolabeled complex exhibited excellent stability at room temperature. Biodistribution of the radiolabeled complex studies in rats showed favorable selective skeletal uptake with rapid clearance from blood along with insignificant accumulation within the other nontargeted organs.
Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).
Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.
The assembly of the ribosome on majority of eukaryotic mRNAs is initiated by the recruitment of eIF4E protein to the mRNA 5' end cap structure. Flowering plants use two eIF4E isoforms, named eIF4E and eIF(iso)4E, as canonical translation initiation factors and possess a homolog of mammalian 4EHP (or eIF4E-2) termed nCBP. Plants from Brassicaceae family additionally conserve a close paralog of eIF4E which in Arabidopsis thaliana has two copies named eIF4E1b and eIF4E1c. In order to assess the efficiency of plant non-canonical (eIF4E1b/1c and nCBP) and canonical (eIF4E and eIF(iso)4E) eIF4E proteins to bind mRNAs we utilized fluorescence titrations to determine accurate binding affinities of five A.thaliana eIF4E isoforms for a series of cap analogs. We found that eIF4E binds cap analogs from 4-fold to 10-fold stronger than eIF(iso)4E, while binding affinities of nCBP and eIF(iso)4E are comparable. Furthermore, eIF4E1c interacts similarly strongly with the cap as eIF4E, but eIF4E1b binds cap analogs ca. 2-fold weaker than eIF4E1c, regardless of the 95% sequence identity between these two proteins. The use of differentially chemically modified cap analogs in binding studies and a detailed analysis of the obtained homology models gave us insight into the molecular characteristic of varying cap-binding abilities of Arabidopsis eIF4E isoforms.
Vascular calcification significantly increases the rates of cardiovascular mortality in hemodialysis (HD) patients. Abnormalities in mineral metabolism may play a role in the pathogenesis of arterial calcification. Whether patients treated with non-calcium-based phosphate binders had reduced aortic vascular calcification compared to those treated with calcium-based phosphate binders is still unclear.
We searched multiple databases for studies published through August 2013 that evaluated the effects of non-calcium-based phosphate binders (NCBP) versus calcium-based phosphate binders (CBP) on cardiovascular calcification and bone remodeling among dialysis patients. We summarized test performance characteristics with the use of forest plots, fixed and random effects models, and Egger regression test.
Eighteen eligible randomized controlled trials totaling 3676 patients were included. Meta-analysis results showed NCBP could significantly attenuate the progression of coronary artery calcification than CBP (WMD: -144.62, 95% CI: -285.62 to -3.63). The serum calcium levels significant lower in NCPB group than in CPB groups (WMD: -0.26, 95% CI: -0.37 to -0.14), but the serum iPTH levels were significantly higher in NCPB groups (WMD: 57.1, 95% CI: 13.42 to 100.78). The osteoid volume and osteoblast numbers were significant higher in NCPB group than in CPB group (WMD: 1.75, 95% CI: 0.78 to 2.73 for osteoid volume; WMD: 4.49, 95% CI: 1.83 to 7.15 for osteoblast numbers). The Egger regression test also showed no potential publication bias (p = 0.725).
Based on available data, NCBPs have equally effective with CBPs for serum phosphate control. But there was significantly lower incidence of coronary artery calcification and a significant higher bone formatting rate in NCBP groups than in CBP groups. So we recommend NCBPs as phosphate binders for HD patients.
To determine if bisphosphonates are associated with reduced risk of acute myocardial infarction (AMI).
A cohort of 14,256 veterans 65 years or older with femoral or vertebral fractures was selected from national administrative databases operated by the US Department of Veterans Affairs and was derived from encounters at Veterans Affairs facilities between October 1, 1998, and September 30, 2006. The time to first AMI was assessed in relationship to bisphosphonate exposure as determined by records from the Pharmacy Benefits Management Database. Time to event analysis was performed using multivariate Cox proportional hazards regression. An adjusted survival analysis curve and a Kaplan-Meier survival curve were analyzed.
After controlling for atherosclerotic cardiovascular disease risk factors and medications, bisphosphonate use was associated with an increased risk of incident AMI (hazard ratio, 1.38; 95% CI, 1.08-1.77; P=.01). The timing of AMI correlated closely with the timing of bisphosphonate therapy initiation.
Our observations in this study conflict with our hypothesis that bisphosphonates have antiatherogenic effects. These findings may alter the risk-benefit ratio of bisphosphonate use for treatment of osteoporosis, especially in elderly men. However, further analysis and confirmation of these findings by prospective clinical trials is required.
PUF family proteins are well-conserved regulators of cell proliferation in different developmental processes. They regulate target mRNAs by promoting degradation or by influencing translation through interaction with the translation initiation machinery. Here we show that Caenorhabditis elegans PUF-8 functions redundantly with the nuclear protein TCER-1 in the post-transcriptional maintenance of at least six germline mRNAs. The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type. These two proteins colocalise at the inner nuclear periphery, and their absence leads to reduced germ cell proliferation and to sterility. A yeast two-hybrid screen of 31 components of the nuclear pore complex and mRNA processing machineries identified seven proteins involved in mRNA export as potential partners of PUF-8. One of these, the nuclear cap-binding protein NCBP-2, colocalises with PUF-8 in the nucleus. A 50 amino acid N-terminal domain of PUF-8 is essential for interaction with NCBP-2 and for PUF-8 to function redundantly with TCER-1. These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance. We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.
Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity.
Obtaining an accurate blood pressure (BP) reading is vital for diagnosing hypertension. However, BP measures taken in the physician's clinic (CBP) are subject to the 'white coat' bias. Measurements taken outside the office using ambulatory (ABP) and home (HBP) monitoring are superior predictors of cardiovascular diseases compared with CBP, but ABP remains underutilized because of the effort and expense involved. Unfortunately, HBP has limitations, including questionable device validity and patient compliance. Thus, it is important to identify feasible alternative techniques to measure BP in the office that will increase the accuracy of the diagnosis.
Auscultatory BP was measured in 249 patients in a nonclinical setting by trained technicians (NCBP); on the following day, patients were taken to their physician (CBP). They were also given an HBP monitor, and a 36 h ABP monitoring. Because ABP is considered the gold standard for prediction of cardiovascular disease, these readings were used as the criterion in a statistical model in which CBP, HBP, and NCBP were entered as predictors. The level of agreement between measurements was estimated.
Multiple regression analysis showed that HBP and NCBP (P < 0.001) explained 94 and 87% of the variance in systolic and diastolic ABP, respectively. The agreement between NCBP and ABP was greater than that between CBP and ABP or between HBP.
When ABP monitoring and HBP monitoring are not options, the NCBP at the clinic can avoid the white coat bias and therefore improve diagnosis.
Although pylorus-preserving gastrectomy (PPG) is performed as a function preserving surgery, patients sometimes suffer from postoperative stasis of the gastric contents after the procedure. Preservation of blood flow and both celiac and pyloric branches of the vagal nerve may decrease the incidence of the stasis.
Patients who underwent PPG at the Cancer Institute Hospital between April 2005 and December 2007 were included in the study. Early surgical results and the incidence of stasis were investigated and compared between patients in whom the celiac branch had been preserved (PPG-CBP group) and those in whom it had not (PPG-nCBP group).
Patients in the PPG-nCBP group had a higher body mass index than patients in the PPG-CBP group (23.9 vs. 22.3 kg/m2; p=0.004), as well as a longer operation time (249.3 vs. 227.0 min; p=0.010). There was no significant difference in the incidence of stasis between the groups (13% vs. 8%; p=0.523).
There was no correlation between preservation of the celiac branch and the incidence of stasis in the first 30 days after PPG. A large randomized control trial investigating both short- and long-term outcomes after PPG is required to elucidate the primary cause of postoperative stasis.
the aim of this study was to determine whether nitrogen-containing bisphosphonate (NCBP) therapy is associated with the prevalence of cardiovascular calcification.
cardiovascular calcification correlates with atherosclerotic disease burden. Experimental data suggest that NCBP might limit cardiovascular calcification, which has implications for disease prevention.
the relationship of NCBP use to the prevalence of aortic valve, aortic valve ring, mitral annulus, thoracic aorta, and coronary artery calcification (AVC, AVRC, MAC, TAC, and CAC, respectively) detected by computed tomography was assessed in 3,710 women within the MESA (Multi-Ethnic Study of Atherosclerosis) with regression modeling.
Analyses were age-stratified, because of a significant interaction between age and NCBP use (interaction p values: AVC p < 0.0001; AVRC p < 0.0001; MAC p = 0.002; TAC p < 0.0001; CAC p = 0.046). After adjusting for age; body mass index; demographic data; diabetes; smoking; blood pressure; cholesterol levels; and statin, hormone replacement, and renin-angiotensin inhibitor therapy, NCBP use was associated with a lower prevalence of cardiovascular calcification in women ≥ 65 years of age (prevalence ratio: AVC 0.68 [95% confidence interval (CI): 0.41 to 1.13]; AVRC 0.65 [95% CI: 0.51 to 0.84]; MAC 0.54 [95% CI: 0.33 to 0.93]; TAC 0.69 [95% CI: 0.54 to 0.88]; CAC 0.89 [95% CI: 0.78 to 1.02]), whereas calcification was more prevalent in NCBP users among the 2,181 women <65 years of age (AVC 4.00 [95% CI: 2.33 to 6.89]; AVRC 1.92 [95% CI: 1.42 to 2.61]; MAC 2.35 [95% CI: 1.12 to 4.84]; TAC 2.17 [95% CI: 1.49 to 3.15]; CAC 1.23 [95% CI: 0.97 to 1.57]).
among women in the diverse MESA cohort, NCBPs were associated with decreased prevalence of cardiovascular calcification in older subjects but more prevalent cardiovascular calcification in younger ones. Further study is warranted to clarify these age-dependent NCBP effects.
To explore the relation between genetic polymorphisms in XPD and risks of chronic benzene poisoning (CBP).
A case-control study was conducted. 152 CBP patients and 152 NCBP workers occupationally exposed to benzene were investigated. Polymerase chain reaction-restrained fragment length polymorphism technique (PCR-RFLP) was applied to detect the single nucleotide polymorphisms (SNPs) at c. 199, c. 201, c. 312 and c. 751 of XPD gene.
No variant alleles was detected at c. 199 and c. 201 of XPD gene. In comparition with the individual genotypes of XPDc. 312Asp/Asp, the risk of CBP suffered from the individual genotype of XPDc. 312Asp/Asn + Asn/Asn decreased a 0.59 fold (ORadj = 0.59, 95% CI = 0.35-0.99, chi2 = 3.99, P < 0.05), when sex, workage and intensity of benzene exposure were adjusted. And in low intensity of benzene exposure group, the risk of CBP suffered from the individual genotypes of XPDc. 312Asp/Asn + Asn/Asn more decreased (ORadj = 0.13, 95% CI = 0.04-0.51, chi2 = 8.93, P < 0.01).
Polymorphism of XPD Asp312Asn could contribute to altered risk of CBP.
Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris, four recessive genes, bc-1, bc-2, bc-3 and bc-u, have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus. In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E. This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F(2) population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3. F(2) plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F(2) plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F(3) plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.
MicroRNA (miRNA) biogenesis proceeds from a primary transcript (pri-miRNA) through the pre-miRNA into the mature miRNA. Here, we identify a role of the Caenorhabditis elegans nuclear export receptor XPO-1 and the cap-binding proteins CBP-20/NCBP-2 and CBP-80/NCBP-1 in this process. The RNA-mediated interference of any of these genes causes retarded heterochronic phenotypes similar to those observed for animals with mutations in the let-7 miRNA or core miRNA machinery genes. Moreover, pre- and mature miRNAs become depleted, whereas primary miRNA transcripts accumulate. An involvement of XPO-1 in miRNA biogenesis is conserved in Drosophila, in which knockdown of Embargoed/XPO-1 or its chemical inhibition through leptomycin B causes pri-miRNA accumulation. Our findings demonstrate that XPO-1/Emb promotes the pri-miRNA-to-pre-miRNA processing and we propose that this function involves intranuclear transport and/or nuclear export of primary miRNAs.
In rat experiments the ability of noncollagen bone proteins (NCBP) in the composition of osteoplactic modified material Gapkol (not tanned in formalin and subjected to vacuum extraction) to increase bone reparation in comparison with traditional Gapkol was studied. Quantitative evaluation was performed on rat parietal bone and qualitative evaluation was performed on rat mandible. It was shown that Gapkol with NCBP (not tanned in formalin and subjected to vacuum extraction) increased reparative osteogenesis.
Fish were collected in late 1995 from 34 National Contaminant Biomonitoring Program (NCBP) stations and 12 National Water Quality Assessment Program (NAWQA) stations in the Mississippi River basin (MRB), and in late 1996 from a reference site in West Virginia. The NCBP sites represented key points (dams, tributaries, etc.) in the largest rivers of the MRB. The NAWQA sites were typically on smaller rivers and were selected to represent dominant land uses in their watersheds. The West Virginia site, which is in an Eastern U.S. watershed adjacent to the MRB, was selected to document elemental concentrations in fish used for other aspects of a larger study and to provide additional contemporaneous data on background elemental concentrations. At each site four samples, each comprising (nominally) 10 adult common carp (Cyprinus carpio, 'carp') or black bass (Micropterus spp., 'bass') of the same sex, were collected. The whole fish were composited by station, species, and gender for analysis of arsenic (As), lead (Pb), and selenium (Se) by atomic absorption spectroscopy and for cadmium (Cd), copper (Cu), and zinc (Zn) by inductively-coupled plasma emission spectroscopy. Concentrations of most of the elements examined were lower in both carp and bass from the reference site, a small impoundment located in a rural area, than from the NCBP and NAWQA sites on rivers and larger impoundments. In contrast, there were few overall differences between NCBP sites NAWQA sites. The 1995 results generally confirmed the continued weathering and re-distribution of these elemental contaminants in the MRB; concentrations declined or were unchanged from 1984-1986 to 1995 at most NCBP sites, thus continuing two-decade trends. Exceptions were Se at Station 77 (Arkansas R. at John Martin Reservoir, CO), where concentrations have been elevated historically and increased slightly (to 3.8-4.7 microg g-(1) in bass and carp); and Pb, Cd, and Zn at Station 67 (Allegheny R. at Natrona, PA), where levels of these metals were high in the past and increased from 1986 to 1995.
Fish were collected in late 1995 from 34 National Contaminant Biomonitoring Program (NCBP) stations and 13 National Water Quality Assessment Program (NAWQA) stations in the Mississippi River basin (MRB) and in late 1996 from a reference site in West Virginia. Four composite samples, each comprising (nominally) 10 adult common carp (Cyprinus carpio) or black bass (Micropterus spp.) of the same sex, were collected from each site and analyzed for organochlorine chemical residues by gas chromatography with electron capture detection. At the NCBP stations, which are located on relatively large rivers, concentrations of organochlorine chemical residues were generally lower than when last sampled in the mid-1980s. Residues derived from DDT (primarily p,p'-DDE) were detected at all sites (including the reference site); however, only traces (<or= 0.02 microg/g) of the parent insecticide (p,p'-DDT) were present, which indicates continued weathering of residual DDT from past use. Nevertheless, concentrations of DDT (as p,p'-DDE) in fish from the cotton-farming regions of the lower MRB were great enough to constitute a hazard to fish-eating wildlife and were especially high at the NAWQA sites on the lower-order rivers and streams of the Mississippi embayment. Mirex was detected at only two sites, both in Louisiana, and toxaphene was found exclusively in the lower MRB. Most cyclodiene pesticides (dieldrin, chlordane, and heptachlor epoxide) were more widespread in their distributions, but concentrations were lower than in the 1980s except at a site on the Mississippi River near Memphis, TN. Concentrations were also somewhat elevated at sites in the Corn Belt. Endrin was detected exclusively at the Memphis site. PCB concentrations generally declined, and residues were detected (>or= 0.05 microg/g) at only 35% of the stations, mostly in the more industrialized parts of the MRB.
Calmodulin-like neuronal Ca2+-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca2+-binding motifs. The guanylate cyclase-activating proteins 1-3 (GCAP1-3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1-2 (GC1-2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class-specific residues for the NCBP subfamilies.
In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.
Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.
As part of the National Contaminant Biomonitoring Program (NCBP, formerly a component of the National Pesticide Monitoring Program), the U.S. Fish and Wildlife Service periodically determined concentrations of organochlorine chemical residues and elemental contaminants in freshwater fish collected from a nationwide network of stations. In late 1986 and early 1987, the last time the network was sampled, a total of 319 composite fish samples were collected from 97 NCBP stations. The samples were analyzed for residues of organochlorine chemicals and the elements As, Cd, Cu, Hg, Pb, Se, and Zn. The mean concentration of total DDT and its homologs (p,p'-constituents) declined from 1984 to 1986, thus continuing a trend that began in 1970. The most persistent DDT homolog (p,p'-DDE) was detected at all stations sampled in 1986, and averaged 74% of total DDT residues, up from 70% in 1974-1979 but essentially unchanged from 1984. Collectively, these findings indicated a low rate of influx and continued weathering of DDT in the environment; nevertheless, DDT concentrations in fish from some stations in the South remained high enough to constitute a threat to piscivorous wildlife. Residues of polychlorinated biphenyls (PCBs) also remained widespread, but a significant downward trend in total PCB concentrations and incidence was evident, and PCB mixtures containing early eluting components were present at fewer stations than in the past. PCB concentrations were generally highest in fish from the industrialized rivers of the Northeast and Midwest and from the Great Lakes. Concentrations of toxaphene also declined, as did its incidence, from 88% of the stations sampled in 1980-1981 to 64% in 1986; however, analytical problems with the 1986 samples may have contributed to the latter. The risks represented by PCBs and toxaphene could not be evaluated on the basis of our data. Among cyclodiene insecticides, dieldrin and chlordane-related residues were the most widespread. Mean concentrations of dieldrin declined through 1986, but remained consistently highest in the Great Lakes. For chlordane-related residues, mean concentrations were lower that reported previously, and trans-nonachlor continued to replace cis-chlordane as the most abundant component. Collectively, these findings suggested a lower rate of chlordane influx to the aquatic environment; however, a point source of cyclodiene insecticides to the Mississippi R. near Memphis, TN, remained evident. Residues of mirex, PCA, BHC isomers, endrin, heptachlor, and HCB were either found at relatively few (< 25%) of the stations sampled in 1986 or were characterized by relatively low concentrations. Concentrations of the herbicide Dacthal (DCPA) were also low, but incidence increased from 46% of the stations sampled in 1984 to 61% in 1986. In general, organochlorine chemical concentrations were lower in 1986 than at any time reported previously. For elemental contaminants, the geometric mean, maximum, and 85th percentile concentrations (respectively, all in microgram/g wet weight) in 1986 samples were as follows: As, 0.083, 1.53, 0.24; Cd, 0.011, 0.32, 0.04; Cu, 0.794, 11.0, 1.7; Hg, 0.087, 0.44, 0.18; Pb, 0.058, 1.90, 0.21; Se, 0.417, 3.41, 0.66; and Zn, 21.191, 94.5, 31.7. Mean concentrations of Cu increased and concentrations of As decreased relative to the 1984 collection, but these changes may reflect subtle differences in the species composition of the 1986 collection relative to other collections; concentrations of both elements differ greatly among fishes. There were no other statistically significant changes from 1984 to 1986; however, mean concentrations of As, Cd, Pb, and Zn declined from 1976, when elemental contaminants in fish were first measured in the NCBP, and 1986. In contrast, mean concentrations of Hg and Se did not change appreciably. Moreover, and in contrast to the other elements measured in 1986, concentrations of Hg and Se were high enough to constitute a threat to pisc
It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA. As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA. In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP. Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library. We designated the proteins encoded by these clones NIPs (NCBP interacting proteins). NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats. We also show that NCBP requires NIP1 for binding to the cap structure. Possible roles of NIPs in cap-dependent nuclear processes are discussed.
It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.
The U.S. Fish and Wildlife Service periodically determines concentrations of organochlorine chemicals in freshwater fish collected from a nationwide network of stations as part of the National Contaminant Biomonitoring Program (NCBP, formerly a part of the National Pesticide Monitoring Program). From late 1984 to early 1985, a total of 321 composite fish samples were collected from 112 stations and analyzed for organochlorine chemical residues. The mean concentrations of total DDT did not change from 1980-81 to 1984, following a period of steady decline through the 1970's; however, the mean concentrations ofp,p'-DDT declined significantly. The most persistent DDT homolog (p,p'-DDE) was detected at 98% of the stations sampled in 1984, and constituted 73% of total DDT residues, up from 70% in 1974-79. Collectively, these findings indicate a low rate of influx and continued weathering of DDT in the environment. Residues of polychlorinated biphenyls (PCBs) also remained widespread, but a significant downward trend in total PCBs was evident, and early eluting PCB components were present at fewer stations than in the past. Mean concentrations of dieldrin have not changed since 1978-79; concentrations remained highest in Hawaii and in the Great Lakes. Toxaphene concentrations declined from 1980-81 to 1984, especially in the Great Lakes, and the incidence of toxaphene declined from 88% of the stations sampled in 1980-81 to 69% in 1984. Mean chordane concentrations did not change from 1980-81 to 1984, following a period of decline; however,trans-nonachlor replacedcis-chlordane as the most abundant component, suggesting a lower influx of chlordane to the aquatic environment. Residues of other organochlorines-mirex, pentachloroanisole (PCA), benzene hexachloride (BHC) isomers, endrin, heptachlor, hexachlorobenzene (HCB), and Dacthal(®) (DCPA)-were either found at relatively few (<25%) of the stations sampled in 1984 or were characterized by relatively low concentrations. In general, organochlorine concentrations were lower in 1984 than at any time reported previously.
From late 1984 to early 1985, the U.S. Fish and Wildlife Service collected a total of 315 composite samples of whole fish from 109 stations nationwide, which were analyzed for arsenic, cadmium, copper, lead, mercury, selenium, and zinc. Geometric mean, maximum, and 85th percentile concentrations (μg/g wet weight) for 1984 samples were as follows: arsenic-0.14, 1.5, 0.27; cadmium-0.03, 0.22, 0.05; copper-0.65, 23.1, 1.0; mercury-0.10, 0.37, 0.17; lead-0.11, 4.88, 0.22; selenium-0.42, 2.30, 0.73; and zinc-21.7, 118.4, 34.2. The mean concentrations of selenium and lead were significantly lower than in the previous NCBP collection (1980-81). Mean concentrations of arsenic and cadmium also declined significantly between 1976, when elemental contaminants in fish were first measured in the NCBP, and 1984. Of greatest significance, lead concentrations declined steadily from 1976 to 1984, suggesting that regulatory measures have successfully reduced the influx of lead to the aquatic environment.
Human neutrophil cobalamin binding protein (NCBP) is located exclusively in the neutrophil secondary granules. The soluble stimuli formlymethionyl-leucyl-phenylalanine and the low-molecular-weight complement fragment C5a both promote the dose-dependent release of NCBP from cytochalasin B-treated neutrophils in vitro. The extracellular discharge of NCBP induced by higher secretagogue is inhibited by prior exposure of neutrophils to the corticosteroids hydrocortisone and methylprednisolone and the nonsteroidal antiinflammatory agents indomethacin and ibuprofen. The four antiinflammatory agents function as competitive antagonists of neutrophil secondary granule discharge with a site of action at or near the cell surface. These findings support the hypothesis that antiinflammatory agents prevent neutrophil activation in vitro by inhibition of stimulus-receptor coupling. The significance of these observations with regard to the in vivo actions of these agents remains uncertain, however.
The ability of noncollagenous bone proteins (NCBP) to inhibit calcium phosphate precipitation in vitro raises the question as to the nature and the relative efficiency of such proteins in vivo. To investigate this question NCBP from young adult sheep bones were fractionated using nondegradative techniques. Their relative activity was measured by their efficiency in preventing hydroxyapatite growth. On a weight (of protein recovered) basis, the activity is about equally divided between the Gla-containing protein, osteocalcin, and a phosphorylated protein essentially the same as osteonectin. On a molecular weight basis, the activity of the phosphorylated protein is almost three times higher than that of the Gla-containing protein. Finally, the inhibitory activity of the phosphorylated protein is alkaline phosphatase sensitive. The properties of this protein could provide a means of regulating the solubility of bone mineral and maintaining an equilibrium of calcium between bone and blood.
The nuclear cap-binding complex (CBC) is a heterodimer composed of CBP20 and CBP80 subunits and has roles in the biogenesis of messenger RNAs (mRNAs), small nuclear RNAs (snRNAs) and microRNAs. CBP20 is a phylogenetically conserved protein that interacts with the 7-methyl guanosine (m7G) cap added to the 5' end of all RNA polymerase II transcripts. CBP80 ensures high affinity binding of the cap by CBP20 and provides a platform for interactions with other factors. Here we characterize an alternative splice variant of CBP20, termed CBP20S. The CBP20S transcript has an in-frame deletion, leading to the translation of a protein lacking most of the RNA recognition motif (RRM). We show that CBP20S is conserved among mammalian species and is expressed in human cell lines and bone marrow cells. Unlike the full-length CBP20, CBP20S does not bind CBP80 or the m7G cap. Nevertheless, CBP20S does bind mRNA, is localized to an active transcription site and redistributed to nucleolar caps upon transcription inhibition. Our results suggest that this novel form CBP20S plays a role in transcription and/or RNA processing independent of CBP80 or the cap.
The use of artificial microRNAs (amiRNAs) is still a relatively new technique in molecular biology with a wide range of applications in life sciences. Here, we describe the silencing of the CBP80/ABH1 gene in Solanum tuberosum with the use of amiRNA. The CBP80/ABH1 protein is part of the Cap Binding Complex (CBC), which is involved in plant responses to drought stress conditions. Transformed plants with a decreased level of CBP80/ABH1 display increased tolerance to water shortage conditions. We describe how to design amiRNA with the Web MicroRNA Designer platform in detail. Additionally, we explain how to perform all steps of a procedure aiming to obtain transgenic potato plants with the use of designed amiRNA, through callus tissue regeneration and Agrobacterium tumefaciens strain LBA4404 as a transgene carrier.
The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.
The genetic basis of hearing loss in humans is relatively poorly understood. In recent years, experimental approaches including laboratory studies of early onset hearing loss in inbred mouse strains, or proteomic analyses of hair cells or hair bundles, have suggested new candidate molecules involved in hearing function. However, the relevance of these genes/gene products to hearing function in humans remains unknown. We investigated whether single nucleotide polymorphisms (SNPs) in the human orthologues of genes of interest arising from the above-mentioned studies correlate with hearing function in children.
577 SNPs from 13 genes were each analysed by linear regression against averaged high (3, 4 and 8 kHz) or low frequency (0.5, 1 and 2 kHz) audiometry data from 4970 children in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth-cohort at age eleven years. Genes found to contain SNPs with low p-values were then investigated in 3417 adults in the G-EAR study of hearing.
Genotypic data were available in ALSPAC for a total of 577 SNPs from 13 genes of interest. Two SNPs approached sample-wide significance (pre-specified at p = 0.00014): rs12959910 in CBP80/20-dependent translation initiation factor (CTIF) for averaged high frequency hearing (p = 0.00079, β = 0.61 dB per minor allele); and rs10492452 in L-plastin (LCP1) for averaged low frequency hearing (p = 0.00056, β = 0.45 dB). For low frequencies, rs9567638 in LCP1 also enhanced hearing in females (p = 0.0011, β = -1.76 dB; males p = 0.23, β = 0.61 dB, likelihood-ratio test p = 0.006). SNPs in LCP1 and CTIF were then examined against low and high frequency hearing data for adults in G-EAR. Although the ALSPAC results were not replicated, a SNP in LCP1, rs17601960, is in strong LD with rs9967638, and was associated with enhanced low frequency hearing in adult females in G-EAR (p = 0.00084).
There was evidence to suggest that multiple SNPs in CTIF may contribute a small detrimental effect to hearing, and that a sex-specific locus in LCP1 is protective of hearing. No individual SNPs reached sample-wide significance in both ALSPAC and G-EAR. This is the first report of a possible association between LCP1 and hearing function.
Osteosarcoma (OS) is a malignant bone tumor very often with pulmonary metastasis and is the main cause of OS mortality. The objective of this study was to screen for possible biomarkers of metastatic OS to explore the mechanisms of pulmonary metastasis of OS through network construction. GSE14359 was downloaded from the Gene Expression Omnibus database, which included 5 samples from conventional OS group with 2 replicates and 4 samples from OS pulmonary metastasis group in duplicate. Differentially expressed genes (DEGs) between two groups were identified by limma packages in R and classical t-test with the threshold of the false discovery rate (FDR) <0.05. The Database for Annotation, Visualization and Integrated Discovery (DAVID) were then used to perform functional annotation (FDR < 0.01). Differential coexpression network was constructed with subspace differential coexpression analysis (SDC), and genes with high degrees in the differential coexpression network were identified. A total of 1344 genes were screened as DEGs, including 677 up- and 667 down-regulated DEGs in the pulmonary metastasis of OS. Thirty-one significantly enriched functions were obtained, such as blood vessel morphogenesis, defense response, cell death and so on. DEGs with high degrees (brain-specific angiogenesis inhibitor 2 (BAI2), formin-like 1 (FMNL1), dual-specificity phosphatase 7 (DUSP7), transient receptor potential melastatin 2 (TRPM2), CBP80/20-dependent translation initiation factor (KIAA0427) and C120rf35) in the differential coexpression network were found. BAI2, FMNL1, DUSP7 and TRPM2 may be useful markers for predicting tumor metastasis and therapeutic targets for the treatment of OS patients with metastasis.
It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5'-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5'UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.
We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.
Histone biogenesis is tightly controlled at multiple steps to maintain the balance between the amounts of DNA and histone protein during the cell cycle. In particular, translation and degradation of replication-dependent histone mRNAs are coordinately regulated. However, the underlying molecular mechanisms remain elusive. Here, we investigate remodeling of stem-loop binding protein (SLBP)-containing histone mRNPs occurring during the switch from the actively translating mode to the degradation mode. The interaction between a CBP80/20-dependent translation initiation factor (CTIF) and SLBP, which is important for efficient histone mRNA translation, is disrupted upon the inhibition of DNA replication or at the end of S phase. This disruption is mediated by competition between CTIF and UPF1 for SLBP binding. Further characterizations reveal hyperphosphorylation of UPF1 by activated ATR and DNA-dependent protein kinase upon the inhibition of DNA replication interacts with SLBP more strongly, promoting the release of CTIF and eIF3 from SLBP-containing histone mRNP. In addition, hyperphosphorylated UPF1 recruits PNRC2 and SMG5, triggering decapping followed by 5'-to-3' degradation of histone mRNAs. The collective observations suggest that both inhibition of translation and recruitment of mRNA degradation machinery during histone mRNA degradation are tightly coupled and coordinately regulated by UPF1 phosphorylation.
During the cell cycle the expression of replication-dependent histones is tightly coupled to DNA synthesis. Histone messenger RNA (mRNA) levels strongly increase during early S-phase and rapidly decrease at the end of it. Here, we review the degradation of replication-dependent histone mRNAs, a paradigm of post-transcriptional gene regulation, in the context of processing, translation, and oligouridylation. Replication-dependent histone transcripts are characterized by the absence of introns and by the presence of a stem-loop structure at the 3' end of a very short 3' untranslated region (UTR). These features, together with a need for active translation, are a prerequisite for their rapid decay. The degradation is induced by 3' end additions of untemplated uridines, performed by terminal uridyl transferases. Such 3' oligouridylated transcripts are preferentially bound by the heteroheptameric LSM1-7 complex, which also interacts with the 3'→5' exonuclease ERI1 (also called 3'hExo). Presumably in cooperation with LSM1-7 and aided by the helicase UPF1, ERI1 degrades through the stem-loop of oligouridylated histone mRNAs in repeated rounds of partial degradation and reoligouridylation. Although histone mRNA decay is now known in some detail, important questions remain open: How is ceasing nuclear DNA replication relayed to the cytoplasmic histone mRNA degradation? Why is translation important for this process? Recent research on factors such as SLIP1, DBP5, eIF3, CTIF, CBP80/20, and ERI1 has provided new insights into the 3' end formation, the nuclear export, and the translation of histone mRNAs. We discuss how these results fit with the preparation of histone mRNAs for degradation, which starts as early as these transcripts are generated.
The cap-binding proteins CBP20 and CBP80 have well-established roles in RNA metabolism and plant growth and development. Although these proteins are thought to be involved in the plant's response to environmental stress, their functions in this process are unclear. Here we demonstrated that Arabidopsis cbp20 and cbp80 null mutants had abnormal leaves and flowers and exhibited increased sensitivity to salt stress. The aberrant phenotypes were more pronounced in the cbp20/80 double mutant. Quantification by iTRAQ (isobaric tags for relative and absolute quantification) identified 77 differentially expressed proteins in the cbp20 and cbp80 lines compared with the wild-type Col-0 under salt stress conditions. Most of these differentially expressed proteins were synergistically expressed in cbp20 and cbp80, suggesting that CBP20 and CBP80 have synergistic roles during the salt stress response. Biochemical analysis demonstrated that CBP20 and CBP80 physically interacted with each other. Further analysis revealed that CBP20/80 regulated the splicing of genes involved in proline and sugar metabolism and that the epigenetic and post-translational modifications of these genes were involved in salt stress tolerance. Our data suggest a link between CBP20/80-dependent protein ubiquitination/sumoylation and the salt stress response.
Multiple endocrine neoplasia type I (MEN1) is an inherited syndrome that includes susceptibility to pancreatic islet hyperplasia. This syndrome results from mutations in the MEN1 gene, which encodes menin protein. Menin interacts with several transcription factors, including JunD, and inhibits their activities. However, the precise mechanism by which menin suppresses gene expression is not well understood. Here, we show that menin interacts with arsenite-resistant protein 2 (ARS2), a component of the nuclear RNA CAP-binding complex that is crucial for biogenesis of certain miRNAs including let-7a. The levels of primary-let-7a (pri-let-7a) are not affected by menin; however, the levels of mature let-7a are substantially decreased upon Men1 excision. Let-7a targets, including Insr and Irs2, pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of insulin receptor substrate 2 (IRS2) to levels observed in Men1-excised cells. Depletion of menin does not affect the expression of Drosha and CBP80, but substantially impairs the processing of pri-miRNA to pre-miRNA. Ars2 knockdown decreased let-7a processing in menin-expressing cells but had little impact on let-7a levels in menin-excised cells. As IRS2 is known to mediate insulin signaling and insulin/mitogen-induced cell proliferation, these findings collectively unravel a novel mechanism whereby menin suppresses cell proliferation, at least partly by promoting the processing of certain miRNAs, including let-7a, leading to suppression of Irs2 expression and insulin signaling.
Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression.
How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5' splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.
Animal replication-dependent histone genes produce histone proteins for the packaging of newly replicated genomic DNA. The expression of these histone genes occurs during S phase and is linked to DNA replication via S-phase checkpoints. The histone RNA-binding protein HBP/SLBP (hairpin-binding protein/stem-loop binding protein), an essential regulator of histone gene expression, binds to the conserved hairpin structure located in the 3'UTR (untranslated region) of histone mRNA and participates in histone pre-mRNA processing, translation and histone mRNA degradation. Here, we report the accumulation of alternatively spliced HBP/SLBP transcripts lacking exons 2 and/or 3 in HeLa cells exposed to replication stress. We also detected a shorter HBP/SLBP protein isoform under these conditions that can be accounted for by alternative splicing of HBP/SLBP mRNA. HBP/SLBP mRNA alternative splicing returned to low levels again upon removal of replication stress and was abrogated by caffeine, suggesting the involvement of checkpoint kinases. Analysis of HBP/SLBP cellular localization using GFP (green fluorescent protein) fusion proteins revealed that HBP/SLBP protein and isoforms lacking the domains encoded by exon 2 and exons 2 and 3 were found in the nucleus and cytoplasm, whereas HBP/SLBP lacking the domain encoded by exon 3 was predominantly localised to the nucleus. This isoform lacks the conserved region important for protein-protein interaction with the CTIF [CBP80/20 (cap-binding protein 80/20)]-dependent initiation translation factor and the eIF4E (eukaryotic initiation factor 4E)-dependent translation factor SLIP1/MIF4GD (SLBP-interacting protein 1/MIF4G domain). Consistent with this, we have previously demonstrated that this region is required for the function of HBP/SLBP in cap-dependent translation. In conclusion, alternative splicing allows the synthesis of HBP/SLBP isoforms with different properties that may be important for regulating HBP/SLBP functions during replication stress.
In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3' end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.
MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain-containing-proteins, NOT2a and NOT2b (previously known as At-Negative on TATA less2 [NOT2] and VIRE2-INTERACTING PROTEIN2, respectively), as components involved in Arabidopsis thaliana miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille domain of DICER-LIKE1 (DCL1), an interaction that is conserved between rice (Oryza sativa) and Arabidopsis thaliana. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of primary miRNAs and mature miRNAs and affects DCL1 but not HYPONASTIC LEAVES1 (HYL1) localization in vivo. In addition, NOT2b protein interacts with polymerase II and other miRNA processing factors, including two cap binding proteins, CBP80/ABH1, CBP20, and SERRATE (SE). Finally, we found that the mRNA levels of some protein coding genes were also affected. Therefore, these results suggest that NOT2 proteins act as general factors to promote the transcription of protein coding as well as miRNA genes and facilitate efficient DCL1 recruitment in miRNA biogenesis.
MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.
The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3'-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated β-actin or eEF2 mRNA to eIF4E-bound polyadenylated β-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3'-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs.
Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.
The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.
Although the importance of abscisic acid (ABA) in plant development and response to abiotic and biotic stresses is well recognized, the molecular basis of the signaling pathway has not been fully elucidated. Mutants in genes related to ABA are widely used as a tool for gaining insight into the mechanisms of ABA signal transduction and ABA-dependent stress response. We used a genetic approach of a suppressor screening in order to decipher the interaction between ABH1 (CBP80) and other components of ABA signaling. ABH1 (CBP80) encodes a large subunit of CBC (CAP BINDING COMPLEX) and the abh1 mutant is drought-tolerant and hypersensitive to ABA during seed germination. The suppressor mutants of abh1 were generated after chemical mutagenesis. The mutant named soa1 (suppressor of abh1 hypersensitivity to ABA 1) displayed an ABA-insensitive phenotype during seed germination. The genetic analysis showed that the soa1 phenotype is dominant in relation to abh1 and segregates as a single locus. Based on soa1's response to a wide spectrum of physiological assays during different stages of development, we used the candidate-genes approach in order to identify a suppressor gene. The molecular analysis revealed that mutation causing the phenotype of soa1 occurred in the ABI4 (ABA insensitive 4) gene. Analysis of pre-miR159 expression, whose processing depends on CBC, as well as targets of miR159: MYB33 and MYB101, which are positive regulators of ABA signaling, revealed a possible link between CBP80 (ABH1) and ABI4 presented here.
Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions of yeast and animal genomes play important roles in key biological processes. Yet, plant lincRNAs remain poorly characterized and how lincRNA biogenesis is regulated is unclear. Using a reproducibility-based bioinformatics strategy to analyze 200 Arabidopsis thaliana transcriptome data sets, we identified 13,230 intergenic transcripts of which 6480 can be classified as lincRNAs. Expression of 2708 lincRNAs was detected by RNA sequencing experiments. Transcriptome profiling by custom microarrays revealed that the majority of these lincRNAs are expressed at a level between those of mRNAs and precursors of miRNAs. A subset of lincRNA genes shows organ-specific expression, whereas others are responsive to biotic and/or abiotic stresses. Further analysis of transcriptome data in 11 mutants uncovered SERRATE, CAP BINDING PROTEIN20 (CBP20), and CBP80 as regulators of lincRNA expression and biogenesis. RT-PCR experiments confirmed these three proteins are also needed for splicing of a small group of intron-containing lincRNAs.
MicroRNAs (miRNAs) are key regulators of gene expression and play critical roles in modulating metabolism, development and physiology in animals and plants. miRNA levels are transcriptionally and post-transcriptionally controlled for their proper function. Recent studies have shown that RNA-binding proteins play important roles in producing miRNAs by affecting the accurate and/or efficient processing of precursors of miRNAs. Many of these RNA-binding proteins also have roles in general RNA metabolism, indicating potential connections between miRNA biogenesis and other RNA metabolism. Here, we focus on the function of several RNA-binding proteins in miRNA biogenesis in Arabidopsis.
SAGA/TFTC is a histone acetyltransferase complex that has a second enzymatic activity because of the presence of a deubiquitination module (DUBm). Drosophila DUBm consists of Sgf11, ENY2 and Nonstop proteins. We show that Sgf11 has other DUBm-independent functions. It associates with Cbp80 component of the cap-binding complex and is thereby recruited onto growing messenger ribonucleic acid (mRNA); it also interacts with the AMEX mRNA export complex and is essential for hsp70 mRNA export, as well as for general mRNA export from the nucleus. Thus, Sgf11 functions as a component of both SAGA DUBm and the mRNA biogenesis machinery.
The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.
In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.
Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5' cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.
The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.
Nuclear cap-binding protein (CBP) 80/20-dependent translation (CT) is one of the targets for miRNA-mediated gene silencing. Here, we provide evidence that human argonaute 2 (Ago2) competes with CBP80/20 for cap-association, inhibiting CT and thus nonsense-mediated mRNA decay (NMD), which is tightly coupled to CT. Tethering of Ago2, but not of Ago2F2V2 which lacks cap-association activity, to the 3'UTR of PTC-containing mRNA abrogates NMD. Immunoprecipitation using CBP80 antibody reveals that Ago2, but not Ago2F2V2, inhibits the binding of CBP80/20 to cap structure. Our observations provide molecular insight into the cross-talk between miRNA-mediated gene silencing, CT, and NMD.
In mammalian cells, two different messenger ribonucleoproteins (mRNPs) serve as templates for protein synthesis. Newly synthesized mRNPs bound by the cap-binding protein heterodimer CBP80-CBP20 (CBC) initially undergo a pioneer round of translation. One purpose of this round of translation is to ensure the quality of gene expression, as exemplified by nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD largely functions to eliminate mRNAs that prematurely terminate translation, although NMD also contributes to proper gene control, and it targets CBC-bound mRNPs. CBC-bound mRNPs are remodeled to eukaryotic translation initiation factor (eIF)4E-bound mRNPs in steps that (1) are a consequence of the pioneer round of translation and (2) occur independently of translation. Rather than supporting NMD, eIF4E-bound mRNPs provide for the bulk of cellular protein synthesis and are the primary targets of mRNA decay mechanisms that conditionally regulate gene expression. Here, we overview cellular processes by which CBC-bound mRNPs are remodeled to eIF4E-bound mRNPs. We also describe the molecular movements of certain factors during NMD in view of the influential role of CBP80.
The nuclear cap-binding complex (CBC), a heterodimer comprised of a 20 kDa subunit (CBP20) and an 80 kDa regulatory subunit (CBP80), binds to nascent RNA polymerase II transcripts and is important throughout different aspects of RNA metabolism. In a recent publication, using a combination of X-ray crystallographic information, mutagenesis studies, small-angle scattering experiments, analytical ultracentrifugation and in vivo assays, we presented evidence that importin-α and importin-β, two nucleocytoplasmic transport proteins, play key roles in regulating the binding of capped RNA by the CBC in cells. A model for how complexes between CBC and the importins cycle in and out of the nucleus and direct the proper positional binding and release of capped RNA is presented here and is discussed in light of recent publications.
In this issue of Molecular Cell, Hwang et al. (2010) show that the cap-binding protein CBP80 promotes nonsense-mediated decay (NMD) at two steps. In this dual capacity, CBP80 may facilitate essential communication between the premature termination codon (PTC) and the exon-junction complex (EJC) to trigger NMD.
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.
Imperfect base-pairing between microRNA (miRNA) and the 3'-untranslated region of target messenger RNA (mRNA) triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 targets cap-binding protein (CBP)80/20-bound mRNAs and exon junction complex-bound mRNAs and inhibits nonsense-mediated mRNA decay (NMD), which is restricted tightly to CBP80/20-bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA-mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post-transcriptional regulation mechanism of gene expression in mammalian cells.
We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.
Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)-protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)-CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon-exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin beta (IMPbeta): Inhibiting the binding of IMPbeta to the complex of CBC-IMPalpha at an mRNA cap using the IMPalpha IBB (IMPbeta-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPbeta and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.
The nuclear cap-binding protein complex (CBC) participates in 5' splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT-PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5' and 3' splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5' splice site.
The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and their dissociation from the CBC in the cytosol, represent essential steps in RNA processing. Here we show how the nucleocytoplasmic transport proteins importin-alpha and importin-beta have key roles in regulating these events. As a first step toward understanding the molecular basis for this regulation, we determined a 2.2-A resolution X-ray structure for a CBC-importin-alpha complex that provides a detailed picture for how importin-alpha binds to the CBP80 subunit of the CBC. Through a combination of biochemical studies, X-ray crystallographic information and small-angle scattering experiments, we then determined how importin-beta binds to the CBC through its CBP20 subunit. Together, these studies enable us to propose a model describing how importin-beta stimulates the dissociation of capped RNA from the CBC in the cytosol following its nuclear export.
During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF.
Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.
Translation of most mRNAs is performed in a cap-dependent manner, requiring a protein complex, the cap complex, to regulate the accessibility of the message to the 40S ribosome. The cap complex initiates protein translation by binding to the 5' cap of an mRNA and recruiting ribosomes to begin translation. Compared to animals and yeast, there are significant plant-specific differences in the regulation of cap-dependent mRNA translation, but these are poorly understood. Here, we purified proteins that bind to the 5' cap during the Arabidopsis growth cycle. The protein profile of the cap-binding complexes varies during the various stages of the growth cycle in suspension culture cells. Using Western blotting, the cap complexes of quiescent cells were found to be composed of only three major proteins: eIF4isoE, which is primarily a cytoplasmic protein, and eIF4E and CBP80, which accumulate in the nucleus. However, when cells proliferate, at least 10 major proteins bind directly or indirectly to the 5' cap. Proteomic, Western blotting and immunoprecipitation data establish that the spectrum of RNA helicases in the cap complexes also changes during the growth cycle. Cap complexes from proliferating cultures mainly contain eIF4A, which associates with at least four cap complexes, but eIF4A is replaced by additional helicases in quiescent cells. These findings suggest that the dynamic and selective recruitment of various proteins to mRNA 5' cap complexes could play an important role in the regulation of gene expression.
The cap-binding protein complex (CBC) binds to the caps of all RNA polymerase II transcripts, and plays an important role in RNA metabolism. We characterized interactions, localization and nuclear-cytoplasmic transport of two subunits of the Arabidopsis thaliana cap-binding protein complex (AtCBC): AtCBP20 and AtCBP80. Using CFP/YFP-tagged proteins, we show that transport of AtCBC from the cytoplasm to the nucleus in the plant cell is different from that described in other eukaryotic cells. We show that the smaller subunit of the complex, AtCBP20, plays a crucial role in the nuclear import of AtCBC. The C-terminal part of AtCBP20 contains two functionally independent nuclear localization signals (NLSs). At least one of these two NLSs is required for the import of CBC into the plant nucleus. The interaction between the A. thaliana CBP20 and CBP80 was also analyzed in detail, using the yeast two-hybrid system and fluorescence resonance energy transfer (FRET) assays. The N-terminal part of AtCBP20 is essential for interaction with AtCBP80. Furthermore, AtCBP80 is important for the protein stability of the smaller subunit of CBC. Based on these data, we propose a model for the nuclear-cytoplasmic trafficking of the CBC complex in plants.
Nonsense-mediated decay (NMD) in eukaryotic cells largely functions as a quality control mechanism by degrading faulty mRNAs that terminate translation prematurely. In recent years it has become evident that NMD also eliminates a subset of naturally occurring mRNA during proper gene expression. The mechanism of NMD in mammalian cells can be distinguished from the mechanism in, for example, Saccharomyces cerevisiae or Caenorhabditis elegans, by its apparent restriction to newly synthesized mRNA during a pioneer round of translation. This dependence can be explained by the need for at least one exon-exon junction complex (EJC) that is deposited on newly synthesized mRNA during the process of pre-mRNA splicing. Additionally, mammalian-cell NMD is promoted by the cap-binding protein heterodimer CBP80/20 that also typifies newly synthesized mRNA. When translation terminates sufficiently upstream of an EJC, the NMD factor Up-frameshift (Upf)1 is thought to join the stable EJC constituent NMD factors Upf2 and Upf3 or Upf3X (also called Upf3a or Upf3b, respectively), and undergo phosphorylation. Phosphorylation appears to trigger translational repression and mRNA decay. Although there are established rules for what generally defines an NMD target in mammalian cells, as with any rule there are exceptions and, thus, the need to experimentally verify individual mRNAs as bona fide targets of NMD. This chapter provides guidelines and protocols for how to define NMD targets using cultured mammalian cells.
Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.
In this study we investigated whether in plants, like in mammals, components of the nuclear cap-binding protein complex (CBC) are involved in nonsense-mediated mRNA decay (NMD). We selected several genes producing at least two alternatively spliced mRNA variants: one with a premature termination codon (PTC+) and another without it (PTC-). For each gene the PTC+/PTC- ratio was calculated using RT-PCR and direct sequencing in four Arabidopsis thaliana lines: wild type, the NMD mutant atupf3-1 and two CBC mutants: cbp20 and abh1. Whereas in the NMD mutant the ratios of PTC+/PTC- splice variants were higher than in wild-type plants, the two CBC mutants investigated showed no change in the PTC+/PTC- ratios. Our results suggest that neither CBP20 nor CBP80 is involved in NMD in A. thaliana.
MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.
While early steps of gene expression, such as transcription preinitiation, are known to often be rate limiting and to be regulated by such stimuli as steroid hormones, the potential impact of downstream steps, including splicing, on the mRNA production rate is unknown. In this work, we studied the effects of the transcriptional stimulus estradiol on cyclin D1, PS2, and c-fos gene expression by measuring the levels of RNA polymerase II on the DNA templates, the levels of nascent transcripts associated with RNA polymerase II, and the levels of unspliced, partially spliced, and fully spliced RNAs. We demonstrated that the efficiency of cotranscriptional splicing of the first intron was higher in the case of cyclin D1 than with PS2 and potentiated the cyclin D1 mRNA production rate. The mechanism involved in cotranscriptional splicing depended on the level of serine 5 phosphorylation of RNA polymerase II at the gene 5' end and on the recruitment of CBP80, one of the two subunits of the cap binding complex, which stimulates splicing of the promoter-proximal intron. Our data indicate that mRNA production from a subset of estradiol-stimulated genes, such as cyclin D1, could occur in a very efficient "assembly line." In contrast, we demonstrated for the first time that despite a strong transcriptional activation of the PS2 gene, the production of mRNA is not optimized owing to inefficient cotranscriptional RNA processing.
The plant hormone abscisic acid (ABA) intricately regulates a multitude of processes during plant growth and development. Recent studies have established a connection between genes participating in various steps of cellular RNA metabolism and the ABA signal transduction machinery. In this chapter we focus on the plant nuclear mRNA cap binding proteins, CBP20 and CBP80. We summarize and report recent findings on their effects on cellular signal transduction networks and mRNA processing events. ABA hypersensitive 1 (abh1) harbors a gene disruption in the Arabidopsis CBP80 gene. Loss-of-function mutation of ABH1 can also result in an early flowering phenotype in the Arabidopsis accession C24. abh1 revealed noncoding cis-natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants with elevated cis-NAT expression in the mutant. abh1 also revealed an influence on the splicing of the MADS box transcription factor Flowering Locus C pre-mRNA, which may result in the regulation of flowering time. Furthermore, new experiments analyzing complementation of cpb20 with site-directed cpb20 mutants provide evidence that the CAP binding activity of CBP20 is essential for the observed cbp-associated phenotypes. In conclusion, mutants in genes participating in RNA processing provide excellent tools to uncover novel molecular mechanisms for the regulation of RNA metabolism and of signal transduction networks in wild-type plants.
The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by apparent stabilization of pri-miRNAs. Whole-genome tiling array analyses reveal that se, abh1/cbp80, and cbp20 mutants also share similar splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, because introns retained in se, abh1/cbp80, and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for nonsense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1.
MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are abundant endogenous small RNAs (smRNAs) that control transcript expression through posttranscriptional gene silencing. Here, we show that concomitant loss of XRN4/EIN5, a 5'-3' exoribonuclease, and ABH1/CBP80, a subunit of the mRNA cap binding complex, results in Arabidopsis plants manifesting myriad developmental defects. We find that ABH1/CBP80 is necessary to obtain proper mature miRNA levels, which suggests this protein affects the miRNA-mediated RNA silencing pathway. Additionally, we show that XRN4/EIN5 affects the levels of a smRNA class that is processed from both sense and antisense strands of approximately 130 endogenous transcripts that apparently are converted to double-stranded RNA (dsRNA) and subsequently processed. We find that the parent transcripts of these smRNAs accumulate in an uncapped form upon loss of XRN4/EIN5, which suggests that uncapped endogenous transcripts can become smRNA biogenesis substrates. Overall, our results reveal unexpected connections between RNA metabolism and silencing pathways.
Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.
Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism; this process removes faulty mRNAs harboring premature termination codons (PTCs). NMD targets newly synthesized mRNAs bound by nuclear cap-binding proteins 80/20 (CBP80/20) and exon junction complex (EJC), the former of which is thought to recruit the ribosome to initiate the pioneer round of translation. After completion of the pioneer round of translation, CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E, which mediates steady-state translation in the cytoplasm. Here, we show that overexpression of eIF4E-T preferentially inhibits cap-dependent steady-state translation, but not the pioneer round of translation. We also demonstrate that overexpression of eIF4E-T or Dcp1a triggers the movement of eIF4E into the processing bodies. These results suggest that the pioneer round of translation differs from steady-state translation in terms of ribosome recruitment.
Nonsense-mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap-binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady-state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.
Nonsense-mediated mRNA decay (NMD) generally eliminates messenger RNAs that prematurely terminate translation and occurs in all eukaryotes that have been studied, although with mechanistic variations. In mammals, NMD seems to be restricted to newly synthesized mRNA that is bound by the cap-binding heterodimer CBP80-CBP20 (CBP80/20) and typically has at least one exon junction complex (EJC) situated downstream of the nonsense codon and added post-splicing. However, mammalian NMD can also target spliced mRNA lacking an EJC downstream of the nonsense codon. Here we provide evidence that this additional pathway, known as failsafe NMD, likewise seems to be restricted to CBP80/20-bound mRNA and does not detectably target its subsequently remodeled product, eIF4E-bound mRNA. Our studies, including analyses of factor dependence, reveal important shared features of the two mammalian-cell NMD pathways as well as fundamental differences between NMD in mammals and Saccharomyces cerevisiae.
The pioneer round of translation plays a role in translation initiation of newly spliced and exon junction complex (EJC)-bound mRNAs. Nuclear cap-binding protein complex CBP80/20 binds to those mRNAs at the 5'-end, recruiting translation initiation complex. As a consequence of the pioneer round of translation, the bound EJCs are dissociated from mRNAs and CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E. Steady-state translation directed by eIF4E allows for an immediate and rapid response to changes in physiological conditions. Here, we show that nonsense-mediated mRNA decay (NMD), which restricts only to the pioneer round of translation but not to steady-state translation, efficiently occurs even during serum starvation, in which steady-state translation is drastically abolished. Accordingly, CBP80 remains in the nucleus and processing bodies are unaffected in their abundance and number in serum-starved conditions. These results suggest that mRNAs enter the pioneer round of translation during serum starvation and are targeted for NMD if they contain premature termination codons.
Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5' exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5' exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.
The mammalian integration site 6 (INT6) protein has been implicated in breast carcinogenesis and characterized as the eIF3e non-core subunit of the translation initiation factor eIF3, but its role in this complex is not known. Here, we show that INT6 knockdown by RNA interference strongly inhibits nonsense-mediated messenger RNA decay (NMD), which triggers degradation of mRNAs with premature stop codons. In contrast to the eIF3b core subunit, which is required for both NMD and general translation, INT6 is only necessary for the former process. Consistent with such a role, immunoprecipitation experiments showed that INT6 co-purifies with CBP80 and the NMD factor UPF2. In addition, several transcripts known to be upregulated by UPF1 or UPF2 depletion were also found to be sensitive to INT6 suppression. From these observations, we propose that INT6, in association with eIF3, is involved in routing specific mRNAs for degradation.
Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100-300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.
Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.
Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.
In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.
Eukaryotic translation initiation factor 4G (eIF4G) plays a critical role in protein expression, and is at the center of a complex regulatory network. Together with the cap-binding protein eIF4E, it recruits the small ribosomal subunit to the 5'-end of mRNA and promotes the assembly of a functional translation initiation complex, which scans along the mRNA to the translation start codon. Human eIF4G contains three consecutive HEAT domains, as well as long unstructured regions involved in multiple protein-protein interactions. Despite the accumulating data about the structure and function of eIF4G, the mechanisms of coordination and regulation of its interactions with other factors have remained largely unknown. Here, we present evidence that eIF4G and the large subunit of the nuclear cap-binding complex, CBP80, share a common origin and domain structure. We propose that the organization of the individual domains in eIF4G and CBP80 could also be conserved. The structure of CBP80, in complex with the nuclear cap-binding protein CBP20, is used to build a model for the mutual orientation of the domains in eIF4G and their interactions with other factors. The organization of the CBP80-CBP20 complex suggests how the activity of eIF4G in translation initiation could be regulated through a dynamic network of overlapping intra- and intermolecular interactions centered around the eIF4G HEAT domains.
Nonsense-mediated mRNA decay (NMD) in mammalian cells targets cap-binding protein 80 (CBP80)-bound mRNA during or after a pioneer round of translation. It is unknown whether eukaryotic translation initiation factor 4G (eIF4G) functions in the pioneer round. We show that baculovirus-produced CBP80 and CBP20 independently interact with eIF4GI. The interactions between eIF4G and the heterodimer CBP80/20 suggest that eIF4G has a function in the pioneer initiation complex rather than merely a presence during remodeling to the steady-state complex. First, NMD is inhibited upon eIF4G cleavage by HIV-2 or poliovirus 2A protease. Second, eIF4GI coimmunopurifies with pre-mRNA, indicating that it associates with transcripts before the pioneer round. Third, eIF4G immunopurifies with Upf NMD factors and eIF4AIII, which are constituents of the pioneer translation initiation complex. We propose a model in which eIF4G serves to connect CBP80/20 with other initiation factors during the pioneer round of translation.
The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2alpha coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2alpha in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2alpha, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.
Studies of nonsense-mediated mRNA decay in mammalian cells have proffered unforeseen insights into changes in mRNA-protein interactions throughout the lifetime of an mRNA. Remarkably, mRNA acquires a complex of proteins at each exon-exon junction during pre-mRNA splicing that influences the subsequent steps of mRNA translation and nonsense-mediated mRNA decay. Complex-loaded mRNA is thought to undergo a pioneer round of translation when still bound by cap-binding proteins CBP80 and CBP20 and poly(A)-binding protein 2. The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.
Processing of nuclear pre-mRNA is an important step in the regulation of gene expression and involves 5(')- and 3(')-end processing, splicing, and editing. Mammalian nuclear pre-mRNAs are assembled in large ribonucleoprotein (lnRNP) complexes, in which the entire population of nuclear pre-mRNA is individually packaged until it is exported to the cytoplasm. The lnRNP particles are supraspliceosomal complexes. They are composed of four spliceosomal substructures and an additional one, which are interconnected by the pre-mRNA, and have an overall mass of 21MDa. The additional substructure was proposed to harbor additional processing activities, such as editing components that were shown to be associated with the lnRNP particles. Here we show that the cap-binding proteins (CBPs), CBP20 and CBP80, are associated with the lnRNP particles, as well as components of the 3(')-end-processing activity. These results, together with our previous demonstration of the association of splicing factors and A-to-I editing enzymes with lnRNP particles, support the view that the lnRNP particles are the nuclear pre-mRNA processing machine. Such a machine is required to execute the nuclear processing steps of the pre-mRNA in an accurate and regulated manner. The supraspliceosomal pre-mRNA processing machine, in which each substructure represents a functional spliceosome, provides a frame onto which the pre-mRNA is folded. It allows juxtaposition of exons about to be spliced, while introns are looped out of each of the respective spliceosomes. This model can account for regulated alternative splicing, which is a major source of protein versatility in mammals.
The nuclear cap-binding complex (CBC) binds the 7-methyl-G(5')ppp(5')N cap structure at the 5' end of pre-messenger and uracil-rich small nuclear RNAs in the nucleus. It mediates interaction of these capped RNAs with various nuclear machineries involved in RNA maturation and is co-exported with them to the cytoplasm. The structure of human CBC, which comprises the subunits CBP20 and CBP80, has previously been determined in a mildly trypsinated form which can no longer bind the cap. Here, the engineering and crystallization of two variant CBCs with deletions in CBP80 which do not affect function are described. A complex with a small N-terminal deletion in CBP80 was crystallized in space group C2 with one complex per asymmetric unit. The crystals diffract to 2 A resolution and give the first structure of intact but cap-free CBC. An additional internal deletion in CBP80 of a prominent solvent-exposed coiled coil gives rise to a more compact complex. This was co-crystallized with the cap analogue m(7)GpppG in two different crystal forms which could grow in the same drop. Form 1 belongs to space group P3(1)21 with one complex per asymmetric unit and diffracts to 2.15 A resolution. Form 2 belongs to space group P2(1)2(1)2(1) with two complexes per asymmetric unit and diffracts to 2.3 A resolution. In both forms, strong extra electron density is observed for the cap analogue and for the N- and C-terminal extensions of CBP20 which was absent or disordered in all previous structures.
The 7-methyl guanosine cap structure of RNA is essential for key aspects of RNA processing, including pre-mRNA splicing, 3' end formation, U snRNA transport, nonsense-mediated decay and translation. Two cap-binding proteins mediate these effects: cytosolic eIF-4E and nuclear cap-binding protein complex (CBC). The latter consists of a CBP20 subunit, which binds the cap, and a CBP80 subunit, which ensures high-affinity cap binding. Here we report the 2.1 A resolution structure of human CBC with the cap analog m7GpppG, as well as the structure of unliganded CBC. Comparisons between these structures indicate that the cap induces substantial conformational changes within the N-terminal loop of CBP20, enabling Tyr 20 to join Tyr 43 in pi-pi stacking interactions with the methylated guanosine base. CBP80 stabilizes the movement of the N-terminal loop of CBP20 and locks the CBC into a high affinity cap-binding state. The structure for the CBC bound to m7GpppG highlights interesting similarities and differences between CBC and eIF-4E, and provides insights into the regulatory mechanisms used by growth factors and other extracellular stimuli to influence the cap-binding state of the CBC.
The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.
Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon-exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense-mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap-binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap-binding protein eIF4E. These and other data indicate that NMD targets CBP80-bound mRNA during a 'pioneer' round of translation, but whether nuclear eIF4E also binds nascent but dead-end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron-containing RNA and the C-terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80-bound but not eIF4E-bound mRNA. Each of these proteins is also detected on CBP80-bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.
In this report we characterize two Arabidopsis thaliana proteins, named AtCBP20 and AtCBP80, that are homologues of human subunits of a nuclear cap-binding protein complex (CBC). AtCBP20 has a calculated molecular mass of 29.9 kDa, and AtCBP80 is a 96.5 kDa protein. AtCBP20 exhibits 68% identity and 82% similarity to human CBP20. Like its human homologue, AtCBP20 contains a canonical RNA binding domain (RBD) with single RNP2 and RNP1 motifs. In addition to the N-terminal part, which is similar to the human protein, AtCBP20 has a long C-terminus rich in arginine, glycine and aspartate residues. The second subunit of the Arabidopsis cap-binding complex, AtCBP80, shows 28% identity and 50% similarity to its homologue from HeLa cells. The protein contains a MIF4G domain at its N-terminus, the feature characteristic to all analyzed CBP80s. This domain, described also in eIF4G and NMD2 proteins, is thought to be involved in protein-protein and also in protein--RNA interactions. Both proteins AtCBP20 and AtCBP80 are encoded by single-copy genes in the A. thaliana genome. The AtCBP20 gene is located on chromosome V, and the AtCBP80 gene is encoded by chromosome II. Among introns identified in the AtCBP20 gene, we discovered an U12 type intervening sequence (an AT-AC intron). This intron is spliced out very efficiently in plants, but when isolated and tested for splicing in tobacco protoplasts, the efficiency of the U12 intron excision was low. Splicing efficiency of the U12 intron is improved by the addition of exon and intron sequences upstream or downstream of the U12 intron. AtCBP20 and AtCBP80 are constitutively expressed in all examined organs of A. thaliana, including roots, stems, leaves and flowers. Interestingly, the steady-state level of both transcripts seem to be very similar in all tissues analyzed.
Nonsense-mediated decay (NMD) eliminates mRNAs that prematurely terminate translation. We used antibody to the nuclear cap binding protein CBP80 or its cytoplasmic counterpart eIF4E to immunopurify RNP containing nonsense-free or nonsense-containing transcripts. Data indicate that NMD takes place in association with CBP80. We defined other components of NMD-susceptible mRNP as CBP20, PABP2, eIF4G, and the NMD factors Upf2 and Upf3. Consistent with the dependence of NMD on translation, the NMD of CBP80-bound mRNA is blocked by cycloheximide or suppressor tRNA. These findings provide evidence that translation can take place in association with CBP80. They also indicate that CBP80-bound mRNA undergoes a "pioneer" round of translation, before CBP80-CBP20 are replaced by eIF4E, and Upf2 and Upf3 proteins dissociate from upstream of exon-exon junctions.
The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II transcripts. It enhances the efficiency of several mRNA maturation steps and is essential for U snRNA nuclear export in multicellular eukaryotes. The 2A crystal structure of human CBC shows that the large subunit, CBP80, comprises three domains, each containing consecutive helical hairpins and resembling the so-called MIF4G domain found in several other proteins involved in RNA metabolism. The small subunit, CPB20, has an RNP fold and associates with the second and third domains of CBP80. Site-directed mutagenesis revealed 4 residues of CBP20 which are critical for cap binding. A model for cap binding is proposed based on these results and the known mode of binding of RNA to RNP domains.
In eukaryotes the majority of mRNAs have an m(7)G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.
Cdc42 is a low molecular weight GTP-binding protein that plays a key regulatory role in a variety of cellular activities. The importance of the coordination of different cell functions by Cdc42 is underscored by the fact that a constitutively active Cdc42 mutant induces cellular transformation. In this study, we describe a novel function for Cdc42: its ability to stimulate pre-messenger RNA splicing. This activity is dependent on cysteine 37 in the effector loop of Cdc42 but is not dependent on cell growth. A likely candidate protein for mediating the Cdc42 effects on pre-mRNA splicing is the nuclear RNA cap-binding complex (CBC), which plays a key role in an early step of cap-dependent RNA splicing. Activation of the CBC by Cdc42 can be inhibited by rapamycin. Additionally, phosphatidylinositol 3-kinase and the Cdc42 effector, pp70 S6 kinase, stimulate the RNA cap-binding activity of the CBC. S6 kinase may directly target the CBC in vivo as it can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are subject to a growth factor-dependent and rapamycin-sensitive phosphorylation in vivo. Together these data suggest the involvement of a Cdc42-S6 kinase pathway in the regulation of RNA splicing, mediated by an increase in capped RNA binding by the CBC, as well as raise the possibility that the effects of Cdc42 on cell growth may be due in part to its regulation of RNA processing.
Computational analysis of sequences of proteins involved in translation initiation in eukaryotes reveals a number of specific domains that are not represented in bacteria or archaea. Most of these eukaryote-specific domains are known or predicted to possess an alpha-helical structure, which suggests that such domains are easier to invent in the course of evolution than are domains of other structural classes. A previously undetected, conserved region predicted to form an alpha-helical domain is delineated in the initiation factor eIF4G, in Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), in the nuclear cap-binding CBP80, and in other, poorly characterized proteins, which is named the NIC (NMD2, eIF4G, CBP80) domain. Biochemical and mutagenesis data on NIC-containing proteins indicate that this predicted domain is one of the central adapters in the regulation of mRNA processing, translation, and degradation. It is demonstrated that, in the course of eukaryotic evolution, initiation factor eIF4G, of which NIC is the core, conserved portion, has accreted several additional, distinct predicted domains such as MI (MA-3 and eIF4G ) and W2, which probably was accompanied by acquisition of new regulatory interactions.
A number of RNA-binding proteins are associated with mRNAs in both the nucleus and the cytoplasm. One of these, Npl3p, is a heterogeneous nuclear ribonucleoprotein-like protein with some similarity to SR proteins and is essential for growth in the yeast S. cerevisiae. Temperature-sensitive alleles have defects in the export of mRNA out of the nucleus (1). In this report, we define a genetic relationship between NPL3 and the nonessential genes encoding the subunits of the cap-binding complex (CBP80 and CBP20). Deletion of CBP80 or CBP20 in combination with certain temperature-sensitive npl3 mutant alleles fail to grow and thus display a synthetic lethal relationship. Further evidence of an interaction between Npl3p and the cap-binding complex was revealed by co-immunoprecipitation experiments; Cbp80p and Cbp20p specifically co-precipitate with Npl3p. However, the interaction of Npl3p with Cbp80p depends on both the presence of Cbp20p and RNA. In addition, we show that Cbp80p is capable of shuttling between the nucleus and the cytoplasm in a manner dependent on the ongoing synthesis of RNA. Taken together, these data support a model whereby mRNAs are co-transcriptionally packaged by proteins including Npl3p and cap-binding complex for export out of the nucleus.
The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.
Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.
In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.
Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T-NLS peptides was co-injected, the nuclear import rate of SV40 T-NLS-containing substrates apparently increased. This up-regulation was reproduced by the co-injection of peptides containing bipartite type NLS of CBP80, but not mutated non-functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth-arrested cells, the nuclear import rate was down-regulated compared to growing cells, the elevation of the functional import rate by co-injected NLS peptides reached the same level as in proliferating cells. This up-regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS-dependent phosphorylation reaction.
Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated on arginine residues. In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p. Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export. Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A)+ RNA, however, is not affected by the lack of methylation. Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80). Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus.
hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.
In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.
Cap structures are added cotranscriptionally to all RNA polymerase II transcripts. They affect several processes including RNA stability, pre-messenger RNA splicing, RNA export from the nucleus and translation initiation. The effect of the cap on translation is mediated by the initiation factor eIF-4F, whereas the effect on pre-mRNA splicing involves a nuclear complex (CBC) composed of two cap binding proteins, CBP80 and CBP20. A role for CBC in the nuclear export of capped RNAs has also been proposed. We report here the characterization of human and Xenopus CBP20s. Antibodies against recombinant CBP20 prevent interaction of CBC with capped RNAs in vitro. Following microinjection into Xenopus oocytes, the antibodies inhibit both pre-mRNA splicing and export of U small nuclear RNAs to the cytoplasm. These results demonstrate that CBC mediates the effect of the cap structure in U snRNA export, and provide direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm.
A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.
Electrical cell-substrate impedance sensing is increasingly being used for label-free and real-time monitoring of changes in cell morphology and number during cell growth, drug screening, and differentiation. In this study, we evaluated the feasibility of using ECIS to monitor C2C12 myoblast differentiation using a fabricated indium tin oxide (ITO) electrode-based chip. C2C12 myoblast differentiation on the ITO electrode was validated based on decreases in the mRNA level of MyoD and increases in the mRNA levels of myogenin and myosin heavy chain (MHC). Additionally, MHC expression and morphological changes in myoblasts differentiated on the ITO electrode were comparable to those in cells in the control culture dish. From the monitoring the integration of the resistance change at 21.5 kHz, the cell differentiation was label-free and real-time detectable in 30 h of differentiation (p < 0.05).
Postnatal growth and repair of skeletal muscle relies upon a population of quiescent muscle precursor cells, called satellite cells that can be activated to proliferate and differentiate into new myofibers, as well as self-renew to replenish the satellite cell population. The balance between differentiation and self-renewal is critical to maintain muscle tissue homeostasis, and alterations in this equilibrium can lead to chronic muscle degeneration. The transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is expressed in Pax7(+) satellite cells of healthy muscle and is downregulated during myoblast differentiation. Persistent expression of C/EBPβ upregulates Pax7, inhibits MyoD, and blocks myogenic differentiation.
Using genetic tools to conditionally abrogate C/EBPβ expression in Pax7(+) cells, we examined the role of C/EBPβ in self-renewal of satellite cells during muscle regeneration.
We find that loss of C/EBPβ leads to precocious differentiation at the expense of self-renewal in primary myoblast and myofiber cultures. After a single muscle injury, C/EBPβ-deficient satellite cells fail to self-renew resulting in a reduction of satellite cells available for future rounds of regeneration. After a second round of injury, muscle regeneration is impaired in C/EBPβ conditional knockout mice compared to wild-type control mice. We find that C/EBPβ can regulate Notch2 expression and that restoration of Notch activity in myoblasts lacking C/EBPβ prevents precocious differentiation.
These findings demonstrate that C/EBPβ is a novel regulator of satellite cell self-renewal during muscle regeneration acting at least in part through Notch2.
We evaluated whether strength training (ST) performed prior to skeletal muscle cryolesion would act as a preconditioning, improving skeletal muscle regeneration and responsiveness to low-level laser therapy (LLLT). Wistar rats were randomly assigned into non-exercised (NE), NE plus muscle lesion (NE + LE), NE + LE plus LLLT (NE + LE + LLLT), strength training (ST), ST + LE, and ST + LE + LLLT. The animals performed 10 weeks of ST (climbing ladder; 3× week; 80% overload). Forty-eight hours after the last ST session, tibialis anterior (TA) cryolesion was induced and LLLT (InGaAlP, 660 nm, 0.035 W, 4.9 J/cm(2)/point, 3 points, spot light 0.028 cm(2), 14 J/cm(2)) initiated and conducted daily for 14 consecutive days. The difference between intergroups was assessed using Student's t test and intragroups by two-way analysis of variance. Cryolesion induced massive muscle degeneration associated with inflammatory infiltrate. Prior ST improved skeletal regeneration 14-days after cryolesion and potentiated the regenerative response to LLLT. Cryolesion induced increased TNF-α levels in both NE + LE and ST + LE groups. Both isolated ST and LLLT reduced TNF-α to control group levels; however, prior ST potentiated LLLT response. Both isolated ST and LLLT increased IL-10 levels with no additional effect. In contrast, increased TA IL-6 levels were restricted to ST and ST + LE + LLLT groups. TA myogenin mRNA levels were not changed by neither prior ST or ST + LLLT. Both prior ST and LLLT therapies increased MyoD mRNA levels and, interestingly, combined therapies potentiated this response. Myf5 mRNA levels were increased only in ST groups. Taken together, our data provides evidences for prior ST potentiating LLLT efficacy in promoting skeletal muscle regeneration.
Though macrophages are essential for skeletal muscle regeneration, which is a complex process, the roles and mechanisms of the macrophages in the process of muscle regeneration are still not fully understood. The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle. One hundred and twelve C57BL/6 mice were randomly divided into muscle contusion and macrophages depleted groups. Their gastrocnemius muscles were harvested at the time points of 12h, 1, 3, 5, 7, 14d post-injury. The changes in skeletal muscle morphology were assessed by hematoxylin and eosin (HE) stain. The gene expression was analyzed by real-time polymerase chain reaction. The data showed that CL-liposomes treatment did affect the expression of myogenic regulatory factors (MyoD, myogenin) after injury. In addition, CL-liposomes treatment decreased the expression of regulatory factors of muscle regeneration (HGF, uPA, COX-2, IGF-1, MGF, FGF6), and increased the expression of inflammatory cytokines (TGF-β1, TNF-α, IL-1β, RANTES) in the late stage of regeneration. Moreover, there were significant correlations between macrophages and some regulatory factors (such as HGF, uPA) for muscle regeneration. These results suggested that macrophages depletion impairs skeletal muscle regeneration, and that the regulatory factors for muscle regeneration may play important roles in this process.
The developmental toxicity of nickel was examined in the embryos of Bombina orientalis, a common amphibian in Korea. Based on a standard frog embryo teratogenesis assay, the LC50 and EC50 for malformation of nickel after 168h of treatment were 33.8μM and 5.4μM, respectively. At a lethal concentration (100μM), nickel treatment decreased the space between gill filaments and caused epithelial swelling and abnormal fusion of gill filaments. These findings suggest that nickel affects the functional development of gills, leading to embryonic death. At sublethal concentrations (1-10μM), nickel produced multiple embryonic abnormalities, including bent tail and tail dysplasia. At 10μM, nickel significantly decreased tail length and tail muscle fiber density in tadpoles, indicating inhibition of myogenic differentiation. Before hatching, the pre-muscular response to muscular response stages (stages 26-31) were the most sensitive period to nickel with respect to tail muscle development. During these stages, MyoD mRNA was upregulated, whereas myogenic regulatory factor 4 mRNA was downregulated by 0.1μM nickel. Calcium-dependent kinase activities in muscular response stage embryos were significantly decreased by nickel, whereas these activities were restored by exogenous calcium. In tadpoles, 10μM nickel significantly decreased the expression of the myosin heavy chain and the 12/101 muscle marker protein in the tail. Expression was restored by exogenous calcium. Our results indicate that nickel affects muscle development by disrupting calcium-dependent myogenesis in developing B. orientalis embryos.
Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.
Quiescent muscle stem cells (MSC) become activated in response to skeletal muscle injury to initiate regeneration. Activated MSCs proliferate and differentiate to repair damaged fibers or self-renew to maintain the pool and insure future regeneration. The balance between self-renewal, proliferation and differentiation is a tightly regulated process controlled by a genetic cascade involving determinant transcription factors such as Pax7, Myf5, MyoD, and MyoG. Recently there have been several reports about the role of arginine methylation as a requirement for epigenetic-mediated control of muscle regeneration. Herein we report that the protein arginine methyltransferase 1 (PRMT1) is expressed in MSCs and that conditional ablation of PRMT1 in MSCs using Pax7(CreERT2) causes impairment of muscle regeneration. Importantly, PRMT1-deficient MSCs have enhanced cell proliferation after injury, but are unable to terminate the myogenic differentiation program, leading to regeneration failure. We identify the co-activator of Six1, Eya1 as a substrate of PRMT1. We show that PRMT1 methylates Eya1 in vitro and loss of PRMT1 function in vivo prevents Eya1 methylation. Moreover, we observe that PRMT1-deficient MSCs have reduced expression of Eya1/Six1 targets MyoD due to disruption of Eya1 recruitment at MyoD promoter and subsequent Eya1-mediated co-activation. These findings suggest that arginine methylation by PRMT1 regulates muscle stem cell fate through the Eya1/Six1/MyoD axis.
Skeletal muscle satellite cells (SC) play an important role in muscle adaptation. In untrained individuals, SC content and activation status has been observed to increase in response to a single bout of exercise. Muscle fiber characteristics change considerably when resistance exercise is performed chronically, but whether training status affects the activity of SC in response to a single bout of exercise remains unknown. We examined the changes in SC content and activation status following a single bout of resistance exercise, prior to and following a 16wk progressive resistance training (RT) program in fourteen young (25±3yr) men. Before and after RT, percutaneous biopsies from the vastus lateralis muscle were taken prior to a single bout of resistance exercise and after 24 and 72h of post-exercise recovery. Muscle fiber size, capillarization, and SC response were determined by immunohistochemistry. Following RT, there was a greater activation of SC after 24h in response to a single bout of resistance exercise (Pre:1.4±0.3,24h:3.1±0.3 Pax7+/MyoD+ cells/100 fibers) as compared to before RT (Pre:1.4±0.3,24h:2.2±0.3 Pax7+/MyoD+ cells/100 fibers, p<0.05); no difference was observed 72h post-exercise. Following 16wk of RT, MyoD mRNA expression increased from basal to 24h after the single bout of exercise (p<0.05); this change was not observed prior to training. Individual capillary-to-fiber ratio (C/Fi) increased in both type I (1.8±0.3 to 2.0±0.3 C/Fi, p<0.05) and type II (1.7±0.3 to 2.2±0.3 C/Fi, p<0.05) fibers in response to RT. Following RT, enhanced activation of SC in response to resistance exercise is accompanied by increases in muscle fiber capillarization.
MyoD is a master regulator of myogenesis with a potent ability to redirect the cell fate of even terminally differentiated cells. Hence, enhancing the activity of MyoD is an important step to maximising its potential utility for in vitro disease modelling and cell replacement therapies. We have previously shown that the reprogramming activity of several neurogenic bHLH proteins can be substantially enhanced by inhibiting their multi-site phosphorylation by proline-directed kinases. Here we have used Xenopus embryos as an in vivo developmental and reprogramming system to investigate the multi-site phospho-regulation of MyoD during muscle differentiation. We show that, in addition to modification of a previously well-characterised site, Serine 200, MyoD is phosphorylated on multiple additional serine/threonine sites during primary myogenesis. Through mutational analysis, we derive an optimally active phospho-mutant form of MyoD that has a dramatically enhanced ability to drive myogenic reprogramming in vivo. Mechanistically, this is achieved through increased protein stability and enhanced chromatin association. Therefore, multi-site phospho-regulation of class II bHLH proteins is conserved across cell lineages and germ layers, and manipulation of phosphorylation of these key regulators may have further potential for enhancing mammalian cell reprogramming.
To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.
In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells.
The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (QM7 MyoD KO #4) through single cell pick-up and expansion. In the undifferentiated condition, Pax7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD QM7 KO#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD QM7 KO#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation.
With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.
Chicken skeletal muscle satellite cells are located between the basement membrane and the sarcolemma of mature muscle fibers. Avian broilers have been genetically selected based on their high growth velocity and large muscle mass. The Wuding chicken is a famous local chicken in Yunnan Province that undergoes non-selection breeding and is slow growing. In this study, we aimed to explore differences in the proliferation and differentiation properties of satellite cells isolated from the two chicken breeds. Using immunofluorescence, hematoxylin-eosin staining and real-time polymerase chain reaction analysis, we analyzed the in vitro characteristics of proliferating and differentiating satellite cells isolated from the two chicken breeds. The growth curve of satellite cells was S-shaped, and cells from Wuding chickens entered the logarithmic phase and plateau phase 1 day later than those from Avian chicken. The results also showed that the two skeletal muscle satellite cell lines were positive for Pax7, MyoD and IGF-1. The expression of Pax7 followed a downward trend, whereas that of MyoD and IGF-1 first increased and subsequently decreased in cells isolated from the two chickens. These data indicated that the skeletal muscle satellite cells of Avian chicken grow and differentiate faster than did those of Wuding chickens. We suggest that the methods of breeding selection applied to these breeds regulate the characteristics of skeletal muscle satellite cells to influence muscle growth.
Muscles of old animals are injured more easily and regenerate poorly, attributed in part to increased levels of circulating pro-inflammatory cytokines. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade is a key mediator of inflammatory cytokine action, and signaling via this pathway is increased in muscles with aging. As a negative regulator of JAK/STAT signaling, a key mediator of myogenic proliferation and differentiation, altered expression of suppressor of cytokine signaling (SOCS3) is likely to have important consequences for muscle regeneration. To model this scenario, we investigated the effect of SOCS3 deletion within mature muscle fibers on injury and repair. We tested the hypothesis that reduced SOCS3 function would alter the inflammatory response and impair muscle regeneration after myotoxic injury.
Mice with a specific deletion of SOCS3 within mature skeletal muscle fibers were used to assess the effect of SOCS3 deletion on muscle injury and repair. Twelve-week-old or 24-month-old SOCS3 muscle-specific knockout (SOCS3 MKO) mice and littermate controls were either left uninjured or injured with a single injection of notexin (10 μg/ml) into the right tibialis anterior (TA) muscle. At 1, 2, 3, 5, 7, or 14 days post-injury, the right TA muscle was excised and subjected to histological, western immunoblotting, and gene expression analyses. Force production and fatigue were assessed in uninjured muscles and at 7 days post-notexin injury.
In uninjured muscles, SOCS3 deletion decreased force production during fatigue but had no effect on the gross or histological appearance of the TA muscles. After notexin injury, deletion of SOCS3 increased STAT3 phosphorylation at day 1 and increased the mRNA expression of the inflammatory cytokine TNF-α, and the inflammatory cell markers F4/80 and CD68 at day 2. Gene expression analysis of the regeneration markers Pax7, MyoD, and Myogenin indicated SOCS3 deletion had no effect on the progression of muscle repair after notexin injury. Inflammation and regeneration were also unchanged in the muscles of 24-month-old SOCS3 MKO mice compared with control.
Loss of SOCS3 expression in mature muscle fibers increased the inflammatory response to myotoxic injury but did not impair muscle regeneration in either adult or old mice. Therefore, reduced SOCS3 expression in muscle fibers is unlikely to underlie impaired muscle regeneration. Further investigation into the role of SOCS3 in other cell types involved in muscle repair is warranted.
MicroRNAs (miRNAs) are highly conserved, short non-coding RNAs that regulate gene expression at the posttranscriptional level. Although many miRNAs are identified in muscles and muscle cells, their individual roles are still not fully understood. In the present study, we investigated a muscle highly-expressed miRNA, miR-127-3p, in C2C12 myoblasts and tissues of goats with different muscle phenotypes (Boer vs Wushan black goats). Our results demonstrated that i) miR-127-3p was extensively expressed in tissues of goats; ii) miR-127-3p was higher expressed in muscle, spleen, heart, and skin in the muscular goats (Boer goats) than the control (Wushan black goats). Then we further characterized the dynamical expression of miR-127-3p, MyoD, MyoG, Myf5, Mef2c, and Myosin in the proliferating and differentiating C2C12 myoblasts at day of 0, 1, 3, 5, and 7 in culture mediums. Especially, we found that miR-127-3p was significantly higher expressed in the proliferating than differentiating cells. Our findings suggest that miR-127-3p probably plays roles in the proliferation and differentiation of myoblasts, which further underlies regulation of muscle phenotype in goats.
TPA Induced Sequence 7 acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. We and others have shown that TIS7 regulates adult myogenesis through MyoD, one of the essential myogenic regulatory factors.
Here, we present data identifying ICln as the specific, novel protein downstream of TIS7 controlling myogenesis. We show that TIS7/ICln epigenetically regulate myoD expression controlling protein methyl transferase activity. In particular, ICln regulates MyoD expression via its interaction with PRMT5 by an epigenetic modification that utilizes symmetrical di-methylation of histone H3 on arginine 8. We provide multiple evidences that TIS7 directly binds DNA, which is a functional feature necessary for its role in transcriptional regulation.
We present here a molecular insight into TIS7-specific control of MyoD gene expression and thereby skeletal muscle differentiation.
Protein 4.1R (4.1R) isoforms are expressed in both cardiac and skeletal muscle. 4.1R is a component of the contractile apparatus. It is also associated with dystrophin at the sarcolemma in skeletal myofibers. However, the expression and function of 4.1R during myogenesis have not been characterized. We now report that 4.1R expression increases during C2C12 myoblast differentiation into myotubes. Depletion of 4.1R impairs skeletal muscle differentiation and is accompanied by a decrease in the levels of myosin heavy and light chains and caveolin-3. Furthermore, the expression of myogenin at the protein, but not mRNA, level is drastically decreased in 4.1R knockdown myocytes. Similar results were obtained using MyoD-induced differentiation of 4.1R(-/-) mouse embryonic fibroblast cells. von Hippel-Lindau (VHL) protein is known to destabilize myogenin via the ubiquitin-proteasome pathway. We show that 4.1R associates with VHL and, when overexpressed, reverses myogenin ubiquitination and stability. This suggests that 4.1R may influence myogenesis by preventing VHL-mediated myogenin degradation. Together, our results define a novel biological function for 4.1R in muscle differentiation and provide a molecular mechanism by which 4.1R promotes myogenic differentiation.
The cdk inhibitor p57(kip2), encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.
Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.
I-mfa (inhibitor of the MyoD family a) is a transcription modulator that binds to MyoD family members and inhibits their transcriptional activities. It is highly expressed in the sclerotome and plays an important role in the patterning of the somite early in development. In this study, the polymorphisms of the bovine I-mfa gene were detected by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and DNA pool sequencing methods in 541 individuals from three Chinese cattle breeds. The results showed that P3 locus had two novel complete linked single nucleotide polymorphisms (NC_007324.4:g.12284A>G and g.12331T>C), resulting in a missense mutation p.S(AGC)113G(GGC) and a synonymous mutation p.H(CAT)128H(CAC), respectively. P4 locus had a novel SNP (NC_007324.4: g.16432C>A), which resulted in a nonsense mutation p.C(TGC)241X(TGA). The statistical analyses indicated that the three SNPs, are associated with the phenotypic traits in Luxi (LX), Qinchuan (QC), and Jiaxian (JX) cattle population (P < 0.05 or P < 0.01). The mutant-type variants were superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to wild-type homozygote. Our results provide evidence that polymorphisms in the I-mfa gene are associated with growth traits and may be used for marker-assisted selection in beef cattle breeding program.
Ca(2+) signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca(2+) stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ε) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ε in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel, mediating store-operated currents. TRPC1ε physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ε-Orai1 complex through TRPC1ε suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ε and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.
The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. Here we implicate Hand1 as a molecular switch that determines whether a trophoblast stem cell continues to proliferate or commits to differentiation. We identify a novel interaction of Hand1 with a protein that contains an I-mfa (inhibitor of myogenic factor) domain that anchors Hand1 in the nucleolus where it negatively regulates Hand1 activity. In the trophoblast stem-cell line Rcho-1, nucleolar sequestration of Hand1 accompanies sustained cell proliferation and renewal, whereas release of Hand1 into the nucleus leads to its activation, thus committing cells to a differentiated giant-cell fate. Site-specific phosphorylation is required for nucleolar release of Hand1, for its dimerization and biological function, and this is mediated by the non-canonical polo-like kinase Plk4 (Sak). Sak is co-expressed in Rcho-1 cells, localizes to the nucleolus during G2 and phosphorylates Hand1 as a requirement for trophoblast stem-cell commitment to a giant-cell fate. This study defines a novel cellular mechanism for regulating Hand1 that is a crucial step in the stem-cell differentiation pathway.
Transient receptor potential canonical 1 (TRPC1) is a transmembrane protein expressed in a range of vertebrate cells including smooth muscle, endothelium, neurones and salivary gland cells. It functions as an element of a mixed cationic Ca(2+)-permeable channel, probably commonly as part of a heterotetrameric assembly involving other related proteins such as TRPC5. Wide-ranging biological roles of TRPC1 are suggested, including regulation of smooth muscle and stem cell proliferation, endothelin-evoked arterial contraction, salivary gland secretion, endothelial permeability, glutamatergic neurotransmission, growth cone turning, neuroprotection, neuronal differentiation, lipid raft integrity and the nuclear factor of activated T-cell transcription factor. The mechanisms by which TRPC1 serves these functions are starting to emerge. At one level, it is apparent that TRPC1 is subcellularly compartmentalised, at least in part in cholesterol-rich caveolae closely associated with sub-plasmalemmal endoplasmic reticulum. At another level, TRPC1 is embedded in a protein complex that can include inositol trisphosphate receptor, homer, calmodulin, caveolin-1, FKBP25, I-mfa, MxA, GluR1alpha, bFGFR-1, G(q/11) protein, phospholipase C-beta/gamma, protein kinase C-alpha and RhoA. It is also apparent that TRPC1 responds to general stimuli-not only depletion of intracellular Ca(2+) stores, but also receptor activation, and membrane stretch. We are at the early stages of understanding of how these various signals and components integrate to form a functional channel, and this article provides a brief overview of current progress.
In transgenic mice, homozygous mutations of trophoblast-specific transcription factors such as Hand1, Mash-2, I-mfa or GCM1 revealed their key regulatory roles in induction, maintenance or differentiation of distinct placental trophoblast subpopulations in vivo. Descriptive studies have shown that several of these factors are also expressed in the human placenta, suggesting that the molecular mechanisms governing trophoblast differentiation could be similar in mice and men. While an increasing number of putative developmental regulators are being identified in the human placenta, little information is available regarding whether the particular factors play an essential role in trophoblast differentiation processes such as formation of anchoring villi, placental bed invasion or syncytialization. However, expression of abundant trophoblast-specific products such as hormones can be regarded as a hallmark of differentiation, suggesting that the factors controlling their transcription could also be involved in the developmental processes of the placenta. Indeed, studies in different model systems revealed that the human homologues of murine trophoblast-specific transcriptional regulators interact with the promoter regions of typical placental genes such as aromatase P450 (CYP19), chorionic gonadotrophin (CG) or placental lactogen (PL). Additionally, the unique combination of more broadly distributed transcription factors of the Sp or Ap-2 protein family in a particular trophoblast cell type is required to govern mRNA expression in a differentiation-dependent manner. Here, we will summarize our present knowledge on these individual transcription factors that are involved in human trophoblast function and differentiation.
We have determined that I-mfa, an inhibitor of several basic helix-loop-helix (bHLH) proteins, and XIC, a Xenopus ortholog of human I-mf domain-containing protein that shares a highly conserved cysteine-rich C-terminal domain with I-mfa, inhibit the activity and DNA binding of the HMG box transcription factor XTcf3. Ectopic expression of I-mfa or XIC in early Xenopus embryos inhibited dorsal axis specification, the expression of the Tcf3/beta-catenin-regulated genes siamois and Xnr3, and the ability of beta-catenin to activate reporter constructs driven by Lef/Tcf binding sites. I-mfa domain proteins can regulate both the Wnt signaling pathway and a subset of bHLH proteins, possibly coordinating the activities of these two critical developmental pathways.
The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability.
Zic3 controls neuroectodermal differentiation and left-right patterning in Xenopus laevis embryos. Here we demonstrate that Zic3 can suppress Wnt/β-catenin signaling and control development of the notochord and Spemann's organizer. When we overexpressed Zic3 by injecting its RNA into the dorsal marginal zone of 2-cell-stage embryos, the embryos lost mesodermal dorsal midline structures and showed reduced expression of organizer markers (Siamois and Goosecoid) and a notochord marker (Xnot). Co-injection of Siamois RNA partially rescued the reduction of Xnot expression caused by Zic3 overexpression. Because the expression of Siamois in the organizer region is controlled by Wnt/β-catenin signaling, we subsequently examined the functional interaction between Zic3 and Wnt signaling. Co-injection of Xenopus Zic RNAs and β-catenin RNA with a reporter responsive to the Wnt/β-catenin cascade indicated that Zic1, Zic2, Zic3, Zic4, and Zic5 can all suppress β-catenin-mediated transcriptional activation. In addition, co-injection of Zic3 RNA inhibited the secondary axis formation caused by ventral-side injection of β-catenin RNA in Xenopus embryos. Zic3-mediated Wnt/β-catenin signal suppression required the nuclear localization of Zic3, and involved the reduction of β-catenin nuclear transport and enhancement of β-catenin degradation. Furthermore, Zic3 co-precipitated with Tcf1 (a β-catenin co-factor) and XIC (I-mfa domain containing factor required for dorsoanterior development). The findings in this report produce a novel system for fine-tuning of Wnt/β-catenin signaling.
The I-mfa domain proteins I-mfa and HIC are considered to be candidate tumor suppressor genes and have been shown to be involved in transcriptional regulation. We show here that I-mfa and HIC specifically interact with SEI-1 through their C-terminal I-mfa domains in vivo. This interaction affects the intracellular localization of I-mfa and requires the region of SEI-1 between 30 and 90 amino acids, which includes its SERTA domain, and results in repression of its intrinsic transcriptional activity. I-mfa also decreases the levels of the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA, the expression of which is coregulated by E2F·DP-1 and SEI-1 in an interaction-dependent manner in vitro. In addition, I-mfa also specifically interacts with other SERTA domain-containing proteins, including SEI-2, SEI-3, SERTAD3 and SERTAD4, through its I-mfa domain in vivo. This interaction also affects the intracellular localization of I-mfa and represses the intrinsic transcriptional activities of SEI-2 and SERTAD3, which are also involved in the E2F-dependent transcription. These data reveal for the first time that I-mfa domain proteins interact with SERTA domain proteins and negatively regulate their transcriptional activity. Because SEI-1, SEI-2 and SERTAD3, whose intrinsic transcriptional activities are repressed by I-mfa, are suggested to be oncogenes, I-mfa domain proteins may be involved in their oncogenic functions by negatively regulating their transcriptional activities.
The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.
In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion.
Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.
Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3beta (GSK-3beta) and to negatively regulate its activity, leading to stimulation of GSK-3beta-dependent beta-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a beta-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3beta complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3beta complex.
Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5' and 3' untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3'-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5'-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.
The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies.
Porcine reproductive and respiratory syndrome (PRRS) virus is an RNA virus that replicates in the cytoplasm, but the viral nucleocapsid (N) protein localizes specifically in the nucleus and nucleolus of virus-infected cells. Nuclear localization of N is non-essential for PRRSV replication in cultured cells but has been shown to modulate the pathogenesis of virus in pigs, suggesting that N plays an accessory role in the nucleus during infection. We identified by yeast two-hybrid screening the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) as a cellular partner for PRRS virus (PRRSV) N protein. This protein is a homolog of human HIC, a recently identified cellular transcription factor. The specific interaction of PRRSV N with HIC was confirmed in cells by mammalian two-hybrid assay and co-immunoprecipitation and in vitro by GST pull-down assay. HIC is a zinc-binding protein and confocal microscopy demonstrated co-localization of N with the HIC-p40 isomer in the nucleus and nucleolus, and in the cytoplasm with HIC-p32, which is the N-terminal truncation of HIC-p40. The porcine homolog of HIC is universally expressed in pig tissues including alveolar macrophages. The interaction of viral capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection.
Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70-80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3' untranslated region (3'UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3'UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3'UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-gamma in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3'UTR in CHO cells.
The auxiliary spliceosomal protein SCNM1 contributes to recognition of nonconsensus splice donor sites. SCNM1 was first identified as a modifier of the severity of a sodium channelopathy in the mouse. The most severely affected strain, C57BL/6J, carries the variant allele SCNM1R187X, which is defective in splicing the mutated donor site in the Scn8a(medJ) transcript. To further probe the in vivo function of SCNM1, we constructed a floxed allele and generated a mouse with constitutive deletion of exons 3-5. The SCNM1Delta3-5 protein is produced and correctly localized to the nucleus, but is more functionally impaired than the C57BL/6J allele. Deficiency of SCNM1 did not significantly alter other brain transcripts. We characterized an ENU-induced allele of Scnm1 and evaluated the ability of wild-type SCNM1 to rescue lethal mutations of I-mfa and Brunol4. The phenotypes of the Scnm1Delta3-5 mutant confirm the role of this splice factor in processing the Scn8a(medJ) transcript and provide a new allele of greater severity for future studies.
The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis but how its developmental activity is regulated is largely unknown. We recently showed that Hand1 is sequestered in the nucleoli of rodent trophoblast stem (TS) cells by the I-mfa domain-containing protein HICp40 and that this is associated with their proliferation and continuing self-renewal. However when these cells commit to differentiate into trophoblast giant (TG) cells, Hand1 is phosphorylated by the polo-like kinase Plk4 (Sak) and released into the nucleus to activate downstream target genes. This event underlies the release of Hand1 from the nucleolus and represents the 'molecular switch' that promotes mitotic cell cycle exit and the onset of endoreduplication. In this brief discussion we examine the wider implications of these findings and address some of the unanswered questions that remain.
The positive transcription elongation factor P-TEFb is a pivotal regulator of gene expression in higher cells. Originally identified in Drosophila, attention was drawn to human P-TEFb by the discovery of its role as an essential cofactor for HIV-1 transcription. It is recruited to HIV transcription complexes by the viral transactivator Tat, and to cellular transcription complexes by a plethora of transcription factors. P-TEFb activity is negatively regulated by sequestration in a complex with the HEXIM proteins and 7SK RNA. The mechanism of P-TEFb release from the inhibitory complex is not known. We report that P-TEFb-dependent transcription from the HIV promoter can be stimulated by the mRNA encoding HIC, the human I-mfa domain-containing protein. The 3'-untranslated region of HIC mRNA is necessary and sufficient for this action. It forms complexes with P-TEFb and displaces 7SK RNA from the inhibitory complex in cells and cell extracts. A 314-nucleotide sequence near the 3' end of HIC mRNA has full activity and contains a predicted structure resembling the 3'-terminal hairpin of 7SK that is critical for P-TEFb binding. This represents the first example of a cellular mRNA that can regulate transcription via P-TEFb. Our findings offer a rationale for 7SK being an RNA transcriptional regulator and suggest a practical means for enhancing gene expression.
Positive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for P-TEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat- and P-TEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication, and suggest that P-TEFb is modulated in the Wnt signaling pathway.
We have previously shown that beta-catenin interacts with a transcription suppressor I-mfa and, through this interaction, canonical Wnt signaling could relieve I-mfa-mediated suppression of myogenic regulatory factors (MRFs). In this study, we found that, based on this interaction, I-mfa-mediated suppression of the Wnt transcription factor T-cell factor/lymphoid enhancing factor-1 (TCF/LEF-1) can also be relieved. Our work showed that knocking down endogenous I-mfa expression mimics canonical Wnt treatment by inducing myogenesis and increasing Wnt reporter gene activity, endogenous Wnt target gene expression and expression of MRFs in P19 cells. More importantly, these I-mfa small interfering RNA (siRNA)-induced effects could be blocked by a dominant-negative mutant of LEF-1, confirming the involvement of the TCF/LEF-1 pathway. In addition, we found that beta-catenin could compete with I-mfa for binding to LEF-1 and relieve the inhibitory effects of I-mfa in overexpression systems. Furthermore, canonical Wnt was able to reduce the levels of endogenous I-mfa associated with LEF-1, while increasing that of I-mfa associated with beta-catenin. All of the evidence supports a conclusion that I-mfa can suppress myogenesis by inhibiting TCF/LEF-1 and that canonical Wnt signaling may relieve the suppression through elevating beta-catenin levels, which in turn relieve I-mfa-mediated suppression.
We studied monosomy and deletions of chromosome 7 in 170 patients with myeloid disorders and we identified a minimal region of loss in 7q31.1 spanning between the D7S2554 and D7S2460 markers. The closest gene to our most deleted microsatellite, D7S2554, is the human I-mfa domain containing (HIC) gene, alias MyoD family inhibitor domain containing (MDFIC). We investigated the involvement of HIC in myeloid neoplasms by screening for mutations the coding regions and the intron-exon boundaries of this gene in 15 patients who presented chromosome 7 deletions in the region of HIC. No mutations were found in the coding region of this gene.
Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.
mPem, a homeobox gene, is expressed in a time and stage specific manner during murine ontogeny. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are not detectable in most of adult tissues, they are present in reproductive system such as testis, epididymis and ovary. This indicates a important role for Pem during embryogenesis and reproductive development. To study the function of mPem protein, we used a GAL4 based yeast two-hybrid assay to screen a 7-day mouse embryo library with full-length of mPem. 3 proteins were found interacting with mPem protein. One of theses is Mdfic. We confirmed the interaction between mPem and Mdfic in yeast and in vitro. Mdfic, MyoD family inhibitor domain containing, encodes the myoD family inhibitor domain (I-mfa domain). The interaction between mPem and Mdfic suggested they maybe form the transcriptional regulator complex to regulate embryo differentiation.
Wnt/beta-catenin signaling plays a critical role in embryonic myogenesis. Here we show that, in P19 embryonic carcinoma stem cells, Wnt/beta-catenin signaling initiates the myogenic process depends on beta-catenin-mediated relief of I-mfa (inhibitor of MyoD Family a) suppression of myogenic regulatory factors (MRFs). We found that beta-catenin interacted with I-mfa and that the interaction was enhanced by Wnt3a. In addition, we found that the interaction between beta-catenin and I-mfa was able to attenuate the interaction of I-mfa with MRFs, relieve I-mfa-mediated suppression of the transcriptional activity and cytosolic sequestration of MRFs, and initiate myogenesis in a P19 myogenic model system that expresses exogenous myogenin. This work reveals a mechanism for the regulation of MRFs during myogenesis by elucidating a beta-catenin-mediated, but lymphoid enhancing factor-1/T cell factor independent, mechanism in regulation of myogenic fate specification and differentiation of P19 mouse stem cells.
The primary function of the HIV-1 regulatory protein Tat, activation of transcription from the viral LTR, is highly regulated by complex interactions between Tat and a number of host cell proteins. Tat nuclear import, a process mediated by importin beta, is a prerequisite for its activity. Here, we report and characterize the interaction of the human inhibitor of MyoD family domain-containing protein (I-mfa), HIC, with Tat at a biochemical and a functional level. This interaction was shown to occur in vivo and in vitro and to involve the nuclear localization signal and the transactivation responsive element-binding domains of Tat and the I-mfa domain of HIC. Coexpression of HIC and Tat resulted in the down-regulation of transactivation of the HIV-1 LTR, and colocalization studies revealed the cytoplasmic sequestration of Tat by HIC. Functionally this sequestration appears to be the underlying mechanism of LTR transcriptional repression by HIC and represents a unique mechanism for the control of Tat activity and regulation of HIV-1 replication.
Tissue-specific class B basic helix-loop-helix (bHLH) transcription factors, dimerising with ubiquitously produced class A bHLH proteins, play a major role in murine trophoblast development. Here, we investigated expression patterns of class A and B bHLH factors in the human placenta and different trophoblast culture systems. Semi-quantitative RT-PCR and RNase protection assay revealed expression of the tissue-restricted factors Hash-2, I-mfa and Stra13 in placentae of early and late pregnancy, in purified villous trophoblasts as well as in invasive trophoblasts isolated from first trimester villous explant cultures. Accordingly, RNA in situ hybridisation localised Hash-2, I-mfa and Stra13 to the trophoblast epithelium, cell columns and extravillous trophoblasts invading maternal decidua. Villous stromal cells in situ and cultivated placental fibroblasts also produced I-mfa and Stra13 but failed to express Hash-2. The widely expressed class A proteins, E12/E47 were absent from all placental cell types while ITF-2 was restricted to placental stromal cells of early and late gestation. In contrast, HEB was identified in all trophoblast cell types using RT-PCR, Western blotting and immunohistochemistry. The negative HLH-regulators Id-1 and Id-2 lacking the DNA-binding domain, were detected in villous stromal cells and different cytotrophoblast subtypes but were absent from the syncytium. The data suggest that a complex interplay of activators (Hash-2, HEB) and repressors (Stra13, I-mfa) could be involved in extravillous trophoblast differentiation whereas downregulation of Id proteins could play a role in syncytialisation.
Siamois is the transcriptional mediator of the dorsal Wnt signaling pathway and is necessary for formation of the Spemann organizer and dorsoanterior development in Xenopus. We have determined that XIC, a Xenopus I-mfa domain protein that regulates Tcf3 binding, is required for dorsoaxial development and specifically for Siamois activity in establishing the dorsal organizer. In loss-of-function studies, we found that embryos injected with a morpholino to XIC mRNA (XIC morphpolino) are missing head structures, neural tube, notochord, and paraxial mesoderm as well as NCAM and XMyoD expression. Although Siamois, Twin, and Xnr3 expression is normal in morpholino-injected embryos, levels of downstream organizer factors, including goosecoid, Xnot, Cerberus, and chordin, are severely reduced. Ectopic axis formation induced by Siamois is repressed by injection of the XIC morpholino and further repressed by coinjection of beta-catenin or a constitutively active Tcf3/HMG/G4A fusion. Activation of reporters driven by the Siamois-responsive proximal element of the goosecoid promoter is inhibited in the presence of the morpholino and can be rescued by murine I-mfa and by a dominant-negative Tcf3. The data indicate a role for XIC in limiting Tcf3-dependent repression of Siamois activities that are required for goosecoid transcription and for dorsal organizer formation.
The Epstein-Barr virus (EBV) BamHI A transcripts are a family of transcripts that are differentially spliced and can be detected in multiple EBV-associated malignancies. Several of the transcripts may encode proteins. One transcript of interest, RK-BARF0, is proposed to encode a 279-amino-acid protein with a possible endoplasmic reticulum-targeting sequence. In this study, the properties of RK-BARF0 were examined through identification of cellular-interacting proteins through yeast two-hybrid analysis and characterization of its expression in EBV-infected cells and tumors. In addition to the interaction previously identified with cellular Notch, it was determined that RK-BARF0 also bound cellular human I-mfa domain-containing protein (HIC), epithelin, and scramblase. An interaction between RK-BARF0 and Notch or epithelin induced proteasome-dependent degradation of Notch and epithelin but not of HIC or scramblase. Low levels of endogenous Notch expression in EBV-positive cell lines may correlate with RK-BARF0 expression. However, a screen of EBV-positive cell lines and tumors with an affinity-purified alpha-RK-BARF0 antiserum did not consistently detect RK-BARF0. These data suggest that while RK-BARF0 may have important cellular functions during EBV infection, and while the phenotype of EBV-positive cells suggest its expression, RK-BARF0 levels may be too low to detect.
The Zic-family proteins control various developmental processes. Previous studies have shown that Zic1, Zic2, and Zic3 can act as transcriptional regulators, and that their functions are repressed by I-mfa, which has been identified as a repressor for basic helix-loop-helix-type transcriptional factors. Here, we investigated the molecular properties of the Zic4 and Zic5 proteins. Zic4/Zic5 showed DNA-binding activity to the Gli-binding sequence, similar to Zic1/Zic2/Zic3 proteins. However, Zic4/Zic5 did not exhibit any significant transcriptional activation ability nor they bind to I-mfa differently from Zic1/Zic2/Zic3. The nuclear localization of Zic4/Zic5 was not affected by the presence of the I-mfa protein, whereas the Zic1/Zic2/Zic3 proteins were translocated to the cytoplasmic compartment in the presence of I-mfa. The difference may be attributable to the dissimilarity of the N-terminal region between the Zic1/Zic2/Zic3 and Zic4/Zic5 proteins, since the binding of the Zic1/Zic2/Zic3 proteins to I-mfa occurs through their N-terminal regions.
Zinc finger proteins belonging to the Zic family control several developmental processes such as patterning of the axial skeleton. Here we mapped the transcriptional regulatory domains in Zic2 protein and identified a protein which specifically binds to one of them. In the mapping experiments, an amino-terminal region was identified as transcriptional regulatory domains. A search for proteins binding to the amino terminal domain of Zic2 revealed that inhibitor of MyoD family (I-mfa) protein, which has been identified as a repressor of myogenic helix-loop-helix class transcription factors, can physically interact with the amino terminal domain. When Zic1-3 and I-mfa proteins were co-expressed in cultured cells, nuclear import of the Zic proteins was inhibited. Consequently, I-mfa inhibited transcriptional activation by the Zic proteins in cultured cells. These results suggest that the physical and functional interaction between Zic and I-mfa proteins can play a role in the vertebrate development.
Depletion of intracellular Ca2+ stores leads to the activation of Ca2+ inflow through store-operated Ca2+ channels. Although the identity of these channels is unknown, there is considerable evidence that the transient receptor potential channel 1 (TRPC1) participates in the formation of these channels. We show that TRPC1 physically interacts with the a-isoform of the inhibitor of the myogenic family (I-mfa), a known inhibitor of basic helix-loop-helix transcription factors, in vitro and in vivo. The interaction is mediated by the C-terminal cytoplasmic tail of TRPC1 and the C-terminal cysteine-rich domain of I-mfa. Using the whole cell configuration of the patch clamp technique, we show that ectopic expression of I-mfa in CHO-K1 cells reduces native store-activated Ca2+ currents, whereas knock-down of endogenous I-mfa in A431 cells by RNA interference enhances these currents. Pipette perfusion of purified recombinant I-mfa rescues the effect of I-mfa knock-down on store-operated conductance. Finally, cell dialysis with a monoclonal antibody specific to TRPC1 results in the suppression of store-activated conductance in cells lacking I-mfa, but not in I-mfa expressing cells. We propose that I-mfa functions as a molecular switch to suppress the store dependence of TRPC1.
Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.
We describe here a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain and involved in gene expression regulation. This family presently includes three members: I-mfa (inhibitor of MyoD family), HIC p40 and HIC p32 (human I-mfa domain-containing protein). I-mfa, by interacting with MyoD family members, represses both transcriptional activation and myogenesis mediated by these factors. HIC two isoforms, HIC p40 and HIC p32, are involved in the positive regulation of Tax-mediated HTLV-I (human T-cell leukemia virus type 1) promoter activation and in the negative regulation of Tat-mediated HIV-1 (human immunodeficiency virus type 1) promoter transcription. The common carboxy-terminal region of HIC p40 and HIC p32, which is clearly involved in these regulations, shares 77% homology with the carboxy-terminal domain of I-mfa. This suggests that I-mfa, HIC p40 and HIC p32 are part of a new family of proteins involved in gene expression regulation and characterized by a specific cysteine-rich carboxy-terminal domain. Moreover, the three proteins present different subcellular localizations: I-mfa and HIC p32 are mainly cytoplasmic while HIC p40 is mainly nucleolar. The specific localization of each member of this new family will be discussed, possibly explaining how they work. Effectively, a mechanism of protein sequestration in a particular compartment, cytoplasm or nucleolus, could be involved in their function, as it is the case for many other proteins. This relationship between sequestration and function regulation will be exemplified for several cellular factors.
I-mfa has been identified as an inhibitor of myogenic basic helix-loop-helix transcription factors, and a related human I-mfa domain-containing protein (HIC) also has been identified as a protein that regulates Tat- and Tax-mediated expression of viral promoters. HIC and I-mfa represent a family of proteins that share a highly conserved cysteine-rich domain, termed the I-mfa domain. We show here that both I-mfa domain proteins, HIC and I-mfa, interacted in vivo with the Axin complex through their C-terminal I-mfa domains. This interaction inhibited Axin-mediated downregulation of free levels of cytosolic beta-catenin. I-mfa and HIC also both directly interacted with lymphocyte enhancer factor (LEF); however, I-mfa but not HIC significantly inhibited reporter constructs regulated by beta-catenin. The overexpression of HIC but not I-mfa decreased the inhibitory effects of Axin on beta-catenin-regulated reporter constructs, while both HIC and I-mfa decreased Axin-mediated c-Jun N-terminal kinase (JNK) activation. These data reveal for the first time that I-mfa domain proteins interact with the Axin complex and affect Axin regulation of both the Wnt and the JNK activation pathways. Interestingly, HIC differs from I-mfa in that I-mfa affects both Axin function and T-cell factor- or LEF-regulated transcription in the Wnt signaling pathway while HIC affects primarily Axin function.
Gonadotropins are essential for ovarian follicular development and differentiation. To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure. cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified. Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist. Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the krüppel-associated box-A domain at the NH(2) terminus. RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary. In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis. Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously. A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the krüppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity. A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1. We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins. Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay. Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis.
I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).
The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.
Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.
The bHLH-repressor protein I-mfa binds to MyoD family members, inhibits their activity, and blocks their nuclear import and binding to DNA. In situ hybridization analysis demonstrated that mouse I-mfa was highly expressed in extraembryonic lineages, in the sclerotome, and subsequently within mesenchymal precursors of the axial and appendicular skeleton, before chondrogenesis occurs. Targeted deletion of I-mfa in a C57Bl/6 background resulted in embryonic lethality around E10.5, associated with a placental defect and a markedly reduced number of trophoblast giant cells. Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced differentiation into trophoblast giant cells. I-mfa interacted with the bHLH protein Mash2, a negative regulator of trophoblast giant cell formation, and inhibited its transcriptional activity in cell culture. In contrast, I-mfa did not interfere with the activity of the bHLH protein Hand1, a positive regulator of giant cell differentiation. Interestingly, I-mfa-null embryos on a 129/Sv background had no placental defect, generally survived to adulthood, and exhibited delayed caudal neural tube closure and skeletal patterning defects that included fusions of ribs, vertebral bodies and abnormal formation of spinous processes. Our results indicate that I-mfa plays an important role in trophoblast and chondrogenic differentiation by negatively regulating a subset of lineage-restricted bHLH proteins.
A number of genes, spanning the evolutionary scale from yeast to mammals, that are involved in spatial and temporal patterning during development contain zinc finger and homeodomain motifs. One such zinc finger/homeodomain protein is Drosophila Zfh-1, a member of the zfh family of Drosophila genes, which is expressed in muscle precursors and is critical for the proper development of muscle. Here we demonstrate that a vertebrate homolog of Zfh-1 (ZEB) is a negative regulator of muscle differentiation. We show that ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription. One target of this repression is the members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation. As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription. This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors. The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression.
Investigating intestinal physiology in vitro remains challenging due to the lack of an effective primary enterocyte culture system. Recently developed protocols for growing organoids containing crypts and villus from adult mouse intestinal epithelium in Matrigel present an attractive alternative to the classical techniques. However, these approaches require the use of sophisticated and expensive serum-free medium supplemented with epithelial growth factor (EGF), Wnt agonist (R-spondin 1), and bone morphogenetic protein inhibitor (Noggin) in high concentrations. Here we demonstrate that is possible to use an isolated chicken embryonic intestinal epithelium to create such an organoid culture. Structures formed in Matrigel matrix in the first two days following isolation survive and enlarge during ensuing weeks. They have the appearance of empty spheres and comprise cells expressing cytokeratin (an epithelial cell marker), villin (a marker of enterocytes), and Sox-9 (a transcription factor characteristic of progenitors and stem cells of intestinal crypts). With chicken embryonic tissue as a source of organoids, prostaglandin E2 is as effective as R-spondin 1 and Noggin in promoting sustained growth and survival of epithelial spheroids.
Transcription factor (TF) signaling regulates gene transcription and requires a complex network of proteins. This network includes co-activators, co-repressors, multiple TFs, histone-modifying complexes, and the basal transcription machinery. It has been widely appreciated that pioneer factors, such as FoxA1 and GATA1, play an important role in opening closed chromatin regions, thereby allowing binding of a secondary factor. In this review we will focus on a newly proposed model wherein multiple TFs, such as steroid receptors (SRs), can function in a pioneering role. This model, termed dynamic assisted loading, integrates data from widely divergent methodologies, including genome wide ChIP-Seq, digital genomic footprinting, DHS-Seq, live cell protein dynamics, and biochemical studies of ATP-dependent remodeling complexes, to present a real time view of TF chromatin interactions. Under this view, many TFs can act as initiating factors for chromatin landscape programming. Furthermore, enhancer and promoter states are more accurately described as energy-dependent, non-equilibrium steady states.
Diamond Blackfan anemia (DBA) is a well known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, <i>GATA1</i>, or <i>TSR2</i>, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the <i>RPS19</i> gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some <i>RPS19</i> missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. We have been interested in the application of Spliceosome Mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, the RNA trans-splicing technology offers several advantages: 1) it obviates the need to deliver the entire cDNA of a normal gene, which significantly reduces the transgene size for packaging and delivery; 2) expression of the corrected gene is completely dependent on expression of the endogenous mutant gene, and genes with large and complex transcriptional elements can be repaired in situ while keeping gene expression under the control of endogenous regulatory elements; 3) RNA trans-splicing utilizes the endogenous spliceosome that exists and functions in all types of cells; 4) RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus, may be capable of treating disorders caused by dominant negative mutations such as in DBA.
Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction.
We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).
We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34(+) hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes.
Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells.
Supplementary data are available at Bioinformatics online.
Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34(+) cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38β did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38β-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and provide evidence suggesting a novel regulatory mechanism for p38α through arginine methylation.
Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.
HDAC1-containing NuRD complex is required for GATA-1-mediated repression and activation.
GATA-1 associated with acetylated HDAC1-containing NuRD complex, which has no deacetylase activity, for gene activation.
Acetylated HDAC1 converts NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation program.
HDAC1 acetylation may function as a master regulator for the activity of HDAC1 containing complexes. Histone deacetylases (HDACs) play important roles in regulating cell proliferation and differentiation. The HDAC1-containing NuRD complex is generally considered as a corepressor complex and is required for GATA-1-mediated repression. However, recent studies also show that the NuRD complex is involved in GATA-1-mediated gene activation. We tested whether the GATA-1-associated NuRD complex loses its deacetylase activity and commits the GATA-1 complex to become an activator during erythropoiesis. We found that GATA-1-associated deacetylase activity gradually decreased upon induction of erythroid differentiation. GATA-1-associated HDAC1 is increasingly acetylated after differentiation. It has been demonstrated earlier that acetylated HDAC1 has no deacetylase activity. Indeed, overexpression of an HDAC1 mutant, which mimics acetylated HDAC1, promotes GATA-1-mediated transcription and erythroid differentiation. Furthermore, during erythroid differentiation, acetylated HDAC1 recruitment is increased at GATA-1-activated genes, whereas it is significantly decreased at GATA-1-repressed genes. Interestingly, deacetylase activity is not required for Mi2 remodeling activity, suggesting that remodeling activity may be required for both activation and repression. Thus, our data suggest that NuRD can function as a coactivator or repressor and that acetylated HDAC1 converts the NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation.
Although MLL-AF9 caused by the chromosomal translocation t(9;11) has a critical role in acute myeloid leukemia, the molecular pathogenesis is poorly understood. Here, we identified that the cell fate determination factor DACH1 is directly up-regulated by MLL-AF9. Recently we showed that the forced expression of DACH1 in myeloid cells induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Consistent with our previous study, ectopic expression of DACH1 contributed to the maintenance of colonogenic activity and blocked the differentiation of myeloid progenitors. Moreover, we here identified an endogenous HOXA9-DACH1 complex mediated by the carboxyl terminus of DACH1 in t(9;11) leukemia cells. qRT-PCR revealed that DACH1 has a stronger transcription-promoting activity with HOXA9 than does PBX2 with HOXA9. Furthermore, C/EBPα and GATA-1 can directly bind to the promoter of DACH1 and act as a transcriptional suppressor. Expression of DACH1 is down-regulated during myeloid differentiation and shows an inverse pattern compared to C/EBPα and GATA-1 expression. However, ectopic expression of C/EBPα and/or GATA-1 could not abrogate the over-expression of DACH1 induced by MLL-AF9. Therefore, we postulate that the inability of C/EBPα and GATA-1 to down-regulate DACH1 expression induced by MLL-AF9 during myeloid differentiation may contribute to t(9;11) leukemogenesis.
There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.
Two mechanisms that play important roles in cell fate decisions are control of a "core transcriptional network" and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.
The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.
Transcription factors are essential for blood cell formation. Mice expressing low levels of c-Myb (c-Myb(low)) have an increased number of bone marrow megakaryocytes (MKs) and corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 (GATA-1(low)) in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. These seemingly opposite roles may affect platelet turnover and thus be of clinical relevance.
To determine how these two transcription factors act together to control megakaryocytopoiesis and platelet formation.
We used a combination of cellular and molecular in vitro assays to examine the ability of bone marrow cells from mice expressing low levels of both c-Myb and GATA-1 (referred to as double(low)) to produce MKs and platelets.
Double(low) cells, or those with low GATA-1 levels in which c-Myb is conditionally deleted, lack the hyperproliferative capacity of GATA-1(low) cells, allowing the cells to proceed towards more committed MKs that are, however, impaired in their capacity to produce fully differentiated cells, as confirmed by the abundance of morphologically aberrant cells that lack the ability to form proplatelets.
c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal maturation. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.
cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior-posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway.
During vertebrate development, the initial wave of hematopoiesis produces cells that help to shape the developing circulatory system and oxygenate the early embryo. The differentiation of primitive erythroid and myeloid cells occurs within a short transitory period, and is subject to precise molecular regulation by a hierarchical cascade of transcription factors. The TALE-class homeodomain transcription factors Meis and Pbx function to regulate embryonic hematopoiesis, but it is not known where Meis and Pbx proteins participate in the hematopoietic transcription factor cascade. To address these questions, we have ablated Meis1 and Pbx proteins in zebrafish, and characterized their molecular effects on known markers of primitive hematopoiesis. Embryos lacking Meis1 and Pbx exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate, and fail to produce visible circulating blood cells. Concomitant with a loss of gata1, Meis1- and Pbx-depleted embryos exhibit downregulated embryonic hemoglobin (hbae3) expression, and possess increased numbers of pu.1-positive myeloid cells. gata1-overexpression rescues hbae3 expression in Pbx-depleted; meis1-morphant embryos, placing Pbx and Meis1 upstream of gata1 in the erythropoietic transcription factor hierarchy. Our study conclusively demonstrates that Meis1 and Pbx act to specify the erythropoietic cell lineage and inhibit myelopoiesis.
GATA-1 and PU.1 are essential hematopoietic transcription factors that control erythromegakaryocytic and myelolymphoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alternate lineage programs. We used immortalized Gata1(-) erythromegakaryocytic progenitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoiesis. Both GATA factors bind the PU.1/Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 binding is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 expression, and low-level expression of its downstream myeloid target genes. Restoration of GATA-1 function induces erythromegakaryocytic differentiation. Concomitantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expression is extinguished. In contrast, when GATA-1 is not present, shRNA knockdown of GATA-2 increases PU.1/Sfpi1 expression by 3-fold and reprograms the cells to become macrophages. Our findings indicate that GATA factors act sequentially to regulate lineage determination during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus.
Although leukemogenic tyrosine kinases (LTKs) activate a common set of downstream molecules, the phenotypes of leukemia caused by LTKs are rather distinct. Here we report the molecular mechanism underlying the development of hypereosinophilic syndrome/chronic eosinophilic leukemia by FIP1L1-PDGFRalpha. When introduced into c-Kit(high)Sca-1(+)Lineage(-) cells, FIP1L1-PDGFRalpha conferred cytokine-independent growth on these cells and enhanced their self-renewal, whereas it did not immortalize common myeloid progenitors in in vitro replating assays and transplantation assays. Importantly, FIP1L1-PDGFRalpha but not TEL-PDGFRbeta enhanced the development of Gr-1(+)IL-5Ralpha(+) eosinophil progenitors from c-Kit(high)Sca-1(+)Lineage(-) cells. FIP1L1-PDGFRalpha also promoted eosinophil development from common myeloid progenitors. Furthermore, when expressed in megakaryocyte/erythrocyte progenitors and common lymphoid progenitors, FIP1L1-PDGFRalpha not only inhibited differentiation toward erythroid cells, megakaryocytes, and B-lymphocytes but aberrantly developed eosinophil progenitors from megakaryocyte/erythrocyte progenitors and common lymphoid progenitors. As for the mechanism of FIP1L1-PDGFRalpha-induced eosinophil development, FIP1L1-PDGFRalpha was found to more intensely activate MEK1/2 and p38(MAPK) than TEL-PDGFRbeta. In addition, a MEK1/2 inhibitor and a p38(MAPK) inhibitor suppressed FIP1L1-PDGFRalpha-promoted eosinophil development. Also, reverse transcription-PCR analysis revealed that FIP1L1-PDGFRalpha augmented the expression of C/EBPalpha, GATA-1, and GATA-2, whereas it hardly affected PU.1 expression. In addition, short hairpin RNAs against C/EBPalpha and GATA-2 and GATA-3KRR, which can act as a dominant-negative form over all GATA members, inhibited FIP1L1-PDGFRalpha-induced eosinophil development. Furthermore, FIP1L1-PDGFRalpha and its downstream Ras inhibited PU.1 activity in luciferase assays. Together, these results indicate that FIP1L1-PDGFRalpha enhances eosinophil development by modifying the expression and activity of lineage-specific transcription factors through Ras/MEK and p38(MAPK) cascades.
Tissue development and function are exquisitely dependent on proper regulation of gene expression, but it remains controversial whether the genomic signals controlling this process are subject to strong selective constraint. While some studies show that highly constrained noncoding regions act to enhance transcription, other studies show that DNA segments with biochemical signatures of regulatory regions, such as occupancy by a transcription factor, are seemingly unconstrained across mammalian evolution. To test the possible correlation of selective constraint with enhancer activity, we used chromatin immunoprecipitation as an approach unbiased by either evolutionary constraint or prior knowledge of regulatory activity to identify DNA segments within a 66-Mb region of mouse chromosome 7 that are occupied by the erythroid transcription factor GATA1. DNA segments bound by GATA1 were identified by hybridization to high-density tiling arrays, validated by quantitative PCR, and tested for gene regulatory activity in erythroid cells. Whereas almost all of the occupied segments contain canonical WGATAR binding site motifs for GATA1, in only 45% of the cases is the motif deeply preserved (found at the orthologous position in placental mammals or more distant species). However, GATA1-bound segments with high enhancer activity tend to be the ones with an evolutionarily preserved WGATAR motif, and this relationship was confirmed by a loss-of-function assay. Thus, GATA1 binding sites that regulate gene expression during erythroid maturation are under strong selective constraint, while nonconstrained binding may have only a limited or indirect role in regulation.
The alpha and beta isoforms of the human thromboxane A(2) (TXA(2)) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm3, respectively. Herein, it was sought to identify factors regulating Prm1 within the megakaryocytic human erythroleukemia 92.1.7 cell line. Through gene deletion and reporter assays, the core Prm1 was localized to between nucleotides -6,320 and -5,895, proximal to the transcription initiation site. Furthermore, two upstream repressor and two upstream activator regions were identified. Site-directed mutagenesis of four overlapping Sp1/Egr1 elements and an NF-E2/AP1 element within the proximal region substantially reduced Prm1 activity. Deletion/mutation of GATA and Ets elements disrupted the upstream activator sequence located between -7,962 and -7,717, significantly impairing Prm1 activity. Electrophoretic mobility shift assays and chromatin immunoprecipitations confirmed that Sp1, Egr1, and NF-E2 bind to elements within the core promoter, whereas GATA-1 and Ets-1 factors bind to the upstream activator sequence (between -7,962 and -7,717). Collectively, these data establish that Sp1, Egr1, and NF-E2 regulate core Prm1 activity in the megakaryocytic-platelet progenitor cells, whereas GATA-1 and Ets-1 act as critical upstream activators, hence providing the first genetic basis for the expression of the human TXA(2) receptor (TP) within the vasculature.
The WGATAR motif is a common nucleotide sequence found in the transcriptional regulatory regions of numerous genes. In vertebrates, these motifs are bound by one of six factors (GATA1 to GATA6) that constitute the GATA family of transcriptional regulatory proteins. Although originally considered for their roles in hematopoietic cells and the heart, GATA factors are now known to be expressed in a wide variety of tissues where they act as critical regulators of cell-specific gene expression. This includes multiple endocrine organs such as the pituitary, pancreas, adrenals, and especially the gonads. Insights into the functional roles played by GATA factors in adult organ systems have been hampered by the early embryonic lethality associated with the different Gata-null mice. This is now being overcome with the generation of tissue-specific knockout models and other knockdown strategies. These approaches, together with the increasing number of human GATA-related pathologies have greatly broadened the scope of GATA-dependent genes and, importantly, have shown that GATA action is not necessarily limited to early development. This has been particularly evident in endocrine organs where GATA factors appear to contribute to the transcription of multiple hormone-encoding genes. This review provides an overview of the GATA family of transcription factors as they relate to endocrine function and disease.
Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes up-regulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1-mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching.
Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation.
We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.
Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.
Mutations of the GATA1 gene on chromosome X have been found in almost all cases of transient myeloproliferative disorder and acute megakaryoblastic leukemia (AMKL) accompanying Down syndrome (DS). Although most GATA1 mutations lead to the expression of GATA1s lacking the N-terminal activation domain, we recently found two novel GATA1 proteins with defects in another N-terminal region. It has been suggested that loss of the N-terminal portion of GATA1 might interfere with physiological interactions with the critical megakaryocytic transcription factor RUNX1, and this would imply that GATA1s is not able to interact properly with RUNX1. However, the interaction domain of GATA1 remains controversial. In this study, we show that GATA1 binds to RUNX1 through its zinc-finger domains, and that the C-finger is indispensable for synergy with RUNX1. All of the patient-specific GATA1 mutants interacted efficiently with RUNX1 and retained their ability to act synergistically with RUNX1 on the megakaryocytic GP1balpha promoter, whereas the levels of transcriptional activities were diverse among the mutants. Thus, our data indicate that physical interaction and synergy between GATA1 and RUNX1 are retained in DS-AMKL, although it is still possible that increased RUNX1 activity plays a role in the development of leukemia in DS.
In addition to their self-renewal capabilities, hematopoietic stem cells guarantee the continuous supply of fully differentiated, functional cells of various types in the peripheral blood. The process which controls differentiation into the different lineages of the hematopoietic system (erythroid, myeloid, lymphoid) is referred to as lineage specification. It requires a potentially multi-step decision sequence which determines the fate of the cells and their successors. It is generally accepted that lineage specification is regulated by a complex system of interacting transcription factors. However, the underlying principles controlling this regulation are currently unknown. Here, we propose a simple quantitative model describing the interaction of two transcription factors. This model is motivated by experimental observations on the transcription factors GATA-1 and PU.1, both known to act as key regulators and potential antagonists in the erythroid vs. myeloid differentiation processes of hematopoietic progenitor cells. We demonstrate the ability of the model to account for the observed switching behavior of a transition from a state of low expression of both factors (undifferentiated state) to the dominance of one factor (differentiated state). Depending on the parameter choice, the model predicts two different possibilities to explain the experimentally suggested, stem cell characterizing priming state of low level co-expression. Whereas increasing transcription rates are sufficient to induce differentiation in one scenario, an additional system perturbation (by stochastic fluctuations or directed impulses) of transcription factor levels is required in the other case.
The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.
GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.
Both nuclear factor erythroid 2 45 kDa subunit (p45) and BTB and CNC homolog 1 (Bach) transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to the musculoaponeurotic fibrosarcoma oncogene (Maf) recognition element (MARE). MARE is known to act as a critical cis-regulatory element of erythroid and megakaryocytic genes. Although detailed analyses of p45-null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of Bach1 and the relationship or redundancy between Bach1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated transgenic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed significant thrombocytopenia associated with impaired maturation of the megakaryocytes, and they developed myelofibrosis. The megakaryocytes in the transgenic mice exhibited reduced proplatelet formation, and the modal ploidy class of megakaryocytes was 2N, indicating the impairment of endomitosis. Transcription of the p45 target genes was down-regulated and we indeed found that BACH1 binds to the thromboxane synthase gene, one of the target genes for p45 in megakaryocytes. These findings thus provide evidence that BACH1 acts as a transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.
The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor beta, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. Here we show that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. We propose that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia.
Vertebrate GATA proteins regulate processes that are vital to development, and each possesses two tandem GATA finger domains: an N-terminal GATA finger and a C-terminal GATA finger. These GATA fingers require Zn(2+) to fold, to bind DNA recognition elements, and to regulate transcription. While the GATA-1 C-terminal finger is necessary and sufficient to bind to single GATA DNA sites, the N-terminal finger interacts with DNA such that the double finger unit (DF domain) has a binding and transactivation profile that is tuned by the DNA-binding site. Co(2+) was used as a spectroscopic probe in a series of competition titrations to determine the affinity of Co(2+) and Zn(2+) for the C-terminal finger from chicken GATA-1 and the double finger from human GATA-1 (referred to in this report as CF and DF). For CF, these experiments yielded K(b)(Co) = 1.0 (+/-1.3) x 10(7) M(-1) and K(b)(Zn) = 2.0 (+/-1.3) x 10(10) M(-1). For DF, these experiments yielded equilibrium constants for the process of two M(2+) binding to form M(2+)(2)-DF of beta(2)(Co) = 2.5 (+/-1.6) x 10(14) M(-2) and beta(2)(Zn) = 6.3 (+/-2.5) x 10(20) M(-2). The ZnS(4) coordination environment of Zn(2+)-bound CF was confirmed with X-ray absorption spectroscopy. A detailed analysis of these data suggests that the N-terminal and C-terminal fingers of DF act as independent and identical Zn(2+)-binding sites and each finger binds Zn(2+) with an affinity equivalent to that of CF.
The locus control region of the beta-globin gene cluster has been used previously to direct erythroid expression of globin genes from retroviral vectors for the purpose of gene therapy. Short erythroid regulatory elements represent a potentially valuable alternative to the locus control region. Among them, the GATA-1 enhancer HS2 was used to replace the retroviral enhancer within the 3'-long terminal repeat (LTR) of the retroviral vector SFCM, converting it into an erythroid-specific regulatory element. In this work, we have functionally studied an additional GATA-1 enhancer, HS1. HS1 participates in the transcriptional autoregulation of GATA-1 through an essential GATA-binding site that is footprinted in vivo. In this work we identified within HS1 a new in vivo footprinted region, and we showed that this sequence indeed binds a nuclear protein in vitro. Addition of HS1 to HS2 within the LTR of SFCM significantly improves the expression of a reporter gene. The deletion of the newly identified footprinted sequence in the retroviral construct further increases expression up to a level almost equal to that of the wild type retroviral LTR, without loss of erythroid specificity, suggesting that this sequence may act as a negative regulatory element. An improved vector backbone, MDeltaN, allows even better expression from the new GATA cassette. These results suggest that substantial improvement of overall expression can be achieved by the combination of multiple changes in both regulatory elements and vectors.
PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each other's function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.
To investigate the effect of the human A gamma-globin gene-173 T-->C mutation on the binding of transacting factors to the promoter and the activity of the promoter.
Electrophoretic mobility shift assay and transient transfection assay were used in this study.
The A gamma-globin gene-173 T-->C mutation decreased the affinity of GATA-1 to the mutant promoter fragment (-201(-)-158) 96% (P < 0.01) and the binding of Oct-1 to the same DNA fragment 55% (P < 0.05). The activity of the mutant promoter was 2-fold (P < 0.05) as strong as that of the normal one in MELGM979 cells. The mutant and the normal promoters exhibited basically same activities in K562 and Hela cells.
The -173 T-->C mutation decreased dramatically the binding of GATA-1, implicating that GA-TA-1 may act as a negative regulator of A gamma-globin gene in adults. The -173 T-->C mutation may enhance the activity of A gamma-globin gene promoter in the adult erythroid cell environment.
The role of cytokines and transcription factors on the regulation of megakaryocy topoiesis and platelet production are reviewed in this article. Megakaryocytopoiesis involves the proliferation and differentiation of megakaryocytic pro genitor cells into immature megakaryocytes, and the differentiation of immature megakaryocytes to mature megakaryocytes which produce platelets. The former is regulated mainly by thrombopoietin (TPO) and to a lesser degree by other cytokines such as interleukin-1 (IL-1), IL-3 and platelet-derived growth factor (PDGF), the later by TPO and probably IL-6 and IL-11. A number of transcription factors have been implicated in the control of megakaryocyte differentiation. GATA-1, FOG-1 and Fli-1 are essential regulators in early- and mid-stages of megakaryocytopoiesis. NF-E2 regulates late-stage of megakaryocytopoiesis and platelet production. However, the platelet release mechanism is poorly understood. Nitric oxide (NO) may act in the stage of platelet release through induction of apoptosis in megakaryocytes.
The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1(KI) (GATA-1 is located on the X-chromosome) or GATA-2(KI/KI) mice, compound GATA-1(KI) GATA-2(KI/KI) mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1(-/-) mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.
Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. We have cloned the Xenopus homolog of LMO-2 and show that it is expressed in a similar pattern to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis.
We have recently demonstrated that a testicular GATA-binding protein, GATA-1, up-regulates the transcription of inhibin alpha-subunit gene through interaction with GATA motifs in the promoter region in MA-10, a mouse Leydig tumor cell line. In this study, we showed that both GATA-1 and GATA-4 also transactivated the transcription from the promoter for the 4.8-kb inhibin/activin beta-B-subunit gene transcripts, beta-B(4.8)-subunit promoter, in two testicular cell lines, MA-10 and MSC-1, which is a mouse Sertoli cell line. The abilities of GATA-1 and GATA-4 interacting with GATA and/or GATA-like sequences to transactivate the beta-B(4.8)-subunit promoter were next examined by mutation analysis. Mutations of GATA or GATA-like sequences caused no apparent effect or only a small decrease in the basal transcriptional activity of this promoter. However, mutation of the GATA motif at -65 markedly decreased 60-70% of the effect of GATA-1 on the transactivation of beta-B(4.8)-subunit promoter in both MA-10 and MSC-1 cells. In addition, mutation of the GATA motif in MSC-1 cells also reduced 40-50% of the effect of GATA-4 to transactivate this promoter. Interestingly, mutation of GATT at -42 caused a 70-90% increase in the transactivation of beta-B(4.8)-subunit promoter by GATA-1 or GATA-4. No significant change in the promoter activity was observed when GATT at -177 or GATC at -201 was mutated. Electrophoretic mobility shift assay confirmed the above observations that these GATA-binding proteins interacted with the GATA motif at -65 and GATT at -42, but not with GATC at -201 or GATT at -177. Serial deletion from the 5'-end of the basal promoter, from -226 to -90, markedly decreased the basal transcription, but increased the effect of GATA-1 on transactivation of the beta-B(4.8)-subunit promoter. In summary, our observations suggest that the two GATA-binding proteins transactivate the beta-B(4.8)-subunit promoter in testicular cells via complicated mechanisms. Both GATA-1 and GATA-4 factors act through the GATA motif at -65 and GATT at -42 to positively and negatively regulate the transcription from this promoter, respectively. Furthermore, GATA-1 may also interact directly or indirectly with DNA sequences at -180 to -90 to regulate the beta-B(4.8)-subunit promoter.
Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells.
We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development.
The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors.
These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units.
Members of the GATA family of zinc-finger transcription factors have critical roles in a variety of cell types. GATA-1, GATA-2 and GATA-3 are required for proliferation and differentiation of several hematopoietic lineages, whereas GATA-4, GATA-5 and GATA-6 activate cardiac and endoderm gene expression programs. Two GATA cofactors have recently been identified. Friend of GATA-1 (FOG-1) interacts with GATA-1 and is expressed principally in hematopoietic lineages, whereas FOG-2 is expressed predominantly in heart and brain. Although gene targeting experiments are consistent with an essential role for FOG-1 as an activator of GATA-1 function, reporter assays in transfected cells indicate that FOG-1 and FOG-2 can act as repressors. We have cloned a Xenopus laevis homologue of FOG that is structurally most similar to FOG-1, but is expressed predominantly in heart and brain, as well as the ventral blood island and adult spleen. Ectopic expression and explant assays demonstrate that FOG proteins can act as repressors in vivo, in part through interaction with the transcriptional co-repressor, C-terminal Binding Protein (CtBP). FOG may regulate the differentiation of red blood cells by modulating expression and activity of GATA-1 and GATA-2. We propose that the FOG proteins participate in the switch from progenitor proliferation to red blood cell maturation and differentiation.
The FLI-1 oncogene, a member of the ETS family of transcription factors, is associated with both normal and abnormal hematopoietic cell growth and lineage-specific differentiation. We have previously shown that overexpression of FLI-1 in pluripotent human hematopoietic cells leads to the induction of a megakaryocytic phenotype. In this report we show that FLI-1 also acts as an inhibitor of erythroid differentiation. Following the induction of erythroid differentiation, pluripotent cells express reduced levels of FLI-1. In contrast, when FLI-1 is overexpressed in these cells, the levels of erythroid markers are reduced. The ability of FLI-1 overexpressing cells to respond to erythroid-specific inducers such as hemin and Ara-C is also inhibited, and the uninduced cells show a reduced level of the erythroid-associated GATA-1 transcription factor mRNA. Furthermore, expression of a GATA-1 promoter-driven reporter construct in K562 cells is inhibited by co-transfection with a construct expressing FLI-1. Our results support the hypothesis that FLI-1 can act both positively and negatively in the regulation of hematopoietic cell differentiation, and that inhibition of GATA-1 expression may contribute to FLI-1-mediated inhibition of erythroid differentiation.
Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBPalpha mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPbeta expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPbeta protein could bind to the potential C/EBP-binding site (bp -90 to -82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPbeta and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPbeta and GATA-1 can stimulate the MBP promoter in the C/EBPbeta and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPbeta and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPbeta or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPbeta. Expression of FOG (riend ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPbeta synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.
We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.
Malignant transformation usually inhibits terminal cell differentiation but the precise mechanisms involved are not understood. PU.1 is a hematopoietic-specific Ets family transcription factor that is required for development of some lymphoid and myeloid lineages. PU.1 can also act as an oncoprotein as activation of its expression in erythroid precursors by proviral insertion or transgenesis causes erythroleukemias in mice. Restoration of terminal differentiation in the mouse erythroleukemia (MEL) cells requires a decline in the level of PU.1, indicating that PU.1 can block erythroid differentiation. Here we investigate the mechanism by which PU.1 interferes with erythroid differentiation. We find that PU.1 interacts directly with GATA-1, a zinc finger transcription factor required for erythroid differentiation. Interaction between PU.1 and GATA-1 requires intact DNA-binding domains in both proteins. PU.1 represses GATA-1-mediated transcriptional activation. Both the DNA binding and transactivation domains of PU.1 are required for repression and both domains are also needed to block terminal differentiation in MEL cells. We also show that ectopic expression of PU.1 in Xenopus embryos is sufficient to block erythropoiesis during normal development. Furthermore, introduction of exogenous GATA-1 in both MEL cells and Xenopus embryos and explants relieves the block to erythroid differentiation imposed by PU.1. Our results indicate that the stoichiometry of directly interacting but opposing transcription factors may be a crucial determinant governing processes of normal differentiation and malignant transformation.
Erythroid Krüppel-like factor (EKLF) is a zinc finger transcription factor required for beta-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression. In an effort to identify factors involved in the expression of this important erythroid-specific regulatory protein, we have isolated the mouse EKLF gene and systematically analyzed the promoter region. Initially, a reporter construct with 1150 base pairs of the EKLF 5'-region was introduced into transgenic mice and shown to direct erythroid-specific expression. We continued the expression studies in erythroid cells and have identified a sequence element consisting of two GATA sites flanking an E box motif. The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each site is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. We further show that GATA-1 binds to the two GATA sites and present evidence for binding of another factor from erythroid cell nuclear extracts to the E box motif. These results are consistent with the formation of a quaternary complex composed of an E box dimer and two GATA-1 proteins binding at a combined GATA-E box-GATA activator element in the distal EKLF promoter.
Normal expression of the human beta-globin domain genes is dependent on at least three types of regulatory elements located within the beta-globin domain: the locus control region (LCR), globin enhancer elements (3'beta and 3'Agamma), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated gamma-globin promoters linked to the 5'HS2 enhancer. We show that in K562 cells, 5'HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 --> +25) gamma promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5'HS2. Mutation of the gamma-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5'HS2. To determine if enhanced expression of gamma-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH gamma-globin gene promoters to 5'HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Agamma-globin gene 3' enhancer to a plasmid containing the gamma-globin gene promoter and 5'HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5'HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5', but not 3', to the gamma-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the gamma-globin promoter and the LCR/5'HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.
In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.
Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX.
EVI1 is a zinc finger oncoprotein that binds via fingers 1-7 to the sequence GACAAGATAA. The target genes on which EVI1 acts are unknown. This binding motif overlaps with that for the GATA transcription factors, (T/A)GATA(A/G), and GATA-1 can bind to and activate transcription via a GACAAGATAA motif. The possibility has been raised that, when overexpressed in leukemogenesis, EVI1 may function by interfering with the differentiation-promoting action of GATA factors. To explore this, we have assessed the affinity of EVI1 for the GATA binding sites derived from erythroid-specific GATA-1 target genes, and found only low affinity interactions. We examined the contacts between EVI1 and DNA by methylation interference studies, which revealed extensive contacts between EVI1 and its binding site. The importance of the contacts for high affinity binding was shown by in vitro quantitative gel shift studies and in vivo cotransfection studies. To examine what types of sequences from mouse genomic DNA bind to EVI1, we isolated and sequenced five EVI1-binding fragments, and each showed the GACAAGATA site. The data presented contribute to our knowledge of the binding specificity of EVI1, and yield a clearer picture of what sequences can, and cannot, act as targets for EVI1 action.
The transcriptional activator, Tax, of human T-cell leukemia virus (HTLV-I) has been considered to interact with cellular proteins to act on target enhancer motifs. Using oligodeoxyribonucleotides containing the tax-responsive element (TAXRE) of the HTLV-I enhancer, we have cloned multiple cDNAs coding for TAXRE-binding proteins (TAXREB), and determined the cDNA and the deduced 200-amino-acid sequences for TAXREB302. The recombinant protein binds to the enhancer DNA by specific interaction to the CRE-like sequence. A single 1.8-kb species of mRNA was detected in cultured cells, as well as in normal human tissues, especially brain and skeletal muscle. The 22-kDa native protein was detected in the cultured-cell lysate by immunoblotting analysis. TAXREB302 does not have structural features common to the CRE-binding protein or activating transcription factor (CREB/ATF) family, but has homology to chicken erythroid transcription factor (Eryf1 or GATA-1), suggesting a possible protein-protein interaction.
The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.
In normal humans the fetal stage-specific gamma-globin genes are silenced after birth and not expressed in the adult. Exceptions are seen in cases of hereditary persistence of fetal haemoglobin (HPFH). These are clinically important because the elevated levels of gamma-globin can alleviate beta-thalassaemia and sickle cell anaemia. One class of mutations is associated with point mutations in the promoter of the gamma-globin genes (non-deletion HPFH), whereas others seem to be caused by large deletions 3' to the gamma-globin genes. To test whether the point mutation found in the Greek non-deletion HPFH (guanine to adenine at nucleotide position -117) is the cause of the raised gamma-globin levels in the adult stage and is not just a linked polymorphism, we engineered this mutation into a gamma-globin gene. When this gene was introduced into mice, the presence of the -117 mutation results in persistence of gamma-globin expression at a high level and a concomitant decrease in beta-globin expression in fetal and adult mice. We show that these changes correlate with the loss of binding of the transcription factor GATA1 to the gamma-globin promoter, suggesting that it may act as a negative regulator of the gamma-globin gene in adults.
Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Lysophosphatidic acid (LPA), a lipid growth factor was previously shown to be a regulator for erythropoietic process through activating LPA receptor 3 (LPA3). However, whether LPA affects megakaryopoiesis remains unclear. In this study, we used K562 leukemia cell line as a model to investigate the roles of LPA in MK differentiation. We demonstrated that K562 cells express both LPA2 and LPA3, and the expression levels of LPA2 are higher than LPA3. Treatment with phorbol 12-myristate 13-acetate, a commonly used inducer of megakaryopoiesis, reciprocally regulates the expressions of LPA2 and LPA3. By pharmacological blockers and knockdown experiments, we showed that activation of LPA2 suppresses whereas, LPA3 promotes megakaryocytic differentiation in K562. The LPA2-mediated inhibition is dependent on β-catenin translocation, whereas reactive oxygen species (ROS) generation is a downstream signal for activation of LPA3. Furthermore, the hematopoietic transcriptional factors GATA-1 and FLI-1, appear to be involved in these regulatory mechanisms. Taken together, our results suggested that LPA2 and LPA3 may function as a molecular switch and play opposing roles during megakaryopoiesis of K562 cells.
We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells). We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c-KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.
The aim of this study was to evaluate cell maturation and the platelet production capacity of the megakaryoblastic DAMI cell line, to characterize platelet-like particles produced and to investigate the mechanisms involved in their production. DAMI cell maturation was induced by phorbol myristate acetate (PMA) and thrombopoietin (TPO). Expression levels of GATA-1, Fli-1 and NF-E2 were evaluated using real-time PCR and western blot. Platelet-like particles were characterized by the presence of GPIb and GPIIb by flow cytometry, while the soluble fragment of GPIb, glycocalicin, was detected by enzyme immunoassay. Dense and alpha granules were evaluated by mepacrine staining and thrombospondin-1 detection, respectively, and by electron microscopy. Functional capacity of platelet-like particles was studied by measuring P-selectin membrane after thrombin stimulation by flow cytometry and actin polymerization using phalloidin-FITC by immunofluorescence. We found that stimulation of DAMI cells with high concentration of PMA and TPO induced the expression of transcription factors GATA-1 and Fli-1 followed by an increase in the isoform a of NF-E2. Mature DAMI cells give rise to extensions resembling proplatelets and later, produce platelet-like particles expressing GPIIb and GPIb on their surface and containing dense and alpha granules, which were confirmed by electron microscopy. Platelet functionality was demonstrated by the increase in P-selectin membrane expression after thrombin stimulation and by their ability to spread on fibrinogen matrices. DAMI cell line induced to differentiate into mature megakaryocytes is able to produce functional platelets providing a suitable model to study the mechanisms involved in platelet generation.
Specific interactions of transcription factors (TFs) with their targets are crucial for specifying gene expression programs during cell differentiation. How specificity is maintained despite limited selectivity of individual TF-DNA interactions is not fully understood. RUNX1 TF is among the most frequently mutated genes in human leukemia and an important regulator of megakaryopoiesis. We used megakaryocytic cell lines to characterize the network of RUNX1 targets and cooperating TFs in differentiating megakaryocytes and demonstrated how dynamic partnerships between RUNX1 and cooperating TFs facilitated regulatory plasticity and specificity during this process. After differentiation onset, RUNX1 directly activated a large number of genes through interaction with preexisting and de novo binding sites. Recruitment of RUNX1 to de novo occupied sites occurred at H3K4me1-marked preprogrammed enhancers. A significant number of these de novo bound sites lacked RUNX motif but were occupied by AP-1 TFs. Reciprocally, AP-1 TFs were up-regulated by RUNX1 after 12-O-tetradecanoylphorbol-13-acetate induction and recruited to RUNX1-occupied sites lacking AP-1 motifs. At other differentiation stages, additional combinatorial interactions occurred between RUNX1 and its coregulators, GATA1 and ETS. The findings suggest that in differentiating megakaryocytic cell lines, RUNX1 cooperates with GATA1, AP-1, and ETS to orchestrate cell-specific transcription programs through dynamic TF partnerships.
Induction of the differentiation of human leukemia cells is a useful strategy in treatment of human leukemia. However, the molecular mechanisms involved in leukemia cell differentiation have not been fully elucidated. Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA binding protein 1 (GATA-1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. Herein we report that Rab7b, a late endosome/lysosome-localized myeloid small GTPase, promotes phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation by increasing nuclear factor κB (NF-κB)-dependent IL-6 production and subsequently enhancing the association of activated signal transducer and activator of transcription 3 (STAT3) with GATA-1. By using PMA-induced megakaryocytic differentiation of leukemia cells as a model, we investigated the roles of Rab7b in megakaryocytic differentiation. We find that Rab7b can potentiate PMA-induced upregulation of megakaryocytic markers, production of IL-6, and activation of NF-κB. Inhibitor of NF-κB and neutralizing antibodies for IL-6 or the IL-6 signaling receptor gp130 can block the effects of Rab7b in megakaryocytic differentiation. In Rab7b-silenced cells, PMA-induced activation of NF-κB, IL-6 production, and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of STAT3 and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may regulate megakaryocytic differentiation.
EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between -211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.
The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.
Tellimagrandin I is a hydrolysable tannin compound widely present in plants. In this study, the effect of tellimagrandin I on chemically induced erythroid and megakaryocytic differentiation was investigated using K562 cells as differentiation model. It was found that tellimagrandin I not only inhibited the hemoglobin synthesis in butyric acid (BA)- and hemin-induced K562 cells with IC50 of 3 and 40microM, respectively, but also inhibited other erythroid differentiation marker including acetylcholinesterase (AChE) and glycophorin A (GPA) in BA-induced K562 cells. Tellimagrandin I also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of CD61 protein, a megakaryocytic marker. RT-PCR analysis showed that tellimagrandin I decreased the expression of erythroid genes (gamma-globin and porphobilinogen deaminase (PBGD)) and related transcription factors (GATA-1 and NF-E2) in BA-induced K562 cells, whereas tellimagrandin I induced the overexpresison of GATA-2 transcription factor that played negative regulation on erythroid differentiation. These results indicated that tellimagrandin I had inhibitory effects on erythroid and megakaryocytic differentiation, which suggested that tannins like tellimagrandin I might influence the anti-tumor efficiency of some drugs and the hematopoiesis processes.
Glutathione S-transferase P1-1 (GSTP1-1) conjugates glutathione to electrophilic compounds and its expression is correlated to chemotherapeutic drug resistance. Results show that GSTP1-1 mRNA as well as protein expressions are increased during Aclarubicin (Acla)- and Doxorubicin (Dox)-induced erythroid differentiation of human K562 cells. In contrast, during megakaryocytic differentiation by 12-O-tetradecanoyl phorbol 13-acetate (TPA), GSTP1-1 expression decreased at both mRNA and protein levels. In order to clarify the molecular mechanisms leading to these variations, we identified a GATA sequence located at -1208 relative to the transcriptional start site of the GSTP1-1 promoter. By gel shift, competition, and supershift analyses we show here the specificity of the GATA-1 binding regulated by both anthracyclines and TPA. Altogether, these results demonstrate for the first time the implication of GATA-1 in differentiation-specific variations of GSTP1-1 expression.
Differentiation induction is a therapeutic principle in acute promyelocytic leukemia (AML) using all- trans retinoic acid. In cell lines with properties of AML M6/M7 (K562 and CMK), differentiation towards megakaryopoietic and erythropoietic phenotypes can be induced in vitro. Transitory myeloproliferative disorder (TMD) is a self-limited disorder of newborn infants with Down syndrome, phenotypically resembling acute myeloid leukemia of megakaryoblastic lineage. Despite spontaneous disappearance of blasts from blood and bone marrow, in about 10% of the patients, overt acute megakaryoblastic leukemia (AML M7) develops up to 4 years later. Recently, mutations of the GATA1 transcription factor have been identified in the megakaryoblastic leukemia of Down syndrome. Here, we studied cells from a patient suffering from megakaryoblastic AML at the age of 2.5 years after spontaneous remission of neonatal TMD. In vitro, terminal differentiation towards a megakaryocyte-like phenotype could be induced by phorbol myristate acetate (PMA), with typical morphological features, upregulation of platelet-specific and downregulation of erythroid antigens, going along with downregulation of c-myc. Whether spontaneous resolution of TMD is a process due to terminal differentiation is still open; however, here we give evidence that in vitro differentiation can be induced even in blasts deriving from an overt AML French-American-British (FAB) M7 after TMD.
The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate-induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC-promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1-regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions -66 and +62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1-stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor alpha1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-beta1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed for fur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions.
Mona/grb2 related adapter downstream of shc is a molecular adapter expressed in platelets, T lymphocytes and myelomonocytic cells. Using human hematopoietic cell lines, we have previously shown that lineage-specific Mona expression is achieved through the production of two transcripts (named 1A and 1B) differing by their 5' untranslated region (5'UTR). Thus, platelets and megakaryocytic cell lines K562 and HEL (Human Erythro-Leukemia) specifically express 1B messenger RNA (mRNA). We report here characterization of the (-2031/+72) genomic region relative to the putative transcription start site of 1B mRNA. We show this region is sufficient to ensure specific reporter gene expression in megakaryocytic cell lines, and that most promoter activity is contained in the (-225/+72) fragment. Electro-mobility shift assay and mutational analyses indicated that GATA-1 and a yet unidentified E-26 family member transcription factor are required for 1B (-2031/+72) promoter activity. Thus, Mona 1B promoter exhibits typical features of megakaryocyte-specific promoters.
Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the alphaV promoter region and are directly involved in the regulation of transcriptional activity of the alphaV gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates alphaV expression during differentiation of pluripotent K562 cells induced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the alphaV gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was protected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract of K562 cells treated with BA revealed an increase in GATA binding activity, which was associated with reduced alphaV mRNA and alphaV protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of alphaV. We conclude that the GATA-1 transcription factor specifically binds to the GATA element in the alphaV gene promoter and negatively regulates alphaV gene expression.
To examine the relationship between cell cycle progression and differentiation in megakaryocytes, variances in the cell cycle during PMA (phorbol 12-myristate-13-acetate)-induced differentiation were analysed in synchronized growing CMK cells. The cells stimulated with PMA in early S phase showed cell cycle arrest in G2 phase. In addition, the cells stimulated with PMA in early G1 phase showed cell cycle arrest in late G1. The expression of gpIIb/IIIa, megakaryocytic differentiation marker, was markedly induced in G2 phase when the cells were stimulated in early S phase, and was also induced in late G1 phase when cells were stimulated in early G1 phase. Therefore, the induction of gpIIb/IIIa expression seems to be associated with cell cycle blockage in both G2 and late G1. Expression of the transcription factor GATA-1 in cells stimulated in early S phase was much higher than that in controls at G2 phase. Furthermore, GATA-1 expression in cells stimulated in early G1 tended to be higher than that in controls at late G1. These periods corresponded to the time that the cell cycle arrest and gpIIb/IIIa expression were induced by PMA. These results suggest that the cell cycle in human megakaryocytic lineage cells may have two regulation points for differentiation.
We investigated megakaryocytic differentiation in a newly-established Ph1-positive leukemic cell line, MC3, which showed tri-lineage immunophenotypes (myeloid antigens2+, CD19(1+) and CD41a1+) and was positive for CD34 and CD38. TPA induced MC3 cells to differentiate to an early stage of megakaryocyte lineage exhibiting an increase in the expression of platelet glycoproteins (GP) IIb/IIIa (CD41a), and an increase in cell size and nuclear ploidy. TPA treatment also enhanced the expression of GPIIb mRNA, and induced the expression of interleukin-6 (IL-6) and its receptor mRNAs, while it did not induce transcripts of the genes IL-11 and mpl ligand, and further decreased the transcript of the mpl gene. Consistent with these findings, MC3 cells treated with TPA showed an increased expression of GATA-1, but not GATA-3 transcripts, whereas those without TPA treatment expressed only the GATA-2 transcript. These results provide an insight into the study for the regulatory mechanism of megakaryocytopoiesis and leukemic cell differentiation.
In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome-positive chronic myeloid leukemia. The NS-Meg cells were positive for alpha-naphthyl acetate esterase and periodic acid-Schiff (PAS) staining and for surface CD4, CD7, CD13, CD34, CD41a, and glycophorin A antigens. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and platelet peroxidase activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha-granules, mitochondria and dense bodies, strongly suggesting platelet production. Erythropoietin (Epo), granulocyte/macrophage colony stimulating factor(GM-CSF), and interleukin 3 (IL-3) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both CD41a and CD61 antigens. Ten-day exposure to Epo induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the Epo receptor (EpoR), for GATA-1, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation.
With erythroid differentiation, committed progenitor cells acquire the ability to respond to erythropoietin (Epo). Epo interacts with target cells through the Epo receptor (Epo-R), whose expression is tightly regulated in a lineage-specific fashion. Epo-R expression is presumed to be progressively activated or repressed as cells progress along the erythroid or the myeloid pathway, respectively. Little is known of the mechanisms that underlie the erythroid-specific expression of the Epo-R gene. GATA-1, the major known transcription factor involved in Epo-R gene regulation, is not erythroid-specific. We have studied the regulation of the expression of the Epo-R gene in two related human Epo-responsive cell lines, UT-7 and UT-7 Epo. These lines express Epo-R at high levels because of amplification of the endogenous gene, which is apparently not rearranged. Treatment for 6 to 24 hours with the tumor promoter, phorbol myristate acetate (PMA), or 24 hours of growth factor starvation (Epo or granulocyte/macrophage colony-stimulating factor [GM-CSF]) decreased or increased the levels of Epo-R mRNA, respectively. In the case of growth factor starvation, the increase (approximately equal to threefold) in the level of Epo-R mRNA correlated directly with an increase in the rate of Epo-R gene transcription as measured by run-off assay. Both increases were observed as early as 3 hours after the growth factor was withdrawn and were reversible; levels of mRNA and transcription rates returned to baseline 3 hours after the cells were reexposed to growth factors. The changes in Epo-R expression after growth factor starvation were coordinated with changes in the level of expression of GATA-1 that were detected both at the mRNA and at the gene transcription level under these conditions (suggesting that GATA-1 was responsible for this upregulation). During PMA treatment, after a transient increase in Epo-R mRNA at 1 hour, a progressive decline in the level of Epo-R mRNA was observed; the level of Epo-R mRNA decreased by 50%, and fell below the level of detection by 6 and 24 hours, respectively. This decrement was explained in part by a fourfold reduction in the rate of gene transcription as well as a reduction (measured as levels of Epo-R mRNA in the presence of actinomycin D) in mRNA stability. The changes in transcription rate occurred in the absence of changes in the level of GATA-1 binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.
Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn(2+)) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G(1) arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation.
Lead (Pb) and cadmium (Cd) are heavy metal toxins that cause many pathophysiologic effects, including anemia. Previous in vitro studies have shown that these metals are able to replace coordinated Zinc (Zn) atoms in the Zn fingers of transcription factors and that this can alter the structure and DNA-binding characteristics of these proteins. This has lead to the hypothesis that one mechanism underlying the toxic effects of Pb and Cd is their ability to alter Zn finger transcription factor function resulting in aberrant target gene expression. A recent report that Pb is able to replace Zn in the Zn fingers of the hematopoietic transcription factor GATA-1 prompted us to address this hypothesis in the setting of MEL cell differentiation. If Pb or Cd is able to inhibit GATA-1 function, this should be detectable through alterations in chemically induced erythroid differentiation and GATA-1-dependent gene expression. Despite a strong rationale for this hypothesis, we have found no significant change in MEL differentiation, the expression of several GATA-1 target genes, or of in vitro and in vivo GATA-1 binding to DNA at concentrations well above those associated with toxic effects in humans. These results argue against the hypothesis that Pb or Cd significantly alters GATA-1 function in vivo.
EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.
The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.
Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers.
GATA1 organizes erythroid and megakaryocytic differentiation by orchestrating the expression of multiple genes that show diversified expression profiles. Here, we demonstrate that GATA1 monovalently binds to a single GATA motif (Single-GATA) while a monomeric GATA1 and a homodimeric GATA1 bivalently bind to two GATA motifs in palindromic (Pal-GATA) and direct-repeat (Tandem-GATA) arrangements, respectively, and form higher stoichiometric complexes on respective elements. The amino-terminal zinc (N) finger of GATA1 critically contributes to high occupancy of GATA1 on Pal-GATA. GATA1 lacking the N finger-DNA association fails to trigger a rate of target gene expression comparable to that seen with the wild-type GATA1, especially when expressed at low level. This study revealed that Pal-GATA and Tandem-GATA generate transcriptional responses from GATA1 target genes distinct from the response of Single-GATA. Our results support the notion that the distinct alignments in binding motifs are part of a critical regulatory strategy that diversifies and modulates transcriptional regulation by GATA1.
Congenital erythropoietic porphyria (CEP) is characterized in most individuals by severe cutaneous photosensitivity with blistering and increased friability of the skin over light-exposed areas. Onset in most affected individuals occurs at birth or early infancy. The first manifestation is often pink to dark red discoloration of the urine. Hemolytic anemia is common and can range from mild to severe, with some affected individuals requiring chronic blood transfusions. Porphyrin deposition may lead to corneal ulcers and scarring, reddish-brown discoloration of the teeth (erythrodontia), and mild bone loss and/or expansion of the bone marrow. The phenotypic spectrum, however, is broad and ranges from non-immune hydrops fetalis in utero to late-onset disease with only mild cutaneous manifestations in adulthood.
The diagnosis of CEP is supported by the biochemical findings of markedly decreased uroporphyrinogen (URO)-synthase activity in erythrocytes and/or markedly increased levels of urinary uroporphyrin I and coproporphyrin I isomers. The diagnosis is confirmed most commonly by identification of biallelic UROS pathogenic variants or on rare occasion by the identification of a hemizygous pathogenic variant in the X-linked gene GATA1.
Treatment of manifestations: There is no FDA-approved treatment for CEP or specific treatment for the photosensitivity. The only effective management is prevention of blistering by avoidance of sun and light exposure, including the long-wave ultraviolet light that passes through window glass or is emitted from artificial light sources. Therefore, the use of protective clothing, wrap-around sun glasses, protective window films, reddish incandescent bulbs, filtering screens for fluorescent lights, and opaque sunscreens containing zinc oxide or titanium oxide is recommended. Wound care is necessary to prevent infection of opened blisters; surgical intervention may be necessary; blood transfusions are necessary when hemolysis is significant. Bone marrow transplantation (BMT) is the only cure for CEP and should be considered in children with severe cutaneous and hematologic involvement. Prevention of primary manifestations: Strict avoidance of sunlight and other long-wave UV light exposure. Prevention of secondary complications: Vitamin D supplementation, immunization for hepatitis A and B. Surveillance: Monitor hematologic indices to assess hemolysis every six months. In those receiving transfusions: monitor for hemolysis more frequently and for iron overload. Monitor hepatic function and vitamin D 25-OH every six to twelve months in all patients. Agents/circumstances to avoid: Avoidance of sunlight and UV light (see Treatment of manifestations). In those with hepatic dysfunction: avoid drugs that may induce cholestasis. Evaluation of relatives at risk: Presymptomatic diagnosis is warranted in relatives at risk for initiation of early intervention (no phototherapy, strict sun protection) and future monitoring for signs of hemolytic anemia. Pregnancy management: Protective filters for artificial lights should be used in the delivery/operating room to prevent phototoxic damage to the mother during delivery. Other: Neither beta-carotene nor phototherapy with narrow-band ultraviolet B radiation has been beneficial.
CEP caused by biallelic UROS pathogenic variants is inherited in an autosomal recessive (AR) manner. CEP caused by a GATA1 pathogenic variant is inherited in an X-linked (XL) manner. AR CEP. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Heterozygotes (carriers) are asymptomatic. Carrier testing for at-risk family members and prenatal testing for pregnancies at increased risk are possible if the pathogenic variants in the family have been identified. XL CEP. If the mother of an affected male is heterozygous for a GATA1 pathogenic variant, the chance of transmitting it in each pregnancy is 50%. Males who inherit the pathogenic variant will be affected; females who inherit the pathogenic variant will be heterozygotes and can be either asymptomatic or have a milder phenotype.
The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling.
Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance.
Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.
Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5-histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST-FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5-NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.
GATA-binding proteins 1 (GATA1) and 2 (GATA2) are zinc-finger transcription factors and belong to the GATA family proteins 1-6. GATA1 interacts with the TP53 tumor suppressor gene, and both GATAs have been shown to be involved in cell growth, apoptosis, and tumorigenesis of several solid tumors. GATA1 and GATA2 expression alterations are associated with poor survival and adverse clinicopathology in prostate and colorectal cancer, while the significance and prognostic value in clear cell renal cell carcinoma (ccRCC) has not been investigated as yet. We investigated relative messenger RNA (mRNA) expression levels of GATA1 and GATA2 in 77 ccRCC and 58 paired adjacent noncancerous renal tissues by quantitative real-time reverse-transcribed PCR. Relative mRNA expression levels were determined using the ΔΔCt method. GATA1 and GATA2 expression levels were significantly decreased in tumor tissues compared with normal tissues (p < 0.001, paired t test). In univariate logistic regression analysis, decreased GATA1 and GATA2 expression levels were associated with advanced tumor disease (p = 0.005 and 0.008), positive distant metastasis (p = 0.03 and 0.001), and lymph node metastasis status (p = 0.011 and 0.038). Reduced expression levels of GATA1 and GATA2 were associated with an increased risk of disease recurrence (p = 0.005 and 0.006; hazard ratio = 0.05 and 0.21). Pairwise bivariate analysis after adjusting for clinicopathological parameters revealed relative mRNA expression of GATA1, but not GATA2, as an independent candidate prognosticator for ccRCC. Our results support that GATA1 and GATA2 are involved in ccRCC tumor biology possibly affecting tumor development and aggressiveness.
The binding of chromatin-associated proteins and incorporation of histone variants correlates with alterations in gene expression. These changes have been particularly well analyzed at the mammalian β-globin locus, where transcription factors such as erythroid Krüppel-like factor (EKLF), which is also known as Krüppel-like factor 1 (KLF1), play a coordinating role in establishing the proper chromatin structure and inducing high-level expression of adult β-globin. We had previously shown that EKLF preferentially interacts with histone H3 and that the H3.3 variant is differentially recruited to the β-globin promoter. We now find that a novel interaction between EKLF and the histone cell cycle regulation defective homolog A (HIRA) histone chaperone accounts for these effects. HIRA is not only critical for β-globin expression but is also required for activation of the erythropoietic regulators EKLF and GATA binding protein 1 (GATA1). Our results provide a mechanism by which transcription factor-directed recruitment of a generally expressed histone chaperone can lead to tissue-restricted changes in chromatin components, structure, and transcription at specific genomic sites during differentiation.
FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers) family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being.
GATA are a family of transcription factors characterized by their ability to bind to the DNA sequence "GATA", and involved in a myriad of cellular processes. GATA1/2/3 factors are known as the hematopoietic GATA factors, which play dominated roles in regulating hematopoiesis. In the present study, a gene encoding GATA transcription factor (designed as CfGATA) was cloned and characterized from the scallop Chlamys farreri. The full-length cDNA of CfGATA is of 2058 bp encoding a predicted polypeptide of 457 amino acids with two conserved zinc finger domains, which shared high similarity with other reported GATA1/2/3 proteins. The mRNA transcripts of CfGATA showed higher expression in gills, hepatopancreas, hemocytes and heart, and the CfGATA protein expressed in HEK293 cells was found to be localized specifically in the nuclei. The recombinant CfGATA protein (rCfGATA) exhibited strong ability to bind specific WGATAR DNA sequence by electrophoretic mobility shift assay in vitro. After CfGATA gene was silenced by RNA interference, the hemocyte renewal rate and circulating total hemocyte count (THC) decreased significantly, which was 7.85-fold and 19.46-fold lower than that of PBS control, respectively (P < 0.05). After LPS stimulation, the expression level of CfGATA mRNA decreased significantly in the hemocytes of PBS or EGFP dsRNA treated scallops, which was accompanied by the increase of hemocyte renewal rate and the reduced circulating THC at 24 h. In contrast, the hemocyte renewal rate and circulating THC did not change significantly in CfGATA gene interfered scallops after LPS stimulation. These results suggested that CfGATA, as a conserved GATA1/2/3 transcription factor, plays essential roles in regulating hemocyte production of scallop.
Promyelocytic leukemia protein (PML) has been implicated as a participant in multiple cellular processes including senescence, apoptosis, proliferation, and differentiation. Studies of PML function in hematopoietic differentiation previously focused principally on its myeloid activities and also indicated that PML is involved in erythroid colony formation. However, the exact role that PML plays in erythropoiesis is essentially unknown. In this report, we found that PML4, a specific PML isoform expressed in erythroid cells, promotes endogenous erythroid genes expression in K562 and primary human erythroid cells. We show that the PML4 effect is GATA binding protein 1 (GATA-1) dependent using GATA-1 knockout/rescued G1E/G1E-ER4 cells. PML4, but not other detected PML isoforms, directly interacts with GATA-1 and can recruit it into PML nuclear bodies. Furthermore, PML4 facilitates GATA-1 trans-activation activity in an interaction-dependent manner. Finally, we present evidence that PML4 enhances GATA-1 occupancy within the globin gene cluster and stimulates cooperation between GATA-1 and its coactivator p300. These results demonstrate that PML4 is an important regulator of GATA-1 and participates in erythroid differention by enhancing GATA-1 trans-activation activity.
The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.
Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148) interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 (gt/gt) mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells) in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 (gt/gt) mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.
The testis zinc finger protein (Tzfp), also known as Repressor of GATA, belongs to the BTB/POZ zinc finger family of transcription factors and is thought to play a role in spermatogenesis due to its remarkably high expression in testis. Despite many attempts to find the in vivo role of the protein, the molecular function is still largely unknown. Here, we address this issue using a novel mouse model with a disrupted Tzfp gene. Homozygous Tzfp null mice are born at reduced frequency but appear viable and fertile. Sertoli cells in testes lacking Tzfp display an increase in Androgen Receptor (AR) signaling, and several genes in the testis, including Gata1, Aie1 and Fanc, show increased expression. Our results indicate that Tzfp function as a transcriptional regulator and that loss of the protein leads to alterations in AR signaling and reduced number of apoptotic cells in the testicular tubules.
Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.
Kaiso zinc finger-containing protein (Kzp), a maternally-derived transcription factor, controls dorsoventral patterning during zebrafish gastrulation. Here, we uncovered a new function for Kzp in zebrafish embryonic primitive hematopoiesis. The depletion of kzp led to defects in primitive hematopoiesis including the development of the erythroid and myeloid lineages. On the other hand, overexpression of kzp caused the ectopic expression of gata1, gata2, and pu.1. Chromosome immunoprecipitation assays revealed that Kzp protein directly binds to gata1, gata2, and pu.1 promoters. Interestingly, the ectopic expression of gata2 was able to rescue the erythroid, but not the myeloid lineage in kzp-depleted zebrafish embryos. gata1 expression controlled by Kzp was dependent on gata2 during primitive erythropoiesis. Our results indicate that Kzp is a critical transcriptional factor for the expression of gata2 and pu.1 to modulate primitive hematopoiesis.
The zinc finger transcription factors GATA1 and GATA2 participate in mast cell development. Although the expression of these factors is regulated in a cell lineage-specific and differentiation stage-specific manner, their regulation during mast cell development has not been clarified. Here, we show that the GATA2 mRNA level was significantly increased while GATA1 was maintained at low levels during the differentiation of mast cells derived from mouse bone marrow (BMMCs). Unlike in erythroid cells, forced expression or small interfering RNA (siRNA)-mediated knockdown of GATA1 rarely affected GATA2 expression, and vice versa, in mast cells, indicating the absence of cross-regulation between Gata1 and Gata2 genes. Chromatin immunoprecipitation assays revealed that both GATA factors bound to most of the conserved GATA sites of Gata1 and Gata2 loci in BMMCs. However, the GATA1 hematopoietic enhancer (G1HE) of the Gata1 gene, which is essential for GATA1 expression in erythroid and megakaryocytic lineages, was bound only weakly by both GATA factors in BMMCs. Furthermore, transgenic-mouse reporter assays revealed that the G1HE is not essential for reporter expression in BMMCs and peritoneal mast cells. Collectively, these results demonstrate that the expression of GATA factors in mast cells is regulated in a manner quite distinct from that in erythroid cells.
Ginsenoside F1 (GF1) is a metabolite produced by hydrolysis of the ginsenoside Re and Rg1 in Panax ginseng. According to various studies, high amounts of ginseng components are absorbed in the metabolized form, which are key constituents responsible for the biological effects of P. ginseng. Recently, GF1 was reported to have beneficial effects on skin. However, there has not been a sound understanding of its antimelanogenic effect and underlying molecular mechanisms. In this study, GF1 reduced α-melanocyte-stimulating hormone-induced melanin secretion in B16F10 cell culture media by 60%. However, it did not suppress intracellular melanin levels, tyrosinase activity and expression. Immunofluorescence assay showed that GF1 had no effect on melanosome transport, but significantly induced dendrite retraction. Pull-down assay demonstrated that GF1 primarily modulates the Rho family GTPases resulting in dendrite retraction. Collectively, these data suggest that GF1 could act as a potent skin-whitening agent.
TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).
Most of the yeast artificial chromosomes (YACs) isolated from the Xp11.23-22 region have shown instability and chimerism and are not a reliable resource for determining physical distances. We therefore constructed a long-range pulsed-field gel electrophoresis map that encompasses approximately 3.5 Mb of genomic DNA between the loci TIMP and DXS146 including a CpG-rich region around the WASP and TFE-3 gene loci. A combined YAC-cosmid contig was constructed along the genomic map and was used for fine-mapping of 15 polymorphic microsatellites and 30 expressed sequence tags (ESTs) or sequence transcribed sites (STSs), revealing the following order: tel-(SYN-TIMP)-(DXS426-ELK1)-ZNF(CA) n-L1-DXS1367-ZNF81-ZNF21-DXS6616- (HB3-OATL1pseudogenes-DXS6950)-DXS6949-DXS694 1-DXS7464E(MG61)-GW1E(EBP)- DXS7927E(MG81)-RBM- DXS722-DXS7467E(MG21)-DXS1011E-WASP-DXS6940++ +-DXS7466E(MG44)-GF1- DXS226-DXS1126-DXS1240-HB1- DXS7469E-(DXS6665-DXS1470)-TFE3-DXS7468E-+ ++SYP-DXS1208-HB2E-DXS573-DXS1331- DXS6666-DXS1039-DXS 1426-DXS1416-DXS7647-DXS8222-DXS6850-DXS255++ +-CIC-5-DXS146-cen. A sequence-ready map was constructed for an 1100-kb gene-rich interval flanked by the markers HB3 and DXS1039, from which six novel ESTs/STSs were isolated, thus increasing the number of markers used in this interval to thirty. This precise ordering is a prerequisite for the construction of a transcription map of this region that contains numerous disease loci, including those for several forms of retinal degeneration and mental retardation. In addition, the map provides the base to delineate the corresponding syntenic region in the mouse, where the mutants scurfy and tattered are localized.
The Gfi1 gene encodes a zinc finger protein which binds DNA and is involved in transcriptional regulation. Gfi1 was assigned to the central portion of mouse Chr 5 by interspecific backcross mapping and to human chromosome band 1p22 and rat chromosome band 14p22 by fluorescence in situ hybridization (FISH). Comparative mapping data presented here describes a new syntenic region between man and rodents.
Obesity, mainly characterized by the excess fat storage, is a global health problem resulting in serious morbidity and mortality. Identification of molecular mechanisms in adipogenic differentiation pathway might lead to development of new strategies for diagnosis, prevention and therapy of obesity and associated diseases. Discovery of new genes and proteins in the differentiation pathway could help to understand the key specific regulators of the adipogenesis. Cytoglobin (Cygb), identified as a new globin family member protein, is expressed in various tissues. Although its interaction with oxygen and nitric oxide indicates the potential role in antioxidant pathways, the exact role remains unclear. In the current study, expression level of Cygb was determined in proliferating and differentiating 3T3-F442A cells by gene expression and protein expression analysis. Results revealed that Cygb expression up-regulated in differentiated cells in parallel with adipogenic differentiation markers; PPARγ, CEBPα and FABP4 expressions. Besides, Cygb overexpression in preadipocytes contributed to the adipogenic differentiation as verified by detection of higher lipid droplets and increased PPARγ, CEBPα and FABP4 expressions with respect to control cells. These findings will shed light on the unknown roles of Cygb in adipogenesis and obesity.
Iron plays the central role in the oxygen transport by the erythrocyte as a constituent of heme and hemoglobin. The importance of iron and heme also resides in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is inactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1 mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses these genes in erythroblasts under iron deficiency to balance the levels of heme and globin. Moreover, an increase in genome-wide DNA methylation was observed in erythroblasts of Bach1-/- mice under iron deficiency. These findings reveal the principle role of iron as a regulator of gene expression in erythroblast maturation and suggest that the iron-heme-Bach1 axis is important for a proper adaptation of erythroblast to iron deficiency to avoid toxic aggregates of non-heme globin.
Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. β2-globin mRNA expression peaked at day 7 after the start of culture. Larval β2-globin was highly expressed in NF56-derived cells, while adult β2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.
The multiple sequence alignment (MSA) of a protein family provides a wealth of information in terms of the conservation pattern of amino acid residues not only at each alignment site but also between distant sites. In order to statistically model the MSA incorporating both short-range and long-range correlations as well as insertions, I have derived a lattice gas model of the MSA based on the principle of maximum entropy. The partition function, obtained by the transfer matrix method with a mean-field approximation, accounts for all possible alignments with all possible sequences. The model parameters for short-range and long-range interactions were determined by a self-consistent condition and by a Gaussian approximation, respectively. Using this model with and without long-range interactions, I analyzed the globin and V-set domains by increasing the "temperature" and by "mutating" a site. The correlations between residue conservation and various measures of the system's stability indicate that the long-range interactions make the conservation pattern more specific to the structure, and increasingly stabilize better conserved residues.
Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory mediator and its expression is up-regulated in chronic obstructive pulmonary disease (COPD). Tristetraprolin (TTP) is implicated in regulation of TNF-α expression; however, whether TTP is involved in cigarette smoke-induced TNF-α expression has not been determined.
TTP expression was examined by western blot analysis in murine alveolar macrophages and alveolar epithelial cells challenged without or with cigarette smoke extract (CSE). TNF-α mRNA stability, and the decay of TNF-α mRNA, were determined by real-time quantitative RT-PCR. TNF-α protein levels were examined at the same time in these cells. To identify the molecular mechanism involved, a construct expressing the human beta-globin reporter mRNA containing the TNF-α 3'-untranslated region was generated to characterize the TTP targeted site within TNF-α mRNA.
CSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-α mRNA stability. Importantly, increased TNF-α mRNA stability due to impaired TTP function resulted in significantly increased TNF-α levels in these cells. Forced TTP expression abrogated the increased TNF-α mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-α mRNA 3'-untranslated region, the data indicate that TTP directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level.
The data demonstrate that cigarette smoke exposure reduces TTP expression and impairs TTP function, resulting in significantly increased TNF-α mRNA stability and excessive TNF-α expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-α expression and inflammatory response induced by cigarette smoke.
Allogeneic hematopoietic stem cell transplantation has been established for several decades as a gene replacement therapy for patients with thalassemia major and now offers very high rates of cure to those who are able to access this therapy. Outcomes have improved tremendously over the last decade even in high-risk patients. The limited data available suggests that the long-term outcome is also excellent with >90% survival but for best results, hematopoietic stem cell transplantation should be offered early before any end organ damage occurs. However, access to this therapy is limited by lack of suitable donors in more than half the patients. Inadequate hematopoietic stem cell transplantation services and the cost of therapy are other reasons for the same, particularly in those parts of the world which have a high prevalence of this condition. As a result <10% of eligible patients are actuallyable to avail this therapy. Other options for curative therapies are therefore needed. Recently, gene correction in autologous hematopoietic stem cells has been successfully established using lentiviral vectors, and several clinical trials have been initiated. A gene editing approach to correct the β globin mutation or disrupt BCL11A to increase fetal hemoglobin production has also been reported and is expected to be introduced in clinical trials soon. Curative possibilities for the major hemoglobin disorders are expanding. Providing access to these therapies around the world would be the challenge.
Although azanucleoside DNA-hypomethylating agents (HMAs) are routinely used for the treatment of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), very few outcome predictors have been established. Expression of the β-like globin gene locus is tightly regulated by DNA methylation, is HMA-sensitive in vitro, and fetal haemoglobin (HbF) expression is under study as a potential biomarker for response of MDS patients to azacitidine. We determined HbF expression in 16 MDS and 36 AML patients receiving decitabine (DAC). Pre-treatment HbF was already elevated (>1·0% of total haemoglobin) in 7/16 and 12/36 patients, and HbF was induced by DAC in 81%/54% of MDS/AML patients, respectively. Elevated pre-treatment HbF was associated with longer median overall survival (OS): 26·6 vs. 8·6 months for MDS (hazard ratio [HR] 8·56, 95% confidence interval [CI] 1·74-42·49, P = 0·008, with similarly longer progression-free and AML-free survival), and 10·0 vs. 2·9 months OS for AML (HR 3·01, 95% CI 1·26-7·22, P = 0·014). In a multivariate analysis, the prognostic value of HbF was retained. Time-dependent Cox models revealed that the prognostic value of treatment-induced HbF induction was inferior to that of pre-treatment HbF. In conclusion, we provide first evidence for in vivo HbF induction by DAC in MDS/AML, and demonstrate prognostic value of elevated pre-treatment HbF, warranting prospective, randomized studies.
Thalassemia is an inherited autosomal recessive blood disorder, caused by mutations in globin genes or their regulatory regions. This results in a reduced rate of synthesis of one of the globin chains that make up haemoglobin. In ß-thalassaemia major there is an underproduction of ß-globin chains combined with excess of free α-globin chains. The excess free α-globin chains precipitate in red blood cells, leading to their destruction (haemolysis) and ineffective erythropoiesis. The conventional approach to treatment is based on the correction of haemoglobin status through regular blood transfusions and iron chelation therapy for iron overload. Although conventional treatment has the capacity to improve the quality of life of people with ß-thalassaemia major, allogeneic hematopoietic stem cell transplantation is the only currently available procedure which has the curative potential. This is an update of a previously published Cochrane Review.
To evaluate the effectiveness and safety of different types of allogeneic hematopoietic stem cell transplantation, in people with severe transfusion-dependant ß-thalassaemia major, ß-thalassaemia intermedia or ß0/+- thalassaemia variants requiring chronic blood transfusion.
We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Haemoglobinopathies Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings.Date of the most recent search: 18 August 2016.
Randomised controlled trials and quasi-randomised controlled trials comparing allogeneic hematopoietic stem cell transplantation with each other or with standard therapy (regular transfusion and chelation regimen).
Two review authors independently screened studies and had planned to extract data and assess risk of bias using standard Cochrane methodologies but no studies were identified for inclusion.
No relevant studies were retrieved after a comprehensive search of the literature.
We were unable to identify any randomised controlled trials or quasi-randomised controlled trials on the effectiveness and safety of different types of allogeneic stem cell transplantation in people with severe transfusion-dependant ß-thalassaemia major or ß0/+- thalassaemia variants requiring chronic blood transfusion. The absence of high-level evidence for the effectiveness of these interventions emphasises the need for well-designed, adequately-powered, randomised controlled clinical trials.
Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women.
This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number.
The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine.
Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women.
Thalassemia is the most common monogenic inherited disease worldwide, affecting individuals originating from many countries to various extents. As the disease requires long-term care, prevention of the homozygous state presents a substantial global disease burden. The comprehensively preventive programs involve carrier detections, molecular diagnostics, genetic counseling, and prenatal diagnosis. Invasive prenatal diagnosis refers to obtaining fetal material by chorionic villus sampling (CVS) at the first trimester, and by amniocentesis or cordocentesis at the second trimester. Molecular diagnosis, which includes multiple techniques that are aimed at the detection of mutations in the α- or β-globin genes, facilitates prenatal diagnosis and definitive diagnosis of the fetus. These are valuable procedures for couples at risk, so that they can be offered options to have healthy offspring. According to local practices and legislation, genetic counseling should accompany the invasive diagnostic procedures, DNA testing, and disclosure of the results. The most critical issue in any type of prenatal molecular testing is maternal cell contamination (MCC), especially when a fetus is found to inherit a particular mutation from the mother. The best practice is to perform MCC studies on all prenatal samples. The recent successful studies of fetal DNA in maternal plasma may allow future prenatal testing that is non-invasive for the fetus and result in significant reduction of invasive diagnostic procedures.
A previous study conducted by our group demonstrated that Radix Astragali compounded with Codonopsis pilosula and Plastrum testudinis was effective in treating pediatric β-thalassemia in a randomized, controlled clinical trial. However, the mechanism of action that underpins this treatment remains to be elucidated. Blood was collected from patients participating in this clinical trial and nucleated red blood cell-enriched mononuclear cells were isolated to facilitate the extraction of RNA and protein. RT-PCR was used to monitor the expression of globin genes and p38 MAPK, and total and phosphorylated p38 MAPK expression was assessed using Western blot analysis. Expression of α-, β-, and Aγ-globin mRNAs was not significantly affected following treatment with R. Astragali or the compounded formulation. However, Gγ-globin mRNA levels increased significantly in both treatment groups (when compared with pretreatment levels) following 12 weeks of treatment. Moreover, posttreatment Gγ-globin expression was significantly higher in both treatment groups compared with the control group. Although neither p38 MAPK mRNA nor protein levels were affected by the treatments, posttreatment phosphorylation of p38 MAPK was significantly increased in the R. Astragali and compounded formulation groups compared with the control group. These data suggest that the molecular mechanisms that underpin the efficacious use of R. Astragali (and its compounded formulation) in pediatric β-thalassemia treatment facilitate the induction of Gγ-globin expression following activation of p38 MAPK.
Thalassemia is a significant health problem worldwide. Prenatal diagnosis is the only effective way to prevent the birth of a fetus with severe thalassemias, which include hemoglobin Bart's hydrops fetalis and thalassemia major. However, accurate prenatal diagnosis depends on the comprehensive consideration of the molecular basis of thalassemias. To make a correct decision, the obstetrician should have a certain understanding of the genetics of thalassemias. Here we present a brief introduction of some fundamental genetic knowledge of thalassemias, including the production of hemoglobin, structure and location of globin genes, hemoglobin switch, epidemiology, clinical classification, molecular and cellular pathology, genotype-phenotype correlation, and genetic modifiers. Furthermore, some unusual clinical cases that cannot be explained by Mendel's laws are described. On the basis of a thorough understanding of the above information, clinicians should have the ability to precisely diagnose thalassemia patients and provide applicable genetic counselling to the affected families.
Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3' UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3' UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3' UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing β-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD.
β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line.
A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 μg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells.
A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones.
The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.
Endothelial Nitric Oxide Synthase (eNOS) is crucial for vascular homeostasis. Polymorphisms T786C and G894T are reportedly affecting eNOS regulation and have been related to various diseases. Sickle Cell Disease (SCD), a clinically diverse chronic hemolytic anemia, implies impaired nitric oxide bioavailability. Aim of this study was to determine eNOS genotype for T786C and G894T polymorphisms in Greek patients with SCD and to elucidate its functional consequences and possible effects on clinical phenotype. Seventy nine steady state cases, most of them compound heterozygous for Sickle cell anemia/beta thalassemia and 48 controls were enrolled. Peripheral blood DNA was extracted and genotyped with PCR-RFLPs and Sanger sequencing. Total RNA was extracted from 14 patients and eNOS mRNA levels were determined by real-time PCR. Genotypes, allele distribution and eNOS mRNA levels did not differ between patients and controls, nor among patients with different beta globin gene mutations. The 786CC genotype was more common in S/S and β(0)/S patients with retinopathy. Moreover 894 TT S/S and β(0)/S patients tended to have higher hematocrit than 894 GG and GT ones. However the T786C eNOS genotype does not seem to affect eNOS mRNA levels, at least in steady state cases. This is the first report describing the effects of eNOS polymorphisms on different forms of SCD, the first enrolling SCD patients of Caucasian origin and the first determining eNOS mRNA levels in peripheral blood from steady-state SCD patients.
Globins are globular proteins for either transport or storage of oxygen which are critical for cellular metabolism. Four globins have been identified in rodent and human brains. Among them, neuroglobin, cytoglobin and hemoglobin chains are constitutively expressed in normal brain, while myoglobin is only expressed in some neurological disorders. Studies on the molecular structure, expression and functional features of these brain globins indicated that they may play crucial roles in maintenance of neural cell survival and activity, including neurons and astrocytes. Their regulation in neurological disorders may help thoroughly understand initiation and progression of ischemia, Alzheimer's disease and glioma, etc. Elucidation of the brain globin functions might remarkably improve medical strategies that sustain neurological homeostasis and treat neurological diseases. Here the expression pattern and functions of brain globins and their involvement in neurological disorders are reviewed.
Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts.
Sickle cell anemia (SCA) is a group of hemoglobin disorders in which the sickle β-globin gene is inherited. It is associated with many complications; most of them are related to thrombotic events.
This study aimed to investigate the association between angiotensin converting enzyme (ACE) insertion/deletion polymorphism and complications of SCA.
A case-control study was conducted in Khartoum state.
A total of 50 patients with SCA and 40 healthy volunteers as a control group were enrolled in this study. Three milliliters of ethylenediamine tetraacetic acid anticoagulated blood were collected from each subject, DNA was extracted by salting-out method, and target DNA regions of the ACE gene were amplified using allele-specific polymerase chain reaction.
Data of this study was analyzed by Statistical Package for Social Sciences. Frequency of qualitative variables was calculated, and correlation was tested by Chi-square test. Regression was used to investigate the association between the polymorphism and complications of SCA.
The frequencies of the DD, ID, and II genotypes were 42%, 50%, and 8%, respectively, for patients, whereas in the control group, it was 80% for DD genotype and 20% for ID, while II genotype was totally absent. The regression analysis showed no statistically significant association between the disease complications and each of the ACE polymorphic genotypes.
No statistically significant association was found between ACE polymorphism and complications of SCA.
The structural and functional conservation of hemoglobin throughout mammals has made the laboratory mouse an exceptionally useful organism in which to study both the protein and the individual globin genes. Early researchers looked to the globin genes as an excellent model in which to examine gene regulation - bountifully expressed and displaying a remarkably consistent pattern of developmental activation and silencing. In parallel with the growth of research into expression of the globin genes, mutations within the β-globin gene were identified as the cause of the β-hemoglobinopathies such as sickle cell disease and β-thalassemia. These lines of enquiry stimulated the development of transgenic mouse models, first carrying individual human globin genes and then substantial human genomic fragments incorporating the multigenic human β-globin locus and regulatory elements. Finally, mice were devised carrying mutant human β-globin loci on genetic backgrounds deficient in the native mouse globins, resulting in phenotypes of sickle cell disease or β-thalassemia. These years of work have generated a group of model animals that display many features of the β-hemoglobinopathies and provided enormous insight into the mechanisms of gene regulation. Substantive differences in the expression of human and mouse globins during development have also come to light, revealing the limitations of the mouse model, but also providing opportunities to further explore the mechanisms of globin gene regulation. In addition, animal models of β-hemoglobinopathies have demonstrated the feasibility of gene therapy for these conditions, now showing success in human clinical trials. Such models remain in use to dissect the molecular events of globin gene regulation and to identify novel treatments based upon the reactivation of developmentally silenced γ-globin. Here, we describe the development of animal models to investigate globin switching and the β-hemoglobinopathies, a field that has paralleled the emergence of modern molecular biology and clinical genetics.
We report the case of a nine-year-old girl, of Moroccan origin, hospitalised for fever and stomach ache. The clinical and biological investigations showed merely a moderate enlargement of the spleen associated with discrete regenerative hemolytic anemia. The etiologic analysis of the hemolysis was completed by the electrophoresis of hemoglobin (Hb) that revealed total absence of HbA, with the presence of 98.7% of HbF and 1.3% of HbA2. These results led to a diagnosis of β0 thalassemia associated with an intermediate phenotype, i.e. beta thalassemia intermedia (BTI). The molecular study of the β-globin gene evidenced a homozygous mutation at codon 35 of the exon 2 TAC>TAA (HBB: c.108C>A) leading to the premature stop codon. The study of several polymorphisms involved in the regulation of the expression of HbF concords with the intermediate phenotype found in the patient. This observation offers an opportunity to redefine BTI and further explore its clinical and genetic specificity.
We have isolated, via differential hybridization screening of a floral cDNA library from sunflower, a cDNA clone that hybridizes to a 1100 nucleotide-long mRNA found exclusively in mature pollen grains. The cDNA encodes a 219 amino acid-long polypeptide containing two potential zinc fingers alternating with two basic domains. A similar organization is found in the erythroid-specific transcription factor Eryf1 from chicken and its murine homolog GF-1. The C-terminus of the protein contains a sixfold repeat of the pentapeptide sequence (S,T,A)(E,D)TQN. These features suggest that the SF3 protein is a transcription factor required for the expression of late pollen genes. The SF3 gene is a member of a multicopy gene family. A genomic copy of the gene has been isolated and sequenced; it is split by four short, AT-rich introns.
We have cloned the gene for the chicken erythroid transcription factor GATA-1 (formerly Eryf1, NF-E1, or GF-1). The gene is composed of six exons, two of which encode the two finger domains of the protein. Transcription of GATA-1 in chicken embryonic erythroid cells initiates from multiple sites clustered approximately 200 base pairs upstream from the start of protein-coding sequence. A number of sequence motifs for known DNA-binding proteins are found to be protected in DNase I-footprinting experiments by either erythroid or brain nuclear extracts or by both. Notably, a cluster of three GATA-1 sites is protected by the erythroid extract, as well as by purified GATA-1. We find that the upstream region of the gene functions as a powerful promoter when transfected into embryonic erythroid cells. In primary chicken embryo fibroblasts the promoter exhibits lower activity, which is increased when the cells are cotransfected with a second plasmid expressing the GATA-1 cDNA. The results suggest that GATA-1 protein plays an autoregulatory role in its own expression.
The areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, encodes a positive-acting regulatory protein with a single putative DNA-binding 'zinc finger' which is remarkably similar to the two 'zinc fingers' of the major regulatory protein of vertebrate erythroid cells (GF-1/Eryf1/NF-E1). The areA-300 mutation alters the specificity of gene activation in that it elevates expression of certain structural genes whilst reducing expression of certain others. It is an 'in-frame' tandem duplication of 417 bp including the entire DNA-binding region. The consequences of areA didactyly are further explored by construction of a double mutant having an altered loop residue in the N-terminal 'finger'.
All asparagine-linked glycans (N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT-1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (β1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT-1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1 kb region upstream from HMT-1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from -33 to -1 positions might act in HMT-1 transcription at basal level and that the region from -200 to -42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200 bp region upstream from HMT-1 ORF might significantly upregulate HMT-1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at -148 to -134 positions should serve as a strong silencer. The control of the HMT-1 transcription by these motifs resided within the 200 bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT-1 expression.
GATA-1, a zinc finger transcription factor, has been demonstrated to play a key role in the progression of leukemia. In this study, we investigate the effects of wogonoside, a naturally bioactive flavonoid derived from Scutellaria baicalensis Georgi, on cell growth and cell cycle in chronic myeloid leukemia (CML) cells, and uncover its underlying mechanisms. The experimental design comprised CML cell lines K562, imatinib-resistant K562 (K562r) cells, and primary CML cells, treated in vitro or in vivo, respectively, with wogonoside; growth and cell cycle were then evaluated. We found that wogonoside could induce growth inhibition and G0/G1 cell cycle arrest in both normal and K562r cells. Wogonoside promotes the expression of GATA-1 and facilitates the binding to methyl ethyl ketone (MEK) and p21 promoter, thus inhibiting MEK/extracellular signal-regulated kinase signaling and cell cycle checkpoint proteins, including CDK2, CDK4, cyclin A, and cyclin D1, and increasing p21 expression. Furthermore, in vivo studies showed that administration of wogonoside decreased CML cells and prolonged survival in NOD/SCID mice with CML cell xenografts. In conclusion, these results clearly revealed the inhibitory effect of wogonoside on the growth in CML cells and suggested that wogonoside may act as a promising drug for the treatment of imatinib-resistant CML.
Eosinophils arise from hematopoietic CD34(+) stem cells in the bone marrow. They acquire IL-5Rα on their surface at a very early stage during eosinophilopoiesis, and differentiate under the strong influence of interleukin (IL)-5. They then exit to the bloodstream, and enter the lung upon exposure to airway inflammatory signals, including eotaxins. In inflamed tissues, eosinophils act as key mediators of terminal effector functions and innate immunity and in linking to adaptive immune responses. Transcription factors GATA-1, CCAAT/enhancer-binding protein, and PU.1 play instructive roles in eosinophil specification from multipotent stem cells through a network of cooperative and antagonistic interactions. Not surprisingly, the interplay of these transcription factors is instrumental in forming the regulatory circuit of expression of eosinophil-specific genes, encoding eosinophil major basic protein and neurotoxin, CC chemokine receptor 3 eotaxin receptor, and IL-5 receptor alpha. Interestingly, a common feature is that the critical cis-acting elements for these transcription factors are clustered in exon 1 and intron 1 of these genes rather than their promoters. Elucidation of the mechanism of eosinophil development and activation may lead to selective elimination of eosinophils in animals and human subjects. Furthermore, availability of a range of genetically modified mice lacking or overproducing eosinophil-specific genes will facilitate evaluation of the roles of eosinophils in the pathogenesis of asthma. This review summarizes eosinophil biology, focusing on development and regulation of eosinophil-specific genes, with a heavy emphasis on the causative link between eosinophils and pathological development of asthma using genetically modified mice as models of asthma.
α-Hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the α-hemoglobin (α-Hb) chains against precipitation. α-Hemoglobin stabilizing protein forms a heterodimer complex with α-Hb, then displaying modified oxygen binding kinetics. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of bone marrow leading to apoptosis and defective erythropoiesis. Several α-Hb variants with a structural abnormality, frequently located in the contact area between α-Hb and AHSP, exhibit instability and a thalassemia-like syndrome when they are associated with another α-thalassemia (α-thal) determinant. We suggest that this disorder could result from a disturbed interaction between the abnormal α-Hb chains and AHSP. Hb Groene Hart (Pro119>Ser) was one of the first examples in which we observed this abnormality. We later verified this mechanism in a list of several variants, now considered as being nondeletional α-thalassemias. Conversely, it was hypothesized from studies on knock-out mice, that a defect affecting AHSP could cause a thalassemia-like syndrome. This was supported in man by studies showing that a decreased expression of AHSP linked to specific genetic clades could act as a modulating factor in some thalassemia phenotypes. It was also supported by our observation of a family from Southeast Asia, in which a child homozygous for an AHSP mutant (Val56>Gly) displayed, in his first year of life, a moderate thalassemia syndrome. This mutant AHSP was expressed in vitro and demonstrated by biochemical and biophysical studies to display a clear defective interaction with α-Hb, which could support the hypothesis that the reb blood cell (RBC) disorders of the child resulted from this abnormality. It therefore appears that AHSP is a factor with a key role in the formation of Hb tetramers and that structural abnormalities, either on the α-Hb or on the AHSP, may act as a thalassemia modulating factor.
LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.
The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hematopoietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dickkopf-1 [Dkk-1], growth factor independent-1 [Gfi-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfi-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development.
Alpha hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the alpha hemoglobin (alpha-Hb) chains against precipitation. AHSP and alpha-Hb form a heterodimer complex. In the absence of AHSP, alpha-Hb oxidizes and precipitates within the erythrocyte precursors of the bone marrow leading to apoptosis and defective erythropoiesis. In vitro the binding of AHSP to ferrous alpha-Hb accelerates oxidation of the heme iron in alpha-Hb, but the complex is more resistant to protein unfolding. AHSP could act as a modulating factor in beta-thalassemia. Recent studies showed more severe thalassemic syndromes in patients with decreased levels of AHSP and in one patient who carried a structurally abnormal AHSP. Some alpha-Hb variants with structural abnormality located in the contact area between alpha-Hb and AHSP exhibit an instability and a thalassemic like syndrome. We suggest that this could result from a disturbed interaction between alpha-Hb variants and AHSP. To study this interaction, we constructed the pGEX-alpha-AHSP vector that co-expressed human alpha-Hb and AHSP. Using this approach, we investigated the alpha42 (C7), alpha104 (G11) and alpha119 (H2) sites, where variants with some thalassemic features have been described. Results obtained with recombinant Groene Hart alpha-Hb and Diamant alpha-Hb, in which proline 119 is replaced by a serine and a leucine, respectively, showed clearly an impaired interaction with AHSP. In contrast, the alpha mutants at the sites 42 and 104 exhibit a normal interaction with AHSP. The CO rebinding kinetics of the AHSP/alpha-Hb(42mutant) complexes were similar to those previously obtained with the AHSP/alpha-Hb(WT) complex, which shows a modified rate that is intermediate to the classical Hb allosteric states.
Erythroid cell production results from passage through cellular hierarchies dependent on differential gene expression under the control of transcription factors responsive to changing niches. We have constructed Genetic Regulatory Networks (GRNs) describing this process, based predominantly on mouse data. Regulatory network motifs identified in E. coli and yeast GRNs are found in combination in these GRNs. Feed-forward motifs with autoregulation generate forward momentum and also control its rate, which is at its lowest in hematopoietic stem cells (HSCs). The simultaneous requirement for multiple regulators in multi-input motifs (MIMs) provides tight control over expression of target genes. Combinations of MIMs, exemplified by the SCL/LMO2 complexes, which have variable content and binding sites, explain how individual regulators can have different targets in HSCs and erythroid cells and possibly also how HSCs maintain stem cell functions while expressing lineage-affiliated genes at low level, so-called multi-lineage priming. MIMs combined with cross-antagonism describe the relationship between PU.1 and GATA-1 and between two of their target genes, Fli-1 and EKLF, with victory for GATA-1 and EKLF leading to erythroid lineage specification. These GRNs are useful repositories for current regulatory information, are accessible in interactive form via the internet, enable the consequences of perturbation to be predicted, and can act as seed networks to organize the rapidly accumulating microarray data.
Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity.
Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.
The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line.
Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML.
Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes.
Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.
The a- and b-globin gene clusters are subject to several levels of regulation. They are expressed exclusively in the erythroid cells, only during defined periods of development and in a perfectly tuned way, assuring, at any stage of ontogeny, a correct balance in the availability of a- and b-globin chains for hemoglobin assembling. Such a tight control is dependent on regulatory regions of DNA located either in proximity or at great distances from the globin genes in a region characterized by the presence of several DNAse I hypersensitive sites and known as the Locus Control Region. All these sequences exert stimulatory, inhibitory or more complex activities by interacting with transcription factors that bridge these regions of DNA to the RNA polymerase machinery. Many of these factors have now been cloned and the corresponding mouse genes inactivated, shading new light on the metabolic pathways they control. It is increasingly recognized that such factors are organized into hierarchies according to the number of genes and circuits they regulate. Some genes such as GATA-1 and 2 are master regulators that act on large numbers of genes at early stage of differentiation whereas others, like EKLF, stand on the lowest step and control only single or limited number of genes at late stages of differentiation. We will review recent data gathered from expression studies in cell cultures, in transgenic or K.O. murine models as well as from a clinical settings. We will also discuss the development of novel theories on the regulation of the a- and b-globin genes and clusters.
We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.
FOG is a multitype zinc finger protein that is essential for megakaryopoiesis, binds to the amino-terminal finger of GATA-1, and modulates the transcription of GATA-1 target genes. Presently investigated are effects of FOG and GATA-1 on the transcription of the megakaryocytic integrin gene, alpha IIb. In GATA-1-deficient FDCER cells (in the presence of endogenous FOG), ectopically expressed GATA-1 activated transcription 3-10-fold both from alpha IIb templates and the endogenous alpha IIb gene. The increased expression of FOG increased reporter construct transcription 30-fold overall. Unexpectedly, alphaIIb gene transcription also was stimulated efficiently upon the ectopic expression in of FOG per se. This occurred in the absence of any detectable expression of GATA-1 and was observed in multiple independent sublines for both the endogenous alpha IIb gene and transfected constructs yet proved to depend largely upon conserved GATA elements 457 and 55 base pairs upstream from the transcriptional start site. In 293 cells, FOG plus GATA-1 but not FOG alone only moderately stimulated alpha IIb transcription, and no direct interactions of FOG with the alpha IIb promoter were detectable. Thus, FOG acts in concert with GATA-1 to stimulate alpha IIb expression but also can act via a GATA-1-independent route, which is proposed to involve additional hematopoietic-restricted cofactors (possibly GATA-2).
To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5'-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation.
We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5'-flanking region of the human embryonic (epsilon) globin gene, located at -270 base pairs 5' to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this epsilon-globin silencer, but also suggest that down-regulation of epsilon-globin gene expression may involve other negative elements flanking the epsilon-globin gene. We have now extended the analysis of epsilon-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5' to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at -3 kilobase pairs (epsilonNRA-I and epsilonNRA-II), flanked by a positive control element, has a strong effect on the epsilon-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within epsilonNRA-II are directly needed for negative control. Both epsilonNRA-I and epsilonNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at -1.7 kilobase pairs (epsilonNRB) does not exhibit general silencer activity as epsilonNRB does not affect transcription activity when used in conjunction with an epsilon-globin minimal promoter. The negative effect of epsilonNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the epsilon-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of epsilon-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, epsilonNRA-I/epsilonNRA-II, epsilonNRB, the epsilon-globin silencer and probably other negative and positive elements in the 5'-flanking region of the epsilon-globin gene.
GATA transcription factors are required for the differentiation of diverse cell types in several species. Recent evidence suggests that their biologic activities may be modulated through interaction with multitype zinc finger proteins, such as Friend of GATA-1 (FOG) and U-shaped (Ush). In cell culture, FOG cooperates with the hematopoietic transcription factor GATA-1 to promote erythroid and megakaryocytic differentiation. We show here that mice lacking FOG die during mid-embryonic development with severe anemia. FOG-/- erythroid cells display a marked, but partial, blockage of maturation, reminiscent of GATA-1- erythroid precursors. In contrast to GATA-1 deficiency, however, megakaryocytes fail to develop in the absence of FOG. Although the FOG-/- erythroid phenotype supports the proposed role of FOG as a GATA-1 cofactor in vivo, the latter finding points to a pivotal, GATA-1-independent requirement for FOG in megakaryocyte development from the bipotential erythroid/megakaryocytic progenitor. We speculate that FOG and other FOG-like proteins serve as complex cofactors that act through both GATA-dependent and GATA-independent mechanisms.
Studies of hematopoietic progenitor cell development in vivo, ex vivo, and in factor-dependent cell lines have shown that c-kit promotes proliferation through synergistic effects with at least certain type 1 cytokine receptors, including the erythropoietin (Epo) receptor. Presently, c-kit is shown to efficiently support both mitogenesis and survival in the FDCP1 cell subline, FDC2. In this system, mitogenic synergy with c-kit was observed for ectopically expressed wild-type Epo receptors (wt-ER), an epidermal growth factor (EGF) receptor/Epo receptor chimera, and a highly truncated Epo receptor construct ER-Bx1. Thus, the Epo receptor cytoplasmic box 1 subdomain appears, at least in part, to mediate mitogenic synergy with c-kit. In studies of potential effectors of this response, Jak2 tyrosine phosphorylation was shown to be induced by Epo, but not by stem cell factor (SCF). In addition and in contrast to signaling in Mo7e and BM6 cell lines, in FDC2-ER cells SCF and Epo each were shown to rapidly activate Pim 1 gene expression. Recently, roles also have been suggested for the nuclear trans-factor GATA-1 in regulating progenitor cell proliferation. In FDC2-ER cells, the ectopic expression of GATA-1 had no detectable effect on Epo inhibition of apoptosis. However, GATA-1 expression did result in a selective and marked inhibition in mitogenic responsiveness to SCF and to a decrease in c-kit transcript expression. These studies of SCF and Epo signaling in FDC2-wt-ER cells serve to functionally map the ERB1 region as a c-kit-interactive domain, suggest that Pim1 might contribute to SCF and Epo mitogenic synergy and support the notion that SCF and Epo may act in opposing ways during red cell differentiation.
Long-range promoter-enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken betaA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein-protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.
Xenopus GATA-6 transcripts are first detected at the beginning of gastrulation in the mesoderm, and subsequent domains of expression include the field of cells shown to have heart-forming potential. In this region, GATA-6 expression continues only in those cells that go on to form the heart; however, a decrease occurs prior to terminal differentiation. Artificial elevation of GATA-6, but not GATA-1, prevents expression of both cardiac actin and heart-specific myosin light chain. This effect is heart-specific because cardiac actin expression is unaffected in somites. Expression of the earlier marker XNkx-2.5 was unaffected and morphological development of the heart was initiated independently of the establishment of the contractile machinery. We conclude that a reduction in the level of GATA-6 is important for the progression of the cardiomyogenic differentiation programme and that GATA-6 may act to maintain heart cells in the precursor state. At later stages, when the elevated GATA-6 levels had decayed, differentiation ensued but the number of cells contributing to the myocardium had increased, suggesting either that the blocked cells had proliferated or that additional cells had been recruited.
The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP. The VBI contributes to both embryonic and adult blood, whereas the DLP contains precursors of adult blood only, which have not been identified previously with molecular markers. The possibility that the GATA-2-expressing cells between the VBI and DLP were haematopoietic progenitors migrating from the VBI to the DLP was ruled out by transplantation analysis. Differential effects of RA on the expression of GATA-1 and GATA-2 suggest that RA has a direct action on haematopoietic differentiation, rather than on the formation of haematopoietic mesoderm.
Our studies of the promoters of the myeloid CSF receptors (M, GM, and G) in cell lines have led to the findings that the promoters are small, and are all activated by the PU.1 and C/EBP proteins. To date, we have only found evidence for involvement of C/EBP alpha, although further experiments will be needed to exclude the role of C/EBP beta and C/EBP delta in receptor gene expression. These studies suggest a model of hematopoiesis (Fig. 2) in which the lineage commitment decisions of multipotential cells are made by the alternative patterns of expression of certain transcription factors, which then activate growth factor receptors which allow those cells to respond to the appropriate growth factor to proliferate and survive. For example, expression of GATA-1 activates its own expression, as well as that of the erythropoietin receptor, inducing these cells to be capable of responding to erythropoietin. Similarly, expression of PU.1 activates its own promoter, and turns on the three myeloid CSF receptors (M, GM, and G), pushing these cells along the pathway of myeloid differentiation. C/EBP proteins, particularly C/EBP alpha, are also critical for myeloid receptor promoter function, and may also act via autoregulatory mechanisms. Murine C/EBP alpha has a C/EBP binding site in its own promoter. Human C/EBP alpha autoregulates its own expression in adipocytes by activating the USF transcription factor. Myeloid genes expressed later during differentiation, such as CD11b, are also activated by PU.1, which is expressed at highest levels in mature myeloid cells, but not by C/EBP alpha, which is downregulated in a differentiated murine myeloid cell line. Consistent with this model are the findings that overexpression of PU.1 in erythroid cells blocks erythroid differentiation, leading to erythroleukemia, and overexpression of GATA-1 in a myeloid line blocks myeloid differentiation. While these findings have provided some framework for understanding myeloid gene regulation, there are a number of critical questions to be addressed in the near future: What is the pattern of expression of the C/EBP proteins during the course of myeloid differentiation and activation of human CD34+ cells? What is the effect of targeted disruption and other mutations of the C/EBP and AML1 proteins on myeloid development and receptor expression? What are the interactions among these three different types of factors (ets, basic region-zipper, and Runt domain proteins) to activate the promoters? What is the effect of translocations, mutations, and alterations in expression of these factors, particularly in different forms of AML?
To elucidate the mechanisms underlying the tissue-restricted expression of GATA factor transcription, we have isolated and analyzed the genomic chicken GATA-3 (cGATA-3) locus. Structural analysis of the clones showed that the cGATA-3 gene consists of six exons which span more than 19 kb. Two trans-activating domains and two Zn finger domains of cGATA-3 were found to be encoded separately by exons 2/3 and 4/5, respectively, indicating that each functional domain of GATA-3 is encoded by a discrete exon. We have determined 1.7 kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites. Activities of presumptive regulatory regions of this gene were assessed by transfecting chimeric constructs into a chicken T cell line MSB-1. The results showed three features of cGATA-3 gene regulation. The basal promoter activity of the cGATA-3 gene is determined by sequences lying between -104 and -29 bp of the promoter region. The upstream region containing the GATA and CACCC elements in close proximity (-1280 to -1152) appeared to act as a negative transcriptional regulator, whereas the region -1151 to -850 acts as a positive regulator. Thus, the expression of cGATA-3 gene is under complex regulation and the mode of regulation of cGATA-3 gene expression is suggested to be different from that of GATA-1 genes.
The gene pannier acts as a repressor of achaete and scute, two transcription factors expressed in discrete subsets of cells at the sites where neural precursors develop. Molecular analysis of mutant alleles revealed the presence of two functional domains within the pannier protein: a zinc finger domain showing homology to the GATA-1 family of vertebrate transcription factors and a domain comprising two putative amphipathic helices. Mutants associated with lesions in the zinc finger domain display an overexpression of achaete and scute and the development of extra neural precursors. Mutant proteins in which the domain including the putative helices is deleted act as hyperactive repressor molecules causing a loss of achaete/scute expression and a loss of neural precursors. Other results suggest that the activity of pannier may be modulated by association with position-specific factors.
GATA-1, a transcription factor of the 'zinc-finger' family, is required for the development of mature erythroid cells and is also highly expressed in the megakaryocytic and mast cell lineages. The helix-loop-helix gene SCL (or TAL) is expressed in the same three hematopoietic lineages as GATA-1. To explore the role of GATA-1 and SCL in hematopoietic differentiation, we introduced a new expression vector bearing each gene into the early myeloid cell line 416B, which could originally differentiate in vivo along the megakaryocytic and granulocytic lineages. Enforced expression of SCL at high levels did not provoke differentiation, but GATA-1 induced the appearance of megakaryocytes as assessed by morphology, the presence of acetylcholinesterase and a polyploid DNA content. Although GATA-1 is thought to stimulate its own transcription in erythrocytes, expression of the endogenous gene was not increased in the megakaryocytic lines; hence GATA-1 may not be autoregulatory in this lineage. Megakaryocytic differentiation was accompanied by a marked decrease in the myeloid surface marker Mac-1. The absence of mast cell or erythroid differentiation suggests that GATA-1 may not be sufficient to provoke maturation along these lineages or that these pathways are impeded in 416B cells. These results demonstrate that a member of the GATA gene family can act as an important regulator of megakaryocytic differentiation.
Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and γ-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.
The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.
Little is known about the transcriptional events underlying megakaryocytic (Mk) differentiation. We sought to identify genes and pathways previously unassociated with megakaryopoiesis and to evaluate the CHRF-288-11 (CHRF) megakaryoblastic cell line as a model system for investigating megakaryopoiesis.
Using DNA microarrays, Q-RT-PCR, and protein-level assays, we compared the dynamic gene expression pattern of phorbol ester-induced differentiation of CHRF cells to cytokine-induced Mk differentiation of human mobilized peripheral blood CD34(+) cells.
Transcriptional patterns of well-known Mk genes were similar between the two systems. CHRF cells constitutively express some early Mk genes including GATA-1. Expression patterns of apoptosis-related genes suggested that increased p53 activity is involved in Mk apoptosis, and this was confirmed by p53-DNA-binding activity data and flow-cytometric analysis of the p53 target gene BBC3. Certain Rho and G-protein-coupled-receptor signaling pathway components were upregulated, including genes not previously associated with Mk cells. Ontological analysis revealed upregulation of defense-response genes, including both known and candidate platelet-derived contributors to inflammation. Upregulation of interferon-responsive genes occurred in the cell line, but not in the primary cells, likely due to a known genetic mutation in the JAK2/STAT5 signaling pathway.
This analysis of megakaryopoiesis, which integrates dynamic gene expression data with protein abundance and activity assays, has identified a number of genes and pathways that may help govern megakaryopoiesis. Furthermore, the transcriptional data support the hypothesis that CHRF cells resemble an early Mk phenotype and, with certain limitations, exhibit genuine transcriptional features of Mk differentiation upon treatment with phorbol esters.
Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 approximately -723) was responsive to PMA-induced repression, while oligo b (-734 approximately -689) and c (-700 approximately -656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 approximately -735), which is regulated by c-Jun.
The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.
Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and platelet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cellular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effects of TPO on these two cell lines. UT-7 and TF-1 cell lines are two factor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly increased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which express different levels of the c-mpl protein (Mpl) were obtained and all became TPO-dependent for their proliferation. The UT-7/c-mpl clones, but not the TF-1/c-mpl clones, were capable of undergoing MK differentiation in response to TPO. This was demonstrated by the increase in MK markers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cytoplasmic alpha-granules, intracellular membranes resembling demarcation membranes which were immunologically labeled with an GPIIb/IIIa anti-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (glycophorin A, beta-globin and EPO receptor) as well as the differentiative activity of EPO in both UT-7/c-mpl and TF-1/c-mpl clones. It is noteworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally suppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1) Mpl expression is necessary but not sufficient for induction of MK differentiation; and (2) induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathways; the pathway involved in the inhibition of erythroid differentiation might be related to a downregulation of GATA-1 expression in TF-1 cells.
Erythropoietin (Epo) is a cytokine known to stimulate proliferation and differentiation of erythroid cells. However, recent gene disruption experiments demonstrated that Epo receptor signaling is not an obligatory step in erythroid differentiation. Here, we describe the role of Epo in proliferation, terminal differentiation, and apoptosis in a novel human Epo-dependent cell line, AS-E2. Upon withdrawal of Epo, the cells ceased to proliferate and underwent apoptotic death. Accompanying this cell death, an increase in the number of hemoglobin-positive cells of approximately 2-fold was observed. This was associated with immediate up-regulation of the GATA-1:GATA-2 ratio and down-regulation of Bcl-xL. Treatment with Epo or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) up-regulated expression of GATA-2 and Bcl-xL, and these elevations were inhibited by inhibitors of protein kinase C (PKC), H7 and H8. HA1004, a structural analogue of H7 but a poor inhibitor of PKC, had no inhibitory effect. Therefore, in AS-E2 cells, it is likely that Epo plays a role in (a) proliferation, (b) inhibition of differentiation, and (c) survival, by maintaining GATA-2 and Bcl-xL expression through activation of PKC.
GATA-1 protein is thought to be a positive regulator of erythroid differentiation. However, ectopic expression of a conditional GATA-2/estrogen receptor chimera was shown to inhibit erythroid differentiation in a hormone-dependent manner, suggesting the negative regulation of erythroid differentiation by GATA-2 protein. Accordingly, we reasoned that the quantitative balance of GATA-1 and GATA-2 protein might affect erythroid differentiation. In this report, we performed specific and quantitative measurements of GATA-1 and GATA-2 protein in a new erythroid cell line, SAM-1, after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). On the basis of these measurements, we show that TPA-induced arrest of erythroid differentiation is coupled with the upregulation of GATA-2 protein, as well as the downregulation of GATA-1 protein. Our results suggest that it is the precise quantitative balance of GATA-1 and GATA-2 protein that regulates erythroid differentiation.
Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.
Dendritic cell-specific transcript (DC-SCRIPT) is a putative DC zinc (Zn) finger-type transcription factor described recently in humans. Here, we illustrate that DC-SCRIPT is highly conserved in evolution and report the initial characterization of the murine ortholog of DC-SCRIPT, which is also preferentially expressed in DC as shown by real-time quantitative polymerase chain reaction, and its distribution resembles that of its human counterpart. Studies undertaken in human embryonic kidney 293 cells depict its nuclear localization and reveal that the Zn finger domain of the protein is mainly responsible for nuclear import. The human and the mouse genes are located in syntenic chromosomal regions and exhibit a similar genomic organization with numerous common transcription factor-binding sites in their promoter region, including sites for many factors implicated in haematopoiesis and DC biology, such as Gfi, GATA-1, Spi-B, and c-Rel. Taken together, these data show that DC-SCRIPT is well-conserved in evolution and that the mouse homologue is more than 80% homologous to the human protein. Therefore, mouse models can be used to elucidate the function of this novel DC marker.
XPA, an essential protein in nucleotide excision repair (NER), interacts with damaged DNA and other proteins (RPA, ERCC1 and TFIIH) to remove a wide variety of chemically and structurally distinct DNA lesions from the eukaryotic genome. To understand the structural basis for the role of XPA in the repair process, the structure of the minimal DNA binding domain of human XPA [XPA-MBD (M98-F219)] was studied by NMR spectroscopy. A three-dimensional structure for XPA-MBD was generated using distance geometry and simulated annealing methods from NOE-based distance restraints, hydrogen bond and Zn-S distance restraints, and dihedral restraints. The structure calculations indicate that XPA-MBD contains elements of well-defined secondary structure interspaced with disordered loops organized into two non-interactive sub-domains: a zinc-binding core (D101-K137) and a loop-rich domain (L138-F219). The zinc-associated core contains an antiparallel beta-sheet (Y102-C105 and K110-M113) and an alpha-helix (C126-K137) separated by a poorly defined turn, reminiscent of the structure of the zinc-binding domain of the chicken erythroid transcription factor GATA-1 when bound to its cognate DNA sequence. The loop-rich domain contains a triple-strand antiparallel beta-sheet (L138-T140, L182-M178 and K163-K167), three loops (K151-L162, N169-D177 and Q208-F219) and three alpha-helices (K141-L150, K183-W194 and Q197-R207). The XPA-MBD structure is discussed in terms of known functions: binding single- and double-stranded DNA and binding RPA.
A change of a universally conserved leucine to valine in the DNA-binding domain of the GATA factor AreA results in inability to activate some AreA-dependent promoters, including that of the uapA gene encoding a specific urate-xanthine permease. Some other AreA-dependent promoters become able to function more efficiently than in the wild-type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild-type and mutant forms of AreA. In vitro binding studies of wild-type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild-type and mutant AreA-DNA complexes derived from the atomic coordinates of the GATA-1-AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA-binding sites, is sufficient to ensure activation of transcription of the uapA gene.
The EVI-1 gene encodes a Zn finger, DNA binding protein previously detected in some acute myelogenous leukemias (AML) and myelodysplasias (MDS), but not in normal marrow or cord blood cells. Experimental studies suggest EVI-1 blocks cellular differentiation by binding to GATA-1 or other specific DNA sequences controlling gene expression, and may be involved in the pathogenesis of some AMLs. To further define potential roles for EVI-1 in leukemia pathogenesis, we studied its regulation in acute promyelocytic leukemias (APL). Seven of 11 APL cases expressed EVI-1 RNA detected by RNA PCR at diagnosis, and expression was detected in two additional cases after treatment with all-trans retinoic acid (ATRA). Two of four cases studied at relapse also expressed EVI-1 RNA. To investigate regulation of EVI-1 expression in APL, we examined its expression in the NB4 APL cell line. NB4 cells did not express EVI-1 under basal conditions, but expressed EVI-1 after ATRA-induced differentiation. When NB4 cells were exposed to ATRA and transferred to cultures with N,N'-hexamethylene-bis-acetamide (HMBA), differentiation occurred but EVI-1 RNA was not detected, indicating that EVI-1 expression was not required for terminal, NB4 differentiation. ATRA-resistant NB4 cells were obtained by continuous culture in gradually increasing concentrations of ATRA. These cells did not express markers of differentiation but continued to express EVI-1 for several weeks even after ATRA withdrawal. To assess whether expression of the APL PML-RAR alpha fusion gene alone was sufficient for ATRA induction of EVI-1, the PML-RAR alpha gene cDNA was expressed in U937 histiocytic lymphoma cells. ATRA treatment of PML-RAR alpha-transfected or control U937 cells did not induce EVI-1 expression. In conclusion, this study demonstrates the EVI-1 gene is consistently expressed in APL cells either constitutively or after ATRA treatment. ATRA represents the first biologically active agent shown to specifically regulate EVI-1 expression in blood cells. In contrast to previous studies in AML and MDS, the pattern of EVI-1 expression suggests it may facilitate rather than inhibit myeloid differentiation during ATRA treatment. However, effects of EVI-1 expression are likely to be complex, and expression in ATRA-resistant APL cells may indicate multiple roles for this gene.
During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.
The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone.
Insulin-like androgenic gland hormone gene (IAG) is a sex regulator specifically expressed in male crustaceans, controlling the male sexual differentiation, spermatogenesis and reproductive strategy. Our previous study reported the cloning and characterization of the prawn Macrobrachium nipponense IAG (MnIAG). In this study, we further identified a 2214-bp MnIAG 5'-flanking region, and analyzed its transcription factor binding sites and transcriptional activity. The results showed that there were two potential promoter core sequences, three TATA boxes and one CAAT box existing in the MnIAG 5'-flanking region as well as many potential transcription factor binding sites, such as SRY, Sox-5, GATA-1, etc. Notably, the transcriptional activity was weak in this region, and a negative regulatory region was found in -604 to -231bp. In addition, we constructed M. nipponense yeast libraries and identified proteins interacting with the MnIAG protein by yeast two hybridization assay. The yeast two-hybrid screening yielded ten positive clones, of which five were annotated by NCBI database, namely heat shock protein 21, NADH dehydrogenase, zinc finger protein, beta-N-acetylglucosaminidase and a hypothetical protein. The identification of MnIAG putative regulatory region and proteins that interact with IAG will facilitate our understanding of the regulatory role of MnIAG and provide a foundation for deep insight into the prawn sex differentiation mechanism and signaling transduction pathways.
We have identified expression of the gene encoding the transcriptional coactivator FOG-1 (Friend of GATA-1; Zfpm1, Zinc finger protein multitype 1) in B lymphocytes. We found that FOG-1 expression is directly or indirectly dependent on the B cell-specific coactivator OBF-1 and that it is modulated during B cell development: expression is observed in early but not in late stages of B cell development. To directly test in vivo the role of FOG-1 in B lymphocytes, we developed a novel embryonic stem cell recombination system. For this, we combined homologous recombination with the FLP recombinase activity to rapidly generate embryonic stem cell lines carrying a Cre-inducible transgene at the Rosa26 locus. Using this system, we successfully generated transgenic mice where FOG-1 is conditionally overexpressed in mature B-cells or in the entire hematopoietic system. While overexpression of FOG-1 in B cells did not significantly affect B cell development or function, we found that enforced expression of FOG-1 throughout all hematopoietic lineages led to a reduction in the number of circulating eosinophils, confirming and extending to mammals the known function of FOG-1 in this lineage.
The human ZNF268 gene encodes a typical KRAB-C2H2 zinc finger protein that may participate in hematopoiesis and leukemogenesis. A recent microarray study revealed that ZNF268 expression continuously decreases during erythropoiesis. However, the molecular mechanisms underlying regulation of ZNF268 during hematopoiesis are not well understood. Here we found that GATA-1, a master regulator of erythropoiesis, repressed the promoter activity and transcription of ZNF268. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that GATA-1 directly bound to a GATA binding site in the ZNF268 promoter in vitro and in vivo. Knockdown of ZNF268 in K562 erythroleukemia cells with specific siRNA accelerated cellular proliferation, suppressed apoptosis, and reduced expression of erythroid-specific developmental markers. It also promoted growth of subcutaneous K562-derived tumors in nude mice. These results suggest that ZNF268 is a crucial downstream target and effector of GATA-1. They also suggest the downregulation of ZNF268 by GATA-1 is important in promoting the growth and suppressing the differentiation of K562 erythroleukemia cells.
The homeostasis of hematopoiesis in the bone marrow is governed by a small number of key transcription factors, including PU.1, GATA-1 and c/EBPα. PU.1, a member of the E-twenty-six family of transcription factors, is indispensable for normal hematopoiesis. Inactivation of PU.1 induces acute leukemia in mice. Recent data suggest that the leukemia-associated fusion protein pro-myelocytic leukemia/retinoic acid receptor alpha (PML/RARα) inhibits PU.1, but the mechanism mediating this inhibition is unclear. Here, we investigated the mechanisms by which the fusion proteins PML/RARα and pro-myelocytic leukemia zinc finger/RARα (PLZF/RARα) (X-RARα) interfere with the function of PU.1. We found that X-RARα proteins functionally inactivate PU.1 by reducing its promoter-binding capacity, resulting in a reduction in PU.1-dependent transcriptional transactivation. In fact, X-RARα proteins directly interact with PU.1, leading to both the sequestration of PU.1 from its target promoters and a reduction in its serine phosphorylation, which is crucial for its promoter binding and transcriptional activity. We found that the functional inactivation of PU.1 could be overcome by the forced overexpression of PU.1 in PML/RARα- or PLZF/RARα-positive murine hematopoietic progenitor cells; evidently, this overexpression rescued the leukemic differentiation block induced by X-RARα proteins. Our data thus provide strong evidence that X-RARα proteins functionally inhibit PU.1, shedding light on the mechanism by which X-RARα proteins induce leukemogenesis.
The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.
GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.
In contrast to the extensive understanding of the zinc finger-DNA interactions, less is known about zinc finger-zinc finger interactions. GATA-1 and Sp1 are transcription factors with zinc finger domains for DNA binding. The interaction between the GATA-1 and Sp1 zinc finger domains is important for synergistic transcriptional effects in erythroid genes. Despite the biological importance of the GATA-1 and Sp1 interaction, the molecular mechanism of the interaction remains unclear. We constructed a series of deletion mutants of the zinc finger domains of GATA-1 and Sp1 to identify the regions within the GATA-1 and Sp1 zinc finger domains that interact. The zinc finger-zinc finger interaction modes were also estimated from calorimetric measurements. This revealed that the interaction between the Sp1 and GATA-1 zinc finger domains was primarily electrostatic, and that the linker region of the Sp1 zinc fingers is important for the association with the GATA-1 zinc finger domains. We propose a new molecular mechanism for zinc finger-zinc finger interactions that should contribute to our understanding of the bio-functional role of the interaction between GATA-1 and Sp1.
Dominant mutations in coding regions of the surfactant protein-C gene, SFTPC, cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. By using a case-control group of infants > or =34 weeks gestation (n = 538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants.
Growth factor-independence 1b (Gfi1b) is a zinc finger transcription factor essential for erythroid and megakaryocytic development. To better understand Gfi1b regulation and to know the implication of the level of expression of this gene in human pathology, we have searched for promoter punctual sequence variations in 214 patients with different hematological diseases. We found two previously unknown congenital mutations at evolutionary conserved GATA and octamer-binding (Oct) transcription factor sites. The Oct site mutation was also found in five relatives of the patient. The GATA motif mutation reduced promoter activity by 50% in vitro, while homozygous patients with the octamer site mutation showed a four-to-five times increase of Gfi1b RNA in platelets. Electrophoretic mobility shift analyses demonstrated that different protein complexes bind to both sites and that binding is reduced by the mutations. Finally, we found that GATA-1 and Oct-1 are the main components of each complex. This study provides evidences of a new mechanism for Gfi1b repression. This is also the first report of Gfi1b mutations with a functional implication; further investigation and follow-up will clarify the involvement of these mutations in hematological disease.
The critical regulator of hematopoiesis GATA-1 recruits diverse coregulators to chromatin, which mediate transcriptional activation and repression. These coregulators include the cell-type-specific multi-zinc finger protein Friend of GATA-1 (FOG-1), the histone acetyltransferase CREB binding protein (CBP), and the key component of the Mediator complex Med1. While FOG-1 is an established GATA-1 coregulator, the importance of interactions between GATA-1 and other coregulators is poorly understood. Furthermore, whether GATA-1 utilizes multiple coregulators at all loci, or if certain coregulators are dedicated to specific loci is unknown. We compared the capacity of GATA-1 to recruit and utilize FOG-1 and Med1 at activated and repressed target genes. Similar to FOG-1, GATA-1 recruited Med1 to activated genes, and the kinetics of FOG-1 and Med1 recruitment were similar. GATA-1 recruited Med1 in Fog1(-/-) cells, indicating that GATA-1-mediated Med1 recruitment is FOG-1-independent. In contrast to FOG-1, GATA-1 evicted Med1 during transcriptional repression. Whereas knocking-down FOG-1 had catastrophic effects on GATA-1-mediated activation and repression, knocking-down Med1 modestly impaired GATA-1 activity only at select loci. These results illustrate both similarities and differences between GATA-1-mediated recruitment of FOG-1 and Med1 to chromatin, with a fundamental difference being the quantitatively greater requirement for FOG-1.
GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.
In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1-responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.
MED-1 is a member of a group of divergent GATA-type zinc finger proteins recently identified in several species of Caenorhabditis. The med genes are transcriptional regulators that are involved in the specification of the mesoderm and endoderm precursor cells in nematodes. Unlike other GATA-type zinc fingers that recognize the consensus sequence (A/C/T)GATA(A/G), the MED-1 zinc finger (MED1zf) binds the larger and atypical site GTATACT(T/C)(3). We have examined the basis for this unusual DNA specificity using a range of biochemical and biophysical approaches. Most strikingly, we show that although the core of the MED1zf structure is similar to that of GATA-1, the basic tail C-terminal to the zinc finger unexpectedly adopts an alpha-helical structure upon binding DNA. This additional helix appears to contact the major groove of the DNA, making contacts that explain the extended DNA consensus sequence observed for MED1zf. Our data expand the versatility of DNA recognition by GATA-type zinc fingers and perhaps shed new light on the DNA-binding properties of mammalian GATA factors.
Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893bp with a 657bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NFkappaB, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12h post-injection with V. anguillarum and then returned to normal between 24h and 48h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V. anguillarum.
ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.
The alpha-chain is a specific component of FcepsilonRI, which is essential for the cell surface expression of FcepsilonRI and the binding of IgE. Recently, two single nucleotide polymorphisms (SNPs) in the alpha-chain promoter, -315C>T and -66T>C, have been shown by statistic studies to associate with allergic diseases. The effect of -66 SNP on GATA-1-mediated promoter activity has been already indicated. In the present study, to investigate roles of the -315 SNP on the alpha-chain promoter functions, the transcription activity was evaluated by reporter assay. The alpha-chain promoter carrying -315T (minor allele) possessed significantly higher transcriptional activity than that of -315C (major allele). EMSA indicated that the transcription factor Sp1, but not Myc-associated zinc finger protein (MAZ), was bound to the -315C allele probe and that a transcription factor belonging to a high mobility group-family bound to the -315T allele probe. The chromatin immunoprecipitation assay suggested that high mobility group 1, 2, and Sp1 bound around -315 of FcepsilonRIalpha genomic DNA in vivo in the human basophil cell line KU812 with -315C/T and in human peripheral blood basophils with -315C/C, respectively. When cell surface expression level of FcepsilonRI on basophils was analyzed by flow cytometry, basophils from individuals carrying -315T allele expressed significantly higher amount of FcepsilonRI compared with those of -315C/C. The findings demonstrate that a -315 SNP significantly affects human FcepsilonRI alpha-chain promoter activity and expression level of FcepsilonRI on basophils by binding different transcription factors to the SNP site.
DNA-binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)(4) metal binding sites which recognize the protein's eponymous binding site, GATA. Unlike other conserved DNA-binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence-specific DNA-binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3' and 5' flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers.
Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner.
Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes.
There is an urgent need to investigate the potential targeted therapy approach for triple-negative breast cancer (TNBC). Our present study reveals that histone deacetylase inhibitors (HDACIs) suberoyl anilide hydroxamic acid (SAHA) and sodium butyrate (NaB) significantly inhibit cell proliferation, arrest cell cycle at G0/G1 phase, and induce mitochondrial related apoptosis of TNBC cells. Further, SAHA and NaB decrease the phosphorylation, protein and mRNA levels of mutant p53 (mtp53) in TNBC cells. While SAHA or NaB has no similar inhibition effect on wild type p53 (wtp53). The inhibition apparently occurs at the level of transcription because the down regulation of precursor p53 transcription is much more rapid (less than 2h) and sharp than that of mature p53. The knockdown of HDAC8, while not HDAC6, inhibits the transcription of mtp53 in TNBC cells. The luciferase assay and ChIP analysis reveal that both SAHA and NaB can reduce the binding of transcription factor Yin Yang 1 (YY1) with the -102 to -96 position of human p53 promoter. Knockdown of YY1 also significantly inhibits the transcription of mtp53 in TNBC cells. Further, SAHA and NaB can inhibit the association of HDAC8 and YY1, increase acetylation of residues 170-200 of YY1, then decrease its transcription activities, and finally suppress YY1 induced p53 transcription. Together, our data establish that SAHA and NaB can be considered as drug candidates for TNBC patients, and HDAC8/YY1/mtp53 signals act as an important target for TNBC treatment.
Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.
Opioid-immune crosstalk occurs when opioid drugs alter the activity of the immune system. In this study, the opioid antagonist β-funaltrexamine (β-FNA) decreases the expression and release of an inflammatory chemokine, interferon-γ inducible protein-10 (CXCL10) from normal human astrocytes stimulated by interleukin 1β (IL-1β). β-FNA decreased CXCL10 by an unknown action that did not involve the mu opioid receptor (MOR). As IL-1β acts through its receptor to activate NF-κB/MAPK signaling pathways which leads to CXCL10 expression and release, key steps in the IL-1β signaling pathways were examined following β-FNA treatment. IL-1β-induced activation of p38 mitogen-activated protein kinases (p38 MAPK) was inhibited by β-FNA as shown by decreased p38 MAPK phosphorylation in treated cells. β-FNA also decreased the levels of activated subunits of NF-κB (p50 and p65) in treated astrocytes. The impact of β-FNA was also observed in proteins that act to negatively regulate NF-κB signaling. IL-1β upregulated the expression of A20, a ubiquitin (Ub)-editing enzyme that dampens NF-κB signaling by altering ubiquination patterns on IL-1 receptor second messengers, and the increase in A20 was significantly inhibited by β-FNA treatment. Inhibition of the Ub-activating enzyme E1 by the inhibitor PYR41 also decreased CXCL10 release, like β-FNA, and concurrent treatment with both PYR41 and β-FNA inhibited CXCL10 more than did either agent alone. In mice, lipopolysaccharide-induced CXCL10 expression in the brain was inhibited by treatment with β-FNA. These findings suggest that β-FNA exerts an anti-inflammatory action in vitro and in vivo that is MOR-independent and possibly due to the alkylating ability of β-FNA.
Alzheimer's disease (AD) is a complex multifactorial disorder with poorly characterized pathogenesis. Our understanding of this disease would thus benefit from an approach that addresses this complexity by elucidating the regulatory networks that are dysregulated in the neural compartment of AD patients, across distinct brain regions. Here, we use a Systems Biology (SB) approach, which has been highly successful in the dissection of cancer related phenotypes, to reverse engineer the transcriptional regulation layer of human neuronal cells and interrogate it to infer candidate Master Regulators (MRs) responsible for disease progression. Analysis of gene expression profiles from laser-captured neurons from AD and controls subjects, using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe), yielded an interactome consisting of 488,353 transcription-factor/target interactions. Interrogation of this interactome, using the Master Regulator INference algorithm (MARINa), identified an unbiased set of candidate MRs causally responsible for regulating the transcriptional signature of AD progression. Experimental assays in autopsy-derived human brain tissue showed that three of the top candidate MRs (YY1, p300 and ZMYM3) are indeed biochemically and histopathologically dysregulated in AD brains compared to controls. Our results additionally implicate p53 and loss of acetylation homeostasis in the neurodegenerative process. This study suggests that an integrative, SB approach can be applied to AD and other neurodegenerative diseases, and provide significant novel insight on the disease progression.
Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.
Matrix metalloproteinase-9 (MMP-9) has been shown to be induced by cytokines including TNF-α and may contribute to bone inflammatory diseases. However, the mechanisms underlying MMP-9 expression induced by TNF-α in MC3T3-E1 cells remain unclear.
We applied gelatin zymography, Western blot, RT-PCR, real-time PCR, selective pharmacological inhibitors of transcription (actinomycin D, Act.D), translation (cycloheximide, CHI), c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), and NF-κB (Bay11-7082), respective siRNAs transfection, promoter assay, immunofluorescence staining, and ELISA to investigate the MMP-9 expression and soluble ICAM-1 (sICAM-1) release induced by TNF-α in MC3T3-E1 cells. Here we demonstrated that TNF-α-induced MMP-9 expression was attenuated by Act.D, CHI, PP1, U0126, SB202190, SP600125, and Bay11-7082, and by the transfection with siRNAs for ERK2, p38 MAPK, and JNK2. TNF-α-stimulated TNFR1, TRAF2, and c-Src complex formation was revealed by immunoprecipitation and Western blot. Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125. TNF-α time-dependently induced MMP-9 promoter activity which was also inhibited by PP1, U0126, SB202190, SP600125, or Bay11-7082. Up-regulation of MMP-9 was associated with the release of sICAM-1 into the cultured medium, which was attenuated by the pretreatment with MMP-2/9i, an MMP-9 inhibitor.
In this study, we demonstrated that TNF-α up-regulates MMP-9 expression via c-Src, MAPKs, and NF-κB pathways. In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells. The interplay between MMP-9 expression and sICAM-1 release may exert an important role in the regulation of bone inflammatory diseases.
As a prototype of the TGF-β superfamily cytokines, TGF-β is well known for its diverse roles in embryogenesis and adult tissue homeostasis. TGF-β evokes cellular responses by signaling mainly through cell membrane receptors and transcription factor R-Smads and Co-Smad (Smad4), while an inhibitory Smad, Smad7, acts as a critical negative regulator of TGF-β signaling. Smad7 antagonizes TGF-β signaling by regulating the stability or activity of the receptors or blocking the DNA binding of the functional R-Smad-Smad4 complex in the nucleus. However, the function of Smad7 in the nucleus is not fully understood. Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor with multiple functions. It has been reported that YY1 can inhibit Smad-dependent transcriptional responses and TGF-β/BMP-induced cell differentiation independently of its DNA binding ability. In this study, we found that Smad7 interacts with YY1 and the interaction is attenuated by TGF-β signaling. Reporter assays and target gene expression analyses revealed that Smad7 and YY1 act in concert to inhibit TGF-β-induced transcription in the nucleus. Furthermore, Smad7 could enhance the interaction of YY1 with the histone deacetylase HDAC1. Consistently, YY1 and HDAC1 augmented the transcription repression activity of Smad7 in Gal4-luciferase reporter analysis. Therefore, our findings define a novel mechanism of Smad7 and YY1 to antagonize TGF-β signaling.
Embryonic cells transcriptionally repress the expression of endogenous and exogenous retroelements. Trim28, a key player in this silencing, is known to act in a large DNA-bound complex, but the other components of the complex are not fully characterized. Here, we show that the zinc finger protein Yin Yang 1 (YY1) is one such component. YY1 binds to the long terminal repeat (LTR) region of both exogenous and endogenous retroviruses (ERVs). Deletion of the YY1-binding site from the retroviral genome leads to a major loss of silencing in embryonic cells and a coordinated loss of repressive histone marks from the proviral chromatin. Depletion of YY1 protein results in marked upregulation of expression of exogenous viruses and of selected ERVs. Finally, we report an embryonic cell-specific interaction between YY1 and Trim28. Our results suggest a major role for YY1 in the silencing of both exogenous retroviruses and ERVs in embryonic cells.
The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.
Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription.
AMP-activated protein kinase (AMPK) is currently known to act as a key regulator of metabolic homeostasis. Several biosynthetic enzymes for fatty acid or glycogen are recognized as the targets of AMPK. In the present study, we investigated the role of AMPK in the interleukin-1 (IL-1)-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. IL-1 induced phosphorylation of AMPK-α (Thr-172), which regulates AMPK activities, and acetyl-CoA carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which suppressed the IL-1-induced phosphorylation of acetyl-CoA carboxylase, increased the release and the mRNA level of IL-6 stimulated by IL-1. Transfection of AMPK siRNA-α also amplified the IL-1-stimulated IL-6 release compared to the control cells. On the other hand, IL-1 elicited the phosphorylation of IκB, which caused subsequent decrease of total level of IκB. Wedelolactone, an inhibitor of IκB kinase, which reduced the phosphorylation both of IκB and NF-κB, significantly enhanced the IL-1-stimulated IL-6 synthesis. Compound C remarkably suppressed the IL-1-induced phosphorylation of IκB. These results strongly suggest that AMPK negatively regulates IL-1-stimulated IL-6 synthesis through the IκB/NF-κB pathway in osteoblasts.
The respiratory epithelium plays a central role in the inflammatory response in asthma and other diseases. Methoxyphenolic compounds are purported to be effective anti-inflammatory agents, but their effects on the airway epithelium have not been well characterized.
Human airway cells were stimulated with TNF-α in the presence or absence of 4-substituted methoxyphenols and resveratrol. The expression of various cytokines was measured by qPCR, ELISAs, and protein arrays. Reactive oxygen species (ROS) production was measured with a reactive fluorescent probe (3',6'-diacetate-2',7'-dichlorofluorescein). Activation of NF-κB was measured by nuclear translocation and phosphorylation. Ribonuclear protein association with mRNA was assessed with a biotin-RNA affinity isolation assay.
Multiple inflammatory mediators were inhibited by methoxyphenols, including: CCL2, CCL5, IL-6, IL-8, ICAM-1, MIF, CXCL1, CXCL10, and Serpin E1. IC50 values were obtained for each compound that showed significant anti-inflammatory activity: diapocynin (20.3 μM), resveratrol (42.7 μM), 2-methoxyhydroquinone (64.3 μM), apocynin (146.6 μM), and 4-amino-2-methoxyphenol (410 μM). The anti-inflammatory activity did not correlate with inhibition of reactive oxygen species production or NF-κB activation. However, methoxyphenols inhibited binding of the RNA-binding protein HuR to mRNA, indicating that they may act post-transcriptionally.
Methoxyphenols demonstrate anti-inflammatory activity in human airway cells. More potent compounds that act via similar mechanisms may have therapeutic potential as novel anti-inflammatory agents.
The transcription factor Yin Yang (YY) 1 controls many divergent cellular processes, including cell proliferation and apoptosis. These are key to cancer development, as a consequence of which its expression has been studied in an increasingly wide range of human cancers, including lymphoma, breast, prostate, colon, ovarian, cervical, and brain cancers, osteosarcoma, rhabdomyosarcoma, and leukemia. It is a regulatory transcription factor for a wide range of genes, including genes involved in control of the cell cycle and apoptosis, and it can act either to upregulate or downregulate downstream gene expression, depending on the cellular environment, cofactors, and the gene targeted. Its expression has been associated with development of a malignant phenotype in some human cancers; tumor progression, including metastasis; and survival. However, as data on its prognostic significance has become available for more human cancers, its role in tumor progression has become controversial; there is conflicting data on its association with outcome, with some studies showing a favorable and others an unfavorable association. This is probably because of the many different roles YY1 plays in control of proliferation and apoptosis, one or the other of which may be more prominent in any given tumor. These studies are reviewed to give an overview of the increasingly recognized importance of YY1 in human tumorigenesis.
Yin Yang (YY) 1 represents the epitome of what is considered to be a "Swiss army knife" transcription factor and regulator. YY1 is a ubiquitous and multifunctional zinc-finger transcription factor member of the Polycomb group protein family, a group of homeobox gene receptors that can act as activators or repressors of transcriptional activity. Furthermore, YY1 can act as a redox sensor, adaptor molecule, and chromatin structure and function regulator. YYl's characteristic function as transcriptional activator and repressor relies on its C2H2 (x4) zinc-finger structural DNA-binding motifs tangled with 2 specific regulatory domains. This structural conformation will render the activity of YY1 susceptible to changes in cellular redox status. YY1 also has been shown to undergo chromatin remodeling via interactions with histone acetyl transferase and histone deacetylase complexes. Both groups modify histones, resulting in altered chromatin structure. Herein, we will discuss the multiple roles and mechanisms of YY1 in the regulation of gene expression, its genetic factor functions, epigenetic regulatory activity, and its role as a redox sensor in the context of malignant neoplastic diseases.
YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes.
miRNAs have the potential to act on diverse downstream genes, and miRNA signatures of HPV-infected tissues may provide insight into HPV-related carcinogenesis. We set out to profile miRNA expression in HPV-infected samples and relate this to histological and grade-specific alterations in the spectrum of cervical carcinogenesis in vivo. A total of 31 miRNAs showed significant and continuous expression along with the progression from normal cervical tissue to cancer, and six of them were validated in 133 samples. By bioinformatics analyses, we established a putative HPV-associated miRNA-mRNA regulatory network, showing that miR-29 is the most highly enriched. We also found that YY1 and CDK6 were both positively correlated with E6/E7 RNA expression and targeted by tumour-suppressive miR-29. Evidence of miR-29 involvement in HPV infection was further verified in patient samples and by various experimental approaches. Taken together, our results suggest that HPVs have oncogenic properties at least in part by reshaping the milieu of cellular miRNAs. miR-29 restrains cell cycle progression and induces apoptosis via YY1 and CDK6 promoting malignant transformation induced by HPV, although the abnormality of miR-29 in HPV-infected cells might be regulated in an indirect way.
Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse.
Evolutionarily conserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA)/(2·CpG) ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements.
We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in epigenetic features and a special response to selective pressure. In addition, our data support the potential role of intronic CpG islands as epigenetic key regulatory elements and suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions.
Chemotherapy of non-Hodgkin's lymphoma is frequently hampered by drug resistance. The monoclonal antibody rituximab specifically targets the CD20 antigen and sensitizes B-cell lymphoma cells to standard anticancer drugs. In the present investigation, we analyzed, whether a combination of rituximab and artesunate may act in a complementary manner and eventually synergize in tumor cell killing. Artesunate is an anti-malarial drug, which also exerts profound activity towards cancer cells. While rituximab alone was minimally cytotoxic, rituximab increased cytotoxicity to artesunate in Ramos cells. Artesunate induced apoptosis, induced Fas/CD95 expression and the formation of reactive oxygen species (ROS) and resulted in a breakdown of mitochondrial membrane potential. This argues for the involvement of both receptor-driven extrinsic and mitochondrial intrinsic routes of apoptosis. Rituximab increased Fas/CD95 expression and ROS formation and decreased mitochondrial membrane potential ultimately leading to increased apoptosis induced by artesunate. The transcription factors YY1 and Sp1 are upstream regulators of apoptosis by controlling the expression of apoptosis-regulating genes. YY1 and Sp1 were down-regulated and Fas/CD95 was up-regulated by rituximab and artesunate indicating that artesunate activated the Fas/CD95 pathway and that rituximab increased the susceptibility of tumor cells to artesunate-induced apoptosis. Furthermore, rituximab affected the expression of antioxidant genes. The antibody decreased artesunate-induced up-regulation of catalase expression and increased artesunate-induced down-regulation of glutathione S-transferase-phi expression. Manganese-dependent superoxide dismutase expression was not changed by artesunate. Antioxidant proteins may help to detoxify artesunate-induced ROS. Rituximab reversed the artesunate-induced expression changes of antioxidant genes and, hence, reduced the detoxification capacity of Ramos cells. The effects of rituximab on antioxidant genes represent a novel mechanism of rituximab for chemosensitization.
In a humid milieu such as mucosal surfaces, pollen grains do not only release allergens but also proinflammatory and immunomodulatory lipids, termed pollen-associated lipid mediators. Among these, the E(1)-phytoprostanes (PPE(1)) were identified to modulate dendritic cell (DC) function: PPE(1) inhibit the DC's capacity to produce IL-12 and enhance DC mediated T(H)2 polarization of naive T cells. The mechanism(s) by which PPE(1) act on DC remained elusive. We thus analyzed candidate signaling elements and their role in PPE(1)-mediated regulation of DC function. Aqueous birch pollen extracts induced a marked cAMP response in DC that could be blocked partially by EP2 and EP4 antagonists. In contrast, PPE(1) hardly induced cAMP and the inhibitory effect on IL-12 production was mostly independent of EP2 and EP4. Instead, PPE(1) inhibited the LPS-induced production of IL-12 p70 by a mechanism involving the nuclear receptor PPAR-gamma. Finally, PPE(1) efficiently blocked NF-kappaB signaling in DCs by inhibiting IkappaB-alpha degradation, translocation of p65 to the nucleus, and binding to its target DNA elements. We conclude that pollen-derived PPE(1) modulate DC function via PPAR-gamma dependent pathways that lead to inhibition of NFkappaB activation and result in reduced DC IL-12 production and consecutive T(H)2 polarization.
The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely studied, Cyclooxygenase-2 (COX-2) is a key enzyme for prostaglandin E2 (PGE(2)) production, which induces the nuclear factor kappa B (NFkappaB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3-E1 osteoblasts were treated with NFkappaB inhibitor BAY 11-7082 and sc791 (a selective COX-2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFkappaB activation and translocation. This induction was not related to COX-2 expression, suggesting that LPS-induced NFkappaB activation is independent of COX-2. It is possible that low concentration of LPS can act as a stimulating factor of the NFkappaB pathway in nonstimulated cells such as osteoblasts. COX-2 is not necessary for the NFkappaB pathway during LPS-induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation.
Human CCAAT/enhancer-binding protein delta (CEBPD) has been reported as a tumor suppressor because it both induces growth arrest involved in differentiation and plays a crucial role as a regulator of pro-apoptotic gene expression. In this study, CEBPD gene expression is down-regulated, and "loss of function" alterations in CEBPD gene expression are observed in cervical cancer and hepatocellular carcinoma. Suppressor of zeste 12 (SUZ12), a component of the polycomb repressive complex 2 (PRC2), silences CEBPD promoter activity, enhancing the methylation of exogenous CEBPD promoter through the proximal CpG islands. Moreover, this molecular approach is consistent with the opposite mRNA expression pattern between SUZ12 and CEBPD in cervical cancer and hepatocellular carcinoma patients. We further demonstrated that Yin-Yang-1 (YY1) physically interacts with SUZ12 and can act as a mediator to recruit the polycomb group proteins and DNA methyltransferases to participate in the CEBPD gene silencing process. Taking these results into consideration, we not only demonstrate the advantage of SUZ12-silenced CEBPD expression in tumor formation but also clarify an in vivo evidence for YY1-mediated silencing paths of SUZ12 and DNA methyltransferases on the CEBPD promoter.
Vacuolar system-associated protein-60 (VASAP-60) constitutes the bovine ortholog of the human "protein kinase C substrate 80K-H" (PRKCSH or 80K-H). We characterized the bovine VASAP-60/PRKCSH gene structure and promoter, identified cis-acting elements controlling VASAP-60 expression, searched for mRNA splice variants, and analyzed mRNA expression in ovarian follicles. Expression of VASAP-60 mRNA showed a 2.4-fold increase (P<0.0001) in granulosa cells of dominant follicles compared to small follicles (2-4 mm) or ovulatory follicles, and no mRNA splice variant was identified. The bovine VASAP-60 gene encompasses 12.5 kb and is composed of 18 exons and 17 introns. Primer extension analysis revealed a single transcription initiation site, and the promoter lacks a TATA box. Promoter activity assays were performed with a series of deletion constructs in different bovine cell lines (endometrial epithelial glandular, kidney epithelial and aortic endothelial) to identify cis-acting elements. The -53/+16 bp fragment (+1 = transcription start site) conferred minimal promoter activity whereas activator and repressor elements were located in the -200/-53 bp and -653/-200 bp fragments, respectively. Analysis of cis-acting elements in the -200/-53 bp activation domain revealed by gel shift assays and chromatin immunoprecipitation assay that transcription factor YY1 binds to VASAP-60 promoter. This study is the first to report that VASAP-60 is up-regulated in granulosa cells of dominant follicles, to document the primary structure of the bovine VASAP-60 gene and promoter, and to demonstrate that YY1 binds to the VASAP-60 proximal promoter and may act as a positive transcriptional regulator.
Synaptotagmin XI (Syt11) is a member of the synaptotagmin family, which is localized in cells either in synaptic vesicles or the cellular membrane, and is known to act as a calcium sensor. The Syt11 gene is located on chromosome locus 1q21-q22, which was previously reported as a major susceptibility locus of familial schizophrenia. Here, we present evidence for an association between the number of 33-bp repeats in the promoter region of the Syt11 gene and schizophrenia. We found that the transcriptional activity of the gene is affected by the number of 33-bp repeats, which include an Sp1 binding site, suggesting that the excessive expression of Syt11 can be associated with schizophrenia. Another (single nucleotide) polymorphism in the Syt11 5'UTR region, where the potent transcription factor YY1 can bind, also affects the transcriptional activity of Syt11.
Amelogenin proteins are essential in the control of enamel biomineralization and the amelogenin gene therefore is spatiotemporally regulated to ensure proper amelogenin protein expression. In this study, we examined the role of sumoylation to alter CCAAT/enhancer-binding protein alpha (C/EBPalpha) activity, and performed a search using a protein/DNA array system for other proteins that act co-operatively with C/EBPalpha to alter amelogenin expression. We observed that C/EBPalpha was modified by sumoylation, and that this modification played an indirect inhibitory role on the regulation of C/EBPalpha activity which appeared to act through other transcription factors. The protein/DNA array allowed us to single out the transcription factor, YY1, which acts in the absence of direct DNA binding to repress both the basal amelogenin promoter activity and C/EBPalpha-mediated transactivation. Taken together, these pathways may account for part of the physiological modulation of the amelogenin gene expression in accordance with tooth developmental and enamel biomineralization requirements.
The multifunctional transcription factor Yin Yang 1 (YY1) is a complex protein that has been shown to play pivotal roles in development, differentiation, cellular proliferation and apoptosis. It can act as a transcriptional repressor, an activator, or an initiator element binding protein that directs and initiates transcription of numerous cellular and viral genes. Because the expression and function of YY1 are known to be intimately associated with cell-cycle progression, the physiological significance of YY1 activity has recently been applied to models of cancer biology. Several lines of evidence imply that YY1 expression and/or activation is associated with tumourigenesis, in addition to its regulatory roles in normal biological processes. However, controversial results also raised and indicated that further studies are still needed to piece all of the seemingly contradictory data into a complete picture. On the basis of YY1 regulations and functions, novel drugs and specific treatment strategies may be developed with new therapeutic applications for tumour patients in the future.
Heme oxygenase (HO)-1 is a stress response protein, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Molecular mechanisms involved in the inducible transcription of ho-1 occurring in response to numerous and diverse stressful conditions have remained elusive. Since the discovery of E1 and E2, the two upstream enhancers regulating induction of ho-1 transcription in 1989, there have been many studies dealing with molecular mechanisms involved in enhancing HO-1 expression. In this commentary, recent advances in our understanding of the mechanisms involved in the induction of HO-1 expression in mammalian cells are summarized with some supportive results reported by others. Currently available data indicate that activation of ho-1 transcription involves both the heme (native substrate)-dependent selective alleviation of repressor and the oxidative stress-dependent activation of transcriptional activator. The stress-released free-heme (HO-1 substrate) from hemoproteins involved in causing oxidative stress itself appears to act as a molecular switch controlling the repressor- activator antagonism on the enhancer sequences of ho-1. Thus, induction of HO-1 appears to operate in a manner like a simple feedback loop. dox Signal. 7, 1674-1687.
The ubiquitous transcription factor Yin Yang 1 (YY1) is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. Mechanisms of action include direct activation or repression, indirect activation or repression via cofactor recruitment, or activation or repression by disruption of binding sites or conformational DNA changes. YY1 activity is regulated by transcription factors and cytoplasmic proteins that have been shown to abrogate or completely inhibit YY1-mediated activation or repression; however, these mechanisms have not yet been fully elucidated. Since expression and function of YY1 are known to be intimately associated with progression through phases of the cell cycle, the physiologic significance of YY1 activity has recently been applied to models of tumor biology. The majority of the data are consistent with the hypothesis that YY1 overexpression and/or activation is associated with unchecked cellular proliferation, resistance to apoptotic stimuli, tumorigenesis and metastatic potential. Studies involving hematopoetic tumors, epithelial-based tumors, endocrine organ malignancies, hepatocellular carcinoma, and retinoblastoma support this hypothesis. Molecular mechanisms that have been investigated include YY1-mediated downregulation of p53 activity, interference with poly-ADP-ribose polymerase, alteration in c-myc and nuclear factor-kappa B (NF-kappaB) expression, regulation of death genes and gene products, and differential YY1 binding in the presence of inflammatory mediators. Further, recent findings implicate YY1 in the regulation of tumor cell resistance to chemotherapeutics and immune-mediated apoptotic stimuli. Taken together, these findings provide strong support of the hypothesis that YY1, in addition to its regulatory roles in normal biologic processes, may possess the potential to act as an initiator of tumorigenesis and may thus serve as both a diagnostic and prognostic tumor marker; furthermore, it may provide an effective target for antitumor chemotherapy and/or immunotherapy.
The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C2 ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E1, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFkappaB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IkappaB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C2 ceramide. Our results suggest that in AST primary cultures, C2 ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFkappaB and Bcl-2 as antiapoptotic factors is discussed.
Prior work has indicated that BMP signals act in concert with FGF8, WNT11 and WNT antagonists to induce the formation of cardiac tissue in the vertebrate embryo. In an effort to understand how these signaling pathways control the expression of key cardiac regulators, we have characterized the cis-regulatory elements of the chick tinman homolog chick Nkx2.5. We find that at least three distinct cardiac activating regions (CARs) of chick Nkx2.5 cooperate to regulate early expression in the cardiac crescent and later segmental expression in the developing heart. In this report, we focus our attention on a 3' BMP-responsive enhancer, termed CAR3, which directs robust cardiac transgene expression. By systematic mutagenesis and gel shift analysis of this enhancer, we demonstrate that GATA4/5/6, YY1 and SMAD1/4 are all necessary for BMP-mediated induction and heart-specific expression of CAR3. Adjacent YY1 and SMAD-binding sites within CAR3 constitute a minimal BMP response element, and interaction of SMAD1/4 with the N terminus of YY1 is required for BMP-mediated induction of CAR3. Our data suggest that BMP-mediated activation of this regulatory region reflects both the induction of GATA genes by BMP signals, as well as modulation of the transcriptional activity of YY1 by direct interaction of this transcription factor with BMP-activated SMADs.
Polycomb group (PcG) genes are required for the stable repression of the homeotic genes and other developmentally regulated genes. Yin Yang 1 (YY1), a vertebrate homolog of the Drosophila PcG pleiohomeotic (Pho), is a multifunctional protein that can act as a repressor or activator of transcription. Xenopus YY1 (XYY1) protein was localized in the central nervous system (CNS), particularly anterior neural tube of tailbud stage embryos. To elucidate the role of endogenous XYY1, loss-of-function studies were performed using XYY1 antisense morpholino oligonucleotide (XYY1 MO). Inhibition of XYY1 function resulted in embryos with antero-posterior axial patterning defects and reduction of head structures. XYY1 MO also reduced the expression of En2, a midbrain/hindbrain junction marker, which was rescued by co-injection of XYY1 mRNA. However, XYY1 MO-injection did not affect the expression of HoxB9, a spinal cord marker. These results suggest that YY1 controls antero-posterior patterning of the CNS during Xenopus embryonic development.
Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs but pho mutants show only mild phenotypes compared with other PcG mutants. We characterize pho-like, a gene encoding a protein with high similarity to Pho. Pho-like binds to Pho-binding sites in vitro and pho-like, pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. We examined the distribution of five PcG proteins on polytene chromosomes from pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, does not cause misexpression of homeotic genes or reporter genes in imaginal disks.
Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells.
Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation).
In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.
During adeno-associated virus (AAV) type 2 productive infections, the p19 promoter of AAV is activated by the AAV Rep78 and Rep68 proteins. Rep-induced activation of p19 depends on the presence of one of several redundant Rep binding elements (RBEs) within the p5 promoter or within the terminal repeats (TR). In the absence of the TR, the p5 RBE and the p19 Sp1 site at position -50 are essential for p19 transactivation. To determine how a Rep complex bound at p5 induces transcription at p19, we made a series of p19 promoter chloramphenicol acetyltransferase constructs in which the p5 RBE was inserted at different locations upstream or downstream of the p19 mRNA start site. The RBE acted like a repressor element at most positions in the presence of both Rep and adenovirus (Ad), and the level of repression increased dramatically as the RBE was inserted closer to the p19 promoter. We concluded that the RBE by itself was not a conventional upstream activation signal and instead behaved like a repressor. To understand how the Rep-RBE complex within p5 activated p19, we considered the possibility that its role was to function as an architectural protein whose purpose was to bring other p5 transcriptional elements to the p19 promoter. In order to address this possibility, we replaced both the p5 RBE and the p19 Sp1 site with GAL4 binding sites. The modified GAL4-containing constructs were cotransfected with plasmids that expressed GAL4 fusion proteins capable of interacting through p53 and T-antigen (T-ag) protein domains. In the presence of Ad and the GAL4 fusion proteins, the p19 promoter exhibited strong transcriptional activation that was dependent on both the GAL4 fusion proteins and Ad infection. This suggested that the primary role of the p5 RBE and the p19 Sp1 sites was to act as a scaffold for bringing transcription complexes in the p5 promoter into close proximity with the p19 promoter. Since Rep and Sp1 themselves were not essential for transactivation, we tested mutants within the other p5 transcriptional elements in the context of GAL4-induced looping to determine which of the other p5 elements was necessary for p19 induction. Mutation of the p5 major late-transcription factor site reduced p19 activity but did not eliminate induction in the presence of the GAL4 fusion proteins. However, mutation of the p5 YY1 site at position -60 (YY1-60) eliminated GAL4-induced transactivation. This implicated the YY1-60 protein complexes in p19 induction by Rep. In addition, both basal p19 activity and activity in the presence of Ad increased when the YY1-60 site was mutated even in the absence of Rep or GAL4 fusion proteins. Therefore, there are likely to be alternative p5-p19 interactions that are Rep independent in which the YY1-60 complex inhibits p19 transcription. We concluded that transcriptional control of the p19 promoter was dependent on the formation of complexes between the p5 and p19 promoters and that activation of the p19 promoter depends largely on the ability of Rep and Sp1 to form a scaffold that positions the p5 YY1 complex near the p19 promoter.
The functional role of the osteoblast nuclear matrix has been a matter of supposition. Its presumed function as an architectural agent of transcription derives primarily from the low solubility of nuclear matrix proteins and their typical localization into discrete subnuclear domains. In addressing how the nuclear matrix regulates skeletal genes, the authors compare Nmp4, Cbfal, and YY1 for the purpose of profiling osteoblast nuclear matrix transcription factors. All three proteins contribute to the transcription of ECM genes and partition into the osteoblast nuclear matrix via a nuclear matrix targeting domain. The authors propose that osteoblast nuclear matrix transcription factors involved in ECM regulation generally have the capacity to alter DNA geometry and reciprocally respond to DNA as an allosteric ligand. This may allow these proteins to adapt to the local nuclear architecture and generate the pattern of regulation specified by that architecture via unmasking of the appropriate transactivation domains. Osteoblast nuclear matrix transcription factors may also act as transcriptional adaptor molecules by supporting the formation of higher order protein complexes along target gene promoters. The genes encoding all three proteins considered here have trinucleotide repeat domains, although the significance of this is unclear. There is no canonical nuclear matrix binding motif, but finger-like structures may be suited for anchoring proteins to discrete subnuclear domains. Finally, the ability to leave the osteoblast nuclear matrix may be as important to the function of some nuclear matrix transcription factors as their association with this subcompartment.
The receptor activator of nuclear factor (NF)-kappaB ligand [RANKL; also known as tumor necrosis factor-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-kappaB (RANK) and act not only as a key factor for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL 2 has a shorter intracellular domain than the original RANKL (RANKL 1), and RANKL 3 lacks a transmembrane domain and was thought to act as a soluble form. In the bone marrow stromal cell line ST2 and preosteoblastic cell line MC3T3-E1, all three RANKL isoforms were detected, but the expression of RANKL 2 was preferentially suppressed by treatment with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone. In young adult thymus, CD4(-)CD8(-) double-negative cells were positive for all three isoforms, CD4(+)CD8(+) double-positive cells were positive for RANKL 1 and RANKL 3 but negative for RANKL 2, and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive cells were positive for all three isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with each RANKL isoform indicated that the three RANKL isoforms were translated, and RANKL 2 protein predominantly stayed in the endoplasmic reticulum and Golgi networks. These results indicate that there are three kinds of RANKL-RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentiation than previously thought.
Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cycle, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-induced HPV-6 E1 promoter and negatively regulates its activity in undifferentiated cells (W. Ai, E. Toussaint, and A. Roman, J. Virol. 73:4220-4229, 1999). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcriptional activator or repressor and that can also inhibit HPV replication in vitro, binds the HPV-6 E1 promoter. EMSAs, using subfragments of the promoter as competitors, showed that the YY1 binding site is located at the 5' end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, monitored with the reporter gene luciferase, was threefold greater than that of the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes showed decreased binding of YY1 to the wild-type promoter. Consistent with this, in differentiated keratinocytes, the activity of the transfected luciferase gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated keratinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated keratinocytes. Both the wild-type and mutated promoters could be negatively regulated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP appear to be negative regulators of the differentiation-induced HPV-6 E1 promoter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.
The regulatory regions for transcriptional control of the MCSF gene are unknown. We examined regulatory control in a 774-bp murine MCSF promoter transfected into MC3T3-E1 osteoblast-like and COS-7 cells. Deletion of upstream sequence from -635 increased basal activity of the promoter by at least four-fold, an increase that was maintained when PU.1, NFkappaB and Egr1/Sp1 consensus sequences were subsequently removed. Mutagenesis identified a suppressor element between -635 and -642 from the transcriptional start site and an oligonucleotide representing this sequence was retarded by nuclear cell protein. TNFalpha (1 ng/ml), PTH (5x10(-8) M), and IL-1alpha (100 pg/ml), which increased MCSF protein secretion, failed to enhance the transcriptional rate of the full-length promoter. TNFalpha was able to stimulate transcription of a heterologous reporter transfected into COS-7 containing multiple copies of the murine MCSF NFkappaB site inserted before a minimal promoter. In contrast, deletion of the same NFkappaB response element increased basal activity in the native promoter. Thus, the NFkappaB sequence may act as a negative regulator in the context of the endogenous promoter. Our results indicate that constitutive transcriptional activity conferred by the MCSF promoter may be damped by a suppressor protein. Transcriptional regulation, however, does not appear to be a major stimulatory mechanism for MCSF secretion.
The transcription factor YY1 is a complex protein that is involved in repressing and activating a diverse number of promoters. Numerous studies have attempted to understand how this one factor can act both as a repressor and an activator in such a wide set of different contexts. The fact that YY1 interacts with a number of key regulatory proteins (e.g. TBP, TFIIB, TAFII55, Sp1, and E1A) has suggested that these interactions are important for determining which particular function of YY1 is displayed at a specific promoter. Two groups of proteins, previously known to function as corepressors and coactivators, that now seem likely to modulate YY1's functions, are the histone deacetylases (HDAC) and histone acetyltransferases (HAT). These two groups of enzymes modify histones, and this modification is proposed to alter chromatin structure. Acetylated histones are typically localized to active chromatin while deacetylated histones colocalize with transcriptionally inactive chromatin. When these enzymes are directed to a promoter through a DNA binding factor such as YY1, that promoter can be activated or repressed. This review will discuss the recent work dealing with the different proteins that interact with YY1, with particular emphasis on ones that modify chromatin, and how they could be involved in regulating YY1's activities.
Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila engrailed gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic, a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.
Human papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Our study shows that sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nt 175-179) and comprising the presumable CCAAC and TATA boxes of the E6 promoter P175 contain a cluster of four motifs similar to the DNA recognition site of the multifunctional cellular transcription factor yin-yang 1 (YY1). Using DNase I footprinting and gel retardation tests it could be demonstrated that three of these motifs indeed act as YY1 binding sites. To test their functional relevance for P175 activity, engineered YY1 binding site mutants were analysed in the context of a P175 test vector using transient expression assays with human keratinocytes. YY1 turned out to exert both positive and negative effects upon the activity of the HPV-8 E6 promoter; binding of YY1 to a site upstream of the promoter's cap-position (BS1) activated transcription, whereas the downstream site (BS2) mediated repression. The second downstream YY1 binding site (BS3) seemed to play an auxiliary role, enhancing the negative effect of YY1 BS2. These observations define YY1 as an important cellular protein involved in the control of E6 oncogene expression of the skin-specific 'high risk' HPV-8 and emphasize the potential regulatory role of sequences located downstream of the transcription start site.
Disruption of Epstein-Barr virus latency is induced by expression of either the BZLF1 (in B cells and epithelial cells) or BRLF1 (in epithelial cells only) immediate-early protein. Regulation of BZLF1 and BRLF1 transcription may therefore modulate the stringency of viral latency. The cellular transcription factor YY1 negatively regulates BZLF1 transcription. Here we show that the BRLF1 promoter (Rp) sequences from -206 to -227 (relative to the mRNA start site) and from -7 to +6 are directly bound by YY1. Mutation of the upstream YY1 binding site increases constitutive Rp activity in epithelial cells and B cells, while mutation of the downstream YY1 binding site does not significantly affect Rp activity. Negative regulation of BZLF1 and BRLF1 transcription by YY1 may act to maintain viral latency.
YY1 is ubiquitously expressed zinc finger DNA binding protein. It can act as a transcriptional repressor or activator and, when binding at the initiator element, as a component of the basal transcription complex. Binding sites for YY1 have been reported in a wide variety of promoters and they exhibit substantial diversity in their sequence. To better understand how YY1 interacts with DNA and to be able to predict the presence of YY1 sites in a more comprehensive fashion, we have selected YY1 binding sites from a random pool of oligonucleotides. The sites display considerable heterogeneity, but contain a conserved 5'-CAT-3' core flanked by variable regions, generating the consensus 5'-(C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c)-3', where the upper case letters represent the preferred base. This high degree of flexibility in DNA recognition can be predicted by modeling the interaction of the four YY1 zinc fingers with DNA and a detailed model for this interaction is presented and discussed.
We have evaluated the potential role of prostaglandins and their second-messenger Cyclic Adenosine Monophosphate (cAMP) in the activation of interleukin-6 (IL-6) promoter regulatory elements leading to IL-6 expression in monocytic cells. We demonstrate that prostaglandins of the E series and their second-messenger cAMP induce the IL-6 promoter in the murine monocytic cell line PU5-1.8. Stimulation with both cAMP and LPS results in a marked synergistic effect. We show that the endogenous IL-6 gene is induced by cAMP as well, even though to a lesser extent than by LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations eliminating potential transcription factor binding sites, including the multiple-response element (MRE), AP-1, NF-IL6, and NF-kappaB binding sites, significantly reduce, but do not completely abrogate, inducibility by cAMP or prostaglandin E1, whereas alterations of four additional putative regulatory elements have no effects. In contrast, LPS-induced promoter activity is almost completely abolished by mutations in the NF-kappaB binding site, suggesting that a single regulatory element is crucial for inducibility by LPS, whereas no individual element is absolutely essential for cAMP signaling. Induction of the AP-1, NF-IL-6, and NF-kappaB elements by cAMP is correlated with the appearance of inducible factors binding to these sites, whereas factors binding to the MRE are constitutively expressed. Our results suggest that cAMP and prostaglandins act through multiple, partially redundant, regulatory elements to induce IL-6 expression in monocytic cells.
The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells.
YY1 is a zinc finger transcription factor which acts as either a repressor or an activator dependent on the promoter context. YY1 is a potent activator of the genuine human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) in HeLa cells, which are known for high-level expression of the HPV-18 early genes. The activating activity of YY1 is dependent on the presence of a newly identified switch region located upstream of the YY1 binding site. Deletion of this region causes YY1 to act as a repressor of HPV-18 promoter activity. In vivo footprinting of the HPV-18 URR and an in vitro electrophoretic mobility shift assay identified proteins binding to the switch region. Site-directed mutagenesis of the switch region and YY1 binding sites suggests that these two regions work in concert to yield high-level HPV-18 URR activity in HeLa cells but not in HepG2 cells, where HPV-18 is almost inactive. These data identified a novel mode of cell type-specific regulation of HPV-18 promoter activity by positive or negative action of YY1, determined by the switch region binding factor(s).
The UCRBP (YY1, delta, NF-E1) protein has been isolated for its ability to bind to the UCR (upstream conserved region) site present in the conserved murine leukemia virus long terminal repeat. UCRBP carries a highly charged N-terminal domain and four C2-H2-type zinc fingers at its C-terminal end. The present study reveals the following results: (i) The UCR site is present in the upstream and/or regulatory regions of numerous mammalian cellular and viral genes to which both recombinant and cellular UCRBP bind. UCR sites are also found in the regulatory regions of repetitive sequences including human LINE-1 elements and mouse intracisternal-A particle sequences. (ii) By immunological and UV cross-linking experiments, we found that two proteins, of approx. 68 kDa and an antigenically related protein of approx. 40 kDa, account for much of the UCR-binding activity in T-lymphocytes. (iii) There is evidence that UCRBP acts as a phosphoprotein. Eight consensus phosphorylation sites are found in the deduced amino-acid sequence of human UCRBP. The cellular UCR-binding activity was abolished by phosphatase treatment, and there is an incremental increase in apparent molecular mass between the cytoplasmic and nuclear forms of the protein, suggesting phosphorylation. (iv) Although UCRBP has been previously shown to act as a transcriptional repressor, we show here that UCRBP can also act as a positive transactivator of a reporter driven by UCR elements when used in co-transfection assays. This transactivation occurred in a dose-restricted manner and was absent at high concentrations of a UCRBP expression plasmid, indicating a complex mode of function.(ABSTRACT TRUNCATED AT 250 WORDS)
Induction of interleukin-6 (IL-6) gene expression is mediated by numerous agents involving all major signal transduction pathways. We have compared the effects of prostaglandins and their second messenger cyclic AMP (cAMP) with the effect of lipopolysaccharide (LPS) on IL-6 gene expression. We demonstrate that secretion of IL-6 is induced by cAMP in murine monocytic PU5-1.8 cells, even though to a lesser extent than by LPS. Nevertheless, cAMP and prostaglandins of the E series in the presence of theophylline induce transcription of the IL-6 promoter more strongly than LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations within four regulatory elements, namely, the multiple response element (MRE), AP-1, NF-IL6, and NF-kappa B sites, significantly reduce, but do not completely abrogate, inducibility by cAMP and prostaglandin E1, whereas alterations of four additional sites have no effects. LPS-induced promoter activity, however, is almost completely abolished by mutations in the NF-kappa B site, suggesting that a single regulatory element is crucial for inducibility by LPS. Stimulation by cAMP is correlated with the binding of inducible factors to the AP-1, NF-IL6, and NF-kappa B elements, whereas factors binding to the MRE are constitutively expressed. Recombinant cAMP response element-binding protein binds to the MRE, indicating a potential role for this factor in the cAMP response. Our results suggest that cAMP and prostaglandins act through multiple, partially redundant regulatory elements to induce IL-6 expression in monocytic cells. Nuclear events that overlap partially with the LPS response but also exhibit distinctive features are involved.
Oncogene expression is generally incompatible with terminal cell differentiation as in myogenesis. We present evidence that this incompatibility can be caused in part by the dual activity of a Kruppel-related zinc finger, YY1 (formerly F-ACT1), in differentially regulating oncogene and muscle-specific gene expression. The c-myc and skeletal alpha-actin gene promoters contain YY1 binding sites thought to act either as positive or negative cis-acting elements. Through manipulating the intracellular level of YY1 by treating primary myoblasts with bromodeoxyuridine (BrdU), which inhibited myogenesis and increased the YY1 protein content, or by transfecting YY1 cDNA expression vector, we show that it can simultaneously inhibit and activate expression of the skeletal alpha-actin and c-myc genes, respectively. The transrepression activity of YY1 solely depends on its C-terminal zinc finger region (amino acids 297-407) while its transactivation function requires an additional N-terminal domain (amino acids 1-90) normally masked in the full-length protein. We propose that the high level of YY1 in proliferating myoblasts might serve to maintain c-myc expression and suppress muscle actin expression, which can then be gradually reversed by downregulating YY1 activity toward myogenesis.
The macrophage-derived cytokine interleukin-1 (IL-1) can provide a second signal with antigen to elicit production of interleukin-2 (IL-2) by helper T cells. The pathway(s) involved remains controversial, with protein kinase C and cyclic AMP (cAMP) invoked as possible second messengers. In the murine thymoma EL4.E1, IL-1 could synergize with the phosphoinositide pathway, because the cells made higher levels of IL-2 in the presence of IL-1 than could be induced by phorbol ester plus calcium ionophore alone. IL-1 is unlikely to act through a sustained increase in cAMP in these cells because it did not raise cAMP levels detectably and because IL-1 and forskolin had opposite effects on IL-2 gene expression. Inducible expression of a transfected reporter gene linked to a cloned fragment of the murine IL-2 gene promoter was initially increased by IL-1 costimulation, implying that IL-1 can increase the rate of transcription of IL-2. The minimal promoter elements required for iL-1 responsiveness were located within 321 bp of the IL-2 RNA cap site, and further upstream sequences to -2800 did not modify this response. IL-1 costimulation resulted in enhanced activity of both an inducible NF-kappa B-like factor and one of two distinct AP-1-like factors that bind to IL-2 regulatory sequences. Neither was induced, however, by IL-1 alone. Another AP-1-like factor and NFAT-1, while inducible in other cell types, were expressed constitutively in the EL4.E1 cells and were unaffected by IL-1. These results are discussed in terms of the combinatorial logic of IL-2 gene expression.
In humans, microsomal epoxide hydrolase (mEH) contributes important biological functions that underlie both detoxification and bioactivation fates arising from exposures to foreign chemicals. Previously, we discovered that human mEH gene transcription is initiated from alternative promoters. The respective transcripts are programmed with tissue specificity and the upstream E1b promoter contributes predominantly to mEH expression. The results presented demonstrate that exposures to the Nrf2 activators, sulforaphane (SFN) and tert-butylhydroquinone (tBHQ), markedly activate E1b transcription in human lung and liver cells. Genomic analyses identified two major DNase I hypersensitive regions (HS-1 and HS-2) within the ~15 kb intervening sequence separating E1b from the downstream E1 promoter. In BEAS-2B cells, the Nrf2 effectors, SFN and tBHQ, selectively activated the more distal HS-2 through an antioxidant response element (ARE). An activator protein 1/12-O-tetradecanoylphorbol-13-acetate interaction was further identified within the HS-2 enhancer that functioned to additionally contribute to ARE-mediated induction responsiveness of the E1b promoter. The results demonstrate that ARE modulation, integrated with additional transcriptional complexes, regulates the tissue-specific expression of mEH and that these processes likely coordinate both the protective and bioactivation functions contributed by mEH activities in human tissues.
The pluripotent P19 embryo carcinoma cell line was studied to determine a signaling pathway regulating MeCP2 expression. P19 cells were induced to differentiate into neurons by RA and express β-III tubulin at one day after induction and synaptophysin by 7 days. MeCP2 was first observed after β-III tubulin expression was detected and continued to rise over the course of differentiation. Both Mecp2 e1 and e2 mRNA forms progressively increased in differentiating cells. MeCP2 expression was increased by tumor necrosis factor (TNF) in early differentiating cells, which was blocked by NFκB inhibitors. TNF did not increase MeCP2 expression in naïve cells. Moreover, TNF did not increase NFκB reporter gene activity in naïve cells even though increases were observed in early differentiating cells. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) increased MeCP2 expression in naïve P19 cells, which was also blocked by NFκB inhibitors. Interestingly, PMA increased NFκB reporter gene activity in naïve cells. Finally, PMA, but not TNF, induced IκBα degradation in naïve P19 cells. Taken together, our data indicates that MeCP2 expression is regulated in part by signaling pathways involving NFκB.
Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.
Principally expressed on the surface of T lymphocytes, the chemokine and HIV receptor CXCR4 has been shown to serve key roles in both chemotaxis and HIV-1-entry into T cells. Understanding the regulation of CXCR4 expression is therefore of paramount importance to further elucidating its endogenous role and contributions to HIV-1 pathogenesis. Using an RNase protection assay (RPA), we have demonstrated that mitogenic stimulation of purified human peripheral blood T lymphocytes (PBL) decreased CXCR4 mRNA relative to unstimulated controls in a calcineurin-dependent manner; an expression pattern mimicked by the chemokine receptor CCR7. A change in transcriptional activity, not in mRNA stability, was required for control of CXCR4 and CCR7 expression. Changes in CXCR4 mRNA expression translated into a stimulation- and calcineurin-dependent decrease in cell surface CXCR4 expression. We have previously demonstrated that CXCR4 mRNA and protein is regulated by cAMP; here we show that calcium and calcineurin signaling pathways modify cAMP-driven changes. Moreover, we provide data supporting a role for the transcription factor YY1 in calcineurin-dependent regulation of CXCR4 expression.
Multiple signal transduction pathways are generally triggered simultaneously by a single extracellular stimulus. As a result, multiple transcription factors (TFs) can be activated downstream to mediate the inducible expression of target genes. Profiling the activation of all TFs will aid in the dissection of the numerous pathways of signal transduction. Tumor necrosis factor alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA) mediate many biological functions, including cell proliferation and apoptosis, by stimulating signaling pathways. Two TFs, nuclear factor kappaB (NFkappaB) and activating factor 1 (AP1), have been identified as targets of both TNFalpha and PMA activation. Here, we describe the use of a protein/DNA array system to identify additional TFs activated by TNFalpha and PMA in HeLa cells. From a total of 150 targeted TFs, six-CREB, E2F, CETP/CRE, c-Rel, MSP1, and Pax6-were identified whose activities, like NFkappaB and AP1, were regulated by both TNFalpha- and PMA-induced pathways. Interestingly, the TF E47 was shown to be specifically activated by TNFalpha but was not affected by treatment with PMA. In addition, GATA, NF-E1, and ISRE were shown to be specifically activated by PMA but not TNFalpha. These findings suggest that TNFalpha and PMA both stimulate unique signaling pathways while mediating transcriptional activation through common pathways.
Histone acetylation alters the chromatin structure and activates the genes that are repressed by histone deacetylation. This investigation demonstrates that treating P3HR1 cells with trichostatin A (TSA) activates the Epstein-Barr virus (EBV) lytic cycle, allowing the virus to synthesize three viral lytic proteins-Rta, Zta and EA-D. Experimental results indicate that TSA and 12-O:-tetradecanoylphorbol-13-acetate synergistically activate the transcription of BRLF1, an immediate-early gene of EBV. Chromatin immunoprecipitation assay reveals that histone H4 at the BRLF1 promoter is acetylated after P3HR1 cells are treated with TSA, suggesting that histone acetylation activates BRLF1 transcription. Furthermore, results in this study demonstrate that mutation of a YY1-binding site in the BRLF1 promoter activates BRLF1 transcription 1.6- and 2.3-fold in P3HR1 cells and C33A cells, respectively. Real time PCR analysis reveals that the mutation also increases the histone acetylation level of the nucleosomes at the BRLF1 promoter 1. 64- and 3.08-fold in P3HR1 and C33A cells, respectively. Results presented herein suggest that histone deacetylation plays an important role in maintaining the viral latency and histone acetylation at the BRLF1 promoter allows the virus to express Rta and to activate the viral lytic cycle.
The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhances transcription of many eukaryotic genes, including that for dopamine beta-hydroxylase (DBH). In the present study, we report identification and characterization of a novel sequence motif residing in the 5'-flanking region of the human DBH gene, which mediates transcriptional induction by TPA. Deletional analyses indicated the promoter region between -223 and -187 base pairs to be critical. Whereas this region does not contain any putative regulatory motifs with significant sequence homology to the AP-1 motif, extensive deletional and site-directed mutational analyses indicated that a sequence between -210 and -199 base pairs, 5'-ATCCGCCTGTCT-3', may represent a novel TPA-response element (TRE). In addition, alteration of the YY1-binding site decreased TPA-mediated induction of the DBH promoter activity, suggesting that contiguous cis-regulatory element(s) cooperate with this novel sequence motif. Furthermore, insertional mutation analyses between the YY1-binding site and the cyclic AMP-responsive element indicated that the stereospecificity of these motifs is important for intact transcriptional induction by TPA. Taken together, these data suggest that transcriptional up-regulation of the human DBH gene in response to TPA requires coordination of a novel TRE (human DBH TRE, hDTRE), cyclic AMP-responsive element, and the YY1-binding site.
Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction.
A 48-bp cis-acting negative element in the Epstein-Barr virus BZLF1 gene P1 promoter has been described previously. By DNase I footprinting experiments, two regions were identified as the protein-binding sites (previously designated site I and site II). In this report, the cellular transcription factor YY1 has been identified as a protein which binds to both of these elements, now designated ZIVA and ZIVB. Both ZIVA and ZIVB conferred cis-acting negative regulation on an enhancerless simian virus 40 promoter. In cotransfection experiments, overexpression of YY1 caused further repression of the enhancerless simian virus 40 promoter containing either the ZIVA or ZIVB element. Cotransfection of a plasmid expressing antisense to YY1 increased the expression of the heterologous promoter containing ZIVA but not ZIVB. In similar experiments carried out with the P1 promoter, overexpression of YY1 caused downregulation of P1 whereas antisense RNA to YY1 caused a slight increase in expression. Analyses of various P1 mutant constructions revealed additional YY1 sites downstream of ZIVB. Overexpression of YY1 also caused downregulation of a P1 mutant with no apparent YY1-binding sites. TPA treatment of Raji cells caused a temporal loss of YY1-binding activity but had no effect on the intracellular levels of YY1 protein. Serum induction of quiescent B cells also caused loss of YY1 binding to the ZIVB site, which was found to be a weak serum response element. In contrast, anti-immunoglobulin G treatment of Akata cells had no effect on either the YY1-binding activity or protein levels. The binding of YY1 to the cis-acting negative elements in infected B cells may play a pivotal role in the maintenance of Epstein-Barr virus latency.
Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.
The human papillomavirus type 18 (HPV-18) promoter contains a TPA responsive element (TRE) which confers TPA responsiveness on a heterologous promoter. In the context of the HPV-18 promoter, however, this AP-1 site is inactive. We have identified a negative regulatory domain in the HPV-18 promoter which represses the constitutive and TPA-induced AP-1 activity. This negative regulatory sequence has been mapped to 44 nucleotides (OL13). We identified this element as a transcriptional silencer based on its ability to interfere with transcriptional initiation. This HPV-18 silencer domain was narrowed down further to 23 nucleotides, the OL13B element, which bears similarity to three other silencer sequences, present in the mouse N-ras gene upstream regulatory region, the mouse albumin gene enhancer and the adeno-associated virus P5 promoter. The transcriptional repressor protein YY1, which negatively regulates the P5 promoter, binds to the HPV-18 silencer with high affinity. Mutation of the YY1 binding site leads to an enhanced activity of the HPV-18 promoter, strongly suggesting that YY1 plays an important role in controlling HPV-18 early gene expression.
We have studied the elements involved in the tetradecanoylphorbol acetate (TPA)-mediated extinction of erythroid-specific genes. We show that transcription driven by a -714/+78-base pair DNA fragment of the erythroid promoter of the human porphobilinogen deaminase gene is down-regulated upon TPA treatment of erythroleukemic cells. Examination of the DNA binding activity of trans-acting factors involved in the expression of the porphobilinogen deaminase erythroid promoter showed (i) a constitutive expression of the CACC binding proteins and (ii) a decrease in DNA binding activity of two tissue-specific factors, NF-E1 and NF-E2. Kinetics experiments indicated that NF-E2 was down-regulated after 1 h of TPA treatment whereas NF-E1 was down-regulated at the protein and mRNA levels only after 5 h of TPA treatment. These results suggest that different pathways, acting via different transcription factors, are involved in the TPA-mediated extinction of erythroid-specific genes.
To investigate the effect of zinc ion on the expression of osteoblastic proteins.
Mice osteoblasts MC3T3-E1 cells were subcultured. Inflammatory environment model was established by tumor necrosis factor α(TNF-α)at a concentration of 10 mg/L. According to different concentration of Zn(2+), the cells were divided into TNF-α group, control group, group A(TNF-α+10(-4) mol/L Zn(2+)), group B(TNF-α+10(-5) mol/L Zn(2+)), group C(TNF-α+10(-6) mol/L Zn(2+)). After 24, 48, and 72 h of culture, cell counting kit-8(CCK-8)assay was used to analyze the proliferation of the cells. ALP activity was examined. Bone morphogenetic protein-2(BMP-2), Runt-related transcription factor 2(RUNX2), Osterix and receptor activator of NF-κB ligand(RANKL)protein levels were determined by Western blotting after 72 h of culture.
The cells grew by adherence after 24 h. After 72 h, the cells grew dense, and the cells showed long spindle shape or irregular shape. The proliferation of osteoblasts in TNF-α group, group B and group C became lower than that in the control group(P<0.05), and was not significantly different between group A and the control group(P >0.05). ALP activity examination demonstrated that the groups cultured for 72 h revealed the highest ALP activity and the most prominent differentation compared with 24 h and 48 h groups. ALP activity was significantly decreased in TNF-α group, group B and group C compared with control group(P<0.05), but was not significantly different between group A and control group(P>0.05). The protein levels of BMP-2, RUNX2 and Osterix were significantly decreased in TNF-α group, group B and group C compared with control group(P<0.05), while showed no significant difference between group A and the control group. Protein level of RANKL was significantly increased in TNF-α group, grope B and group C compared with control group(P<0.05), while showed no significant difference between group A and control group.
The concentration of 10(-4) mol/L Zn(2+) can significantly increase the expression of osteoblastic proteins such as ALP, BMP-2, RUNX2, Osterix and decrease the expression of RANKL in mice osteoblasts in TNF-α inflammatory environment.
Titanium is biocompatible with bodily tissues. However, the formation of ROS on the titanium surfaces might have negative response of the activity of the surroundings cells. Terrein was isolated from Penicullium sp. 20135 and found to reduce the effects of LPS-induced inflammation. This study examined the role of Terrein on the biocompatibility of titanium to determine if it can help improve osseointegration. MC-3T3 E1 cells were grown on titanium surfaces. The biocompatibility of Terrein was examined by adding it directly to the culture media at the indicated concentration. The cells on the titanium surface produced excessive ROS and decreased the activity of Cu/Zn SOD and Mn SOD. Moreover, the cells had higher activity towards oxidative stress molecules, such as MAPK, FAK and iNOS expression. In addition, MC-3T3 E1 osteoblast-like cells promoted osteoclast differentiation but reduced osteoblast differentiation and mineralization on the titanium surface. Interestingly, the cells given the Terrein treatment showed higher resistance towards oxidative stress through the up-regulation of ERK1/2 and FAK activity but the down-regulation of SAPK/JNK and iNOS activity. Moreover, Terrein promoted osteoblast differentiation and bone mineralization to elevate the activity of ALP, SPARC and down-regulate RANKL expression after blocking NF-κB translocation from the cytosol to the nucleus. In conclusion, the presence of Terrein on titanium surfaces increases osteoblast cell growth without inflammation. Moreover, Terrein, as a putative antioxidant agent, may enhance osseointegration by decreasing the level of ROS and having a potentially synergistic effect on osteoblast differentiation.
CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements ("chromatin insulators") including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon-intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.
The preferential screening of cDNA libraries derived from the mouse osteoblastic cell line MC3T3-E1 has yielded a cDNA clone encoding a 442-amino-acid protein designated STAP (signal transduction and adaptor protein), which contains several motifs shared among transcription factors and adaptors such as a Zn-finger like motif, a proline-rich domain, and a PEST sequence. The amino acid sequence homology search also reveals that STAP is identical to a mouse oxidative stress protein, A170, and has 90% homology with a human p62 protein that binds to the tyrosine kinase p56(lck) SH2 domain. Northern blot analysis indicated a broad expression profile of STAP mRNA in various tissues and cell lines. In MC3T3-E1 cells, STAP mRNA was induced by treatment with TGF-beta, but not with BMP-2 or GDF-5. Analysis of the mouse STAP gene isolated from the genomic library revealed that the STAP gene spans a region of over 11 kb and comprises eight exons. The transcription start site was identified by primer extension analysis to be located 35 bp upstream from the translation initiation site. Sequencing analysis of the 5' flanking region of the STAP gene revealed multiple consensus motifs/sequences for several DNA binding transcription factors. The STAP gene had a TATA box, but no CCAAT box. Potential Sp1, AP-1, NF-E2, MyoD, and NF-kappaB binding sites were found in the 5' flanking region (1.4 kb) of the STAP gene.
The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.
Cu/Zn-superoxide dismutase (SOD1) catalyses the dismutation of superoxide radicals and neutralizes the oxidative effects of various chemicals. Deletion analysis of the upstream region of the rat SOD1 gene revealed that the promoter contains a positive regulatory element (PRE) and a negative regulatory element (NRE), which encompass the regions from -576 to -412 and from -412 to -305 respectively from the site of initiation of transcription. These DNA elements showed enhancer and silencer activities respectively in the natural context and in a heterologous promoter system. Using an electrophoretic-mobility-shift assay and a supershift assay with a specific antibody, the cis-elements of the PRE and NRE were identified as binding sites for transcription factors Elk1 and YY1 (Ying-Yang 1) respectively. Consistent with the presumed roles of the PRE and NRE, Elk1 increased SOD1 gene transcription about 4-5-fold, whereas YY1 exerted a negative effect of about 6-fold. Mutations of the Elk1- and YY1-binding sites led to diminution and elevation respectively of transcriptional activities, both in the natural context and in heterologous promoter systems. These results suggest that the transcription factors Elk1 and YY1, binding in the PRE and NRE respectively, co-ordinate the expression of the SOD1 gene.
Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells.
We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2.
YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as opposed to YY1, suggesting that YY2 exerts unique contributions to gene regulation. YY2 binding was not generally associated with gene promoters. However, several YY2 binding sites are linked to long noncoding RNA (lncRNA) genes and we show that the expression levels of a few of those are Yy2-dependent.
Abscisic acid (ABA) plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we identified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 (YY1), as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yy1 mutant was more sensitive to ABA and NaCl than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYY1 can bind the ABA REPRESSOR1 (ABR1) promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and salt-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 (ABI4) might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis.
Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation. YY1 effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The role of the multifunctional transcription factor Yin Yang 1 (YY1) in tissue development is poorly understood. In the present, we investigated YY1a role in developing zebrafish on PSR-mediated apoptotic cell engulfment during organic morphogenesis.
YY1a is first expressed 0.5 h post-fertilization (hpf), in the whole embryo 12 hpf, and in brain, eyes, and heart 72 hpf by in situ hybridization assay. The nucleotide sequence of zebrafish YY1a transcription factor (clone zfYY1a; HQ 166834) was found to be similar to that of zebrafish YY1a (99 % sequence identity; NM 212617). With the loss-of-function assay, YY1a knockdown by a morpholino oligonucleotide led to downregulation of the phosphatidylserine engulfing receptor zfPSR during embryonic segmentation and to the accumulation of a large number of dead apoptotic cells throughout the entire early embryo, especially in the posterior area. Up to 24 hpf, these cells interfered with embryonic cell migration and cell-cell interactions that normally occur in the brain, heart, eye, and notochord. Finally, with gain-of-function assay, defective morphants could be rescued by injecting both YY1a mRNA and PSR mRNA and trigger resumption of normal development.
Taken together, our results suggest that YY1a regulates PS receptor expression that linked to function of PSR-phagocyte mediated apoptotic cell engulfment during development, especially the development of organs such as the brain and heart. YY1a/PSR-mediated engulfing system may involve in diseases.
This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF.
Yin Yang 1 (YY1) is a zinc finger protein that functions as a transcriptional activator or repressor and participates in multiple biological processes, including development and tumorigenesis. To investigate the role of YY1 in developing T cells, we used mouse models that depleted YY1 at two distinct stages of thymocyte development. When YY1 was depleted in CD4(-)CD8(-) double-negative thymocytes, development to the CD4(+)CD8(+) double-positive stage was impaired, due to increased apoptosis that prevented expansion of post-β-selection thymocytes. When YY1 was depleted in double-positive thymocytes, they underwent increased cell-autonomous apoptosis in vitro and displayed a shorter lifespan in vivo, as judged by their ability to undergo secondary Vα-to-Jα recombination. Mechanistically, we found that the increased apoptosis in YY1-deficient thymocytes was attributed to overexpression of p53, because concurrent loss of p53 completely rescued the developmental defects of YY1-deficient thymocytes. These results indicated that YY1 functions as a critical regulator of thymocyte survival and that it does so by suppressing the expression of p53.
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the induction of antioxidant gene expression and protects cells against oxidative injury. However, there are controversial findings regarding the roles of Nrf2 on bone metabolism under oxidative stress. The role of Nrf2 on the differentiation of radiation-exposed osteoblasts is also unclear. We investigated whether Nrf2 negatively or positively affects osteoblast differentiation in response to irradiation. Irradiation inhibited osteoblast differentiation of MC3T3-E1 cells in a dose-dependent manner. This inhibition was evidenced by the irradiation-mediated decreases in bone-like nodule formation, alkaline phosphatase (ALP) activity, calcium accumulation, and expression of osteoblast markers, such as ALP, osteocalcin, osteopontin, bone sialoprotein, osterix, and Runx2. These reductions were accompanied by increased induction of Nrf2 and heme oxygenase-1 (HO-1), accumulation of cellular oxidants, and depletion of antioxidant defense enzymes. siRNA-mediated silencing of Nrf2 markedly reversed the negative effect of irradiation on osteoblast differentiation of the cells, leading to a decrease in HO-1 and an increase in Runx2 levels. Irradiation-mediated decreases in the levels of Runx2 and osteocalcin mRNA, but not of Nrf2 protein, were also significantly inhibited by HO-1 inhibitor, zinc protoporphyrin IX. Furthermore, N-acetyl cysteine restored all of the changes induced by irradiation to near-normal levels in the cells. These results demonstrate that irradiation inhibits osteoblast differentiation and mineralization of MC3T3-E1 cells through the oxidative stress-mediated activation of Nrf2/HO-1 pathway.
Interleukin (IL)-4 acts on T cells as a growth and activation factor, and promotes the differentiation of type 2 T helper cells. In T cells, expression of the gene encoding IL-4 is regulated by inducible or constitutive factors. Yin-Yang (YY)-1 is one of constitutive transcription factors binding to the IL-4 promoter. The recently identified YY2 protein is similar to YY1, with both sharing high levels of homology in their zinc finger motifs. However, the role of YY2 in T cells is unclear. YY1 and YY2 were constitutively expressed in EL4 T cells, and their expression was not dependent on stimulation. IL-4 promoter (-741/+56 fragment) activity was enhanced by YY1, but inhibited by YY2. The enhanced IL-4 promoter activity by YY1 was reduced by simultaneous expression of YY2. In addition, the DNA binding affinity of YY1 to the IL-4 promoter was adversely affected by YY2. Our results suggest that YY1 and YY2 exert opposing effects on the IL-4 promoter as they compete for the same DNA binding sites.
Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.
Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.
Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPβ (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPβ from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.
Biomineralization is an important and ubiquitous process in organisms. The shell formation of mollusks is a typical biomineral physical activity and is used as a canonical model in biomineralization research. Most recent studies focused on the identification of matrix proteins involved in shell formation; however, little is known about their transcriptional regulation mechanism, especially the transcription factors involved in shell formation. In this study, we identified a homolog of the YY-1 transcriptional factor from Pinctada fucata, named Pf-YY-1, and characterized its expression pattern and biological functions. Pf-YY-1 has a typical zinc finger motif highly similar to those in humans, mice, and other higher organisms, which indicated its DNA-binding capability and its function as a transcription factor. Pf-YY-1 is ubiquitously expressed in many tissues, but at a higher level in the mantle, which suggested a role in biomineralization. The expression pattern of Pf-YY-1 during pearl sac development was quite similar to, and was synchronized with, those of Prisilkin-39, ACCBP, and other genes involved in biomineralization, which also suggested its function in biomineralization.
YY1 (Yin Yang 1) is a zinc finger protein with an essential role in various biological functions via DNA- and protein-protein interactions with numerous partners. YY1 is involved in the regulation of a broad spectrum of cellular processes such as embryogenesis, proliferation, tumorigenesis, and snRNA transcription. The more than 100 reported targets of the YY1 protein suggest that it contains intrinsically disordered regions that are involved in such diverse interactions. Here, we present a study of the structural properties of human YY1 using several biochemical and biophysical techniques (fluorescence, circular dichroism, gel filtration chromatography, proteolytic susceptibility) together with various bioinformatics approaches. To facilitate our exploration of the YY1 structure, the full-length protein as well as an N-terminal fragment (residues 1-295) and the C-terminal DNA binding domain were used. We found the N-terminus to be a non-compact fragment of YY1 with little residual secondary structure and lacking a well-defined tertiary structure. The results of our study indicate that YY1 belongs to the family of intrinsically disordered proteins (IDPs), which exist natively in a partially unfolded conformation.
Esophageal squamous cell carcinoma (ESCC), one of the most common gastrointestinal tumors, is known for its high mortality rate. microRNAs (miRNAs) have been reported to play important regulatory roles in cancer metastasis and progression. miR-34a has been demonstrated to be associated with the development of and metastasis in certain types of cancer via various target genes, but its function and targets in ESCC are unknown. The aim of this study was to examine whether the expression of miR-34a was significantly decreased in ESCC tissues, compared with normal esophageal tissues using RT-PCR and western blot analysis. The results showed that miR-34a overexpression increased apoptosis and decreased clonogenic formation, but inhibited invasion and migration in ESCC cells by suppressing MMP-2 and -9 expression. Yin Yang-1 (YY1), a widely distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins, was found to be a direct target of miR-34a in ESCC cell lines. Rescue experiments indicated that the suppressive effect of miR-34a on invasion and migration was mediated by activating YY1 expression. Results of the present study showed that miR-34a is associated with ESCC migration and provides a potential therapeutic and diagnostic target for ESCC.
Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal β-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.
Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general.
Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Yin Yang 1 (YY1) is a ubiquitous and multifunctional zinc-finger transcription factor that plays important biological functions in cell homeostasis and tumorigenesis. The purpose of this study was to investigate the expression of YY1 in different ESCC tissues and the potential relationship with clinicopathological features.
One hundred and four ESCC tissues were collected in this study. The protein levels of YY1 were measured by immunohistochemistry. TE-1 cell invasion in vitro was assessed using the Transwell assay.
There were no obvious differences between expression levels in patients over age 64 and those younger than 64, and no noticeable distinction was observed between males and females. However, the YY1 protein level was significantly higher in ESCC tissues with lymph node metastasis than those without lymph node metastasis (P=0.042). Furthermore, the expression of the YY1 protein was stronger in stage III-IV patients than in stage I-II patients (P=0.002), but the protein levels between different histological grades (well, moderate, or poor) showed no statistical significance. Similarly, there was no difference in YY1 expression in patients with or without lymphatic invasion. The Transwell assay revealed that the overexpression of YY1 promoted the invasion ability of TE-1 cells and the inhibition of YY1 could reverse this promotion.
YY1 expression was associated with TNM stage and lymph node metastasis, suggesting that YY1 can influence human esophageal cancer progression and metastasis.
The genomic binding of CTCF is highly conserved across mammals, but the mechanisms that underlie its stability are poorly understood. One transcription factor known to functionally interact with CTCF in the context of X-chromosome inactivation is the ubiquitously expressed YY1. Because combinatorial transcription factor binding can contribute to the evolutionary stabilization of regulatory regions, we tested whether YY1 and CTCF co-binding could in part account for conservation of CTCF binding.
Combined analysis of CTCF and YY1 binding in lymphoblastoid cell lines from seven primates, as well as in mouse and human livers, reveals extensive genome-wide co-localization specifically at evolutionarily stable CTCF-bound regions. CTCF-YY1 co-bound regions resemble regions bound by YY1 alone, as they enrich for active histone marks, RNA polymerase II and transcription factor binding. Although these highly conserved, transcriptionally active CTCF-YY1 co-bound regions are often promoter-proximal, gene-distal regions show similar molecular features.
Our results reveal that these two ubiquitously expressed, multi-functional zinc-finger proteins collaborate in functionally active regions to stabilize one another's genome-wide binding across primate evolution.
Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignancies worldwide. Ying Yang 1 (YY1), a ubiquitously expressed GLI-Krüppel zinc finger transcription factor, plays a regulatory role in a variety of fundamental biological processes, such as embryonic development, growth, apoptosis, differentiation and oncogenic transformation. The purpose of this study was to investigate the expression of YY1 in normal and cancerous esophageal tissues and its function in ESCC development. We found that the expression of YY1 mRNA was significantly increased in the tumor tissues, compared with the para-tissues or normal esophageal tissues. The increased expression of YY1 in tumor samples was further confirmed by immunohistochemistry. Furthermore, the overexpression of YY1 conferred radioresistance to the ESCC TE-1 cells and resulted in markedly reduced cell proliferation. Accordingly, the small interfering RNA-mediated silencing of YY1 expression in TE-1 cells resulted in increased proliferation by enhancing the binding of P21 to Cyclin D1 and CDK4, a protein complex known to mediate cell cycle progression. Moreover, besides P21, heme oxygenase-1 (HO-1) was identified as a YY1 downstream effector, as YY1 stimulated HO-1 expression in esophageal cancer cells. YY1 mediated biological function through transcription of HO-1. Forced expression of HO-1 could moderately suppress proliferation of TE-1 cells. The expression of YY1 significantly correlated with that of HO-1 in ESCC tissues. Taken together, we demonstrated overexpression of YY1 in esophageal carcinoma and identified HO-1 as its target.
NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.
In the present research, we have studied an influence of enhanced expression TRIM14 on alphavirus Sindbis (SINV, Togaviridae family) infection. In the HEK293 cells transfected with human trim14 gene (HEK-trim14), SINV yield after infection was decreased 1000-10,000 times (3-4 lg of TCD50/ml) at 24 h p.i. and considerably less (1-2 lg of TCD50/ml) at 48 h p.i. Analysis of the expression of 43 genes directly or indirectly involved in innate immune machine in HEK-trim14 non-infected cells comparing with the control (non-transfected) HEK293 cells revealed that stable trim14 transfection in HEK293 cells caused increased transcription of 18 genes (ifna, il6 (ifnβ2), isg15, raf-1, NF-kB (nf-kb1, rela, nf-kb2, relb), grb2, grb3-3, traf3ip2, junB, c-myb, pu.1, akt1, tyk2, erk2, mek2) and lowered transcription of 3 genes (ifnγ, gata1, il-17a). The similar patterns of genes expression observe in SINV-infected non-transfected HEK293 cells. However, SINV infection of HEK-trim14 cells caused inhibition of the most interferon cascade genes as well as subunits of transcription factor NF-κB. Thus, stable enhanced expression of trim14 gene in cells activates the transcription of many immunity genes and suppresses the SINV reproduction, but SINV infection of HEK-trim14 cells promotes inhibition of some genes involved in innate immune system.
Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-β superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.
Pierre Robin Sequence (PRS) is usually classified into syndromic and nonsyndromic groups, with a further subclassification of the nonsyndromic group into isolated PRS and PRS with additional anomalies (PRS-Plus). The aim of this research is to provide an accurate phenotypic characterisation of nonsyndromic PRS, specifically the PRS-Plus subgroup. We sought to examine the frequency of sequence variants in previously defined conserved noncoding elements (CNEs) in the putative enhancer region upstream of SOX9, the regulation of which has been associated with PRS phenotypes. We identified 141 children with nonsyndromic PRS at the Royal Children's Hospital, Melbourne from 1985 to 2012 using 2 databases. Clinical and demographic data were extracted by file review and children categorized as 'isolated PRS' or 'PRS-Plus'. A subset of children with PRS-Plus was selected for detailed phenotyping and DNA sequencing of the upstream SOX9 CNEs. We found 83 children with isolated PRS and 58 with PRS-Plus. The most common PRS-Plus malformations involved the musculoskeletal and ocular systems. The most common coexisting craniofacial malformation was choanal stenosis/atresia. We identified 10 children with a family history of PRS or cleft palate. We found a single nucleotide substitution in a putative GATA1-binding site in one patient, but it was inherited from his phenotypically unaffected mother. PRS-Plus represents a broad phenotypic spectrum with uncertain pathogenesis. Dysmorphology assessment by a clinical geneticist is recommended. SOX9 CNE sequence variants are rare in our cohort and are unlikely to play a significant role in the pathogenesis of PRS-Plus.
Terminal erythroid differentiation is tightly coordinated with cell cycle exit, which is regulated by cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19(INK4d), a member of CDKI family, is abundantly expressed in erythroblasts and that p19(INK4d) knockdown delayed erythroid differentiation, inhibited cell growth, led to increased apoptosis and generation of abnormally nucleated late stage erythroblasts. Unexpectedly, p19(INK4d) knockdown did not affect cell cycle. Rather it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. As GATA1 protein is protected by nuclear HSP70, we examined the effects of p19(INK4d) knockdown on HSP70 and found that p19(INK4d) knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced ERK activation. Further biochemical analysis revealed that p19(INK4d) directly binds to Raf kinase inhibitor PEBP1 and that p19(INK4d) knockdown increased the expression of PEBP1 that in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19(INK4d) via a novel PEBP1-pERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.
In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology.
Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KO(DC)), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KO(DC) mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KO(DC) DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.
Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi-lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down-regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi-lineage hematopoiesis in the early yolk sac of developing embryos. J. Cell. Physiol. 232: 323-330, 2017. © 2016 Wiley Periodicals, Inc.
Transient abnormal myelopoesis is mostly self-resolving and has a good prognosis, but some patients subsequently die of liver fibrosis. We report the case of an infant with Down syndrome who developed life-threatening liver fibrosis at the same time as the blasts were about to disappear. This patient also had a marked increase in eosinophils, which were possibly harboring a GATA1 mutation and were expressing a high level of platelet-derived growth factor-B mRNA; these may have been involved in the development of liver fibrosis. Low-dose cytosine arabinoside therapy effectively treated both hypereosinophilia and liver fibrosis.
Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the seven enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.
DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.
Transient myeloproliferative disorder (TMD) is a leukemia type that occurs typically in newborns. In Down syndrome, TMD is referred to as transient abnormal myelopoiesis (TAM).(32) Recently, transientness has also been reported in acute myeloid leukemia patients with germline trisomy 21 mosaicism, and even in cases with somatic trisomy 21, with or without GATA1 mutations. TMD cases without trisomy 21 are rare, and recurrent genetic aberrations that aid in clinical decision-making are scarcely described. We describe here a TMD patient without trisomy 21 or GATA1 mutation in whom single-nucleotide polymorphism analysis of leukemic blasts revealed a novel combined submicroscopic deletion (5q31.1-5q31.3 and 8q23.2q24).
Lymphatic system plays an important role in maintaining the fluid homeostasis and normal immune responses, anatomic or functional obstruction of which leads to lymphedema, and treatments for therapeutic lymphangiogenesis are efficiency for secondary lymphedema. Total saponins of panaxnotoginseng (PNS) are a mixture isolated from Panaxnotoginseng (Burkill) F.H.Chen, which has been used as traditional Chinese medicine in China for treatment of cardio- and cerebro-vascular diseases. The aim of this study was to determine the effect and mechanism of PNS on lymphangiogenesis.
The Tg (fli1: egfp; gata1: dsred) transgenic zebrafish embryos were treated with different concentrations of PNS (10, 50, 100μM) for 48h with or without the 6h pretreatment of the 30μM Vascular endothelial growth factors receptor (VEGFR)-3 kinase inhibitor, followed with morphological observation and lympangiogenesis of thoracic duct assessment. The effect of PNS on cell viability, migration, tube formation and Vascular endothelial growth factors (VEGF)-C mRNA and protein expression of lymphatic endothelial cells (LECs) were determined. The role of phosphatidylinositol-3 (PI-3)-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2 pathways, c-Jun N-terminal kinase (JNK) and P38 mitogen activated protein kinases (MAPK) signaling in PNS-induced VEGF-C expression of LECs by using pharmacological agents to block each signal.
PNS promotes lymphangiogenesis of thoracic duct in zebrafish with or without VEGFR3 Kinase inhibitor pre-impairment. PNS promotes proliferation, migration and tube formation of LECs. The tube formation induced by PNS could be blocked by VEGFR3 Kinase inhibitor. PNS induce VEGF-C expression of LEC, which could be blocked by ERK1/2, PI3K and P38MAPK signaling inhibitors.
PNS activates lymphangiogenesis both in vivo and in vitro by up-regulating VEGF-C expression and activation of ERK1/2, PI3K and P38MAPK signaling. These findings provide a novel insight into the role of PNS in lymphangiogenesis and suggest that it might be an attractive and suitable therapeutic agent for treating secondary lymphedema or other lymphatic system impairment related disease.
MiR-592 has been identified as a neural-enriched microRNA, plays an important role in mNPCs differentiation, could induce astrogliogenesis differentiation arrest or/and enhance neurogenesis in vitro Previous studies showed that long noncoding RNAs (lncRNAs) were involved in the neuronal development and activity. To investigate the role of miR-592 in neurogenesis, we described the expression profile of lncRNAs in miR-592 knockout mouse embryonic stem cells (mESCs) and the corresponding normal mESCs by microarray. By the microarray analysis and luciferase reporter assays, we demonstrated that lncRNA - AK048794, regulated by transcription factor GATA1, functioned as a competing endogenous RNA (ceRNA) for miR-592 and led to the de-repression of its endogenous target FAM91A1, which is involved in mESC pluripotency maintenance. Taken together, these observations imply that AK048794 modulated the expression of multiple genes involved in mESC pluripotency maintenance by acting as a ceRNA for miR-592, which may build up the link between the regulatory miRNA network and mESC pluripotency.
Children with constitutional trisomy 21 (Down syndrome (DS)) have a unique predisposition to develop myeloid leukaemia of Down syndrome (ML-DS). This disorder is preceded by a transient neonatal preleukaemic syndrome, transient abnormal myelopoiesis (TAM). TAM and ML-DS are caused by co-operation between trisomy 21, which itself perturbs fetal haematopoiesis and acquired mutations in the key haematopoietic transcription factor gene GATA1. These mutations are found in almost one third of DS neonates and are frequently clinically and haematologcially 'silent'. While the majority of cases of TAM undergo spontaneous remission, ∼10 % will progress to ML-DS by acquiring transforming mutations in additional oncogenes. Recent advances in the unique biological, cytogenetic and molecular characteristics of TAM and ML-DS are reviewed here.
Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice toward the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p < 0.01) differences in hepatic gene and lincRNA expression in vaccination-protected vs. non-vaccinated mice toward the end of crisis phase. In vaccination-protected mice, infections induce up-regulations of 276 genes and 40 lincRNAs and down-regulations of 200 genes and 43 lincRNAs, respectively, by >3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by <100- and >10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria.
Anaemia or decreased blood haemoglobin is the most common blood disorder often characterized by reduced red blood cell (RBC) numbers. RBCs are produced from differentiation and commitment of haematopoietic stem cells to the erythroid lineage by a process called erythropoiesis. Coordination of erythropoietin receptor signalling with several erythroid transcription factors including GATA1 is essential for this process. A number of additional players that are critical for RBC production have been identified in recent years. Major technological advances, such as the development of RNA interference, genetically modified animals, including zebrafish, and imaging flow cytometry have led to these discoveries; the emergence of -omics approaches in combination with the optimization of ex vivo erythroid cultures have also produced a more comprehensive understanding of erythropoiesis. Here we summarize studies describing novel regulators of erythropoiesis that modulate erythroid cell production in the context of human erythroid disorders involving hypoxia, iron regulation, immune-related molecules, and the transcription factor FOXO3.
The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.
Children with Down syndrome (DS) are at increased risk of developing distinctive clonal myeloid disorders, including transient abnormal myelopoiesis (TAM) and myeloid leukaemia of DS (ML-DS). TAM connotes a spontaneously resolving congenital myeloproliferative state observed in 10%-20% of DS newborns. Following varying intervals of apparent remission, a proportion of children with TAM progress to develop ML-DS in early childhood. Therefore, TAM and ML-DS represent a biological continuum. Both disorders are characterised by recurring truncating somatic mutations of the GATA1 gene, which are considered key pathogenetic events.
We herein report, to our knowledge, the first observation on the frequency and nature of GATA1 gene mutations in a cohort of Malaysian children with DS-associated TAM (n = 9) and ML-DS (n = 24) encountered successively over a period of five years at a national referral centre.
Of the 29 patients who underwent GATA1 analysis, GATA1 mutations were observed in 15 (51.7%) patients, including 6 (75.0%) out of 8 patients with TAM, and 9 (42.9%) of 21 patients with ML-DS. All identified mutations were located in exon 2 and the majority were sequence-terminating insertions or deletions (66.7%), including several hitherto unreported mutations (12 out of 15).
The low frequency of GATA1 mutations in ML-DS patients is unusual and potentially indicates distinctive genomic events in our patient cohort.
The dynamic and reversed expression of GATA1 and GATA2 are essential for proper erythroid differentiation. Our previous work demonstrates that LSD1, a histone H3K4 demethylase, represses GATA2 expression at late stage of erythroid differentiation. K562 and MEL cells were used and cultured in Roswell Park Memorial Institute-1640 medium (RPMI) and Dulbecco's modified Eagle's medium (DMEM), respectively. Western blot assay was used to examine the GATA1, GATA2, TAL1, HDAC1, HDAC2, CoREST and β-actin protein. The immunoprecipitation assay and GST pull-down assay were employed to detect the precipitated protein complexes and investigate the interaction between the proteins. The small interfering RNA (siRNA) and nonspecific control siRNA were synthesized to silence the target genes. Double fluorescence immunostaining was used to observe the association of LSD1 with GATA2 in K562 cells. The results indicated that knockdown of LSD1 in K562 cell causes increased H3K4 di-methylation at GATA1 locus and activates GATA1 expression, demonstrating that LSD1 represses GATA1 expression through LSD1-mediated histone demethylation. Upon induced erythroid differentiation of K562 cells, the interaction between GATA2 and LSD1 is decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 is increased, which forms a complex that efficiently suppresses GATA2 expression. In conclusion, these observations reveal an elegant mechanism to modulate GATA1 and GATA2 expression during erythroid differentiation. While LSD1 mainly forms complex with GATA2 to repress GATA1 expression in hematopoietic progenitor cells, it mostly forms complex with TAL1 to repress GATA2 expression in differentiated cells.
To explore the influence of GATA-1 on expression of EpoR in bone marrow CD71+ cells of rat model with high altitude polycythemia (HAPC).
Forty-eight male SD rats were randomly divided into normal control and HAPC model group. HAPC model was established at the altitude of 4 300 meters in the natural environment, and verified by bone marrow cell counts and hematological parameters. Myeloid CD71+ cells were separated by the density gradient centrifugation combined with magnetic activated cell sorting. The expression of EpoR on cell membrane was detected by flow cytometry and cell immunofluorescence. The expression changes of GATA-1 and EpoR mRNA and protein were detected by Q-PCR and Western blot, respectively. CD71+ cells were cultured under normoxia and hypoxia, respectively. After transfection for 96 h, the optimal interference sequence GATA-1 shRNA1 was selected. And the mRNA and protein expression level of GATA-1 and EpoR were detected by Q-PCR and Western blot respectively.
The animal model with HAPC was established successfully and comfirmed by the bone marrow cell counting and the hematologic parameters in comparison with that of the normal control. EpoR expression on the myeloid CD71+ cell membrane in HAPC group was significantly higher than that in normal control (P<0.05). The expression of GATA-1 and EpoR in myeloid CD71+ cells of HAPC group was higher than that in control group (P<0.05). The mRNA and protein expression of GATA-1 and EpoR in two groups positively correlated (control group, r=0.929, P<0.01, r=0.802, P<0.05; HAPC group, r=0.822, P<0.05, r=0.839, P<0.01). However, the mRNA and protein expression of EpoR at normoxia and hypoxia was significantly lower than that in negative control group after interfernce with GATA-1 shRNA1 for 96 h (P<0.05). And the expression of GATA-1 and EpoR under hypoxia was higher than that in normoxia.
The effect of GATA-1 on EpoR expression may be correlated with the pathogenesis of HAPC.
Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by a paucity of erythroid progenitors. We summarized the clinical and genetic features of 104 DBA patients in a single-center retrospective study in China. Data of DBA patients who received consultations at our center from 2003 to 2015 were analyzed retrospectively. Genes encoding 10 ribosomal proteins (RPs) and GATA1 were sequenced for mutation detection. Our cohort was composed of 65 males and 39 females. Congenital malformations were observed in 19 patients. Mutations of the RP genes were detected in 58.3 % patients. Twenty different mutations were first reported. Thirty-four patients received prednisone combined with CsA therapy, and improvement was observed in 20 cases. During follow-up for a median 39 months, 33.7 % of the patients achieved remission, 41.3 % of the patients were persistently transfusion independent, 21.7 % of the patients were transfusion dependent, and three patients died. The patient group with detected mutations had a younger age of disease onset, a higher malformation rate, and tended to have a lower remission rate and a higher transfusion-dependence rate. Prednisone in combination with cyclosporine A can be a second-line choice for DBA patients. Differences were detected between DBA patients with and without detectable mutations in the genes studied.
Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.
The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice in vivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.
GATA1 mutations and trisomy 21 are inextricably linked in the neonatal leukemia of children with Down syndrome (DS). A recent report by Banno et al. (2016) sheds new light on the mechanism of the synergy between these genetic alterations by modeling hematopoietic abnormalities with patient-derived induced pluripotent stem cells.
During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.
Endothelial cell (EC) apoptosis plays a vital role in the pathogenesis of atherosclerosis in patients with diabetes mellitus (DM), but the underlying mechanism remains unclear. Cellular repressor of E1A-stimulated genes (CREG) is a novel gene reported to be involved in maintaining the homeostasis of ECs. Therefore, in the present study, we investigated the role of CREG in high glucose/high palmitate-induced EC apoptosis and to decipher the upstream regulatory mechanism underlying the transcriptional regulation of CREG.
The expression of CREG and the rate of apoptosis were assessed in lower-limb atherosclerotic lesions from patients with type 2 DM (T2DM). Primary human umbilical vein endothelial cells (HUVECs) were isolated and cultured in a high glucose/high palmitate medium (25 mmol/L D-glucose, 0.4 mmol/L palmitate), and the over-expression and knock-down of CREG were performed in HUVECs to determine the role of CREG in EC apoptosis. The upstream regulatory mechanism of CREG was identified using a promoter-binding transcription-factor profiling array, chromatin immunoprecipitation (ChIP) assay and a mutation analysis.
Compared with normal arteries from non-diabetic patients, reduced CREG expression and increased apoptosis were found in the endothelium of atherosclerotic lesions from patients with T2DM. In vitro treatment of HUVECs with a high glucose/high palmitate medium also resulted in decreased CREG expression and increased apoptosis. Moreover, high glucose/high palmitate induced-HUVEC apoptosis was increased by the knock-down of CREG and rescued by the over-expression of CREG. We also demonstrated that GATA1 was able to bind to the promoter of the human CREG gene. A deletion mutation at -297/-292 in the CREG promoter disrupted GATA1 binding and reduced the activation of CREG transcription by approximately 83.3%. Finally, the overexpression of GATA1 abrogated the high glucose/high palmitate-induced apoptosis in HUVECs.
The over-expression of CREG inhibits high glucose/high palmitate-induced apoptosis in HUVECs. CREG is transcriptionally upregulated by GATA1. Thus, CREG might be a potential therapeutic target for intervention of vascular complications related to diabetes.
SCL, a transcription factor of the basic helix-loop-helix family, is a master regulator of hematopoiesis. Scl specifies lateral plate mesoderm to a hematopoietic fate and establishes boundaries by inhibiting the cardiac lineage. A combinatorial interaction between Scl and Vegfa/Flk1 sets in motion the first wave of primitive hematopoiesis. Subsequently, definitive hematopoietic stem cells (HSCs) emerge from the embryo proper via an endothelial-to-hematopoietic transition controlled by Runx1, acting with Scl and Gata2. Past this stage, Scl in steady state HSCs is redundant with Lyl1, a highly homologous factor. However, Scl is haploinsufficient in stress response, when a rare subpopulation of HSCs with very long term repopulating capacity is called into action. SCL activates transcription by recruiting a core complex on DNA that necessarily includes E2A/HEB, GATA1-3, LIM-only proteins LMO1/2, LDB1, and an extended complex comprising ETO2, RUNX1, ERG, or FLI1. These interactions confer multifunctionality to a complex that can control cell proliferation in erythroid progenitors or commitment to terminal differentiation through variations in single component. Ectopic SCL and LMO1/2 expression in immature thymocytes activates of a stem cell gene network and reprogram cells with a finite lifespan into self-renewing preleukemic stem cells (pre-LSCs), an initiating event in T-cell acute lymphoblastic leukemias. Interestingly, fate conversion of fibroblasts to hematoendothelial cells requires not only Scl and Lmo2 but also Gata2, Runx1, and Erg, indicating a necessary collaboration between these transcription factors for hematopoietic reprogramming. Nonetheless, full reprogramming into self-renewing multipotent HSCs may require additional factors and most likely, a permissive microenvironment.
Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis.
The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of β-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.
Molecular oxygen sustains aerobic life, but it also serves as the substrate for oxidative stress, which has been associated with the pathogenesis of disease and with aging. Compared with mice housed in normoxia (21% O2), reducing ambient oxygen to 10% O2 (hypoxia) resulted in increased hematopoietic stem cell (HSC) function as measured by bone marrow (BM) cell engraftment onto lethally irradiated recipients. The number of BM c-Kit(+)Sca-1(+)Lin(-) (KSL) cells as well as the number of cells with other hematopoietic stem and progenitor cell markers were increased in hypoxia mice, whereas the BM cells' colony-forming capacity remained unchanged. KSL cells from hypoxia mice showed a decreased level of oxidative stress and increased expression of transcription factor Gata1 and cytokine receptor c-Mpl, consistent with the observations of increased erythropoiesis and enhanced HSC engraftment. These observations demonstrate the benefit of a hypoxic HSC niche and suggest that hypoxic conditions can be further optimized to preserve stem cell integrity in vivo.
Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.
Leukemia often results in severe anemia, which may significantly contribute to patient mortality and morbidity. However, the mechanisms underlying defective erythropoiesis in leukemia have not been fully elucidated. In this study, we demonstrated that insufficient erythropoiesis in an immunocompetent acute myeloid leukemia (AML) murine model was due to reduced proliferation of megakaryocyte erythroid progenitors and increased apoptosis of erythroblasts. Colony-forming cell assays indicated that the leukemic bone marrow (BM) plasma inhibited erythroid colony formation, whereas they had no inhibitory effect on other types of colonies. Cytokine array analysis demonstrated that the chemokine CCL3 was elevated in the plasma of AML mice and patients. CCL3 inhibited erythroid differentiation of hematopoietic stem cells, common myeloid progenitors and especially megakaryocytic-erythroid progenitors. Administration of the CCR1 antagonist partially recovered the yield of erythroid colonies in the presence of CCL3 or leukemic BM plasma. Mechanistically, we observed an increase of p38 phosphorylation and subsequent downregulation of GATA1 after CCL3 treatment. Furthermore, knockdown of CCL3 attenuated leukemic progression and alleviated anemia. Therefore, our results demonstrate that elevated CCL3 in the leukemic environment suppresses erythropoiesis via CCR1-p38 activation, suggesting a novel mechanism for the erythroid defects observed in leukemia.
Acute erythroid leukemia (AEL) is characterized by lower incidence, poorer prognosis and worse survival than other types of leukemia and results from collaboration of malignant proliferation and erythroid differentiation blockage. The expression, function and therapeutic significance of noncoding RNAs in AEL have not been well studied. Here, we show that one miRNA cluster, including miR-23a, -27a and -24, is dramatically downregulated in AEL patients. Restoration of miR-23a, -27a and -24 expression induces apoptosis and erythropoiesis, inhibits adverse growth and partly relieves the leukemic symptoms of AEL patients. At the whole-genome scale, we identify that miR-23a, -27a and -24 synergistically target multiple members of the oncogenic gp130-JAK1-Stat3 pathway, and thus reinforce their inhibition on the cascade to regulate cell proliferation and apoptosis. Importantly, Ruxolitinib, a JAK1 inhibitor, could rescue the phenotypic changes induced by miR-23a, -27a and -24 inhibitors. Furthermore, miR-23a cluster-mediated-inactivation of the JAK1-Stat3 pathway promotes the expression and activity of GATA1 via inhibiting PU.1, thereby improving erythroid differentiation. Collectively, we reveal an important regulatory circuit comprising GATA1, the miR-23a cluster and gp130-JAK1-Stat3 pathway, that synergistically facilitates apoptosis and erythropoiesis and restrains adverse proliferation, indicating the therapeutic significance of miR-23a, -27a and -24 for AEL treatment.
The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.
Diamond-Blackfan anemia (DBA) in its classic form is characterized by a profound normochromic and usually macrocytic anemia with normal leukocytes and platelets, congenital malformations in up to 50% of affected individuals, and growth retardation in 30% of affected individuals. The hematologic complications occur in 90% of affected individuals during the first year of life. The phenotypic spectrum ranges from a mild form (e.g., mild anemia, no anemia with only subtle erythroid abnormalities, physical malformations without anemia) to a severe form of fetal anemia resulting in nonimmune hydrops fetalis. DBA is associated with an increased risk for acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), and solid tumors including osteogenic sarcoma.
The diagnosis is established in a proband when all four of the following diagnostic criteria are present: Age younger than one year. Macrocytic anemia with no other significant cytopenias. Reticulocytopenia. Normal marrow cellularity with a paucity of erythroid precursors. Other causes of bone marrow failure (e.g., Fanconi anemia, Pearson syndrome, dyskeratosis congenita, human immunodeficiency virus infection) need to be considered and ruled out as appropriate. DBA has been associated with pathogenic variants in sixteen genes that encode ribosomal proteins and in GATA1 and TSR2. A pathogenic variant in one of these eighteen genes is identified in approximately 65% of individuals with DBA.
Treatment of manifestations: Corticosteroid treatment, recommended in children older than age twelve months, initially improves the red blood cell count in approximately 80% of affected individuals. Chronic transfusion with packed red blood cells is initially necessary while the diagnosis is made and in those not responsive to corticosteroids. Hematopoietic stem cell transplantation (HSCT), the only curative therapy for the hematologic manifestations of DBA, is often recommended for those who are transfusion dependent or develop other cytopenias. Treatment of malignancies should be coordinated by an oncologist. Chemotherapy must be given cautiously as it may lead to prolonged cytopenia and subsequent toxicities. Prevention of secondary complications: Transfusion-related iron overload is the most common complication in transfusion-dependent individuals. Iron chelation therapy with deferasirox orally or desferrioxamine subcutaneously is recommended after ten to 12 transfusions. Corticosteroid-related side effects must also be closely monitored, especially as related to risk for infection, growth retardation, and loss of bone density in growing children. Often individuals will be placed on transfusion therapy if these side effects are intolerable. Surveillance: Complete blood counts several times a year; bone marrow aspirate/biopsy periodically to evaluate morphology and cellularity in the event of another cytopenia or a change in response to treatment. In steroid-dependent individuals: monitor blood pressure and (in children) growth. Agents/circumstances to avoid: Deferiprone for the treatment of iron overload, which has led to severe neutropenia in a few individuals with DBA; infection (especially those on corticosteroids). Evaluation of relatives at risk: Molecular genetic testing of at-risk relatives of a proband with a known pathogenic variant allows for early diagnosis and appropriate monitoring for bone marrow failure, physical abnormalities, and related cancers.
DBA is most often inherited in an autosomal dominant manner; GATA1-related and TSR2-related DBA are inherited in an X-linked manner. Approximately 40% to 45% of individuals with autosomal dominant DBA have inherited the pathogenic variant from a parent; approximately 55% to 60% have a de novo pathogenic variant. Each child of an individual with autosomal dominant DBA has a 50% chance of inheriting the pathogenic variant. Males with GATA1 or TSR2-related DBA pass the pathogenic variant to all of their daughters and none of their sons. Women heterozygous for a GATA1 or TSR2 pathogenic variant have a 50% chance of transmitting the pathogenic variant in each pregnancy: males who inherit the pathogenic variant will be affected; females who inherit the pathogenic variant will be carriers and will usually not be affected. Carrier testing of at-risk female relatives is possible if the GATA1 or TSR2 pathogenic variant has been identified in the family. Prenatal testing for pregnancies at increased risk is possible if the familial pathogenic variant has been identified.
Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.
Cytogenetic analyses of a consecutive series of 67 paediatric (median age 8 years; range 0-17) de novo acute myeloid leukaemia (AML) patients revealed aberrations in 55 (82%) cases. The most common subgroups were KMT2A rearrangement (29%), normal karyotype (15%), RUNX1-RUNX1T1 (10%), deletions of 5q, 7q and/or 17p (9%), myeloid leukaemia associated with Down syndrome (7%), PML-RARA (7%) and CBFB-MYH11 (5%). Single nucleotide polymorphism array (SNP-A) analysis and exon sequencing of 100 genes, performed in 52 and 40 cases, respectively (39 overlapping), revealed ≥1 aberration in 89%; when adding cytogenetic data, this frequency increased to 98%. Uniparental isodisomies (UPIDs) were detected in 13% and copy number aberrations (CNAs) in 63% (median 2/case); three UPIDs and 22 CNAs were recurrent. Twenty-two genes were targeted by focal CNAs, including AEBP2 and PHF6 deletions and genes involved in AML-associated gene fusions. Deep sequencing identified mutations in 65% of cases (median 1/case). In total, 60 mutations were found in 30 genes, primarily those encoding signalling proteins (47%), transcription factors (25%), or epigenetic modifiers (13%). Twelve genes (BCOR, CEBPA, FLT3, GATA1, KIT, KRAS, NOTCH1, NPM1, NRAS, PTPN11, SMC3 and TP53) were recurrently mutated. We conclude that SNP-A and deep sequencing analyses complement the cytogenetic diagnosis of paediatric AML.
Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.
Transcription factors Runx1, CBFβ and GATA1/2/3 play essential roles in regulating hematopoietic development during embryogenesis of vertebrate. In previous study, the orthologous genes of Runt, CBFβ and GATA1/2/3 have been identified from scallop Chlamys farreri and proved to have conserved function in regulating hemocyte production. Here, these three transcription factors were selected as hematopoietic markers to explore potential developmental events of hematopoiesis during ontogenesis of scallop. The transcripts of CfRunt, CfCBFβ and CfGATA were detected abundantly after 32-cell embryo, trochophore and morula stage, and reached to a peak level in 32-cell embryos and D-shaped veligers, pediveligers or gastrula respectively. Further whole-mount immunofluorescence assay showed that the immunoreactivity of CfRunt was firstly observed at 32-cell stage and then its distribution was specialized gradually to the mesoderm during gastrulation. By trochophore, the expression of CfRunt, CfCBFβ and CfGATA proteins occurred coincidently in two specific symmetry cell mass located bilaterally on prototroch, and then disappeared rapidly in D-shaped or umbonal vliger, respectively. However, remarkable expressions of the three transcription factors were observed consistently in a new sinus structure appeared at the dorsal anterior side of D-shaped and umbonal veliger. After bacterial challenge, the mRNA expression levels of the three transcription factors were up-regulated or down-regulated significantly in trochophore, D-shaped veliger and pediveliger, indicating the available hematopoietic regulation in scallop larvae. The results revealed that scallop might experience two waves of hematopoiesis during early development, which occurred in the bilateral symmetry cell mass of trochophore and the sinus structure of veliger.
GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.
Patients who survive initial burn injury are susceptible to nosocomial infections. Anemia of critical illness is a compounding factor in burn patients that necessitates repeated transfusions, which further increase their susceptibility to infections and sepsis. Robust host response is dependent on an adequate number and function of monocytes/macrophages and dendritic cells. In addition to impaired RBC production, burn patients are prone to depletion of dendritic cells and an increase in deactivated monocytes. In steady-state hematopoiesis, RBCs, macrophages, and dendritic cells are all generated from a common myeloid progenitor within the bone marrow. We hypothesized in a mouse model of burn injury that an increase in myeloid-specific transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B at the common myeloid progenitor stage steers their lineage potential away from the megakaryocyte erythrocyte progenitor production and drives the terminal fate of common myeloid progenitors to form macrophages vs. dendritic cells, with the consequences being anemia, monocytosis, and dendritic cell deficits. Results indicate that, even though burn injury stimulated bone marrow hematopoiesis by increasing multipotential stem cell production (Lin(neg)Sca1(pos)cKit(pos)), the bone marrow commitment is shifted away from the megakaryocyte erythrocyte progenitor and toward granulocyte monocyte progenitors with corresponding alterations in peripheral blood components, such as hemoglobin, hematocrit, RBCs, monocytes, and granulocytes. Furthermore, burn-induced V-maf musculoaponeurotic fibrosarcoma oncogene homolog B in common myeloid progenitors acts as a transcriptional activator of M-CSFR and a repressor of transferrin receptors, promoting macrophages and inhibiting erythroid differentiations while dictating a plasmacytoid dendritic cell phenotype. Results from small interfering RNA and gain-of-function (gfp-globin transcription factor 1 retrovirus) studies indicate that targeted interventions to restore V-maf musculoaponeurotic fibrosarcoma oncogene homolog B/globin transcription factor 1 balance can mitigate both immune imbalance and anemia of critical illness.
There is much current interest in the role of the platelet storage pool of α-granule proteins both in hemostasis and non-hemostatic events. As well as in the arrest of bleeding, the secreted proteins participate in wound healing, inflammation, and innate immunity while in pathology they may be actors in arterial thrombosis and atherosclerosis as well as cancer and metastasis. For a long time, gray platelet syndrome (GPS) has been regarded as the classic inherited platelet disorder caused by an absence of α-granules and their contents. While NBEAL2 is the major source of mutations in GPS, other gene variants may give rise to significant α-granule deficiencies in platelets. These include GATA1, VPS33B, or VIPAS39 in the arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome and now GFI1B. Nevertheless, many phenotypic differences are associated with mutations in these genes. This critical review was aimed to assess genotype/phenotype variability in disorders of platelet α-granule biogenesis and to urge caution in grouping all genetic defects of α-granules as GPS. Am. J. Hematol. 91:714-718, 2016. © 2016 Wiley Periodicals, Inc.
Congenital erythropoietic porphyria (CEP) is a rare genetic disease resulting from the remarkable deficient activity of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthetic pathway. This enzyme defect results in overproduction of the non-physiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I. The predominant clinical characteristics of CEP include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. The severity of clinical manifestations is markedly heterogeneous among patients; and interdependence between disease severity and porphyrin amount in the tissues has been pointed out. A more pronounced endogenous production of porphyrins concomitant to activation of ALAS2, the first and rate-limiting of the haem synthesis enzymes in erythroid cells, has also been reported. CEP is inherited as autosomal recessive or X-linked trait due to mutations in UROS or GATA1 genes; however an involvement of other causative or modifier genes cannot be ruled out.
The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.
Children with Down syndrome (DS) have a markedly increased risk of leukemia. They are at particular risk of acute megakaryoblastic leukemia, known as myeloid leukemia associated with DS (ML-DS), the development of which is closely linked to a preceding temporary form of neonatal leukemia called transient abnormal myelopoiesis (TAM). Findings from recent clinical and laboratory studies suggest that constitutional trisomy 21 and GATA1 mutation(s) cause TAM, and that additional genetic alteration(s) including those in epigenetic regulators and signaling molecules are involved in the progression from TAM to ML-DS. Thus, this disease progression represents an important model of multi-step leukemogenesis. The present review focuses on the evolutionary process of TAM to ML-DS, and advances in the understanding of perturbed hematopoiesis in DS with respect to GATA1 mutation and recent findings, including cooperating genetic events, are discussed.
Invertebrates rely on the efficient innate immune mechanisms against invaders, in which the continuous production of hemocytes (hematopoiesis) is indispensable. In the present study, the hematopoietic tissue (HPT) from Chinese mitten crab Eriocheir sinensis was identified and characterized. It was a thin and non-transparent sheet located at the dorsolateral side of the stomach, which was composed of a series of ovoid lobules. Each lobule was surrounded by connective tissue containing a large amount of spherical cells with big nucleus. In HPT, the cells were full of mitochondria and granules, and DNA replication was detected in some cells by EdU labeling technique. Cell proliferation was observed in HPT by transmission electron microscope (TEM). The distribution of two transcription factors, GATA1 and RUNX1, were examined by human GATA1 and RUNX1 antibodies, respectively. Three homologues of RUNX1 were detected in the HPT while no signal of RUNX1 was observed in hemocytes, and GATA1 was detected in both HPT and some hemocytes. The mRNA transcript of a novel hematopoiesis related cytokine EsAst was detected in hepatopancreas and hemocytes, but it was no detectable in HPT. The mRNA expression level of EsAst in hepatopancreas was 1.38-fold higher than that in hemocytes. Total hemocytes counts were related to the mRNA expression level of EsAst post Aeromonas hydrophila challenge. The results suggested that the stem cells in the hematopoietic tissue of Chinese mitten crab E. sinensis were regulated by transcriptional and humoral factors to generate hemocytes.
Today the regulatory role of microRNAs (miRs) is well characterized in many diverse cel- lular processes. MiR-based regulation is categorized under epigenetic regulatory mecha- nisms. These small non-coding RNAs participate in producing and maturing erythrocytes, expressing hematopoietic factors and regulating expression of globin genes by post-tran- scriptional gene silencing. The changes in expression of miRs (miR-144/-320/-451/-503) in thalassemic/sickle cells compared with normal erythrocytes may cause clinical severity. According to the suppressive effects of certain miRs (miR-15a/-16-1/-23a/-26b/-27a/-451) on a number of transcription factors [myeloblastosis oncogene (MYB), B-cell lymphoma 11A (BCL11A), GATA1, Krüppel-like factor 3 (KLF3) and specificity protein 1 (Sp1)] during β globin gene expression, It has been possible to increasing γ globin gene expression and fetal hemoglobin (HbF) production. Therefore, this strategy can be used as a novel therapy in infusing HbF and improving clinical complications of patients with hemoglobi- nopathies.
Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF) is the most important regulator of tumor angiogenesis. However, how transcription factors interact with histone-modifying enzymes to regulate VEGF transcription and tumor angiogenesis remains unclear. Here, we show that transcription factor GATA1 associates with the histone methyltransferase SET7 to promote VEGF transcription and breast tumor angiogenesis. Using chromatin immunoprecipitation assay, we found that GATA1 was required for recruitment of SET7, RNA polymerase II and transcription factor II B to VEGF core promoter. GATA1 enhanced breast cancer cell (MCF7, ZR75-1 and MDA-MB-231)-secreted VEGF via SET7, which promoted vascular endothelial cell (HUVEC) proliferation, migration and tube formation. SET7 was required for GATA1-induced breast tumor angiogenesis and growth in nude mice. Immunohistochemical staining showed that expression of GATA1 and SET7 was upregulated and positively correlated with VEGF expression and microvessel number in 80 breast cancer patients. GATA1 and SET7 are independent poor prognostic factors in breast cancer. Our data provide novel insights into VEGF transcriptional regulation and suggest GATA1/SET7 as cancer therapeutic targets.
Children with Down syndrome (DS) have a significantly increased risk of childhood leukemia, in particular acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (DS-ALL). A pre-leukemia, called transient myeloproliferative disorder (TMD), characterised by a GATA binding protein 1 (GATA1) mutation, affects up to 30% of newborns with DS. In most cases, the pre-leukemia regresses spontaneously, however one-quarter of these children will go on to develop AMKL or myelodysplastic syndrome (MDS) . AMKL and MDS occurring in young children with DS and a GATA1 somatic mutation are collectively termed myeloid leukemia of Down syndrome (ML-DS). This model represents an important multi-step process of leukemogenesis, and further study is required to identify therapeutic targets to potentially prevent development of leukemia. DS-ALL is a high-risk leukemia and mutations in the JAK-STAT pathway are frequently observed. JAK inhibitors may improve outcome for this type of leukemia. Genetic and epigenetic studies have revealed likely candidate drivers involved in development of ML-DS and DS-ALL. Overall this review aims to identify potential impacts of new research on how we manage children with DS, pre-leukemia and leukemia.
Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.
In hematopoietic system development, PU.1 and GATA-1 as lineage-specific transcription factors (TF) are expressed in common myeloid progenitors. The cross antagonism between them ascertains gene expression programs of monocytic and erythroid cells, respectively. This concept in transdifferentiation approaches has not been well considered yet, especially in intralineage conversion systems. To demonstrate whether PU.1 suppression induces monocyte lineage conversion into red blood cells, a combination of three PU.1-specific siRNAs was implemented to knock down PU.1 gene expression and generate the balance in favor of GATA-1 expression to induce erythroid differentiation. For this purpose, monocytes were isolated from human peripheral blood and transfected by PU.1 siRNAs. In transfected monocytes, the rate of PU.1 expression in mRNA level was significantly decreased until 0.38 ± 0.118 when compared to untreated monocytes at 72 h (p value ≤0.05) which resulted in significant overexpression of GATA1 of 16.1 ± 0.343-fold compared to the untreated group (p value ≤0.01). Subsequently, overexpression of hemoglobin (α 13.26 ± 1.34-fold; p value≤0.0001) and β-globin (37.55 ± 16.56-fold; p value≤0.0001) was observed when compared to control groups. The results of western immunoblotting confirm those findings too. While, reduced expression of monocyte, CD14 gene, was observed in qRT-PCR and flow cytometry results. Our results suggest that manipulating the ratio of the two TFs in bifurcation differentiation pathways via applying siRNA technology can possibly change the cells' fate as a safe way for therapeutics application.
Leukemia arises through an evolutionary process of somatic mutation and selection. Transient abnormal myelopoiesis (TAM) is a clonal pre-leukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism underlying leukemogenesis, a xenograft model of TAM was established using NOG mice. Serial engraftment after cell transplantation from a TAM patient who developed ML-DS a year later showed the self-renewal capacity of these cells. We detected a GATA1 mutation but no copy number alterations (CNAs) in the primary patient sample by conventional genomic sequencing and CNA profiling. However, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, both of which are clinically associated with ML-DS. Detailed genetic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with various leukemia-initiating potentials already exist in the neonatal TAM phase, and that ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model could be a valuable tool for gaining insight into the processes underlying leukemogenesis.
We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.
During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.
SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish.
Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation.
Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.
Acute myeloid leukemia (AML) 1-Evi-1 is a chimeric gene generated by the t (3; 21) (q26; q22) translocation, which leads into malignant transformation of hematopoietic stem cells by unclear mechanisms. This in vivo study aimed to establish a stable line of zebrafish expressing the human RUNX1-Evi-1 fusion gene under the control of a heat stress-inducible bidirectional promoter, and investigate its roles in hematopoiesis and hematologic malignancies.
We introduced human RUNX1-Evi-1 fusion gene into embryonic zebrafish through a heat-shock promoter to establish Tg(RE:HSE:EGFP) zebrafish. Two males and one female mosaic F0 zebrafish embryos (2.1%) were identified as stable positive germline transgenic zebrafish.
The population of immature myeloid cells and hematopoietic blast cells were accumulated in peripheral blood and single cell suspension from kidney of adult Tg(RE:HSE:EGFP) zebrafish. RUNX1-Evi-1 presented an intensive influence on hematopoietic regulatory factors. Consequently, primitive hematopoiesis was enhanced by upregulation of gata2 and scl, while erythropoiesis was downregulated due to the suppression of gata1. Early stage of myelopoiesis was flourishing with the high expression of pu.1, but it was inhibited along with the low expression of mpo. Microarray analysis demonstrated that RUNX1-Evi-1 not only upregulated proteasome, cell cycle, glycolysis/gluconeogenesis, tyrosine metabolism, drug metabolism, and PPAR pathway, but also suppressed transforming growth factor β, Jak-STAT, DNA replication, mismatch repair, p53 pathway, JNK signaling pathway, and nucleotide excision repair. Interestingly, histone deacetylase 4 was significantly up-regulated. Factors in cell proliferation were obviously suppressed after 3-day treatment with histone deacetylase inhibitor, valproic acid. Accordingly, higher proportion of G1 arrest and apoptosis were manifested by the propidium iodide staining.
RUNX1-Evi-1 may promote proliferation and apoptosis resistance of primitive hematopoietic cell, and inhibit the differentiation of myeloid cells with the synergy of different pathways and factors. VPA may be a promising choice in the molecular targeting therapy of RUNX1-Evi-1-related leukemia.
Recent reports of recurrent mutations in childhood acute myeloid leukemia (AML) have identified potential targets for new therapeutic strategies. Acute promyelocytic leukemia (APL) is characterized commonly by a fusion between the PML gene and the RARA gene, genes targetable by arsenic (ATO) and retinoic acid (ATRA), respectively. A mutation in GATA1, common in AML of Down syndrome (ML-DS), renders cells more susceptible to cytarabine and anthracyclines, thus permitting targeted dose reductions to preserve high survival rates while reducing toxicity. In all other patients, Ras pathway mutations, KMT2A and other methyltransferase mutations, FLT3 mutations, and KIT mutations are all relatively common in childhood AML and all are potentially "druggable". The focus of this review is on those therapies likely to be clinically available in the near future. The preclinical and clinical data providing a rationale for testing in children of specific agents in children is discussed. Whether the expression of a potential target is sufficient to predict response to a targeted therapy is an open question in childhood AML. Development of clinical trials to evaluate targeted therapies in small molecularly defined subsets of AML will be the next great challenge for all cooperative groups in North America and Europe.
RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.
Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for β-thalassemia (β-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors' binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for β-thal patients.
To investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism.
The fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, β-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, β-tubulin and vimentin protein expression levels.
Benzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, β-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, β-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, β-tubulin and vimentin in the cells.
Simulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.
The hemangioblast is a progenitor cell with the capacity to give rise to both hematopoietic and endothelial progenitors. Currently, the regulatory mechanisms underlying hemangioblast formation are being elucidated, whereas those controllers for the selection of hematopoietic or endothelial fates still remain a mystery. To answer these questions, we screened for zebrafish mutants that have defects in the hemangioblast expression of Gata1, which is never expressed in endothelial progenitors. One of the isolated mutants, it627, showed not only down-regulation of hematopoietic genes but also up-regulation of endothelial genes. We identified the gene responsible for the it627 mutant as the zebrafish homolog of Lys-specific demethylase 1 (LSD1/KDM1A). Surprisingly, the hematopoietic defects in lsd1(it627) embryos were rescued by the gene knockdown of the Ets variant 2 gene (etv2), an essential regulator for vasculogenesis. Our results suggest that the LSD1-dependent shutdown of Etv2 gene expression may be a significant event required for hemangioblasts to initiate hematopoietic differentiation.
ChIP-seq experiments are widely used to detect and study DNA-protein interactions, such as transcription factor binding and chromatin modifications. However, downstream analysis of ChIP-seq data is currently restricted to the evaluation of signal intensity and the detection of enriched regions (peaks) in the genome. Other features of peak shape are almost always neglected, despite the remarkable differences shown by ChIP-seq for different proteins, as well as by distinct regions in a single experiment.
We hypothesize that statistically significant differences in peak shape might have a functional role and a biological meaning. Thus, we design five indices able to summarize peak shapes and we employ multivariate clustering techniques to divide peaks into groups according to both their complexity and the intensity of their coverage function. In addition, our novel analysis pipeline employs a range of statistical and bioinformatics techniques to relate the obtained peak shapes to several independent genomic datasets, including other genome-wide protein-DNA maps and gene expression experiments. To clarify the meaning of peak shape, we apply our methodology to the study of the erythroid transcription factor GATA-1 in K562 cell line and in megakaryocytes.
Our study demonstrates that ChIP-seq profiles include information regarding the binding of other proteins beside the one used for precipitation. In particular, peak shape provides new insights into cooperative transcriptional regulation and is correlated to gene expression.
Erythroid development and differentiation from multiprogenitor cells into red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, cooperate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are juxtaposed along the DNA and their cognate DNA binding site across the mouse genome remains unclear. We applied high-resolution ChIP-exo (chromatin immunoprecipitation followed by 5'-to-3' exonuclease treatment and then massively parallel DNA sequencing) to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak pairing, were used to determine high-confidence binding locations by ChIP-exo. We identified ∼10,000 GATA1 and ∼15,000 TAL1 locations, which were essentially confirmed by ChIP-seq (chromatin immunoprecipitation followed by massively parallel DNA sequencing). Of these, ∼4,000 locations were bound by both GATA1 and TAL1. About three-quarters of them were tightly linked to a partial E-box located 7 or 8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs that reflect their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7 or 8 bp upstream of a WGATAA motif across thousands of genomic locations.
Thalidomide was shown to stimulate erythropoiesis and increase hemoglobin level in multiple myeloma patients, but way of such activity remains unclear. The aim of the study was to investigate the mechanisms of thalidomide stimulating effect on erythroid differentiation.
Hematopoietic stem cells were isolated from bone marrow aspirates taken from myeloma patients and cultured with or without thalidomide. Then the generation of erythroid cells and the expression of STAT5, GATA-1, GATA-2, selected caspases and Bcl-2 family proteins in erythroid cells were assessed using flow cytometry and real-time PCR.
The generation of erythroblasts was higher in thalidomide than in control cultures (63.9% vs. 55.8%, p < 0.001). The expression of caspase 3 (cytometry 947.3 vs. 1021.0, p = 0.025; PCR 12.9 vs. 16.3, p = 0.025) and caspase 8 (cytometry 1050.8 vs. 1168.5, p = 0.033; PCR 16.2 vs. 17.8, p = 0.004) was significantly lower in thalidomide than in control cultures. The expression of STAT5 (cytometry 331.5 vs. 276.1, p = 0.015; PCR 24.3 vs. 21.1, p = 0.003) and GATA-1 (cytometry 259.7 vs. 232.0, p = 0.027; PCR 18.9 vs. 16.5, p = 0.003) was higher in thalidomide than in control cultures.
Our results suggest that thalidomide enhances expression of STAT5 in response of erythroid cells to erythropoietin and as a result of caspase 3 suppression. Moreover it may exert inhibitory effect on an external pathway of caspases activation with consequent decreased degradation of GATA-1 transcription factor by downstream caspases.
Because of an extra copy of the Aβ precursor protein gene on chromosome 21, Down syndrome (DS) individuals develop high levels of Aβ peptides and Alzheimer disease-like brain amyloidosis early in life. Here we show that the γ-secretase activating protein (GSAP), a key enzyme in amyloidogenesis, is increased in DS brains and specifically regulated at the transcriptional level by GATA1 transcription factor. The discovery of this novel pathway has translational implications for DS, because pharmacological inhibition of GSAP is an attractive and viable Aβ-lowering therapeutic strategy for this disorder.
The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies.
Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-β release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-β content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-β reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-β-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-β content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.
The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and β-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.
The generation of blood cellular components from hematopoietic stem cells is important for the therapy of a broad spectrum of hematological disorders. In recent years, several lines of evidence suggested that certain nutrients, vitamins and flavonoids may have important roles in controlling the stem cell fate decision by maintaining their self-renewal or stimulating the lineage-specific differentiation. In this study, main olive leaf phytochemicals oleuropein (Olp), apigenin 7-glucoside (Api7G) and luteolin 7-glucoside (Lut7G) were investigated for their potential effects on hematopoietic stem cell differentiation using both phenotypic and molecular analysis. Oleuropein and the combination of the three compounds enhanced the differentiation of CD34+ cells into myelomonocytic cells and lymphocytes progenitors and inhibited the commitment to megakaryocytic and erythroid lineages. Treatment with Lut7G stimulated both the erythroid and the myeloid differentiation, while treatment with Api7G specifically induced the differentiation of CD34+ cells towards the erythroid lineage and inhibited the myeloid differentiation. Erythroid differentiation induced by Api7G and Lut7G treatments was confirmed by the increase in hemoglobin genes expressions (α-hemoglobin, β-hemoglobin and γ-hemoglobin) and erythroid transcription factor GATA1 expression. As revealed by microarray analysis, the mechanisms underlying the erythroid differentiation-inducing effect of Api7G on hematopoietic stem cells involves the activation of JAK/STAT signaling pathway. These findings prove the differentiation-inducing effects of olive leaf compounds on hematopoietic stem cells and highlight their potential use in the ex vivo generation of blood cells.
FcεRI, which is composed of α, β, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-β1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-β1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-β/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIβ, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIβ, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-β1 was mimicked by forced expression of Ehf, suggesting that TGF-β1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.
Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression.
The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-β signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-β superfamily revealed signs of positive selection in TGF-β1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-β signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations. Transcriptome analysis revealed the striking dysregulation of the transforming growth factor β (TGFβ) signaling pathway in DBA lines. Expression of TGFβ target genes, such as TGFBI, BAMBI, COL3A1 and SERPINE1 was significantly increased in the DBA iPSCs. We quantified intermediates in canonical and non-canonical TGFβ pathways and observed a significant increase in the levels of the non-canonical pathway mediator p-JNK in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. Surprisingly, nuclear levels of SMAD4, a mediator of canonical TGFβ signaling, were decreased in DBA cells due to increased proteolytic turnover. We also observed the up-regulation of TGFβ1R, TGFβ2, CDKN1A and SERPINE1 mRNA, and the significant decrease of GATA1 mRNA in the primitive multilineage progenitors. In summary our observations identify for the first time a dysregulation of the TGFβ pathway in the pathobiology of DBA.
Although Down syndrome (DS) patients have a higher risk of developing transient myeloproliferative disorder (TMD) and acute leukemia, very little data is available on long-term outcome in Taiwanese patients. The current study was designed to determine the clinical characteristics and treatment outcome of DS patients with TMD or acute leukemia (AL).
In 25 consecutive DS patients with TMD or AL enrolled from 1990 to 2012, clinical manifestations and treatment protocols were investigated and GATA1 (GATA binding protein 1) mutations were identified. Among 16 DS-acute myeloid leukemia (DS-AML) patients, clinical outcomes were compared between survivors and nonsurvivors.
Most of our DS patients had TMD (32%), acute megakaryoblastic leukemia (24%), or acute erythromegakaryoblastic leukemia (16%). The median follow-up time was 22.5 months (1-230 months). The age was younger and the hemoglobin (Hb) level and platelet count were higher in TMD patients than in leukemia patients. Among DS-AML patients, the Hb level was higher in survivors than nonsurvivors (8.8 ± 2.7 g/dL vs. 5.8 ± 2.4 g/dL; p = 0.044) and the age was older in relapsed patients than in nonrelapsed patients (43.8 ± 18 months old vs. 21.6 ± 8.6 months old; p = 0.025). The 3-year overall survival (OS) rate was 44%, higher in patients receiving appropriate chemotherapy than in those receiving inadequate treatment (63.6% vs. 0%, p = 0.001), and higher in those diagnosed with TMD or AL after 2008 than before 2008 (33.3% vs. 75%; p = 0.119).
Outcome in DS-AML patients is optimal if appropriate treatment is provided. With modification of the treatment strategy in 2008, OS increased in Taiwan.
Acute megakaryocytic leukemia (AMegL) is a biologically heterogenous subtype of acute myeloid leukemia (AML) that arises from megakaryocytes. Improvements in the accuracy of diagnosing AMegL as well as interest in the molecular analysis of leukemias have led to an increased amount of data available on this rare AML subtype. In this review, we will analyze the diverse molecular features unique to AMegL and how they have influenced the development of novel treatment strategies, including polyploidization. The review will also consider the data available on clinical outcomes in AMegL and how it is a poor individual prognostic factor for AML. Finally, the role of allogeneic hematopoietic stem cell transplant in AMegL will be explored.
Children with trisomy 21 are prone to developing hematologic disorders, including transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). The papulovesicular eruption of TMD provides an important clue to the diagnosis. In contrast, AMKL rarely has associated cutaneous findings. We report the case of a 22-month-old child with trisomy 21 who presented with the acute onset of diffusely scattered and crusted papules, plaques, and vesicles. A thorough infectious evaluation was negative and the patient was unresponsive to empiric antibiotic and antiinflammatory therapies. Complete blood count (CBC) was notable for mild pancytopenia, with a normal peripheral smear. Two weeks later he was reassessed and found to have a population of blasts on repeat CBC. Subsequent evaluation ultimately led to a diagnosis of AMKL. This is the first reported case of a cutaneous eruption in a young child with Down syndrome and transformed AMKL. When children with trisomy 21 present with the acute onset of crusted papules and vesicles that cannot be accounted for by an infectious etiology, a diagnosis of AMKL should be considered even in the absence of a history of TMD.
Acute megakaryoblastic leukemia (AMKL) comprises between 4% and 15% of newly diagnosed pediatric acute myeloid leukemia patients. AMKL in children with Down syndrome (DS) is characterized by a founding GATA1 mutation that cooperates with trisomy 21, followed by the acquisition of additional somatic mutations. In contrast, non-DS-AMKL is characterized by chimeric oncogenes consisting of genes known to play a role in normal hematopoiesis. CBFA2T3-GLIS2 is the most frequent chimeric oncogene identified to date in this subset of patients and confers a poor prognosis.
A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied.
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS that has been linked with defects in regulatory T cell function. Therefore, strategies to selectively target pathogenic cells via enhanced regulatory T cell activity may provide therapeutic benefit. Kv1.3 is a voltage-gated potassium channel expressed on myelin-reactive T cells from MS patients. Kv1.3-knockout (KO) mice are protected from experimental autoimmune encephalomyelitis, an animal model of MS, and Kv1.3-KO Th cells display suppressive capacity associated with increased IL-10. In this article, we demonstrate that myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells exhibit a unique regulatory phenotype characterized by high CD25, CTLA4, pSTAT5, FoxO1, and GATA1 expression without a corresponding increase in Foxp3. These phenotypic changes result from increased signaling through IL-2R. Moreover, myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells can ameliorate experimental autoimmune encephalomyelitis following transfer to wild-type recipients in a manner that is partially dependent on IL-2R and STAT5 signaling. The present study identifies a population of Foxp3(-) T cells with suppressive properties that arises in the absence of Kv1.3 and enhances the understanding of the molecular mechanism by which these cells are generated. This increased understanding could contribute to the development of novel therapies for MS patients that promote heightened immune regulation.
Myeloid malignancy with Down syndrome (ML-DS) is estimated to have a step-wise leukemogenesis including GATA1 mutation. Trisomy 21 is essential for ML-DS; however, we do not know exactly which gene or genes located on chromosome 21 are necessary for the ML-DS. We report a female infant with transient myeloproliferative disorder (TMD) and partial trisomy 21. SNP array analysis showed 10 Mb amplification of 21q22.12-21q22.3, which included DYRK1A, ERG, and ETS but not the RUNX1 gene. With two other reported TMD cases having partial trisomy 21, DYRK1A, ERG, and ETS were the most likely genes involved in collaboration with the GATA1 mutation.
The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, β-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.
The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli.
Oogenesis is essential for female gamete production in mammals. The total number of ovarian follicles is determined early in life and production of ovarian oocytes is thought to stop during the lifetime. However, the molecular mechanisms underling oogenesis, particularly autophagy regulation in the ovary, remain largely unknown. Here, we reveal an important MYBL2-VDAC2-BECN1-BCL2L1 pathway linking autophagy suppression in the developing ovary. The transcription factors GATA1 and MYBL2 can bind to and activate the Vdac2 promoter. MYBL2 regulates the spatiotemporal expression of VDAC2 in the developing ovary. Strikingly, in the VDAC2 transgenic pigs (Sus scrofa/Ss), VDAC2 exerts its function by inhibiting autophagy in the ovary. In contrast, Vdac2 knockout promotes autophagy. Moreover, VDAC2-mediated autophagy suppression is dependent on its interactions with both BECN1 and BCL2L1 to stabilize the BECN1 and BCL2L1 complex, suggesting VDAC2 as an autophagy suppressor in the pathway. Our findings provide a functional connection among the VDAC2, MYBL2, the BECN1-BCL2L1 pathway and autophagy suppression in the developing ovary, which is implicated in improving female fecundity.
Insulin/IGF-1 signaling (IIS) pathway is known to control growth, development and reproduction. Insulin-like peptide mediated body size plasticity in Drosophila melanogaster has been reported. Here, our studies showed that IIS pathway and nutrition regulate growth and maturation of the male accessory gland (MAG) in the red flour beetle, Tribolium castaneum. The size of MAG increased from day 1 to day 5 post-adult emergence (PAE). This increase in the size of MAG is contributed by an increase in cell size, but not cell number. The growth of MAG was impaired after double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes coding for ILP3, InR, Chico, PI3k, AKT, and GATA1 involved in IIS pathway. Interestingly, starvation showed similar effects on the growth and maturation of MAG. The phenotypes observed in animals where IIS signaling pathway genes were knocked down are similar to the phenotypes observed after starving beetles for 5 days PAE. These data suggest that nutrition signals working through IIS pathway regulate maturation of MAG by promoting the growth of MAG cells.
Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.
The thrombopoietic environment of the neonate is established during prenatal life; therefore, a comprehensive understanding of platelet-forming cell development during embryogenesis is critical to understanding the etiology of early-onset thrombocytopenia. The recent discovery that the first platelet-forming cells of the conceptus are not megakaryocytes (MKs) but diploid platelet-forming cells (DPFCs) revealed a previously unappreciated complexity in thrombopoiesis. This raises important questions, including the following. When do conventional MKs appear? Do pathogenic genetic lesions of adult MKs affect DPFCs? What role does myeloproliferative leukemia virus (MPL), a key regulator of adult megakaryopoiesis, play in prenatal platelet-forming lineages? We performed a comprehensive study to determine the spatial and temporal appearance of prenatal platelet-forming lineages. We demonstrate that DPFCs originate in the yolk sac and then rapidly migrate to other extra- and intraembryonic tissues. Using gene disruption models of Gata1 and Nfe2, we demonstrate that perturbing essential adult MK genes causes an analogous phenotype in the early embryo before the onset of hematopoietic stem/progenitor cell-driven (definitive) hematopoiesis. Finally, we present the surprising finding that DPFC and MK commitment from their respective precursors is MPL independent in vivo but that completion of MK differentiation and establishment of the prenatal platelet mass is dependent on MPL expression.
Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4(+) T cell count in the setting of HIV/AIDS or primary immunodeficiency. Non-HIV-infected individuals taking immunosuppressive drug regimens targeting T cell activation are also susceptible. Given the crucial role of CD4(+) T cells in host defense against Pneumocystis, we used RNA sequencing of whole lung early in infection in wild-type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild-type mice, a strong eosinophil signature was observed at day 14 post Pneumocystis challenge, and eosinophils were increased in the bronchoalveolar lavage fluid of wild-type mice. Furthermore, eosinophilopoiesis-deficient Gata1(tm6Sho)/J mice were more susceptible to Pneumocystis infection when compared with BALB/c controls, and bone marrow-derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1(-/-) mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. The pIL5-treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden, compared with mice treated with control plasmid. In addition, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57BL/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1(tm6Sho)/J mice. Taken together, these results demonstrate that an early role of CD4(+) T cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development in the treatment of Pneumocystis pneumonia in the immunosuppressed population.
Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies.
TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included.
Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.
Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(1716)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(1716)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(1716)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.
The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility.
Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.
The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.
In vertebrates, heart pumping is required for cardiac morphogenesis and altering myocardial contractility leads to abnormal intracardiac flow forces and valve defects. Among the different mechanical cues generated in the developing heart, oscillatory flow has been proposed to be an essential factor in instructing endocardial cell fate toward valvulogenesis and leads to the expression of klf2a, a known atheroprotective transcription factor. To date, the mechanism by which flow forces are sensed by endocardial cells is not well understood. At the onset of valve formation, oscillatory flows alter the spectrum of the generated wall shear stress (WSS), a key mechanical input sensed by endothelial cells. Here, we establish that mechanosensitive channels are activated in response to oscillatory flow and directly affect valvulogenesis by modulating the endocardial cell response. By combining live imaging and mathematical modeling, we quantify the oscillatory content of the WSS during valve development and demonstrate it sets the endocardial cell response to flow. Furthermore, we show that an endocardial calcium response and the flow-responsive klf2a promoter are modulated by the oscillatory flow through Trpv4, a mechanosensitive ion channel specifically expressed in the endocardium during heart valve development. We made similar observations for Trpp2, a known Trpv4 partner, and show that both the absence of Trpv4 or Trpp2 leads to valve defects. This work identifies a major mechanotransduction pathway involved during valve formation in vertebrates.
To systematically review current evidence regarding prenatal diagnosis and management of transient abnormal myelopoiesis (TAM) in fetuses with trisomy 21. A novel case of GATA1-positive TAM, in which following serial in utero blood transfusion clinical improvement and postnatal remission were observed, is included.
A systematic search of electronic databases (inception to October 2014) and reference lists, hand-searching of journals and expert contact. All confirmed cases of prenatal TAM were included for analysis. Data on study characteristics, design and quality were obtained.
Of 73 potentially relevant citations identified, 22 studies were included, describing 39 fetuses. All studies included comprised single case or small cohort studies; overall quality was 'very low'. Fetal/neonatal outcome was poor; 12 stillbirths (30.8%), 4 neonatal deaths (10.2%) and 7 infant deaths (17.9%). In two cases, the pregnancy was terminated (5.1%). TAM was primarily detected in the third trimester (79.4%), and in 14 a retrospective diagnosis was made postpartum. Ultrasound features indicative of TAM included hepatomegaly±splenomegaly (79.5%), hydrops fetalis (30.8%), pericardial effusion (23.1%) and aberrant liquor volume (15.4%). When performed, liver function tests were abnormal in 91.6% of cases.
Prenatal TAM presents a challenging diagnosis, and prognosis is poor, with consistently high mortality. A low threshold to measure haematological and biochemical markers is advised when clinical features typical of TAM are detected in the context of trisomy 21. Larger prospective studies are warranted to accurately ascertain the role of GATA1 analysis and potential value of prenatal therapy.
Based on our previous study showing the inhibition of lenkemia T cell proliferation by down-regulating PPP2R5C expression, this study was aimed to analyze the influence of down-regulating PPP2R5 expression via RNA interference on genes relatied with TAL1 signaling pathway by using gene chip technique.
The PPP2R5C-siRNA799 was transduced into Jurkat cells by nucleofection, the total RNA was isolated from treated Jurkat cells after culture for 48 hours; the target sequences were prepared by revevse transcription after mRNA purification, and were hybridized with affymetrix gene expression profile chip 3' IVT. The original image data were collected using affymetrix gene chip scanner 3 000, and the gene expression profile was analyzed using gene spring GX 11.0 soflware.
The expression of all 26 genes related with TAL1 signaling pathway was changed, out of which the expression of 15 genes were up-regulated and the expression of 11 genes was down-regulated in PPP2R5C-siRNA 799-transfected Jurkat cells. The genes with significantly up-regulated expression were GATA1, TCF4, XRCC6 and TCF3, while the genes with significantly down-regulated expression were SIN3A and RUNX1.
The down-regulation of PPP2R5C gene expression in Jurkat cells via RNA interference to a certain degree can inhibit TAL1 signaling pathway genes, thereby suppresses the proliferation of Jurkat cells.
Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous ribosomopathy and inherited bone marrow failure syndrome characterized by anemia, reticulocytopenia, and decreased erythroid precursors in the bone marrow with an increased risk of malignancy and, in approximately 50%, physical abnormalities.
We retrospectively analyzed clinical data from 77 patients with DBA born in the Russian Federation from 1993 to 2014. In 74 families there was one clinically affected individual; in only three instances a multiplex family was identified. Genomic DNA from 57 DBA patients and their first-degree relatives was sequenced for mutations in RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a, and GATA1.
Severe anemia presented before 8 months of age in all 77 patients; before 2 months in 61 (78.2%); before 4 months in 71 (92.2%). Corticosteroid therapy was initiated after 1 year of age in the majority of patients. Most responded initially to steroids, while 5 responses were transient. Mutations in RP genes were detected in 35 of 57 patients studied: 15 in RPS19, 6 in RPL5, 3 in RPS7, 3 each in RPS10, RPS26, and RPL11 and 1 each in RPS24 and RPL35a; 24 of these mutations have not been previously reported. One patient had a balanced chromosomal translocation involving RPS19. No mutations in GATA1 were found.
In our cohort from an ethnically diverse population the distribution of mutations among RP genes was approximately the same as was reported by others, although within genotypes most of the mutations had not been previously reported.
Despite recent advances in the treatment of children with acute megakaryoblastic leukemia (AMKL) using intensified treatment protocols, clear prognostic indicators, and treatment recommendations for this acute myeloid leukemia (AML) subgroup are yet to be defined. Here, we report the outcome of 97 pediatric patients with de novo AMKL (excluding Down syndrome [DS]) enrolled in the prospective multicenter studies AML-BFM 98 and AML-BFM 04 (1998-2014). AMKL occurred in 7.4 % of pediatric AML cases, at younger age (median 1.44 years) and with lower white blood cell count (mean 16.5 × 10(9)/L) as compared to other AML subgroups. With 60 ± 5 %, children with AMKL had a lower 5-year overall survival (5-year OS; vs. 68 ± 1 %, P log rank = 0.038). Yet, we achieved an improved 5-year OS in AML-BFM 04 compared to AML-BFM 98 (70 ± 6 % vs. 45 ± 8 %, P log rank = 0.041). Allogeneic hematopoietic stem cell transplantation in first remission did not provide a significant survival benefit (5-year OS 70 ± 11 % vs. 63 ± 6 %; P Mantel-Byar = 0.85). Cytogenetic data were available for n = 78 patients. AMKL patients with gain of chromosome 21 had a superior 5-year OS (80 ± 9 %, P log rank = 0.034), whereas translocation t(1;22)(p13;q13) was associated with an inferior 5-year event-free survival (38 ± 17 %, P log rank = 0.04). However, multivariate analysis showed that treatment response (bone marrow morphology on day 15 and 28) was the only independent prognostic marker (RR = 4.39; 95 % CI, 1.97-9.78). Interestingly, GATA1-mutations were detected in six patients (11 %) without previously known trisomy 21. Thus, AMKL (excluding DS) remains an AML subgroup with inferior outcome. Nevertheless, with intensive therapy regimens, a steep increase in the survival rates was achieved.
Epidemiological studies indicate that vitamin D exerts a protective effect on the development of various solid cancers. However, concerns have been raised regarding the potential deleterious role of high vitamin D levels in the development of esophageal adenocarcinoma (EAC). This study investigated genetic variation in the vitamin D receptor (VDR) in relation to its expression and risk of Barrett esophagus (BE) and EAC. VDR gene regulation was investigated by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and gel shift assays. Fifteen haplotype tagging single-nucleotide polymorphisms (SNPs) of the VDR gene were analyzed in 858 patients with reflux esophagitis (RE), BE or EAC and 202 healthy controls. VDR mRNA expression was higher in BE compared with squamous epithelium. VDR protein was located in the nucleus in BE. An rs1989969T/rs2238135G haplotype was identified in the 5' regulatory region of the VDR gene. It was associated with an approximately two-fold reduced risk of RE, BE and EAC. Analysis of a replication cohort was done for BE that confirmed this. The rs1989969T allele causes a GATA-1 transcription factor binding site to appear. The signaling of GATA-1, which is regarded as a negative transcriptional regulator, could explain the findings for rs1989969. The rs2238135G allele was associated with a significantly reduced VDR expression in BE; for the rs1989969T allele, a trend in reduced VDR expression was observed. We identified a VDR haplotype associated with reduced esophageal VDR expression and a reduced incidence of RE, BE and EAC. This VDR haplotype could be useful in identifying individuals who benefit most from vitamin D chemoprevention.
Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45-c-Kit-CD71+/-Ter119+ erythroid cells and downregulated Gata1, Klf1 and β-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45-c-Kit-CD71+/-Ter119+ cells and downregulated β-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.
The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.
The erythropoietin (Epo) receptor (EpoR) is expressed in the brain and was shown to have neuroprotective effects against brain damage in animal models. A recent study indicated that EpoR and its activity are the downstream effectors of Klotho for cytoprotection in the kidney. Thus, we propose that Klotho can stimulate the expression of EpoR in neuronal cells to enhance Epo-mediated protection. H19-7 hippocampal neuronal cells were treated with recombinant Klotho. In H19-7 cells, Klotho increased the expression of both the EpoR protein and mRNA. Klotho also enhanced the transcription activity of the EpoR promoter in H19-7 cells. Moreover, Klotho augmented the Epo-triggered phosphorylation of Jak2 and Stat5 and protected H19-7 cells from hydrogen peroxide cytotoxicity. The silencing of EpoR abolished the protective effect of Klotho against peroxide-induced cytotoxicity. Finally, the silencing of GATA1 diminished the Klotho-induced increase in EpoR protein and mRNA expression as well as its promoter activity. In conclusion, Klotho increased EpoR expression in neuronal cells through GATA1, thereby enabling EpoR to function as a cytoprotective protein against oxidative injury.
The gene regulatory networks in which two lineage-affiliated transcription factors, such as GATA1 and PU.1, inhibit each other but activate themselves so as to regulate the choice between alternative cell fates have been extensively studied. These simple networks can generate bistability and explain the transitions between the alternative cell fates. The commitment of a progenitor cell to a new fate corresponds to the occurrence of different types of bifurcations, depending on if a system is symmetrical and how perturbations affect the system. Here we take a general modeling and analyzing approach and show that the lateral inhibition with symmetry and asymmetry can lead to different bifurcation dynamics. Especially, if cell fate decision-making is initiated with asymmetry or symmetry-breaking perturbations, a progenitor cell pre-patterns itself into a polarized cell, depending on the asymmetry or symmetry-breaking perturbations. This study may help us understand the fundamental features of binary cell fate decisions more clearly and further apply to a wider range of decision-making processes.
Our previous study on the dynamic transcriptomes activated during human erythropoiesis suggested that transcription factor forkhead box O3 (FOXO3) possibly plays a role in erythroid differentiation. Functional studies in human cell line TF-1 indicated that FOXO3 knockdown repressed erythropoietin (EPO)-induced erythroid differentiation by activating promoter region of B-cell translocation gene 1 (BTG1), thereby regulating its expression. In zebrafish, injection of foxo3b-specific morpholinos (foxo3b MO) resulted in reduced globin (hbae1 and hbbe2) and gata1 gene expression. Transcriptome analyses of erythroid lineage cells isolated from the control and foxo3b morphants revealed the dynamic regulation of foxo3b. Further study suggested that BTG1 is partially responsible for FOXO3 regulation in erythroid differentiation of TF-1 cells but is inconsequential in zebrafish. Taken together, we found that FOXO3 plays an important role in erythroid differentiation in both human TF-1 cells and zebrafish, but the mechanism underlying this regulation still remains unclear.
Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247-406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.
Although thyroid hormone is a known stimulator of erythropoietic differentiation, severe anemia is sometimes observed in patients with hyperthyroidism and this mechanism is not fully understood. The aim of this study was to investigate the effect of triiodothyronine (T3) on hemin-induced erythropoiesis. Human erythroleukemia K562 cells were used as an erythroid differentiation model. Cell differentiation was induced by hemin and the effect of pre-incubation with T3 (0.1 to 100 nM) was analyzed by measuring the benzidine-positive rate, hemoglobin content, CD71 expression (transferrin receptor), and mRNA expression for transcription factors related to erythropoiesis and thyroid hormone receptors (TRs). Hemin, a promoter of erythroid differentiation, increased the levels of mRNAs for TRα, TRβ, and retinoid X receptor α (RXRα), as well as those for nuclear factor-erythroid 2 (NFE2), GATA-binding protein 1 (GATA1) and GATA-binding protein 2 (GATA2). Lower concentrations of T3 had a stimulatory effect on hemin-induced hemoglobin production (1 and 10 nM), CD71 expression (0.1 nM), and α-globin mRNA expression (1 nM), while a higher concentration of T3 (100 nM) abrogated the stimulatory effect on these parameters. T3 at 100 nM did not affect cell viability and proliferation, suggesting that the abrogation of erythropoiesis enhancement was not due to toxicity. T3 at 100 nM also significantly inhibited expression of GATA2 and RXRα mRNA, compared to 1 nM T3. We conclude that a high concentration of T3 attenuates the classical stimulatory effect on erythropoiesis exerted by a low concentration of T3 in hemin-induced K562 cells.
Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.
GATA transcription factors are evolutionarily conserved transcriptional regulators that recognize promoter elements with a G-A-T-A core sequence. In comparison to animal genomes, the GATA transcription factor family in plants is comparatively large with approximately 30 members. Here, we review the current knowledge on B-GATAs, one of four GATA factor subfamilies from Arabidopsis thaliana. We show that B-GATAs can be subdivided based on structural features and their biological function into family members with a C-terminal LLM- (leucine-leucine-methionine) domain or an N-terminal HAN- (HANABA TARANU) domain. The paralogous GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED) and CGA1/GNL (CYTOKININ-INDUCED GATA1/GNC-LIKE) are introduced as LLM-domain containing B-GATAs from Arabidopsis that control germination, greening, senescence, and flowering time downstream from several growth regulatory signals. Arabidopsis HAN and its monocot-specific paralogs from rice (NECK LEAF1), maize (TASSEL SHEATH1), and barley (THIRD OUTER GLUME) are HAN-domain-containing B-GATAs with a predominant role in embryo development and floral development. We also review GATA23, a regulator of lateral root initiation from Arabidopsis that is closely related to GNC and GNL but has a degenerate LLM-domain that is seemingly specific for the Brassicaceae family. The Brassicaceae-specific GATA23 and the monocot-specific HAN-domain GATAs provide evidence that neofunctionalization of B-GATAs was used during plant evolution to expand the functional repertoire of these transcription factors.
Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.
GATA1 is a key transcription factor for erythropoiesis. GATA1 gene expression is strictly regulated at the transcriptional level. While the regulatory mechanisms governing mouse Gata1 (mGata1) gene expression have been studied extensively, how expression of the human GATA1 (hGATA1) gene is regulated remains to be elucidated. To address this issue, we generated hGATA1 bacterial artificial chromosome (BAC) transgenic mouse lines harboring a 183-kb hGATA1 locus covering the hGATA1 exons and distal flanking sequences. Transgenic hGATA1 expression coincides with endogenous mGata1 expression and fully rescues hematopoietic deficiency in mGata1 knockdown mice. The transgene exhibited copy number-dependent and integration position-independent expression of hGATA1, indicating the presence of chromatin insulator activity within the transgene. We found a novel insulator element at 29 kb 5' to the hGATA1 gene and refer to this element as the 5' CCCTC-binding factor (CTCF) site. Substitution mutation of the 5' CTCF site in the hGATA1 BAC disrupted the chromatin architecture and led to a reduction of hGATA1 expression in splenic erythroblasts under conditions of stress erythropoiesis. Our results demonstrate that expression of the hGATA1 gene is regulated through the chromatin architecture organized by 5' CTCF site-mediated intrachromosomal interactions in the hGATA1 locus.
CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.
Celiac disease (CD) is an autoimmune disease induced by an autoimmune reaction to indigested gluten, which occurs in genetically predisposed population. The etiology of CD is linked to innate and adaptive immunity, mostly mediated by lymphocytes, especially T cells, infiltrating into the small intestinal wall. The subpopulations of T cells that infiltrate inflamed intestinal tissues comprise various CD4+ T cells and CD8+ T cells. The plethora of T cell subtypes activated in CD leads to simultaneous activation of different signaling cascades including GATA1, NF-kB, JAK or STAT5 the activity of which may be modified by diet or drugs. It was recently showed that food allergens may accelerate CD by altering the interaction between IL-15 and CD4+ T cells in the activation of CD8+ T cells. Increased levels of cytokines like IL-15 are considered to play a role in CD development. Furthermore it was showed that some drugs like tofacitinib or ruxolitinib may influence CD by blocking IL-15 signaling and CD8+ T cell activity. This mini-review will summarize the current knowledge on the role of CD4+ T cell and CD8+ T cell in clinical and experimental CD and will describe how T cell-activated signaling pathways and locally released proteins may be influenced by dietary factors and drugs used in CD treatment.
During the maturation phase of mammalian erythroid differentiation, highly proliferative cells committed to the erythroid lineage undergo dramatic changes in morphology and function to produce circulating, enucleated erythrocytes. These changes are caused by equally dramatic alterations in gene expression, which in turn are driven by changes in the abundance and binding patterns of transcription factors such as GATA1. We have studied the dynamics of GATA1 binding by ChIP-seq and the global expression responses by RNA-seq in a GATA1-dependent mouse cell line model for erythroid maturation, in both cases examining seven progressive stages during differentiation. Analyses of these data should provide insights both into mechanisms of regulation (early versus late targets) and the consequences in cell physiology (e.g. distinctive categories of genes regulated at progressive stages of differentiation). The data are deposited in the Gene Expression Omnibus, series GSE36029, GSE40522, GSE49847, and GSE51338.
Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.
The NFE2 transcription factor was identified over 25 years ago. The NFE2 protein forms heterodimers with small MAF proteins, and the resulting complex binds to regulatory elements in a large number of target genes. In contrast to other CNC transcription family members including NFE2L1 (NRF1), NFE2L2 (NRF2) and NFE2L3 (NRF3), which are widely expressed, earlier studies had suggested that the major sites of NFE2 expression are hematopoietic cells. Based on cell culture studies it was proposed that this protein acts as a critical regulator of globin gene expression. However, the knockout mouse model displayed only mild erythroid abnormalities, while the major phenotype was a defect in megakaryocyte biogenesis. Indeed, absence of NFE2 led to severely impaired platelet production. A series of recent data, also summarized here, shed new light on the various functional roles of NFE2 and the regulation of its activity. NFE2 is part of a complex regulatory network, including transcription factors such as GATA1 and RUNX1, controlling megakaryocytic and/or erythroid cell function. Surprisingly, it was recently found that NFE2 also has a role in non-hematopoietic tissues, such as the trophoblast, in which it is also expressed, as well as the bone, opening the door to new research areas for this transcription factor. Additional data showed that NFE2 function is controlled by a series of posttranslational modifications. Important strides have been made with respect to the clinical significance of NFE2, linking this transcription factor to hematological disorders such as polycythemias.
We encountered a case of neonatal acute megakaryoblastic leukemia not associated with Down syndrome (DS). Molecular cytogenetic analysis of leukemic blast cells indicated that increased blast cell status was caused by transient abnormal myelopoiesis with trisomy 21 and GATA1 mutation. Based on these molecular cytogenetic data, intensive chemotherapy was avoided, and the patient was successfully cured with low-dose cytarabine. Morphologically, leukemic blast cells of acute megakaryoblastic leukemia in a non-DS neonate are indistinguishable from a blast cell of transient abnormal myelopoiesis. The possibility of transient abnormal myelopoiesis should be carefully considered before intensive chemotherapy is adopted.
Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
Autophagy is a process that leads to the degradation of unnecessary or dysfunctional cellular components and long-lived protein aggregates. Erythropoiesis is a branch of hematopoietic differentiation by which mature red blood cells (RBCs) are generated from multi-potential hematopoietic stem cells (HSCs). Autophagy plays a critical role in the elimination of mitochondria, ribosomes and other organelles during erythroid terminal differentiation. Here, the modulators of autophagy that regulate erythroid differentiation were summarized, including autophagy-related (Atg) genes, the B-cell lymphoma 2 (Bcl-2) family member Bcl-2/adenovirus E1B 19 kDa interacting protein 3-like (Nix/Binp3L), transcription factors globin transcription factor 1 (GATA1) and forkhead box O3 (FoxO3), intermediary factor KRAB-associated protein1 (KAP1), and other modulators, such as focal adhesion kinase family-interacting protein of 200-kDa (FIP200), Ca2+ and 15-lipoxygenase. Understanding the modulators of autophagy in erythropoiesis will benefit the autophagy research field and facilitate the prevention and treatment of autophagy-related red blood cell disorders.
GATA1 is a master transcriptional regulator of the differentiation of several related myeloid blood cell types, including erythrocytes and megakaryocytes. Germ-line mutations that cause loss of full length GATA1, but allow for expression of the short isoform (GATA1s), are associated with defective erythropoiesis in a subset of patients with Diamond Blackfan Anemia. Despite extensive studies of GATA1s in megakaryopoiesis, the mechanism by which GATA1s fails to support normal erythropoiesis is not understood. In this study, we used global gene expression and chromatin occupancy analysis to compare the transcriptional activity of GATA1s to GATA1. We discovered that compared to GATA1, GATA1s is less able to activate the erythroid gene expression program and terminal differentiation in cells with dual erythroid-megakaryocytic differentiation potential. Moreover, we found that GATA1s bound to many of its erythroid-specific target genes less efficiently than full length GATA1. These results suggest that the impaired ability of GATA1s to promote erythropoiesis in DBA may be caused by failure to occupy erythroid-specific gene regulatory elements.
The roles of the GATA4 and GATA6 transcription factors in testis development were examined by simultaneously ablating Gata4 and Gata6 with Sf1Cre (Nr5a1Cre). The deletion of both genes resulted in a striking testicular phenotype. Embryonic Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) (conditional double mutant) testes were smaller than control organs and contained irregular testis cords and fewer gonocytes. Gene expression analysis revealed significant down-regulation of Dmrt1 and Mvh. Surprisingly, Amh expression was strongly up-regulated and remained high beyond postnatal day 7, when it is normally extinguished. Neither DMRT1 nor GATA1 was detected in the Sertoli cells of the mutant postnatal testes. Furthermore, the expression of the steroidogenic genes Star, Cyp11a1, Hsd3b1, and Hsd17b3 was low throughout embryogenesis. Immunohistochemical analysis revealed a prominent reduction in cytochrome P450 side-chain cleavage enzyme (CYP11A1)- and 3β-hydroxysteroid dehydrogenase-positive (3βHSD) cells, with few 17α-hydroxylase/17,20 lyase-positive (CYP17A1) cells present. In contrast, in postnatal Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) testes, the expression of the steroidogenic markers Star, Cyp11a1, and Hsd3b6 was increased, but a dramatic down-regulation of Hsd17b3, which is required for testosterone synthesis, was observed. The genes encoding adrenal enzymes Cyp21a1, Cyp11b1, Cyp11b2, and Mcr2 were strongly up-regulated, and clusters containing numerous CYP21A2-positive cells were localized in the interstitium. These data suggest a lack of testis functionality, with a loss of normal steroidogenic testis function, concomitant with an expansion of the adrenal-like cell population in postnatal conditional double mutant testes. Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) animals of both sexes lack adrenal glands; however, despite this deficiency, males are viable in contrast to the females of the same genotype, which die shortly after birth.
Transient abnormal myelopoiesis (TAM) is a disorder of Down syndrome newborns characterized by megakaryocytic blasts indistinguishable from acute myeloid leukemia (AML), which undergoes spontaneous remission. Acquired GATA1 mutations are present in blasts of both TAM and the subsequent AML which sometimes develops. We present a unique case of a newborn with leukemic megakaryoblasts indistinguishable from those of TAM who had neither extra material from chromosome 21 in the germline or blasts, nor evidence of GATA1 mutations. These findings suggest there are other genetic abnormalities that can lead to TAM besides GATA1 mutation in the setting of trisomy 21. Pediatr Blood Cancer 2015;62:353-355. © 2014 Wiley Periodicals, Inc.
Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.
Among neonates with Down syndrome (DS) and transient leukemia (TL), hyperleukocytosis (white blood cell [WBC] count >100 × 10(9) /L) is associated with increased blood viscosity, respiratory failure due to pulmonary hypertension, multiorgan failure, and increased risk of early death. There have been no previous studies focusing on the effects of exchange transfusion (ExT) on WBC count, respiratory status, and other parameters in TL patients with hyperleukocytosis.
An observational retrospective study was carried out at a single center of all five DS neonates with TL, GATA1 mutations, and hyperleukocytosis, born at a median gestational age of 34 weeks (range, 30-38 weeks) with birthweight 2556 g (range, 1756-3268 g) during a 24 month study period between September 2011 and August 2013. All five neonates underwent ExT at a median age of 2 days (range, 0-5 days) before initiation of other cytoreductive therapy with cytarabine, which was carried out in two patients.
All patients required respiratory support before ExT. After ExT, respiration status improved in all five patients: WBC count (mean) decreased by 85% from 143 × 10(9) /L to 21 × 10(9) /L. None developed tumor lysis syndrome. Three survived and two died: one hydrops fetalis neonate born at gestational week 30 died at age 5 days, and another died eventually from acute gastroenteritis 40 days after leaving hospital at the age of 155 days with complete remission. Two of the three surviving neonates developed acute megakaryocytic leukemia at age 90 days and 222 days.
ExT was very effective in improving hyperleukocytosis and may have had favorable effects on respiration.
Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.
Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. In vivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroid differentiation.
The nephrotoxicity of aristolochic acid (AA) is well known, but information regarding the attenuation of AA-induced toxicity is limited. The aim of the present study was to study the nephroprotective effects of resveratrol (Resv) and ursolic acid (UA) in a zebrafish model. We used two transgenic lines, Tg(wt1b:EGFP) and Tg(gata1:DsRed), to evaluate the nephroprotective effects of Resv and UA by recording subtle changes in the kidney and red blood cell circulation. Our results demonstrated that both Resv and UA treatment can attenuate AA-induced kidney malformations and improve blood circulation. Glomerular filtration rate assays revealed that both Resv and UA treatment can restore renal function (100% for Mock; 56.1% ± 17.3% for AA-treated; 80.2% ± 11.3% for Resv+AA; and 83.1% ± 8.1% for UA+AA, n = 15). Furthermore, real-time RT-PCR experiments showed that pre-treatment with either Resv or UA suppresses expression of pro-inflammatory genes. In conclusion, our findings reveal that AA-induced nephrotoxicities can be attenuated by pre-treatment with either Resv or UA. Therefore, we believe that zebrafish represent an efficient model for screening AA-protective natural compounds.
Transient abnormal myelopoiesis (TAM), a clonal proliferation of predominantly megakaryocytic precursor cells, affects 4%-10% of newborns with Down syndrome. Approximately 20%-30% of TAM survivors are at risk of development of acute myeloid leukemia (myeloid leukemia associated with Down syndrome, ML-DS). We report unusual placental findings in a female infant with trisomy 21 born at 38 weeks of gestation. In line with previous descriptions of placental pathology in infants with TAM, abundant blast-like cells were present in the lumen of chorionic and stem villous vessels. In addition, there was multifocal extensive infiltration of the wall of chorionic vessels by maturing myeloid cells in a pattern reminiscent of TAM- or leukemia-associated systemic infiltration. The clinical significance of this unusual choriovascular involvement of the placenta in TAM is undetermined.
Polycomb repressive complex 2 (PRC2) plays crucial roles in transcriptional regulation and stem cell development. However, the context-specific functions associated with alternative subunits remain largely unexplored. Here we show that the related enzymatic subunits EZH1 and EZH2 undergo an expression switch during blood cell development. An erythroid-specific enhancer mediates transcriptional activation of EZH1, and a switch from GATA2 to GATA1 controls the developmental EZH1/2 switch by differential association with EZH1 enhancers. We further examine the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and reveal the existence of an EZH1-SUZ12 subcomplex lacking EED. EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin, and positively regulate gene expression. Loss of EZH2 expression leads to repositioning of EZH1 to EZH2 targets. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical functions during blood stem cell specification.
The onset of hematopoiesis in mammals is defined by generation of primitive erythrocytes and macrophage progenitors in embryonic yolk sac. Laboratories have met the challenge of transient and swiftly changing specification events from ventral mesoderm through multipotent progenitors and maturing lineage-restricted hematopoietic subtypes, by developing powerful in vitro experimental models to interrogate hematopoietic ontogeny. Most importantly, studies of differentiating embryonic stem cell derivatives in embryoid body and stromal coculture systems have identified crucial roles for transcription factor networks (e.g. Gata1, Runx1, Scl) and signaling pathways (e.g. BMP, VEGF, WNT) in controlling stem and progenitor cell output. These and other relevant pathways have pleiotropic biological effects, and are often associated with early embryonic lethality in knockout mice. Further refinement in subsequent studies has allowed conditional expression of key regulatory genes, and isolation of progenitors via cell surface markers (e.g. FLK1) and reporter-tagged constructs, with the purpose of measuring their primitive and definitive hematopoietic potential. These observations continue to inform attempts to direct the differentiation, and augment the expansion, of progenitors in human cell culture systems that may prove useful in cell replacement therapies for hematopoietic deficiencies. The purpose of this review is to survey the extant literature on the use of differentiating murine embryonic stem cells in culture to model the developmental process of yolk sac hematopoiesis.
Despite numerous circumstantial evidences, the pathogenic role of TGF-β in primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms, is still unclear because of the modest (2-fold) increases in its plasma levels observed in PMF patients and in the Gata1(low) mouse model. Whether myelofibrosis is associated with increased bioavailability of TGF-β bound to fibrotic fibres is unknown. Transmission electron-microscopy (TEM) observations identified that spleen from PMF patients and Gata1(low) mice contained megakaryocytes with abnormally high levels of TGF-β and collagen fibres embedded in their cytoplasm. Additional immuno-TEM observations of spleen from Gata1(low) mice revealed the presence of numerous activated fibrocytes establishing with their protrusions a novel cellular interaction, defined as peripolesis, with megakaryocytes. These protrusions infiltrated the megakaryocyte cytoplasm releasing collagen that was eventually detected in its mature polymerized form. Megakaryocytes, engulfed with mature collagen fibres, acquired the morphology of para-apoptotic cells and, in the most advanced cases, were recognized as polylobated heterochromatic nuclei surrounded by collagen fibres strictly associated with TGF-β. These areas contained concentrations of TGF-β-gold particles ~1000-fold greater than normal and numerous myofibroblasts, an indication that TGF-β was bioactive. Loss-of-function studies indicated that peripolesis between megakaryocytes and fibrocytes required both TGF-β, possibly for inducing fibrocyte activation, and P-selectin, possibly for mediating interaction between the two cell types. Loss-of-function of TGF-β and P-selectin also prevented fibrosis. These observations identify that myelofibrosis is associated with pathological increases of TGF-β bioavailability and suggest a novel megakaryocyte-mediated mechanism that may increase TGF-β bioavailability in chronic inflammation.
GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using a Gata1 bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1(ΔdbGATA) mice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossed Gata1(ΔdbGATA) mice with Gata2 hypomorphic mutant mice (Gata2(fGN/fGN) mice), the Gata1(ΔdbGATA)::Gata2(fGN/fGN) compound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion on Gata1 expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATA locus. These results thus reveal a novel role of the dbGATA motif in the maintenance of Gata1 expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.
Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.
KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the β-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the β-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the β-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the β-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping.
Current ChIP-seq studies are interested in comparing multiple epigenetic profiles across several cell types and tissues simultaneously for studying constitutive and differential regulation. Simultaneous analysis of multiple epigenetic features in many samples can gain substantial power and specificity than analyzing individual features and/or samples separately. Yet there are currently few tools can perform joint inference of constitutive and differential regulation in multi-feature-multi-condition contexts with statistical testing. Existing tools either test regulatory variation for one factor in multiple samples at a time, or for multiple factors in one or two samples. Many of them only identify binary rather than quantitative variation, which are sensitive to threshold choices.
We propose a novel and powerful method called dCaP for simultaneously detecting constitutive and differential regulation of multiple epigenetic factors in multiple samples. Using simulation, we demonstrate the superior power of dCaP compared to existing methods. We then apply dCaP to two datasets from human and mouse ENCODE projects to demonstrate its utility. We show in the human dataset that the cell-type specific regulatory loci detected by dCaP are significantly enriched near genes with cell-type specific functions and disease relevance. We further show in the mouse dataset that dCaP captures genomic regions showing significant signal variations for TAL1 occupancy between two mouse erythroid cell lines. The novel TAL1 occupancy loci detected only by dCaP are highly enriched with GATA1 occupancy and differential gene expression, while those detected only by other methods are not.
Here, we developed a novel approach to utilize the cooperative property of proteins to detect differential binding given multivariate ChIP-seq samples to provide better power, aiming for complementing existing approaches and providing new insights in the method development in this field.
Erythropoiesis is one of the best understood examples of cellular differentiation. Morphologically, erythroid differentiation proceeds in a nearly identical fashion between humans and mice, but recent evidence has shown that networks of gene expression governing this process are divergent between species. We undertook a systematic comparative analysis of six histone modifications and four transcriptional master regulators in primary proerythroblasts and erythroid cell lines to better understand the underlying basis of these transcriptional differences. Our analyses suggest that while chromatin structure across orthologous promoters is strongly conserved, subtle differences are associated with transcriptional divergence between species. Many transcription factor (TF) occupancy sites were poorly conserved across species (∼25% for GATA1, TAL1, and NFE2) but were more conserved between proerythroblasts and cell lines derived from the same species. We found that certain cis-regulatory modules co-occupied by GATA1, TAL1, and KLF1 are under strict evolutionary constraint and localize to genes necessary for erythroid cell identity. More generally, we show that conserved TF occupancy sites are indicative of active regulatory regions and strong gene expression that is sustained during maturation. Our results suggest that evolutionary turnover of TF binding sites associates with changes in the underlying chromatin structure, driving transcriptional divergence. We provide examples of how this framework can be applied to understand epigenomic variation in specific regulatory regions, such as the β-globin gene locus. Our findings have important implications for understanding epigenomic changes that mediate variation in cellular differentiation across species, while also providing a valuable resource for studies of hematopoiesis.
Balancing stem cell self-renewal and initiation of lineage specification programs is essential for the development and homeostasis of the hematopoietic system. We have specifically ablated geminin in the developing murine hematopoietic system and observed profound defects in the generation of mature blood cells, leading to embryonic lethality. Hematopoietic stem cells (HSCs) accumulated in the fetal liver following geminin ablation, while committed progenitors were reduced. Genome-wide transcriptome analysis identified key HSC transcription factors as being upregulated upon geminin deletion, revealing a gene network linked with geminin that controls fetal hematopoiesis. In order to obtain mechanistic insight into the ability of geminin to regulate transcription, we examined Hoxa9 as an example of a key gene in definitive hematopoiesis. We demonstrate that in human K562 cells geminin is associated with HOXA9 regulatory elements and its absence increases HOXA9 transcription similarly to that observed in vivo. Moreover, silencing geminin reduced recruitment of the PRC2 component SUZ12 to the HOXA9 locus and resulted in an increase in RNA polymerase II recruitment and H3K4 trimethylation (H3K4me3), whereas the repressive marks H3K9me3 and H3K27me3 were reduced. The chromatin landscape was also modified at the regulatory regions of HOXA10 and GATA1. K562 cells showed a reduced ability to differentiate to erythrocytes and megakaryocytes upon geminin silencing. Our data suggest that geminin is indispensable for fetal hematopoiesis and regulates the generation of a physiological pool of stem and progenitor cells in the fetal hematopoietic system.
Children with Down syndrome (DS) and acute leukemias acute have unique biological, cytogenetic, and intrinsic factors that affect their treatment and outcome. Myeloid leukemia of Down syndrome (ML-DS) is associated with high event-free survival (EFS) rates and frequently preceded by a preleukemia condition, the transient abnormal hematopoiesis (TAM) present at birth. For acute lymphoblastic leukemia (ALL), their EFS and overall survival are poorer than non-DS ALL, it is important to enroll them on therapeutic trials, including relapse trials; investigate new agents that could potentially improve their leukemia-free survival; and strive to maximize the supportive care these patients need.
Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia.
X-linked thrombocytopenia with thalassemia (XLTT) is caused by the mutation 216R > Q in exon 4 of the GATA1 gene. Male hemizygous patients display macrothrombocytopenia, splenomegaly, and a β-thalassemia trait. We describe two XLTT families where three males were initially misdiagnosed as having primary myelofibrosis (PMF) and all five investigated males showed mild-moderate bone marrow (BM) reticulin fibrosis. Comparative investigations were performed on blood samples and BM biopsies from males with XLTT, PMF patients and healthy controls. Like PMF, XLTT presented with high BM microvessel density, low GATA1 protein levels in megakaryocytes, and elevated blood CD34+ cell counts. But unlike PMF, the BM microvessel pericyte coverage was low in XLTT, and no collagen fibrosis was found. Further, as evaluated by immunohistochemistry, expressions of the growth factors VEGF, AGGF1, and CTGF were low in XLTT megakaryocytes and microvessels but high in PMF. Thus, although the reticulin fibrosis in XLTT might simulate PMF, opposing stromal and megakaryocyte features may facilitate differential diagnosis. Additional comparisons between these disorders may increase the understanding of mechanisms behind BM fibrosis in relation to pathological megakaryopoiesis.
Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements.
Functional consequences to which vertebrate GATA transcription factors contribute in the adult brain remain largely an open question. The present study examines how human GATA-1 and GATA-2 (hGATA-1 and hGATA-2) are linked to neuronal differentiation and depressive behaviors in rats. We investigated the effects of adeno-associated viral expression of hGATA-1 and hGATA-2 (AAV-hGATA1 and AAV-hGATA2) in the dentate gyrus (DG) of the dorsal hippocampus on dendrite branching and spine number. We also examined the influence of AAV-hGATA1 and AAV-hGATA2 infusions into the dorsal hippocampus on rodent behavior in models of depression. Viral expression of hGATA-1 and hGATA-2 cDNA in rat hippocampal neurons impaired dendritic outgrowth and spine formation. Moreover, viral-mediated expression of hGATA-1 and hGATA-2 in the dorsal hippocampus caused depressive-like deficits in the forced swim test and learned helplessness models of depression, and decreased the expression of several synapse-related genes as well as spine number in hippocampal neurons. Conversely, shRNA knockdown of GATA-2 increased synapse-related gene expression, spine number, and dendrite branching. The results demonstrate that hGATA-1 and hGATA-2 expression in hippocampus is sufficient to cause depressive like behaviors that are associated with reduction in spine synapse density and expression of synapse-related genes.
Combinatorial actions of relatively few transcription factors control hematopoietic differentiation. To investigate this process in erythro-megakaryopoiesis, we correlated the genome-wide chromatin occupancy signatures of four master hematopoietic transcription factors (GATA1, GATA2, TAL1, and FLI1) and three diagnostic histone modification marks with the gene expression changes that occur during development of primary cultured megakaryocytes (MEG) and primary erythroblasts (ERY) from murine fetal liver hematopoietic stem/progenitor cells. We identified a robust, genome-wide mechanism of MEG-specific lineage priming by a previously described stem/progenitor cell-expressed transcription factor heptad (GATA2, LYL1, TAL1, FLI1, ERG, RUNX1, LMO2) binding to MEG-associated cis-regulatory modules (CRMs) in multipotential progenitors. This is followed by genome-wide GATA factor switching that mediates further induction of MEG-specific genes following lineage commitment. Interaction between GATA and ETS factors appears to be a key determinant of these processes. In contrast, ERY-specific lineage priming is biased toward GATA2-independent mechanisms. In addition to its role in MEG lineage priming, GATA2 plays an extensive role in late megakaryopoiesis as a transcriptional repressor at loci defined by a specific DNA signature. Our findings reveal important new insights into how ERY and MEG lineages arise from a common bipotential progenitor via overlapping and divergent functions of shared hematopoietic transcription factors.
We used mouse ENCODE data along with complementary data from other laboratories to study the dynamics of occupancy and the role in gene regulation of the transcription factor TAL1, a critical regulator of hematopoiesis, at multiple stages of hematopoietic differentiation. We combined ChIP-seq and RNA-seq data in six mouse cell types representing a progression from multilineage precursors to differentiated erythroblasts and megakaryocytes. We found that sites of occupancy shift dramatically during commitment to the erythroid lineage, vary further during terminal maturation, and are strongly associated with changes in gene expression. In multilineage progenitors, the likely target genes are enriched for hematopoietic growth and functions associated with the mature cells of specific daughter lineages (such as megakaryocytes). In contrast, target genes in erythroblasts are specifically enriched for red cell functions. Furthermore, shifts in TAL1 occupancy during erythroid differentiation are associated with gene repression (dissociation) and induction (co-occupancy with GATA1). Based on both enrichment for transcription factor binding site motifs and co-occupancy determined by ChIP-seq, recruitment by GATA transcription factors appears to be a stronger determinant of TAL1 binding to chromatin than the canonical E-box binding site motif. Studies of additional proteins lead to the model that TAL1 regulates expression after being directed to a distinct subset of genomic binding sites in each cell type via its association with different complexes containing master regulators such as GATA2, ERG, and RUNX1 in multilineage cells and the lineage-specific master regulator GATA1 in erythroblasts.
The liver plays a central role in the maintenance of homeostasis and health in general. However, there is substantial inter-individual variation in hepatic gene expression, and although numerous genetic factors have been identified, less is known about the epigenetic factors.
By analyzing the methylomes and transcriptomes of 14 fetal and 181 adult livers, we identified 657 differentially methylated genes with adult-specific expression, these genes were enriched for transcription factor binding sites of HNF1A and HNF4A. We also identified 1,000 genes specific to fetal liver, which were enriched for GATA1, STAT5A, STAT5B and YY1 binding sites. We saw strong liver-specific effects of single nucleotide polymorphisms on both methylation levels (28,447 unique CpG sites (meQTL)) and gene expression levels (526 unique genes (eQTL)), at a false discovery rate (FDR) < 0.05. Of the 526 unique eQTL associated genes, 293 correlated significantly not only with genetic variation but also with methylation levels. The tissue-specificities of these associations were analyzed in muscle, subcutaneous adipose tissue and visceral adipose tissue. We observed that meQTL were more stable between tissues than eQTL and a very strong tissue-specificity for the identified associations between CpG methylation and gene expression.
Our analyses generated a comprehensive resource of factors involved in the regulation of hepatic gene expression, and allowed us to estimate the proportion of variation in gene expression that could be attributed to genetic and epigenetic variation, both crucial to understanding differences in drug response and the etiology of liver diseases.
Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.
Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.
Transient abnormal myelopoiesis occurs exclusively in patients with Down syndrome (constitutional trisomy 21), manifests in the neonatal period, and is characterized by circulating megakaryoblasts with varied degrees of multisystem organ involvement. In most cases, this process resolves spontaneously by 3 to 6 months of age, but for some, the disease can be fatal. Affected patients are particularly prone to develop acute megakaryoblastic leukemia in early childhood. Somatic GATA1 mutations are believed to be pivotal in the development of transient abnormal myelopoiesis and have proven to be a marker of clonal identity in its evolution to megakaryoblastic leukemia. We describe a study case of transient abnormal myelopoiesis and review the clinical manifestations, laboratory features, natural history, molecular genetics, and postulated disease pathogenesis of this disorder.
GATA1 mutations are found almost exclusively in children with myeloid proliferations related to Down syndrome (DS). Here, we report two phenotypically and cytogenetically normal children with acute megakaryoblastic leukemia (AMKL) whose blasts had both acquired trisomy 21 and GATA1 mutation. Patient 1 was diagnosed with transient abnormal myelopoiesis in the neonatal period. Following spontaneous improvement of the disease, leukemic blasts increased 7 months later. He received less intensive chemotherapy, and he is now 6 years old in complete remission. Patient 2 was diagnosed with AMKL at the age of 18 months. Although he received intensive chemotherapy and a cord blood transplantation, he died without gaining remission. In both cases, trisomy 21 and GATA1 mutation were detected only in leukemic blasts, but not in germline samples. Based on a literature review, we identified reports describing 14 non-DS AMKL with GATA1 mutation and acquired trisomy 21. Of those, 12 cases were diagnosed during the neonatal period, whereas the remaining 2 cases were diagnosed at the age of 22 and 31 months, respectively.
These cases suggest that GATA1 mutation may cooperate with the additional chromosome 21 in developing myeloid proliferations even in non-DS patients.
Congenital erythropoietic porphyria (CEP) is a rare genetic disease that is characterized by a severe cutaneous photosensitivity causing unrecoverable deformities, chronic hemolytic anemia requiring blood transfusion program, and by fatal systemic complications. A correct and early diagnosis is required to develop a management plan that is appropriate to the patient's needs. Recently only one case of X-linked CEP had been reported, describing the trans-acting GATA1-R216W mutation. Here, we have characterized two novel X-linked CEP patients, both with misleading hematological phenotypes that include dyserythropoietic anemia, thrombocytopenia, and hereditary persistence of fetal hemoglobin. We compare the previously reported case to ours and propose a diagnostic paradigm for this variant of CEP. Finally, a correlation between phenotype variability and the presence of modifier mutations in loci related to disease-causing gene is described.
Congenital myeloproliferative disorders and transient leukemic disorders have been described in the perinatal period in infants with trisomy 21 (Down syndrome). We report a novel case of a neonate with trisomy 21 with GATA1-mutated congenital myeloproliferative disorder complicated by placental fetal thrombotic vasculopathy featuring chorionic vessel leukemic thrombi, fetal circulation vascular injuries, and large aggregates of avascular villi. These thrombotic and vasculopathic changes within the placenta are likely a reflection of the hypercoagulable state caused by the myeloproliferative disorder. Placental fetal thrombotic vasculopathy is associated with adverse outcomes for the infant, and should be documented during formal pathological examination of the placenta.
GATA-1, an X-linked gene, encodes a transcription factor that plays a role in erythropoiesis and megakaryopoiesis. GATA-1 mutations have been associated with various diseases, such as X-linked thrombocytopenia. ALAS2 is an X-linked erythroid-specific isoenzyme expressed during erythropoiesis. Mutations of ALAS2 were associated with X-linked sideroblastic anemia. We report a case of newborn twin boy with anemia and thrombocytopenia at birth. A bone marrow biopsy at 4 months of age showed marked dyserythropoiesis, dysmegakaryopoiesis, and rare ringed sideroblasts. Gene sequencing study showed a previously reported mutation in GATA-1 at c.622G>A location (G208R) and a novel ALAS2 mutation at c.1436G>A location (R479Q).
Children with Down syndrome (DS) are at a substantially increased risk to develop acute myeloid leukemia (AML). This increase in incidence is tempered, however, by favorable overall survival rates of approximately 80%, whereas survival for non-DS children with similar leukemic subtypes is <35%. In this review, the clinical studies that have contributed to this overall high survival will be presented and their individual successes will be discussed. Important issues including intensity of treatment regimens, the role of bone marrow transplants and prognostic indicators will be reviewed. In particular, the roles of high- vs low- vs very low-dose cytarabine will be discussed, as well as potential therapeutic options in the future and the direction of the field over the next 5 years. In summary, children with DS and AML should be treated with a moderate-intensity cytarabine-based regimen with curative intent.
While we have considerable understanding of the transcriptional networks controlling mammalian cell differentiation, our knowledge of posttranscriptional regulatory events is very limited. Using differentiation of primary erythroid cells as a model, we show that the sequence-specific mRNA-binding protein Cpeb4 is strongly induced by the erythroid-important transcription factors Gata1 and Tal1 and is essential for terminal erythropoiesis. By interacting with the translation initiation factor eIF3, Cpeb4 represses the translation of a large set of mRNAs, including its own mRNA. Thus, transcriptional induction and translational repression combine to form a negative feedback loop to control Cpeb4 protein levels within a specific range that is required for terminal erythropoiesis. Our study provides an example of how translational control is integrated with transcriptional regulation to precisely control gene expression during mammalian cell differentiation.
Fetal stem cells isolated from umbilical cord blood (UCB) possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs) associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs) with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1) and 30 TFs were activated by day 56 (Profile-2). Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1) and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34+ stem cells including prolonged γ-globin expression combined with unique TFNs support novel mechanisms controlling the γ/β-globin switch during UCB-derived erythropoiesis.
GATA-1(low/low) mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG(-/-) mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA-1(low/low) mice have increased OPG levels. Here, we sought to determine whether GATA-1 knockdown in OPG(-/-) mice could rescue the observed osteoporotic bone phenotype. GATA-1(low/low) mice were bred with OPG(-/-) mice and bone phenotype assessed. GATA-1(low/low) × OPG(-/-) mice have increased cortical bone porosity, similar to OPG(-/-) mice. Both OPG(-/-) and GATA-1(low/low) × OPG(-/-) mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG(-/-) and GATA-1(low/low) × OPG(-/-) femurs are weaker and less stiff than C57BL/6 or GATA-1(low/low) femurs. Notably, GATA-1(low/low) × OPG(-/-) mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA-1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA-1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone.
We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature messenger RNA (mRNA) (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage, and surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth.
Infants with Down syndrome (DS) are at a high risk of developing transient abnormal myelopoiesis (TAM). A GATA1 mutation leading to the production of N-terminally truncated GATA1 (GATA1s) in early megakaryocyte/erythroid progenitors is linked to the onset of TAM and cooperated with the effect of trisomy 21 (Ts21). To gain insights into the underlying mechanisms of the progression to TAM in DS patients, we generated human pluripotent stem cells harbouring Ts21 and/or GATA1s by combining microcell-mediated chromosome transfer and genome editing technologies. In vitro haematopoietic differentiation assays showed that the GATA1s mutation blocked erythropoiesis irrespective of an extra chromosome 21, while Ts21 and the GATA1s mutation independently perturbed megakaryopoiesis and the combination of Ts21 and the GATA1s mutation synergistically contributed to an aberrant accumulation of skewed megakaryocytes. Thus, the DS model cells generated by these two technologies are useful in assessing how GATA1s mutation is involved in the onset of TAM in patients with DS.
β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.
Children with constitutional trisomy 21 (cT21, Down Syndrome, DS) are at a higher risk for both myeloid and B-lymphoid leukaemias. The myeloid leukaemias are often preceded by a transient neonatal pre-leukaemic syndrome, Transient Abnormal Myelopoiesis (TAM). TAM is caused by cooperation between cT21 and acquired somatic N-terminal truncating mutations in the key haematopoietic transcription factor GATA1. These mutations, which are not leukaemogenic in the absence of cT21, are found in almost one-third of neonates with DS. Analysis of primary human fetal liver haematopoietic cells and of human embryonic stem cells demonstrates that cT21 itself substantially alters human fetal haematopoietic development. Consequently, many haematopoietic developmental defects are observed in neonates with DS even in the absence of TAM. Although studies in mouse models have suggested a pathogenic role of deregulated expression of several chromosome 21-encoded genes, their role in human leukaemogenesis remains unclear. As cT21 exists in all embryonic cells, the molecular basis of cT21-associated leukaemias probably reflects a complex interaction between deregulated gene expression in haematopoietic cells and the fetal haematopoietic microenvironment in DS.
Wogonin, a flavonoid derived from Scutellaria baicalensis Georgi, has been demonstrated to be highly effective in treating hematologic malignancies. In this study, we investigated the anticancer effects of wogonin on K562 cells, K562 imatinib-resistant cells, and primary patient-derived CML cells. Wogonin up-regulated transcription factor GATA-1 and enhanced binding between GATA-1 and FOG-1, thereby increasing expression of erythroid-differentiation genes. Wogonin also up-regulated the expression of p21 and induced cell cycle arrest. Studies employing benzidine staining and analyses of cell surface markers glycophorin A (GPA) and CD71 indicated that wogonin promoted differentiation of K562, imatinib-resistant K562, and primary patient-derived CML cells. Wogonin also enhanced binding between GATA-1 and MEK, resulting in inhibition of the growth of CML cells. Additionally, in vivo studies showed that wogonin decreased the number of CML cells and prolonged survival of NOD/SCID mice injected with K562 and imatinib-resistant K562 cells. These data suggested that wogonin induces cycle arrest and erythroid differentiation in vitro and inhibits proliferation in vivo.
Changes in cis-regulatory element composition that result in novel patterns of gene expression are thought to be a major contributor to the evolution of lineage-specific traits. Although transcription factor binding events show substantial variation across species, most computational approaches to study regulatory elements focus primarily upon highly conserved sites, and rely heavily upon multiple sequence alignments. However, sequence conservation based approaches have limited ability to detect lineage-specific elements that could contribute to species-specific traits. In this paper, we describe a novel framework that utilizes a birth-death model to trace the evolution of lineage-specific binding sites without relying on detailed base-by-base cross-species alignments. Our model was applied to analyze the evolution of binding sites based on the ChIP-seq data for six transcription factors (GATA1, SOX2, CTCF, MYC, MAX, ETS1) along the lineage toward human after human-mouse common ancestor. We estimate that a substantial fraction of binding sites (∼58-79% for each factor) in humans have origins since the divergence with mouse. Over 15% of all binding sites are unique to hominids. Such elements are often enriched near genes associated with specific pathways, and harbor more common SNPs than older binding sites in the human genome. These results support the ability of our method to identify lineage-specific regulatory elements and help understand their roles in shaping variation in gene regulation across species.
The aim of the present study was to investigate the underlying molecular mechanisms of rheumatoid arthritis (RA) using microarray expression profiles from osteoarthritis and RA patients, to improve diagnosis and treatment strategies for the condition. The gene expression profile of GSE27390 was downloaded from Gene Expression Omnibus, including 19 samples from patients with RA (n=9) or osteoarthritis (n=10). Firstly, the differentially expressed genes (DEGs) were obtained with the thresholds of |logFC|>1.0 and P<0.05, using the t‑test method in LIMMA package. Then, differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) were screened with q<0.25 by the differential coexpression analysis and differential regulation analysis of gene expression microarray data package. Secondly, pathway enrichment analysis for DCGs was performed by the Database for Annotation, Visualization and Integrated Discovery and the DCLs associated with RA were selected by comparing the obtained DCLs with known transcription factor (TF)-targets in the TRANSFAC database. Finally, the obtained TFs were mapped to the known TF-targets to construct the network using cytoscape software. A total of 1755 DEGs, 457 DCGs and 101988 DCLs were achieved and there were 20 TFs in the obtained six TF-target relations (STAT3-TNF, PBX1‑PLAU, SOCS3-STAT3, GATA1-ETS2, ETS1-ICAM4 and CEBPE‑GATA1) and 457 DCGs. A number of TF-target relations in the constructed network were not within DCLs when the TF and target gene were DCGs. The identified TFs may have an important role in the pathogenesis of RA and have the potential to be used as biomarkers for the development of novel diagnostic and therapeutic strategies for RA.
Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.
Functional relationship networks, which summarize the probability of co-functionality between any two genes in the genome, could complement the reductionist focus of modern biology for understanding diverse biological processes in an organism. One major limitation of the current networks is that they are static, while one might expect functional relationships to consistently reprogram during the differentiation of a cell lineage. To address this potential limitation, we developed a novel algorithm that leverages both differentiation stage-specific expression data and large-scale heterogeneous functional genomic data to model such dynamic changes. We then applied this algorithm to the time-course RNA-Seq data we collected for ex vivo human erythroid cell differentiation.
Through computational cross-validation and literature validation, we show that the resulting networks correctly predict the (de)-activated functional connections between genes during erythropoiesis. We identified known critical genes, such as HBD and GATA1, and functional connections during erythropoiesis using these dynamic networks, while the traditional static network was not able to provide such information. Furthermore, by comparing the static and the dynamic networks, we identified novel genes (such as OSBP2 and PDZK1IP1) that are potential drivers of erythroid cell differentiation. This novel method of modeling dynamic networks is applicable to other differentiation processes where time-course genome-scale expression data are available, and should assist in generating greater understanding of the functional dynamics at play across the genome during development.
The network described in this article is available at http://guanlab.ccmb.med.umich.edu/stageSpecificNetwork.
Docking protein 2 (Dok2) is an adapter protein which is involved in hematopoiesis. However, it still remains unclear how Dok2 functions in regulation of transcription of hematopoietic genes. To address this issue, we knocked-down Dok2 mRNA in mouse erythroleukemia cells which highly express Dok2 intrinsically.
Mouse erythroleukemia cells were transfected with Dok2 siRNA for 24 h and gene expression of erythroid differentiation-related genes, such as GATA binding protein 1 (Gata1), Krüppel-like factor 1 (Klf1), α-globin and β-globin were assessed by real-time polymerase chain reaction.
Among the tested genes, expression of Klf1 exhibited a 1.94-fold increase when compared to the control 24 h after transfection. Immunocytochemistry and chromatin immunoprecipitation assays revealed that Dok2 protein localizes in the nucleus and binds to the promoter region of Klf1 gene.
Dok2 is able to control Klf1 expression by transcriptional regulation through directly binding to its promoter region.
Leukemia cell lines are utilized as tools for molecular analysis. Their implementation in therapy will require standards for quality control, including appropriate selection criteria for functional analysis and efficacy determination.
Characteristics of six human leukemia cell lines -Kasumi-1, NB-4, MOLM-13, MV-4-11, K562, and Jurkat cells-were investigated using multiple color analysis of surface antigen expression and comparative analysis of gene expression.
Differentiation states of Kasumi-1 and MOLM-13 cells are colony-forming units-granulocyte/macrophage equivalent cells to myeloblasts with comparatively high Growth factor independent-1(GFI1) and Transcription factor PU.1 (PU.1) expression, respectively. NB4 and MV-4-11 express high levels of CCAAT/enhancer-binding protein-alpha (CEBPα) and differentiate from myeloblasts to pro-monocytes and myeloblasts, respectively. K562 cells are colony-forming units-erythroid equivalent cells to erythroblasts, with the highest expression of GATA-binding factor 2 (GATA2), GATA1 and Friend of gata-1 (FOG1). Jurkat cells are pro-T to mature T-cells with the highest Neurogenic locus notch-1 homolog protein 1 (NOTCH1) expression.
Our study gives a useful guideline of standards for appropriate usage of leukemia cell lines for examining novel targets in vitro.
Growth factor independence 1 (Gfi-1) is a part of the transcriptional network that regulates the development of adult hematopoietic stem and progenitor cells. Gfi-1-null (Gfi-1(-/-)) mice have reduced numbers of hematopoietic stem cells (HSCs), impaired radioprotective function of hematopoietic progenitor cells (HPCs), and myeloid and erythroid hyperplasia. We found that the development of HPCs and erythropoiesis, but not HSC function, was rescued by reducing the expression of inhibitor of DNA-binding protein 2 (Id2) in Gfi-1(-/-) mice. Analysis of Gfi-1(-/-);Id2(+/-) mice revealed that short-term HSCs, common myeloid progenitors (CMPs), erythroid burst-forming units, colony-forming units in spleen, and more differentiated red cells were partially restored by reducing Id2 levels in Gfi-1(-/-) mice. Moreover, short-term reconstituting cells, and, to a greater extent, CMP and megakaryocyte-erythroid progenitor development, and red blood cell production (anemia) were rescued in mice transplanted with Gfi-1(-/-);Id2(+/-) bone marrow cells (BMCs) in comparison with Gfi-1(-/-) BMCs. Reduction of Id2 expression in Gfi-1(-/-) mice increased the expression of Gata1, Eklf, and EpoR, which are required for proper erythropoiesis. Reducing the levels of other Id family members (Id1 and Id3) in Gfi-1(-/-) mice did not rescue impaired HPC function or erythropoiesis. These data provide new evidence that Gfi-1 is linked to the erythroid gene regulatory network by repressing Id2 expression.
α-Synuclein is highly expressed in neural tissue and during erythropoiesis, where the key erythroid regulator GATA1 has been found to modulate its expression. While specific α-synuclein (SNCA) mutations are known to cause autosomal dominant familial Parkinson's disease, its wild-type function remains under debate. To investigate the role of α-synuclein in murine hematopoiesis and erythropoiesis, we utilized Snca knock-out mice and analyzed erythroid compartments for maturation defects, in vivo erythrocyte survival, and erythrocyte-based reactive oxygen species (ROS) and nitric oxide synthase (NOS) levels. Our findings show that while bone marrow and spleen erythropoiesis and peripheral blood erythrocyte survival in Snca(-/-) mice was comparable to controls, the levels of ROS and of NOS-2 were significantly decreased in mature erythrocytes in these animals. These results indicate a role for α-synuclein in regulating oxidative stress in erythrocytes in vivo and could open new avenues for the investigation of its function in non-neural tissue.
The JAK2V617F mutation is found in the majority of patients with myeloproliferative neoplasms (MPNs). Transgenic expression of the mutant gene causes MPN-like phenotypes in mice. We have produced JAK2V617F mice with p53 null background. Some of these mice developed acute erythroleukemia. From one of these mice, we derived a cell line designated J53Z1. J53Z1 cells were stained positive for surface markers CD71 and CD117 but negative for Sca-1, TER-119, CD11b, Gr-1, F4/80, CD11c, CD317, CD4, CD8a, CD3e, B220, CD19, CD41, CD42d, NK-1.1, and FceR1. Real time PCR analyses demonstrated expressions of erythropoietin receptor EpoR, GATA1, and GATA2 in these cells. J53Z1 cells grew rapidly in suspension culture containing fetal bovine serum with a doubling time of ∼18 hours. When transplanted into C57Bl/6 mice, J53Z1 cells induced acute erythroleukemia with massive infiltration of tumor cells in the spleen and liver. J53Z1 cells were responsive to stimulation with erythropoietin and stem cell factor and were selectively inhibited by JAK2 inhibitors which induced apoptosis of the cells. Together, J53Z1 cells belong to the erythroid lineage, and they may be useful for studying the role of JAK2V617F in proliferation and differentiation of erythroid cells and for identifying potential therapeutic drugs targeting JAK2.
In this issue of Blood, Gagne et al describe a cohort of 362 patients clinically classified as having Diamond-Blackfan anemia (DBA), in which 175 (48%) were found to have mutations and deletions in ribosomal protein genes or GATA1, and 8 of the remaining patients (2.2% overall) had mitochondrial gene deletions consistent with Pearson marrow-pancreas syndrome (PS). The authors propose that all patients with presumptive DBA should be tested for mitochondrial DNA (mtDNA) deletion during their initial genetic evaluation.
The clinical and molecular findings of 77 cases of neonatal leukemia (NL) and 380 of infant leukemia (IL) were selected to distinguish features between NL and IL. Somatic gene mutations associated with acute leukemia including FLT3, RAS and PTPN11 were revisited. There were 42 cases of congenital leukemia associated with Down syndrome (DS) and 39 of these cases presented features of acute myeloid leukemia (AML)-M7. Twenty-seven of the DS cases underwent spontaneous remission and were reclassified as a transient myeloproliferative disorder. GATA1 mutations were found in 70% of these cases. In non-DS, frequent abnormalities were MLL rearrangements, mainly MLL-AFF1 in acute lymphoblastic leukemia and MLL-MLLT3 in AML. The FLT3 mutation was not found, while RAS (n = 4) and PTPN11 (n = 2) mutations were identified and reported for the first time in NL. There was substantial evidence to support that somatic abnormalities occur in utero. Thus, congenital leukemia is a good model for understanding leukemogenesis.
Emerging evidence has shown that microRNAs have key roles in regulating various normal physiological processes, whereas their deregulated expression is correlated with various diseases. The miR-146 family includes miR-146a and miR-146b, with a distinct expression spectrum in different hematopoietic cells. Recent work indicated that miR-146a has a close relationship with inflammation and autoimmune diseases. miR-146-deficient mice have developed some abnormal hematopoietic phenotypes, suggesting the potential functions of miR-146 in hematopoietic development. In this study, we found that miR-146b was consistently up-regulated in both K562 and CD34(+) hematopoietic stem/progenitor cells (HSPCs) undergoing either erythroid or megakaryocytic differentiation. Remarkably, erythroid and megakaryocytic maturation of K562 cells was induced by excess miR-146b but inhibited by decreased miR-146b levels. More importantly, an mRNA encoding receptor tyrosine kinase, namely platelet-derived growth factor receptor α (PDGFRA), was identified and validated as a direct target of miR-146b in hematopoietic cells. Gain-of-function and loss-of-function assays showed that PDGFRA functioned as a negative regulator in erythroid and megakaryocytic differentiation. miR-146b could ultimately affect the expression of the GATA-1 gene, which is regulated by HEY1 (Hairy/enhancer-of-split related with YRPW motif protein 1), a transcriptional repressor, via inhibition of the PDGFRA/JNK/JUN/HEY1 pathway. Lentivirus-mediated gene transfer also demonstrated that the overexpression of miR-146b promoted erythropoiesis and megakaryocytopoiesis of HSPCs via its regulation on the PDGFRA gene and effects on GATA-1 expression. Moreover, we confirmed that the binding of GATA-1 to the miR-146b promoter and induction of miR-146b during hematopoietic maturation were dependent on GATA-1. Therefore, miR-146b, PDGFRA, and GATA-1 formed a regulatory circuit to promote erythroid and megakaryocytic differentiation.
A large gap exists in our understanding of the course of differentiation from mesoderm to definitive hematopoietic stem cells (HSCs). Previously, we reported that Runx1(+) cells in embryonic day 7.5 (E7.5) embryos contribute to the hemogenic endothelium in the E10.5 aorta-gonad-mesonephros (AGM) region and HSCs in the adult bone marrow. Here, we show that two Runx1(+) populations subdivided by Gata1 expression exist in E7.5 embryos. The hemogenic endothelium and the HSCs are derived only from the Runx1(+)Gata1(-) population. A subset of this population moves from the extra- to intraembryonic region during E7.5-E8.0, where it contributes to the hemogenic endothelium of the dorsal aorta (DA). Migration occurs before the heartbeat is initiated, and it is independent of circulation. This suggests a developmental trajectory from Runx1(+) cells in the E7.5 extraembryonic region to definitive HSCs via the hemogenic endothelium.
Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.
Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.
Patients with physical findings suggestive of Treacher Collins syndrome (TCS) or mandibulofacial dysostosis (MFD) and macrocytic anemia diagnostic of Diamond-Blackfan anemia (DBA) have been reported. Disease-causing genes have been identified for TCS and other MFDs. Mutations in several ribosomal protein genes and the transcription factor GATA1 result in DBA. However, no disease-causing mutation had been identified in the reported patients with the combination of TCS/MFD and DBA phenotype, and we hypothesized that pathogenic mutations in a single gene could be identified using whole exome analysis. We studied probands from six unrelated families. Combining exome analysis and Sanger sequencing, we identified likely pathogenic mutations in 5/6 families. Two mutations in unrelated families were seen in RPS26, the known DBA10 gene. One variant was predicted to affect mRNA splicing, and the other to lead to protein truncation. In another family a likely pathogenic X-linked mutation affecting a highly conserved residue was found in TSR2, which encodes a direct binding partner of RPS26. De novo mutations affecting the RPS28 start codon were found in two unrelated probands, identifying RPS28 as a novel disease gene. We conclude that the phenotype combining features of TCS with DBA is genetically heterogeneous. Each of the pathogenic variants identified is predicted to impede ribosome biogenesis, which in turn could result in altered cell growth and proliferation, causing abnormal embryologic development, defective erythropoiesis and reduced growth. The phenotype combining TCS/MFD and DBA is highly variable, overlaps with DBA and lies within the phenotypic spectrum of ribosomopathies. © 2014 Wiley Periodicals, Inc.
The somatic mutation theory of cancer proposes that cancer begins with a somatic mutation occurring at random in a single cell that then passes the mutation to its progeny, generating a clone of premalignant cells. This clone leads to a full malignant tumor through additional mutations and selection processes. Strikingly, the best-documented human model of early oncogenesis, i.e., transient myeloproliferative disorder followed by acute megakaryoblastic leukemia (AMKL) in infants with Down syndrome (DS, or trisomy 21), exhibits important discrepancies with the SMT. Somatic mutations in megakaryocytic precursors occur at least 100,000 times more frequently in the GATA1 gene in fetuses with DS compared to the general population. Further, mutations are limited to GATA1 only; the general mutation rate does not significantly differ between individuals with DS and euploid individuals. Importantly, the mutations are also lineage-specific, occurring only in the megakaryocytic lineage, and proliferative anomalies of the megakaryocytic lineage are observed before the occurrence of GATA1 mutations. Thus, GATA1 mutations in fetuses with DS cannot be random events occurring in normal cells. Here, transcription-associated mutagenesis is proposed as the mechanism by which the earliest mutations of AMKL occur in DS. Transcription-associated mutagenesis is observed in non-dividing cells when a gene is over-expressed. The over-expression of GATA1 in the megakaryocytic lineage in DS fetal liver cells is proposed to be the cause of targeted GATA1 somatic mutations. As transcription-associated mutagenesis is a universal process, this mechanism may also apply to early oncogenesis in other situations, including after birth and following exposure to a carcinogenic agent. Thus, this hypothesis represents a new avenue for understanding and exploring oncogenesis in the context of DS and in other disease states.
C/EBPα is expressed preferentially in myeloid compared with lymphoid or erythroid cells and directs myeloid lineage specification. C/EBPα is also expressed at lower levels in HSCs and in several nonhematopoietic tissues. The Cebpa gene has a conserved, 450-bp segment at +37 kb that harbors enhancer-specific epigenetic marks and is activate in a myeloid cell line. Herein, we characterize transgenic C57BL/6 mice, in which the Cebpa enhancer and 845-bp promoter regulate a hCD4 reporter. FACS analysis, in vitro colony assays, and in vivo competitive and secondary transplantation revealed that myeloid but not MEPs or lymphoid progenitors and also functional LT-HSCs are found almost exclusively in the Cebpa-hCD4(+) compared with hCD4(-) marrow population. hCD4(+) CMP yielded predominantly myeloid, whereas hCD4(-) CMP generated mainly Meg/E colonies. Providing insight into control of CMP maturation, Cebpa and Pu.1 RNAs were preferentially expressed in hCD4(+) CMP, Scl, Gata2, Gata1, Klf1, Ets1, and Fli1 predominated in hCD4(-) CMP, and Runx1, Myb, HoxA9, and Erg levels were similar in both. Cebpa-hCD4 transgene expression was lacking in multiple nonhematopoietic tissues. In summary, the +37-kb Cebpa enhancer and promoter are sufficient for marrow myeloid progenitor and LT-HSC-specific expression.
Transient myeloproliferative disorder (TMD) is a clonal proliferation of megakaryoblasts, typically occurring in newborns with Down syndrome. It is believed that TMD occurs in the presence of GATA1 mutation together with trisomy 21. However, a limited number of patients with TMD but without Down syndrome have been reported, all with a blast population with numeric or rarely structural chromosome 21 abnormalities. We present the first case of a newborn boy with a TMD without trisomy 21 and without any of the mentioned molecular or cytogenetic abnormalities. This case report suggests that unknown disease mechanisms may provoke or mimic TMD. This case report is followed by a concise review of the literature discussing the different entities and pathomechanisms of TMD and acute megakaryocytic leukaemia in patients with or without Down syndrome.
The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation.
The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)-V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1(-/-) double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1(+/+) mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit-erythroid and reduced colony-forming unit-megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.
Hematopoiesis is an exquisitely regulated process in which stem cells in the developing embryo and the adult generate progenitor cells that give rise to all blood lineages. Master regulatory transcription factors control hematopoiesis by integrating signals from the microenvironment and dynamically establishing and maintaining genetic networks. One of the most rudimentary aspects of cell type-specific transcription factor function, how they occupy a highly restricted cohort of cis-elements in chromatin, remains poorly understood. Transformative technologic advances involving the coupling of next-generation DNA sequencing technology with the chromatin immunoprecipitation assay (ChIP-seq) have enabled genome-wide mapping of factor occupancy patterns. However, formidable problems remain; notably, ChIP-seq analysis yields hundreds to thousands of chromatin sites occupied by a given transcription factor, and only a fraction of the sites appear to be endowed with critical, non-redundant function. It has become en vogue to map transcription factor occupancy patterns genome-wide, while using powerful statistical tools to establish correlations to inform biology and mechanisms. With the advent of revolutionary genome editing technologies, one can now reach beyond correlations to conduct definitive hypothesis testing. This review focuses on key discoveries that have emerged during the path from single loci to genome-wide analyses, specifically in the context of hematopoietic transcriptional mechanisms.
Growth factor independence 1b (GFI1B) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b, we used conditionally deficient mice that harbor floxed Gfi1b alleles and inducible (Mx-Cre, Cre-ERT) or erythroid specific (EpoR-Cre) Cre expressing transgenes. In contrast to the germline knockout, EpoR-Cre mediated erythroid specific ablation of Gfi1b allows full gestation, but causes perinatal lethality with very few mice surviving to adulthood. Both the embryonic deletion of Gfi1b by EpoR-Cre and the deletion in adult mice by Mx-Cre or Cre-ERT leads to reduced numbers of erythroid precursors, perturbed and delayed erythroid maturation, anemia and extramedullary erythropoiesis. Global expression analyses showed that the Hba-x, Hbb-bh1 and Hbb-y embryonic globin genes were upregulated in Gfi1b deficient TER119+ fetal liver cells over the gestation period from day 12.5-17.5 p.c. and an increased level of Hbb-bh1 and Hbb-y embryonic globin gene expression was even maintained in adult Gfi1b deficient mice. While the expression of Bcl11a, a regulator of embryonic globin expression was not affected by Gfi1b deficiency, the expression of Gata1 was reduced and the expression of Sox6, also involved in globin switch, was almost entirely lost when Gfi1b was absent. These findings establish Gfi1b as a regulator of embryonic globin expression and embryonic and adult erythroid maturation.
We report a rare case of transient abnormal myelopoiesis (TAM) in a phenotypically normal neonate. The presence of a palpable hepatomegaly prompted in-depth laboratory tests, which revealed the presence of severe hyperleukocytosis, with blast cells present in a peripheral blood smear. Although no signs of Down syndrome were present, we suspected TAM. Further analysis identified a mutation in GATA1 along with the unique finding of two different trisomic cell lines, detected upon karyotyping; one with trisomy 21 only, and one with trisomies 21 and 22, which was present in a subpopulation of peripheral blood cells. These genetic abnormalities disappeared by the age of 6 months. The presence of two different trisomic clones may be an evidence of the polyclonal nature of TAM in this patient.
Abstract Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating-circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin-coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)-R1/2, VE-cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF-R1/2, VE-cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium-related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran-treated EPCs; however, those of hematopoietic- and antiangiogenic-related transcription factors TAL1, RUNX1, c-MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran-exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.
Trisomy 21 alters fetal liver hematopoiesis and, in combination with somatic globin transcription factor 1 (GATA1) mutations, leads to development of transient myeloproliferative disease in newborns. However, little is known about the morphological hematopoietic changes caused by trisomy 21 in the fetus, and to date, the exact onset of GATA1 mutations remains uncertain. Therefore, we analyzed fetal liver hematopoiesis from second trimester pregnancies in trisomy 21 and screened for GATA1 mutations. We examined 57 formalin-fixed and paraffin-embedded fetal liver specimens (49 harboring trisomy 21 and 8 controls) by immunohistochemistry for CD34, CD61, factor VIII, and glycophorin A. GATA1 exon 2 was sequenced in fetal livers and corresponding nonhematologic tissue. Cell counts of megakaryocytes (P = .022), megakaryocytic precursors (P = .021), and erythroid precursors were higher in trisomy 21 cases. CD34-positive hematopoietic blasts showed no statistically significant differences. No mutation was detected by GATA1 exon 2 sequencing in fetal livers from 12 to 25 weeks of gestation. Our results suggest that GATA1 exon 2 mutations occur late in trisomy 21 fetal hematopoiesis. However, trisomy 21 alone provides a proliferative stimulus of fetal megakaryopoiesis and erythropoiesis. CD34-positive precursor cells are not increased in trisomy 21 fetal livers.
Erythroid differentiation-associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34(+) cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34(+) cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO-treated CD34(+) cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG-induced erythroid differentiation. ChIP-PCR experiments confirmed that GATA1 and EDAG co-occupy GATA1-targeted genes in primary erythroid cells and in vivo. ChIP-seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP-Seq and GATA1 ChIP-Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1-activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation.
Pearson marrow pancreas syndrome (PS) is a multisystem disorder caused by mitochondrial DNA (mtDNA) deletions. Diamond-Blackfan anemia (DBA) is a congenital hypoproliferative anemia in which mutations in ribosomal protein genes and GATA1 have been implicated. Both syndromes share several features including early onset of severe anemia, variable nonhematologic manifestations, sporadic genetic occurrence, and occasional spontaneous hematologic improvement. Because of the overlapping features and relative rarity of PS, we hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. Here, we evaluated patient DNA samples submitted for DBA genetic studies and found that 8 (4.6%) of 173 genetically uncharacterized patients contained large mtDNA deletions. Only 2 (25%) of the patients had been diagnosed with PS on clinical grounds subsequent to sample submission. We conclude that PS can be overlooked, and that mtDNA deletion testing should be performed in the diagnostic evaluation of patients with congenital anemia.
GATA1-related cytopenia is characterized by thrombocytopenia and/or anemia ranging from mild to severe. One or more of the following may also be present: platelet dysfunction, mild β-thalassemia, neutropenia, and congenital erythropoietic porphyria (CEP) in males. Thrombocytopenia typically presents in infancy as a bleeding disorder with easy bruising and mucosal bleeding (e.g., epistaxis). Anemia ranges from minimal (mild dyserythropoiesis) to severe (hydrops fetalis requiring in utero transfusion). At the extreme end of the clinical spectrum, severe hemorrhage and/or erythrocyte transfusion dependence are life long; at the milder end, anemia and the risk for bleeding decrease spontaneously with age. Female carriers may have mild to moderate symptoms, such as menorrhagia.
Diagnostic laboratory findings usually include macrothrombocytopenia (low number of platelets that are larger than normal) and/or anemia with red cell indices that may be micro-, normo- or macrocytic. Defects in platelet aggregation in response to agonists may be seen. In some cases electron microscopy reveals reduced numbers of platelet alpha granules and dysplastic features in platelets and megakaryocytes. GATA1 is the only gene in which pathogenic variants are known to cause GATA1-related cytopenia.
Treatment of manifestations: Platelet transfusions for moderate to severe epistaxis, gingival bleeding, or gastrointestinal bleeding; no specific treatment for mild symptoms (easy bruisability); erythrocyte transfusions when anemia is symptomatic (fatigue, tachycardia). Prevention of primary manifestations: For severe cases, bone marrow transplantation (BMT) can be curative. Surveillance: monitoring complete blood counts (with frequency depending on disease severity) to inform supportive care; monitoring those undergoing repeated erythrocyte transfusions for iron overload. Agents/circumstances to avoid: Those with thrombocytopenia should avoid antiplatelet agents including aspirin and nonsteroidal anti-inflammatory agents (NSAIDs) (e.g., ibuprofen). Those with thrombocytopenia and/or platelet aggregation defects should avoid contact sports or activities with a high risk of trauma. Evaluation of relatives at risk: If a GATA1 pathogenic variant has been identified in the family, complete blood counts and molecular genetic testing of at-risk relatives can be offered. At-risk relatives who choose not to have molecular genetic testing should have complete blood counts to evaluate for thrombocytopenia, anemia, or neutropenia. Pregnancy management: For pregnancies in which a male fetus is known to be affected: monitoring by a high-risk obstetric practice; serial specialized ultrasounds to screen for signs of fetal anemia or bleeding; in utero transfusion if the affected fetus is severely anemic.
GATA1-related cytopenia is inherited in an X-linked manner. If the mother of an affected male has a GATA1 pathogenic variant, the chance of transmitting it in each pregnancy is 50%. Affected males pass the pathogenic variant to all of their daughters and none of their sons. Carrier testing for at-risk family members and prenatal testing for pregnancies at increased risk are possible once the pathogenic variant has been identified in the family.
Protein tyrosine phosphatases (PTPs) are involved in hematopoiesis, but the function of many PTPs is not well characterized in vivo. Here, we have identified Ptpn9a, an ortholog of human PTPN9, as a crucial regulator of erythroid cell development in zebrafish embryos. ptpn9a, but not ptpn9b, was expressed in the posterior lateral plate mesoderm and intermediate cell mass - two primitive hematopoietic sites during zebrafish embryogenesis. Morpholino-mediated knockdown of ptpn9a caused erythrocytes to be depleted by inhibiting erythroid cell maturation without affecting erythroid proliferation and apoptosis. Consistently, both dominant-negative PTPN9 (with mutation C515S) and siRNA against PTPN9 inhibited erythroid differentiation in human K562 cells. Mechanistically, depletion of ptpn9 in zebrafish embryos in vivo or in K562 cells in vitro increased phosphorylated STAT3, and the hyper-phosphorylated STAT3 entrapped and prevented the transcription factors GATA1 and ZBP-89 (also known as ZNF148) from regulating erythroid gene expression. These findings imply that PTPN9 plays an important role in erythropoiesis by disrupting an inhibitory complex of phosphorylated STAT3, GATA1 and ZBP-89, providing new cellular and molecular insights into the role of ptpn9a in developmental hematopoiesis.
Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86(-) CD150(+) CD48(-) HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150(HI) CD86(-) CD18(L)°CD41(+) and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation.
Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome. As with the other rare inherited bone marrow failure syndromes, the study of these disorders provides important insights into basic biology and, in the case of DBA, ribosome biology; the disruption of which characterizes the disorder. Thus DBA serves as a paradigm for translational medicine in which the efforts of clinicians to manage DBA have informed laboratory scientists who, in turn, have stimulated clinical researchers to utilize scientific discovery to provide improved care. In this review we describe the clinical syndrome Diamond Blackfan anemia and, in particular, we demonstrate how the study of DBA has allowed scientific inquiry to create opportunities for progress in its understanding and treatment.
Most heritable anemias are caused by mutations in genes encoding globins, red blood cell (RBC) membrane proteins, or enzymes in the glycolytic and hexose monophosphate shunt pathways. A less common class of genetic anemia is caused by mutations that alter the functions of erythroid transcription factors (TFs). Many TF mutations associated with heritable anemia cause truncations or amino acid substitutions, resulting in the production of functionally altered proteins. Characterization of these mutant proteins has provided insights into mechanisms of gene expression, hematopoietic development, and human disease. Mutations within promoter or enhancer regions that disrupt TF binding to essential erythroid genes also cause anemia and heritable variations in RBC traits, such as fetal hemoglobin content. Defining the latter may have important clinical implications for de-repressing fetal hemoglobin synthesis to treat sickle cell anemia and β thalassemia. Functionally important alterations in genes encoding TFs or their cognate cis elements are likely to occur more frequently than currently appreciated, a hypothesis that will soon be tested through ongoing genome-wide association studies and the rapidly expanding use of global genome sequencing for human diagnostics. Findings obtained through such studies of RBCs and associated diseases are likely generalizable to many human diseases and quantitative traits.
The high-affinity IgE receptor, FcεRI, which is composed of α-, β-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of β mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIβ). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIβ, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIβ transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.
GATA transcription factor family members GATA1 and GATA2 play crucial roles in the regulation of lineage-restricted genes during erythroid differentiation. GATA1 is indispensable for survival and terminal differentiation of erythroid, megakaryocytic and eosinophilic progenitors, whereas GATA2 regulates proliferation and maintenance of hematopoietic stem and progenitor cells. Expression levels of GATA1 and GATA2 are primarily regulated at the transcriptional level through auto- and reciprocal regulatory networks formed by these GATA factors. The dynamic and strictly controlled change of expression from GATA2 to GATA1 during erythropoiesis has been referred to as GATA factor switching, which plays a crucial role in erythropoiesis. The regulatory network comprising GATA1 and GATA2 gives rise to the stage-specific changes in Gata1 and Gata2 gene expression during erythroid differentiation, which ensures specific expression of early and late erythroid genes at each stage. Recent studies have also shed light on the genome-wide binding profiles of GATA1 and GATA2, and the significance of epigenetic modification of Gata1 gene during erythroid differentiation. This review summarizes the current understanding of network regulation underlying stage-dependent Gata1 and Gata2 gene expressions and the functional contribution of these GATA factors in erythroid differentiation.
Some Kampo medicines that are herbal and traditional in Japan have had beneficial effects when given to patients with anemia. However, molecular mechanisms underlying their effects are unclear. To address this question, four Kampo medicines used to treat anemia-ninjin'yoeito (NYT), shimotsuto (SMT), juzentaihoto (JTT), and daibofuto (DBT)-were tested separately using in vitro cultures of mouse bone marrow mononuclear cells. Among them, NYT was most effective in stimulating cell proliferation and up-regulating Myc expression. Flow cytometry analysis indicated that, among hematopoietic components of those cultures, myeloid cells expressing CD45/Mac-1/Gr-1/F4/80 increased in number, but Ter119/CD71 erythroid cells did not. Accordingly, real-time PCR analysis showed up-regulation of the myeloid gene Pu.1, whereas the erythroid genes Gata1 and Klf1 were down-regulated. Overall, these findings provide molecular evidence that NYT accelerates myelopoiesis but not erythropoiesis in vitro.
Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1(-/y)), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1(-/y) bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5' end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit(+) FcεRIα(+) mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.
MicroRNAs (miRNAs) represent a class of small non-coding regulatory RNAs that play important roles in normal hematopoiesis, including erythropoiesis. Although studies have identified several miRNAs that regulate erythroid commitment and differentiation, we do not understand the mechanism by which the crucial erythroid transcription factors, GATA-1and NF-E2 directly regulate and control differentiation via miRNA pathways. In this study, we identified miR-199b-5p as a key regulator of human erythropoiesis, and its expression was up-regulated during the erythroid differentiation of K562 cells. Furthermore, the increase of miR-199b-5p in erythroid cells occurred in a GATA-1- and NF-E2-dependent manner during erythrocyte maturation. Both GATA-1 and NF-E2 bound upstream of the miR-199b gene locus and activated its transcription. Forced expression of miRNA-199b-5p in K562 cells affected erythroid cell proliferation and maturation. Moreover, we identified c-Kit as a direct target of miR-199b-5p in erythroid cells. Taken together, our results establish a functional link among the erythroid transcription factors GATA-1/NF-E2, miR-199b-5p and c-Kit, and provide new insights into the coupling of transcription and post-transcription regulation in erythroid differentiation.
Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.
To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+β) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.
Although endocrine therapy impedes estrogen-ER signaling pathway and thus reduces breast cancer mortality, patients remain at continued risk of relapse after tamoxifen or other endocrine therapies. Understanding the mechanisms of endocrine resistance, particularly the role of transcriptional regulation is very important and necessary.
We propose a two-step workflow based on linear model to investigate the significant differences between MCF7 and OHT cells stimulated by 17β-estradiol (E2) respect to regulatory transcription factors (TFs) and their interactions. We additionally compared predicted regulatory TFs based on RNA polymerase II (PolII) binding quantity data and gene expression data, which were taken from MCF7/MCF7+E2 and OHT/OHT+E2 cell lines following the same analysis workflow. Enrichment analysis concerning diseases and cell functions and regulatory pattern analysis of different motifs of the same TF also were performed.
The results showed PolII data could provide more information and predict more recognizably important regulatory TFs. Large differences in TF regulatory mode were found between two cell lines. Through verified through GO annotation, enrichment analysis and related literature regarding these TFs, we found some regulatory TFs such as AP-1, C/EBP, FoxA1, GATA1, Oct-1 and NF-κB, maintained OHT cells through molecular interactions or signaling pathways that were different from the surviving MCF7 cells. From TF regulatory interaction network, we identified E2F, E2F-1 and AP-2 as hub-TFs in MCF7 cells; whereas, in addition to E2F and E2F-1, we identified C/EBP and Oct-1 as hub-TFs in OHT cells. Notably, we found the regulatory patterns of different motifs of the same TF were very different from one another sometimes.
We inferred some regulatory TFs, such as AP-1 and NF-κB, cooperated with ER through both genomic action and non-genomic action. The TFs that were involved in both protein-protein interactions and signaling pathways could be one of the key resistant mechanisms of endocrine therapy and thus also could be new treatment targets for endocrine resistance. Our flexible workflow could be integrated into an existing analytical framework and guide biologists to further determine underlying mechanisms in human diseases.
Hidden Markov models (HMMs) are flexible and widely used in scientific studies. Particularly in genomics and genetics, there are multiple distinct regimes in the genome within each of which the relationships among multivariate features are distinct. Examples include differential gene regulation depending on gene functions and experimental conditions, and varying combinatorial patterns of multiple transcription factors. We developed a software package called MRHMMs (Multivariate Regression Hidden Markov Models and the variantS) that accommodates a variety of HMMs that can be flexibly applied to many biological studies and beyond. MRHMMs supplements existing HMM software packages in two aspects. First, MRHMMs provides a diverse set of emission probability structures, including mixture of multivariate normal distributions and (logistic) regression models. Second, MRHMMs is computationally efficient for analyzing large data-sets generated in current genome-wide studies. Especially, the software is written in C for the speed advantage and further amenable to implement alternative models to meet users' own purposes.
http://sourceforge.net/projects/mrhmms/
Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.
Mammals express thousands of long noncoding (lnc) RNAs, a few of which are known to function in tissue development. However, the entire repertoire of lncRNAs in most tissues and species is not defined. Indeed, most lncRNAs are not conserved, raising questions about function. We used RNA sequencing to identify 1109 polyadenylated lncRNAs expressed in erythroblasts, megakaryocytes, and megakaryocyte-erythroid precursors of mice, and 594 in erythroblasts of humans. More than half of these lncRNAs were unannotated, emphasizing the opportunity for new discovery through studies of specialized cell types. Analysis of the mouse erythro-megakaryocytic polyadenylated lncRNA transcriptome indicates that ~75% arise from promoters and 25% from enhancers, many of which are regulated by key transcription factors including GATA1 and TAL1. Erythroid lncRNA expression is largely conserved among 8 different mouse strains, yet only 15% of mouse lncRNAs are expressed in humans and vice versa, reflecting dramatic species-specificity. RNA interference assays of 21 abundant erythroid-specific murine lncRNAs in primary mouse erythroid precursors identified 7 whose knockdown inhibited terminal erythroid maturation. At least 6 of these 7 functional lncRNAs have no detectable expression in human erythroblasts, suggesting that lack of conservation between mammalian species does not predict lack of function.
Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC.
Red blood cells (RBCs), which constitute the most abundant cell type in the body, come in two distinct flavors- primitive and definitive. Definitive RBCs in mammals circulate as smaller, anucleate cells during fetal and postnatal life, while primitive RBCs circulate transiently in the early embryo as large, nucleated cells before ultimately enucleating. Both cell types are formed from lineage-committed progenitors that generate a series of morphologically identifiable precursors that enucleate to form mature RBCs. While definitive erythroid precursors mature extravascularly in the fetal liver and postnatal marrow in association with macrophage cells, primitive erythroid precursors mature as a semi-synchronous cohort in the embryonic bloodstream. While the cytoskeletal network is critical for the maintenance of cell shape and the deformability of definitive RBCs, little is known about the components and function of the cytoskeleton in primitive erythroblasts. Erythropoietin (EPO) is a critical regulator of late-stage definitive, but not primitive, erythroid progenitor survival. However, recent studies indicate that EPO regulates multiple aspects of terminal maturation of primitive murine and human erythroid precursors, including cell survival, proliferation, and the rate of terminal maturation. Primitive and definitive erythropoiesis share central transcriptional regulators, including Gata1 and Klf1, but are also characterized by the differential expression and function of other regulators, including myb, Sox6, and Bcl11A. Flow cytometry-based methodologies, developed to purify murine and human stage-specific erythroid precursors, have enabled comparative global gene expression studies and are providing new insights into the biology of erythroid maturation.
TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the β-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the β-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the (G)γ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the β-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the (G)γ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes.
In this issue of Blood, Alvarez-Dominguez et al use a combination of genomics technology, bioinformatics, and functional analyses to provide new insights into our understanding of the role of long noncoding RNAs (lncRNAs) in erythropoiesis.1 This is an initial step forward in our understanding of the many roles of lncRNAs in normal and perturbed erythropoiesis. lncRNAs have recently emerged as critical, multifunctional regulators of cellular gene expression.
Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform.
Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hbαe1, hbβe1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3'-UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1.
Studies implicate a potential role for EAF1 in MLL-ELL induced leukemogenesis; however the biological function of EAF1 in this process remains unknown. In this study, we show that knockdown of zebrafish eaf1 by morpholinos caused serious defects in both primitive and definitive hematopoiesis. Using microarray analysis, we identified foxo3b as a target gene suppressed by eaf1. Ectopic expression of foxo3b in embryos mimicked the phenotypes exhibited in eaf1 morphants, except that foxo3b had no effect on runx1 and c-myb expression while eaf1 morphants did not express these markers in the ventral wall of dorsal aorta. Subsequent experiments showed that a dominant negative form of Foxo3b (dn-foxo3b) partially restored primitive hematopoietic defects in eaf1 morphants, suggesting that foxo3b might serve as a key factor for mediating eaf1 function in primitive hematopoiesis. Furthermore, we observed that foxo3b inhibited the transcriptional activity of gata1 and spi1 through protein-protein interaction. Our findings not only suggest a function of eaf1 on hematopoiesis in vivo, but also reveal a novel regulatory pathway, eaf1-foxo3b-gata1/spi1, that may shed light on the role of EAF1 in MLL-ELL induced leukemogenesis.
Epigenetic modifications introduce an additional layer of regulation that drastically expands the instructional capability of the human genome. The regulatory consequences of DNA methylation is context dependent; it can induce, enhance, and suppress gene expression, or have no effect on gene regulation. Therefore, it is essential to account for the genomic location of its occurrence and the protein factors it associates with to improve our understanding of its function and effects. Here, we use ENCODE ChIP-seq and DNase I hypersensitivity data, along with large-scale breast cancer genomic data from The Cancer Genome Atlas (TCGA) to computationally dissect the intricacies of DNA methylation in regulation of cancer transcriptomes. In particular, we identified a relationship between estrogen receptor α (ERα) activity and DNA methylation patterning in breast cancer. We found compelling evidence that methylation status of DNA sequences at ERα binding sites is tightly coupled with ERα activity. Furthermore, we predicted several transcription factors including FOXA1, GATA1, and SUZ12 to be associated with breast cancer by examining the methylation status of their binding sites in breast cancer. Lastly, we determine that methylated CpGs highly correlated with gene expression are enriched in regions 1kb or more downstream of TSSs, suggesting more significant regulatory roles for CpGs distal to gene TSSs. Our study provides novel insights into the role of ERα in breast cancers.
The RNA-binding protein Elavl1 (also known as HuR) regulates gene expression at the posttranscriptional level. Early embryonic lethality of the mouse knockout challenges investigation into hematopoietic functions for Elavl1. We identified 2 zebrafish elavl1 genes, designated elavl1a (the predominant isoform during embryogenesis) and elavl1b. Knockdown of Elavl1a using specific morpholinos resulted in a striking loss of primitive embryonic erythropoiesis. Transcript levels for early hematopoietic regulatory genes including lmo2 and scl are unaltered, but levels of gata1 transcripts, encoding a key erythroid transcription factor, are significantly reduced in elavl1a morphants. Other mesoderm markers are mostly unchanged by depletion of Elav1a. The 3'-untranslated region (UTR) of gata1 contains putative Elavl1a-binding sites that support robust expression levels when fused to a transfected luciferase reporter gene, and Elavl1a binds the gata1 3'-UTR sequences in a manner dependent on these sites. Moreover, expression of a transgenic reporter specifically in developing embryonic erythroid cells is enhanced by addition of the gata1 3'UTR with intact Elavl1-binding sites. Injection of gata1 messenger RNA partially rescues the erythropoiesis defect caused by Elavl1 knockdown. Our study reveals a posttranscriptional regulatory mechanism by which RNA-binding protein Elavl1a regulates embryonic erythropoiesis by maintaining appropriate levels of gata1 expression.
GATA1 mutations are tightly associated with transient myeloproliferative disorder (TMD) and acute megakaryoblstic leukemia (AMKL) in children with Down syndrome. Numerous genes are altered in GATA-1-deficient megakaryocytes, which may contribute to the hyperproliferation and abnormal terminal differentiation of these malignant cells. In this study, we demonstrate that Pstpip2 is a GATA-1-repressed gene that controls megakaryopoiesis. Ectopic expression of PSTPIP2 impaired megakaryocytic differentiation as evidenced by a decrease of CD41 expression and reduced DNA content in K562 cells. PSTPIP2 overexpression also caused enhanced activation of Src family kinases and subsequently reduced ERK phosphorylation. Consistently, PSTPIP2 knockdown showed the opposite effect on differentiation and signaling. Moreover, the W232A mutant of PSTPIP2, defective in its interaction with PEST family phosphatases that recruit c-Src terminal kinase (CSK) to suppress Src family kinases, failed to inhibit differentiation and lost its ability to enhance Src family kinases or reduce ERK phosphorylation. In fact, the W232A mutant of PSTPIP2 promoted megakaryocyte differentiation. These observations suggest that PSTPIP2 recruiting PEST phosphatases somehow blocked CSK activity and led to enhanced activation of Src family kinases and reduced ERK phosphorylation, which ultimately repressed megakaryocyte differentiation. Supporting this idea, PSTPIP2 interacted with LYN and the expression of a dominant negative LYN (LYN DN) overwhelmed the inhibitory effect of PSTPIP2 on differentiation and ERK signaling. In addition, a constitutively active LYN (LYN CA) normalized the enhanced megakaryocyte differentiation and repressed ERK signaling in PSTPIP2 knockdown cells. Finally, we found that PSTPIP2 repressed ERK signaling, differentiation, and proliferation and verified that PSTPIP2 upregulation repressed megakaryocyte development in primary mouse bone marrow cells. Our study thus reveals a novel mechanism by which dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte proliferation and differentiation in pathogenesis of related diseases.
Isolated congenital asplenia is characterized by the absence of a spleen at birth without any other developmental defect. Isolated congenital asplenia is a rare and life-threatening disease that predisposes patients to severe bacterial infections. The first and main genetic etiology of isolated congenital asplenia was discovered in 2013. Mutations in the gene RPSA, which encodes ribosomal protein SA, cause more than half of the cases of isolated congenital asplenia. These disease-causing mutations lead to haploinsufficiency of RPSA. Haploinsufficiency of genes encoding other ribosomal proteins have been reported to cause other developmental defects in humans, and in model organisms like the fly or the mouse. About half of the patients with Diamond-Blackfan anemia, which is a well-characterized ribosomopathy, present developmental defects such as craniofacial defects, cardiac defects or thumb abnormalities. The mechanism of pathogenesis linking mutations in ribosomal proteins, which are highly and ubiquitously expressed, to specific developmental defects remains to be elucidated. One hypothesis is that the ribosome, and ribosomal proteins in particular, regulate the expression of specific genes during development.
The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.
Megakaryocyte morphogenesis employs a "hypertrophy-like" developmental program that is dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback-regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large, inactive 7SK small nuclear ribonucleoprotein particle (snRNP) complexes. Here, we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis that is mediated by dissolution of the 7SK snRNP complex. In this pathway, calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here, we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.
GATA1-PU.1 genetic switch is a paradigmatic genetic switch that governs the differentiation of progenitor cells into two different fates, erythroid and myeloid fates. In terms of dynamical model representation of these fates or lineages corresponds to stable attractor and choosing between the attractors. Small asymmetries and stochasticity intrinsically present in all genetic switches lead to the effect of delayed bifurcation which will change the differentiation result according to the timing of the process and affect the proportion of erythroid versus myeloid cells. We consider the differentiation bifurcation scenario in which there is a symmetry-breaking in the bifurcation diagrams as a result of asymmetry in external signaling. We show that the decision between two alternative cell fates in this structurally symmetric decision circuit can be biased depending on the speed at which the system is forced to go through the decision point. The parameter sweeping speed can also reduce the effect of asymmetry and produce symmetric choice between attractors, or convert the favorable attractor. This conversion may have important contributions to the immune system when the bias is in favor of the attractor which gives rise to non-immune cells.
The selective and temporal DNA methylation plays an important role in the self-renewal and differentiation of hematopoietic stem cells (HSCs), but the molecular mechanism that controls the dynamics of DNA methylation is not understood. Here, we report that the PIAS1 epigenetic pathway plays an important role in regulating HSC self-renewal and differentiation. PIAS1 is required for maintaining the quiescence of dormant HSCs and the long-term repopulating capacity of HSC. Pias1 disruption caused the abnormal expression of lineage-associated genes. Bisulfite sequencing analysis revealed the premature promoter demethylation of Gata1, a key myeloerythroid transcription factor and a PIAS1-target gene, in Pias1(-/-) HSCs. As a result, Pias1 disruption caused the inappropriate induction of Gata1 in HSCs and common lymphoid progenitors (CLPs). The expression of other myeloerythroid genes was also enhanced in CLPs and lineage-negative progenitors, with a concurrent repression of B cell-specific genes. Consistently, Pias1 disruption caused enhanced myeloerythroid, but reduced B lymphoid lineage differentiation. These results identify a novel role of PIAS1 in maintaining the quiescence of dormant HSCs and in the epigenetic repression of the myeloerythroid program.
ETV7 is a human oncoprotein that cooperates with Eμ-MYC to promote pre-B-cell leukemia in mice. It is normally expressed in the bone marrow and fetal liver and is upregulated in primary leukemia, suggesting that it is involved in proper hematopoiesis and leukemogenesis. ETV7 has been deleted in most rodents, but is conserved in all other vertebrates, including the zebrafish, Danio rerio. In this report, we characterize the function of the zebrafish etv7 gene during erythropoiesis. Our results demonstrate that etv7 regulates the expression of the zebrafish lanosterol synthase (lss) gene, an essential gene in the cholesterol synthesis pathway. Furthermore, morpholino knockdown of etv7 leads to loss of hemoglobin-containing red blood cells, a phenotype that can be rescued by injection of exogenous cholesterol. We conclude that etv7 is essential for normal red blood cell development through regulation of the lss gene and the cholesterol synthesis pathway.
Vascular complications are the leading cause of mortality and morbidity in patients with diabetes. However, proper animal models of diabetic vasculopathy that recapitulate the accelerated progression of vascular lesions in human are unavailable. In the present study, we developed a zebrafish model of diabetic vascular complications and the methodology for quantifying vascular lesion formation real-time in the living diabetic zebrafish.
Wild type zebrafish (AB) and transgenic zebrafish lines of fli1:EGFP, lyz:EGFP, gata1:dsRed, double transgenic zebrafish of gata1:dsRed/fli1:EGFP were exposed to high cholesterol diet and 3% glucose (HCD-HG) for 10 days. The zebrafish model with HCD-HG treatment was characterized by significantly increased tissue levels of insulin, glucagon, glucose, total triglyceride and cholesterol. Confocal microscopic analysis further revealed that the diabetic larvae developed clearly thickened endothelial layers, distinct perivascular lipid depositions, substantial accumulations of inflammatory cells in the injured vasculature, and a decreased velocity of blood flow. Moreover, the vascular abnormalities were improved by the treatment of pioglitazone and metformin.
A combination of high cholesterol diet and high glucose exposure induces a rapid onset of vascular complications in zebrafish similar to the early atherosclerotic vascular injuries in mammalian diabetic models, suggesting that zebrafish may be used as a novel animal model for diabetic vasculopathy.
Transient leukemia (TL) is evident in 5-10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34(+)-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1(Δe2) knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
In this issue of Blood, Roberts et al report the comprehensive screening of a large cohort of Down syndrome neonates for the transient abnormal myelopoiesis (TAM) disorder based on blood cell morphology review and screening for GATA1 mutations, the signature genetic marker of TAM.
Transcription factor (TF) binding to DNA can be modeled in a number of different ways. It is highly debated which modeling methods are the best, how the models should be built and what can they be applied to. In this study a linear k-mer model proposed for predicting TF specificity in protein binding microarrays (PBM) is applied to a high-throughput SELEX data and the question of how to choose the most informative k-mers to the binding model is studied. We implemented the standard cross-validation scheme to reduce the number of k-mers in the model and observed that the number of k-mers can often be reduced significantly without a great negative effect on prediction accuracy. We also found that the later SELEX enrichment cycles provide a much better discrimination between bound and unbound sequences as model prediction accuracies increased for all proteins together with the cycle number. We compared prediction performance of k-mer and position specific weight matrix (PWM) models derived from the same SELEX data. Consistent with previous results on PBM data, performance of the k-mer model was on average 9%-units better. For the 15 proteins in the SELEX data set with medium enrichment cycles, classification accuracies were on average 71% and 62% for k-mer and PWMs, respectively. Finally, the k-mer model trained with SELEX data was evaluated on ChIP-seq data demonstrating substantial improvements for some proteins. For protein GATA1 the model can distinquish between true ChIP-seq peaks and negative peaks. For proteins RFX3 and NFATC1 the performance of the model was no better than chance.
Elucidation of the genetic pathways that control red blood cell development has been a central goal of erythropoiesis research over the past decade. Notably, data from several recent studies have provided new insights into the regulation of erythroid gene transcription. Transcription profiling demonstrates that erythropoiesis is mainly controlled by a small group of lineage-restricted transcription factors [Gata binding protein 1 (Gata1), T cell acute lymphocytic leukemia 1 protein (Tal1), and Erythroid Kruppel-like factor (EKLF; henceforth referred to as Klf1)]. Binding-site mapping using ChIP-Seq indicates that most DNA-bound Gata1 and Tal1 proteins are contained within higher order complexes (Ldb1 complexes) that include the nuclear adapters Ldb1 and Lmo2. Ldb1 complexes regulate Klf1, and Ldb1 complex-binding sites frequently colocalize with Klf1 at erythroid genes and cis-regulatory elements, indicating strong functional synergy between Gata1, Tal1, and Klf1. Together with new data demonstrating that Ldb1 can mediate long-range promoter-enhancer interactions, these findings provide a foundation for the first comprehensive models of the global regulation of erythroid gene transcription.
The hemangioblast is a multipotential progenitor, which is derived from the mesoderm and can further differentiate into hematopoietic and endothelial lineages. The molecular mechanism governing the specification of hemangioblasts is fundamental to regenerative medicine based on embryonic stem cells for the treatment of various hematologic and vascular diseases. Here we show that aggf1 acts at the top of the genetic regulatory hierarchy in the specification of hemangioblasts in zebrafish. Knockdown of aggf1 expression decreases expression of endothelial cell-specific markers (cdh5, admr) and disrupts primitive hematopoiesis as shown by a decreased number of erythroid cells and reduced expression of gata1 (marker for erythroid progenitors) and pu.1 (myeloid progenitors). Aggf1 knockdown also decreases expression of runx1 and c-myb, indicating that it is required for specification of hematopoietic stem cells (definitive hematopoiesis). Aggf1 knockdown led to dramatically reduced expression of hemangioblast markers fli1, etsrp, lmo2, and scl, and hematopoietic/endothelial defects in aggf1 morphants were rescued by messenger RNA for scl, fli-vp16, or etsrp. Taken together, these data indicate that aggf1 is involved in differentiation of both hematopoietic and endothelial lineages and that aggf1 acts upstream of scl, fli1, and etsrp in specification of hemangioblasts.
Transient neonatal leukemia occurs almost exclusively in Down syndrome babies. We report here the unusual case of a newborn without Down syndrome who presented neonatal transient leukemia and who achieved spontaneously complete remission. Trisomy 21 and GATA1 mutation were both present in leukemic cells. While close follow-up is advised since true leukemia may develop later, the patient is still in remission for 2.5 years. We performed a literature review of 15 other similar cases.
Our case of transient leukemia without Down syndrome and the literature review highlight the important role of trisomy 21 and GATA1 mutation in the development of transient neonatal leukemia.
The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin β3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.
In this issue of Blood, Takai et al provide some tantalizing clues on how expression of the GATA1 transcription factor, a master regulator of erythroid/megakaryocytic differentiation, is suppressed in the hematopoietic stem/progenitor cell (HSPC) compartment.
It has been reported that patients with Down syndrome (DS) frequently develop transient myeloproliferative disorder (TMD) and less commonly myeloid leukemia in DS (ML-DS). We examined the pathogenetic relationship of these conditions with somatic mutations of the GATA1 gene in children with both TMD and ML-DS. To determine the incidence of GATA1 mutations in a cohort of DS patients and the applicability of these mutations as a clonal marker to detect minimal residual disease, we screened 198 samples of 169 patients with DS for mutations in GATA1 exon 2 by direct sequencing. Novel mutations were detected in four of the 169 DS patients (2 with TMD and 2 with ML-DS). We examined spontaneous remission and response to therapy in TMD and ML-DS patients and concluded that these mutations can be used as stable markers in PCR analysis to monitor these events.
TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.
Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis.
Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.
To investigate the origin of hematopoietic progenitors contained in the stromal vascular fraction (SVF) of human adipose tissue.
Tissue samples obtained from lipectomies were subjected to enzymatic digestion with collagenase to obtain a single-cell suspension. The centrifuged cell pellet, termed SVF, was separated immunomagnetically into CD45(+) and CD45(-) cells and cultured in serum-free medium containing hematopoietic cytokines. The freshly isolated and cultured cells were evaluated to determine their ability to form hematopoietic colony-forming units in clonogenic assays and for the expression of certain hematopoietic transcription factors by reverse transcription-polymerase chain reaction; the gene expression level was compared to that in CD34(+) hematopoietic progenitor cells from cord blood (CB) and adult peripheral blood (PB). To characterize erythroid progenitors, burst-forming units-erythroid (BFU-E) were developed in a semisolid medium under different culture conditions, and the hemoglobin composition and globin gene expression in the erythroid colonies were determined.
The transcription factors SCL/TAL1, RUNX1, RUNX2 and GATA2 were expressed in both the CD45(+) and CD45(-) SVF populations; however, in contrast to our observations in the CD34(+) cells from CB and adult PB, GATA1 was not detected. Nevertheless, GATA1 could be detected in the SVF cells after seven days in culture, whereas its expression was upregulated in the CB CD34(+) cells. The analysis of BFU-E-derived colonies revealed that virtually all erythroid cells produced by SVF cells expressed fetal hemoglobin, and the γ-globin mRNA levels ranged between those obtained in the adult- and neonatal-derived erythroid cells. Moreover, the SVF-derived erythroid cells synthesized similar levels of α- and β-globin mRNA, whereas the α-globin transcript levels were consistently higher those of β-globin in the cells derived from CB or PB CD34(+) cells. Furthermore, although the cellular distribution of hemoglobin in the erythroid cells derived from the CD34(+) cells obtained from hematopoietic tissues was dependent on the presence or absence of serum in the culture medium, this did not affect the SVF-derived erythroid cells.
Our results demonstrate that hematopoietic progenitors in SVF have molecular and functional features that differ from those exhibited by circulating progenitors, suggesting the possibility of a different origin.
Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1(low) mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1(low) mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1(low) mice were crossed with P-sel(null) mice according to standard genetic protocols and Gata1(low)P-sel(wt), Gata1(low)P-sel(null) and Gata1(WT)P-sel(null) or Gata1(wt)P-sel(wt) (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1(low) mice. Moreover, platelet microparticles are reduced in Gata1(low) mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1(low) mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1(low) mice significantly higher frequency of thrombotic events was seen in adult and old Gata1(low) mice compared to Gata1(low)P-sel(null) mice. Thus, presence of the P-selectin null trait rescued Gata1(low) mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1(low) mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.
Developmental processes of hematopoietic cells are orchestrated by transcriptional networks. GATA-1, the founding member of the GATA family of transcription factors, has been demonstrated to play crucial roles in the differentiation of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remains elusive. Here we show that basophils abundantly express Gata1 mRNAs, and that siRNA-mediated knockdown of Gata1 resulted in impaired production of IL-4 by basophils in response to the stimulation with IgE plus antigens. ΔdblGATA mice that carry the mutated Gata1 promoter and are widely used for functional analysis of eosinophils owing to their selective loss of eosinophils showed a decreased number of basophils with reduced expression of Gata1 mRNAs. The number of basophil progenitors in bone marrow was reduced in these mice, and the generation of basophils from their bone marrow cells in culture with IL-3 or thymic stromal lymphopoietin was impaired. ΔdblGATA basophils responded poorly ex vivo to stimulation with IgE plus antigens compared with wild-type basophils as assessed by degranulation and production of IL-4 and IL-6. Moreover, ΔdblGATA mice showed impaired responses in basophil-mediated protective immunity against intestinal helminth infection. Thus, ΔdblGATA mice showed numerical and functional aberrancy in basophils in addition to the known deficiency of eosinophils. Our findings demonstrate that GATA-1 plays a key role in the generation and function of basophils and underscore the need for careful distinction of the cell lineage responsible for each phenotype observed in ΔdblGATA mice.
The stem cell protein SALL4 plays a critical role in hematopoiesis by regulating the cell fate. In primitive hematopoietic precursors, it activates or represses important genes via recruitment of various epigenetic factors such as DNA methyltransferases, and histone deacylases. Here, we demonstrate that LSD1, a histone lysine demethylase, also participates in the trans-repressive effects of SALL4. Based on luciferase assays, the amine oxidase domain of LSD1 is important in suppressing SALL4-mediated reporter transcription. In freshly isolated adult mouse bone marrows, both SALL4 and LSD1 proteins are preferentially expressed in undifferentiated progenitor cells and co-localize in the nuclei. Further sequential chromatin immunoprecipitation assay confirmed that these two factors share the same binding sites at the promoter regions of important hematopoietic regulatory genes including EBF1, GATA1, and TNF. In addition, studies from both gain- and loss-of-function models revealed that SALL4 dynamically controls the binding levels of LSD1, which is accompanied by a reversely changed histone 3 dimethylated lysine 4 at the same promoter regions. Finally, shRNA-mediated knockdown of LSD1 in hematopoietic precursor cells resulted in altered SALL4 downstream gene expression and increased cellular activity. Thus, our data revealed that histone demethylase LSD1 may negatively regulate SALL4-mediated transcription, and the dynamic regulation of SALL4-associated epigenetic factors cooperatively modulates early hematopoietic precursor proliferation.
The discovery that an abnormally activated JAK-STAT signaling pathway is central to the pathogenesis of myeloproliferative neoplasms has promoted the clinical development of small-molecule JAK2 inhibitors. These agents have shown remarkable efficacy in disease control, but do not induce molecular remission; on the other hand, interferon holds the promise to target the putative hematopoietic progenitor cell initiating the disease. The presence of additional molecular abnormalities indicates a high molecular complexity of myeloproliferative neoplasms, and the need for simultaneously targeting different targets. Several drugs are currently under study as single agents and in combination. This review briefly describes the several in vitro and in vivo models of myeloproliferative neoplasms that are being used as preclinical models for drug development.
We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.
The transcription factor GATA1 is essential for erythroid and megakaryocytic cell differentiation. Gata1 hematopoietic regulatory domain (G1HRD) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo. G1HRD contains a promoter-proximal enhancer composed of a GATA-palindrome motif, four CP2-binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and β-galactosidase expression are driven by wild-type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis-acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter (TDR) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA-palindrome motif and the CACCC boxes. Mutation of CP2-binding sites also moderately decreased G1HRD activity. The combined mutation of the CP2-binding sites and the GATA-palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.
Elevated reactive oxygen species (ROS) and oxidative damage occur in the red blood cells (RBCs) of SOD1-deficient C57BL/6 mice. This leads to autoimmune responses against RBCs in aged mice that are similar to autoimmune hemolytic anemia (AIHA). We examined whether a SOD1 deficiency and/or the human SOD1 transgene (hSOD1) would affect phenotypes of AIHA-prone New Zealand Black (NZB) mice by establishing three congenic strains: those lacking SOD1, those expressing hSOD1 under a GATA-1 promoter, and those lacking mouse SOD1 but expressing hSOD1. Levels of intracellular ROS and oxidative stress markers increased, and the severity of the AIHA phenotype was aggravated by a SOD1 deficiency. In contrast, the transgenic expression of hSOD1 in an erythroid cell-specific manner averted most of the AIHA phenotype evident in the SOD1-deficient mice and also ameliorated the AIHA phenotype in the mice possessing intrinsic SOD1. These data suggest that oxidative stress in RBCs may be an underlying mechanism for autoimmune responses in NZB mice. These results were consistent with the hypothetical role of reactive oxygen species in triggering the autoimmune reaction in RBCs and may provide a novel approach to mitigating the progression of AIHA by reducing oxidative stress.
The prognostic impact of mutations in the CCAAT/enhancer binding protein α (CEBPA) gene was evaluated in the context of concomitant molecular mutations and cytogenetic aberrations in acute myeloid leukemia (AML). CEBPA was screened in a cohort of 2296 adult AML cases. Of 244 patients (10.6%) with CEBPA mutations, 140 cases (6.1%) were single-mutated (CEBPAsm) and 104 cases (4.5%) were double-mutated (CEBPAdm). Cytogenetic analysis revealed normal karyotype in 172/244 (70.5%) of CEBPAmut cases, whereas in 72/244 cases (29.5%) at least one cytogenetic aberration was detected. Concurrent molecular mutations were seen less frequently in CEBPAdm than in CEBPAsm AML cases (69.2% vs 88.6% P<0.001). In detail, the spectrum of concurrent mutations was different in both groups with the frequent occurrence of GATA1 and WT1 mutations in CEBPAdm patients. In contrast, FLT3-ITD, NPM1, ASXL1 and RUNX1 mutations were detected more frequently in CEBPAsm cases. Favorable outcome was restricted to CEBPAdm cases and remained an independent prognostic factor for a favorable outcome in multivariate analysis (hazard ratio: 0.438, P=0.020). Outcome in CEBPAsm cases strongly depended on concurrent FLT3-ITD. In conclusion, we propose that only CEBPAdm should be considered as an entity in the WHO classification of AML and should be clearly distinguished from CEBPAsm AML.
Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome-related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).
The aim of the present study was to investigate the role of each estrogen receptors on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats. Activation of ESR1 by 17β-estradiol (E2) and ESR1-selective agonist PPT increased CCND1 expression, and this effect was dependent on NF-kB activation. E2 and the ESR2-selective agonist DPN, but not PPT, increased, in a PI3K and CREB-dependent manner, the expression of CDKN1B and the transcription factors GATA-1 and DMRT1. Analyzing the expression of ESR1 and ESR2 in different stages of development of Sertoli cells, we observed that the ESR1/ESR2 ratio decreased with age, and this ratio seems to be important to determine the end of cell proliferation and the start of cell differentiation. In Sertoli cells from 15-day-old rats, the ESR1/ESR2 ratio favors the effect of ESR1 and the activation of this receptor increased [Methyl-(3)H]thymidine incorporation. We propose that in Sertoli cells from 15-day-old rats E2 modulates Sertoli cell proliferation through ESR1/NF-kB-mediated increase of CCND1, and cell cycle exit and differentiation through ESR2/CREB-mediated increase of CDKN1B, GATA-1 and DMRT1. The present study reinforces the important role of estrogen for normal testis development.
Transcriptional networks orchestrate complex developmental processes, and such networks are commonly instigated by master regulators for development. By now, considerable progress has been made in elucidating GATA factor-dependent genetic networks that control red blood cell development. Here we reported that GATA-1 and GATA-2 co-regulated the expression of two microRNA genes, microRNA-27a and microRNA-24, with critical roles in regulating erythroid differentiation. In general, GATA-2 occupied the miR-27a≈24 promoter and repressed their transcription in immature erythroid progenitor cells. As erythropoiesis proceeded, GATA-1 directly activated miR-27a≈24 transcription, and this involved a GATA-1-mediated displacement of GATA-2 from chromatin, a process termed 'GATA switch'. Furthermore, the mature miR-27a and miR-24 cooperatively inhibited GATA-2 translation and favoured the occupancy switch from GATA-2 to GATA-1, thus completing a positive feedback loop to promote erythroid maturation. In line with the essential role of GATA factors, ectopic expression of miR-27a or miR-24 promoted erythropoiesis in human primary CD34+ haematopoietic progenitor cells and mice, whereas attenuated miR-27 or miR-24 level led to impaired erythroid phenotypes in haematopoietic progenitor cells and zebrafish. Taken together, these data integrated micro RNA expression and function into GATA factor coordinated networks and provided mechanistic insight into a regulatory circuit that comprised GATA1/2 switch and miR-27a/24 in erythropoiesis.
GATA1 is a master regulator of hematopoietic differentiation, but Gata1 expression is inactivated in hematopoietic stem cells (HSCs). Using a bacterial artificial chromosome containing the Gata1 gene modified with green fluorescent protein (GFP) reporter, we explored the function of the 3.7-kb Gata1 upstream region (GdC region) that harbors 3 core cis-elements: Gata1 hematopoietic enhancer, double GATA-motif, and CACCC-motif. Transgenic GFP expression directed by the Gata1-BAC faithfully recapitulated the endogenous Gata1 expression pattern. However, deletion of the GdC-region eliminated reporter expression in all hematopoietic cells. To test whether the combination of the core cis-elements represents the regulatory function of the GdC-region, we replaced the region with a 659-bp minigene that linked the three cis-elements (MG-GFP). The GFP reporter expression directed by the MG-GFP BAC fully recapitulated the erythroid-megakaryocytic Gata1 expression. However, the GFP expression was aberrantly increased in the HSCs and was associated with decreases in DNA methylation and abundant GATA2 binding to the transgenic MG-GFP allele. The 3.2-kb sequences interspaced between the Gata1 hematopoietic enhancer and the double GATA-motif were able to recruit DNA methyltransferase 1, thereby exerting a cis-repressive function in the HSC-like cell line. These results indicate that the 3.2-kb interspacing sequences inactivate Gata1 by maintaining DNA-methylation in the HSCs.
Transient abnormal myelopoiesis (TAM), a preleukemic disorder unique to neonates with Down syndrome (DS), may transform to childhood acute myeloid leukemia (ML-DS). Acquired GATA1 mutations are present in both TAM and ML-DS. Current definitions of TAM specify neither the percentage of blasts nor the role of GATA1 mutation analysis. To define TAM, we prospectively analyzed clinical findings, blood counts and smears, and GATA1 mutation status in 200 DS neonates. All DS neonates had multiple blood count and smear abnormalities. Surprisingly, 195 of 200 (97.5%) had circulating blasts. GATA1 mutations were detected by Sanger sequencing/denaturing high performance liquid chromatography (Ss/DHPLC) in 17 of 200 (8.5%), all with blasts >10%. Furthermore low-abundance GATA1 mutant clones were detected by targeted next-generation resequencing (NGS) in 18 of 88 (20.4%; sensitivity ∼0.3%) DS neonates without Ss/DHPLC-detectable GATA1 mutations. No clinical or hematologic features distinguished these 18 neonates. We suggest the term "silent TAM" for neonates with DS with GATA1 mutations detectable only by NGS. To identify all babies at risk of ML-DS, we suggest GATA1 mutation and blood count and smear analyses should be performed in DS neonates. Ss/DPHLC can be used for initial screening, but where GATA1 mutations are undetectable by Ss/DHPLC, NGS-based methods can identify neonates with small GATA1 mutant clones.
Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.
Benzene is a common occupational hazard as well as a widespread pollutant. Its metabolites play important roles in its toxicity to the hematopoietic system, but little is known about how benzene metabolites affect erythropoiesis. Our previous study demonstrated that benzene metabolites, including phenol and hydroquinone, inhibited hemin-induced erythroid differentiation of K562 cells. In present study, to elucidate the role of DNA methylation in benzene metabolites-induced inhibition on erythroid differentiation, it was investigated whether DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR), was able to prevent benzene metabolites inhibiting hemin-induced erythroid differentiation in K562 cells, and the methylation levels of erythroid-specific genes in benzene metabolites-treated K562 cells were analyzed by Quantitative MassARRAY methylation analysis platform. It was found that treatment of K562 cells with 5-aza-CdR completely prevented phenol and hydroquinone inhibiting hemin-induced hemoglobin synthesis and hemin-induced expression of erythroid specific genes, including α- and β-globin, erythroid porphobilinogen deaminase and GATA binding protein 1 (GATA-1). Consistently, the exposure to benzene metabolites caused an increase in DNA methylation levels at a few CpG sites in some erythroid specific genes, including α-globin gene and α-cluster HS40 element, β-globin gene and HS core sequence in LCR of β-globin gene cluster, erythroid porphobilinogen deaminase gene, and GATA-1 gene. These results indicated that DNA methylation played a role in benzene metabolites inhibiting hemin-induced erythroid differentiation of K562 cells via down-regulating transcription of some erythroid related genes.
The establishment and maintenance of cell type-specific transcriptional programs require an ensemble of broadly expressed chromatin remodeling and modifying enzymes. Many questions remain unanswered regarding the contributions of these enzymes to specialized genetic networks that control critical processes, such as lineage commitment and cellular differentiation. We have been addressing this problem in the context of erythrocyte development driven by the transcription factor GATA-1 and its coregulator Friend of GATA-1 (FOG-1). As certain GATA-1 target genes have little to no FOG-1 requirement for expression, presumably additional coregulators can mediate GATA-1 function. Using a genetic complementation assay and RNA interference in GATA-1-null cells, we demonstrate a vital link between GATA-1 and the histone H4 lysine 20 methyltransferase PR-Set7/SetD8 (SetD8). GATA-1 selectively induced H4 monomethylated lysine 20 at repressed, but not activated, loci, and endogenous SetD8 mediated GATA-1-dependent repression of a cohort of its target genes. GATA-1 used different combinations of SetD8, FOG-1, and the FOG-1-interacting nucleosome remodeling and deacetylase complex component Mi2β to repress distinct target genes. Implicating SetD8 as a context-dependent GATA-1 corepressor expands the repertoire of coregulators mediating establishment/maintenance of the erythroid cell genetic network, and provides a biological framework for dissecting the cell type-specific functions of this important coregulator. We propose a coregulator matrix model in which distinct combinations of chromatin regulators are required at different GATA-1 target genes, and the unique attributes of the target loci mandate these combinations.
Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the rate-limiting enzyme for heme biosynthesis in erythroid cells, and a missense mutation of the ALAS2 gene is associated with congenital sideroblastic anemia. However, the gene responsible for this form of anemia remains unclear in about 40% of patients. Here, we identify a novel erythroid-specific enhancer of 130 base pairs in the first intron of the ALAS2 gene. The newly identified enhancer contains a cis-acting element that is bound by the erythroid-specific transcription factor GATA1, as confirmed by chromatin immunoprecipitation analysis in vivo and by electrophoretic mobility shift assay in vitro. A promoter activity assay in K562 human erythroleukemia cells revealed that the presence of this 130-base pair region increased the promoter activity of the ALAS2 gene by 10-15-fold. Importantly, two mutations, each of which disrupts the GATA-binding site in the enhancer, were identified in unrelated male patients with congenital sideroblastic anemia, and the lower expression level of ALAS2 mRNA in bone marrow erythroblasts was confirmed in one of these patients. Moreover, GATA1 failed to bind to each mutant sequence at the GATA-binding site, and each mutation abolished the enhancer function on ALAS2 promoter activity in K562 cells. Thus, a mutation at the GATA-binding site in this enhancer may cause congenital sideroblastic anemia. These results suggest that the newly identified intronic enhancer is essential for the expression of the ALAS2 gene in erythroid cells. We propose that the 130-base pair enhancer region located in the first intron of the ALAS2 gene should be examined in patients with congenital sideroblastic anemia in whom the gene responsible is unknown.
The high frequency of a unique neonatal preleukaemic syndrome, transient abnormal myelopoiesis (TAM), and subsequent acute myeloid leukaemia in early childhood in patients with trisomy 21 (Down syndrome) points to a specific role for trisomy 21 in transforming foetal haematopoietic cells. N-terminal truncating mutations in the key haematopoietic transcription factor GATA1 are acquired during foetal life in virtually every case. These mutations are not leukaemogenic in the absence of trisomy 21. In mouse models, deregulated expression of chromosome 21-encoded genes is implicated in leukaemic transformation, but does not recapitulate the effects of trisomy 21 in a human context. Recent work using primary human foetal liver and bone marrow cells, human embryonic stem cells and iPS cells shows that prior to acquisition of GATA1 mutations, trisomy 21 itself alters human foetal haematopoietic stem cell and progenitor cell biology causing multiple abnormalities in myelopoiesis and B-lymphopoiesis. The molecular basis by which trisomy 21 exerts these effects is likely to be extremely complex, to be tissue-specific and lineage-specific and to be dependent on ontogeny-related characteristics of the foetal microenvironment.
Eosinophil-produced cytokines have been shown to participate in the maintenance of antigen-specific plasma cells (PC) in bone marrow (BM), suggesting that eosinophils are required in the development and/or maintenance of alloantibody responses posttransplant. To test this hypothesis, we sensitized eosinophil-deficient ΔdblGATA1 mice and wild-type (WT) control mice with allogeneic splenocytes or with allogeneic heart grafts and compared the kinetics and titers of serum donor-specific antibodies (DSA), as well as BM and spleen CD130 + B220 low PC populations between groups. Spleen cells from naïve ΔdblGATA1 BALB/c mice contained higher percentages of PC than WT without detectable differences in BM PCs. After sensitization with allogeneic splenocytes, BALB/c ΔdblGATA1 mice contained fewer BM PCs but more splenic PCs compared to controls. These differences were associated with modestly lower titers of serum DSA 4 and 12 weeks after sensitization but secondary immunizations induced similar increases in both groups. Moreover, the kinetics and strength of DSA did not differ in WT and ΔdblGATA1 BALB/c mice transplanted with B6 cardiac allografts, nor did they differ in transplanted ΔdblGATA1 and WT mice on a B6 background. Therefore, eosinophils are not required for alloantibody formation or maintenance in mice and are thus unlikely to be effective targets for antibody desensitization.
Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes.
GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.
It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.
About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%-30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage-specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN-responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/β IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/β antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN-responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage-specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.
The ChIP-seq technique enables genome-wide mapping of in vivo protein-DNA interactions and chromatin states. Current analytical approaches for ChIP-seq analysis are largely geared towards single-sample investigations, and have limited applicability in comparative settings that aim to identify combinatorial patterns of enrichment across multiple datasets. We describe a novel probabilistic method, jMOSAiCS, for jointly analyzing multiple ChIP-seq datasets. We demonstrate its usefulness with a wide range of data-driven computational experiments and with a case study of histone modifications on GATA1-occupied segments during erythroid differentiation. jMOSAiCS is open source software and can be downloaded from Bioconductor 1.
The physiological switch in expression of the embryonic, fetal, and adult β-like globin genes has garnered enormous attention from investigators interested in transcriptional mechanisms and the molecular basis of hemoglobinopathies. These efforts have led to the discovery of cell type-specific transcription factors, unprecedented mechanisms of transcriptional coregulator function, genome biology principles, unique contributions of nuclear organization to transcription and cell function, and promising therapeutic targets. Given the vast literature accrued on this topic, this article will focus on the master regulator of erythroid cell development and function GATA-1, its associated proteins, and its frontline role in controlling hemoglobin synthesis. GATA-1 is a crucial regulator of genes encoding hemoglobin subunits and heme biosynthetic enzymes. GATA-1-dependent mechanisms constitute an essential regulatory core that nucleates additional mechanisms to achieve the physiological control of hemoglobin synthesis.
Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30-50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60-65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.
Latent transforming growth factor β-binding protein 4 (LTBP4) is an extracellular matrix molecule that is a member of important connective tissue networks and is needed for the correct folding and the secretion of TGF-β1. LTBP4 is downregulated in carcinomas of various tissues. Here we show that LTBP4 is also downregulated in adenocarcinomas and squamous cell carcinomas of the esophagus in vitro and in vivo. Re-expression of LTBP4 in esophageal cancer cell lines reduced cell migration ability, whereas cell viability and cell proliferation remained unchanged. Hypermethylation of the promoter regions of the two main human LTBP4 transcriptional forms, LTBP4L and LTBP4S, was found to be involved in LTBP4 silencing. Detailed investigations of the methylation patterns of the promoter regions of LTBP4L and LTBP4S identified GATA1, SP1, E2F4 and SMAD3 as potential transcription factors involved in LTBP4 expression. In in vitro transcription factor activity studies we discovered E2F4 as novel powerful regulator for LTBP4S expression.
Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.
Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.
Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.
We previously reported that IL-3 signaling induces phosphorylation of GATA-1 at the serine²⁶ position, which contributes to IL-3-mediated anti-apoptotic response. Here, we demonstrate that phosphorylation of GATA-1 at serine²⁶ is also transiently induced in cells of the erythroid lineage (primary erythroblasts and erythrocyte-committed progenitors [EPs]) by erythropoietin (EPO), the principal cytokine regulating erythropoiesis. To examine whether phosphorylation of GATA-1 at serine²⁶ would have any impact on erythropoiesis, mutant mice carrying either a glutamic acid (GATA-1(S26E)) or alanine (GATA-1(S26A)) substitution at serine²⁶ were generated. Neither GATA-1(S26E) nor GATA-1(S26A) mice showed any significant difference from control mice in peripheral blood cell composition under either steady state or stress conditions. The erythroblast differentiation in both mutant mice also appeared to be normal. However, a moderate reduction in the CFU-E progenitor population was consistently observed in the bone marrow of GATA-1(S26E), but not GATA-1(S26A) mice, suggesting that such defect was compensated for within the bone marrow. Surprisingly, reduced CFU-E progenitor population in GATA-1(S26E) mice was mainly due to EPO-induced growth suppression of GATA-1(S26E) EPs, albeit in the absence of EPO these cells manifested a survival advantage. Further analyses revealed that EPO-induced growth suppression of GATA-1(S26E) EPs was largely due to the proliferation block resulted from GATA-1(S26E)-mediated transcriptional activation of the gene encoding the cell cycle inhibitor p21(Waf1/Cip1). Taken together, these results suggest that EPO-induced transient phosphorylation of GATA-1 at serine²⁶ is dispensable for erythropoiesis. However, failure to dephosphorylate this residue following its transient phosphorylation significantly attenuates the colony-forming activity of EPs.
Hematopoiesis - the process by which blood cells are formed - has been studied intensely for over a century using a variety of model systems. There is conservation of the overall hematopoietic process between vertebrates, although some differences do exist. Over the last decade, the zebrafish has come to the forefront as a new model in hematopoiesis research, as it allows the use of large-scale genetics, chemical screens and transgenics. This comparative approach to understanding hematopoiesis has led to fundamental knowledge about the process and to the development of new therapies for disease. Here, we provide a broad overview of vertebrate hematopoiesis. We also highlight the benefits of using zebrafish as a model.
Missense mutations in transcription factor GATA1 underlie a spectrum of congenital red blood cell and platelet disorders. We investigated how these alterations cause distinct clinical phenotypes by combining structural, biochemical, and genomic approaches with gene complementation systems that examine GATA1 function in biologically relevant cellular contexts. Substitutions that disrupt FOG1 cofactor binding impair both gene activation and repression and are associated with pronounced clinical phenotypes. Moreover, clinical severity correlates with the degree of FOG1 disruption. Surprisingly, 2 mutations shown to impair DNA binding of GATA1 in vitro did not measurably affect in vivo target gene occupancy. Rather, one of these disrupted binding to the TAL1 complex, implicating it in diseases caused by GATA1 mutations. Diminished TAL1 complex recruitment mainly impairs transcriptional activation and is linked to relatively mild disease. Notably, different substitutions at the same amino acid can selectively inhibit TAL1 complex or FOG1 binding, producing distinct cellular and clinical phenotypes. The structure-function relationships elucidated here were not predicted by prior in vitro or computational studies. Thus, our findings uncover novel disease mechanisms underlying GATA1 mutations and highlight the power of gene complementation assays for elucidating the molecular basis of genetic diseases.
Erythropoiesis is finely regulated by two major cytokines, stem cell factor (SCF) and erythropoietin (Epo). Decrease levels of Epo result in caspase activation and erythroid progenitors apoptosis. However, normal erythroid cell maturation requests caspase activation and cleavage of various caspase substrates, except the erythroid transcription factor GATA-1, that is protected by interaction with the chaperone HSP70 in the nucleus. Therefore, molecular abnormalities associated with decrease of HSP70 expression in the nucleus may result in ineffective erythropoiesis characterized by apoptosis and impaired maturation of erythroid precursors. These findings open new potential targeted therapies for erythroid disorders.
To investigate function of the Lim-only protein(LMO2) in hemangioblast generated from murine embryonic stem cells differentiation to hematopoietic cells.
The hemangioblast-specific expression vector with lmo2 or green fluorescence protein gene was constructed, respectively. The murine embryonic stem cells were transfected by the hemangioblast-specific expression vectors. The neomycin-resistance ES cell clones were obtained after having been screened by G418. The cell clones were spontaneously differentiated into embryo bodies(EB) containing hemangioblast.Expression of the hematopoietic genes was investigated by real-time reverse transcription-ploymerase chain reaction during EB differentiation.For the EB cells, blast-cloning forming cells analysis and blood-colony forming unit analysis were then performed, respectively. The numbers of the blasts were counted during hematopoietic differentiation.
The hemangioblast-specific expression vector with lmo2 or green fluorescence protein was transfected into ES cells.The neomycin-resistance ES cells generated EBs from 2.5 days to 10 days.Real time reverse transcription-ploymerase chain reaction analysis indicated that overexpression of lmo2 increased the expression of hematopoietic genes(gata1, tal1, Β-h1, and Β-major globin) during EB formation.Blast-cloning forming cells analysis showed that the numbers of the blasts generated by ES/lmo2 was 2-or 3-fold than those in the controls.The total numbers of the blood-colony forming unit or the numbers of the erythrocyte colony-forming unit generated by ES/lmo2 were 2.5 times or 3 times, respectively, when compared with the controls.
LMO2 enhances the proliferation and differentiation of hemangioblasts.
Balanced and precisely controlled processes between self-renewal and differentiation of hematopoietic stem cells (HSCs) into all blood lineages are critical for vertebrate definitive hematopoiesis. However, the molecular mechanisms underlying the maintenance and differentiation of HSCs have not been fully elucidated. Here, we show that zebrafish Ddx46, encoding a DEAD-box RNA helicase, is expressed in HSCs of the caudal hematopoietic tissue (CHT). The number of HSCs expressing the molecular markers cmyb or T-cell acute lymphocytic leukemia 1 (tal1) was markedly reduced in Ddx46 mutants. However, massive cell death of HSCs was not detected, and proliferation of HSCs was normal in the CHT of the mutants at 48 h postfertilization. We found that myelopoiesis occurred, but erythropoiesis and lymphopoiesis were suppressed, in Ddx46 mutants. Consistent with these results, the expression of spi1, encoding a regulator of myeloid development, was maintained, but the expression of gata1a, encoding a regulator of erythrocyte development, was downregulated in the mutants. Taken together, our results provide the first genetic evidence that zebrafish Ddx46 is required for the multilineage differentiation of HSCs during development, through the regulation of specific gene expressions.
Erythropoiesis is dependent on the lineage-specific transcription factors Gata1, Tal1, and Klf1. Several erythroid genes have been shown to require all 3 factors for their expression, suggesting that they function synergistically; however, there is little direct evidence for widespread cooperation. Gata1 and Tal1 can assemble within higher-order protein complexes (Ldb1 complexes) that include the adapter molecules Lmo2 and Ldb1. Ldb1 proteins are capable of coassociation, and long-range Ldb1-mediated oligomerization of enhancer- and promoter-bound Ldb1 complexes has been shown to be required for β-globin gene expression. In this study, we generated a genomewide map of Ldb1 complex binding sites that revealed widespread binding at erythroid genes and at known erythroid enhancer elements. Ldb1 complex binding sites frequently colocalized with Klf1 binding sites and with consensus binding motifs for other erythroid transcription factors. Transcriptomic analysis demonstrated a strong correlation between Ldb1 complex binding and Ldb1 dependency for gene expression and identified a large cohort of genes coregulated by Ldb1 complexes and Klf1. Together, these results provide a foundation for defining the mechanism and scope of Ldb1 complex activity during erythropoiesis.
The hematopoietic stem cell (HSC) compartment is heterogeneous, yet our understanding of the identities of different HSC subtypes is limited. Here we show that platelet integrin CD41 (αIIb), currently thought to only transiently mark fetal HSCs, is expressed on an adult HSC subtype that accumulates with age. CD41+ HSCs were largely quiescent and exhibited myeloerythroid and megakaryocyte gene priming, governed by Gata1, whereas CD41- HSCs were more proliferative and exhibited lymphoid gene priming. When isolated without the use of blocking antibodies, CD41+ HSCs possessed long-term repopulation capacity on serial transplantations and showed a marked myeloid bias compared with CD41- HSCs, which yielded a more lymphoid-biased progeny. CD41-knockout (KO) mice displayed multilineage hematopoietic defects coupled with decreased quiescence and survival of HSCs, suggesting that CD41 is functionally relevant for HSC maintenance and hematopoietic homeostasis. Transplantation experiments indicated that CD41-KO-associated defects are long-term transplantable, HSC-derived and, in part, mediated through the loss of platelet mass leading to decreases in HSC exposure to important platelet released cytokines, such as transforming growth factor β1. In summary, our data provide a novel marker to identify a myeloid-biased HSC subtype that becomes prevalent with age and highlights the dogma of HSC regulation by their progeny.
The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes.
In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element.
Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element.
These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.
Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown.
We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells.
The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.
Epidermal Langerhans cells (LC) expressing the high-affinity receptor for IgE (FcεRI) play a key role in atopic dermatitis (AD). AD skin is highly colonized with Staphylococcus aureus (S.a.), which are sensed by Toll-like receptor 2 (TLR2). We hypothesized that TLR2 may impact on the expression of FcεRI on LC.
To study a putative impact of TLR2 signaling on FcεRI, we analyzed FcεRI and known transcription factors of the receptor after ligand binding to TLR2.
We generated LC from CD34(+) progenitors in vitro (CD34LC) expressing FcεRI and TLR2 as well as its partners TLR1 and TLR6. The expression of FcεRI and known transcription factors of the receptor was analyzed on the protein and RNA level by flow cytometry, Western blotting, and real-time PCR.
For CD34LC from 123 donors, we observed a high heterogeneity in FcεRI surface expression correlating with mRNA level of its α-chain. Stimulation of TLR1/2 or TLR2/6 dramatically down-regulated FcεRI on protein and mRNA level of both α- and γ-chain. Further analysis of putative transcription factors for FCER1A revealed the lack of GATA1 in CD34LC, weak expression of ELF1 and YY1, and high expression of PU.1. While ELF1 and YY1 appeared to be little affected by TLR2 engagement, PU.1 was significantly down-regulated.
Taken together, our findings show that in human, LC ligation of TLR2 by S.a.-derived products down-regulates FcεRI and its transcription factor PU.1, thus suggesting that FcεRI is controlled by PU.1 in these cells.
Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.
We report the genomic occupancy profiles of the key hematopoietic transcription factor GATA-1 in pro-erythroblasts and mature erythroid cells fractionated from day E12.5 mouse fetal liver cells. Integration of GATA-1 occupancy profiles with available genome-wide transcription factor and epigenetic profiles assayed in fetal liver cells enabled as to evaluate GATA-1 involvement in modulating local chromatin structure of target genes during erythroid differentiation. Our results suggest that GATA-1 associates preferentially with changes of specific epigenetic modifications, such as H4K16, H3K27 acetylation and H3K4 di-methylation. Furthermore, we used random forest (RF) non-linear regression to predict changes in the expression levels of GATA-1 target genes based on the genomic features available for pro-erythroblasts and mature fetal liver-derived erythroid cells. Remarkably, our prediction model explained a high proportion of 62% of variation in gene expression. Hierarchical clustering of the proximity values calculated by the RF model produced a clear separation of upregulated versus downregulated genes and a further separation of downregulated genes in two distinct groups. Thus, our study of GATA-1 genome-wide occupancy profiles in mouse primary erythroid cells and their integration with global epigenetic marks reveals three clusters of GATA-1 gene targets that are associated with specific epigenetic signatures and functional characteristics.
The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.
Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.
Erythropoietin is the principal regulator of erythropoiesis and promotes the survival, proliferation and differentiation of erythroid progenitor cells in mammals. In this study we report on the molecular and functional characterization of erythropoietin from the goldfish. Quantitative expression analysis of goldfish epo revealed the highest mRNA levels in heart, followed by brain, liver, spleen and kidney tissues. There was no marked change of epo expression in goldfish primary kidney macrophage cultures, as progenitor cell to macrophage development progressed, indicating that erythropoietin is not involved in monopoiesis. Recombinant goldfish erythropoietin induced proliferation of progenitor cells in a dose-dependent manner, and up-regulated the expression of erythroid transcription factors gata1 and lmo2 in progenitor cells. Furthermore, recombinant goldfish erythropoietin stimulated erythroid colony formation in a dose-dependent manner and promoted survival of erythroid progenitor cells as colony-forming cells. Our results demonstrate that the function of erythropoietin in the goldfish is similar to that of mammals and suggest a highly conserved mechanism of early erythrocyte development in lower and higher vertebrates.
Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.
The intense physiologic demand to generate vast numbers of red blood cells requires the establishment of a complex genetic network by the master regulatory transcription factor GATA-1 and its coregulators. This network dictates the genesis of enucleated erythrocytes by orchestrating the survival, proliferation, and differentiation of progenitor cells. In addition to the crucial GATA-1 coregulator Friend of GATA-1 (FOG-1), a component of the Mediator complex, Med1, facilitates GATA-1-dependent transcription at select target genes and controls erythropoiesis. It is not known to what extent Med1 contributes to GATA-1 function or whether Med1 controls a large or restricted cohort of genes that are not regulated by GATA-1. Using a genetic complementation assay in GATA-1-null erythroid cells, we demonstrate that Med1 and another Mediator component, Med25, regulate a restricted cohort of genes that are predominantly not controlled by GATA-1. Most of these genes were not regulated by Med1 in fibroblasts. Loss-of-function analyses with GATA-1-independent Med1 target genes indicate that Rrad, which encodes a small GTPase induced during human erythropoiesis, conferred erythroid cell survival. Thus, while Med1 is a context-dependent GATA-1 coregulator, it also exerts specialized functions in erythroid cells to control GATA-1-independent, cell-type-specific genes, which include candidate regulators of erythroid cell development and function.
Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.
The NXF (nuclear export factor) family members are implicated in the transport of mRNA from the nucleus to the cytoplasm. Recently, some members of the NXF family have been reported to play divergent functional roles, such as post-transcriptional regulation, translational control, regulation of mRNA stability and trafficking. However, little is known about the roles of NXF3 in spermatogenesis. In the present study, we found that mouse NXF3, specifically expressed in principal cells in segment II of the caput epididymis, as well as Sertoli cells in the mouse testis, was required to mediate TGF-β (transforming growth factor β)-induced down-regulation of Tgfb3/TGF-β3 mRNA expression and protein secretion in Sertoli cells. In addition, NXF3 was also involved in TGF-β-induced transcriptional regulation of other genes associated with Sertoli cell maturation and the restructuring of the Sertoli cell BTB (blood-testis barrier), such as Gata1 (GATA-binding protein 1), Wt1 (Wilms's tumour homologue 1), Cldn11 (claudin11) and Cdkn1a (cyclin-dependent kinase inhibitor 1A or p21(Cip1)). The transcriptional regulation of NXF3 was mediated through physical interaction with STRAP (serine/threonine kinase receptor-associated protein), where NXF3 inhibited the complex formation among Smad7, STRAP and activated type I TGF-β receptor. Taken together, our data provide mechanistic insights into the roles of NXF3 in TGF-β-mediated expression of Tgfb3 and other genes. NXF3 may be implicated in Sertoli cell maturation and the extensive restructuring of the Sertoli cell BTB.
Dicer, a member of the RNase III family, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs. Recent evidence indicates that DICER1 expression levels vary among different solid tumors and decreased or increased DICER1 expression has been associated with aggressive cancers. In this study, we assessed DICER1 expression levels in acute myeloid leukemia (AML) and investigated its biological effects and transcriptional regulation in leukemia cell lines. We demonstrated that DICER1 was overexpressed in AML patients and leukemia cell lines by real-time quantitative PCR and western blot analysis. A functional assay demonstrated that the silencing of DICER1 inhibited cell proliferation and promoted apoptosis in leukemia cell lines. We also demonstrated that DICER1 was upregulated by the hematopoietic transcription factor, GATA1, through luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays. These data suggest that DICER1 plays an important role in AML and the finding that the upregulation of DICER1 is induced by GATA1 may provide a framework for the understanding of differential DICER1 expression levels in multiple types of cancer.
The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a 'spin embryoid body' method in serum-free differentiation medium.
Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells.
We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.
miRNAs play important roles in many biological processes, including erythropoiesis. Although several miRNAs regulate erythroid differentiation, how the key erythroid regulator, GATA-1, directly orchestrates differentiation through miRNA pathways remains unclear. In this study, we identified miR-23a as a key regulator of erythropoiesis, which was upregulated both during erythroid differentiation and in GATA-1 gain-of-function experiments, as determined by miRNA expression profile analysis. In primary human CD34+ hematopoietic progenitor cells, miR-23a increased in a GATA-1-dependent manner during erythroid differentiation. Gain- or loss-of-function analysis of miR-23a in mice or zebrafish demonstrated that it was essential for normal morphology in terminally differentiated erythroid cells. Furthermore, a protein tyrosine phosphatase, SHP2, was identified as a downstream target of miR-23a that mediated its regulation of erythropoiesis. Taken together, our data identify a key GATA-1-miRNA axis in erythroid differentiation.
The role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at -110 bp and -299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the -110 and -299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the -110 bp CpG site in the MMP13 promoter inhibited its HIF-2α-driven transactivation and decreased HIF-2α binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2α, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2α transactivation, indicating that the -299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms.
The requirement that leukemic Gata1 mutations be present in cells harboring trisomy 21 led to the discovery that overexpression of ERG drives aberrant megakaryopoiesis. Given that constitutive PI3K/AKT signaling is a frequent component of hematologic malignancies and the relationship between AKT and Notch in this lineage, we studied the crosstalk between AKT signaling and ERG in megakaryopoiesis. We discovered that constitutive AKT signaling is associated with a dramatic increase in apoptosis of WT megakaryocytes (MKs), but that overexpression of ERG blocks AKT-induced death. We further found that Gata1 mutations protect MKs from activated AKT-induced apoptosis. As a consequence, however, the enhanced signaling inhibits differentiation of Gata1 mutant, but not WT, MKs. Gata1 mutant cells that overexpress ERG with hyperactive AKT are characterized by diminished FOXO1/3a expression and an increased dependency on the c-Jun pathway similar to that seen in acute megakaryoblastic leukemia (AMKL) cell lines, acute myeloid leukemia (AML) with knockdown of FOXO3a, or AML with expression of myristoylated Akt. Additionally, we found that the AKT allosteric inhibitor MK2206 caused reduced cell viability and proliferation of AMKL cell lines. The contribution of aberrant AKT signaling during the ontogeny of Down syndrome-transient myeloproliferative disorder/AMKL indicates that AKT is a therapeutic target in this form of AML.
Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.
Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.
The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.
Cell type-specific transcription factors regulate the repertoire of genes expressed in a cell and thereby determine its phenotype. The differentiation of megakaryocytes, the platelet progenitors, from hematopoietic stem cells is a well-known process that can be mimicked in culture. However, the efficient formation of platelets in culture remains a challenge. Platelet formation is a complicated process including megakaryocyte maturation, platelet assembly and platelet shedding. We hypothesize that a better understanding of the transcriptional regulation of this process will allow us to influence it such that sufficient numbers of platelets can be produced for clinical applications. After an introduction to gene regulation and platelet formation, this review summarizes the current knowledge of the regulation of platelet formation by the transcription factors EVI1, GATA1, FLI1, NFE2, RUNX1, SRF and its co-factor MKL1, and TAL1. Also covered is how some platelet disorders including myeloproliferative neoplasms, result from disturbances of the transcriptional regulation. These disorders give us invaluable insights into the crucial role these transcription factors play in platelet formation. Finally, there is discussion of how a better understanding of these processes will be needed to allow for efficient production of platelets in vitro.
Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.
Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.
An extremely premature male neonate presented with an unusual multisystem dysfunction within the first 24 to 48 hours of life. The unfolding of clinical events and investigations revealed a transient myeloproliferative disorder (TMD). TMD was the main indication for karyotyping of this premature infant without clinical symptoms of Down syndrome. The awareness of TMD in a newborn warrants karyotype analysis to look for trisomy 21 and a close surveillance because of its potential progression to true leukaemia.
scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.
Abnormal expression of microRNAs (miRNAs) has been implicated in carcinogenesis. Here we report a novel BCR (breakpoint cluster region)-ABL (c-abl oncogene 1, non-receptor tyrosine kinase)/GATA1/microRNA-138 (miR-138) circuitry in chronic myeloid leukemia (CML). miR-138 expression is downregulated in K562 cells and primary CML samples, which is restored after imatinib treatment. The tumor suppressor activity of miR-138 is demonstrated by the induction of cell cycle arrest at G0/G1, inhibition of cell proliferation and colony forming unit granulocyte-macrophage colony formation and enhanced imatinib-induced apoptosis in K562 and Ku812 cells overexpressing miR-138. Moreover, overexpression of miR-138 led to the downregulation of BCR-ABL. Based on luciferase assay, ABL and BCR-ABL are shown to be the target genes regulated by miR-138. Furthermore, miR-138 binding to ABL was shown to localize to the coding region instead of 3'-untranslated regions (3'-UTR) of ABL mRNA. In addition, CCND3 is another target of miR-138, which represses CCND3 expression by binding to its 3'-UTR. Finally, upregulation of miR-138 upon imatinib treatment is associated with the enhancement of GATA1 activity, which binds to the miR-138 promoter. In conclusion, miR-138 is a tumor suppressor miRNA underexpressed in CML. miR-138 represses expression of both BCR-ABL and CCND3 via binding to the coding region and 3'-UTR, respectively. miR-138 expression is activated by GATA1, which in turn is repressed by BCR-ABL. Therefore, miR-138, by virtue of a BCR-ABL/GATA1/miR-138 circuitry, is a tumor suppressor miRNA implicated in the pathogenesis of CML and its clinical response to imatinib.
Histone deacetylases (HDACs) regulate multiple developmental processes and cellular functions. However, their roles in blood development have not been determined, and in Xenopus laevis a specific function for HDACs has yet to be identified. Here, we employed the class I selective HDAC inhibitor, valproic acid (VPA), to show that HDAC activity is required for primitive hematopoiesis.
VPA treatment during gastrulation resulted in a complete absence of red blood cells (RBCs) in Xenopus tadpoles, but did not affect development of other mesodermal tissues, including myeloid and endothelial lineages. These effects of VPA were mimicked by Trichostatin A (TSA), a well-established pan-HDAC inhibitor, but not by valpromide, which is structurally similar to VPA but does not inhibit HDACs. VPA also caused a marked, dose-dependent loss of primitive erythroid progenitors in mouse yolk sac explants at clinically relevant concentrations. In addition, VPA treatment inhibited erythropoietic development downstream of bmp4 and gata1 in Xenopus ectodermal explants.
These findings suggest an important role for class I HDACs in primitive hematopoiesis. Our work also demonstrates that specific developmental defects associated with exposure to VPA, a significant teratogen in humans, arise through inhibition of class I HDACs.
Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n = 5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an ex vivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.
Cyclin A1 is a male germ cell-specific cell cycle regulator that is essential for spermatogenesis. It is unique among the cyclins by virtue of its highly restricted expression in vivo, being present in pachytene and diplotene spermatocytes and not in earlier or later stages of spermatogenesis. To begin to understand the molecular mechanisms responsible for this narrow window of expression of the mouse cyclin A1 (Ccna1) gene, we carried out a detailed analysis of its promoter. We defined a 170-bp region within the promoter and showed that it is involved in repression of Ccna1 in cultured cells. Within this region we identified known cis-acting transcription factor binding sequences, including an Sp1-binding site and two GATA1-binding sites. Neither Sp1 nor GATA1 is expressed in pachytene spermatocytes and later stages of germ cell differentiation. Sp1 is readily detected at earlier stages of spermatogenesis. Site-directed mutagenesis demonstrated that neither factor alone was sufficient to significantly repress expression driven by the Ccna1 promoter, while concurrent binding of Sp1, and most likely GATA1 and possibly additional factors was inhibitory. Occupancy of Sp1 on the Ccna1 promoter and influence of GATA1-dependent cis-acting elements was confirmed by ChIP analysis in cell lines and most importantly, in spermatogonia. In contrast with many other testis-specific genes, the CpG island methylation status of the Ccna1 promoter was similar among various tissues examined, irrespective of whether Ccna1 was transcriptionally active, suggesting that this regulatory mechanism is not involved in the restricted expression of Ccna1.
Intraductal papillary mucinous neoplasm (IPMN) is a type of tumor that grows within the pancreatic ducts. It is a progress from hyperplasia to intraductal adenoma (IPMA), to noninvasive carcinoma, and ultimately to invasive carcinoma (IPMC). The objective of this study was to explore the molecular mechanism of the progression from IPMA to IPMC. By using the GSE19650 affymetrix microarray data accessible from Gene Expression Omnibus (GEO) database, we first identified the differentially expressed genes (DEGs) between IPMA and IPMC, followed by the protein-protein interaction and single-nucleotide polymorphism (SNP) analysis of the DEGs. Our study identified thousands of DEGs which involved regulation of cell cycle and apoptosis in this progression from IPMA to IPMC. Protein-protein interaction network construction found that MYC, IL6ST, NR3C1, CREBBP, GATA1 and LRP1 might play an important role in the progression. Furthermore, the SNP analysis confirmed the association between BRAC1 and pancreas cancer. In conclusion, our data provide a comprehensive bioinformatics analysis of genes and pathways which may be involved in the progression of IPMN from IPMA to IPMC.
Carcinoma-associated fibroblasts (CAFs) contribute to both tumor growth and cancer progression. In this report, we applied an emerging transcription factor (TF) activity array to fibroblasts to capture the activity of the intracellular signaling network and to define a signature that distinguishes mammary CAFs from normal mammary fibroblasts. Normal fibroblasts that restrained cancer cell invasion developed into an invasion-promoting CAF phenotype through exposure to conditioned medium from MDA-MB-231 breast cancer cells. A myofibroblast-like CAF cell line expressing high levels of smooth muscle actin was compared to normal mammary fibroblasts before and after induction. Comparison of TF activity profiles for all three fibroblast types identified a TF activity signature common to CAFs which included activation of reporters for TFs ELK1, GATA1, retinoic acid receptor (RAR), serum response factor (SRF), and vitamin D receptor (VDR). Additionally, CAFs resembling myofibroblasts, relative to normal fibroblasts, had elevated activation corresponding to NF-kappaB, RUNX2, and YY1, and distinct activity patterns for several differentiation-related TF reporters. Induction of CAFs by exposure of normal fibroblasts to conditioned medium from MDA-MB-231 cells resulted in increased activation of reporters for HIF1, several STAT TFs, and proliferation-related TFs such as AP1. Myofibroblast-like CAFs and induced normal mammary fibroblasts promoted invasion of breast cancer cells by distinct mechanisms, consistent with their distinct patterns of TF activation. The TF activity profiles of CAF subtypes provide an overview of intracellular signaling associated with the induction of a pro-invasive stroma, and provide a mechanistic link between the microenvironmental stimuli and phenotypic response.
The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations.
The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.
Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.
Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR) that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs) or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq).
In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58%) of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context.
Here we present the first genome-wide inventory of GR-binding sites in a neuronal context. These results provide an exciting first global view into neuronal GR targets and the neuron-specific modes of GR action and potentially contributes to our understanding of glucocorticoid action in the brain.
About a quarter of children with Down syndrome and transient abnormal myelopoiesis (TAM) progress to acute megakaryoblastic leukemia (AMKL). We describe isolated dysmegakaryopoiesis despite complete resolution of TAM in an 18-month-old girl, who developed AMKL 6 months later.
In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.
During erythropoiesis, erythropoietin stimulates induction of erythroid transcription factors that activate expression of erythroid genes including the erythropoietin receptor (EPO-R) that results in increased sensitivity to erythropoietin. DNA binding of the basic helix-loop-helix transcription factor, TAL1/SCL, is required for normal erythropoiesis. A link between elevated TAL1 and excessive erythrocytosis is suggested by erythroid progenitor cells from a patient that exhibits unusually high sensitivity to erythropoietin with concomitantly elevated TAL1 and EPO-R expression. We found that TAL1 regulates EPO-R expression mediated via three conserved E-box binding motifs (CAGCTG) in the EPO-R 5' untranslated transcribed region. TAL1 increases association of the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to the region that includes the transcription start site and the 5' GATA and 3' E-box motifs flanking the EPO-R transcription start site suggesting that TAL1 promotes accessibility of this region. Nucleosome shifting has been demonstrated to facilitate TAL1 but not GATA-1 binding to regulate target gene expression. Accordingly, we observed that with induced expression of EPO-R in hemotopoietic progenitor cells, nucleosome phasing shifts to increase the linker region containing the EPO-R transcription start site and TAL1 binds to the flanking 5' GATA and 3' E-box regions of the promoter. These data suggest that TAL1 binds to the EPO-R promoter to activate EPO-R expression and provides a potential link to elevated EPO-R expression leading to hypersensitivity to erythropoietin and the resultant excessive erythrocytosis.
Children with trisomy 21 have a unique predisposition to develop a megakaryoblastic proliferative disease of varying severity during their first 3 months of life. This disorder exists in no other children or adults without the presence of trisomy 21 and only occurs in the fetal or neonatal period of life. Its spontaneous resolution in most cases further delineates it from otherwise indistinguishable neonatal leukaemias. The identification that GATA1 mutations are the leukaemogenic source along with three recently reported prospective clinical trials now provide a clearer understanding of this haematopoietic disorder. These recent advances in this enigmatic disorder, now known as Transient Myeloproliferative Disorder, are reviewed here in order to bring clarity to the breadth of organ involvement, the range of severity, the risk factors for mortality, the therapeutic options for severe manifestations, the natural course of spontaneous resolution regardless of therapy, and the elucidation of the subsequent risk for myeloid leukaemia.
The transcription factor Foxp3 participates dominantly in the specification and function of Foxp3(+)CD4(+) regulatory T cells (T(reg) cells) but is neither strictly necessary nor sufficient to determine the characteristic T(reg) cell signature. Here we used computational network inference and experimental testing to assess the contribution of other transcription factors to this. Enforced expression of Helios or Xbp1 elicited distinct signatures, but Eos, IRF4, Satb1, Lef1 and GATA-1 elicited exactly the same outcome, acting in synergy with Foxp3 to activate expression of most of the T(reg) cell signature, including key transcription factors, and enhancing occupancy by Foxp3 at its genomic targets. Conversely, the T(reg) cell signature was robust after inactivation of any single cofactor. A redundant genetic switch thus 'locked in' the T(reg) cell phenotype, a model that would account for several aspects of T(reg) cell physiology, differentiation and stability.
Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
This review describes the genetics of unusual blood group phenotypes, particularly those with altered expression of Lutheran antigens, and how this area of study has informed our understanding of erythropoiesis in general and haemoglobin switching in particular.
Mutations in erythroid transcription factors GATA1 (GATA1 binding protein 1) and KLF1 (Kruppel-like factor 1) cause benign and disease phenotypes in humans [X-linked Lu(a-b-) phenotype, In(Lu) blood group phenotype, hereditary persistence of foetal haemoglobin, borderline HbA(2), and congenital dyserythropoietic anaemia (CDA)]. These studies explain the occurrence of rare blood group phenotypes with simultaneous altered expression of antigens from several blood group systems and illuminate the role of KLF1 in gamma and delta globin gene regulation.
The study of rare blood group phenotypes is a potent tool for discovery of mutations in human genes. Elucidation of the molecular basis of the rare In(Lu) phenotype revealed the first mutations in human KLF1. Subsequently, numerous additional mutations have been described, one of which causes a rare form of CDA. Analysis of the X-linked Lu(a-b-) phenotype revealed a mutation in the C-terminal domain of human GATA1. The apparent sensitivity of the Lutheran glycoprotein to alterations in GATA1 and KLF1 activity suggest that it could be a useful biomarker of erythroid transcription factor mutation.
In mammals, hematopoietic homeostasis is maintained by a fine-tuned balance among the self-renewal, proliferation, differentiation and survival of hematopoietic stem cells and their progenies. Each process is also supported by the delicate balance of the expression of multiple genes specific to each process. GATA1 is a transcription factor that comprehensively regulates the genes that are important for the development of erythroid and megakaryocytic cells. Accumulating evidence supports the notion that defects in GATA1 function are intimately linked to hematopoietic disorders. In particular, the somatic mutation of the GATA1 gene, which leads to the production of N-terminally truncated GATA1, contributes to the genesis of transient myeloproliferative disorder and acute megakaryoblastic leukemia in infants with Down syndrome. Similarly, a mutation in the GATA1 regulatory region that reduces GATA1 expression is involved in the onset of erythroid leukemia in mice. In both cases, the accumulation of immature progenitor cells caused by GATA1 dysregulation underlies the pathogenesis of the leukemia. This review provides a summary of multi-step leukemogenesis with a focus on GATA1 dysfunction.
Human erytholeukemia K562 cells are induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, sodium butyrate and 1-β-d-arabinofuranosylcytosine. We have investigated the induction of erythroid differentiation of K562 cells by glutamine depletion. When K562 cells were cultured in glutamine-minus medium, the induction of hemoglobin synthesis, accompanied by those of heme-biosynthetic enzymes and erythroid transcriptional factors, was observed. This induction was dependent on the temporally marked decrease of intracellular level of glutathione, followed by the marked activation of p38MAPK and SAPK/JNK, but not ERK. Under glutamine-deficient conditions, the treatment of K562 cells with sodium butyrate resulted in the marked enhancement of the induction of heme biosynthesis. Glutamine depletion also accelerated the expressions of erythroid-related factors including α-globin and heme-biosynthetic enzymes, GATA-1 and NF-E2, in sodium butyrate-induced K562 cells. The transcriptional activity of β-globin gene promoter-reporter was markedly enhanced by these treatments, indicating that glutamine deficiency in combination with sodium butyrate treatment gives high efficiency of chemical-induced differentiation in the hematopoiesis process.
A child was admitted to our hospital for repair of a ventricular septal defect (VSD) characterized by a predominantly right-to-left shunt and a severe stenosis of the right ventricular outflow tract (Tetralogy of Fallot). Severe congenital anemia (hemoglobin 72 g/L), thrombocytopenia (42×G/L) and profound platelet dysfunction led a stem cell defect to be suspected. X-linked thrombocytopenia (GATA-1 mutation) was diagnosed. GATA-1 defect may complicate medical interventions due to excessive bleeding and partial or complete bone marrow failure. Maintaining a platelet count of 100 G/L and a maximal clot firmness (EXTEM-MCF) >50 mm allowed repair of the congenital heart defect without bleeding or hematological complications. Anemia and thrombocytopenia persisted after cardiac surgery, while the spontaneous bleeding tendency improved.
Because the interaction between omega-3 fatty acids and mast cells has remained largely unknown in allergies, we investigated whether omega-3 fatty acids affect the activation of mast cells by examining Th2-associated cytokine production and possible molecular mechanisms. Alpha-linolenic acid and its metabolites including eicosapentaenoic acid and decosahexaenoic acid induced a dramatic decrease in the production of interleukin (IL)-4, IL-5 and IL-13 in a dose-dependent manner, as well as mRNA expression of their genes, in activated MC/9 mast cells and bone marrow-derived mast cells. The effects were comparable to those of cyclosporin A (1 μM), a well-known immunosuppressive agent. Nuclear expression of GATA binding protein-1 (GATA-1) and GATA binding protein-2 (GATA-2), essential transcription factors for mast cell activation, was also greatly suppressed. However, their mRNA expressions were not affected. In P815 mast cells, which do not express GATA-1, the suppressive effects on cytokines were abolished. On the contrary, omega-3 fatty acids had less significant effects on IL-4 and IL-5 and resulted in a slight decrease in IL-13 production in EL-4 T cells. Finally, oral administration of fish oil containing high level of omega-3 fatty acids significantly reduced the severity of dermatitis and the thickening of epidermis/dermis in a NC/Nga murine atopic model. The number of cells expressing CD117(+) and FcεRIα(+) was greatly decreased and GATA-1 expression in the cells was also diminished. Taken together, omega-3 fatty acids might target mast cells to a greater extent than T cells to suppress Th2 cytokine expression by inhibiting GATAs for alleviation of allergic disease.
Tissue-specific transcription patterns are preserved throughout cell divisions to maintain lineage fidelity. We investigated whether transcription factor GATA1 plays a role in transmitting hematopoietic gene expression programs through mitosis when transcription is transiently silenced. Live-cell imaging revealed that a fraction of GATA1 is retained focally within mitotic chromatin. ChIP-seq of highly purified mitotic cells uncovered that key hematopoietic regulatory genes are occupied by GATA1 in mitosis. The GATA1 coregulators FOG1 and TAL1 dissociate from mitotic chromatin, suggesting that GATA1 functions as platform for their postmitotic recruitment. Mitotic GATA1 target genes tend to reactivate more rapidly upon entry into G1 than genes from which GATA1 dissociates. Mitosis-specific destruction of GATA1 delays reactivation selectively of genes that retain GATA1 during mitosis. These studies suggest a requirement of mitotic "bookmarking" by GATA1 for the faithful propagation of cell-type-specific transcription programs through cell division.
The joint application of clinical and genetic investigation to patients with inherited thrombocytopenias, as well as the availability of new methods for studying megakaryopoiesis, has greatly expanded the knowledge of these disorders in the last few years with regard to their etiology, pathogenesis and clinical aspects. In particular, new diseases have been described, as deriving from mutations in the genes FLNA, TUBB1, ITGA2/ITGB3, ANKRD26, CYCS, and ABCG5 or ABCG8. Moreover, forms previously considered separate entities were found to be different clinical aspects of a single disease. For instance, identification of MYH9 as the gene whose mutations cause the May-Hegglin anomaly led to the recognition that Sebastian platelet syndrome, Epstein syndrome, and Fechtner syndrome derive from mutations of the same gene and describe overlapping disorders. Despite these advances, knowledge of hereditary thrombocytopenias is still far from satisfactory because for approximately half of the patients it is not possible to formulate a definite diagnosis in that their illnesses has not yet been described. In this review, we provide a systematic description of hereditary thrombocytopenias as we know them today, giving special attention to genetic aspects.
Previous imaging and postmortem studies have reported a lower brain volume and a smaller size and density of neurons in the dorsolateral prefrontal cortex (dlPFC) of subjects with major depressive disorder (MDD). These findings suggest that synapse number and function are decreased in the dlPFC of patients with MDD. However, there has been no direct evidence reported for synapse loss in MDD, and the gene expression alterations underlying these effects have not been identified. Here we use microarray gene profiling and electron microscopic stereology to reveal lower expression of synaptic-function–related genes (CALM2, SYN1, RAB3A, RAB4B and TUBB4) in the dlPFC of subjects with MDD and a corresponding lower number of synapses. We also identify a transcriptional repressor, GATA1, expression of which is higher in MDD and that, when expressed in PFC neurons, is sufficient to decrease the expression of synapse-related genes, cause loss of dendritic spines and dendrites, and produce depressive behavior in rat models of depression.
GATA1 is a hematopoietic transcription factor essential for expression of most genes encoding erythro-megakaryocytic proteins, i.e., globins and platelet glycoproteins. A role for GATA1 as a cell proliferation regulator has been proposed, as some of its bona fide targets comprise global regulators, such as c-KIT or c-MYC, or cell cycle factors, i.e., CYCLIN D or p21CIP1. In this study, we describe that GATA1 directly regulates the expression of replication licensing factor CDC6. Using reporter transactivation, electrophoretic mobility shift and chromatin immunoprecipitation assays, we show that GATA1 stimulates CDC6 transcription by binding to a canonical binding site located within a 166bp enhancer region upstream CDC6 promoter. This evolutionary conserved GATA binding site conforms to recently described chromatin occupancy rules, i.e., preferred bases within core WGATAR (TGATAA), 5' and 3' flanking bases (GGTGATAAGG) and distance to the transcription initiation site. We also found adjacent conserved binding sites for ubiquitously expressed transcription factor CP2, needed for GATA activity on CDC6 enhancer. Our results add to the growing evidence for GATA1 acting as a direct transcriptional regulator of the cell cycle machinery, thus linking cell proliferation control and specific gene expression programs during lineage differentiation.
Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23–68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.
The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.
Transgenic mice represent a unique opportunity in biomedical research to discover the genes underlying disease and understand how manipulating the function of single genes and proteins alters physiology in a whole animal system. These advances in biomedical research may accelerate the time between when basic discoveries are made and when the research can be 'translated', that is, when the research will positively impact the lives of patients. The purpose of this article is to present some examples of promising mouse models of human diseases.
Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans-particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL) where GATA1FL mutations are an essential driver for disease pathogenesis.
Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation.
We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI.
These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.
KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1(+/+) and Klf1(-/-) erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.
Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues.
Deficiency of the transcription factor MafB, which is normally expressed in macrophages, can underlie cellular dysfunction associated with a range of autoimmune diseases and arteriosclerosis. MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory cis-elements (motifs) offers a robust approach.
Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5'AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs.
These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation.
RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133:4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.
The transcription factor Hairy Enhancer of Split 1 (HES1), a downstream effector of the Notch signaling pathway, is an important regulator of hematopoiesis. Here, we demonstrate that in primary erythroid cells, Hes1 gene expression is transiently repressed around proerythroblast stage of differentiation. Using mouse erythroleukemia cells, we found that the RNA interference (RNAi)-mediated depletion of HES1 enhances erythroid cell differentiation, suggesting that this protein opposes terminal erythroid differentiation. This is also supported by the decreased primary erythroid cell differentiation upon HES1 upregulation in Ikaros-deficient mice. A comprehensive analysis led us to determine that Ikaros favors Hes1 repression in erythroid cells by facilitating recruitment of the master regulator of erythropoiesis GATA-1 alongside FOG-1, which mediates Hes1 repression. GATA-1 is then necessary for the chromatin binding of the NuRD remodeling complex ATPase MI-2, the transcription factor GFI1B, and the histone H3K27 methyltransferase EZH2 along with Polycomb repressive complex 2. We show that EZH2 is required for the transient repression of Hes1 in erythroid cells. In aggregate, our results describe a mechanism whereby GATA-1 utilizes Ikaros and Polycomb repressive complex 2 to promote Hes1 repression as an important step in erythroid cell differentiation.
Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy and favorable prognosis. Because little research has been focused on refractory/relapsed (R/R) cases, we conducted a retrospective analysis for R/R ML-DS. Among ML-DS patients diagnosed between 2000 and 2010 in Japan, 26 relapsed (25 in the BM and 1 in the skin), and 3 refractory patients were enrolled. The male/female ratio was 18/11. The median age at initial diagnosis of ML-DS was 2 years, and the median time to relapse was 8.6 months. Each patient initially had been treated with ML-DS-specific protocols. Thirteen of the 26 patients achieved complete remission with various kinds of reinduction chemotherapies; 2 of 8 survived without further recurrence after receiving allogeneic hematopoietic stem cell transplantation, and 4 of 5 maintained complete remissions with chemotherapy alone. Treatment failures mostly were associated with disease progression rather than treatment-related toxicities. The 3-year OS rate was 25.9% ± 8.5%. A longer duration from initial diagnosis to relapse was a significant favorable prognostic factor (P < .0001). We conclude that clinical outcome for patients with R/R ML-DS generally are unfavorable, even in those receiving hematopoietic stem cell transplantation. Novel methods to identify poor prognostic factors for ML-DS are necessary.
GATA-1 and its cofactor FOG-1 are required for the differentiation of erythrocytes and megakaryocytes. In contrast, mast cell development requires GATA-1 and the absence of FOG-1. Through genome-wide comparison of the chromatin occupancy of GATA-1 and a naturally occurring mutant that cannot bind FOG-1 (GATA-1(V205G)), we reveal that FOG-1 intricately regulates the chromatin occupancy of GATA-1. We identified GATA1-selective and GATA-1(V205G)-selective binding sites and show that GATA-1, in the absence of FOG-1, occupies GATA-1(V205G)-selective sites, but not GATA1-selective sites. By integrating ChIP-seq and gene expression data, we discovered that GATA-1(V205G) binds and activates mast cell-specific genes via GATA-1(V205G)-selective sites. We further show that exogenous expression of FOG-1 in mast cells leads to displacement of GATA-1 from mast cell-specific genes and causes their downregulation. Together these findings establish a mechanism of gene regulation whereby a non-DNA binding cofactor directly modulates the occupancy of a transcription factor to control lineage specification.
Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis.
Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.
Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.
Hematopoietic development occurs in complex microenvironments and is influenced by key signaling events. Yet how these pathways communicate with master hematopoietic transcription factors to coordinate differentiation remains incompletely understood. The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. It is also the most frequent target of genetic alterations in human leukemia. Here, we report that RUNX1 is phosphorylated by Src family kinases (SFKs) and that this occurs on multiple tyrosine residues located within its negative regulatory DNA-binding and autoinhibitory domains. Retroviral transduction, chemical inhibitor, and genetic studies demonstrate a negative regulatory role of tyrosine phosphorylation on RUNX1 activity in Mk and CD8 T-cell differentiation. We also demonstrate that the nonreceptor tyrosine phosphatase Shp2 binds directly to RUNX1 and contributes to its dephosphorylation. Last, we show that RUNX1 tyrosine phosphorylation correlates with reduced GATA1 and enhanced SWI/SNF interactions. These findings link SFK and Shp2 signaling pathways to the regulation of RUNX1 activity in hematopoiesis via control of RUNX1 multiprotein complex assembly.
Increasing evidence points to endoglin (Eng), an accessory receptor for the transforming growth factor-β superfamily commonly associated with the endothelial lineage, as an important regulator of the hematopoietic lineage. We have shown that lack of Eng results in reduced numbers of primitive erythroid colonies as well as downregulation of key hematopoietic genes. To determine the effect of Eng overexpression in hematopoietic development, we generated a doxycycline-inducible embryonic stem cell line. Our results demonstrate that induction of Eng during embryoid body differentiation leads to a significant increase in the frequency of hematopoietic progenitors, in particular, the erythroid lineage, which correlated with upregulation of Scl, Gata1, Runx1, and embryonic globin. Interestingly, activation of the hematopoietic program happened at the expense of endothelial and cardiac cells, as differentiation into these mesoderm lineages was compromised. Eng-induced enhanced erythroid activity was accompanied by high levels of Smad1 phosphorylation. This effect was attenuated by addition of a bone morphogenetic protein (BMP) signaling inhibitor to these cultures. Among the BMPs, BMP4 is well known for its role in hematopoietic specification from mesoderm by promoting expression of several hematopoietic genes, including Scl. Because Scl is considered the master regulator of the hematopoietic program, we investigated whether Scl would be capable of rescuing the defective hematopoietic phenotype observed in Eng(-/-) embryonic stem cells. Scl expression in Eng-deficient embryonic stem cells resulted in increased erythroid colony-forming activity and upregulation of Gata1 and Gata2, positioning Eng upstream of Scl. Taken together, these findings support the premise that Eng modulates the hematopoietic transcriptional network, most likely through regulation of BMP4 signaling.
Diamond-Blackfan anemia (DBA) is a hypoplastic anemia characterized by impaired production of red blood cells, with approximately half of all cases attributed to ribosomal protein gene mutations. We performed exome sequencing on two siblings who had no known pathogenic mutations for DBA and identified a mutation in the gene encoding the hematopoietic transcription factor GATA1. This mutation, which occurred at a splice site of the GATA1 gene, impaired production of the full-length form of the protein. We further identified an additional patient carrying a distinct mutation at the same splice site of the GATA1 gene. These findings provide insight into the pathogenesis of DBA, showing that the reduction in erythropoiesis associated with the disease can arise from causes other than defects in ribosomal protein genes. These results also illustrate the multifactorial role of GATA1 in human hematopoiesis.
Mutations in numerous genes encoding ribosomal proteins (RPs) occur in 50%-70% of individuals with Diamond-Blackfan anemia (DBA), establishing the disease as a ribosomopathy. As described in this issue of JCI, Sankaran, Gazda, and colleagues used genome-wide exome sequencing to study DBA patients with no detectable mutations in RP genes. They identified two unrelated pedigrees in which the disease is associated with mutations in GATA1, which encodes an essential hematopoietic transcription factor with no known mechanistic links to ribosomes. These findings ignite an interesting and potentially emotional debate on how we define DBA and whether the term should be restricted to pure ribosomopathies. More generally, the work reflects the powerful knowledge and controversies arising from the deluge of data generated by new genetic technologies that are being used to analyze human diseases.
BCR/ABL expression is the key characteristic of chronic myeloid leukaemia (CML). The progression of CML is associated with genomic instability. Systematic analysis of DNA damage signalling in the presence of BCR/ABL thus offers opportunities to identify mechanisms of leukaemic progression. We therefore undertook a quantitative phosphoproteomics study to test whether BCR/ABL expression could globally affect the response to genotoxic stress signalling. Etoposide-induced DNA damage was chosen and the concentration and time of exposure determined such that apoptosis was not associated with the study. More than 1100 phosphoentities were identified. BCR/ABL was shown to significantly alter the response to etoposide in many cases. These include sites on MDC1, a key component of DNA repair, and Hemogen. Hemogen is a transcriptional target of HOXB4 and GATA1, two transcription factors involved in haemopoietic development, and is overexpressed in acute myeloid leukaemia. To validate Hemogen phosphorylation, absolute quantification using an isotopomeric standard and selected reaction monitoring was performed. This revealed a strong correlation with isobaric tagging data and offering quantification at about 10 fmol per million cells. Furthermore we found that multiple protein phosphorylation changes mediated by BCR/ABL were connected to the increased activation of NFκB, a key survival transcription factor, after etoposide exposure.
Chromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts, in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.
We have investigated the role of erythroid transcription factors mRNA expression in patients with acute myeloid leukemia (AML) in the context of cytogenetic and other prognostic molecular markers, such as FMS-like Tyrosine Kinase 3 (FLT3), Nucleophosmin 1 (NPM1), and CCAAT/enhance-binding protein α (CEBPA) mutations. Further validation of Erythroid Krüppel-like Factor (EKLF) mRNA expression as a prognostic factor was assessed.We evaluated GATA binding protein 1 (GATA1), GATA binding protein 2 (GATA2), EKLF and Myeloproliferative Leukemia virus oncogen homology (cMPL) gene mRNA expression in the bone marrow of 65 AML patients at diagnosis, and assessed any correlation with NPM1, FLT3 and CEBPA mutations. EKLF-positive AML was associated with lower WBC in peripheral blood (P = 0.049), a higher percentage of erythroblasts in bone marrow (p = 0.057), and secondary AMLs (P = 0.036). High expression levels of EKLF showed a trend to association with T-cell antigen expression, such as CD7 (P = 0.057). Patients expressing EKLF had longer Overall Survival (OS) and Event Free Survival (EFS) than those patients not expressing EKLF (median OS was 35.61 months and 19.31 months, respectively, P = 0.0241; median EFS was 19.80 months and 8.03 months, respectively, P = 0.0140). No correlation of GATA1, GATA2, EKLF and cMPL levels was observed with FLT-3 or NPM1 mutation status. Four of four CEBPA mutated AMLs were EKLF positive versus 10 of 29 CEBPA wild-type AMLs; three of the CEBPA mutated, EKLF-positive AMLs were also GATA2 positive. There were no cases of CEBPA mutations in the EKLF-negative AML group. In conclusion, we have validated EKLF mRNA expression as an independent predictor of outcome in AML, and its expression is not associated with FLT3-ITD and NPM1 mutations. EKLF mRNA expression in AML patients may correlate with dysregulated CEBPA.
It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny.
Hematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells.
In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH).
This broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes.
We have analyzed publicly available K562 Hi-C data, which enable genome-wide unbiased capturing of chromatin interactions, using a Mixture Poisson Regression Model and a power-law decay background to define a highly specific set of interacting genomic regions. We integrated multiple ENCODE Consortium resources with the Hi-C data, using DNase-seq data and ChIP-seq data for 45 transcription factors and 9 histone modifications. We classified 12 different sets (clusters) of interacting loci that can be distinguished by their chromatin modifications and which can be categorized into two types of chromatin linkages. The different clusters of loci display very different relationships with transcription factor-binding sites. As expected, many of the transcription factors show binding patterns specific to clusters composed of interacting loci that encompass promoters or enhancers. However, cluster 9, which is distinguished by marks of open chromatin but not by active enhancer or promoter marks, was not bound by most transcription factors but was highly enriched for three transcription factors (GATA1, GATA2 and c-Jun) and three chromatin modifiers (BRG1, INI1 and SIRT6). To investigate the impact of chromatin organization on gene regulation, we performed ribonucleicacid-seq analyses before and after knockdown of GATA1 or GATA2. We found that knockdown of the GATA factors not only alters the expression of genes having a nearby bound GATA but also affects expression of genes in interacting loci. Our work, in combination with previous studies linking regulation by GATA factors with c-Jun and BRG1, provides genome-wide evidence that Hi-C data identify sets of biologically relevant interacting loci.
Spontaneous remission in 2 children with myelofibrosis, one with megakaryocytic acute myeloblastic leukemia and t(1;22) (with recurrence later) and one with Down syndrome and GATA1 mutation (permanent), are described. One had sepsis and was treated with antibiotics and blood products, whereas the other received only blood products. Remission was spontaneous, without chemotherapy treatment. Possible explanations for these outcomes include immunologic response to sepsis by a leukemia-specific T-cell response or the release of various cytokines, such as tumor necrosis factor and interleukin-2, during infections. Natural killer and cytotoxic T cells transfused with blood products might have also triggered an immune response.
Carbonic anhydrase 1 (Car1), an early specific marker of the erythroid differentiation, has been used to distinguish fetal and adult erythroid cells since its production closely follows the γ- to β-globin transition, but the molecular mechanism underlying transcriptional regulation of Car1 is unclear. Here, we show that Car1 mRNA decreases significantly when erythroid differentiation is induced in MEL cells. The Ldb1 protein complex including GATA1/SCL/LMO2 binds to the Car1 promoter in uninduced cells and reduced enrichment of the complex during differentiation correlates with loss of Car1 expression. Knockdown of Ldb1 results in a reduction of Ser2 phosphorylated RNA Pol II and Cdk9 at the Car1 promoter region, suggesting that Ldb1 is required for recruitment of Pol II as well as the transcription regulator P-TEFb to enhance elongation of Car1 transcripts. Taken together, these data show that Ldb1 forms a regulatory complex to maintain Car1 expression in erythroid cells.
The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.
Hyperthermia treatment has at times been associated with increased platelet levels in humans. The heat shock protein HSP70, which can be induced by hyperthermia in megakaryocytes and erythrocytes, was recently shown to protect GATA-1 from degradation and to be required for erythroid differentiation. Based on these findings, we hypothesize that mild hyperthermia (MH), such as fever (39°C), could impact the differentiation of hematopoietic progenitors into erythrocytes and their subsequent maturation. Cell growth and erythroid differentiation increased dramatically in cord blood CD34(+) cell cultures incubated under MH. Erythroid maturation was also strongly promoted, which resulted in an increased proportion of hemoglobinized and enucleated erythroids. The rise in erythroid development was traced to a strong synergistic activity between MH and erythropoietin (EPO). The molecular basis for this potent synergy appears to originate from the capacity of MH to increase the basal activation of several signaling molecules downstream of the EPO receptor and the transcriptional activity of GATA-1. Moreover, the potent impact of MH on erythroid development was found be dependent on increased intracellular levels of reactive oxygen species. Thus, fever-like temperatures can promote the differentiation of progenitors along the erythroid lineage and accelerate their maturation through normal regulatory circuitry.
Nfil3, a transcription factor that has an array of functions in immune cells, has been described as key regulator of CD8α(+) dendritic cell and natural killer cell development in mice. In this report we show that Nfil3 is enriched in the myeloid compartment of adult zebrafish including eosinophils. Knockdown of Nfil3 in pu.1:GFP embryos resulted in a reduced number of myeloid cells as early as 24h post-fertilization, while erythropoiesis was unaffected. Using mpx and fms-fluorescent transgenic fish we found that all myeloid cell lineages, and in particular macrophages, had reduced numbers at 4days post-fertilization. This was reflected by less myeloid cells accumulating at a wound site. Pu.1, l-plastin, csf1r and mpx had reduced expression in Nfil3 morphants while runx1, gata1 and rag1 were unaffected. Collectively, these results describe a conserved expression pattern of Nfil3 in evolutionarily divergent species and indicate that Nfil3 is central to myeloid lineage commitment.
Transcription factor GATA1 regulates the expression of a cluster of genes important for hematopoietic cell differentiation toward erythroid and megakaryocytic lineages. Three functional domains have been identified in GATA1, a transactivation domain located in the N terminus (N-TAD) and two zinc finger domains located in the middle of the molecule. Although N-TAD is known as a solitary transactivation domain for GATA1, clinical observations in Down syndrome leukemia suggest that there may be additional transactivation domains. In this study, we found in reporter co-transfection assays that transactivation activity of GATA1 was markedly reduced by deletion of the C-terminal 95 amino acids without significant attenuation of the DNA binding activity or self-association potential. We therefore generated transgenic mouse lines that expressed GATA1 lacking the C-terminal region (GATA1-ΔCT). When we crossed these transgenic mouse lines to the Gata1-deficient mouse, we found that the GATA1-ΔCT transgene rescued Gata1-deficient mice from embryonic lethality. The embryos rescued with an almost similar level of GATA1-ΔCT to endogenous GATA1 developed beyond embryonic 13.5 days, showing severe anemia with accumulation of immature erythroid cells, as was the case for the embryos rescued by endogenous levels of GATA1 lacking N-TAD (GATA1-ΔNT). Distinct sets of target genes were affected in the embryos rescued by GATA1-ΔCT and GATA1-ΔNT. We also found attenuated GATA1 function in cell cycle control of immature megakaryocytes in both lines of rescued embryos. These results thus demonstrate that GATA1 has two independent transactivation domains, N-TAD and C-TAD. Both N-TAD and C-TAD retain redundant as well as specific activities for proper hematopoiesis in vivo.
Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.
The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation.
We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified.The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.
The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.
Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71(+)Ter119(+) erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Scl, Gata1, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng(+)Flk-1(+) progenitors coexpressed TGFβ and BMP receptors and target genes. Furthermore, Eng(+)Flk-1(+) cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFβ superfamily ligands BMP4, BMP2, or TGF-β1. These data demonstrate that the E(+)F(+) fraction at E7.5 represents mesodermal cells competent to respond to TGFβ1, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling.
Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (<5 years old) to prevent overtreatment. Treatment with reduced intensity seems sufficient in children trisomy 21 mosaic and ML-DS.
Both endothelial and erythroid cells are generated in the intermediate cell mass (ICM) during zebrafish embryogenesis, but the nature of the genes that contribute to the processes of erythrocyte maturation and blood vessel network formation is not fully understood. From our in situ-based screening, we have identified a novel factor, Vap (Vascular Associated Protein) that is predominantly expressed in the ICM, and subsequently enriched in endothelial cells. Vap expression in the ICM was drastically suppressed in the cloche mutant that has defects in both vasculogenesis and hematopoiesis, whereas Vap expression was not affected in the vlad tepes/gata1 mutant. Knockdown of Vap using anti-sense morpholinos (VAP-MO) not only resulted in decreased numbers of erythrocytes but also in the strong suppression of hemoglobin production. Further, we found that Vap knockdown caused the disorganization of the intersegmental vessels (ISVs), which show irregular branching. We propose that Vap plays an important role in the maturation of endothelial and erythroid cells in zebrafish.
The mouse posterior primitive streak at neural plate/headfold stages (NP/HF, ~7.5 dpc-8 dpc) represents an optimal window from which hemangioblasts can be isolated. We performed immunohistochemistry on this domain using established monoclonal antibodies for proteins that affect blood and endothelial fates. We demonstrate that HoxB4 and GATA1 are the first set of markers that segregate independently to endothelial or blood populations during NP/HF stages of mouse embryonic development. In a subset of cells, both proteins are co-expressed and immunoreactivities appear mutually excluded within nuclear spaces. We searched for this particular state at later sites of hematopoietic stem cell emergence, viz., the aorta-gonad-mesonephros (AGM) and the fetal liver at 10.5-11.5 dpc, and found that only a rare number of cells displayed this character. Based on this spatial-temporal argument, we propose that the earliest blood progenitors emerge either directly from the epiblast or through segregation within the allantoic core domain (ACD) through reduction of cell adhesion and pSmad1/5 nuclear signaling, followed by a stochastic decision toward a blood or endothelial fate that involves GATA1 and HoxB4, respectively. A third form in which binding distributions are balanced may represent a common condition shared by hemangioblasts and HSCs. We developed a heuristic model of hemangioblast maturation, in part, to be explicit about our assumptions.
Children with Down syndrome (DS) have an increased risk of Acute Myeloid Leukaemia (ML-DS), particularly megakaryoblastic leukaemia, which is clonally -related to the neonatal myeloproliferative syndrome, Transient Abnormal Myelopoiesis (TAM) unique to infants with DS. Molecular, biological, and clinical data indicate that TAM is initiated before birth when fetal liver haematopoietic cells trisomic for chromosome 21 acquire mutations in GATA1. TAM usually resolves spontaneously by 6 months; however 20-30% subsequently develop ML-DS harbouring the same GATA1 mutation(s). This review focuses on recent studies describing haematological, clinical and biological features of TAM and discusses approaches to diagnose, treat and monitor minimal residual disease in TAM. An important unanswered question is whether ML-DS is always preceded by TAM as it may be clinically and possibly haematologically 'silent'. We have briefly discussed the role of population-based screening for TAM and development of treatment strategies to eliminate the preleukaemic TAM clone, thereby preventing ML-DS.
The discovery of the JAK2V617F mutation in most patients with Ph-negative myeloproliferative neoplasms has led to the development of JAK2 kinase inhibitors. However, JAK2 inhibitor therapy has shown limited efficacy and dose-limiting hematopoietic toxicities in clinical trials. In the present study, we describe the effects of vorinostat, a small-molecule inhibitor of histone deacetylase, against cells expressing JAK2V617F and in an animal model of polycythemia vera (PV). We found that vorinostat markedly inhibited proliferation and induced apoptosis in cells expressing JAK2V617F. In addition, vorinostat significantly inhibited JAK2V617F-expressing mouse and human PV hematopoietic progenitors. Biochemical analyses revealed significant inhibition of phosphorylation of JAK2, Stat5, Stat3, Akt, and Erk1/2 in vorinostat-treated, JAK2V617F-expressing human erythroleukemia (HEL) cells. Expression of JAK2V617F and several other genes, including GATA1, KLF1, FOG1, SCL, C/EPBα, PU.1, and NF-E2, was significantly down-regulated, whereas the expression of SOCS1 and SOCS3 was up-regulated by vorinostat treatment. More importantly, we observed that vorinostat treatment normalized the peripheral blood counts and markedly reduced splenomegaly in Jak2V617F knock-in mice compared with placebo treatment. Vorinostat treatment also decreased the mutant allele burden in mice. Our results suggest that vorinostat may have therapeutic potential for the treatment of PV and other JAK2V617F-associated myeloproliferative neoplasms.
The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.
Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a ~3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.
Mast cell degranulation is a dynamic, highly organized process involving numerous signaling molecules and enzymes. Although the molecular mechanisms underlying antigen-mediated mast cell degranulation have been studied intensively, little is known about the transcriptional control of this process. Here, we show that the hematopoietic transcription factors GATA1 and GATA2 are involved in mast cell degranulation through the control of phospholipase C-γ1 (PLC-γ1) expression. Knockdown of GATA1 and/or GATA2 by specific siRNA significantly reduced antigen-induced degranulation and Ca(2+) mobilization in the rat basophilic leukemia cell line RBL-2H3. RT-PCR analyses showed that PLC-γ1 expression was significantly decreased by this GATA factor repression. Other GATA factor targets, such as the previously reported α and β subunits of the high-affinity IgE receptor (FcεRI), were unaffected. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that GATA factors directly activate PLC-γ1 gene transcription through a conserved GATA-binding motif that resides in the 5'-upstream sequence. Furthermore, we show evidence that the PLC-γ1 expression is regulated by GATA2 in mast cells derived from mouse bone marrow. These data indicate that PLC-γ1 is a target gene of GATA factors in mast cells and provide evidence that GATA1 and GATA2 control antigen-mediated mast cell degranulation by regulating the expression of PLC-γ1.
Individuals with Down syndrome (DS; also known as trisomy 21) have a markedly increased risk of leukemia in childhood but a decreased risk of solid tumors in adulthood. Acquired mutations in the transcription factor-encoding GATA1 gene are observed in nearly all individuals with DS who are born with transient myeloproliferative disorder (TMD), a clonal preleukemia, and/or who develop acute megakaryoblastic leukemia (AMKL). Individuals who do not have DS but bear germline GATA1 mutations analogous to those detected in individuals with TMD and DS-AMKL are not predisposed to leukemia. To better understand the functional contribution of trisomy 21 to leukemogenesis, we used mouse and human cell models of DS to reproduce the multistep pathogenesis of DS-AMKL and to identify chromosome 21 genes that promote megakaryoblastic leukemia in children with DS. Our results revealed that trisomy for only 33 orthologs of human chromosome 21 (Hsa21) genes was sufficient to cooperate with GATA1 mutations to initiate megakaryoblastic leukemia in vivo. Furthermore, through a functional screening of the trisomic genes, we demonstrated that DYRK1A, which encodes dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A, was a potent megakaryoblastic tumor-promoting gene that contributed to leukemogenesis through dysregulation of nuclear factor of activated T cells (NFAT) activation. Given that calcineurin/NFAT pathway inhibition has been implicated in the decreased tumor incidence in adults with DS, our results show that the same pathway can be both proleukemic in children and antitumorigenic in adults.
Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5' deleted promoter-reporter constructs and luciferase assay illustrated the region -159/-100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region -159/-100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.
Transient myeloproliferative disorder (TMD) of the newborn and acute megakaryoblastic leukaemia (AMKL) in children with Down syndrome (DS) represent paradigmatic models of leukaemogenesis. Chromosome 21 gene dosage effects and truncating mutations of the X-chromosomal transcription factor GATA1 synergize to trigger TMD and AMKL in most patients. Here, we report the occurrence of TMD, which spontaneously remitted and later progressed to AMKL in a patient without DS but with a distinct dysmorphic syndrome. Genetic analysis of the leukaemic clone revealed somatic trisomy 21 and a truncating GATA1 mutation. The analysis of the patient's normal blood cell DNA on a genomic single nucleotide polymorphism (SNP) array revealed a de novo germ line 2·58 Mb 15q24 microdeletion including 41 known genes encompassing the tumour suppressor PML. Genomic context analysis of proteins encoded by genes that are included in the microdeletion, chromosome 21-encoded proteins and GATA1 suggests that the microdeletion may trigger leukaemogenesis by disturbing the balance of a hypothetical regulatory network of normal megakaryopoiesis involving PML, SUMO3 and GATA1. The 15q24 microdeletion may thus represent the first genetic hit to initiate leukaemogenesis and implicates PML and SUMO3 as novel components of the leukaemogenic network in TMD/AMKL.
Trib1 has been identified as a myeloid oncogene in a murine leukemia model. Here we identified a TRIB1 somatic mutation in a human case of Down syndrome-related acute megakaryocytic leukemia. The mutation was observed at well-conserved arginine 107 residue in the pseudokinase domain. This R107L mutation remained in leukocytes of the remission stage in which GATA1 mutation disappeared, suggesting the TRIB1 mutation is an earlier genetic event in leukemogenesis. The bone marrow transfer experiment showed that acute myeloid leukemia development was accelerated by transducing murine bone marrow cells with the R107L mutant in which enhancement of ERK phosphorylation and C/EBPα degradation by Trib1 expression was even greater than in those expressing wild-type. These results suggest that TRIB1 may be a novel important oncogene for Down syndrome-related acute megakaryocytic leukemia.
GATA1 plays essential roles in erythroid gene expression. The N-terminal finger of GATA1 (GATA1-Nf) is important for association with FOG1. Substitution mutations in GATA1-Nf, such as GATA1(V205M) that diminish the GATA1-FOG1 association, have been identified in human thrombocytopenia and anemia cases. A mouse model of human thrombocytopenia has been established using a transgenic complementation rescue approach; GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of GATA1(V205G), but rescued adult mice suffered from severe thrombocytopenia. In this study, we examined GATA1-deficient mice rescued with GATA1(V205G) at a comparable level to endogenous GATA1. Mice rescued with this level of GATA1(V205G) rarely survive to adulthood. Rescued newborns suffered from severe anemia and jaundice accompanied with anisocytosis and spherocytosis. Expression of Slc4a1, Spna1, and Aqp1 genes (encoding the membrane proteins band-3, α-spectrin, and aquaporin-1, respectively) were strikingly diminished, whereas expression of other canonical GATA1-target genes, such as Alas2, were little affected. Lack of these membrane proteins provoked perturbation of membrane skeleton. Importantly, the red cells exhibited increased reactive oxygen species accumulation. These results thus demonstrate that the loss of the GATA1-FOG1 interaction causes a unique combination of membrane protein deficiency and disturbs the function of GATA1 in maintaining erythroid homeostasis.
ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA.
The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein.
Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.
The transcription factor p45 nuclear factor-erythroid-derived 2 (NF-E2) plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythroid differentiation in vivo. Absence of p45 NF-E2 in mice leads to a twofold increase in serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely, late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels, as well as an increased proportion of erythroid cells arrested at the G(1) stage of cell cycle in p45 NF-E2-deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.
Gene expression profiling of airway epithelial and inflammatory cells can be used to identify genes involved in environmental asthma.
Airway epithelia and inflammatory cells were obtained via bronchial brush and bronchoalveolar lavage (BAL) from 39 subjects comprising three phenotypic groups (nonatopic nonasthmatic, atopic nonasthmatic, and atopic asthmatic) 4 hours after instillation of LPS, house dust mite antigen, and saline in three distinct subsegmental bronchi. RNA transcript levels were assessed using whole genome microarrays.
Baseline (saline exposure) differences in gene expression were related to airflow obstruction in epithelial cells (C3, ALOX5AP, CCL18, and others), and to serum IgE (innate immune genes and focal adhesion pathway) and allergic-asthmatic phenotype (complement genes, histone deacetylases, and GATA1 transcription factor) in inflammatory cells. LPS stimulation resulted in pronounced transcriptional response across all subjects in both airway epithelia and BAL cells, with strong association to nuclear factor-κB and IFN-inducible genes as well as signatures of other transcription factors (NRF2, C/EBP, and E2F1) and histone proteins. No distinct transcriptional profile to LPS was observed in the asthma and atopy phenotype. Finally, although no consistent expression changes were observed across all subjects in response to house dust mite antigen stimulation, we observed subtle differences in gene expression (e.g., GATA1 and GATA2) in BAL cells related to the asthma and atopy phenotype.
Our results indicate that among individuals with allergic asthma, transcriptional changes in airway epithelia and inflammatory cells are influenced by phenotype as well as environmental exposures.
DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.
Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.
Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.
Normal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3-mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.
The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.
Although adults with Down syndrome (DS) show a decreased incidence of cancer compared to individuals without DS, children with DS are at an increased risk of leukemia. Nearly half of these childhood leukemias are classified as acute megakaryoblastic leukemia (AMKL), a relatively rare subtype of acute myeloid leukemia (AML). Here, we summarize the clinical features of myeloid leukemia in DS, review recent research on the mechanisms of leukemogenesis, including the roles of GATA1 mutations and trisomy 21, and discuss treatment strategies. Given that trisomy 21 is a relatively common event in hematologic malignancies, greater knowledge of how the genes on chromosome 21 contribute to DS-AMKL will increase our understanding of a broader class of patients with leukemia.
Polo-like kinase 2 (Plk2) is a member of the serine/threonine protein kinase family involved in cell-cycle regulation and cellular response to stresses. It is of great interest to investigate the molecular mechanisms that control the expression of Plk2. Here, using real-time PCR and Western blot assays, we show that trichostatin A (TSA), a histone deacetylase inhibitor, upregulated Plk2 mRNA and protein expression in the human osteosarcoma MG-63 cell line. Luciferase activity analysis of the truncated Plk2 promoter indicated that the region from -1220 to -830 of the Plk2 promoter was sensitive to TSA. Moreover, using the electrophoresis mobility shift assay and chromatin immunoprecipitation assay, we identified two GATA-1 responsive elements at positions -1051 and -949, to which GATA-1 binding was enhanced by TSA under in vitro and in vivo conditions. Immunoprecipitation and Western blot showed that the levels of acetylated GATA-1 were increased with TSA in MG-63 cells, consistent with their binding affinities to the GATA-1 responsive elements. In summary, these data demonstrate that acetylation plays a crucial role in Plk2 expression and acetylation of GATA-1 by TSA treatment may upregulate their DNA-binding affinities, resulting in the activation of Plk2 promoter. These results may contribute to the understanding of the molecular mechanism of Plk2 regulation.
Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood.
We generated etsrp:gfp transgenic zebrafish and used them to identify regulatory regions and transcription factors critical for etsrp expression and angioblast specification from mesoderm.
To investigate the mechanisms that initiate angioblast cell transcription during embryogenesis, we have performed promoter analysis of the etsrp locus in zebrafish. We describe three enhancer elements sufficient for endothelial gene expression when place in front of a heterologous promoter. The deletion of all 3 regulatory regions led to a near complete loss of endothelial expression from the etsrp promoter. One of the enhancers, located 2.3 kb upstream of etsrp contains a consensus FOX binding site that binds Foxc1a and Foxc1b in vitro by EMSA and in vivo using ChIP. Combined knockdown of foxc1a/b, using morpholinos, led to a significant decrease in etsrp expression at early developmental stages as measured by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization. Decreased expression of primitive erythrocyte genes scl and gata1 was also observed, whereas pronephric gene pax2a was relatively normal in expression level and pattern.
These findings identify mesodermal foxc1a/b as a direct upstream regulator of etsrp in angioblasts. This establishes a new molecular link in the process of mesoderm specification into angioblast.
Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome (DS) is characterized by circulating blast cells in the blood. TAM usually resolves spontaneously, but several studies have associated this condition with early death, focusing on the development of effective treatments. We report the case of a neonate with DS who had TAM and novel GATA1 mutation. Although the patient eventually died of hepatic failure, exchange blood transfusion and low-dose cytarabine treatment dramatically improved pulmonary hypertension and acute renal failure refractory to conventional therapy. Such a blast-reducing approach might be useful for improving circulatory disturbances in neonates with DS and TAM.
While acute megakaryoblastic leukaemia (AMKL) occurs in children with (DS-AMKL) and without (paediatric non-DS-AMKL) Down syndrome, it can also affect adults without DS (adult non-DS-AMKL). We have analysed these subgroups of patients (11 children with DS-AMKL, 12 children and four adults with non-DS-AMKL) for the presence of molecular lesions, including mutations and chromosomal abnormalities studied by sequencing and single nucleotide polymorphism array-based karyotyping, respectively. In children, AMKL was associated with trisomy 21 (somatic in non-DS-AMKL), while numerical aberrations of chromosome 21 were only rarely associated with adult AMKL. DS-AMKL was also associated with recurrent somatic gains of 1q (4/11 DS-AMKL patients). In contrast to trisomy 21 and gains of 1q, other additional chromosomal lesions were evenly distributed between children and adults with AMKL. A mutational screen found GATA1 mutations in 11/12 DS-AMKL, but mutations were rare in paediatric non-DS-AMKL (1/12) and adult AMKL (0/4). JAK3 (1/11), JAK2 (1/11), and TP53 mutations (1/11) were found only in patients with DS-AMKL. ASXL1, IDH1/2, DNMT3A, RUNX1 and CBL mutations were not found in any of the patient group studied, while NRAS mutation was identified in two patients with paediatric non-DS-AMKL.
Subacute thyroiditis (SAT) is an extremely rare complication of influenza vaccination. Several infectious agents have been related with SAT. It is also well known the association between HLA-B35 and the development of SAT. We describe a case of subacute thyroiditis and dyserythropoesis occurring shortly after administration of an influenza vaccine in a 55-year-old man with history of diabetes and psoriasis, family history of autoimmunity without clinical evidence of acute viral infection prior to the onset of symptoms. We propose that, the events occurring in the patient may be explained as result of complex interactions between the individual genetic background and environmental exposure to infectious agents that generated a pro-inflammatory status, where the vaccine was the trigger for the subsequent alterations in thyroid and bone marrow. These findings highlight the importance of immunogenetic factors involved in response to vaccination that is the central theme in the growing field of 'vaccinomics'.
Acute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s.
Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays.
Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation.
Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes.
The GATA family of transcription factors, including the founding member, GATA-1, have an important role in gene regulation. GATA-1 is integral to successful hematopoiesis. A wide variety of mutations in GATA-1 affect its function, as well as its interaction with its cofactors (especially Friend of GATA) and the genes upon which GATA-1 acts. Here we review the known mutations, focusing on the specific alterations within the amino acid sequence, the resulting effect on hematopoietic development, and the clinical manifestations that result. Attention is also paid to the relationship between Trisomy 21, also known as Down syndrome, and the phenomenon of a truncated GATA-1, named GATA-1s. The evidence for specific interaction between GATA-1 and chromosome 21, which may explain the correlation between these two mutations, is briefly reviewed.
Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling energy metabolism, is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest, without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers, Nanog and stage-specific embryonic antigen-1, in the presence of leukemia inhibitory factor, without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently, an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation, an effect inhibited by MG132, a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs, it increased expression levels of erythroid cell lineage markers (Ter119, GATA1, Klf1, Hbb-b, and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR, endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs.
Previous cytogenetic studies of myeloid and acute lymphoblastic leukemias in children with Down syndrome (ML-DS and DS-ALL) have revealed significant differences in abnormality patterns between such cases and acute leukemias in general. Also, certain molecular genetic aberrations characterize DS-related leukemias, such as GATA1 mutations in ML-DS and deregulation of the CRLF2 gene in DS-ALL. Whether microdeletions/microduplications also vary between DS and non-DS cases is presently unclear. To address this issue, we performed single nucleotide polymorphism array analyses of eight pediatric ML-DS and 17 B-cell precursor DS-ALL. In the ML-DS cases, a total of 29 imbalances (20 gains and nine losses) and two partial uniparental isodisomies (pUPDs) were detected. None of the 11 small (defined as <10 Mb) imbalances were recurrent, nor were the pUPDs, whereas of the 18 large aberrations, three were recurrent-dup(1q), +8 and +21. In contrast, several frequent changes were identified in the DS-ALL cases, which harbored 82 imbalances (30 gains and 52 losses) and four pUPDs. Of the 40 large changes, 28 were gains and 12 losses, with +X, dup(Xq), dup(1q), del(7p), dup(8q), del(9p), dup(9p), del(12p), dup(17q), and +21 being recurrent. Of the 40 microdeletions identified, several targeted specific genes, with the following being repeatedly deleted: BTG1 and CDKN2A/B (29% of cases), ETV6, IKZF1, PAX5 and SERP2 (18%), and BTLA, INPP4B, P2RY8, and RB1 (12%). Loss of the SERP2 and INPP4B genes, encoding the stress-associated endoplasmic reticulum protein family member 2 and the inositol polyphosphate 4-phosphatase-II, respectively, has previously never been implicated in leukemia. Although deletions of the other genes have been associated with ALL, the high frequency of BTG1 loss is a novel finding. Such deletions may characterize a clinical subgroup of DS-ALL, comprising mainly boys with a high median age. In conclusion, ML-DS and DS-ALL are genetically distinct, with mainly gains in ML-DS and deletions in DS-ALL. Furthermore, DS-ALL is characterized by several recurrent gene deletions, with BTG1 loss being particularly frequent.
Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.
BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.
Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.
Peroxiredoxin 5 (Prx5) is a member of a family consisting of six antioxidant enzymes. Prx5 is ubiquitously expressed in various tissues including mitochondria and peroxisomes, implying that Prx5 functions as a regulator of the cellular oxidation state. Prx5 plays a critical role in protecting cells from oxidative stress by inhibiting the accumulation of reactive oxygen species and cell death. Overexpression of Prx5 in mammary tissue is associated with poor prognosis of breast cancer. The present study was conducted to elucidate the regulatory mechanisms of Prx5 gene expression and the physiological relevance of Prx5 in breast cancer cell survival. Analysis of the promoter region of the Prx5 gene and reporter gene assays revealed the promoter region critical for Prx5 gene regulation to which the novel negative transcription regulator, GATA1 binds in human breast cancer cell lines. GATA1 was further confirmed as a potential transcription factor for Prx5 gene expression in another set of transfection assays using a reporter gene construct that contained a mutated GATA1 binding site. Direct binding of GATA1 to the Prx5 promoter was demonstrated using chromatin immunoprecipitation and electrophoretic mobility shift assays. Finally, functions of Prx5 associated with apoptosis were examined in two human breast cancer cell lines displaying high levels of either Prx5 or GATA1 expression. Knockdown of GATA1 led to increased expression of Prx5 and inhibition of apoptosis. Our data suggest that Prx5 may protect cells from oxidative stress-mediated apoptosis in a GATA1-regulated manner.
The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the (A)γ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.
Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)-like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule-like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.
Hmgn2 (high mobility group nucleosomal 2), a ubiquitous nucleosome-binding protein that unfolds chromatin fibres and enhances DNA replication, reportedly regulates differentiation of epithelial and mesenchymal cells. To investigate how Hmgn2 regulates HC (haemopoietic cell) differentiation, we quantified Hmgn2 expression in HCs of mouse FL (fetal liver) during erythroid differentiation. Hmgn2 expression levels were >10-fold higher in immature erythroid progenitors than in mature erythroid cells, suggesting that Hmgn2 antagonizes erythroid differentiation. To address this issue, Hmgn2 were transfected into both Friend erythroleukaemia cells and FL HCs. There was a 3.3-fold decrease in relatively mature c-Kit(+)/CD71(+) erythroid cells, a 2.9-fold increase in immature c-Kit(+)/CD71(-) erythroid cells in transfected Friend cells, a 1.1-fold decrease in relatively mature CD71(+)/Ter119(+) erythroid cells, and a 1.7-fold increase in relatively immature c-Kit(+)/CD71(+) erythroid cells in FL HCs accompanied by down-regulation of genes encoding the erythroid transcription factors, Gata1 and Klf1. Two days after Hmgn2 transfection of Friend erythroleukaemia cells, the number of S-phase cells increased, whereas the number of cells in G(1) decreased, while that of mitotic cells remained unchanged. We conclude that ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro, which may be due to enhancement of DNA replication and/or blocking entry of mitosis at S-phase.
We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.
Repetitive sequences consist of >50% of mammalian genomic DNAs and among these SINEs (short interspersed nuclear elements), e.g. B1 elements, account for 8% of the mouse genome. In an effort to delineate the molecular mechanism(s) involved in the blockade of the in vitro differentiation program of MEL (murine erythroleukaemia) cells by treatment with methylation inhibitors, we detected a DNA region of 559 bp in chromosome 7 located downstream of the 3'-end of the β(major) globin gene (designated B1-559) with unique characteristics. We have fully characterized this B1-559 region that includes a B1 element, several repeats of ATG initiation codons and consensus DNA-binding sites for erythroid-specific transcription factors NF-E2 (nuclear factor-erythroid-derived 2), GATA-1 and EKLF (erythroid Krüppel-like factor). Fragments derived from B1-559 incubated with nuclear extracts form protein complexes in both undifferentiated and differentiated MEL cells. Transient reporter-gene experiments in MEL and human erythroleukaemia K-562 cells with recombinant constructs containing B1-559 fragments linked to HS-2 (hypersensitive site-2) sequences of human β-globin gene LCR (locus control region) indicated potential cooperation upon erythropoiesis and globin gene expression. The possible interaction between the B1-559 region and β(major) globin gene transcriptional activation upon execution of erythroid MEL cell differentiation programme is discussed.
Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.
Aquaporin 1 (Aqp1) is a water channel protein, expressed widely in microvascular endothelial cells and implicated in mammalian tumor angiogenesis. However, its developmental expression has not yet been characterized in great detail. An enhancer trap screen was performed using a Tol2-derived GFP reporter in zebrafish embryos. An insertional Et(GBT-B1)tpl1 line was identified that has reporter insertion in the vicinity of the aqp1a gene. We further characterized the embryonic expression pattern of this GFP reporter line, as well as that of endogenous aqp1a. Both endogenous aqp1a and reporter GFP expression were restricted to the vascular endothelial cells within the dorsal aorta, cranial, intersegmental and other secondary vessels, but were absent in the axial venous vasculature. In addition, endogenous aqp1a expression was observed in both primitive and definitive hematopoietic erythroid progenitors, as well as in the otic vesicle, swim bladder, pneumatic duct, intestine and a subset of neurons within the retina and the midbrain-hindbrain region. We further show that gata1 and etsrp/etv2 function is required for hematopoietic and endothelial aqp1a expression, respectively. Aqp1a expression is restricted to endothelial and erythroid cells during early embryogenesis. The transgenic Et(GBT-B1)tpl1 line recapitulates endogenous endothelial aqp1a expression. Because currently very few reporter lines can differentiate between arterial and venous endothelial cells, the Et(GBT-B1)tpl1 transgenic line and characterization of the aqp1a expression pattern will be useful for future studies of endothelial and arterial-venous differentiation.
β-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including β-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of β-lapachone on zebrafish embryonic heart development.
Embryos were treated with β-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation.
We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in β-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of β-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of β-lapachone-treated embryos. Reduction in wall shear stress was uncovered in β-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by β-lapachone is mediated through an NQO1- and calcium-dependent pathway.
The novel finding of this study is that β-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.
Acquired trisomy 21 is one of the most common numerical abnormalities in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), and MDS/MPN; however, little is known about its pathogenic impact, accompanying submicroscopic changes, and its relation to other clinical features. Furthermore, previous studies addressing this issue have mainly focused on cases in which +21 was part of a complex karyotype.
We ascertained the incidence of +21, both as a sole change (T21s) and irrespective of additional changes (T21all), in relation to disease type, morphologic subgroup, gender, and age in all published AML, MDS, MPN, and MDS/MPN cases. Furthermore, single nucleotide polymorphism (SNP) array analysis was performed on 11 myeloid malignancies with T21s, followed by mutation analysis of the FGFR1, FLT3, GATA1, JAK2, KIT, NPM1, NRAS, RUNX1, and TET2 genes.
The frequencies of T21s and/or T21all varied significantly among the AML, MDS, MPN, and MDS/MPN cases, among the AML and MPN subtypes, and in relation to the age of the AML, MDS, and MPN patients. In the 11 cases analyzed by SNP array, a total of nine genomic imbalances, comprising seven deletions and two duplications, were identified in six cases; none of the alterations were recurrent. Partial uniparental disomies (UPDs) were found in five cases; two recurrent UPDs were identified, namely UPD4q and UPD7q. Mutations in NPM1, RUNX1, and TET2 were detected in five cases, three of which harbored a pathogenic RUNX1 mutation. The TET2 mutation was found in one of the cases with UPD4q.
The results show that trisomy 21-positive myeloid malignancies are clinically highly variable and that they display a heterogeneous pattern of copy number alterations and mutations.
STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by sub-functionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development.
A state-dependent dynamic network is a collection of elements that interact through a network, whose geometry evolves as the state of the elements changes over time. The genome is an intriguing example of a state-dependent network, where chromosomal geometry directly relates to genomic activity, which in turn strongly correlates with geometry. Here we examine various aspects of a genomic state-dependent dynamic network. In particular, we elaborate on one of the important ramifications of viewing genomic networks as being state-dependent, namely, their controllability during processes of genomic reorganization such as in cell differentiation.
Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.
Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.
One case of acute megakaryoblastic leukemia (AMKL) with trisomy 21, trisomy 14 and unmutated GATA1 gene in a phenotypically normal girl was reported. The patient experienced transient myelodysplasia before the onset of AMKL. The bone marrow blasts manifested typical morphology of megakaryoblast both by the May-Giemsa staining and under the electronic microscopy. Leukemic cells were positive for CD13, CD33, CD117, CD56, CD38, CD41 and CD61 in flow cytometry analysis. Cytogenetic study showed karyotype of 48, XX, +14, +21 in 40% metaphases. Known mutations of GATA1 gene in Down syndrome or acquired trisomy 21 were not detected in this case.
miRNAs are small non-coding RNAs with average length of ~21 bp. miRNA formation seems to be dependent upon multiple factors besides Drosha and Dicer, in a tissue/stage-specific manner, with interplay of several specific binding factors. In the present study, we have investigated transcription factor binding sites in and around the genomic sequences of precursor miRNAs and RNA-binding protein (RBP) sites in miRNA precursor sequences, analysed and tested in comprehensive manner. Here, we report that miRNA precursor regions are positionally enriched for binding of transcription factors as well as RBPs around the 3' end of mature miRNA region in 5' arm. The pattern and distribution of such regulatory sites appears to be a characteristic of precursor miRNA sequences when compared with non-miRNA sequences as negative dataset and tested statistically.When compared with 1 kb upstreamregions, a sudden sharp peak for binding sites arises in the enriched zone near the mature miRNA region. An expression-data-based correlation analysis was performed between such miRNAs and their corresponding transcription factors and RBPs for this region. Some specific groups of binding factors and associated miRNAs were identified. We also identified some of the overrepresented transcription factors and associated miRNAs with high expression correlation values which could be useful in cancer-related studies. The highly correlated groups were found to host experimentally validated composite regulatory modules, in which Lmo2-GATA1 appeared as the predominant one. For many of RBP-miRNAs associations, coexpression similarity was also evident among the associated miRNA common to given RBPs, supporting the Regulon model, suggesting a common role and common control of these miRNAs by the associated RBPs. Based on our findings, we propose that the observed characteristic distribution of regulatory sites in precursor miRNA sequence regions could be critical inmiRNA transcription, processing, stability and formation and are important for therapeutic studies. Our findings also support the recently proposed theory of self-sufficient mode of transcription by miRNAs, which states that miRNA transcription can be carried out in host-independent mode too.
The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation of gene expression in cell lineage differentiation and maturation. Most GATA1-regulated events require GATA1 to bind FOG1, and essentially all GATA1-activated genes are cooccupied by a TAL1/E2A/LMO2/LDB1 complex; however, it is not known whether FOG1 and TAL1/E2A/LMO2/LDB1 are simultaneously recruited by GATA1. Our structural data reveal that the FOG1-binding domain of GATA1, the N finger, can also directly contact LMO2 and show that, despite the small size (< 50 residues) of the GATA1 N finger, both FOG1 and LMO2 can simultaneously bind this domain. LMO2 in turn can simultaneously contact both GATA1 and the DNA-binding protein TAL1/E2A at bipartite E-box/WGATAR sites. Taken together, our data provide the first structural snapshot of multiprotein complex formation at GATA1-dependent genes and support a model in which FOG1 and TAL1/E2A/LMO2/LDB1 can cooccupy E-box/WGATAR sites to facilitate GATA1-mediated activation of gene activation.
NUP98-HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment-related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98-HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98-HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1 expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98-HOXA9. Following irradiation, NUP98-HOXA9-expressing embryos showed increased numbers of cells in G2-M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98-HOXA9-induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98-HOXA9-transgenic fish developed a myeloproliferative neoplasm (MPN) at 19-23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high-throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.
Genetic defects of platelet function give rise to mucocutaneous bleeding of varying severity because platelets fail to fulfil their haemostatic role after vessel injury. Abnormalities of pathways involving glycoprotein (GP) mediators of adhesion (Bernard-Soulier syndrome, platelet-type von Willebrand disease) and aggregation (Glanzmann thrombasthenia) are the most studied and affect the GPIb-IX-V complex and integrin αIIbβ3, respectively. Leukocyte adhesion deficiency-III combines Glanzmann thrombasthenia with infections and defects of kindlin-3, a mediator of integrin activation. Agonist-specific deficiencies in platelet aggregation relate to mutations of primary receptors for ADP (P2Y(12)), thromboxane A(2) (TXA2R) and collagen (GPVI); however, selective abnormalities of intracellular signalling pathways remain better understood in mouse models. Defects of secretion from δ-granules are accompanied by pigment defects in the Hermansky-Pudlak and Chediak-Higashi syndromes; they concern multiple genes and protein complexes involved in secretory organelle biogenesis and function. Quebec syndrome is linked to a tandem duplication of the urokinase plasminogen activator (PLAU) gene while locus assignment to chromosome 3p has advanced the search for the gene(s) responsible for α-granule deficiency in the gray platelet syndrome. Defects of α-granule biosynthesis also involve germline VPS33B mutations in the ARC (arthrogryposis, renal dysfunction and cholestasis) syndrome. A mutation in transmembrane protein 16F (TMEM16F) has been linked to a defective procoagulant activity and phosphatidylserine expression in the Scott syndrome. Cytoskeletal dysfunction (with platelet anisotrophy) occurs not only in the Wiskott-Aldrich syndrome but also in filamin A deficiency or MYH9-related disease while GATA1 mutations or RUNX1 haploinsufficiency can affect expression of multiple platelet proteins.
Interplays among lineage-specific nuclear proteins, chromatin modifying enzymes, and the basal transcription machinery govern cellular differentiation, but their dynamics of action and coordination with transcriptional control are not fully understood. Alterations in chromatin structure appear to establish a permissive state for gene activation at some loci, but they play an integral role in activation at other loci. To determine the predominant roles of chromatin states and factor occupancy in directing gene regulation during differentiation, we mapped chromatin accessibility, histone modifications, and nuclear factor occupancy genome-wide during mouse erythroid differentiation dependent on the master regulatory transcription factor GATA1. Notably, despite extensive changes in gene expression, the chromatin state profiles (proportions of a gene in a chromatin state dominated by activating or repressive histone modifications) and accessibility remain largely unchanged during GATA1-induced erythroid differentiation. In contrast, gene induction and repression are strongly associated with changes in patterns of transcription factor occupancy. Our results indicate that during erythroid differentiation, the broad features of chromatin states are established at the stage of lineage commitment, largely independently of GATA1. These determine permissiveness for expression, with subsequent induction or repression mediated by distinctive combinations of transcription factors.
Enhancers of transcription activate transcription via binding of sequence-specific transcription factors to their target sites in chromatin. In this report, we identify GATA1-bound distal sites genome-wide and find a global reorganization of the nucleosomes at these potential enhancers during differentiation of hematopoietic stem cells (HSCs) to erythrocytes. We show that the catalytic subunit BRG1 of BAF complexes localizes to these distal sites during differentiation and generates a longer nucleosome linker region surrounding the GATA1 sites by shifting the flanking nucleosomes away. Intriguingly, we find that the nucleosome shifting specifically facilitates binding of TAL1 but not GATA1 and is linked to subsequent transcriptional regulation of target genes.
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.
Overexpression of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)-κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti-inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit-erythroid colony formation in human CD34(+) cells. CD34(+) cells pre-incubated with resveratrol for 72 h in the presence of TNFα inhibited NF-κB activation via decreased NF-κB nuclear localization without altering total NF-κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34(+) cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα-mediated NF-κB activation and promote erythropoiesis in primary human CD34(+) cells.
The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.
The roles of Sry-related HMG box (Sox) genes in zebrafish hematopoiesis are not clearly defined. In this study, we have characterized the sequence homology, gene expression, hematopoietic functions, and regulation of sox genes in F group (SoxF) in zebrafish embryos.
Expression of zebrafish SoxF genes were analyzed by whole-mount in situ hybridization, reverse transcription polymerase chain reaction, and real-time reverse transcription polymerase chain reaction of erythroid cells obtained from Tg(gata1:GFP) embryos by fluorescence-activated cell sorting. Roles of SoxF genes were analyzed in zebrafish embryos using morpholino knockdown and analyzed by whole-mount in situ hybridization and real-time reverse transcription polymerase chain reaction. Embryo patterning and vascular development were analyzed.
All members, except sox17, contained a putative β-catenin binding site. sox7 and 18 expressed primarily in the vasculature. sox17 expressed in the intermediate cell mass and its knockdown significantly reduced primitive erythropoiesis at 18 hours post-fertilization (hpf). Definitive hematopoiesis was unaffected. Concomitant sox7 and sox18 knockdown disrupted vasculogenesis and angiogenesis, but not hematopoiesis. sox32 knockdown delayed medial migration of hematopoietic and endothelial progenitors at 18 hpf and abolished cmyb expression at the caudal hematopoietic tissue at 48 hpf. These defects could be prevented by delaying its knockdown using a caged sox32 morpholino uncaged at 10 hpf. Knockdown of SoxF genes significantly upregulated their own expression and that of sox32 also upregulated sox18 expression.
sox17 helped to maintain primitive hematopoiesis, whereas sox7 and sox18 regulated angiogenesis and vasculogenesis. sox32 affected both vascular and hematopoietic development through its effects on medial migration of the hematopoietic and endothelial progenitors.
Children with Down syndrome (DS) up to the age of 4 years are at a 150-fold excess risk of developing myeloid leukemia (ML-DS). Approximately 4%-5% of newborns with DS develop transient myeloproliferative disorder (TMD). Blast cell structure and immunophenotype are similar in TMD and ML-DS. A mutation in the hematopoietic transcription factor GATA1 is present in almost all cases. Here, we show that simple techniques detect GATA1 mutations in the largest series of TMD (n = 134; 88%) and ML-DS (n = 103; 85%) cases tested. Furthermore, no significant difference in the mutational spectrum between the 2 disorders was seen. Thus, the type of GATA1 sequence mutation is not a reliable tool and is not prognostic of which patients with TMD are probable to develop ML-DS.
Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra β-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous β-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in 'jackpot' cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression.
Neonates with Down syndrome (DS) are predisposed to developing transient abnormal myelopoiesis (TAM) and acute myeloid leukemia (AML) associated with DS. However, there is a paucity of data on hematological aberrations and GATA1 mutations in neonates with DS in East Asian populations.
Total 109 patients with DS who had one or more CBCs obtained were enrolled. The molecular analysis of the GATA1 gene performed in 10 patients (three TAM, three AML associated with DS at diagnosis, one remission case of AML associated with DS and three DS without TAM or AML).
East Asian DS neonates showed low frequency of thrombocytopenia, uncommon neutrophilia and higher prevalence rate of TAM compared to previous reports from western countries. GATA1 mutations were identified in almost all TAM and AML associated with DS samples, but were not detected in the samples from DS without TAM or AML associated with DS.
East Asian DS neonates and children showed distinctive spectrum of hematological abnormalities.
The c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells.
To identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling.
We identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3.
These findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.
One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 μM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.
The microgravity environment of spaceflight leads to a series of changes in the human blood system. The aim of the present study was to examine the influence of simulated microgravity on the differentiation of CD34+ cells and to explore whether transcription factor GATA-1, required for the terminal differentiation of committed erythroid progenitor cells, is involved in this process.
CD34+ cells were cultured in the simulated microgravity conditions created by a rotary cell-culture system (RCCS). The effects of simulated microgravity on the differentiation and apoptosis of CD34+ cells were analyzed using flow cytometry and propidium iodide (PI) staining, respectively. Expression of GATA-1 mRNA in CD34+ cells was determined by real-time quantitative PCR.
In the RCCS group, GlyA+ (glycophorin A) expression was lower and CD33+ expression higher than in the 1-g liquid control group (22.21% +/- 3.02% and 60.05% +/- 3.08%, vs. 52.12% +/- 1.92% and 18.87% +/- 1.41%, respectively). The proportion of differentiated cells in the 1-g methylcellulos e group (Gly+% = 54.39% +/- 2.86%, CD33+% = 21.09% +/- 3.19%) was similar to that in the 1-g liquid control group. As shown by real-time quantitative PCR, the relative expression of GATA-1 mRNA in the RCCS group was only 20% of that in the -g control group. CONCLUSIONSs: The differentiation of CD3+ cells, and especially erythroid differentiation, was inhibited by simulated microgravity by a mechanism that appears to involve the suppression of GATA-1 mRNA expression. The results of this study may be useful in understanding the critical effect of simulated microgravity on the pathogenesis of space anemia.
GATA-1 and NF-E2 are erythroid specific activators that bind to the β-globin locus. To explore the roles of these activators in transcription of the human fetal stage specific γ-globin genes, we reduced GATA-1 and p45/NF-E2 using shRNA in erythroid K562 cells. GATA-1 or p45/NF-E2 knockdown inhibited the transcription of the γ-globin genes, hypersensitive site (HS) formation in the LCR and chromatin loop formation of the β-globin locus, but histone acetylation across the locus was decreased only in the case of GATA-1 knockdown. In p45/NF-E2 knockdown cells, GATA-1 binding was maintained at the LCR HSs and γ-globin promoter, but NF-E2 binding at the LCR HSs was reduced by GATA-1 knockdown regardless of the amount of p45/NF-E2 in K562 cells. These results indicate that histone acetylation is dependent on GATA-1 binding, but the binding of GATA-1 is not sufficient for the γ-globin transcription, HS formation and chromatin loop formation and NF-E2 is required. This idea is supported by the distinctive binding pattern of CBP and Brg1 in the β-globin locus. Furthermore GATA-1-dependent loop formation between HS5 and 3'HS1 suggests correlation between histone modifications and chromatin looping.
Hematopoiesis involves an elaborate regulatory network of transcription factors that coordinates the expression of multiple downstream genes, and maintains homeostasis within the hematopoietic system through the accurate orchestration of cellular proliferation, differentiation and survival. As a result, defects in the expression levels or the activity of these transcription factors are intimately linked to hematopoietic disorders, including leukemia. The GATA family of nuclear regulatory proteins serves as a prototype for the action of lineage-restricted transcription factors. GATA1 and GATA2 are expressed principally in hematopoietic lineages, and have essential roles in the development of multiple hematopoietic cells, including erythrocytes and megakaryocytes. Moreover, GATA2 is crucial for the proliferation and maintenance of hematopoietic stem cells and multipotential progenitors. In this review, we summarize the current knowledge regarding the biological properties and functions of the GATA2 transcription factor in normal and malignant hematopoiesis.
Hematopoiesis is evolutionarily conserved from zebrafish to mammals, and this includes both primitive and definitive waves during embryogenesis. Primitive hematopoiesis is dominated by erythropoiesis with limited myelopoiesis. Protein sumoylation, a ubiquitination-like posttranslational protein modification, is implicated in a variety of biochemical processes, most notably in transcriptional repression. We show here that the loss of 6 small ubiquitin-related modifier (SUMO) paralogs triggers a sharp up-regulation of the myeloid-specific marker mpo and down-regulation of the erythroid-specific marker gata1 in myelo-erythroid progenitor cells (MPCs) in the intermediate cell mass (ICM) during primitive hematopoiesis. Accordingly, in transgenic zebrafish lines, hyposumoylation expands myelopoiesis at the expense of erythropoiesis. A SUMO-CCAAT/enhancer-binding protein α (SUMO-C/ebpα) fusion restores the normal myelopoiesis/erythropoiesis balance, suggesting that sumoylation status of C/ebpα contributes to myelo-erythroid lineage determination. Our results therefore implicate sumoylation in early lineage determination and reveal the possible molecular mechanism underlying the puzzling biased primitive hematopoiesis in vertebrates.
Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors--GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL--in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.
Recent data demonstrate that small synthetic compounds specifically targeting bromodomain proteins can modulate the expression of cancer-related or inflammatory genes. Although these studies have focused on the ability of bromodomains to recognize acetylated histones, it is increasingly becoming clear that histone-like modifications exist on other important proteins, such as transcription factors. However, our understanding of the molecular mechanisms through which these modifications modulate protein function is far from complete. The transcription factor GATA1 can be acetylated at lysine residues adjacent to the zinc finger domains, and this acetylation is essential for the normal chromatin occupancy of GATA1. We have recently identified the bromodomain-containing protein Brd3 as a cofactor that interacts with acetylated GATA1 and shown that this interaction is essential for the targeting of GATA1 to chromatin. Here we describe the structural basis for this interaction. Our data reveal for the first time the molecular details of an interaction between a transcription factor bearing multiple acetylation modifications and its cognate recognition module. We also show that this interaction can be inhibited by an acetyllysine mimic, highlighting the importance of further increasing the specificity of compounds that target bromodomain and extraterminal (BET) bromodomains in order to fully realize their therapeutic potential.
Ribosomal proteins (RPs), structural components of the ribosome involved in protein synthesis, are of significant importance in all organisms. Previous studies have suggested that some RPs may have other functions in addition to assembly of the ribosome. The small ribosomal subunits RPS7, has been reported to modulate the mdm2-p53 interaction. To further investigate the biological functions of RPS7, we used morpholino antisense oligonucleotides (MO) to specifically knockdown RPS7 in zebrafish. In RPS7-deficient embryos, p53 was activated, and its downstream target genes and biological events were induced, including apoptosis and cell cycle arrest. Hematopoiesis was also impaired seriously in RPS7-deficient embryos, which was confirmed by the hemoglobin O-dianisidine staining of blood cells, and the expression of scl, gata1 and α-E1 globin were abnormal. The matrix metalloproteinase (mmp) family genes were also activated in RPS7 morphants, indicating that improper cell migration might also cause development defects. Furthermore, simultaneously knockdown of the p53 protein by co-injecting a p53 MO could partially reverse the abnormal phenotype in the morphants. These results strengthen the hypothesis that specific ribosomal proteins regulate p53 and that their deficiency affects hematopoiesis. Moreover, our data implicate that RPS7 is a regulator of matrix metalloproteinase (mmp) family in zebrafish system. These specific functions of RPS7 may provide helpful clues to study the roles of RPs in human disease.
Oligonucleotide array comparative genomic hybridization, karyotype and fluorescence in situ hybridization analyses were employed to delineate the cytogenetic abnormalities in a case of pediatric acute megakaryoblastic leukemia. Here we present a unique genetic profile that includes bi-allelic deletions within 13q14, where the retinoblastoma tumor suppressor gene (RB1) resides, as well as isolated trisomy 21 without a concomitant mutation in the hematopoietic transcription factor GATA1s and translocation (17;22), that does not involve the megakaryoblastic leukemia 1 (MKL1) gene located on chromosome 22. Alteration of the RB1 gene is most likely the critical leukemogenic event in this patient.
Acetylation of histones triggers association with bromodomain-containing proteins that regulate diverse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 "reads" acetyl marks on nuclear factors to promote their stable association with chromatin.
Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/β-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.
Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins, including SMAD1. In previous work, we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly, we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely, when Smad1 is depleted in FlK1(+) mesoderm, at a stage after hemangioblast commitment, the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis, including Gata1, Runx1 and Eklf, rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity, indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism, hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore, this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors.
Many physiological perturbations can cause anemia. In cancer patients, activation of the immune system leads to the production of proinflammatory cytokines including tumor necrosis factor alpha (TNFα), that have been shown to inhibit red-cell production via poorly understood mechanisms. Treatment of anemia by human recombinant erythropoietin (EPO) is strongly suspected to induce tumor growth. This study focuses on the mechanisms involved in TNFα-mediated inhibition of erythropoiesis. CD34(+) hematopoietic stem/progenitor cells (HSPCs) were isolated from human cord blood. Erythropoiesis was achieved in vitro by stimulating cells with EPO. We show that TNFα clearly affected erythroid development, as assessed by May-Grünwald/Giemsa staining, flow cytometry analysis and fluorescent microscopy. The amount of hemoglobin-producing cells as well as the expression of GATA-1 target erythro-specific genes (EPO receptor, glycophorin A and globins) was found decreased after TNFα treatment of HSPC. In correlation, TNFα induced the expression of the transcription factors GATA-2 and PU.1, described as inhibitors of erythropoiesis. In this regard, TNFα promoted the formation of the GATA-1/PU.1 complex that has been reported to block the transcriptional activity of GATA-1. Our results clearly demonstrate that TNFα prevents EPO-mediated erythropoiesis of HSPC as an early event, by directly affecting erythroid cell development.
Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23, MYB was a good candidate gene. Our molecular investigations, based on fluorescence in situ hybridization and rapid amplification of cDNA ends, revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together, these results establish, for the first time, a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia.
Recent studies suggest that the hematopoietic and cardiac lineages have close ontogenic origins, and that an early mesodermal cell population has the potential to differentiate into both lineages. Studies also suggest that specification of these lineages is inversely regulated. However, the transcriptional networks that govern the cell fate specification of these progenitors are incompletely defined.
Here, we show that Nkx2-5 regulates the hematopoietic/erythroid fate of the mesoderm precursors early during cardiac morphogenesis. Using transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1, in the Nkx2-5-null embryos. We further observed that overexpression of Nkx2-5 with an Nkx2-5-inducible embryonic stem cell system significantly repressed Gata1 gene expression and suppressed the hematopoietic/erythroid potential, but not the endothelial potential, of the embryonic stem cells. This suppression was cell-autonomous, and was partially rescued by overexpressing Gata1. In addition, we demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene.
Our results demonstrate that the hematopoietic/erythroid cell fate is suppressed via Nkx2-5 during mesodermal fate determination, and that the Gata1 gene is one of the targets that are suppressed by Nkx2-5.
The transcription factors PU.1 and GATA-1 are known to be important in the development of blood progenitor cells. Specifically they are thought to regulate the differentiation of progenitor cells into the granulocyte/macrophage lineage and the erythrocyte/megakaryocite lineage. While several mathematical models have been proposed to investigate the interaction between the transcription factors in recent years, there is still debate about the nature of the progenitor state in the dynamical system, and whether the existing models adequately capture new knowledge about the interactions gleaned from experimental data. Further, the models utilise different formalisms to represent the genetic regulation, and it appears that the resulting dynamical system depends upon which formalism is adopted. In this paper we analyse the four existing models, and propose an alternative model which is shown to demonstrate a rich variety of dynamical systems behaviours found across the existing models, including both bistability and tristability required for modelling the undifferentiated progenitors.
Classical swine fever (CSF) causes severe disease in pigs, characterized by hemorrhage, fever, and leucopenia. A primary target of the virus is endothelial cells, where a pro-inflammatory and pro-coagulant response occurs with downregulation of gap junctional communication; these changes establish a basis for haemostatic imbalance. The aim of this study was to gain an understanding of the effect of classical swine fever virus (CSFV) on endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability. Porcine aortic endothelial cells (PAECs) were infected with CSFV at different multiplicity of infection (M.O.I.) for 48 h. Downregulation of the transcription and translation levels of eNOS was detected by semi-quantitative RT-PCR, immunoconfocal microscopy, and western blotting. This was accompanied by a reduction in NO bioavailability and attenuation of angiogenesis. Without influence from the progeny virus titer, the decrease in eNOS protein was reversed by an ERK inhibitor (PD98059) and two PI3/Akt inhibitors (LY294002 and wortmannin). In addition, we found that the transcription factors AP1, Sp1, and GATA1/2 may be involved in the downregulation of eNOS promoter activity. In conclusion, infection of PAECs with CSFV attenuated the expression of eNOS and reduced NO bioavailability through activation of the ERK and PI3/Akt pathways.
Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine. Use of iPSC lines for future hematotherapy will require examination of their hematopoietic potential. Adult skin fibroblast somatic cells constitute a source of iPSCs that can be accessed clinically without ethical issues. Here, we used different methods to compare mesodermal and hematopoietic potential by embryoid body formation of five iPSC lines established from adult mouse tail-tip fibroblasts (TTFs). We observed variation in proliferation and in expression of genes (Brachyury, Tbx1, Gata1, Klf1, Csf1r) and proteins (Flk1, Ter119 and CD45) among TTF-derived lines. 256H18 iPSCs showed highest proliferation and most efficient differentiation into mesodermal and hematopoietic cells, while expression levels of the pluripotency genes Oct3/4, Sox2, Klf4 and Nanog were lowest among lines analyzed. By contrast, the 212B2 line, transduced with c-Myc, showed lowest proliferation and differentiation potential, although expression levels of Oct3/4, Sox2 and Klf4 were highest. Overall, we find that mesodermal and hematopoietic potential varies among iPSCs from an identical tissue source and that c-Myc expression likely underlies these differences.
Identification of hematopoietic progenitor cells in the zebrafish (Danio rerio) has been hindered by a lack of functional assays to gauge proliferative potential and differentiation capacity. To investigate the nature of myeloerythroid progenitor cells, we developed clonal methylcellulose assays by using recombinant zebrafish erythropoietin and granulocyte colony-stimulating factor. From adult whole kidney marrow, erythropoietin was required to support erythroid colony formation, and granulocyte colony-stimulating factor was required to support the formation of colonies containing neutrophils, monocytes, and macrophages. Myeloid and erythroid colonies showed distinct morphologies and were easily visualized and scored by their expression of lineage-specific fluorescent transgenes. Analysis of the gene-expression profiles after isolation of colonies marked by gata1:DsRed or mpx:eGFP transgenes confirmed our morphological erythroid and myeloid lineage designations, respectively. The majority of progenitor activity was contained within the precursor light scatter fraction, and more immature precursors were present within the lymphoid fraction. Finally, we performed kinetic analyses of progenitor activity after sublethal irradiation and demonstrated that recovery to preirradiation levels occurred by 14 days after irradiation. Together, these experiments provide the first report of clonal hematopoietic progenitor assays in the zebrafish and establish the number, characteristics, and kinetics of myeloerythroid progenitors during both steady-state and stress hematopoiesis.
Benzene is a common occupational hazard and a ubiquitous environmental pollutant. Benzene exposure at the levels even below 1ppm still showed hematotoxicity. It is widely accepted that the metabolites of benzene play important roles in the benzene toxicity to the hematopoietic system, but little is known about the effects of benzene metabolites on erythropoiesis. In present study, erythroid progenitor-like K562 cells were used to determine the effects of phenolic metabolites of benzene, including phenol, hydroquinone and 1,2,4-benzenetriol, on the erythroid differentiation. After the treatment with these benzene metabolites at the concentrations with no obvious cytotoxicity, the hemin-induced hemoglobin synthesis in K562 cells decreased in a concentration- and time-dependent manner, and the expression of CD71 and GPA protein on the surface of K562 cells was also inhibited. The reverse transcription-PCR was used to determine the mRNA level of the erythroid related genes in the K562 cells that were treated with benzene metabolites. The hemin-induced expression of globin genes, including α-, β- and γ-globin genes, and the gene encoding the heme synthesis enzyme porphobilinogen deaminase was inhibited by benzene metabolites. When the K562 cells were pretreated with benzene metabolites, the hemin-induced expression of two transcription factor genes GATA-1 and NF-E2 was distinctly reduced, and the pre-treatment with benzene metabolites promoted the decrease of the mRNA level of transcription factor gene GATA-2 by hemin. These results indicated that benzene metabolites inhibited the hemin-induced erythroid differentiation through affecting the transcription of the erythroid related genes.
A poorly understood problem in genetics is how the three-dimensional organization of the nucleus contributes to establishment and maintenance of transcriptional networks. Genetic loci can reside in chromosome "territories" and undergo dynamic changes in subnuclear positioning. Such changes appear to be important for regulating transcription, although many questions remain regarding how loci reversibly transit in and out of their territories and the functional significance of subnuclear transitions. We addressed this issue using GATA-1, a master regulator of hematopoiesis implicated in human leukemogenesis, which often functions with the coregulator Friend of GATA-1 (FOG-1). In a genetic complementation assay in GATA-1-null cells, GATA-1 expels FOG-1-dependent target genes from the nuclear periphery during erythroid maturation, but the underlying mechanisms are unknown. We demonstrate that GATA-1 induces extrusion of the β-globin locus away from its chromosome territory at the nuclear periphery, and extrusion precedes the maturation-associated transcriptional surge and morphological transition. FOG-1 and its interactor Mi-2β, a chromatin remodeling factor commonly linked to repression, were required for locus extrusion. Erythroid Krüppel-like factor, a pivotal regulator of erythropoiesis that often co-occupies chromatin with GATA-1, also promoted locus extrusion. Disruption of transcriptional maintenance did not restore the locus subnuclear position that preceded activation. These results lead to a model for how a master developmental regulator relocalizes a locus into a new subnuclear neighborhood that is permissive for high level transcription as an early step in establishing a cell type-specific genetic network. Alterations in the regulatory milieu can abrogate maintenance without reversion of locus residency back to its original neighborhood.
A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult β-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" βH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with βH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human β-globin locus, like human fetal erythroblasts, express predominately human γ-, low β-, and no ε-globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. We conclude that yolk sac-derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.
Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.
Steroid hormone biosynthesis requires the steroidogenic acute regulatory protein (STAR). STAR is part of a protein complex that transports cholesterol through the mitochondrial membrane where steroidogenesis begins. Several transcription factors participate to direct the proper spatiotemporal and hormonal regulation of the Star gene in Leydig cells. Mechanistically, this is believed to involve the functional interplay between many of these factors. Here we report a novel transcriptional cooperation between GATA factors and cJUN on the mouse Star and human STAR promoters in MA-10 Leydig cells. This cooperation was observed with different GATA members (GATA1, 4, and 6), whereas only cJUN could cooperate with GATA factors. GATA/cJUN transcriptional cooperation on the Star promoter is mediated via closely juxtaposed GATA and AP-1 binding motifs. Mutation of all functional GATA and cJUN elements abolished GATA/cJUN cooperation, which is in agreement with previous data reporting a direct interaction between GATA4 and cJUN in a heterologous system. These data add valuable new insights that further define the molecular mechanisms that govern Star transcription in steroidogenic cells of the testis.
Lineage fate decisions underpin much of development as well as tissue homeostasis in the adult. A mechanistic paradigm for such decisions is the erythroid versus myeloid fate decision controlled by cross-antagonism between gata1 and pu.1 transcription factors. In this study, we have systematically tested this paradigm in blood-producing populations in zebrafish embryos, including the haematopoietic stem cells (HSCs), and found that it takes a different form in each population. In particular, gata1 activity varies from autostimulation to autorepression. In addition, we have added a third member to this regulatory kernel, tif1γ (transcription intermediate factor-1γ). We show that tif1γ modulates the erythroid versus myeloid fate outcomes from HSCs by differentially controlling the levels of gata1 and pu.1. By contrast, tif1γ positively regulates both gata1 and pu.1 in primitive erythroid and prodefinitive erythromyeloid progenitors. We therefore conclude that the gata1/pu.1 paradigm for lineage decisions takes different forms in different cellular contexts and is modulated by tif1γ.
Mast cells constitutively express Notch1 and Notch2 on the cell surface. Notch ligand Dll1 (Delta-like 1) stimulation induces MHC class II expression in mast cells and renders them as antigen-presenting cells. However, nothing is known about the mechanism by which Notch signaling induces MHC class II expression in mast cells. MHC class II genes are regulated by the class II transactivator (CIITA). In mice, transcription of the CIITA gene is controlled by three cell type-specific promoters (pI, pIII, and pIV). Here, we show that CIITA expression induced by Dll1 stimulation in mouse bone marrow-derived mast cells (BMMCs) depends critically on the signal mediated by Notch1 and that the most dominant promoter in Notch signaling-mediated CIITA expression in BMMCs is pIII, which is a lymphoid lineage-specific promoter. ChIP assays indicated that Notch signaling increased the binding of the transcription factor PU.1 to CIITA pIII in BMMCs. The knockdown of PU.1 expression using a specific siRNA suppressed Notch signaling-mediated CIITA expression, suggesting that PU.1 contributes to the expression of MHC class II induced by Notch signaling in mast cells. Furthermore, we show that a portion of freshly isolated splenic mast cells express MHC class II and that the most dominant promoter of CIITA in mast cells is pIII. These findings indicate that activation of CIITA pIII plays an important role in MHC class II expression in mast cells.
Multiple observations support the existence of developmental differences in megakaryocytopoiesis. We have previously shown that neonatal megakaryocyte (MK) progenitors are hyperproliferative and give rise to MKs smaller and of lower ploidy than adult MKs. Based on these characteristics, neonatal MKs have been considered immature. The molecular mechanisms underlying these differences are unclear, but contribute to the pathogenesis of disorders of neonatal megakaryocytopoiesis. In the present study, we demonstrate that low-ploidy neonatal MKs, contrary to traditional belief, are more mature than adult low-ploidy MKs. These mature MKs are generated at a 10-fold higher rate than adult MKs, and result from a developmental uncoupling of proliferation, polyploidization, and terminal differentiation. This pattern is associated with up-regulated thrombopoietin (TPO) signaling through mammalian target of rapamycin (mTOR) and elevated levels of full-length GATA-1 and its targets. Blocking of mTOR with rapamycin suppressed the maturation of neonatal MKs without affecting ploidy, in contrast to the synchronous inhibition of polyploidization and cytoplasmic maturation in adult MKs. We propose that these mechanisms allow fetuses/neonates to populate their rapidly expanding bone marrow and intravascular spaces while maintaining normal platelet counts, but also set the stage for disorders restricted to fetal/neonatal MK progenitors, including the Down syndrome-transient myeloproliferative disorder and the thrombocytopenia absent radius syndrome.
To report clinical experiences and cytogenetic findings of transient myeloproliferative disorder (TMD) in neonates with and without Down syndrome (DS).
GATA1 gene was screened in DNA samples from neonates presenting with TMD during their leukemic and remission status.
Six neonates (2 phenotypically normal and 4 DS) born in the past 6 years had presented with TMD; all had trisomy 21 during leukemic status. Two DS infants died during early infancy, one of hepatic failure and one of cardiac complication. One non-DS infant evolved into myelodysplastic syndrome (MDS) and acute leukemia since 14 months old. Three other patients have not developed true leukemia after follow-up of 8, 9, and 70 months, respectively. The authors detected mutations within exon 2 of GATA1 gene in 3 DS and 2 non-DS infants. All these mutations disappeared after remission of TMD, but an identical mutation was detected in one non-DS patient when evolving into MDS. Trisomy 21 was confined to leukemic clone in non-DS patients.
TMD should be considered in case of congenital leukemia with megakaryoblastic features and accompanied by trisomy 21 and GATA1 mutation. Both DS and non-DS patients will possibly develop true leukemia within few years.
The transcription factor GATA1 regulates multiple genes in erythroid lineage cells. However, the means by which GATA1 regulates the expression of target genes during erythropoiesis remains to be elucidated. Three mechanisms have been postulated for the regulation of genes by GATA1. First, individual target genes may have multiple discrete thresholds for cellular GATA1. GATA1 has a dynamic expression profile during erythropoiesis, thus the expression of a set of GATA1 target genes may be triggered at a given stage of differentiation by cellular GATA1. Second, the expression of GATA1 target genes may be modified, at least in part, by GATA2 occupying the GATA-binding motifs. GATA2 is expressed earlier in erythropoiesis than GATA1, and prior GATA2 binding may afford GATA1 access to GATA motifs through epigenetic remodeling and thus facilitate target gene expression. Third, other regulatory molecules specific to each target gene may function cooperatively with GATA1. If GATA1 is required for the expression of such cofactors, a regulatory network will be formed and relevant gene expression will be delayed. We propose that the stage-specific regulation of erythroid genes by GATA1 is tightly controlled through a combination of these mechanisms in vivo.
IL-1R antagonist (IL-1Ra) is required for adequate host defense in invasive pneumococcal disease (IPD). The minor allele of an IL1RN gene (C/T) promoter polymorphism (rs4251961) has been shown to be associated with decreased IL-1Ra production in healthy adults. We genotyped 299 children with IPD, and examined 19 IL1RN haplotype-tagging single-nucleotide polymorphisms. Human embryonic kidney HEK293(T) cells were transfected with the promoter reporter plasmid pGL3p containing either allelic variant C (pGL3pCC) or T (pGL3pTT) with or without cotransfection with an expression construct overexpressing the globin transcription factor GATA-1. Plasma IL-1Ra concentrations were significantly higher in nonsurvivors compared with survivors (p < 0.0005), and the C allele of rs4251961 was associated with a significant increase in plasma IL-1Ra concentrations (p = 0.01) during the acute illness of IPD. These findings were validated in a cohort of 276 treatment-naive HIV-infected adults, with borderline significance (p = 0.058). Functional analyses demonstrated that the activity of the promoter constructs containing the T allele increased ~6-fold as compared with basal activity, and that containing the C allele by ~9-fold (p < 0.001) in the presence of GATA-1. Our findings suggest that the IL-1Ra single-nucleotide polymorphism rs4251961 plays a key role in the pathophysiology of IPD and in other human infections.
Mitoferrin 1 (Mfrn1; Slc25a37) and mitoferrin 2 (Mfrn2; Slc25a28) function as essential mitochondrial iron importers for heme and Fe/S cluster biogenesis. A genetic deficiency of Mfrn1 results in a profound hypochromic anemia in vertebrate species. To map the cis-regulatory modules (CRMs) that control expression of the Mfrn genes, we utilized genome-wide chromatin immunoprecipitation (ChIP) datasets for the major erythroid transcription factor GATA-1. We identified the CRMs that faithfully drive the expression of Mfrn1 during blood and heart development and Mfrn2 ubiquitously. Through in vivo analyses of the Mfrn-CRMs in zebrafish and mouse, we demonstrate their functional and evolutionary conservation. Using knockdowns with morpholinos and cell sorting analysis in transgenic zebrafish embryos, we show that GATA-1 directly regulates the expression of Mfrn1. Mutagenesis of individual GATA-1 binding cis elements (GBE) demonstrated that at least two of the three GBE within this CRM are functionally required for GATA-mediated transcription of Mfrn1. Furthermore, ChIP assays demonstrate switching from GATA-2 to GATA-1 at these elements during erythroid maturation. Our results provide new insights into the genetic regulation of mitochondrial function and iron homeostasis and, more generally, illustrate the utility of genome-wide ChIP analysis combined with zebrafish transgenesis for identifying long-range transcriptional enhancers that regulate tissue development.
Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human β-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.
To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2.
We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45.
We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45.
NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.
The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α₂δ₂) and fetal hemoglobin (HbF, α₂γ₂) both inhibit the polymerization of hemoglobin S, which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α₂β₂) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1, and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34(+) cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a new genetic therapeutic approach to β-hemoglobinopathies.
The PU.1 and GATA1 genes play an important role in the differentiation of blood stem cells. The protein levels expressed by these genes are thought to be regulated by a self-excitatory feedback loop for each gene and a cross-inhibitory feedback loop between the two genes. A mathematical model that captures the dynamical interaction between these two genes reveals that constant levels of self-excitation and cross-inhibition allow the most self-exciting or cross-inhibiting gene to dominate the system. However, since biological systems rarely exist in an unchanging equilibrium, we modeled this gene circuit using discrete time-dependent changes in the parameters in lieu of steady state parameters. These time-dependent parameters lead to new phenomena, including the development of new limit cycles and basins of attraction. These phenomena are not present in models using constant parameter values. Our findings suggest that even small perturbations in the PU.1 and GATA1 feedback loops may substantially alter the gene expression and therefore the cell phenotype. These time-dependent parameter models may also have implications for other gene systems and provide new ways to understand the mechanisms of cellular differentiation.
Recent studies have showed that transcription elongation factors regulate early development. Foggy/Spt5 is a subunit of DRB sensitivity-inducing factor, which negatively and positively regulates transcription elongation. Here, we report that the positive function of Foggy/Spt5 is required for gata1 expression during zebrafish embryonic hematopoiesis. Antisense morpholino oligonucleotide (MO)-mediated knockdown of foggy/spt5 has led to a reduction in the expression of gata1 and the gata1 target genes alas2 and hbae3 and inhibited proper hemoglobin production. By contrast, expression of hematopoietic stem cell and endothelial markers, including scl, lmo2, gata2, fli-1, and flk-1, and expression of biklf, whose product directs gata1 expression via its direct binding to the gata1 promoter, were unaltered, suggesting that gata1 is a functionally important target gene of Foggy/Spt5. The MO-mediated gata1 repression was relieved by forced expression of wild-type foggy/spt5, but not by a mutant lacking the positive function. Therefore, this study provides evidence that Foggy/Spt5 plays an important role in gata1 gene expression and erythropoiesis through its transcriptional activation domain.
Microtubules play important roles in mitotic spindle assembly and chromosome segregation to maintain normal cell cycle progression. A number of microtubule-associated proteins have been identified in epithelial and neural cell cultures; however, their physiological significance is not well characterized due to the lack of appropriate in vivo animal models. Nucleolar spindle-associated protein (NuSAP) is a microtubule-binding protein and is reported to be involved in mitosis by cell culture studies. In this report, we identified the zebrafish homologue of human NuSAP and investigated its expression profile and functions. Using in situ hybridization, we demonstrated that transcripts of zebrafish nusap1 are specifically expressed in the retina, forebrain, hindbrain and neural crest. When the in vivo expression of nusap1 was knocked down through antisense oligonucleotide morpholino technology, the morphants of nusap1 showed impaired morphogenesis in the trunk and yolk extension, implying the involvement of Nusap1 in cell migration. Mechanistic studies revealed that nusap1 morphants have an altered expression pattern of neural crest markers crestin and sox9b, but normal expression of blood vessel and notochord markers gata1 and shh. In addition, nusap1 mRNA injection caused serious apoptosis in retina and hindbrain tissue, and these phenotypes can be rescued by co-injection of morpholino against nusap1. These observations not only suggest a role for Nusap1 in connecting apoptosis with cell migration, but also provide strong evidences that Nusap1 is potentially involved in morphogenesis in vertebrates.
Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis.
We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype.
Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects.
During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.
Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.
Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined.
In this study, we show that ALCAM expression is silenced in erythromegakaryocytic progenitor cell lines. In agreement with this finding, the ALCAM promoter is occupied by GATA-1 in vivo, and a cognate motif at -850 inhibited promoter activity in K562 and MEG-01 cells. Gain-of-function studies showed that ALCAM clusters K562 cells in a process that requires PKC. Induction of megakaryocytic differentiation in K562 clones expressing ALCAM activated PKC-δ and triggered apoptosis.
There is a lineage-specific silencing of ALCAM in bi-potential erythromegakaryocytic progenitor cell lines. Marked apoptosis of ALCAM-expressing K562 clones treated with PMA suggests that aberrant ALCAM expression in erythromegakaryocytic progenitors may contribute to megakaryocytopenia.
Oogenesis and primordial follicle formation are tightly linked processes, requiring organized and precisely timed communication between somatic and germ cells. Deviations in ovarian cell cross talk, or aberrant gene expression within one of the cell populations, can lead to follicle loss or dysfunction, resulting in infertility. Expression of GATA-like protein-1 (GLP-1) in ovarian somatic cells is required for normal fertility in female mice, as GLP-1 deficiency leads to the absence of oocytes at birth. However, the timing and nature of this germ cell loss is not well understood. In this study, we characterize the embryonic germ cell loss in GLP-1 null mice. Quantitative PCR demonstrates that ovarian Glp-1 mRNA is expressed in a bimodal pattern during embryogenesis, peaking at E13.5-14.5 and again at birth. In contrast, adult ovaries express low but detectable levels of Glp-1 mRNA. Analysis of developing GLP-1 null mouse ovaries shows that germ cells are appropriately specified and migrate normally to nascent gonads. Upon arrival at the gonad, precocious loss of germ cells begins at around E13.5. This loss is completed by birth and is accompanied by defects in the expression of genes associated with meiotic entry. Interestingly, somatic pregranulosa cells still form basement membranes surrounding germ line cysts and express mRNA encoding paracrine signaling molecules that communicate with oocytes, albeit at lower levels than normal. Together, these data imply that the somatic cell protein GLP-1 is not necessary for many pregranulosa cell functions but is required for germ cell survival.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) function as signal transducers and control many different physiologic processes. G proteins can be grouped into four families: Gs, Gi, Gq and G12. Gα13 belongs to the G12 family. In zebrafish, there are two isoforms of Gα13: Gα13a and Gα13b. We show here that knockdown of Gα13b in zebrafish results in hematopoietic and angiogenic defects. The Gα13b morphants don't show complete loss of expression of gata1, pu.1 or flk until 35 hpf suggests that Gα13b is closely related to the development of hematopoietic cells. Further studies reveal that blood cells and vascular endothelial cells have undergone apoptosis through a p53-dependent pathway in Gα13b-depleted embryos. Injection of p53 morpholino could partially rescue the phenotype of Gα13b morphants. These data possibly demonstrate a new role for Gα13 in cell survival.
Thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) are accepted to be important molecules in the development and maintenance of allergic diseases. Although several types of histamine H(1) receptor antagonist (antihistamine) have been developed and used for the treatment of allergic diseases, the influence of antihistamines on TARC and MDC production is not well understood.
The present study was undertaken to examine the influence of antihistamines on TARC and MDC production from CD14+ cells after antigenic stimulation in vitro.
CD14+ cells prepared from patients with pollinosis to Japanese cedar pollen were stimulated with specific allergen extracted from Japanese cedar pollen (Cry j 1) in the presence of azelastine (AZE), ketotifen (KET), fexofenadine (FEX) and oxatomide (OXA) for 6 days. TARC and MDC levels in culture supernatants were examined by ELISA. We also examined the influence of FEX on TARC and MDC mRNA expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and transcription factor activation in CD14+ cells after Cry j 1 stimulation.
FEX at 250 ng/ml, which is almost equal to therapeutic blood levels, caused a significant inhibition of TARC and MDC production.However, AZE, OXA and KET required higher concentrations than their therapeutic blood levels to suppress production of these factors. FEX at 250 ng/ml also suppressed NF-κB activation, phosphorylation of p38 MAPK and extracellular signal-regulated kinases 1 and 2 and expression of mRNA for TARC and MDC.
These results suggest that antihistamines, especially FEX, suppress CC chemokine production from CD14+ cells through interference with antigen-mediated signaling and result in favorable modification of allergic disease states or conditions.
A relatively small cadre of lineage-restricted transcription factors largely orchestrates erythropoiesis, but how these nuclear factors interact to regulate this complex biology is still largely unknown. However, recent technological advances, such as chromatin immunoprecipitation (ChIP) paired with massively parallel sequencing (ChIP-seq), gene expression profiling, and comprehensive bioinformatic analyses, offer new insights into the intricacies of red cell molecular circuits.
Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones.
Acute leukemias in children with Down syndrome (DS) are characterized by unique clinical and biological features. Notable among DS children with acute myeloid leukemia (AML), is the high frequency of the acute megakaryocytic leukemia (AMkL) subtype, which uniformly harbor somatic mutations in the transcription factor GATA1 gene. DS patients with AML, and in particular AMkL, have event-free survival rates of 80-100% in contrast to event-free survival rates of less than 35% for non-DS children with AMkL. DS children with acute lymphoblastic leukemia have a more heterogeneous disease, with approximately 30% of the patients having somatic JAK2 mutations, heightened methotrexate sensitivity and higher rates of treatment-related toxicities. These features highlight a striking relationship between genes localized to chromosome 21, leukemogenesis and sensitivity to leukemia chemotherapy agents.
Preeclampsia is a major obstetrical complication affecting maternal and fetal health. While it is clear that there is a substantial placental contribution to preeclampsia pathogenesis, the maternal contribution is less well characterized. We therefore performed a genome-wide transcriptome analysis to explore disease-associated changes in maternal gene expression patterns in peripheral blood mononuclear cells (PBMCs).
Preeclampsia was defined as gestational hypertension, proteinuria and hyperurecimia. Total RNA was isolated from PBMCs obtained from women with uncomplicated pregnancies (n = 5) and women with preeclamptic pregnancies (n = 5). Gene expression analysis was carried out using Agilent oligonucleotide microarrays. Biological pathway analysis was undertaken using Ingenuity Pathway Analysis software. Quantitative real-time PCR (QRTPCR) was performed to validate the gene expression changes of selected genes in normotensive and preeclamptic patients (n = 12 each).
We identified a total of 368 genes that were differentially expressed in women with preeclampsia compared to normal controls with false discovery rate (FDR) controlled at 10%. In follow up experiments we further analyzed the expression levels of a number of genes that were identified as altered by the microarray data including survivin (BIRC5), caveolin (CAV1), GATA binding protein-1 (GATA1), signal tranducer and activator of transcription 1 (STAT1), E2F transcription factor-1 (E2F1), fibronectin-1 (FN1), interleukin-4 (IL-4), matrix metalloprotease-9 (MMP-9) and WAP four disulfide domain protein (WFDC-1) by QRTPCR. Additionally we performed immuno blot analysis and zymography to verify some of these candidate genes at the protein level. Computational analysis of gene function identified an anti-proliferative and altered immune function cellular phenotype in severe preeclamptic samples.
We have characterized the genome-wide mRNA expression changes associated with preeclampsia-specific genes in circulating maternal blood cells at the time of delivery. In addition to providing information relating to the biological basis of the preeclampsia phenotype, our data provide a number of potential biomarkers for use in the further characterization of this disease.
Developmental control mechanisms often use multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. We analyzed the function of components of a complex containing master regulators of hematopoiesis (GATA-1 and Scl/TAL1) and the non-DNA binding components ETO2, the LIM domain protein LMO2, and the chromatin looping factor LDB1. Surprisingly, we discovered that ETO2 and LMO2 regulate distinct target-gene ensembles in erythroid cells. ETO2 commonly repressed GATA-1 function via suppressing histone H3 acetylation, although it also regulated methylation of histone H3 at lysine 27 at select loci. Prior studies defined multiple modes by which GATA-1 regulates target genes with or without the coregulator Friend of GATA-1 (FOG-1). LMO2 selectively repressed genes that GATA-1 represses in a FOG-1-independent manner. As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is unknown, this mechanistic link has important biological and pathophysiological implications. The demonstration that ETO2 and LMO2 exert qualitatively distinct functions at endogenous loci illustrates how components of complexes containing master developmental regulators can impart the capacity to regulate unique cohorts of target genes, thereby diversifying complex function.
The most common translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus. The LMO2 insertions predisposed these children to T-ALL, yet how LMO2 contributes to T cell transformation remains unclear. The LIM (Lin 11, Isl-1, Mec-3) domain containing LMO2 protein regulates erythropoiesis as part of a large transcriptional complex consisting of LMO2, TAL1, E47, GATA1 and LDB1 that recognizes bipartite E-box-GATA1 sites on target genes. Similarly, a TAL1/E47/LMO2/LDB1 complex is observed in human T-ALL and Tal1 and Lmo2 expression in mice results in disease acceleration. To address the mechanism(s) of Tal1/Lmo2 synergy in leukemia, we generated Lmo2 transgenic mice and mated them with mice that express wild-type Tal1 or a DNA-binding mutant of TAL1. Tal1/Lmo2 and MutTAL1/Lmo2 bitransgenic mice exhibit perturbations in thymocyte development due to reduced E47/HEB transcriptional activity and develop leukemia with identical kinetics. These data demonstrate that the DNA-binding activity of Tal1 is not required to cooperate with Lmo2 to cause leukemia in mice and suggest that Lmo2 may cooperate with Tal1 to interfere with E47/HEB function(s).
The Meis1 protein represents an important cofactor for Hox and Pbx1 and is implicated in human and murine leukemias. Though much is known about the role of meis1 in leukemogenesis, its function in normal hematopoiesis remains largely unclear. Here we characterized the role of the proto-oncogene, meis1, during zebrafish primitive and definitive hematopoiesis.
Zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells and Sudan black to quantify neutrophils. The numbers of other cells (scl-, gata1- and alas2-positive cells) were also quantified by measuring the corresponding stained areas of the embryos. We used anti-Meis1 antibody and whole mount immunohistochemistry to determine the pattern of expression of Meis1 during zebrafish development and then analyzed the functional role of Meis1 by knocking-down the meis1 gene.
Using antisense morpholino oligomers to interrupt meis1 expression we found that, although primitive macrophage development could occur unhampered, posterior erythroid differentiation required meis1, and its absence resulted in a severe decrease in the number of mature erythrocytes. Furthermore a picture emerged that meis1 exerts important effects on later stages of erythrocyte maturation and that these effects are independent of gata1, but under the control of scl. In addition, meis1 morpholino knock-down led to dramatic single arteriovenous tube formation. We also found that knock-down of pbx1 resulted in a phenotype that was strikingly similar to that of meis1 knock-down zebrafish.
These results imply that meis1, jointly with pbx1, regulates primitive hematopoiesis as well as vascular development.
Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.
To identify molecular effects of the antineoplastic agent protein kinase C inhibitor 412 (PKC412) (midostaurin), we applied gene expression profiling in zebrafish using whole-genome microarrays. Behavioral, developmental, and physiological effects were investigated in order to analyze for correlations between altered gene expression profiles with effects on development and physiology. Zebrafish blastula-stage embryos were exposed for 6 days postfertilization to nominal levels of 2 and 40 μg/l PKC412. Among the 259 and 511 altered transcripts at both concentrations, respectively, the expressions of genes involved in the circadian rhythm were further investigated. Alteration of swimming behavior was not observed. Pathways of interest affected by PKC412 were angiogenesis, apoptosis, DNA damage response, and response to oxidative stress. Angiogenesis was analyzed in double-transgenic zebrafish embryos Tg(fli1a:EGFP)y1;Tg(gata1:dsRed)sd2; no major defects were induced by PKC412 treatment at both concentrations. Apoptosis occurred in olfactory placodes of embryos exposed to 40 μg/l, and DNA damage was induced at both PKC412 concentrations. However, there were no significant effects on reactive oxygen species formation. This study leads to the conclusion that PKC412-induced alterations of gene transcripts are partly paralleled by physiological effects at high, but not at low PKC412 concentrations expected to be of environmental relevance.
Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs), its function during hematopoiesis was unclear. Here, we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB, 1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all, 14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos, of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs, but none of them, even in combined aldh1a2 and aldh1a3 knockdown, recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion, DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined.
The transcriptional regulator GATA1 is crucially involved in megakaryocytopoiesis and erythropoiesis. Mutations of the gene which is located on the X chromosome have been associated with platelet and red blood cell abnormalities. We identified a family with a GATA1 (G208R) mutation in whom a low male birth rate and frequent miscarriages among heterozygous females suggested increased fetal death in male hemizygotes. Female mutation carriers had normal or near normal hemoglobin levels and platelet counts ranging from normal to severely reduced, probably reflecting skewed X chromosome inactivation. Platelets were dimorphous, and thrombocytopenia was associated with erythroblastosis. The only living male mutation carrier had severe macrothrombocytopenia with life-threatening bleeding episodes, moderate to severe anemia, eosinopenia, skeletal abnormalities, and abundant extramedullary hematopoiesis. Long-term sequelae in the 50-year-old patient included unilateral nephrectomy following misinterpretation of paraspinal hematopoiesis as renal cancer, spinal stenosis which was possibly favored by progressive bone marrow expansion, and severe secondary gout.
Survivin is an inhibitor of apoptosis protein family member that has an essential role in cellular proliferation as a component of the chromosome passenger complex. Survivin is highly expressed in embryos and in proliferating adult tissues, but it is not expressed in most differentiated cells. During tumorigenesis, however, survivin expression is dramatically upregulated. Although many studies have shown that survivin is required for cancer cells, the extent to which survivin contributes to the initiation of tumors is unknown. Here we show that transgenic mice that overexpress survivin in hematopoietic cells are at an increased risk of hematologic tumors. In examining how survivin might contribute to tumorigenesis, we observed that hematopoietic cells engineered to overexpress survivin are less susceptible to apoptosis. We conclude that survivin may promote tumorigenesis by imparting a survival advantage to cells that acquire additional genetic lesions.
Neonatal transient myeloproliferative disorder and acute megakaryoblastic leukaemia of Down syndrome are considered different manifestations of the same disease. In most cases, transient myeloproliferative disorders require no treatment, while acute megakaryoblastic leukaemia of Down's syndrome is characterised by an increased sensitivity to chemotherapy and its treatment should be adapted with a reduction in dose intensity. Both entities share specific mutations at exón 2 of the transcription factor GATA1.
We analysed biological features and GATA1 mutations in 4 patients with transient abnormal myelopoiesis (2) and acute megakaryoblastic leukaemia (2) including one phenotypically normal trisomy 21 mosaicism. We found abnormal GATA1 mutated clones in each case, and a specific point mutation at exón 2 was detected in three cases. Given the heterogeneous phenotype of megakaryoblastic blasts and the low percentage of blasts at presentation, the recognition of GATA1 mutations was helpful for diagnosis. In addition, molecular remission was established in 2 patients after subsequent normal mutational GATA1 analysis.
We conclude that GATA1 mutational study is a useful tool for the diagnosis and management of trisomy 21 associated myeloproliferative disorders.
We present a cytogenetically normal neonate who developed transient abnormal myelopoiesis. The blasts showed trisomy 21. In contrast, fibroblasts, and PHA-stimulated peripheral blood demonstrated normal diploid line on extensive karyotyping. Direct sequencing of the DNA derived from the peripheral blood at overt disease revealed splice site mutation in the boundary of GATA1 exon 2. The patient received three courses of chemotherapy leading to complete remission. During the complete remission, there was neither mutation of GATA1 exon 2 nor trisomy 21, confirming somatic nature of both abnormalities. The patient is now free from the disease 12 months after remission. This case emphasizes the significance of trisomy 21 as the cause of transient abnormal myelopoiesis in Down syndrome.
Gene regulation commonly involves interaction among DNA, proteins and biochemical conditions. Using chromatin immunoprecipitation (ChIP) technologies, protein-DNA interactions are routinely detected in the genome scale. Computational methods that detect weak protein-binding signals and simultaneously maintain a high specificity yet remain to be challenging. An attractive approach is to incorporate biologically relevant data, such as protein co-occupancy, to improve the power of protein-binding detection. We call the additional data related with the target protein binding as supporting tracks.
We propose a novel but rigorous statistical method to identify protein occupancy in ChIP data using multiple supporting tracks (PASS2). We demonstrate that utilizing biologically related information can significantly increase the discovery of true protein-binding sites, while still maintaining a desired level of false positive calls. Applying the method to GATA1 restoration in mouse erythroid cell line, we detected many new GATA1-binding sites using GATA1 co-occupancy data.
http://stat.psu.edu/ approximately yuzhang/pass2.tar.
Targeted disruption of the Fli1 gene results in embryonic lethality. To dissect the roles of functional domains in Fli1, we recently generated mutant Fli1 mice that express a truncated Fli1 protein (Fli1(ΔCTA)) that lacks the carboxy-terminal regulatory (CTA) domain. Heterozygous Fli1(ΔCTA) mice are viable, while homozygous mice have reduced viability. Early postnatal lethality accounts for 30% survival of homozygotes to adulthood. The peripheral blood of these viable Fli1(ΔCTA)/Fli1(ΔCTA) homozygous mice has reduced platelet numbers. Platelet aggregation and activation were also impaired and bleeding times significantly prolonged in these mutant mice. Analysis of mRNA from total bone marrow and purified megakaryocytes from Fli1(ΔCTA)/Fli1(ΔCTA) mice revealed downregulation of genes associated with megakaroyctic development, including c-mpl, gpIIb, gpIV, gpIX, PF4, NF-E2, MafG, and Rab27B. While Fli1 and GATA-1 synergistically regulate the expression of multiple megakaryocytic genes, the level of GATA-1 present on a subset of these promoters is reduced in vivo in the Fli1(ΔCTA)/Fli1(ΔCTA) mice, providing a possible mechanism for the impared transcription observed. Collectively, these data showed for the first time a hemostatic defect associated with the loss of a specific functional domain of the transcription factor Fli1 and suggest previously unknown in vivo roles in megakaryocytic cell differentiation.
Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.
Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.
The model organism Danio rerio, also known as the zebrafish, is an excellent system for studying the developmental process of hematopoiesis. It is an ideal model for in vivo imaging, and it is useful for large-scale genetic screens. These have led to the discovery of previously unknown players in hematopoiesis, as well as helped our understanding of hematopoietic development. In this review, we will summarize hematopoiesis in the zebrafish and discuss how genetic approaches using the zebrafish system have helped to build our current knowledge in the field of hematopoiesis.
Erythropoiesis in adult mammals is characterized by the progressive maturation of hematopoietic stem cells to lineage-specific progenitors, to morphologically identifiable precursors which enucleate to form mature erythrocytes. In contrast, primitive erythropoiesis is characterized by the appearance within the yolk sac of a transient, lineage-restricted progenitor population which generates a wave of erythroid precursors. These precursors undergo progressive maturation in the bloodstream, characterized by nuclear condensation and embryonic hemoglobin accumulation. This process is dependent on erythropoietin signaling through its cognate receptor, as well as the function of several erythroid-specific transcription factors, including GATA1 and EKLF. Targeted disruption of genes in the mouse that result in failure of the emergence or maturation of the primitive erythroid lineage leads to early fetal death, indicating that the primitive erythroid lineage is necessary for survival of the mammalian embryo. While it was thought for over a century that primitive erythroid cells were uniquely nucleated mammalian red cells, it is now recognized that they, like their definitive erythroid counterparts, enucleate to form reticulocytes and pyrenocytes. This surprising finding indicates that the primitive erythroid lineage is indeed mammalian, rather than non-mammalian, in character.
Severe malaria anemia is characterized by inhibited/altered erythropoiesis and presence of hemozoin-(HZ)-laden bone-marrow macrophages. HZ mediates peroxidation of unsaturated fatty acids and production of bioactive aldehydes such as 4-hydroxynonenal (HNE). HZ-laden human monocytes inhibited growth of cocultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Cocultivation with HZ or treatment with low micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. After HZ/HNE treatment, 2 critical proteins in cell-cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G₁-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resultant decreased expression of cyclin A and D2 retarded cell-cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrinR1, stem cell factorR, interleukin-3R, and erythropoietinR. The reduced receptor expression and the impaired cell-cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin. Present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia.
Oncogene-mediated transformation of hematopoietic cells has been studied extensively, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay of signaling pathways and oncogenes is essential for developing targeted cancer therapies. Here, we address the vexing issue of how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by the triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21. We demonstrate overactivity of insulin-like growth factor (IGF) signaling in authentic human DS-AMKL and in a DS-AMKL mouse model generated through retroviral insertional mutagenesis. Fetal but not adult megakaryocytic progenitors are dependent on this pathway. GATA1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. Failure of a direct GATA1-E2F interaction in mutated GATA1s converges with overactive IGF signaling to promote cellular transformation of DS fetal progenitors, revealing a complex, fetal stage-specific regulatory network. Our study underscores context-dependent requirements during oncogenesis, and explains resistance to transformation of ostensibly similar adult progenitors.
Transcriptional networks orchestrate complex developmental processes. Such networks are commonly instigated by master regulators of development. Considerable progress has been made in elucidating GATA factor-dependent genetic networks that control blood cell development. GATA-2 is required for the genesis and/or function of hematopoietic stem cells, whereas GATA-1 drives the differentiation of hematopoietic progenitors into a subset of the blood cell lineages. GATA-1 directly represses Gata2 transcription, and this involves GATA-1-mediated displacement of GATA-2 from chromatin, a process termed a GATA switch. GATA switches occur at numerous loci with critical functions, indicating that they are widely utilized developmental control tools.
TAL1 plays pivotal roles in vascular and hematopoietic developments through the complex with LMO2 and GATA1. Hemangioblasts, which have a differentiation potential for both endothelial and hematopoietic lineages, arise in the primitive streak and migrate into the yolk sac to form blood islands, where primitive hematopoiesis occurs. ZFAT (a zinc-finger gene in autoimmune thyroid disease susceptibility region/an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook) was originally identified as an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook, and is highly conserved among species. ZFAT is thought to be a critical transcription factor involved in immune-regulation and apoptosis; however, developmental roles for ZFAT remain unknown. Here we show that Zfat-deficient (Zfat(-/-)) mice are embryonic-lethal, with impaired differentiation of hematopoietic progenitor cells in blood islands, where ZFAT is exactly expressed. Expression levels of Tal1, Lmo2, and Gata1 in Zfat(-/-) yolk sacs are much reduced compared with those of wild-type mice, and ChIP-PCR analysis revealed that ZFAT binds promoter regions for these genes in vivo. Furthermore, profound reduction in TAL1, LMO2, and GATA1 protein expressions are observed in Zfat(-/-) blood islands. Taken together, these results suggest that ZFAT is indispensable for mouse embryonic development and functions as a critical transcription factor for primitive hematopoiesis through direct-regulation of Tal1, Lmo2, and Gata1. Elucidation of ZFAT functions in hematopoiesis might lead to a better understanding of transcriptional networks in differentiation and cellular programs of hematopoietic lineage and provide useful information for applied medicine in stem cell therapy.
Self-renewal and differentiation of hematopoietic stem cells (HSCs) are balanced by the concerted activities of the fibroblast growth factor (FGF), Wnt, and Notch pathways, which are tuned by enzyme-mediated remodeling of heparan sulfate proteoglycans (HSPGs). Sulfatase modifying factor 1 (SUMF1) activates the Sulf1 and Sulf2 sulfatases that remodel the HSPGs, and is mutated in patients with multiple sulfatase deficiency. Here, we show that the FGF signaling pathway is constitutively activated in Sumf1(-/-) HSCs and hematopoietic stem progenitor cells (HSPCs). These cells show increased p-extracellular signal-regulated kinase levels, which in turn promote beta-catenin accumulation. Constitutive activation of FGF signaling results in a block in erythroid differentiation at the chromatophilic erythroblast stage, and of B lymphocyte differentiation at the pro-B cell stage. A reduction in mature myeloid cells and an aberrant development of T lymphocytes are also seen. These defects are rescued in vivo by blocking the FGF pathway in Sumf1(-/-) mice. Transplantation of Sumf1(-/-) HSPCs into wild-type mice reconstituted the phenotype of the donors, suggesting a cell autonomous defect. These data indicate that Sumf1 controls HSPC differentiation and hematopoietic lineage development through FGF and Wnt signaling.
In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.
We present the first transcriptional regulatory element found in a PAH gene intron. The element is located in the PAH gene intron 8, acts as an enhancer specifically in the hepatoma cell line, and binds GATA-1 transcription factor. Herein the presented data could unlock a new area for the analysis of PAH gene expression and could contribute to refining genotype-phenotype correlation.
Erythropoietic and megakaryocytic programs are specified from multipotential progenitors by the transcription factor GATA1. FOG1, a GATA1-interaction partner, is critical for GATA1 function in several contexts by bringing multiple complexes into association with GATA1 to facilitate activation or repression of target genes. To further elucidate regulation of these associations by cellular and extracellular cues, we examined FOG1 for post-translational modifications. We found that FOG1 is SUMOylated and phosphorylated in erythroid cells in a differentiation-dependent manner. Removal of the SUMOylation sites in FOG1 does not impair nuclear localization, protein stability, or chromatin occupancy. However, SUMOylation of FOG1 modulates interactions with C-terminal binding protein family members, specifically promoting CTBP1 binding. Phosphorylation of FOG1 modulates SUMOylation and, therefore, indirectly regulates the CTBP interaction. Post-translational modification of FOG1 may contribute to control of co-occupancy by CTBP family members, the NuRD complex, and GATA1 at differentially regulated genes.
 Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive.
 Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34(+) -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis.
 In UT-7/mpl cells, anagrelide (1μm) did not interfere with MPL-mediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3μm) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes.
 Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events.
Ldb1 and erythroid partners SCL, GATA-1, and LMO2 form a complex that is required to establish spatial proximity between the β-globin locus control region and gene and for transcription activation during erythroid differentiation. Here we show that Ldb1 controls gene expression at multiple levels. Ldb1 stabilizes its erythroid complex partners on β-globin chromatin, even though it is not one of the DNA-binding components. In addition, Ldb1 is necessary for enrichment of key transcriptional components in the locus, including P-TEFb, which phosphorylates Ser2 of the RNA polymerase C-terminal domain for efficient elongation. Furthermore, reduction of Ldb1 results in the inability of the locus to migrate away from the nuclear periphery, which is necessary to achieve robust transcription of β-globin in nuclear transcription factories. Ldb1 contributes these critical functions at both embryonic and adult stages of globin gene expression. These results implicate Ldb1 as a factor that facilitates nuclear relocation for transcription activation.
Coordination of cellular processes through the establishment of tissue-specific gene expression programs is essential for lineage maturation. The basic helix-loop-helix hemopoietic transcriptional regulator TAL1 (formerly SCL) is required for terminal differentiation of red blood cells. To gain insight into TAL1 function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that TAL1 coordinates expression of genes in most known red cell-specific processes. The majority of TAL1's genomic targets require direct DNA-binding activity. However, one-fifth of TAL1's target sequences, mainly among those showing high affinity for TAL1, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by TAL1 identified CAGNTG as TAL1-preferred E-box motif in erythroid cells. Novel motifs were also characterized that may help distinguish activated from repressed genes and suggest a new mechanism by which TAL1 may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of TAL1, to sequences occupied by TAL1 suggests that TAL1's binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors.
Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we showed that overexpression of EDAG in a myeloid cell line 32D led to an erythroid phenotype with increased number of benzidine-positive cells and up-regulation of erythroid specific surface marker TER119. The megakaryocytic specific marker CD61 was also induced significantly. Using a genome-wide microarray analysis and a twofold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, NF-E2, Gfi-1b, hemogen, SCL, hemoglobin alpha, beta and megakaryocytic gene GPIX were increased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of these genes. Taken together, EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.
To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf(164) isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.
BCL11A is a major regulator of fetal hemoglobin production. Reduced levels of BCL11A have been shown to delay switching from fetal to adult hemoglobin, suggesting that it acts as a stage-specific repressor of gamma globin expression. We have carried out a survey of BCL11A binding in the globin, BCL11A and GATA1 loci by ChIP-on-chip analysis in primary human erythroid cells. We found strong occupancy in both alpha and beta globin upstream regulatory regions as well as in regions involved in switching and hereditary persistence of fetal hemoglobin. Genetic studies have identified a restricted 14kb region in BCL11A intron 2 as being highly associated with HbF levels. Strong GATA-1 binding and acetylated histone H3 was found in this area, which could be indicative of a regulatory element, changes in which might be responsible for the overall regulation of BCL11A. We also observed BCL11A and GATA-1 binding in a known auto-regulatory promoter element of the GATA1 locus.
Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (Delta dblGATA) mice lack eosinophils.
Ovalbumin (OVA)-reactive Th clones were transferred into Delta dblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge.
The number of BALF eosinophils was significantly increased in WT mice, but not detectable in Delta dblGATA mice. BHR was also induced in WT mice, but significantly attenuated in Delta dblGATA mice.
Eosinophils are involved in T-cell-mediated BHR.
KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.
Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2α protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.
Lineage-determination transcription factors coordinate cell differentiation and proliferation by controlling the synthesis of lineage-specific gene products as well as cell cycle regulators. GATA-1 is a master regulator of erythropoiesis. Its role in regulating erythroid-specific genes has been extensively studied, whereas its role in controlling genes that regulate cell proliferation is less understood. Ectopic expression of GATA-1 in erythroleukemia cells releases the block to their differentiation and leads to terminal cell division. An early event in reprogramming the erythroleukemia cells is induction of the cyclin-dependent kinase inhibitor p21. Remarkably, ectopic expression of p21 also induces the erythroleukemia cells to differentiate. We now report that GATA-1 directly regulates transcription of the p21 gene in both erythroleukemia cells and normal erythroid progenitors. Using reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we show that GATA-1 stimulates p21 gene transcription by binding to consensus binding sites in the upstream region of the p21 gene promoter. This activity is also dependent on a binding site for Sp1/KLF-like factors near the transcription start site. Our findings indicate that p21 is a crucial downstream gene target and effector of GATA-1 during red blood cell terminal differentiation.
The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.
A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells.
We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression.
Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad-a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models.
The discovery of JAK2 mutations in Philadelphia-negative myeloproliferative neoplasms has prompted investigators to evaluate mutation-targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1(low) mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27(Kip1) activity and is treatable by Aplidin, a cyclic depsipeptide that activates p27(Kip1) in several cancer cells. Aplidin restored expression of Gata1 and p27(Kip1) in Gata1(low) hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF-beta/VEGF levels released in the microenvironment by immature Gata1(low) megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin-treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1(low) progenitor cells remained low. These results indicate that Aplidin effectively alters the natural history of myelofibrosis in Gata1(low) mice and suggest this drug as candidate for clinical evaluation in PMF.
Small ubiquitin-like modifier (SUMO) modification of proteins (SUMOylation) and deSUMOylation have emerged as important regulatory mechanisms for protein function. SENP1 (SUMO-specific protease) deconjugates SUMOs from modified proteins. We have created SENP1 knockout (KO) mice based on a Cre-loxP system. Global deletion of SENP1 (SENP1 KO) causes anemia and embryonic lethality between embryonic day 13.5 and postnatal day 1, correlating with erythropoiesis defects in the fetal liver. Bone marrow transplantation of SENP1 KO fetal liver cells to irradiated adult recipients confers erythropoiesis defects. Protein analyses show that the GATA1 and GATA1-dependent genes are down-regulated in fetal liver of SENP1 KO mice. This down-regulation correlates with accumulation of a SUMOylated form of GATA1. We further show that SENP1 can directly deSUMOylate GATA1, regulating GATA1-dependent gene expression and erythropoiesis by in vitro assays. Moreover, we demonstrate that GATA1 SUMOylation alters its DNA binding, reducing its recruitment to the GATA1-responsive gene promoter. Collectively, we conclude that SENP1 promotes GATA1 activation and subsequent erythropoiesis by deSUMOylating GATA1.
By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.
The human beta-globin genes are expressed in a developmentally controlled fashion. Studies on the molecular mechanisms underlying the stage-specific regulation of globin genes have been fueled by the clinical benefit of elevated fetal gamma-globin expression in patients with sickle cell anemia and thalassemia. Recent reports suggested a role of the hematopoietic transcription factor GATA-1, its cofactor FOG-1, and the associated chromatin remodeling complex NuRD in the developmental silencing of HBG1 and HBG2 gene expression. To examine whether FOG-1 via NuRD controls HBG1 and HBG2 silencing in vivo, we created mice in which the FOG-1/NuRD complex is disrupted (A. Miccio et al., EMBO J. 29:442-456, 2010) and crossed these with animals carrying the entire human beta-globin gene locus as a transgene. We found that the FOG-1/NuRD interaction is dispensable for the silencing of human HBG1 and HBG2 expression. In addition, mutant animals displayed normal silencing of the endogenous embryonic globin genes. In contrast, a significant reduction of adult-type human and murine globin gene expression was found in adult bone marrows of mutant animals. These results suggest that, unexpectedly, NuRD is required for FOG-1-dependent activation of adult-type globin gene expression but is dispensable for human gamma-globin silencing in vivo.
An isolated pericardial effusion was observed during a routine prenatal ultrasound in a fetus of 30 and 3/7 weeks gestation. Amniocentesis was performed and revealed a trisomy 21. After prenatal counseling, the parents opted for termination of the pregnancy at 32 weeks. Postmortem examination confirmed the presence of a pericardial effusion, without structural cardiac anomalies, and showed the development of ascites and subcutaneous edema. Histological examination showed an infiltrate of megakaryoblasts and irregular, dysplastic megakaryocytes confined to the epicardium, the pericardial lymph nodes, and the pancreas, consistent with a myeloid proliferation related to Down syndrome. Sequencing of exons 2 and 3 of the GATA1 gene from the umbilical cord blood and from megakaryoblast infiltrate showed no mutation. A high incidence of chromosomal abnormalities, in particular trisomy 21, has been described in fetuses with pericardial effusion. However, myeloid proliferation related to Down syndrome without GATA1 mutations is extremely rare. To our knowledge, only one such case has been reported to date. We present here a 2nd case, which further supports the hypothesis that hyperproliferation of megakaryocytes in a subset of Down syndrome patients may be initiated by events other than GATA1 mutations.
An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of alpha-granules. The mechanism that sorts the alpha-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the alpha-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the alpha-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different alpha-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1(low) megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1(low) is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1(low), the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate alpha-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the alpha-granule content in response to external stimuli.
Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.
Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34(+) human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.
To evaluate the effects of the transcription factor GATA-1 on determining cell fate between dendritic cell (DC) and mast cell (MC) lineages, GATA-1 was exogenously expressed in bone marrow-derived (BM) DCs. Exogenous expression of GATA-1 by a retrovirus in BMDCs inhibited expression of CD11c, CD80, CD86, and major histocompatibility complex class II with suppression of antigen-presenting ability and morphological changes toward granulocyte-like cells. Transcription of MC proteases and c-kit was markedly upregulated by GATA-1. Expression of IRF-4 and -8 was markedly suppressed, whereas PU.1 mRNA level was not affected by GATA-1. Chromatin immunoprecipitation assay showed that recruitment of PU.1 on the IRF-8 promoter was reduced in GATA-1-expressing DCs. These results indicate that GATA-1 suppresses PU.1 function but not PU.1 transcription. Thus, GATA-1 appears to determine cell fate by regulating several cell-specific transcription factors.
The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), epsilon-globin, and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1, and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus.
This study was designed to characterize the differential protein expression in the progeny of human liver cells surviving exposure to ionizing radiation. The progeny of irradiated cells were derived from a human liver cell line exposed to 0, 2, 4, or 6 Gy of (60)Co gamma-irradiation. Total protein of the cells was extracted by two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. In total, 42 differentially expressed proteins from the progeny of irradiated cells were screened, of which 17 were identified by matrix assistant laser desorption ion-top flight-mass spectrometry (MALDI-TOF-MS) analysis. There were 4 upregulated and 13 downregulated proteins detected. The upregulated expression of two proteins, mitochondrial heat-shock 60-kD protein (HSP60) and globin transcription factor 1 (GATA-1), was further confirmed by immunoblotting. Database search revealed that these differentially expressed proteins may function in cell cycle regulation, cytoskeleton maintenance, stress response, and tumor metastasis, indicating an effect of radiation-induced genomic instability (RIGI) in the progeny of irradiated cells. Analysis on functional roles of the screened proteins may provide insight into further mechanistic investigations underlying molecular events induced by RIGI.
Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3-mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis.
CD34(+) cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA.
In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61(+)GPA(-) megakaryocytic and a CD61(+)GPA(+) mixture of megakaryocytic and erythroid precursors from CD34(+) hematopoietic precursors at a pharmacological concentration (100 microg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61(-)GPA(+) erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61(-)GPA(+) erythroid precursors. In serum-containing cultures, only low numbers of CD61(+) or GPA(+) cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34(+) cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF.
These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease.
The authors report the first case of transient myeloproliferative disorder (TMD) in a neonate with trisomy 12. The clinical course consisted of respiratory distress since birth with probability of transient tachypnea of newborn, but routine investigation revealed total leukocyte count of 56000/microL with 91% blasts, which returned to normal spontaneously during the subsequent 3 weeks. GTG-banded karyotype from peripheral blood was done to detect any mutation, specifically trisomy 21, but the proband revealed trisomy 12 and denaturing polyacrylamide gel electrophoresis (PAGE) detected mutation in exon 2 of GATA1. The condition has been described in association with Down syndrome (trisomy 21) but never with trisomy 12. This case demonstrates the importance of knowing this entity so that it is not erroneously diagnosed as a leukemic process. This is extremely important because most cases of TMD resolve spontaneously within a few weeks to months and do not require treatment other than supportive measures. A search of the literature did not reveal any similar case.
Paclitaxel, an antitumoral drug, was used in a single dose (29 mg/kg i.p.) as an injury agent for inducing transient suppression of hematopoiesis in a murine experimental model during 10days. The aim of this study focuses on erythropoietin (EPO) receptor, GATA binding protein 1 (globin transcription factor 1) (GATA-1) and erythroid Krüppel-like factor (EKLF) expressions related to the apoptotic events triggered by paclitaxel in bone marrow and the subsequent in vivo erythropoietic recovery. Results showed a massive impairment of erythropoiesis early post paclitaxel administration (1-2 days), which involved induction of high Bax/Bcl-x(L) ratio, caspase-3 activation, disruptions of the medullar niche and cell death by both apoptosis and necrosis. EPO receptor over-expression was noticed from day 3 onwards. It prompted the subsequent up-regulations of GATA-1 and EKLF transcription factors as well as of the anti apoptotic protein Bcl-x(L), crucial proteins in driving erythropoiesis. This study suggests that EPO receptor recovery is necessary for the subsequent bone marrow ability to accomplish the erythroid program through the modulation of apoptotic and survival events after a single paclitaxel insult. These findings contribute to new insights into the molecular mechanisms involved during in vivo erythropoiesis post paclitaxel administration. Therefore, the detailed knowledge of the injury elicited by this drug on red blood cell production may have clinical relevance to explore new therapeutic approaches.
Primary myelofibrosis (PMF) belongs to the Philadelphia-negative myeloproliferative neoplasms and is a hematological disorder caused by abnormal function of the hematopoietic stem cells. The disease manifests itself with a plethora of alterations, including anemia, splenomegaly and extramedullary hematopoiesis. Its hallmarks are progressive marrow fibrosis and atypical megakaryocytic hyperplasia, two distinctive features used to clinically monitor disease progression. In an attempt to investigate the role of abnormal megakaryocytopoiesis in the pathogenesis of PMF, several transgenic mouse models have been generated. These models are based either on mutations that interfere with the extrinsic (thrombopoietin and its receptor, MPL) and intrinsic (the GATA1 transcription factor) control of normal megakaryocytopoiesis, or on known genetic lesions associated with the human disease. Here we provide an up-to-date review on the insights into the pathobiology of human PMF achieved by studying these animal models, with particular emphasis on results obtained with Gata1(low) mice.
GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1(Delta)(neo)(Delta)(HS)) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1(Delta)(neo)(Delta)(HS) megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes.
Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in long-term proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34(+) cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent.
Eosinophil/basophil (Eo/B) progenitor phenotype and function in cord blood (CB) are associated with atopic risk at birth and infant clinical outcomes. Molecular analyses of eosinophil-basophil differentiation events could identify clinically predictive biomarkers. To determine CB kinetic patterns of Eo/B lineage-associated gene expression (GATA-1, MBP1 and IL-5R alpha) after IL-5 stimulation, CB non-adherent mononuclear cells were isolated from random fresh and frozen samples and incubated in the presence of recombinant human interleukin-5. Some underwent CD34+ positive selection using magnetic cell separation. At various time-points, mRNA expression of GATA-1, MBP1 and IL-5R alpha (total transcripts) was determined utilizing multiplex quantitative polymerase chain reaction (Q-PCR). Relative expression levels of the IL-5R alpha soluble vs. transmembrane isoforms were also analyzed. Stimulation of the non-adherent mononuclear cells with IL-5 resulted in early up-regulation of GATA-1, peaking at 48 h, followed by decreasing expression and down-regulation by 96 h. The CD34+ enriched population demonstrated an equivalent expression pattern (r = 0.963, p = 0.0349). MBP1 mRNA expression [non-adherent mononuclear cells (NAMNCs) and CD34+ alike; r = 0.988, p = 0.012] was slowly up-regulated in response to IL-5, maximal at 96 h. Total IL-5R alpha expression appeared stable over the time-course, mediated by differential expression of the soluble and transmembrane isoforms (i.e., initial increase in the transmembrane contribution followed by a predominance of the soluble isoform by 48-72 h). Multiplex Q-PCR analysis of mRNA from CB demonstrates expression of critical eosinophil-basophil lineage-specific events that are consistent with current understanding of eosinophil differentiation and maturation. The non-adherent mononuclear cell population provides a surrogate signal for the CD34+ progenitor population.
Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.
To identify the regulatory sequences driving Gata1 expression in conventional dendritic cells (cDC).
The number and expression levels of Gata1, Gata1-target genes and hypersensitive site (HS) 2 (the eosinophil-specific enhancer)-driven green fluorescent protein (GFP) reporter of cDCs from mice lacking HS1 (the erythroid/megakaryocytic-specific enhancer, Gata1(low) mutation) and wild-type littermates, as well as the response to lipopolysaccharide of ex vivo-generated wild-type and Gata1(low) DCs were investigated.
cDC maturation was associated with bell-shaped changes in Gata1 expression that peaked in cDCs precursors from blood. The Gata1(low) mutation did not affect Gata1 expression in cDC precursors and these cells expressed the HS2-driven reporter, indicating that Gata1 expression is HS2-driven in these cells. By contrast, the Gata1(low) mutation reduced Gata1 expression in mature cDCs and these cells did not express GFP, indicating that mature cDCs express Gata1 driven by HS1. In blood, the number of cDC precursors expressing CD40/CD80 was reduced in Gata1(low) mice, while CD40(pos)/CD80(pos) cDC precursors from wild-type mice expressed the HS2-GFP reporter, suggesting that Gata1 expression in these cells is both HS1- and HS2-driven. In addition, the antigen and accessory molecules presentation process induced by lipopolysaccharide in ex vivo-generated wild-type DC was associated with increased acetylated histone 4 occupancy of HS1, while ex vivo-generated Gata1(low) cDCs failed to respond to lipopolysaccharide, suggesting that HS1 activation is required for cDC maturation.
These results identify a dynamic pattern of Gata1 regulation that switches from an HS1 to an HS2-dependent phase during the maturation of cDCs associated with the antigen-presentation process in the blood.
The present study aimed to identify optimal treatment intensity in children with mosaic Down syndrome (DS) and acute megakaryoblastic leukemia (AMKL). A retrospective review of AMKL patients was undertaken to identify mosaic DS children. Between November 1992 and November 2007, seven children were diagnosed as mosaic DS and AMKL. The median age at diagnosis was 29 months (range 4-34 months). Three patients had a past history of transient abnormal myelopoiesis. UPN1-4 were treated with intermediate-dose cytarabine and UPN4 received additional one course of high-dose cytarabine. All of these patients were remained in first CR. UPN5-7 were treated with high-dose cytarabine according to the AML99 protocol. UPN5 with GATA1 mutation suffered from acute pneumonia and pancreatitis and discontinued chemotherapy. UPN7 relapsed after cessation of chemotherapy and was rescued with allo-PBSCT. The cumulative doses of cytarabine were 3.5-10.65 g/m(2) in the UPN1-4 and 40.4-78.4 g/m(2) in the UPN5-7. The 8-year overall survival was 100% and the 8-year event-free survival 85.7%, respectively. Our retrospective study reveals that patients with mosaic DS and AMKL have a good prognosis. Reduction in intensity may work in patients with mosaic DS as well as with AML-DS.
Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.
Children with constitutional trisomy 21 or Down's syndrome (DS) are predisposed to develop myeloid leukemia (ML) at a young age. DS-ML is frequently preceded by transient leukemia (TL), a spontaneously resolving accumulation of blasts during the newborn period. Somatic mutations of GATA1 in the blasts of TL and DS-ML likely function as an initiating event. We hypothesized that the phenotypic difference between TL and DS-ML is due to a divergent functional repertoire of the leukemia-initiating cells. Using an NOD/SCID model, we found that cells initiating DS-ML engrafted, disseminated to distant bone marrow sites, and propagated the leukemic clone in secondary recipients. In contrast, TL cells lacked the ability to expand and to migrate, but were able to persist in the recipient bone marrow. We found some evidence of genomic progression with 1 of 9 DS-ML samples and none of 11 TL samples harboring a mutation of N-RAS. The findings of this pilot study provide evidence for the functional impact of second events underlying the transformation of TL into DS-ML and a needed experimental tool for the functional testing of these promoting events.
Efforts have been made to understand how erythroid differentiation is regulated, and recent discoveries have clarified that lineage-specific transcription factor networks are essential for proper differentiation of erythroid cells. The transcription factors GATA1 and GATA2 are involved in such networks that regulate erythroid gene expression. Importantly, expression of Gata1 and Gata2 genes is also under the control of such regulatory networks. The present review is focused on the mechanism of Gata1 and Gata2 gene regulation during erythropoiesis and the physiological significance of their dynamic regulation.
Gata1 and Gata2 genes are regulated by multiple transcription factors, including their own products GATA1 and GATA2. GATA1 and GATA2 recognize specific regulatory GATA motifs, and their expression levels change dynamically during erythroid differentiation, leading to diversified gene expression during erythropoiesis.
Strict regulations of the Gata1 and Gata2 genes are critical for proper lineage commitment and development of erythroid cells. It has been shown in transgenic mouse analyses that cis-acting GATA binding motifs are critical for the expression of Gata1 and Gata2 genes. Furthermore, expression of Gata1 and Gata2 genes along with a set of erythroid genes appeared to be regulated by GATA factor switching.
Children with trisomy 21/Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the related transient leukemia (DS-TL). The factors on human chromosome 21 (Hsa21) that confer this predisposing effect, especially in synergy with consistently mutated transcription factor GATA1 (GATA1s), remain poorly understood. Here, we investigated the role of Hsa21-encoded miR-125b-2, a microRNA (miRNA) overexpressed in DS-AMKL/TL, in hematopoiesis and leukemogenesis. We identified a function of miR-125b-2 in increasing proliferation and self-renewal of human and mouse megakaryocytic progenitors (MPs) and megakaryocytic/erythroid progenitors (MEPs). miR-125b-2 overexpression did not affect megakaryocytic and erythroid differentiation, but severely perturbed myeloid differentiation. The proproliferative effect of miR-125b-2 on MEPs accentuated the Gata1s mutation, whereas growth of DS-AMKL/TL cells was impaired upon miR-125b repression, suggesting synergism during leukemic transformation in GATA1s-mutated DS-AMKL/TL. Integrative transcriptome analysis of hematopoietic cells upon modulation of miR-125b expression levels uncovered a set of miR-125b target genes, including DICER1 and ST18 as direct targets. Gene Set Enrichment Analysis revealed that this target gene set is down-regulated in DS-AMKL patients highly expressing miR-125b. Thus, we propose miR-125b-2 as a positive regulator of megakaryopoiesis and an oncomiR involved in the pathogenesis of trisomy 21-associated megakaryoblastic leukemia.
To identify genetic factors that interact with social environments to impact human health, we used a bioinformatic strategy that couples expression array-based detection of environmentally responsive transcription factors with in silico discovery of regulatory polymorphisms to predict genetic loci that modulate transcriptional responses to stressful environments. Tests of one predicted interaction locus in the human IL6 promoter (SNP rs1800795) verified that it modulates transcriptional response to beta-adrenergic activation of the GATA1 transcription factor in vitro. In vivo validation studies confirmed links between adverse social conditions and increased transcription of GATA1 target genes in primary neural, immune, and cancer cells. Epidemiologic analyses verified the health significance of those molecular interactions by documenting increased 10-year mortality risk associated with late-life depressive symptoms that occurred solely for homozygous carriers of the GATA1-sensitive G allele of rs1800795. Gating of depression-related mortality risk by IL6 genotype pertained only to inflammation-related causes of death and was associated with increased chronic inflammation as indexed by plasma C-reactive protein. Computational modeling of molecular interactions, in vitro biochemical analyses, in vivo animal modeling, and human molecular epidemiologic analyses thus converge in identifying beta-adrenergic activation of GATA1 as a molecular pathway by which social adversity can alter human health risk selectively depending on individual genetic status at the IL6 locus.
Trisomy of human chromosome 21 (Hsa21) results in Down syndrome (DS), a disorder that affects many aspects of physiology, including hematopoiesis. DS children have greatly increased rates of acute lymphoblastic leukemia and acute megakaryoblastic leukemia (AMKL); DS newborns present with transient myeloproliferative disorder (TMD), a preleukemic form of AMKL. TMD and DS-AMKL almost always carry an acquired mutation in GATA1 resulting in exclusive synthesis of a truncated protein (GATA1s), suggesting that both trisomy 21 and GATA1 mutations are required for leukemogenesis. To gain further understanding of how Hsa21 contributes to hematopoietic abnormalities, we examined the Tc1 mouse model of DS, which carries an almost complete freely segregating copy of Hsa21, and is the most complete model of DS available. We show that although Tc1 mice do not develop leukemia, they have macrocytic anemia and increased extramedullary hematopoiesis. Introduction of GATA1s into Tc1 mice resulted in a synergistic increase in megakaryopoiesis, but did not result in leukemia or a TMD-like phenotype, demonstrating that GATA1s and trisomy of approximately 80% of Hsa21 perturb megakaryopoiesis but are insufficient to induce leukemia.
Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS-3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS-25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS-25/6 or mHS-3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS-3.5 alone is sufficient for megakaryocytic development, mHS-3.5 and mHS-25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.
The functions of parathyroid hormone-related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock-in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp(Delta/Delta)) was developed. Pthrp(Delta/Delta) mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, beta-catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/Ink4a, P21, GSK-3beta, Il-6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down-regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment.
The SCL (TAL1) transcription factor is a critical regulator of haematopoiesis and its expression is tightly controlled by multiple cis-acting regulatory elements. To elaborate further the DNA elements which control its regulation, we used genomic tiling microarrays covering 256 kb of the human SCL locus to perform a concerted analysis of chromatin structure and binding of regulatory proteins in human haematopoietic cell lines. This approach allowed us to characterise further or redefine known human SCL regulatory elements and led to the identification of six novel elements with putative regulatory function both up and downstream of the SCL gene. They bind a number of haematopoietic transcription factors (GATA1, E2A LMO2, SCL, LDB1), CTCF or components of the transcriptional machinery and are associated with relevant histone modifications, accessible chromatin and low nucleosomal density. Functional characterisation shows that these novel elements are able to enhance or repress SCL promoter activity, have endogenous promoter function or enhancer-blocking insulator function. Our analysis opens up several areas for further investigation and adds new layers of complexity to our understanding of the regulation of SCL expression.
Eosinophils contribute to the pathophysiology of allergic and infectious diseases, albeit their molecular functions remain unknown. Mature eosinophils are identified by their unique morphology and staining characteristics. However, it is difficult to fractionate living eosinophils by flow cytometry because these granulocytes express multiple cell surface markers that are shared by other cells of hematopoietic or non-hematopoietic origin. In this study, we describe a flow cytometry-based method to enumerate and fractionate eosinophils by developmental stages. To fractionate these cell types, we used transgenic mouse lines that express fluorescent proteins under control of the Gata1 gene hematopoietic regulatory region (Gata1-HRD), which is exclusively active in Gata1-expressing hematopoietic cells, including eosinophils. As expected, mature eosinophils were highly enriched in the fluorescent reporter-expressing subfraction of bone marrow myeloid cells that were negatively selected by using multiple antibodies against B220, CD4, CD8, Ter119, c-Kit and CD71. Cytochemical analyses of flow-sorted cells identified the cells in this fraction as eosinophils harboring eosinophilic granules. Additionally, expression of eosinophil-specific genes, for instance eosinophil enzymes and the IL-5 receptor alpha gene, were specifically detected in this fraction. The expression of these eosinophil-specific genes increased as the cells differentiated. This method for enrichment of bone marrow eosinophils is applicable to fractionation of eosinophils and bronchoalveolar lavage fluid from transgenic mice with atopic asthma. Thus, both pathological and developmental stages of eosinophils are efficiently fractionated by this flow cytometry-based method using Gata1-HRD transgenic reporter mice. This study, therefore, proposes a useful means to study the experimental allergic and inflammatory systems.
One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.
The X-linked Gata1(low) mutation in mice induces strain-restricted myeloproliferative disorders characterized by extramedullary hematopoiesis in spleen (CD1 and DBA/2) and liver (CD1 only). To assess the role of the microenvironment in establishing this myeloproliferative trait, progenitor cell compartments of spleen and marrow from wild-type and Gata1(low) mice were compared. Phenotype and clonal assay of non-fractionated cells indicated that Gata1(low) mice contain progenitor cell numbers 4-fold lower and 10-fold higher than normal in marrow and spleen, respectively. However, progenitor cells prospectively isolated from spleen, but not from marrow, of Gata1(low) mice expressed colony-forming function in vitro. Therefore, calculation of cloning activity of purified cells demonstrated that the total number of Gata1(low) progenitor cells was 10- to 100-fold lower than normal in marrow and >1,000 times higher than normal in spleen. This observation indicates that Gata1(low) hematopoiesis is favored by the spleen and is in agreement with our previous report that removal of this organ induces wild-type hematopoiesis in heterozygous Gata1(low/+) females (Migliaccio et al., 2009, Blood 114:2107). To clarify if rescue of wild-type hematopoiesis by splenectomy prevented extramedullary hematopoiesis in liver, marrow cytokine expression profile and liver histopathology of splenectomized Gata1(low/+) females were investigated. After splenectomy, the marrow expression levels of TGF-beta, VEGF, osteocalcin, PDGF-alpha, and SDF-1 remained abnormally high while Gata1(low) hematopoiesis was detectable in liver of both CD1 and DBA/2 mutants. Therefore, in the absence of the spleen, Gata1(low) hematopoiesis is supported by the liver suggesting that treatment of myelofibrosis in these animals requires the rescue of both stem cell and microenvironmental functions.
In this paper, we characterized expression of GATA1 and FLI1 gene promoters in thrombocytes of zebrafish transgenic lines, G1-GM2 and TG(fli1:EGFP)y1 that carry transgenes of GATA1 and FLI1 gene promoters driving GFP. We found two discrete populations of thrombocytes verified by morphology, labeled with GFP in both G1-GM2 and TG(fli1:EGFP)y1 lines: (1) the more intensely labeled GFP+ thrombocyte, and (2) the less intensely labeled GFP+ thrombocytes. The more intensely labeled GFP+ thrombocyte in G1-GM2 line and the less intensely labeled GFP+ thrombocytes in the TG(fli1:EGFP)y1 line corresponded to young thrombocytes. These results showed that young thrombocytes have higher GATA1 promoter activity, while mature thrombocytes have more FLI1 gene promoter transcription. This finding suggests that there is a gradual loss of GATA1 and gain of FLI1 expression as the thrombocytes mature, and this overexpression of FLI1 may help maintain the thrombocyte lineage. Furthermore, the presence of transcriptional factors similar to those found in megakaryocytes raises the possibility that vertebrate thrombocytes may be the forerunners of mammalian megakaryocytes and, therefore, could serve as a model to study megakaryocyte maturation.
Children with Down syndrome (DS) have a unique form of acute megakaryocytic leukemia (AMKL) characterized by the presence of mutations in the GATA1 gene leading to increased chemosensitivity and a favorable outcome. We describe an 8-month-old male with DS who was diagnosed with AMKL without a mutation in the GATA1 gene. The patient was treated according to the DS-AML-regimen but his disease progressed and he succumbed 9 months later. This rare case of DS AMKL without a GATA1 mutation with an unfavorable outcome suggests that GATA1 testing may play a useful role in initial stratification.
Expression of survivin, a member of the inhibitor of apoptosis protein family, is elevated in human cancers and considered as a new therapeutic target. Mechanism upregulating survivin expression in tumour cells is poorly understood. In this study, we show that breast cancer patients harbouring a polymorphism G235A in the survivin promoter present a higher level of survivin expression. This polymorphism creates a binding site for the transcription factor GATA-1 inducing a second GATA-1-binding site in survivin promoter. At the mRNA level, GATA-1 was present in breast carcinomas and adjacent normal tissues, whereas the protein was only detected in carcinomas by western blot and immunohistochemistry. Transfection of wild-type and different constitutively active GATA-1 mutants (serine 26, 178 or 310) showed that only phospho-serine 26 GATA-1 was able to increase survivin expression. This increase was higher in G235A than in G235G cell lines. Phospho-serine 26 GATA-1 bound directly survivin promoter, with a stronger interaction in G235A than in G235G polymorphism indicating that both GATA-1-binding sites are functional. These data identify GATA-1 as a key feature in tumour aggressiveness by enhancing survivin expression and delineate its targeting as a possible new therapeutic strategy in breast carcinomas.
Phenotypic rescue experiments have been commonly used in zebrafish since it is convenient to study the causality of mutant phenotypes just by injecting mRNA into embryos. However, this strategy is only effective for phenotypes at early embryonic stages due to mRNA instability. For later developmental stages, DNA constructs are used to express exogenous genes, while it is usually ineffective owing to the problem of mosaicism. This study attempted to solve the problem by using Tol2-mediated transgenesis. As a model case, we used vlad tepes (vlt), a zebrafish gata1 mutant, whose phenotypes have never been able to be rescued at later stages by transient rescue experiments. Blood cell-specific transgenic expression of gata1 was driven by its own promoter/enhancer elements. The co-injection of a Tol2-donor plasmid containing gata1 cDNA and transposase mRNA efficiently rescued the bloodless phenotypes of vlt even in day 12 larvae when definitive erythropoiesis took place with primitive erythropoiesis. This Tol2-mediated rescue is therefore considered to be a quick and easy method for analyzing the mutant phenotypes in zebrafish.
Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fog(ki/ki)) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fog(ki/ki) MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fog(ki/ki) MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.
GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1 independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.
Mutations of GATA1, leading to aberrant expression of a truncated form of GATA1 (called GATA1s), are present in transient leukaemia (TL) in neonates with Down syndrome. Using these molecular markers of TL, we investigated the growth and differentiation potential of TL blasts in the presence of hematopoietic growth factors (HGFs). Interleukin-3, stem cell factor and granulocyte-macrophage colony-stimulating factor potently stimulated the growth of TL blast progenitors and induced differentiation towards basophil/mast cell lineages, whereas thrombopoietin induced differentiation towards megakaryocytes. GATA1s was expressed in TL blasts in all five patients examined but was down-regulated during differentiation induced by these HGFs, while full-length GATA1 was not expressed throughout the culture. GATA1 mutations were detected in TL blasts in four patients, including one patient with two distinct mutations. The cells of this patient exhibited identical and only mutated sequences both before and after culture with HGFs, confirming the leukemic cell origin of these differentiated cells. Erythroid differentiation of TL blasts was not evident with any HGFs. These data indicate that TL blasts have the potential to grow and differentiate towards particular hematopoietic lineages in the presence of specific HGFs and that the down-regulation of GATA1s might be involved in blast cell differentiation.
Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs.
Down syndrome (DS), which occurs once in every 800 births, is associated with a trisomy on locus 21. Among the many aberrations caused by DS, including shortened stature and distorted facies, are several blood dyscrasias, including childhood leukemias-namely, acute myeloid leukemia (AML) and acute lymphoblastic, or lymphocytic, leukemia (ALL). One focus of the diagnosis of ALL is to distinguish it from AML.The benefits of immunophenotyping extend to treatment as well. ALL is associated with an inherited trisomy 21 in DS children (ALL-DS) and with acquired trisomies, +21, 8, and 13, in non-DS children (ALL-NDS). The differences in treatment, outcome, and prognosis between ALL-DS and ALL-NDS can be attributed to the interaction of their respective trisomies with several genetic mutations, including one on the GATA1 growth factor transcription gene. Other mutations are the gene fusion at TEL/AML1, and a new mutation found, which labels the Janus Kinase gene or JAK2 as on oncogenic precursor, which when associated with the B-cell precursor gene or BCP is highly leukomogenic. The treatments for the 2 groups have been based on quality of risk, with ALL-DS children having the highest risk along with the poorest prognosis, but alterations in medication regimens have brought treatment outcomes to near equality. It is worthwhile to study the trisomy 21 because in the future it may provide an understanding of all blood dyscrasias.
In vitro studies have suggested that connexin43 (cx43) expression is of particular importance during establishment and regeneration of the mammalian hematopoietic system. However, little is known about its in vivo functions during hematopoiesis due to the embryonic lethality of mammalian knockout models. In this study, we observed that zebrafish cx43 is not only expressed in the eyes, cerebellum, heart, and vasculature, but also expressed, albeit at low levels, in intermediate cell mass (ICM, the primitive hematopoietic site). Knockdown of cx43 leads to vacuolization in the wedge of the ICM and an apparent reduction in the number of circulating blood cells, but does not affect their cellular morphology. Whole-mount in situ hybridization analysis revealed that the hemangioblastic marker flk-1 and the primitive hematopoietic markers lmo2 and scl are basically maintained at normal levels in cx43 morphant embryos at 12-13 h postfertilization (hpf) compared with the con-MO injected embryos. However, subsequent expression of the definitive hematopoietic stem cell (HSC) marker c-myb was severely downregulated in the ventral wall of the dorsal aorta of cx43-depleted embryos at 36 hpf. Furthermore, we confirmed this phenotype by injection of cx43-MO into Tg(gata1:EGFP) embryos. Together, our results show that cx43 contributes to late primitive and definitive hematopoiesis in zebrafish embryos.
The transcriptional co-factor Friend of GATA1 (FOG-1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N-terminus. To test the importance of FOG-1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG-1/NuRD interaction (FOG-1(R3K5A)). Homozygous FOG-1(R3K5A) mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG-1(R3K5A/R3K5A) megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG-1/NuRD interaction is required for the earlier described 'GATA Switch'. In addition, ablation of FOG-1/NuRD interaction led to inappropriate expression of mast cell and eosinophil-specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG-1(R3K5A/R3K5A) megakaryocytes, suggesting that FOG-1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG-1/NuRD interaction for the re-enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.
Down syndrome is characterized by multiple phenotypic manifestations associated with trisomy of chromosome 21. The transient myeloproliferative disorder and acute megakaryocytic leukemia associated with Down syndrome are uniquely associated with mutations in the transcription factor GATA1; however, the identity of trisomic genes on chromosome 21 that predispose to these hematologic disorders remains unknown. Using a loss-of-function allele, we show that specific reduction to functional disomy of the Erg gene corrects the pathologic and hematologic features of myeloproliferation in the Ts(17(16))65Dn mouse model of Down syndrome, including megakaryocytosis and progenitor cell expansion. Our data provide genetic evidence establishing the need for Erg trisomy for myeloproliferation in Ts(17(16))65Dn mice and imply that increased ERG gene dosage may be a key consequence of trisomy 21 that can predispose to malignant hematologic disorders in Down syndrome.
The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of leukemia patients. Expression of a CALM-AF10 transgene results in leukemia, with prolonged latency and incomplete penetrance, suggesting that additional events are necessary for leukemic transformation. CALM-AF10 mice infected with the MOL4070LTR retrovirus developed acute leukemia, and ligation-mediated polymerase chain reaction was used to identify retroviral insertions at 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. A total of 26% (11 of 42) of the mice had retroviral integrations near Zeb2, a transcriptional corepressor leading to overexpression of the Zeb2-transcript. A total of 91% (10 of 11) of mice with Zeb2 insertions developed B-lineage acute lymphoblastic leukemia, suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. More than half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2, and Ras pathway mutations. We searched for Nras, Kras, and Ptpn11 point mutations in the CALM-AF10 leukemic mice. Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10.
Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly, GFI1B expression is tightly regulated during erythropoiesis, but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2, a high-mobility group HMG protein, as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and, to a lesser extent, of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly, knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation.
Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G(1) phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G(1) phase of the cell cycle, the period during which the decision to differentiate is made.
The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data provide insights into GATA-1-mediated gene regulation in vivo.
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.
GATA transcription factors interact with FOG proteins to regulate tissue development by activating and repressing transcription. FOG-1 (ZFPM1), a co-factor for the haematopoietic factor GATA-1, binds to the NuRD co-repressor complex through a conserved N-terminal motif. Surprisingly, we detected NuRD components at both repressed and active GATA-1/FOG-1 target genes in vivo. In addition, while NuRD is required for transcriptional repression in certain contexts, we show a direct requirement of NuRD also for FOG-1-dependent transcriptional activation. Mice in which the FOG-1/NuRD interaction is disrupted display defects similar to germline mutations in the Gata1 and Fog1 genes, including anaemia and macrothrombocytopaenia. Gene expression analysis in primary mutant erythroid cells and megakaryocytes (MKs) revealed an essential function for NuRD during both the repression and activation of select GATA-1/FOG-1 target genes. These results show that NuRD is a critical co-factor for FOG-1 and underscore the versatile use of NuRD by lineage-specific transcription factors to activate and repress gene transcription in the appropriate cellular and genetic context.
Irradiation or chemotherapy that suspend normal spermatogenesis is commonly used to treat various cancers. Fortunately, spermatogenesis in many cases can be restored after such treatments but knowledge is limited about the re-initiation process. Earlier studies have described the cellular changes that happen during recovery from irradiation by means of histology. We have earlier generated gene expression profiles during induction of spermatogenesis in mouse postnatal developing testes and found a correlation between profiles and the expressing cell types. The aim of the present work was to utilize the link between expression profile and cell types to follow the cellular changes that occur during post-irradiation recovery of spermatogenesis in order to describe recovery by means of gene expression.
Adult mouse testes were subjected to irradiation with 1 Gy or a fractionated radiation of two times 1 Gy. Testes were sampled every third or fourth day to follow the recovery of spermatogenesis and gene expression profiles generated by means of differential display RT-PCR. In situ hybridization was in addition performed to verify cell-type specific gene expression patterns.
Irradiation of mice testis created a gap in spermatogenesis, which was initiated by loss of A1 to B-spermatogonia and lasted for approximately 10 days. Irradiation with 2 times 1 Gy showed a more pronounced effect on germ cell elimination than with 1 Gy, but spermatogenesis was in both cases completely reconstituted 42 days after irradiation. Comparison of expression profiles indicated that the cellular reconstitution appeared equivalent to what is observed during induction of normal spermatogenesis.
The data indicates that recovery of spermatogenesis can be monitored by means of gene expression, which could aid in designing radiation treatment regimes for cancer patients leading to better restoration of spermatogenesis.
We describe a patient, who developed during the first week of life petechiae and hematomas caused by severe thrombocytopenia and gastrointestinal bleeding due to multiple small gastric hemangiomata. Bone marrow examination showed hypermegakaryocytosis and dysmegakaryopoiesis. Alloimmune thrombocytopenia was excluded. Only 3 y later, platelet counts normalized and bleedings disappeared but small skin hemangiomata remained. Electron microscopy showed enlarged round platelets with a paucity of alpha granules similar as in GATA1-deficient patients but no GATA1 mutation was found. Immunoblot analysis showed a strong interaction between patient Igs and recombinant GATA1, GATA2, and the N finger (Nf) of GATA1. The lymphocyte transformation test with recombinant GATA1Nf was positive. In vitro culturing of normal CD34 cells with purified patient Igs showed a decreased number of megakaryocyte colonies but an increased overall size of the colonies compared with control Igs. Mice injected with patient Igs showed a reduced platelet count compared with mice injected with control Igs. Thrombopoiesis was also reduced after injection of patient Igs in transgenic zebrafish compared with control Igs. In conclusion, this study is the first report of an anti-GATA1 autoantibody leading to severe thrombocytopenia and gastrointestinal bleeding from multiple pinpoint hemangiomata.
GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4-6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5(tm)(2)(Eem)), and we show that although Gata5(-/-) mice are viable, Gata4(+/-)5(-/-) mutants die at mid-gestation and exhibit profound cardiovascular defects, including abnormalities of cardiomyocyte proliferation and cardiac chamber maturation. These results demonstrate functional redundancy between Gata4 and Gata5 during cardiac development and implicate Gata5 as a candidate modifier gene for congenital heart disease.
The transcription factor GATA1 regulates an extensive program of gene activation and repression during erythroid development. However, the associated mechanisms, including the contributions of distal versus proximal cis-regulatory modules, co-occupancy with other transcription factors, and the effects of histone modifications, are poorly understood. We studied these problems genome-wide in a Gata1 knockout erythroblast cell line that undergoes GATA1-dependent terminal maturation, identifying 2616 GATA1-responsive genes and 15,360 GATA1-occupied DNA segments after restoration of GATA1. Virtually all occupied DNA segments have high levels of H3K4 monomethylation and low levels of H3K27me3 around the canonical GATA binding motif, regardless of whether the nearby gene is induced or repressed. Induced genes tend to be bound by GATA1 close to the transcription start site (most frequently in the first intron), have multiple GATA1-occupied segments that are also bound by TAL1, and show evolutionary constraint on the GATA1-binding site motif. In contrast, repressed genes are further away from GATA1-occupied segments, and a subset shows reduced TAL1 occupancy and increased H3K27me3 at the transcription start site. Our data expand the repertoire of GATA1 action in erythropoiesis by defining a new cohort of target genes and determining the spatial distribution of cis-regulatory modules throughout the genome. In addition, we begin to establish functional criteria and mechanisms that distinguish GATA1 activation from repression at specific target genes. More broadly, these studies illustrate how a "master regulator" transcription factor coordinates tissue differentiation through a panoply of DNA and protein interactions.
GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene.
GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, our data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF.
The transcription factor Gata1 is required for the development of erythrocytes and megakaryocytes. Previous studies with a complementation rescue approach showed that the zinc finger domains are required for both primitive and definitive hematopoiesis. Here we report a novel zebrafish gata1 mutant with an N-ethyl-N-nitrosourea-induced point mutation in the C-finger (gata1(T301K)). The Gata1 protein with this mutation bound to its DNA target sequence with reduced affinity and transactivated inefficiently in a reporter assay. gata1(T301K/T301K) fish had a decreased number of erythrocytes during primitive hematopoiesis but normal adult hematopoiesis. We crossed the gata1(T301K/T301K) fish with those carrying the R339X mutation, also known as vlad tepes (vlt), which abolishes DNA binding and transactivation activities. As we reported previously, gata1(vlt/vlt) embryos were "bloodless" and died approximately 11 to 15 days after fertilization. Interestingly, the gata1(T301K/vlt) fish had nearly a complete block of primitive hematopoiesis, but they resumed hematopoiesis between 7 and 14 days after fertilization and grew to phenotypically normal fish with normal adult hematopoiesis. Our findings suggest that the impact of Gata1 on hematopoiesis correlates with its DNA-binding ability and that primitive hematopoiesis is more sensitive to reduction in Gata1 function than definitive hematopoiesis.
Hematopoietic transcription factors GATA-1 and PU.1 bind each other on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that coexpress GATA-1 and PU.1 are blocked at the blast stage but respond to molecular removal (downregulation) of PU.1 or addition (upregulation) of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells, we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and core-binding factor, beta subunit (Cbfb), which encode two key hematopoietic transcription factors. Inhibition of GATA-1 by small interfering RNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Significant derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we show that either activation of PU.1 or inhibition of GATA-1 efficiently reverses the transcriptional block imposed by GATA-1 and leads to the activation of a myeloid transcriptional program directed by PU.1.
Splenectomized mice express progressively increased numbers of platelets in the blood and reduced numbers of megakaryocytes in the marrow with age. The megakaryocytes in the marrow of these animals express reduced levels of Gata1, a transcription factor necessary for their maturation. In addition, the marrow from these animals expresses greater levels of cytokines (TGF-beta, PDGF-alpha, and VEGF) known to be produced at high levels by megakaryocytes expressing reduced levels of Gata1. This high level of cytokine expression is in turn associated with active osteoblast proliferation localized to areas of the femur, where megakaryocytes expressing reduced Gata1 levels are also found. These results confirm the role of megakaryocytes as regulator of bone formation in mice and suggest that a cross-talk between the spleen and marrow may regulate the total numbers of hemopoietic niches present in an animal.
To understand the mechanism in lymphoid development, it is critical to identify developmental intermediates downstream of hematopoietic stem cells, including cells responsible for seeding the thymus. Although early studies showed that hematopoietic stem cells choose either the myeloid-erythroid or the lymphoid pathway, recent data suggest that myelo-lymphoid precursors lacking erythroid potential exist in early hematopoiesis before cells are fully committed to the lymphoid lineage. We here summarize the phenotype and functional properties of such progenitors and the current developmental map for the lymphoid lineage.
The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV.
Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.
Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019).
The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.
DNA sequence motifs and epigenetic modifications contribute to specific binding by a transcription factor, but the extent to which each feature determines occupancy in vivo is poorly understood. We addressed this question in erythroid cells by identifying DNA segments occupied by GATA1 and measuring the level of trimethylation of histone H3 lysine 27 (H3K27me3) and monomethylation of H3 lysine 4 (H3K4me1) along a 66 Mb region of mouse chromosome 7. While 91% of the GATA1-occupied segments contain the consensus binding-site motif WGATAR, only approximately 0.7% of DNA segments with such a motif are occupied. Using a discriminative motif enumeration method, we identified additional motifs predictive of occupancy given the presence of WGATAR. The specific motif variant AGATAA and occurrence of multiple WGATAR motifs are both strong discriminators. Combining motifs to pair a WGATAR motif with a binding site motif for GATA1, EKLF or SP1 improves discriminative power. Epigenetic modifications are also strong determinants, with the factor-bound segments highly enriched for H3K4me1 and depleted of H3K27me3. Combining primary sequence and epigenetic determinants captures 52% of the GATA1-occupied DNA segments and substantially increases the specificity, to one out of seven segments with the required motif combination and epigenetic signals being bound.
Histone deacetylases (HDACs) are globally implicated in the growth and differentiation of mammalian cells; however, relatively little is known about their specific roles in hematopoiesis. In this study, we investigated the expression of HDACs in human hematopoietic cells and their functions during hematopoiesis. The expression of HDACs was very low in hematopoietic progenitor cells, which was accompanied by histone hyperacetylation. HDACs were detectable in more differentiated progenitors and erythroid precursors but down-regulated in mature myeloid cells especially granulocytes. In contrast, acute myeloid leukemias showed HDAC overexpression and histone hypoacetylation. Transcription of the HDAC1 gene was repressed by CCAAT/enhancer binding proteins during myeloid differentiation, and activated by GATA-1 during erythro-megakaryocytic differentiation. Small interfering RNA-mediated knockdown of HDAC1 enhanced myeloid differentiation in immature hematopoietic cell lines and perturbed erythroid differentiation in progenitor cells. Myeloid but not erythro-megakaryocytic differentiation was blocked in mice transplanted with HDAC1-overexpressing hematopoietic progenitor cells. These findings suggest that HDAC is not merely an auxiliary factor of genetic elements but plays a direct role in the cell fate decision of hematopoietic progenitors.
The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.
Two major pathways of human myeloid dendritic cell (DC) subset differentiation have previously been delineated. Langerhans cells (LCs) reside in epithelia in the steady state, whereas monocytes can provide dendritic cells (DCs) on demand in response to inflammatory signals. Both DC subset pathways arise from shared CD14+ monocyte precursors, which in turn develop from myeloid committed progenitor cells. However, the underlying hematopoietic mechanisms still remain poorly defined. Here, we demonstrate that the vitamin D(3) receptor (VDR) is induced by transforming growth factor beta1 during LC lineage commitment and exerts a positive role during LC generation. In contrast, VDR is repressed during interleukin-4 (IL-4)-dependent monocyte-derived DC (moDC) differentiation. We identified GATA-1 as a repressor of VDR. GATA-1 is induced by IL-4 in moDCs. Forced inducible expression of GATA-1 mimics IL-4 in redirecting moDC differentiation and vice versa, GATA-1 knockdown arrests moDC differentiation at the monocyte stage. Moreover, ectopic GATA-1 expression stabilizes the moDC phenotype under monocyte-promoting conditions in the presence of vitamin D3 (VD3). In summary, human myeloid DC subset differentiation is inversely regulated by GATA-1 and VDR. GATA-1 mediates the repression of VDR and enables IL-4-dependent moDC differentiation. Conversely, VDR is induced downstream of transforming growth factor beta1 and is functionally involved in promoting LC differentiation.
The patterns of malignancies in Down syndrome (DS) are unique and highlight the relationship between chromosome 21 and cancer. DS children have a approximately 10- to 20-fold higher risk for developing acute lymphoblastic leukemia and acute myeloid leukemia (AML), as compared with non-DS children, although they do not have a uniformly increased risk of developing solid tumors. DS children with acute lymphoblastic leukemia frequently experience higher levels of treatment-related toxicity and inferior event-free survival rates, as compared with non-DS children. DS children also develop AML with unique features and have a 500-fold increased risk of developing the AML subtype, acute megakaryocytic leukemia (AMkL; M7). Nearly 10% of DS newborns are diagnosed with a variant of AMkL, the transient myeloproliferative disorder, which can resolve spontaneously without treatment; event-free survival rates for DS patients with AMkL ranges from 80% to 100%, in comparison with <30% for non-DS children with AMkL. In addition, somatic mutations of the GATA1 gene have been detected in nearly all DS TMD and AMkL cases and not in leukemia cases in non-DS children. GATA1 mutations are key factors linked to both leukemogenesis and the high cure rates of DS AMkL patients. Identifying the mechanisms that account for the high event-free survival rates of DS AMkL patients may ultimately improve AML treatment as well. Examining leukemogenesis in DS children may identify factors linked to the general development of childhood leukemia and lead to potential new therapeutic strategies to fight this disease.
Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.
Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.
Two GATA1-related leukemias have been described: one is an erythroleukemia that develops in mice as a consequence of diminished expression of wild-type GATA1, whereas the other is an acute megakaryoblastic leukemia (AMKL) that arises in Down syndrome children as a consequence of somatic N-terminal truncation (DeltaNT) of GATA1. We discovered that mice expressing the shortened GATA1 protein (DeltaNTR mice) phenocopies the human transient myeloproliferative disorder (TMD) that precedes AMKL in Down syndrome children. In perinatal livers of the DeltaNTR mutant mice, immature megakaryocytes accumulate massively, and this fraction contains cells that form hyperproliferative megakaryocytic colonies. Furthermore, showing good agreement with the clinical course of TMD in humans, DeltaNTR mutant mice undergo spontaneous resolution from the massive megakaryocyte accumulation concomitant with the switch of hematopoietic microenvironment from liver to bone marrow/spleen. These results thus demonstrate that expression of the GATA1/Gata1 N-terminal deletion mutant per se induces hyperproliferative fetal megakaryopoiesis. This mouse model serves as an important means to clarify how impaired GATA1 function contributes to the multi-step leukemogenesis.
Erythropoietic and megakaryocytic programs are directed by the transcription factor GATA1. Friend of GATA1 (FOG1), a protein interaction partner of GATA1, is critical for GATA1 function in multiple contexts. Previous work has shown that FOG1 recruits two multi-protein complexes, the nucleosome remodeling domain (NuRD) complex and a C-terminal binding protein (CTBP)-containing complex, into association with GATA1 to mediate activation and repression of target genes. To elucidate mechanisms that might differentially regulate the association of FOG1, as well as GATA1, with these two complexes, we characterized a previously unrecognized translational isoform of FOG1. We found that an N-terminally truncated version of FOG1 is produced from an internal ATG and that this isoform, designated FOG1S, lacks the nucleosome remodeling domain-binding domain, altering the complexes with which it interacts. Both isoforms interact with the C-terminal binding protein complex, which we show also contains lysine-specific demethylase 1 (LSD1). FOG1S is preferentially excluded from the nucleus by unknown mechanisms. These data reveal two novel mechanisms for the regulation of GATA1 interaction with FOG1-dependent protein complexes through the production of two translational isoforms with differential interaction profiles and independent nuclear localization controls.
Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-beta-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-beta-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is compromised.
Several anticancer drugs with diverse chemical structures can induce differentiation of cancer cells. This study was undertaken to explore the potential contribution of caspase-3 to pharmacologically-induced differentiation of K562 cells.
We assessed differentiation by measuring the expression of glycophorin A and haemoglobin synthesis in K562 cells treated with low concentrations of doxorubicin, hydroxyurea, cytosine arabinoside, cisplatin and haemin. Caspase-3 activation, mitochondrial membrane potential dissipation and viability were assessed by FACS. GATA-1-binding activity was evaluated by EMSA.
Treatment of K562 cells with low concentrations of the tested drugs activated caspase-3 but did not trigger detectable apoptosis. Instead, elevated levels of haemoglobin-positive and glycophorin A/caspase-3-double-positive cells were observed, suggesting involvement of caspase-3 in drug-induced differentiation. Inhibition of caspase-3 activity significantly reduced the ability of K562 cells to execute the differentiation programme. Mitochondrial membrane potential dissipation was observed, indicating involvement of the mitochondrial pathway. Binding activity of GATA-1, transcription factor responsible for differentiation and cell survival, was not diminished by increased caspase-3 activity during drug-stimulated differentiation.
Our results could explain how anticancer drugs, with diverse structures and modes of action, can stimulate erythroid differentiation in leukaemic cells with appropriate genetic backgrounds. Our findings imply that some similarities exist between pharmacologically-induced differentiation of erythroleukaemic cells and normal erythropoiesis, both involving caspase-3 activation at high levels of anti-apoptotic protein Bcl-X(L) and chaperone protein Hsp70 (heat shock protein 70). Therefore, the functions of caspase-3, unrelated to cell death, can be extended to pharmacologically-induced differentiation of some cancer cells.
GATA-2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Dysregulation of GATA-2 is a hallmark of acute megakaryoblastic leukemia in children with Down syndrome, a malignancy that is defined by the combination of trisomy 21 and a GATA1 mutation. Here, we show that GATA-2 is required for normal megakaryocyte development as well as aberrant megakaryopoiesis in Gata1 mutant cells. Furthermore, we demonstrate that GATA-2 indirectly controls cell cycle progression in GATA-1-deficient megakaryocytes. Genome-wide microarray analysis and chromatin immunoprecipitation studies revealed that GATA-2 regulates a wide set of genes, including cell cycle regulators and megakaryocyte-specific genes. Surprisingly, GATA-2 also negatively regulates the expression of crucial myeloid transcription factors, such as Sfpi1 and Cebpa. In the absence of GATA-1, GATA-2 prevents induction of a latent myeloid gene expression program. Thus, GATA-2 contributes to cell cycle progression and the maintenance of megakaryocyte identity of GATA-1-deficient cells, including GATA-1s-expressing fetal megakaryocyte progenitors. Moreover, our data reveal that overexpression of GATA-2 facilitates aberrant megakaryopoiesis.
The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron.
A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis.
Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway.
Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.
Hematopoiesis, the dynamic process of blood cell development, is regulated by the activity of the bone morphogenetic protein (BMP) signaling pathway and by many transcription factors. However, the molecules and mechanisms that regulate BMP/Smad signaling in hematopoiesis are largely unknown. Here, we show that the Integrator complex, an evolutionarily conserved group of proteins, functions in zebrafish hematopoiesis by modulating Smad/BMP signaling. The Integrator complex proteins are known to directly interact with RNA polymerase II to mediate 3' end processing of U1 and U2 snRNAs. We have identified several subunits of the Integrator complex in zebrafish. Antisense morpholino-mediated knockdown of the Integrator subunit 5 (Ints5) in zebrafish embryos affects U1 and U2 snRNA processing, leading to aberrant splicing of smad1 and smad5 RNA, and reduced expression of the hematopoietic genes stem cell leukemia (scl, also known as tal1) and gata1. Blood smears from ints5 morphant embryos show arrested red blood cell differentiation, similar to scl-deficient embryos. Interestingly, targeting other Integrator subunits also leads to defects in smad5 RNA splicing and arrested hematopoiesis, suggesting that the Ints proteins function as a complex to regulate the BMP pathway during hematopoiesis. Our work establishes a link between the RNA processing machinery and the downstream effectors of BMP signaling, and reveals a new group of proteins that regulates the switch from primitive hematopoietic stem cell identity and blood cell differentiation by modulating Smad function.
Children with Down syndrome (DS) have a marked increase in susceptibility to Acute Megakaryoblastic Leukaemia (DS-AMKL) and the closely linked neonatal preleukaemic syndrome, Transient Myeloproliferative Disorder (DS-TMD). The distinct stages of DS-TMD and DS-AMKL provide an excellent tractable model to study leukaemogenesis. This review focuses on recent studies describing clinical, haematological and biological features of DS-AMKL and DS-TMD. The findings from these studies suggest that mutations in the key haemopoietic regulator GATA1 (GATA binding protein 1) in DS-AMKL and DS-TMD may be useful in diagnosis and assessing minimal residual disease. These findings raise the possibility of population-based screening strategies for DS-TMD and the development of treatment to eliminate the preleukaemic TMD clone to prevent DS-AMKL. Advances in our understanding of perturbed haemopoiesis in DS, the role of GATA1 and of cooperating mutations are also discussed. These findings have implications for leukaemia biology more broadly given the frequency of acquired trisomy in other human leukaemias.
For the erythroid cell lineage development in vertebrates, GATA-1 transcription factor is essential. In our report, we have demonstrated that the approximate developmental status of erythrocytes and the progression of blood formation can be studied non-invasively in GATA-1:DsRed transgenic zebrafish (Danio rerio) embryo and larva by characterization of fluorescence luminance spectra. The study was carried out for animals maintained under normoxic and hypoxic (152 and 20 torr PO(2) respectively) conditions up to 10 days post-fertilization (dpf) and total blood cell concentrations and fluorescent cells' percentage were determined for this purpose. The erythroids were classified into five intensity stages (IS) on the basis of their fluorescence intensity. The luminescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf to 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The total blood cell concentration dropped after 8 dpf in contrast to hypoxic group which showed further increasing trend. The fluorescent cells' percentage in normoxic group was generally higher as compared to the hypoxic ones. Our method successfully defined various stages of erythroid development. An effort was also made to correlate our luminance data (GATA-1 expression) and total blood cell concentrations with Epo mRNA production. Quantitative RT-PCR of 2-15 dpf old zebrafish was carried out for this purpose. Normoxic animals showed 1-3 Epo mRNA copies per ng RNA in contrast to the hypoxic larvae that showed remarkable fluctuation of 1 to 12 Epo mRNA copies per ng RNA during development. The blood volume (aortic diameter) and production time scale proved to be important factors to define the relationship of Epo mRNA with total blood cell concentration and GATA-1 protein expression respectively.
Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1(low) mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.
AGGF1 is an angiogenic factor, and its deregulation is associated with a vascular malformation consistent with Klippel-Trenaunay syndrome (KTS). This study defines the molecular mechanism for transcriptional regulation of AGGF1 expression. Transcription of AGGF1 starts at two nearby sites, -367 and -364 bp upstream of the translation start site. Analyses of 5'- and 3'-serial promoter deletions defined the core promoter/regulatory elements, including two repressor sites (from -1971 to -3990 and from -7521 to -8391, respectively) and two activator sites (a GATA1 consensus binding site from -295 to -300 and a second activator site from -129 to -159). Both the GATA1 site and the second activator site are essential for AGGF1 expression. A similar expression profile was found for GATA1 and AGGF1 in cells (including various endothelial cells) and tissues. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that GATA1 was able to bind to the AGGF1 DNA in vitro and in vivo. Overexpression of GATA1 increased expression of AGGF1. We identified one rare polymorphism -294C>T in a sporadic KTS patient, which is located in the GATA1 site, disrupts binding of GATA1 to DNA, and abolishes the GATA1 stimulatory effect on transcription of AGGF1. Knockdown of GATA1 expression by siRNA reduced expression of AGGF1, and resulted in endothelial cell apoptosis and inhibition of endothelial capillary vessel formation and cell migration, which was rescued by purified recombinant human AGGF1 protein. These results demonstrate that GATA1 regulates expression of AGGF1 and reveal a novel role for GATA1 in endothelial cell biology and angiogenesis.
Transient myeloproliferative disorder (TMD) is a hematologic abnormality usually associated with Down syndrome that may present with a skin eruption in addition to typical systemic findings. We report a case of a patient with TMD and a vesiculopustular eruption without the phenotypic characteristics of Down syndrome who was found to have mosaic trisomy 21. Mutations of the globin transcription factor 1 gene, GATA1, are associated with both TMD and acute megakaryocytic leukemia. Transient myeloproliferative disorder typically presents with pancytopenia, hepatosplenomegaly, and immature circulating white blood cells, and affects approximately 10% of neonates with Down syndrome. These abnormalities rapidly regress within the first few months of life. However, 20% to 30% of neonates with Down syndrome and TMD later develop leukemia. The tumor antigen PRAME (preferentially expressed antigen in melanoma) may serve as a marker for leukemic transformation. We report an illustrative case to alert clinicians about this uncommon cause of vesiculopustular eruption in a neonate without the phenotypic characteristics of Down syndrome and review the clinical findings and laboratory studies that aid in accurate diagnosis.
HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23 is associated with elevated fetal hemoglobin levels and has pleiotropic effects on several hematologic parameters. To investigate potential regulatory activity in the region, we have measured sensitivity of the sequences to DNase I cleavage that identified 3 tissue-specific DNase I hypersensitive sites in the core intergenic interval. Chromatin immunoprecipitation with microarray (ChIP-chip) analysis showed strong histone acetylation in a defined interval of 65 kb corresponding to the core HBS1L-MYB intergenic region in primary human erythroid cells but not in non-MYB-expressing HeLa cells. ChIP-chip analysis also identified several potential cis-regulatory elements as strong GATA-1 signals that coincided with the DNase I hypersensitive sites present in MYB-expressing erythroid cells. We suggest that HMIP contains regulatory sequences that could be important in hematopoiesis by controlling MYB expression. This study provides the functional link between genetic association of HMIP with control of fetal hemoglobin and other hematologic parameters. We also present a large-scale analysis of histone acetylation as well as RNA polymerase II and GATA-1 interactions on chromosome 6q, and alpha and beta globin gene loci. The data suggest that GATA-1 regulates numerous genes of various functions on chromosome 6q.
Growth Factor Independent-1B (Gfi-1B) is a transcriptional repressor that plays critical roles in the control of erythropoiesis and megakaryopoiesis. Gfi-1B expression was described to be repressed by an autoregulatory feedback control loop. Here, we show that Gfi-1 transcription is positively regulated early after induction of erythroid differentiation and remains highly active to late erythroblasts. Using chromatin immunoprecipitation assays in CD34+ cells from human cord blood, we found that Gfi-1 and GATA-2 in immature progenitors and then Gfi-1B and GATA-1 in erythroblasts are bound to the Gfi-1B promoter as well as to the promoter of c-myc, a known Gfi-1B target gene. Surprisingly, this Gfi-1/GATA-2-Gfi-1B/GATA-1 switch observed at erythroblast stages is associated to an increase in the Gfi-1B transcription whereas it triggers repression of c-myc transcription. Accordingly, analysis of chromatin modification patterns shows that HDAC, CoREST, and LSD1 are recruited to the c-myc promoter leading to appearance of repressive chromatin marks. In contrast, the Gfi-1B promoter remains associated with a transcriptionally active chromatin configuration as highlighted by an increase in histone H3 acetylation and concomitant release of the LSD1 and CoREST corepressors. The repressive function of Gfi-1B therefore depends on the nature of the proteins recruited to the target gene promoters and on chromatin modifications. We conclude that Gfi-1B behaves as a lineage-affiliated gene with an open chromatin configuration in multipotent progenitors and sustained activation as cells progress throughout erythroid differentiation.
How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human diseases associated with GATA-1 mutations.
The TP73 gene gives rise to transactivation domain-p73 isoforms (TAp73) as well as DeltaNp73 variants with a truncated N terminus. Although TAp73alpha and -beta proteins are capable of inducing cell cycle arrest, apoptosis, and differentiation, DeltaNp73 acts in many cell types as a dominant-negative repressor of p53 and TAp73. It has been proposed that p73 is involved in myeloid differentiation, and its altered expression is involved in leukemic degeneration. However, there is little evidence as to which p73 variants (TA or DeltaN) are expressed during differentiation and whether specific p73 isoforms have the capacity to induce, or hinder, this differentiation in leukemia cells. In this study we identify GATA1 as a direct transcriptional target of TAp73alpha. Furthermore, TAp73alpha induces GATA1 activity, and it is required for erythroid differentiation. Additionally, we describe a functional cooperation between TAp73 and DeltaNp73 in the context of erythroid differentiation in human myeloid cells, K562 and UT-7. Moreover, the impaired expression of GATA1 and other erythroid genes in the liver of p73KO embryos, together with the moderated anemia observed in p73KO young mice, suggests a physiological role for TP73 in erythropoiesis.
Denaturing high-performance liquid chromatography (dHPLC) was developed to screen DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. In this study, we have evaluated the dHPLC screening method and direct sequencing for the detection of GATA1 mutations in peripheral blood and bone marrow aspirates samples from children with Down syndrome (DS). Cases were ascertained consecutively as part of an epidemiological study of DS and hematological disorders in Brazil. A total of 130 samples corresponding to 115 children with DS were analysed using dHPLC and direct sequencing methods to detect mutations in GATA1 exons 2, 3 and 4 gene sequences. The overall detection rate of sequencing and dHPLC screening methods was similar. Twenty mutations were detected in exon 2 and one mutation in exon 3 (c.231_232 dupGT) sequences of acute megakaryoblastic leukemia and transient leukemia samples. Four GATA1 mutations were newly described [c.155C > G; c.156_178 del23 bp; c.29_30 del GG; c.182C > A and c.151A > T,c.153_162 del 10 bp). Out of four, three had single nucleotide change. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.
We previously demonstrated a silencing role for signal transducers and activators of transcription 3 (STAT3) in gamma-globin gene regulation in primary erythroid cells. Recently, GATA-1, a key transcription factor involved in hematopoietic cell development, was shown to directly inhibit STAT3 activity in vivo. Therefore, we completed studies to determine if interactions between these two factors influence gamma-globin gene expression.
Chromatin immunoprecipitation assay was used to ascertain in vivo protein binding at the gamma-globin 5' untranslated region (5'UTR); protein-protein interactions were examined by coimmunoprecipitation analysis. In vitro protein-DNA binding were completed using surface plasmon resonance and electrophoretic mobility shift assay. Activity of a luciferase gamma-globin promoter reporter and levels of gamma-globin messenger RNA and fetal hemoglobin in stable K562 cell lines overexpressing STAT3 and GATA-1, were used to determine the influence of the STAT3/GATA-1 interaction on gamma-globin gene expression.
We observed interaction between STAT3 and GATA-1 in K562 and mouse erythroleukemia cells in vivo at the gamma-globin 5'UTR by chromatin immunoprecipitation assay. Electrophoretic mobility shift assay performed with a 41-base pair gamma-globin DNA probe (gamma41) demonstrated the presence of STAT3 and GATA-1 proteins in complexes assembled at the gamma-globin 5'UTR. A consensus STAT3 DNA probe inhibited GATA-1-binding in a concentration-dependent manner, and the converse was also true. Enforced STAT3 expression augmented its binding at the gamma-globin 5'UTR in vivo and silenced gamma-promoter-driven luciferase activity. Stable enforced STAT3 expression in K562 cells reduced endogenous gamma-globin messenger RNA level. This effect was reversed by GATA-1.
These data provide evidence that GATA-1 can reverse STAT3-mediated gamma-globin gene silencing in erythroid cells.
Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light-dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light-dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light-dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.
Previous investigations from our laboratory identified the ultrastructural pathology and cytochemistry of macrothrombocytes (MTC) from patients with the X-linked, G208S varient of the GATA-1 mutation.A subsequent biochemical study of the MTC cytoskeletal proteins using polyacrylamide gel electrophoresis and western blot analysis revealed the MTC were deficient in the high-molecular weight, actin binding protein, talin. The present study has used immunofluorescent techniques to further characterize the talin deficiency. Results confirm that the GATA-1, G208S MTC are deficient in talin, and what little is present relocates to the undersurface of the plasma membrane following activations where it associates with adhesion plaques.
The congenital dyserythropoietic anemias are rare recessive disorders characterized by erythroblast multinuclearity, ineffective erythropoiesis, anemia and iron overload. In this perspective article, Drs. Iolascon and Delaunay examine genetic and clinical aspects of these inherited disorders.
ARR19 (androgen receptor corepressor of 19 kDa), which encodes for a leucine-rich protein, is expressed abundantly in the testis. Further analyses revealed that ARR19 was expressed in Leydig cells, and its expression was differentially regulated during Leydig cell development. Adenovirus-mediated overexpression of ARR19 in Leydig cells inhibited testicular steroidogenesis, down-regulating the expression of steroidogenic enzymes, which suggests that ARR19 is an antisteroidogenic factor. Interestingly, cAMP/luteinizing hormone attenuated ARR19 expression in a fashion similar to that of GATA-1, which was previously reported to be down-regulated by cAMP. Sequence analysis of the Arr19 promoter revealed the presence of two putative GATA-1 binding motifs. Further analyses with 5' deletion and point mutants of putative GATA-1 binding motifs showed that these GATA-1 binding sites were critical for high promoter activity. CREB-binding protein coactivated GATA-1 and markedly increased the activity of the Arr19 promoter. Both GATA-1 and CREB-binding proteins occupied the GATA-1 motifs within the Arr19 promoter, which was repressed by cAMP treatment. Altogether, these findings demonstrate that ARR19 is the target gene of GATA-1 and suggest that ARR19 gene expression in testicular Leydig cells is regulated by luteinizing hormone/cAMP signaling via the control of GATA-1 expression, resulting in the control of testicular steroidogenesis.
We report a male infant with Down syndrome who had a transient myeloproliferative disorder associated with skin lesions. He was transferred to a neonatal intensive care unit because of low body weight, fetal edema, disseminated intravascular coagulation, and 10% blast cells in the peripheral blood. On postnatal day (PD) 1, erythema with small papules, vesicles, and pustules appeared on the entire body. A smear preparation from the pustules on PD 2 showed 10% blast cells. A biopsy specimen taken on PD 5 revealed subcorneal pustules containing neutrophils and eosinophils. Genetic analyses detected a somatic mutation (197G>T, Glu295Stop) in exon 2 of GATA-1. On PD 10, the eruptions resolved spontaneously and the population of blast cells in peripheral blood decreased to 1%. The number of blast cells in pustules decreased markedly after three days. Therefore, we recommend that cytologic examination should be performed as early as possible.
Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.
Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia.
The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng.
The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection.
Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.
Transcription factors originally identified as drivers of erythroid differentiation subsequently became linked to megakaryopoiesis, reflecting the shared parentage of red cells and platelets. The divergent development of megakaryocytic and erythroid progenitors relies on signaling pathways that impose lineage-specific transcriptional programs on non-lineage-restricted protein complexes. One such signaling pathway involves RUNX1, a transcription factor upregulated in megakaryocytes and downregulated in erythroid cells. In this pathway, RUNX1 engages the erythro-megakaryocytic master regulator GATA-1 in a megakaryocytic transcriptional complex whose activity is highly dependent on the P-TEFb kinase complex. The implications of this pathway for normal and pathological megakaryopoiesis are discussed.
The nuclear location and relocation of genes play crucial regulatory roles in gene expression. SATB1, a MAR-binding protein, has been found to regulate beta-like globin genes through chromatin remodeling. In this study, we generated K562 cells over-expressing wild-type or nuclear matrix targeting sequences (NMTS)-deficient SATB1 and found that like wild-type SATB1, NMTS-deficient SATB1 induces out loop of beta-globin cluster from its chromosome territory (CT), while it is unable to associate the cluster with the nuclear matrix as wild-type SATB1 does and had no regulatory functions to the beta-globin cluster. Besides, our data showed that the transacting factor occupancies and chromatin modifications at beta-globin cluster were differentially affected by wild-type and NMTS-deficient SATB1. These results indicate that SATB1 regulates beta-like globin genes at the nuclear level interlaced with chromatin and DNA level, and emphasize the nuclear matrix binding activity of SATB1 to its regulatory function.
Transcription factor GATA1 plays a critical role in erythropoiesis through the integrated regulation of cell proliferation, differentiation, and apoptosis. In Gata1.05 gene knockdown mice, Gata1 expression deteriorates to 5% of wild-type allelic expression, a level insufficient for supporting normal erythropoiesis and one that leads to accumulation of erythroid progenitors that are readily transformed into erythroblastic leukemia. Serial engraftment of leukemic cells into primary or subsequent nude mice reconstituted complete leukemic phenotype in recipient. To delineate characteristics of leukemic stem cells (LSCs), we analyzed LSCs of Gata1.05 leukemia, which have a potential to reestablish leukemia in mice.
Leukemic cells isolated from the first recipient mice of Gata1.05 leukemia cells were divided into two fractions using Hoechst dye. Fractionated cells were transplanted into second recipient, or analyzed gene expression profiles and cell-cycle status. Consequences of 5-fluorouracil (5-FU) treatment on leukemic cells in vivo were studied.
LSCs were enriched in the Hoechst dye-excluded side population (SP), and leukemic cells in the SP population (LSP cells) were morphologically and immunophenotypically indistinguishable from other leukemic cells. However, expression of hematopoietic stem cell (HSC)-related genes was upregulated in the LSP cells. In cell-cycle analyses, LSP cells were quiescent like HSCs, but reentry into the cell cycle was stimulated by 5-FU treatment. Nonetheless, 5-FU treatment established a point of newly adjusted equilibrium in the LSP cells and the cells never recovered to their previous quiescent state.
Based on this observation, distinct self-renewal regulatory mechanisms in LSCs may be considered as one of the causes of worsening of the features of leukemia after injury and relapse.
TEL/ETV6 located at chromosome 12p13 encodes a member of the E26 transformation-specific family of transcription factors. TEL is known to be rearranged in a variety of leukemias and solid tumors resulting in the formation of oncogenic chimeric protein. Tel is essential for maintaining hematopoietic stem cells in the bone marrow. To understand the role of TEL in erythropoiesis, we generated transgenic mice expressing human TEL under the control of Gata1 promoter that is activated during the course of the erythroid-lineage differentiation (GATA1-TEL transgenic mice). Although GATA1-TEL transgenic mice appeared healthy up to 18 months of age, the level of hemoglobin was higher in transgenic mice compared to non-transgenic littermates. In addition, CD71+/TER119+ and c-kit+/CD41+ populations proliferated with a higher frequency in transgenic mice when bone marrow cells were cultured in the presence of erythropoietin and thrombopoietin, respectively. In transgenic mice, enhanced expression of Alas-e and beta-major globin genes was observed in erythroid-committed cells. When embryonic stem cells expressing human TEL under the same Gata1 promoter were differentiated into hematopoietic cells, immature erythroid precursor increased better compared to controls as judged from the numbers of burst-forming unit of erythrocytes. Our findings suggest some roles of TEL in expanding erythroid precursors and accumulating hemoglobin.
The transcription factors GATA-1 and GATA-2 have been implicated in promoting differentiation of eosinophilic leukocytes. In this study, we examined the roles of GATA-1 and GATA-2 in activating transcription of the secretory ribonuclease, the eosinophil-derived neurotoxin (EDN/RNase 2). Augmented expression of both GATA-1 and GATA-2 was detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Deletion or mutation of one or both of the two consensus GATA-binding sites in the extended 1000-bp 5' promoter of the EDN gene resulted in profound reduction in reporter gene activity. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicate that GATA-1 and GATA-2 proteins bind to both functional GATA consensus sequences in the EDN promoter. Interestingly, RNA silencing of GATA-1 alone had no impact on EDN expression; silencing of GATA-2 resulted in diminished expression of EDN, and also diminished expression of GATA-1 in both butyric acid-induced HL-60 clone 15 cells and in differentiating human eosinophils derived from CD34(+) hematopoietic progenitors. Likewise, overexpression of GATA-2 in uninduced HL-60 clone 15 cells resulted in augmented transcription of both EDN and GATA-1. Taken together, our data suggest that GATA-2 functions directly via interactions with the EDN promoter and also indirectly, via its ability to regulate the expression of GATA-1 in differentiating eosinophils and eosinophil cell lines.
We report a sixteen-year-old boy with Down syndrome and relapse of AML (M7), who has been in complete remission (CR) more than 12 months after bone marrow transplantation (BMT) from an HLA-matched sibling donor. Because monosomy 7 was detected at onset of AML and he experienced relapse after the treatment of AML 99 Down protocol, his prognosis was considered very poor. However, he achieved CR following chemotherapy that included high-dose AraC and BMT from an HLA-matched sibling donor without severe complication. He has remained in CR for more than 12 months after BMT. In this case, GATA1 mutation was not detected at either onset or relapse of AML and it is suggested that this case is in a different risk group than the usual Down syndrome patient with AML showing GATA1 mutation.
Children with Down syndrome and acute megakaryoblastic leukemia (DS-AMKL) have been shown to have increased sensitivity to cytarabine based chemotherapy. The excellent prognosis in patients with DS-AMKL may be due to mutations in the GATA1 gene leading to reduced expression of the enzyme cytidine deaminase. This leads to a decreased ability to convert cytarabine into its inactive metabolite, resulting in high intracellular concentration of this cytotoxic agent. We report two cases of DS-AMKL with GATA1 mutations who had poor outcome. These patients had high expression levels of cytidine deaminase mRNA transcripts. We speculate that other factors can affect overall outcome in patients with DS-AMKL irrespective of the presence of GATA1 mutations.
All-trans retinoic acid (ATRA) induces granulocytic maturation of WEHI-3B D+ leukemia cells and LiCl enhances this maturation, while WEHI-3B D- cells are non-responsive to ATRA. Transfection of SCL, expressed in D- but absent in D+ cells, into D+ cells, caused resistance to ATRA, while transfection of GATA-1 into D+ cells produced resistance to the combination of ATRA and LiCl. SCL expression in D+ cells did not induce the expression of c-Kit, a putative target gene for SCL. LiCl, known to inhibit some kinases by displacing Mg2+, did not affect tyrosine kinase activity of the cytoplasmic domain of c-Kit.
The hematopoietic transcription factor GATA-1 regulates the expression of several genes associated with differentiation of erythroid cells. We show here the inhibitory effect of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, on hemoglobinization and erythroid transcription factor GATA-1 expression in erythroleukemia (HEL) as well as in chronic myelogenous leukemia (K562) cells, which were induced to differentiate towards the erythroid lineage after aclacinomycin (Acla), doxorubicin (Dox) or hemin (HM) treatment. As a result, we observed i) a decreased expression of Friend of GATA-1 (FOG-1), an essential cofactor of GATA-1 transcription factor, ii) a downregulation of GATA-1 by proteasomal degradation and iii) a reduced acetylation level of GATA-1 in HM-induced K562 cells after TNF treatment. As a result, these modifications i) decreased the level of GATA-1/FOG-1 complex, ii) unsettled the GATA-1/GATA-2 balance, iii) reduced GATA-1 transcriptional activity and iv) inhibited erythroid marker gene expression (glycophorin A, erythropoietin receptor, gamma-globin) independently of the cell line or the inducer used. These data provided new insights into the role of GATA-1 regulation in TNFalpha-mediated inhibition of erythroid differentiation in erythroleukemia.
Although oncogenic functions and the clinical significance of Wilms tumor 1 (WT1) have been extensively studied in acute leukemia, the regulatory mechanism of its transcription still remains to be determined. We found a significant correlation among the amounts of WT1, GATA-1 and GATA-2 mRNAs from leukemia and solid tumor cell lines. Overexpression and small interfering RNA (siRNA) transfection experiments of GATA-1 and GATA-2 showed that these GATA transcription factors could induce WT1 expression. Promoter analysis showed that the 5' promoter did not explain the different WT1 mRNA levels between cell lines. The 3' enhancer, especially the distal sites out of six putative GATA binding sites located within the region, but not the intron 3 enhancer, were essential for the WT1 mRNA level. Electrophoretic mobility shift assay (EMSA) showed both GATA-1 and GATA-2 bound to these GATA sites. Besides acute leukemia cell lines, solid tumor cell lines including, TYK-nu-cPr also showed a high level of WT1 mRNA. We showed that GATA-2 expression is a determinant of WT1 mRNA expression in both TYK-nu-cPr cells and HL60 cells without GATA-1 expression. Taken together, these results suggest that GATA-1 and/or GATA-2 binding to a GATA site of the 3' enhancer of WT1 played an important role in WT1 gene expression.
TAM receptors (Tyro3, Axl and Mer) are expressed in hematopoietic tissues. The roles of the three receptors in hematopoiesis are, however, largely unknown. We investigated the role of TAM receptors in regulating erythropoiesis.
Single and double mutant mice for Axl and Mer were used in the study. Cellularity of bone marrow and spleen, hematologic parameters, flow cytometry analysis of erythroid cell maturation, erythropoietic response to acute hemolytic anemia, bone marrow transplantation and the expression of erythropoisis were analyzed to evaluate the function of Axl and Mer in erythropoiesis.
Axl and Mer, but not Tyro3, were constitutively expressed in developing erythroid cells. Mice lacking Axl and Mer (Axl(-/-)Me(-/-)) had impaired erythropoiesis in bone marrow and expanded splenic erythropoiesis. We found an inhibition of differentiation at the transition from erythroid progenitors to proerythroblasts in Axl(-/-)Mer(-/-) mice. These mice exhibited a low rate of erythropoietic response to acute anemia induced by phenylhydrazine. Bone marrow transplantation studies showed that the impaired erythropoiesis in Axl(-/-)Mer(-/-) mice is erythroid cell-autonomous. TAM receptors may influence erythropoiesis through the regulation of GATA-1 erythropoietin receptor and EpoR expression in erythroid progenitors. Notably, mice lacking single Axl or Mer exhibited normal erythropoiesis in steady-state conditions.
Axl and Mer play an important role in regulating erythropoiesis. This finding provides a novel insight into the mechanism of erythropoiesis.
EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of approximately 10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro.
Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies.
Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking.
The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.
The transcription factors PU.1 and GATA-1 antagonize each other in common myeloid progenitors and their relative abundance is thought to decide whether the cell follows the erythrocyte/megakaryocyte lineage or the granulocyte/macrophage lineage. We propose a kinetic model for the PU.1-GATA-1 interaction, analyse its phase space and interpret the results of our analysis. The conclusions have broader implications for the modelling of cell-fate selection.
The ultrastructural pathology of GATA-1, V205M and G208S macrothrombocytes was discussed in earlier investigations. This study has used the same technology to evaluate macrothrombocytes from a patient with the GATA-1, R216Q mutation. Some of the pathological features observed in macrothrombocytes from patients with the V205M and G208S variations including hypo- and agranular platelets, tubular inclusions and platelets within platelets, as well as platelets within platelets within platelets were identified. However, tubular membrane sheets in megakaryocytes and platelets of the V205M and G208S types and large groups of platelets attached to platelets to form megathrombocytes were not observed. The unique pathology of the megathrombocytes from this patient was the near absence of dense bodies in his giant cells. Storage Pool Deficiency, together with large platelets, defective adhesion and aggregation of his macrocytes under shear stress to vWF and collagen and defective clot retraction may contribute to the pathogenesis of his bleeding disorder.
The dynamic packaging of DNA into chromatin is a fundamental step in the control of diverse nuclear processes. Whereas certain transcription factors and chromosomal components promote the formation of higher-order chromatin loops, the co-regulator machinery mediating loop assembly and disassembly is unknown. Using mice bearing a hypomorphic allele of the BRG1 chromatin remodeler, we demonstrate that the Brg1 mutation abrogated a cell type-specific loop between the beta-globin locus control region and the downstream beta major promoter, despite trans-acting factor occupancy at both sites. By contrast, distinct loops were insensitive to the Brg1 mutation. Molecular analysis with a conditional allele of GATA-1, a key regulator of hematopoiesis, in a novel cell-based system provided additional evidence that BRG1 functions early in chromatin domain activation to mediate looping. Although the paradigm in which chromatin remodelers induce nucleosome structural transitions is well established, our results demonstrating an essential role of BRG1 in the genesis of specific chromatin loops expands the repertoire of their functions.
ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis.
Hematopoietic stem cell lineage choices are decided by genetic networks that are turned ON/OFF in a switch-like manner. However, prior to lineage commitment, genes are primed at low expression levels. Understanding the underlying molecular circuitry in terms of how it governs both a primed state and, at the other extreme, a committed state is of relevance not only to hematopoiesis but also to developmental systems in general. We develop a computational model for the hematopoietic erythroid-myeloid lineage decision, which is determined by a genetic switch involving the genes PU.1 and GATA-1. Dynamical models based upon known interactions between these master genes, such as mutual antagonism and autoregulation, fail to make the system bistable, a desired feature for robust lineage determination. We therefore suggest a new mechanism involving a cofactor that is regulated as well as recruited by one of the master genes to bind to the antagonistic partner that is necessary for bistability and hence switch-like behavior. An interesting fallout from this architecture is that suppression of the cofactor through external means can lead to a loss of cooperativity, and hence to a primed state for PU.1 and GATA-1. The PU.1-GATA-1 switch also interacts with another mutually antagonistic pair, C/EBPalpha-FOG-1. The latter pair inherits the state of its upstream master genes and further reinforces the decision due to several feedback loops, thereby leading to irreversible commitment. The genetic switch, which handles the erythroid-myeloid lineage decision, is an example of a network that implements both a primed and a committed state by regulating cooperativity through recruitment of cofactors. Perturbing the feedback between the master regulators and downstream targets suggests potential reprogramming strategies. The approach points to a framework for lineage commitment studies in general and could aid the search for lineage-determining genes.
Survivin is an inhibitor of apoptosis and its role in embryonic development is not completely understood. In zebrafish, survivin undergoes gene duplication. Survivin1 (sur1) has been shown to mediate angiogenesis but not hematopoiesis. In this study, we examined survivin2 (sur2) with particular reference to its role in primitive hematopoiesis during zebrafish development. sur2 was expressed predominantly in the intermediate cell mass (ICM, site of primitive hematopoiesis). Morpholino (MO) targeting at intron1-exon2 junction of sur2 significantly reduced green fluorescent protein(+) (erythroid) cell population in transgenic Tg (gata1:gfp) embryos at 18 h post-fertilization (h.p.f.; wild type: 4.49+/-0.15%; Sur2(MO) embryos: 2.22+/-0.12%, P=0.02). Molecular targeting was confirmed by reverse transcription-PCR and MO specificity by successful sur2 mRNA rescue. sur2 MO also downregulated genes associated with hematopoietic stem cells (scl, lmo2), erythroid (gata1, alpha- and beta-embryonic hemoglobins) as well as early (pu.1) and late (mpo, l-plastin) myelomonocytic lineages at 12 and 18 h.p.f. This was associated with an increase in apoptosis in the ICM and alteration of cell-cycle status of erythroid cells. Both effects were caspase dependent. In conclusion, sur2 is important in maintaining hematopoietic stem and lineage committed cells during zebrafish development, by virtue of its antiapoptotic activity in a caspase dependent and cell autonomous fashion.
Children with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL). In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.
Somatic CD34+ hematopoietic stem/precursor cells (HSPCs) give rise to hematopoietic cells and endothelial cells and have been used in clinical applications. Understanding the genes responsible for stemness and how they interact with each other will help us to manipulate these cells more efficiently in the future.
We performed microarray analysis on human CD34+ HSPCs and on two different progeny cell types, i.e. microvascular endothelial cells and peripheral blood mononuclear cells. Systems biology and advanced bioinformatics tools were used to help clarify the genetic networks associated with these stem cell genes.
We identified CD34+ HSPC genes and found that they were involved in critical biologic processes such as cell cycle regulation, chromosome organization, and DNA repair. We also identified a novel precursor gene cluster on chromosome 19p13.3. Analysis of HSPC-enriched genes using systems biology tools revealed a complex genetic network functioning in CD34+ cells, in which several genes acted as hubs to maintain the stability (such as GATA1) or connectivity (such as hepatic growth factor) of the whole network.
This study provides the foundation for a more detailed understanding of CD34+ HSPCs.
Although acquired mutations in the GATA1 gene have been reported for Down syndrome-related acute megakaryoblastic leukemia (DS-AMKL) in Caucasians, this is the first report of a Korean Down syndrome patient with AMKL carrying a novel mutation of the GATA1 gene. A 3-yr-old Korean girl with Down syndrome was admitted to our hospital complaining of pallor and fever. The findings of a peripheral blood smear and bone marrow study were compatible with the presence of AMKL. A chromosome study showed 48,XX,-7,+21c,+21,+r[3]/47,XX,+21c[17]. Following GATA1 gene mutation analysis, a novel mutation, c.145dupG (p.Ala49GlyfsX18), was identified in the N-terminal activation domain of the GATA1 gene. This mutation caused a premature termination at codon 67 and expression of an abnormal GATA-1 protein with a defective N-terminal activation domain, and the absence of full-length GATA-1 protein. This case demonstrates that a leukemogenic mechanism for DS-AMKL is contributed by a unique collaboration between overexpressed genes from trisomy 21 and an acquired GATA1 mutation previously seen in Caucasians and now in a Korean patient.
Mostly studied in murine models, Ikaros--a factor that positively or negatively controls gene transcription--was first described as essential to lymphopoiesis until its involvement in numerous hematopoietic lineages was documented. Indeed, Ikaros is also active in murine erythropoiesis and more precisely during fetal to adult globin switching. Recently, these observations were confirmed by our team in a human context. We here review some of the most important characteristics of Ikaros, and detail more precisely how defects of Ikaros activity either by gene inactivation or mutation in mice, or by forced expression of dominant negative isoforms in human precursor cells, modify the erythroid differentiation. An increased cell death, together with decreased cell proliferation, decreased expression of erythroid-specific genes including GATA1, and a delay in fetal to adult globin switching were observed. At the same time, myeloid differentiation was slightly favoured thus suggesting that Ikaros could be involved in the control of the myeloid/erythroid commitment.
During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.
To explore the effect of basic fibroblast growth factor 1 (bFGF1) during embryonic development on hematopoiesis, to study the expression of FGF1, vascular endothelial growth factor receptor (KDR), CD133, CD34 and transcription factors Ihh, SCL, GATA-1, GATA-2 and PU. 1 in the yolk sac, and to learn about the role and relationships of FGF1, hematopoietic cells and transcription factors during embryonic hematopoiesis.
10 microm sections and total RNA were prepared from 107 human embryos aged 3-12 weeks. Immunohischemical SP staining and RT-PCR were performed.
The yolk sac blood islands of human 3 approximately 12 weeks embryos consisted of peripheral vascular endothelial cells and central hematopoietic cells. The expression of FGF1 was firstly found in visceral mesoderm around periphery of yolk sac blood island at day 16, while was little inside it. KDR was not or lowly expressed and CD34 and CD133 were not expressed then. The expression increased, gray value decreased and staining enhanced at day 21. Strong staining of CD34+, CD133+ and KDR+ cells were found in blood island and mesoderm at day 30, their gray values changed from 156 +/- 16, 173 +/- 18 and 160 +/- 14 to 53 +/- 7, 52 +/- 6 and 69 +/- 8 respectively. FGF1 expression was strong positive, the gray value declined dramatically from 161 +/- 13 to 40 +/- 5. Some positive cells formed vessel-like structure along the periphery of blood island. Moderate expression of CD34+, CD133+, KDR+ cells increased at day 45, the cells aggregated into mass in blood island and FGF1+ cells did the same in blood island, while little in mesoderm. Its gray valve was increased. After 7 weeks, CD133+, KDR+, CD34+ cells significantly decreased their gray values increased, the staining became week. FGF1 was weakly expressed in yolk sac and its gray value increased to 179 +/- 22. RT-PCR showed Ihh, SCL, GATA-1 and GATA-2 were expressed at different time in yolk sac. PU. 1 were not expressed at day 16, and then expressed.
The hematopoietic properties of yolk sac may be dependent on signaling through FGF receptors and FGF1 plays an important role in hematopoietic stem cell homeostasis. The FGF pathway regulates primitive hematopoiesis by modulating transcription factors such as Gata1 expression level and activity.
Down syndrome (DS) persons are born with various hematopoietic abnormalities, ranging from relatively benign, such as neutrophilia and macrocytosis, to a more severe transient myeloproliferative disorder (TMD). In most cases, these abnormalities resolve in the first few months to years of life. However, sometimes the TMD represents a premalignant disease that develops into acute megakaryocytic leukemia (AMKL), usually in association with acquired GATA1 mutations. To gain insight into the mechanisms responsible for these abnormalities, we analyzed the hematopoietic development of the Ts1Cje mouse model of DS. Our analyses identified defects in mature blood cells, including macrocytosis and anemia, as well as abnormalities in fetal liver and bone marrow stem and progenitor cell function. Despite these defects, the Ts1Cje mice do not develop disease resembling either TMD or AMKL, and this was not altered by a loss of function allele of Gata1. Thus, loss of Gata1 and partial trisomy of chromosome 21 orthologs, when combined, do not appear to be sufficient to induce TMD or AMKL-like phenotypes in mice.
GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.
A transient abnormal myelopoiesis was observed in a newborn with Down syndrome. Cytogenetic study revealed multiple oligoclonal abnormalities: 47,XY,inv(6)(p23q21),+21c[3]/47,XY,der(7)t(1;7)(q25;p15),+21c[1]/47,XY,del(13)(q?),+21c[1]/47,XY,+21c[15]. Ten months after the patient achieved remission, the transient abnormal myelopoiesis evolved to an acute megakaryoblastic leukemia. Cytogenetic study revealed only a single clonal abnormality, 47,XY,der(7)t(1;7)(q25;p15),+21c, identical to one of the structural changes seen at birth. Sequence analysis of the GATA1 gene revealed a deletion-insertion mutation within the exon 2 introducing a stop codon after Arg 64. It may be that the der(7)t(1;7)(q25;p15) abnormality played some selective role in the development of acute megakaryoblastic leukemia in this patient. To our knowledge, the present case is unique in demonstrating a subclone with der(7)t(1;7)(q25;p15) evolving to acute leukemia.
The aim of this study was to evaluate the biological correlation and prognostic impact of Gata-1, Gata-2, EKLF, and c-MPL transcript level in a group of 41 acute myeloid leukemia (AML) patients. Gata-1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata-1 expression on the probability of achieving complete remission has been confirmed. Gata-2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c-MPL was associated with CD34+ AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML.
Currently, the origin of autoimmune diseases is considered to be multifactorial. Genetic predisposition, immune system malfunction or even backfire, hormonal regulation, and environmental factors, i.e. infections, all play important roles in the pathogenesis of autoimmune diseases such as the antiphospholipid syndrome (APS). New drugs and strategies aimed at preventing infections could further improve the outcome of APS and other autoimmune diseases.
In this concise report, we describe the history and evolution of childhood acute leukemia studies in Brazil, and the application if key biomarkers for clinical trials and epidemiological studies over the past 8 years. Highlights of each ongoing study are summarized. A Brazilian network integrating hospitals and scientific institutions from all country regions has been established. This organization is made possible through informatics and computer networking, and the standardization of pathological reviews including immunophenotyping and molecular characterization of childhood leukemias. The unique characteristics of the Brazilian population combined with a large clinical and epidemiologic framework for patient ascertainment has enabled large-scale epidemiological studies on childhood leukemia in Brazil.
Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.
GATA-1 controls hematopoietic development by activating and repressing gene transcription, yet the in vivo mechanisms that specify these opposite activities are unknown. By examining the composition of GATA-1-associated protein complexes in a conditional erythroid rescue system as well as through the use of tiling arrays we detected the SCL/TAL1, LMO2, Ldb1, E2A complex at all positively acting GATA-1-bound elements examined. Similarly, the SCL complex is present at all activating GATA elements in megakaryocytes and mast cells. In striking contrast, at sites where GATA-1 functions as a repressor, the SCL complex is depleted. A DNA-binding defective form of SCL maintains association with a subset of active GATA elements indicating that GATA-1 is a key determinant for SCL recruitment. Knockdown of LMO2 selectively impairs activation but not repression by GATA-1. ETO-2, an SCL-associated protein with the potential for transcription repression, is also absent from GATA-1-repressed genes but, unlike SCL, fails to accumulate at GATA-1-activated genes. Together, these studies identify the SCL complex as a critical and consistent determinant of positive GATA-1 activity in multiple GATA-1-regulated hematopoietic cell lineages.
An X-linked mutation in the GATA-1 transcription factor, G208S, causes macrothrombocytopenia and serious bleeding problems in affected male family members. The unique ultrastructural pathology of their platelets was described previously. The present investigation has evaluated the cytoskeletal proteins of the GATA-1, G208S macrothrombocytes of two male patients by page gel electrophoresis and Western blot analysis. The 235-245 KD cytoskeletal protein, Talin, was absent from their (PAGE) gels and undetectable by a specific talin antibody on Western blots.
The zebrafish is an established model system for studying the embryonic emergence of tissues and organs, including the hematopoietic system. We hypothesized that key signaling pathways controlling embryonic hematopoiesis continue to be important in the adult, and we sought to develop approaches to test this in zebrafish, focused on the bone morphogenetic protein (BMP) signaling pathway. Functions for this pathway in adult hematopoiesis have been challenging to probe in other models.
Several approaches tested the function of BMP signaling during adult zebrafish hematopoiesis. First, we evaluated steady-state hematopoiesis in adult fish that are heterozygous for mutant alleles of Smad5, or are homozygous for mutant alleles, and rescued to adulthood by injection of RNA encoding Smad5. Second, we tested the relative ability of smad5 mutant fish to recover from hemolytic anemia. Third, we generated a transgenic line that targets the expression of a dominant-negative BMP receptor to adult-stage Gata1+ progenitor cells.
Adult fish with a strong mutant smad5 allele are anemic at steady state and, in addition, respond to hemolytic anemia with kinetics that are altered compared to wild-type fish. Fish expressing a mutant BMP receptor in early Gata1+ definitive progenitors generate excessive eosinophils.
Our study provides proof of principle that regulation of adult hematopoiesis can be studied in zebrafish by altering specific pathways. We show that the BMP signaling pathway is relevant for adult hematopoiesis to maintain steady state erythropoiesis, control the erythropoietic response following stress anemia, and to generate normal numbers of eosinophils.
To study the molecular pharmacological basis of the YiSui ShenXu Granule, a complex prescription of the Chinese traditional medicine used to treat beta-thalassemia.
Real-time quantitative PCR method had been applied to analyze the genes expression: gamma-globin, Ckit, EpoR, Spi, FKLF, GATA1 and GATA2 in K562 cell treated and untreated with this complex prescription and its each single herbal medicine.
The results showed that this complex prescription increased the gamma-globin, EpoR, Spi and FKLF expression and decreased the Ckit, GATA1 and GATA2 expression. And all single herbal medicines of this complex prescription could change some of those gene expressions, but not the same as the complex prescription. Even that, this study results indicated that the YiSui ShenXu Granule has its molecular pharmacological basis in treating beta-thalassemia.
Enhanced differentiation of human embryonic stem cells (HESCs), induced by genetic modification could potentially generate a vast number of diverse cell types. Such genetic modifications have frequently been achieved by over-expression of individual regulatory proteins. However, careful evaluation of the expression levels is critical, since this might have important implications for the differentiation potential of HESCs. To date, attempts to promote osteogenesis by means of gene transfer into HESCs using the early bone "master" transcription factor osterix (Osx) have not been reported. In this study, we attained HESC subpopulations expressing two significantly different levels of Osx, following lentiviral gene transfer. Both subpopulations exhibited spontaneous differentiation and reduced expression of markers characteristic of the pluripotent phenotype, such as SSEA3, Tra1-60, and Nanog, In order to promote bone differentiation, the cells were treated with ascorbic acid, beta-glycerophosphate and dexamethasone. The high level of Osx, compared to endogenous levels found in primary human osteoblasts, did not enhance osteogenic differentiation, and did not up-regulate collagen I expression. We show that the high Osx levels instead induced the commitment towards the hematopoietic-endothelial lineage-by up-regulating the expression of CD34 and Gata1. However, low levels of Osx up-regulated collagen I, bone sialoprotein and osteocalcin. Conversely, forced high level expression of the homeobox transcription factor HoxB4, a known regulator for early hematopoiesis, promoted osteogenesis in HESCs, while low levels of HoxB4 lead to hematopoietic gene expression.
GATA-1 is one of the six members of the GATA gene family, a group of related transcription factors discovered in the 1980s. In the past few decades, the crucial role of GATA-1 in normal human hematopoiesis has been delineated. As would be expected, mutations in GATA-1 have subsequently been found to have important clinical significance, and are directly linked to deregulated formation of certain blood cell lineages. This paper reviews the functional consequences of GATA-1 mutations by linking specific errors in the gene, or its downstream protein products, to documented human diseases. These five human diseases are: X-linked thrombocytopenia (XLT), X-linked thrombocytopenia with thalassemia (XLTT), congenital erythropoietic porphyria (CEP), transient myeloproliferative disorder (TMD) and acute megarakaryoblastic leukemia (AMKL) associated with Trisomy 21, and, lastly, a particular subtype of anemia associated with the production of GATA-1s, a shortened, mutant isoform of the wild-type GATA-1. The different phenotypic expressions associated with GATA-1 mutations illustrate the integral function of the transcription factor in overall body homeostasis. Furthermore, these direct genotype-phenotype correlations reinforce the importance of unraveling the human genome, as such connections may lead to important therapeutic or preventive therapies.
Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.
The first wave of erythropoiesis in amniotic animals generates all primitive erythrocytes and takes place exclusively in yolk sac mesoderm. It is less clear, however, to what extent and for how long the yolk sac contributes to the second wave of erythropoiesis which gives rise to definitive erythrocytes for later embryonic and adult use. Here, we examine the initiation, duration, and site of definitive erythrocyte formation in chicken yolk sac. We show that the earliest definitive erythrocytes are generated in yolk sac venous vessels surrounding major arteries at embryonic day (E) 4-4.5, and that mature definitive erythrocytes enter circulating at E4.5-E5. This takes place at a time when yolk sac vasculature remodels extensively to generate paired arterial/venous vessels. The yolk sac remains the predominant site for definitive erythropoiesis from E5 to E10, and continues to generate definitive erythrocytes at least until E15. Similar to primitive erythropoiesis, definitive erythropoiesis in the yolk sac is accompanied by the expression of transcriptional regulators gata1, scl, and lmo2. Furthermore, our data suggest that one main source of definitive erythropoietic cells is the pre-existing vascular endothelial cells. It remains unclear whether yolk sac derived hematopoietic progenitors that do not undergo erythropoiesis in the yolk sac may take up intraembryonic niches and contribute to erythropoietic stem cell population after hatching.
To identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.
Maltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.
MBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.
LMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.
Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.
Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.
To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated).
One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients.
CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes.
Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.
Children with Down's syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Down's syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Down's syndrome.
JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Down's syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Down's syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells.
Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Down's syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Down's syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders.
A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia.
Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.
The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been linked to inflammation- and cancer-related anemia, which reduces both quality of life and prognosis of patients. The aim of this study was to reveal molecular mechanisms linked to the inhibition of erythroid differentiation by TNFalpha. In this study, we showed that the inhibition of erythropoietin (Epo)-mediated differentiation by TNFalpha lead to a downregulation of hemoglobin synthesis and was correlated to a modulation of key erythroid transcription factors. Thus, a reverse of the transcription factor GATA-1/GATA-2 balance normally present during erythropoiesis, as well as a downregulation of the cofactor of GATA-1, friend of GATA-1 (FOG-1), and the coregulating transcription factor nuclear factor erythroid 2 (NF-E2) was observed after TNFalpha treatment. Moreover, we showed a reduction of GATA-1/FOG-1 interaction due to a reduced transcription of GATA-1 and a proteasome-dependent FOG-1 degradation after TNFalpha treatment. These changes led to an inhibition of erythroid gene expression including Epo receptor (EpoR), alpha- and gamma-globin, erythroid-associated factor (ERAF), hydroxymethylbilane synthetase (HMBS), and glycophorin A (GPA). An analysis of distinct signaling pathway activations then revealed an activation of p38 by TNF, as well as a corresponding involvement of this mitogen-activated protein kinase (MAPK) in the cytokine-dependent inhibition of erythroid differentiation. Indeed the p38 inhibitor, SB203580, abrogated the inhibitory effect of TNFalpha on the major erythroid transcription factor GATA-1 as well as erythroid marker expression in Epo-induced TF-1 cells. Overall, these data contribute to a better understanding of cytokine-dependent anemia, by giving first hints about key erythroid transcription factor modulations after TNFalpha treatment as well as an involvement of p38 in the inhibition of erythroid differentiation.
IFN consensus sequence binding protein (Icsbp) (IFN response factor-8) is a hematopoietic transcription factor with dual functions in myelopoiesis and immunity. In this study, we report a novel role of Icsbp in regulating the development of eosinophils. Loss of Icsbp in mice leads to a reduction of eosinophils in different tissues. During parasite infection with the nematode Nippostrongylus brasiliensis, Icsbp-deficient mice fail to mount eosinophilia despite a vigorous IL-5 response. Numbers of phenotypically defined eosinophil progenitors are decreased and those progenitors have, on a per-cell basis, reduced eosinophil differentiation potential. The transcription factor Gata1, crucial for eosinophil development, is reduced expressed in committed eosinophil progenitors in wells as mature eosinophils. These findings identify Icsbp as a novel transcription factor critical for the development of the eosinophil lineage.
With-no-lysine kinase-4 (WNK4), a member of the serine-threonine protein kinase family, acts as a multifunctional regulator of diverse ion transporters. Therefore, it is interesting to investigate the mechanisms that control its expression. We have previously demonstrated that glucocorticoid downregulates human WNK4 (hWNK4) expression through the negative glucocorticoid responsive element. Here, using real-time PCR and Western blot assays, we show that trichostatin A (TSA), a histone deacetylase inhibitor, upregulated hWNK4 mRNA and protein expression in human embryo kidney 293 cells. Analysis of the transcriptional activity of a series of the truncated hWNK4 promoters by luciferase assay indicated that the region -484 to -337 of the hWNK4 promoter was sensitive to TSA, and a GATA-1 binding motif was identified at position -426 using TRANSFAC-TESS program. Moreover, using electrophoresis mobility shift assay and chromatin immunoprecipitation assay, the GATA-1 binding affinity to the hWNK4 promoter was shown to increase with TSA under in vitro and in vivo conditions. Immunoprecipitation and Western blot analyses showed that the levels of acetylated GATA-1 were increased with TSA, in agreement with changes in its DNA-binding affinity. These findings indicate that TSA induces hWNK4 expression, at least in part, by increasing GATA-1 acetylation, and thereby its binding to the GATA-1 responsive element, within the hWNK4 promoter.
In an earlier study, a cDNA was cloned that showed abundant expression in the eye at postnatal day (P)2 but was downregulated at P10; it was named ODAG (ocular development-associated gene). Its biological function was examined by generating and analyzing transgenic mice overexpressing ODAG (ODAG Tg) in the eye and by identifying ODAG-binding proteins.
Transgenic mice were generated by using the mouse Crx promoter. EGFP was designed to be coexpressed with transgenic ODAG, to identify transgene-expressing cells. Overexpression of ODAG was confirmed by Northern and Western blot analysis. IOP was measured with a microneedle technique. The eyes were macroscopically examined and histologically analyzed. EGFP expression was detected by confocal microscope. Proteins associated with ODAG were isolated by pull-down assay in conjugation with mass spectrometry.
Macroscopically, ODAG Tg exhibited gradual protrusion of the eyeballs. The mean IOP of ODAG Tg was significantly higher than that of wild-type (WT) littermates. Histologic analysis exhibited optic nerve atrophy and impaired retinal development in the ODAG Tg eye. EGFP was expressed highly in the presumptive outer nuclear layer and weakly in the presumptive inner nuclear layer in the ODAG Tg retina. Rab6-GTPase-activating protein (Rab6-GAP) and its substrate, Rab6, were identified as ODAG-binding proteins.
Deregulated expression of ODAG in the eye induces elevated intraocular pressure and optic nerve atrophy and impairs retinal development, possibly by interfering with the Rab6/Rab6-GAP-mediated signaling pathway. These results provide new insights into the mechanisms regulating ocular development, and ODAG Tg would be a novel animal model for human diseases caused by ocular hypertension.
The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.
Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.
Human parvovirus B19 infection is responsible for a wide range of human diseases ranging from mild erythema infectiosum in immunocompetent children to fetal loss in primary infected pregnant women and aplastic anemia or lethal cytopenias in adult immunocompromised patients. Since persistent viral infection is responsible for an autoimmune response and clinical symptoms can mimic autoimmune inflammatory disorders, parvovirus B19 is the object of intense efforts to clarify whether it is also able to trigger autoimmune diseases. Indeed the virus has been implicated as the causative or the precipitating agent of several autoimmune disorders including rheumatoid arthritis, systemic lupus, antiphospholipid syndrome, systemic sclerosis and vasculitides. Molecular mimicry between host and viral proteins seems to be the main mechanism involved in the induction of autoimmunity. By means of a random peptide library approach, we have identified a peptide that shares homology with parvovirus VP1 protein and with human cytokeratin. Moreover the VP peptide shares similarity with the transcription factor GATA1 that plays an essential role in megakaryopoiesis and in erythropoiesis. These new data sustain the role played by molecular mimicry in the induction of cross-reactive (auto)antibodies by parvovirus B19 infection.
Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines.
Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal-truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% +/- 4% vs 17.1% +/- 3%, CD34(+)CD38(+) cells; P < .001) with common myeloid progenitors (19.6% +/- 2% vs 44.0% +/- 7%) and granulocyte-monocyte (GM) progenitors (15.8% +/- 4% vs 34.5% +/- 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34(+) cells was markedly increased (78% +/- 7% vs 15% +/- 3%) affecting megakaryocyte-erythroid ( approximately 7-fold higher) and GM and colony-forming unit-granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.
The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.
Haem-regulated eIF2alpha kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.
Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse alpha-globin gene cluster and proved the extensive interaction between HS26 and alpha-globin genes. In addition, we have identified a candidate alpha1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.
Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach.
Evolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP.
Overall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells.
The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.
Semenogelin (SEMG) I is a cancer-testis antigen expressed in myeloma cells. SEMG I expression is upregulated by IL-4, IL-6 and 5-azacytidine. In this study, we set out to define the core promoter sequence needed for the expression of SEMG I in myeloma cells. We found that nucleotide sequences spanning the two putative GATA-1 binding domains are vital for the primary regulation of SEMG I promoter function while the other parts of the promoter sequence are responsible for the fine adjustment of the core promoter function. The core promoter sequence is responsive to the enhancing effect of IL-4 and IL-6.
The Runx1/AML1 transcription factor is required for the generation of hematopoietic stem cells and is one of the most frequently targeted genes in human leukemia. Runx1-deficient mice die around embryonic day (E)12.5 due to severe hemorrhage in the central nervous system and the complete absence of definitive hematopoietic cells. Since mice lacking the heterodimeric partner of Runx1, PEBP2beta/CBFbeta, are almost identical in phenotype to Runx1 (-/-) mice, PEBP2beta was believed to be essential for the in vivo function of Runx1. Here we show that transgenic overexpression of Runx1 partially rescues the lethal phenotype of PEBP2beta-deficient mice at E12.5. Some of the rescued mice escaped from the severe hemorrhage at E11.5-12.5, although definitive hematopoiesis was not restored. Thus, PEBP2beta-independent Runx1 activation can occur in vivo. This observation sheds new light on the mechanism(s) that regulate the activity of Runx transcription factors.
GATA-1 is an erythroid activator that binds beta-globin gene promoters and DNase I hypersensitive sites (HSs) of the beta-globin locus control region (LCR). We investigated the direct role of GATA-1 interaction at the LCR HS2 enhancer by mutating its binding sites within minichromosomes in erythroid cells. Loss of GATA-1 in HS2 did not compromise interaction of NF-E2, a second activator that binds to HS2, nor was DNase I hypersensitivity at HS2 or the promoter of a linked epsilon-globin gene altered. Reduction of NF-E2 using RNAi confirmed the overall importance of this activator in establishing LCR HSs. However, recruitment of the histone acetyltransferase CBP and RNA pol II to HS2 was diminished by GATA-1 loss. Transcription of epsilon-globin was severely compromised with loss of RNA pol II from the transcription start site and reduction of H3 acetylation and H3K4 di- and tri-methylation in coding sequences. In contrast, widespread detection of H3K4 mono-methylation was unaffected by loss of GATA-1 in HS2. These results support the idea that GATA-1 interaction in HS2 has a prominent and direct role in co-activator and pol II recruitment conferring active histone tail modifications and transcription activation to a target gene but that it does not, by itself, play a major role in establishing DNase I hypersensitivity.
The adult erythron is maintained via dynamic modulation of erythroblast survival potentials. Toward identifying novel regulators of this process, murine splenic erythroblasts at 3 developmental stages were prepared, purified and profiled. Stage-to-stage modulated genes were then functionally categorized, with a focus on apoptotic factors. In parallel with BCL-X and NIX, death-associated protein kinase-2 (DAPK2) was substantially up-modulated during late erythropoiesis. Among hematopoietic lineages, DAPK2 was expressed predominantly in erythroid cells. In a Gata1-IE3.9int-DAPK2 transgenic mouse model, effects on steady-state reticulocyte and red blood cell (RBC) levels were limited. During hemolytic anemia, however, erythropoiesis was markedly deficient. Ex vivo ana-lyses revealed heightened apoptosis due to DAPK2 at a Kit(-)CD71(high)Ter119(-) stage, together with a subsequent multifold defect in late-stage Kit(-)CD71(high)Ter119(+) cell formation. In UT7epo cells, siRNA knock-down of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated. DAPK2 therefore comprises a new candidate attenuator of stress erythropoiesis.
Inhibitors of DNA binding (Id) family members are key regulators of cellular differentiation and proliferation. These activities are related to the ability of Id proteins to antagonize E proteins and other transcription factors. As negative regulators of E proteins, Id proteins have been implicated in lymphocyte development. Overexpression of Id1, Id2, or Id3 has similar effects on lymphocyte development. However, which Id protein plays a physiologic role during lymphocyte development is not clear. By analyzing Id2 knock-out mice and retroviral transduced hematopoietic progenitors, we demonstrated that Id2 is an intrinsic negative regulator of B-cell development. Hematopoietic progenitor cells overexpressing Id2 did not reconstitute B-cell development in vivo, which resembled the phenotype of E2A null mice. The B-cell population in bone marrow was significantly expanded in Id2 knock-out mice compared with their wild-type littermates. Knock-down of Id2 by shRNA in hematopoietic progenitor cells promoted B-cell differentiation and induced the expression of B-cell lineage-specific genes. These data identified Id2 as a physiologically relevant regulator of E2A during B lymphopoiesis. Furthermore, we identified a novel Id2 function in erythroid development. Overexpression of Id2 enhanced erythroid development, and decreased level of Id2 impaired normal erythroid development. Id2 regulation of erythroid development is mediated via interacting with transcription factor PU.1 and modulating PU.1 and GATA-1 activities. We conclude that Id2 regulates lymphoid and erythroid development via interaction with different target proteins.
In this article, whole mount in situ hybridization is used to examine early blood vessel and blood cell development in the embryos of the brown trout Salmo trutta lacustris. cDNAs encoding for the angiogenic markers fli1 and flk1, and for the hematopoietic markers gata1 and gata2, were identified from an expressed sequence tag library of rainbow trout. Results show that fli1, flk1 and gata2 are activated in bilateral bands of the lateral trunk mesoderm before the onset of somitogenesis, shortly followed by gata1. These bands then converge toward the ventral midline to form the intermediate cell mass (ICM) (anterior ICM). Subsequent axial vasculogenesis and initial blood cell formation involve a clear spatial separation of fli1 and gata gene expression. Fli1 staining is most intense within the axial vessel (dorsal aorta, posterior cardinal vein) forming and lateral ICM cells, whereas binding of gata1 and gata2 probes becomes confined to the central portion of ICM cells beneath the dorsal aorta. This is followed by a first wave of angiogenesis, indicated by expression of fli1 and flk1. This gives rise to the intersegmental, dorsal longitudinal anastomotic and intestinal vessels. Further angiogenesis and hematopoiesis are activated in the "posterior ICM" of the tail. Here, the absence of gata1 indicates that hematopoiesis in this tissue generates myeloid rather than erythroid cells. The results supplement and validate previous, now historical morphological work in salmonids, thus aiding the elucidation of a comprehensive general scheme of angiogenic and hematopoietic development in the teleost embryo.
Growth factor independence 1 (GFI1) is important for maturation of mammalian lymphocytes and neutrophils and maintenance of adult hematopoietic stem cells (HSCs). The role of GFI1 in embryonic hematopoiesis is less well characterized. Through an enhancer trap screen and bioinformatics analysis, we identified a zebrafish homolog of Gfi1 (named gfi1.1) and analyzed its function during embryonic development. Expression of both an endogenous gfi1.1 gene and a GFP reporter gene inserted near its genomic locus was detected in hematopoietic cells of zebrafish embryos. Morpholino (MO) knockdown of gfi1.1 reduced expression of scl, lmo2, c-myb, mpo, rag1, gata1 and hemoglobin alpha embryonic-1 (hbae1), as well as the total amount of embryonic hemoglobin, but increased expression of pu.1 and l-plastin. Under the same conditions, MO injection did not affect the markers involved in vascular and pronephric development. Conversely, overexpression of gfi1.1 via mRNA injection enhanced expression of gata1 but inhibited expression of pu.1. These findings suggest that Gfi1.1 plays a critical role in regulating the balance of embryonic erythroid and myeloid lineage determination, and is also required for the differentiation of lymphocytes and granulocytes during zebrafish embryogenesis.
Phenotypic cell-to-cell variability within clonal populations may be a manifestation of 'gene expression noise', or it may reflect stable phenotypic variants. Such 'non-genetic cell individuality' can arise from the slow fluctuations of protein levels in mammalian cells. These fluctuations produce persistent cell individuality, thereby rendering a clonal population heterogeneous. However, it remains unknown whether this heterogeneity may account for the stochasticity of cell fate decisions in stem cells. Here we show that in clonal populations of mouse haematopoietic progenitor cells, spontaneous 'outlier' cells with either extremely high or low expression levels of the stem cell marker Sca-1 (also known as Ly6a; ref. 9) reconstitute the parental distribution of Sca-1 but do so only after more than one week. This slow relaxation is described by a gaussian mixture model that incorporates noise-driven transitions between discrete subpopulations, suggesting hidden multi-stability within one cell type. Despite clonality, the Sca-1 outliers had distinct transcriptomes. Although their unique gene expression profiles eventually reverted to that of the median cells, revealing an attractor state, they lasted long enough to confer a greatly different proclivity for choosing either the erythroid or the myeloid lineage. Preference in lineage choice was associated with increased expression of lineage-specific transcription factors, such as a >200-fold increase in Gata1 (ref. 10) among the erythroid-prone cells, or a >15-fold increased PU.1 (Sfpi1) (ref. 11) expression among myeloid-prone cells. Thus, clonal heterogeneity of gene expression level is not due to independent noise in the expression of individual genes, but reflects metastable states of a slowly fluctuating transcriptome that is distinct in individual cells and may govern the reversible, stochastic priming of multipotent progenitor cells in cell fate decision.
Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 Delta dblGATA mice (eosinophil-null mice via the Delta DblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.
Comparison of normal erythroblasts and erythroblasts from persons with the rare In(Lu) type of Lu(a-b-) blood group phenotype showed increased transcription levels for 314 genes and reduced levels for 354 genes in In(Lu) cells. Many erythroid-specific genes (including ALAS2, SLC4A1) had reduced transcript levels, suggesting the phenotype resulted from a transcription factor abnormality. A search for mutations in erythroid transcription factors showed mutations in the promoter or coding sequence of EKLF in 21 of 24 persons with the In(Lu) phenotype. In all cases the mutant EKLF allele occurred in the presence of a normal EKLF allele. Nine different loss-of-function mutations were identified. One mutation abolished a GATA1 binding site in the EKLF promoter (-124T>C). Two mutations (Leu127X; Lys292X) resulted in premature termination codons, 2 (Pro190LeufsX47; Arg319GlufsX34) in frameshifts, and 4 in amino acid substitution of conserved residues in zinc finger domain 1 (His299Tyr) or domain 2 (Arg328Leu; Arg328His; Arg331Gly). Persons with the In(Lu) phenotype have no reported pathology, indicating that one functional EKLF allele is sufficient to sustain human erythropoiesis. These data provide the first description of inactivating mutations in human EKLF and the first demonstration of a blood group phenotype resulting from mutations in a transcription factor.
Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.
We have analyzed single nucleotide polymorphisms (SNPs) in the XPC, XPA, and XPG genes of the nucleotide excision repair (NER) pathway in the Indian population. In the XPC gene we observed nine polymorphisms in the coding region, four polymorphisms in the intronic region, and two polymorphisms in the 5' untranslated region (UTR). In the XPA gene we observed one frequent SNP (allele frequency 0.48) within the 5' UTR at the 1665 position in a large proportion of the sample. In addition, we observed three novel heterozygous polymorphisms (a C to A transversion at position 1523 and a G to A transition at positions 1418 and 1458, with an allele frequency of 0.004) within the promoter region. In silico PCR analysis demonstrated that all three novel polymorphisms lie within a putative CpG island and that the variation at position 1418 falls within the potential GATA1/2/3 transcription factor(s) binding site and also within the negative control element. We performed a gel retardation assay with HeLa cell nuclear extract with an oligonucleotide encompassing this region. One of the alleles found at position 1458 of the XPA gene showed a significant change in protein-DNA interaction. In the XPG gene we found five polymorphisms in the coding region and one each in the 5' UTR of exon 1 and in intron 13.
Experimental perturbation of signaling or transcription factor networks has been used to study the developmental potential of lymphoid progenitors, lineage-committed precursors and mature lymphocytes. Common lymphoid progenitors and uncommitted pro-T cells can be efficiently diverted into myeloid or erythroid lineages by ectopic cytokine signaling or retroviral expression of the myeloid C/EBPalpha or erythroid GATA1 transcription factor. Forced C/EBPalpha expression furthermore induces direct transdifferentiation of immature thymocytes or B cells into macrophages. Notably, conditional inactivation of the B cell commitment factor Pax5 is sufficient to convert mature B cells into functional T cells via dedifferentiation to uncommitted progenitors. Together these experiments have uncovered an unanticipated developmental plasticity of lymphocytes, which may account for lineage switches observed in human malignancies.
According to the currently prevailing perception, sporadic cancer arises as a result of somatic mutations in a cell that lead to its uncontrolled proliferation. It is generally accepted that somatic mutations occur randomly and that they are generally either "spontaneous" or due to various external agents (physical or chemical carcinogens). Constitutional genetic conditions, which modify cancer risk, may become instrumental in opening new concepts in carcinogenesis. For instance, evidence gathered from Down syndrome (DS) individuals has challenged the above perceived explanation since somatic mutations in these patients markedly differ from those arisen in the general population, and do not seem to occur at random. There is no global increase or decrease of somatic mutations in all genes in DS. Instead, specific mutations have been detected on particular loci, depending on the tissue type and the age of the DS person. In the context of constitutional trisomy 21, where cancer have a very particular and striking distribution, biochemical, physiological and architectural imbalances in specific tissues may be responsible for a vulnerable state leading to carcinogenesis. Conversely, in tissues of DS patients which seem resistant to cancer, the proliferative and maturation state of the cells would lead to a refractory state of neoplastic transformation. Experimentally, we observed that the proliferation of tumor cells in extracellular matrix produced by trisomic 21 fibroblasts appears to be inhibited when compared to that of those placed on an extracellular matrix produced by euploid fibroblasts. These observations challenge the current somatic mutation theory of carcinogenesis, and strongly suggest instead a critical role of cells and tissues in the microenvironment where carcinogenesis occurs.
The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However, the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis, as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver, we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies, we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population, followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis.
Increased fetal hemoglobin (Hb F; alpha(2)gamma(2)) production in adults can ameliorate the clinical severity of sickle cell disease and beta-thalassemia major. Thus, understanding the regulation of gamma-globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt -675 and -526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of gamma-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing gamma-globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of gamma-globin gene expression in adults.
Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.
Unregulated activation of mast cells can contribute to the pathogenesis of inflammatory and allergic diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. Absence of mast cells in animal models can lead to impairment in the innate immune response to parasites and bacterial infections. Aberrant clonal accumulation and proliferation of mast cells can result in a variety of diseases ranging from benign cutaneous mastocytosis to systemic mastocytosis or mast cell leukemia. Understanding mast cell differentiation provides important insights into mechanisms of lineage selection during hematopoiesis and can provide targets for new drug development to treat mast cell disorders. In this review, we discuss controversies related to development, sites of origin, and the transcriptional program of mast cells.
We report a case of pediatric acute megakaryocytic leukemia (AMKL) showing 48,XX,+21,+21 as a sole acquired cytogenetic abnormality without the mutation of GATA1 gene. A physical examination showed a phenotypically normal female. Bone marrow findings showed diffuse infiltration of leukemic blasts having scanty cytoplasm with budding blebs and prominent nucleoli, which were negative for myeloperoxide (MPO) stain, Sudan black B stain and periodic acid-Schiff stain. Immunophenotyping of leukemic cells revealed positive expression of CD34, CD13, CD33, CD117, CD41, CD61, CD7 and negative expression of TdT, anti-MPO, CD64, CD56, CD2, CD3, CD5, CD10, CD19, CD20 and CD22. A fluorescence in situ hybridization analysis showed four distinct AML1 signals in 284 of 300 interphase nuclei. The entire six exons of the GATA1 gene (7757bp) were directly sequenced. We could not find any mutations, including known polymorphisms, which are known to be involved in transient myeloproliferative disorder and acute megakaryocytic leukemia of Down syndrome. After achieving complete remission, the tetrasomy 21 disappeared.
Regulatory mechanisms of human hematopoiesis remain largely uncharacterized. Through expression profiling of prospectively isolated stem and primitive progenitor cells as well as committed progenitors from cord blood (CB), we identified MLLT3 as a candidate regulator of erythroid/megakaryocytic (E/Meg) lineage decisions. Through the analysis of the hematopoietic potential of primitive cord blood cells in which MLLT3 expression has been knocked down, we identify a requirement for MLLT3 in the elaboration of the erythroid and megakaryocytic lineages. Conversely, forced expression of MLLT3 promotes the output of erythroid and megakaryocytic progenitors, and analysis of MLLT3 mutants suggests that this capacity of MLLT3 depends on its transcriptional regulatory activity. Gene expression and cis-regulatory element analyses reveal crossregulatory interactions between MLLT3 and E/Meg-affiliated transcription factor GATA-1. Taken together, the data identify MLLT3 as a regulator of early erythroid and megakaryocytic cell fate in the human system.
A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34(+)Sca-1(+)c-Kit(+) MPP fraction. The GATA-1(+) MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1(+) MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1(+) and PU.1(+) MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.
The journeys that hematopoietic cells take to differentiate from long-term stem cells into committed cells have recently been a topic of debate. In this issue of Cell Stem Cell, Arinobu et al. (2007) and Pronk et al. (2007) provide new insights into the paths traveled by hematopoietic progenitors.
In a mouse experimental asthma model, the administration of bacterial lipopolysaccharide (LPS), particularly at low doses, enhances the levels of ovalbumin (OVA)-induced eosinophilic airway inflammation. In an effort to clarify the cellular and molecular basis for the LPS effect, we demonstrate that the OVA-induced eosinophilic inflammation in the lung is dramatically increased by administration of LPS at the priming phase in wild-type mice, whereas such an increase was not observed in mast cell deficient mice. Adoptive transfer of bone marrow-derived mast cells (BMMC) from wild type but not from Toll-like receptor 4 (TLR4)-deficient mice restored the increased eosinophilic inflammation in mast cell-deficient mice. Moreover, in vitro analysis revealed that treatment of BMMC with LPS resulted in sustained up-regulation of GATA1 expression and increased production of Th2 cytokines (IL-4, IL-5, and IL-13) upon restimulation. Thus, mast cells appear to control allergic airway inflammation after their activation and modulation through TLR4-mediated induction of GATA1 proteins and subsequent increase in Th2 cytokine production.
GATA1 is a prototypical lineage-restricted transcription factor that is central to the correct differentiation, proliferation and apoptosis of erythroid and megakaryocytic cells. Mutations in GATA1 can generate a truncated protein, which contributes to the genesis of transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukaemia (AMKL) in infants with Down syndrome. Similarly, Gata1 knockdown to 5% of its wild-type level causes high incidence of erythroid leukaemia in mice. The GATA1-related leukaemias in both human and mouse could provide important insights into the mechanism of multi-step leukaemogenesis. Efforts are afoot to produce mouse models that are reflective of TMD and AMKL.
Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.
The differentiation of embryonic stem (ES) cells offers a powerful approach to study mechanisms implicated in cell fate decision. A major hurdle, however, is to promote the directed and efficient differentiation of ES cells toward a specific lineage. Here, we define in serum-free media the minimal factor requirement controlling each step of the differentiation process, resulting in the production of highly enriched hematopoietic progenitors. Four factors - Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) - are sufficient to drive the selective and efficient differentiation of mouse ES cells to hematopoiesis. Each of these factors appears to regulate a step of the process: Bmp4 promotes the very efficient formation of mesoderm; bFGF and activin A induce the differentiation of these mesodermal precursors to the hemangioblast fate; and VEGF is required for the production of fully committed hematopoietic progenitors. The stimulation of mesodermal precursors by bFGF and activin A switches on very rapidly the hematopoietic program, allowing us to dissect the molecular events leading to the formation of the hemangioblast. Runx1, Scl (Tal1) and Hhex expression is upregulated within 3 hours of stimulation, whereas upregulation of Lmo2 and Fli1 is observed later. Interestingly, increased expression levels of genes such as cMyb, Pu.1 (Sfpi1), Gata1 and Gata2 are not observed at the onset of hemangioblast commitment. This stepwise control of differentiation is extremely efficient, giving rise to a very high frequency of hematopoietic precursors, and provides an optimal system for understanding the molecular machineries involved in blood progenitor commitment.
Evidences indicate that locus control region (LCR) of beta-globin spatially closes to the downstream active gene promoter to mediate the transcriptional activation by looping. DNA binding proteins may play an important role in the looping formation. NF-E2 is one of the key transcription factors in beta-globin gene transcriptional activation. To shed light on whether NF-E2 is involved in this process, DS19MafKsiRNA cell pools were established by specifically knocked down the expression of MafK/NF-E2 p18, one subunit of NF-E2 heterodimer. In the above cell pools, it was observed that the occupancy efficiency of NF-E2 on beta-globin gene locus and the expression level of beta-globin genes were decreased. H3 acetylation, H3-K4 methylation and the deposition of RNA polymerase II, but not the recruitment of GATA-1, were also found reduced at the beta-globin gene cluster. Chromosome Conformation Capture (3C) assay showed that the cross-linking frequency between the main NF-E2 binding site HS2 and downstream structural genes was reduced compared to the normal cell. This result demonstrated that MafK/NF-E2 p18 recruitment was involved in the physical proximity of LCR and active beta-globin genes upon beta-globin gene transcriptional activation.
MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.
The zinc finger transcription factor GATA-1 requires direct physical interaction with the cofactor friend of GATA-1 (FOG-1) for its essential role in erythroid and megakaryocytic development. We show that in the mast cell lineage, GATA-1 functions completely independent of FOG proteins. Moreover, we demonstrate that FOG-1 antagonizes the fate choice of multipotential progenitor cells for the mast cell lineage, and that its down-regulation is a prerequisite for mast cell development. Remarkably, ectopic expression of FOG-1 in committed mast cell progenitors redirects them into the erythroid, megakaryocytic, and granulocytic lineages. These lineage switches correlate with transcriptional down-regulation of GATA-2, an essential mast cell GATA factor, via switching of GATA-1 for GATA-2 at a key enhancer element upstream of the GATA-2 gene. These findings illustrate combinatorial control of cell fate identity by a transcription factor and its cofactor, and highlight the role of transcriptional networks in lineage determination. They also provide evidence for lineage instability during early stages of hematopoietic lineage commitment.
The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal "on" state.
To characterize childhood acute megakaryoblastic leukaemia (AMKL), we reviewed 45 children with AMKL diagnosed between 1986 and 2005 at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. Twenty-four patients (53%) had AMKL associated with Down syndrome (DS-AMKL) and 21 (47%) had non-DS-AMKL. The median age of the DS-AMKL patients was 21 months (range, 8-38 months) and that of non-DS-AMKL patients was 15 months (range, 2-185 months). The morphology of blast cells was categorized into three groups according to the stage of megakaryocyte maturation. The blast cells were more immature in DS-AMKL than in non-DS-AMKL in terms of morphology and immunophenotyping. Cytogenetic abnormalities of leukaemic cells were classified into seven categories: normal karyotype including constitutional trisomy 21 in DS-AMKL; numerical abnormalities only; t(1;22)(p13;q13); 3q21q26 abnormalities; t(16;21)(p11;q22); -5/del(5q) and/or -7/del(7q); and other structural changes. The outcome of children with either DS-AMKL or non-DS-AMKL is excellent. The 10-year overall survival estimate was 79% [95% confidence interval (CI): 54-90] for DS-AMKL and 76% (95% CI: 58-91) for non-DS-AMKL (P = 0.81) with a median follow-up of 78 months (range, 20-243 months). Our study shows the diverse heterogeneity of childhood AMKL and the need for subclassification according to cytogenetic and morphological features.
The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre-mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.
Differentiation of hematopoietic stem and progenitor cells is an intricate process controlled in large part at the level of transcription. While some key megakaryocytic transcription factors have been identified, the complete network of megakaryocytic transcriptional control is poorly understood. Using global gene expression microarray analysis, Gene Ontology-based functional annotations, and a novel interlineage comparison with parallel, isogenic granulocytic cultures as a negative control, we closely examined the mRNA level of transcriptional regulators in megakaryocytes derived from human mobilized peripheral blood CD34(+) hematopoietic cells. This approach identified 199 differentially expressed transcription factors or transcriptional regulators. We identified and detailed the transcriptional kinetics of most known megakaryocytic transcription factors including GATA1, FLI1, and MAFG. Furthermore, many genes with transcription factor activity or transcription factor binding activity were identified in megakaryocytes that had not previously been associated with that lineage, including BTEB1, NR4A2, FOXO1A, MEF2C, HDAC5, VDR, and several genes associated with the tumor suppressor p53 (HIPK2, FHL2, and TADA3L). Protein expression and nuclear localization were confirmed in megakaryocytic cells for four of the novel candidate megakaryocytic transcription factors: FHL2, MXD1, E2F3, and RFX5. In light of the hypothesis that transcription factors expressed in a particular differentiation program are important contributors to such a program, these data substantially expand our understanding of transcriptional regulation in megakaryocytic differentiation of stem and progenitor cells.
Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologs of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In this study, we show that primitive erythrocytes in Runx1(-/-) mice display abnormal morphology and reduced expression of Ter119, Erythroid Kruppel-like factor (EKLF, KLF1), and GATA-1. These results suggest that mouse Runx1 plays a role in the development of both primitive and definitive hematopoietic cells.
A complete understanding of the transcriptional regulation of developmental lineages requires that all relevant factors be identified. Here, we have taken a proteomic approach to identify novel proteins associated with GATA-1, a lineage-restricted zinc finger transcription factor required for terminal erythroid and megakaryocytic maturation. We identify the Krüppel-type zinc finger transcription factor ZBP-89 as being a component of multiprotein complexes involving GATA-1 and its essential cofactor Friend of GATA-1 (FOG-1). Using chromatin immunoprecipitation assays, we show that GATA-1 and ZBP-89 cooccupy cis-regulatory elements of certain erythroid and megakaryocyte-specific genes, including an enhancer of the GATA-1 gene itself. Loss-of-function studies in zebrafish and mice demonstrate an in vivo requirement for ZBP-89 in megakaryopoiesis and definitive erythropoiesis but not primitive erythropoiesis, phenocopying aspects of FOG-1- and GATA-1-deficient animals. These findings identify ZBP-89 as being a novel transcription factor involved in erythroid and megakaryocytic development and suggest that it serves a cooperative function with GATA-1 and/or FOG-1 in a developmental stage-specific manner.
The level of survivin was reported to be scarce in mouse megakaryocytes (MKs) compared with erythroid cells. Considering this finding and previously reported in vitro data showing decreased MK ploidy upon retroviral-mediated overexpression of survivin, we sought to examine whether ectopic survivin expression in the MK lineage might alter ploidy level in vivo. Here we report the generation of 2 tissue specific hematopoietic transgenic mouse models, one expressing survivin in both the erythroid and MK lineages and the other expressing survivin solely in the MK lineage. Survivin protein overexpression was confirmed in MKs and erythrocytes. Surprisingly, analysis of both transgenic mouse lines showed no detectable changes in MK number, ploidy level, and platelet and erythrocyte counts, as compared with control mice. We conclude that elevated survivin expression does not alter MK/erythroid lineage development and that elevated survivin, alone, does not interfere with MK ploidy in vivo.
The specification and differentiation of hematopoietic stem cells into red blood cells requires precise coordination by multiple transcription factors. Most genes important for erythroid maturation are regulated by the Gata family of DNA-binding proteins. Previously, we identified three novel genes kelch-repeat containing protein (krcp), kiaa0650, and testhymin/glucocorticoid inducible transcript 1 (glcci1) to be expressed in erythroid cells in a Gata-independent manner, and we sought to further understand how these transcripts are regulated during zebrafish hematopoiesis.
We employed a loss-of-function approach, using combinations of antisense morpholinos to hematopoietic transcription factors and assayed for changes in gene expression in zebrafish embryos.
Upon examination of embryos deficient for Gata1, Gata2, Biklf, and/or Scl, we found distinct gene combinations were required for expression of the novel genes. While krcp expression was dependent upon Gata1 and Biklf, kiaa0650 expression was greatly reduced and glcci1 was maintained in Gata1/Gata2/Biklf-deficient embryos. As with the gata1 gene, kiaa0650 and krcp required Scl for blood expression. Although reduced, glcci1 was expressed in posterior blood precursors in the absence of Scl and Gata2.
This work identifies glcci1 as having Scl-independent expression in the posterior hematopoietic mesoderm, suggesting that its posterior expression is activated by factors upstream or parallel to Scl and Gata2. Additionally, these studies establish that blood gene expression programs are regulated by transcription factors acting in combination during erythroid maturation.
Enhancers can regulate designate promoters over long distances by forming chromatin loops. Whether chromatin loops are lost or reconfigured during gene repression is largely unexplored. We examined the chromosome conformation of the Kit gene that is expressed during early erythropoiesis but is downregulated upon cell maturation. Kit expression is controlled by sequential occupancy of two GATA family transcription factors. In immature cells, a distal enhancer bound by GATA-2 is in physical proximity with the active Kit promoter. Upon cell maturation, GATA-1 displaces GATA-2 and triggers a loss of the enhancer/promoter interaction. Moreover, GATA-1 reciprocally increases the proximity in nuclear space among distinct downstream GATA elements. GATA-1-induced transitions in chromatin conformation are not simply the consequence of transcription inhibition and require the cofactor FOG-1. This work shows that a GATA factor exchange reconfigures higher-order chromatin organization, and suggests that de novo chromatin loop formation is employed by nuclear factors to specify repressive outcomes.
GATA-1 and GATA-2 control proliferation and differentiation of hematopoietic progenitor cells via transcriptional regulation. In this issue of Molecular Cell, Jing et al. (2008) demonstrate that GATA factor exchange on the Kit locus directs a transcriptional switch by reconfiguring chromatin loops.
Histone deacetylase (HDAC) inhibitors repress interleukin-2 (IL-2) gene expression in T cells and possess immunosuppressive activity in vivo. In addition to its immunosuppressive activity, HDAC inhibitors block GATA binding protein-1 (GATA-1) gene expression in megakaryocytes and elicit thrombocytopenia. In this report we state that for a given immunosuppressive dose of HDAC inhibitor, the ratio of GATA-1 reporter gene activity relative to IL-2 reporter gene assay (G/I ratio of measured IC(50)) can be predictive of a HDAC inhibitor's thrombocytopenic effect. This study utilized nine HDAC inhibitors at a minimal effective dose in a rat heterotopic cardiac transplantation model and the resultant G/I ratios and platelet depletion rates were highly correlated (r=0.933). These results indicate that calculation of G/I ratio can be a novel method for selecting immunosuppressive HDAC inhibitor having minimal thrombocytopenic effect which will benefit the search for new immunosuppressants of greater safety and efficacy.
To assess whether alterations in the stromal cell-derived factor-1 (SDF-1)/CXCR4 occur in patients with primary myelofibrosis (PMF) and in Gata1 low mice, an animal model for myelofibrosis, and whether these abnormalities might account for increased stem/progenitor cell trafficking.
In the mouse, SDF-1 mRNA levels were assayed in liver, spleen, and marrow. SDF-1 protein levels were quantified in plasma and marrow and CXCR4 mRNA and protein levels were evaluated on stem/progenitor cells and megakaryocytes purified from the marrow. SDF-1 protein levels were also evaluated in plasma and in marrow biopsy specimens obtained from normal donors and PMF patients.
In Gata1 low mice, the plasma SDF-1 protein was five times higher than normal in younger animals. Furthermore, SDF-1 immunostaining of marrow sections progressively increased with age. Similar abnormalities were observed in PMF patients. In fact, plasma SDF-1 levels in PMF patients were significantly higher (by twofold) than normal (p < 0.01) and SDF-1 immunostaining of marrow biopsy specimens demonstrated increased SDF-1 deposition in specific areas. In two of the patients, SDF-1 deposition was normalized by curative therapy with allogenic stem cell transplantation. Similar to what already has been reported for PMF patients, the marrow from Gata1 low mice contained fewer CXCR4 pos CD117 pos cells and these cells expressed low levels of CXCR4 mRNA and protein.
Similar abnormalities in the SDF-1/CXCR4 axis are observed in PMF patients and in the Gata1 low mice model of myelofibrosis. We suggest that these abnormalities contribute to the increased stem/progenitor cell trafficking observed in this mouse model as well as patients with PMF.
GATA-1 is a hematopoietic transcription factor that is essential for the terminal maturation of proerythroblasts, megakaryocytic cells and mast cells. The erythroid-specific promoter of the human GATA-1 gene directs the high expression of a reporter gene in K562 cells. Multiple putative transcription factor binding sites were identified in the promoter from the -860 to the -1 base pair (bp). For a better understanding of the transcriptional control of human GATA-1 gene expression, we tested the transcriptional activity of a series of deletions from the 5' end of the 860-bp promoter. A region between -221 and -128 bp retains most of the transcriptional activity of the full-length promoter. Deletion of the CGCCC box at -195 bp reduced reporter gene activity to 60.4%. Further deletion of the CACCC box at -173 bp nearly abolished reporter gene expression, indicating that the CACCC box is more critical. In vitro experiments of electrophoretic mobility shifts and in vivo studies using chromatin immuno-precipitation (ChIP) assays show that the Sp1/Sp3 proteins bind the CACCC site in the nuclei of K562 cells. Coincidently, hyperacetylation of histones in the GATA-1 erythroid promoter was also shown by ChIP assay. Co-transfection of Sp1 expression plasmids and plasmids with a wild-type promoter showed enhanced reporter gene activity in a dose-dependent manner. The combined data demonstrate that Sp1/Sp3, but not EKLF, is involved in the activation of the GATA-1 erythroid promoter, and that histones H3 and H4 are highly acetylated in this promoter region for an actively transcribed GATA-1 gene in K562 cells in which EKLF is barely detectable.
We investigated erythroid-specific expression of the human PPOX gene. This gene encodes protoporphyrinogen oxidase, which is involved in synthesizing heme for red blood cells and heme as a cofactor for the respiratory cytochromes. In vitro luciferase transfection assays in human uninduced and hemin induced erythroleukemic K562 cells showed that the presence of exon 1 increased promoter activity fourfold as compared to reporter constructs lacking this exon. This transcriptional regulation was mediated by two GATA-1 sites in exon 1. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that both GATA sites were able to bind GATA-1 in vitro and in vivo. Exon 1 did not affect promoter activity in human hepatoma HepG2 cells and U937 monocytic cells but its presence decreased promoter activity in HeLa human cervical carcinoma cells. We conclude that the GATA-1 binding sites in exon 1 constitute key regulatory elements in differential expression of PPOX in erythroid and non-erythroid cells.
Most bilaterian animals have evolved a through gut that is regionally specialized along the anterior-posterior axis. In the polychaete annelid, Capitella sp. I, the alimentary canal is subdivided into a buccal cavity, pharynx, esophagus, midgut, and hindgut. Members of the Fox and GATA families of transcription factors have conserved functions in patterning ectodermal and endodermal gut components. We have isolated and characterized expression of one FoxA gene (CapI-foxA) and four GATA genes (CapI-gataB1, CapI-gataB2, CapI-gataB3, and CapI-gataA1) from Capitella sp. I. Both gene families are expressed in the developing gut of this polychaete. CapI-foxA, an ortholog of the FoxA subgroup, is expressed in vegetal hemisphere micromeres of cleavage-stage embryos, in multiple blastomeres within and surrounding the blastopore during gastrulation, and throughout morphogenesis of the pharynx, esophagus, and hindgut. The CapI-gataB genes group within the vertebrate GATA4/5/6 subfamily, appear to be products of lineage-specific gene duplication, and are expressed in specific domains of endomesoderm. CapI-gataB1 is expressed in endoderm precursors and throughout developing midgut endoderm, and is particularly prominent at anterior and posterior midgut boundaries. CapI-gataB2 is co-expressed with CapI-gataB1 in midgut endoderm, and is also expressed in visceral mesoderm. CapI-gataB3 is limited to and coexpressed with CapI-gataB2 in visceral mesoderm. CapI-gataA1 groups within the vertebrate GATA1/2/3 subfamily and is expressed primarily in ectodermal tissues of the brain, ventral nerve cord, lateral trunk, and both pharyngeal and esophageal regions of the foregut. Collectively, the CapI-foxA and CapI-gata genes show patterns of expression that span almost the entire length of the developing alimentary canal, consistent with a role in gut development.
AML1-ETO is one of the most common chromosomal translocation products associated with acute myelogenous leukemia (AML). Patients carrying the AML1-ETO fusion gene exhibit an accumulation of granulocyte precursors in the bone marrow and the blood. Here, we describe a transgenic zebrafish line that enables inducible expression of the human AML1-ETO oncogene. Induced AML1-ETO expression in embryonic zebrafish causes a phenotype that recapitulates some aspects of human AML. Using this highly tractable model, we show that AML1-ETO redirects myeloerythroid progenitor cells that are developmentally programmed to adopt the erythroid cell fate into the granulocytic cell fate. This fate change is characterized by a loss of gata1 expression and an increase in pu.1 expression in myeloerythroid progenitor cells. Moreover, we identify scl as an early and essential mediator of the effect of AML1-ETO on hematopoietic cell fate. AML1-ETO quickly shuts off scl expression, and restoration of scl expression rescues the effects of AML1-ETO on myeloerythroid progenitor cell fate. These results demonstrate that scl is an important mediator of the ability of AML1-ETO to reprogram hematopoietic cell fate decisions, suggesting that scl may be an important contributor to AML1-ETO-associated leukemia. In addition, treatment of AML1-ETO transgenic zebrafish embryos with a histone deacetylase inhibitor, Trichostatin A, restores scl and gata1 expression, and ameliorates the accumulation of granulocytic cells caused by AML1-ETO. Thus, this zebrafish model facilitates in vivo dissection of AML1-ETO-mediated signaling, and will enable large-scale chemical screens to identify suppressors of the in vivo effects of AML1-ETO.
Whereas the transcription factors GATA-1 and GATA-2 function both uniquely and redundantly to control blood cell development, the process termed hematopoiesis, mechanisms underlying their unique versus common functions are poorly understood. We used two independent assays to demonstrate that GATA-1 is considerably more stable than GATA-2 in multiple cellular contexts, even though both factors are subject to degradation via the ubiquitin-proteasome system. Studies with GATA factor mutants and novel chimeric GATA factors provided evidence that both GATA-1 and GATA-2 have highly unstable zinc finger core modules. The GATA-1 and GATA-2 N-termini both confer stabilization to their respective zinc finger core modules. In contrast, the GATA-1 and GATA-2 C-termini confer stabilization and destabilization, respectively. As GATA-2 stabilization via proteasome inhibition impairs the capacity of GATA-1 to displace GATA-2 from endogenous chromatin sites, we propose that differential GATA factor stability is an important determinant of chromatin target site occupancy and therefore the establishment of genetic networks that control hematopoiesis.
Acute myeloid leukemia (AML) in Down syndrome (DS) children has several unique features including a predominance of the acute megakaryocytic leukemia (AMkL) phenotype, higher event-free survivals compared to non-DS children using cytosine arabinoside (ara-C)/anthracycline-based protocols and a uniform presence of somatic mutations in the X-linked transcription factor gene, GATA1. Several chromosome 21-localized transcription factor oncogenes including ETS2 may contribute to the unique features of DS AMkL. ETS2 transcripts measured by real-time RT-PCR were 1.8- and 4.1-fold, respectively, higher in DS and non-DS megakaryoblasts than those in non-DS myeloblasts. In a doxycycline-inducible erythroleukemia cell line, K562pTet-on/ETS2, induction of ETS2 resulted in an erythroid to megakaryocytic phenotypic switch independent of GATA1 levels. Microarray analysis of doxycycline-induced and doxycycline-uninduced cells revealed an upregulation by ETS2 of cytokines (for example, interleukin 1 and CSF2) and transcription factors (for example, TAL1), which are key regulators of megakaryocytic differentiation. In the K562pTet-on/ETS2 cells, ETS2 induction conferred differences in sensitivities to ara-C and daunorubicin, depending on GATA1 levels. These results suggest that ETS2 expression is linked to the biology of AMkL in both DS and non-DS children, and that ETS2 acts by regulating expression of hematopoietic lineage and transcription factor genes involved in erythropoiesis and megakaryopoiesis, and in chemotherapy sensitivities.
Long-range interactions between distant regulatory elements, such as enhancers, and their target genes underlie the specificity of gene expression in many developmentally regulated gene families. NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long-range interactions. Here, we show that NLI/Ldb1 and erythroid-binding partners GATA-1/SCL/LMO2 bind in vivo to the beta-globin locus control region (LCR). The C-terminal LIM interaction domain of NLI is required for formation of the complex on chromatin. Loss of the LIM domain converts NLI into a dominant-negative inhibitor of globin gene expression, and knockdown of NLI by using shRNA results in failure to activate beta-globin expression. Kinetic studies reveal that the NLI/GATA-1/SCL/LMO2 complex is detected at the beta-globin promoter coincident with RNA Pol II recruitment, beta-globin transcription, and chromatin loop formation during erythroid differentiation, providing evidence that NLI facilitates long-range gene activation.
To find out how GATA-1 and GATA-2 behave in the bone marrow of patients with Monge's disease.
The levels of mRNA in mononuclear cells (MNC) and proteins of GATA-1 and GATA-2 in the bone marrow of patients with Monge's disease and controls were determined by RT-PCR and immune cytolysis chemical method.
(1) All patients and controls expressed GATA-1 mRNA (Monge's disease 1.033 +/- 0.146, Control 0.458 +/- 0.076) and GATA-2 mRNA (Monge's disease 0.451 +/- 0.073, Control 0.185 +/- 0.074). All patients expressed both GATA-1 (positive cell counts 77.3 +/- 33.3, positive score 135.4 +/- 75.4) and GATA-2 ( positive cell counts 29.4 +/- 11.4, positive score 48.4 +/- 19.7). All the controls expressed GATA-1 (positive cell counts 18.1 +/- 11.3, positive score 24.2 +/- 13.4) while 12 of 20 controls expressed GATA-2 ( positive cell counts 5.4 +/- 3.0, positive score 7.3 +/- 4.2). The expression of mRNA and proteins of GATA-1 and GATA-2 in Monge's disease were higher than in controls (P < 0.01). (2) There was a positive correlation between GATA-1 and Hb (P < 0.01), as did between mRNA and proteins of GATA-1 and GATA-2. (3) Both the proteins of GATA-1 and GATA-2 located only in the cytoplasm but not the nucleus.
Two of inherent genes, GATA-1 and GATA-2 which were expressed at higher levels in patients with Monge's disease than in controls might play significant roles in the pathogenesis of Monge's disease.
Available data suggest that gene regulation by the Gata-1 Hematopoietic Regulatory Domain (Gata-1-HRD) is limited to cells derived from the erythroid lineage. This characteristic makes Gata-1-HRD a candidate for control of cre expression in conditional knock-in and knock-out models in which erythroid-specific gene expression is essential. To characterize the specificity of Gata-1 HRD regulation of cre, transgenic mice expressing improved cre recombinase (iCre) under the control of Gata-1-HRD were generated. The founders were crossbred with mice that have an inactive loxP-containing beta-galactosidase gene that can be rescued by the cre recombinase. The beta-galactosidase activity was detected in the marrow of this crossbred mouse, but no activity was observed in other organs. To identify the cre expressing cells in marrow, double-immunostaining of marrow sections with anti-beta-galactosidase, and antibodies against various hematopoietic lineage markers or erythropoietin receptor (epor) was performed. The epor positive cells in marrow expressed beta-galactosidase, but megakaryocytic precursors and nonerythroid epor-positive cells in brain and spleen did not. We conclude that when cre is under control of Gata-1-HRD, its expression/function is limited to erythroid progenitors. The knock-in and knock-out models utilizing Gata-1-HRD-iCre, can be explored for the studies of erythroid-specific gene expression.
The prognosis of leukemia developed in Down syndrome (DS) patients has improved markedly. Most DS leukemia occurs before 3 years of age and is classified as acute megakaryocytic leukemia (AMKL). Mutations in the GATA1 gene have been found in almost all DS patients with AMKL. In contrast, it has been shown that occurrence of DS acute myeloid leukemia (DS-AML) after 3 years of age may indicate a higher risk for a poor prognosis, but its frequency is very low. Age is one of the significant prognostic indicators in DS-AML. The prognostic factor of gene alterations has not been reported in older DS-AML patients. We here describe the case of a 7-year-old DS boy with AML-M2, who had no history of transient abnormal myelopoiesis or any clinical poor prognostic factors, such as high white blood cell counts or extramedullary infiltration. We molecularly analyzed the GATA1, FLT3, MLL-partial tandem duplication, NRAS, and RUNX1 (previously AML1) genes and did not detect any alterations. The patient has lived for more than 5 years after treatment on the AML99-Down protocol in Japan. This suggests that a patient lacking these genes alterations might belong to a subgroup of older DS-AML patients with good prognosis. Accumulation of more data on older pediatric DS-AML patients is needed.
Friend of GATA-1 (FOG-1) is a binding partner of GATA-1, a zinc finger transcription factor with crucial roles in erythroid, megakaryocytic, and mast-cell differentiation. FOG-1 is indispensable for the function of GATA-1 during erythro/megakaryopoiesis, but FOG-1 is not expressed in mast cells. Here, we analyzed the role of FOG-1 in mast-cell differentiation using a combined experimental system with conditional gene expression and in vitro hematopoietic induction of mouse embryonic stem cells. Expression of FOG-1 during the progenitor period inhibited the differentiation of mast cells and enhanced the differentiation of neutrophils. Analysis using a mutant of PU.1, a transcription factor that positively or negatively cooperates with GATA-1, revealed that this lineage skewing was caused by disrupted binding between GATA-1 and PU.1, which is a prerequisite for mast-cell differentiation. However, FOG-1 expression in mature mast cells brought approximately a reversible loss of the mast-cell phenotype. In contrast to the lineage skewing, the loss of the mast-cell phenotype was caused by down-regulation of MITF, a basic helix-loop-helix transcription factor required for mast-cell differentiation and maturation. These results indicate that FOG-1 inhibits mast-cell differentiation in a differentiation stage-dependent manner, and its effects are produced via different molecular mechanisms.
Thrombocytosis is associated with inflammation, and certain inflammatory cytokines, including IFN-gamma, stimulate megakaryocyte and platelet production. However, the roles of IFN-gamma and its downstream effector STAT1 in megakaryocyte development are poorly understood. We previously reported that STAT1 expression was significantly downregulated in Gata1-knockdown murine megakaryocytes, which also have impaired terminal maturation. Here, we show that ectopic expression of STAT1, or its target effector IRF-1, rescued multiple defects in Gata1-deficient megakaryopoiesis in mice, inducing polyploidization and expression of a subset of platelet-expressing genes. Enforced expression of STAT1, IRF-1, or GATA-1 enhanced phosphorylation of STAT1, STAT3, and STAT5 in cultured Gata1-deficient murine megakaryocytes, with concomitant megakaryocyte maturation. In contrast, enhanced thrombopoietin signaling, conferred by enforced expression of constitutively active JAK2 or c-MPL, induced phosphorylation of STAT3 and STAT5, but not STAT1, and failed to rescue megakaryocyte maturation. Finally, megakaryocytes from Stat1(-/-) mice were defective in polyploidization. Together, these findings reveal a unique role for STAT1 in megakaryopoiesis and provide new insights into how GATA-1 regulates this process. Our studies elucidate potential mechanisms by which various inflammatory disorders can cause elevated platelet counts.
Previous investigations from our laboratory have characterized the ultrastructural and cytochemical pathology of platelets in male members of a family with X-linked GATA-1 G208S macrothrombocytoenia. A large proportion of their circulating platelets were hypogranular macrothrombocytes, resembling cells from patients with the Gray Platelet Syndrome. However, most of the GATA-1 macrothrombocytes contained some alpha granules, and a small number had as many as are present in normal platelets. GATA-1 macrothrombocytes also contained tubular inclusions formed from elements of the dense tubular system wrapped around each other like scrolls. Many macrothrombocytes contained flat tubular membrane sheets connected to channels of the open canalicular system, platelets in platelets and platelets attached to platelets forming very large macrothrombocytes. The present study has examined one obligate and three potential female carriers in this family. Thin sections of their platelets examined in the electron microscope revealed features consistent with the pathology observed in male family members. Most of their platelets were normal-sized, discoid cells containing the usual complement of alpha and delta storage organelles and channels of the dense tubular system and OCS. However, a significant number of giant platelets containing the usual frequency of alpha and delta granules and hypogranular and agranular giant platelets were observed. The frequency of the macrothrombocytes varied in each of the four women studied, but were present in all. The ability of their platelets to bind multimers of vWF, in contrast to male family members, did not differ from normal controls. Near normal as well as normal platelet counts and the ability of their platelets to bind vWF multimers may protect them from the serious bleeding problems of males with the X-linked GATA-1 G208S mutation. Our findings indicate that obligate female carriers of the GATA-1 gene can be detected by examination of their platelets in the electron microscope and distinguished from the pathology of other giant platelet disorders.
Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34(+) cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34(+) cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.
In agar cultures of marrow cells from adult female BALB/c chimeric GATA-1(Plt13/+) mice, a high frequency of unusual dispersed colonies was noted. Analysis showed that these were colonies of mast cells and that mast cell colony-forming cells (progenitors) could be detected in clonal cultures of adult marrow, neonatal marrow, or fetal liver if the combined stimulus of stem cell factor and interleukin-3 was used. Mast cell progenitors were in active cell cycle and showed an extensive capacity for self-generation. Mast cell colonies both from control GATA-1(+/+) mice and GATA-1(Plt13/+) mice could generate growth factor-dependent cloned cell lines that grew for >18 months. Surprisingly, the majority of the excessive numbers of mast cell progenitors in chimeric GATA-1(Plt13/+) mice were transcribing the inactive Plt13 allele of GATA-1, suggesting that GATA-1 normally acts to restrict the emergence of committed mast cell progenitors. In sharp contrast, all eosinophil progenitors in these mice were transcribing the normal GATA-1 allele. No excess tissue mast cells were observed in GATA-1(Plt13/+) mice, suggesting that the excess mast cell progenitors in these mice might be generating mast cells with a defective in vivo proliferative or tissue homing capacity.
Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than unaffected children, and most leukemia cases with DS present as acute megakaryocytic leukemia (AMKL). At least 10% of neonates with DS develop transient myeloproliferative disorder (TMD), and 20% to 30% of patients with TMD develop AMKL. Mutations in the GATA1 gene are identified not only in AMKL patients but also in TMD patients; however, sequential analysis of GATA1 is not often performed in the same patients. We describe a child with DS who developed TMD followed by AMKL and have identified different mutations in the GATA1 gene during the course of TMD and AMKL. Distinct clones were associated with the development of TMD and AMKL in this patient.
Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development.
When the orphan nuclear receptors TR2 and TR4, the DNA-binding subunits of the DRED repressor complex, are forcibly expressed in erythroid cells of transgenic mice, embryos exhibit a transient mid-gestational anemia as a consequence of a reduction in the number of primitive erythroid cells. GATA-1 mRNA is specifically diminished in the erythroid cells of these TR2/TR4 transgenic embryos as it is in human CD34(+) progenitor cells transfected with forcibly expressed TR2/TR4. In contrast, in loss-of-function studies analyzing either Tr2- or Tr4-germline-null mutant mice or human CD34(+) progenitor cells transfected with force-expressed TR2 and TR4 short hairpin RNAs (shRNAs), GATA-1 mRNA is induced. An evolutionarily conserved direct repeat (DR) element, a canonical binding site for nuclear receptors, was identified in the GATA1 hematopoietic enhancer (G1HE), and TR2/TR4 binds to that site in vitro and in vivo. Mutation of that DR element led to elevated Gata1 promoter activity, and reduced promoter responsiveness to cotransfected TR2/TR4. Thus, TR2/TR4 directly represses Gata1/GATA1 transcription in murine and human erythroid progenitor cells through an evolutionarily conserved binding site within a well-characterized, tissue-specific Gata1 enhancer, thereby providing a mechanism by which Gata1 can be directly silenced during terminal erythroid maturation.
It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.
Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.
Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute leukemia. An estimated 10% of newborns with DS develop Transient Myeloproliferative Disease (TMD) or Transient Leukemia (TL), a clonal accumulation of megakaryoblasts that resolves spontaneously within months. Acute megakaryoblastic leukemia (AMKL) develops in approximately 20% of cases of TMD/TL by 4 years of age. Both the blasts of AMKL and TMD/TL in DS harbor somatic mutations of GATA1, an essential transcriptional regulator of megakaryocytic differentiation. The distinct phenotypes of megakaryoblastic leukemia in DS are a unique biological model of the incremental process of leukemic transformation.
Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, and SRD5A1 in Jurkat; GH up-regulated SRD5A1 and GHR in K562. GATA1 expression was down-regulated in IGF-1-treated HL60, ESR2 was down-regulated in nandrolone-treated Jurkat, and AR and PGR were down-regulated in GH-treated Jurkat. This pilot study shows the potential of molecular biology-based strategies in anti-doping controls.
Ikaros--a factor that positively or negatively controls gene transcription--is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)--a known dominant--negative protein that interferes with normal Ikaros activity-in cord blood or apheresis CD34(+) cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. In secondary cultures, expression of Ik6 favored reversion of sorted and phenotypically defined erythroid cells into myeloid cells, and prevented reversion of myeloid cells into erythroid cells. We conclude that Ikaros is involved in human adult or fetal erythroid differentiation as well as in the commitment between erythroid and myeloid cells.
When a human leukemic cell line, HT93 was incubated with all-trans retinoic acid (ATRA), IL-5, or both, this cell line was differentiated into eosinophic lineage, in that an eosinophilic specific granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO) appeared. Both CD11b and CC chemokine receptor, CCR3 expression were upregulated, while CD71 expression was downregulated by ATRA or ATRA+IL-5. Concomitantly, marked production of eotaxin-2/CCL24 was observed, but no production of eotaxin-1/CCL11 and eotaxin-3/CCL26 was detected. Since only 20 to 30% cells incubated with ATRA became positive for CCR3, CCR3(+) population was enriched by a magnetic activated cell sorter (MACS). Enriched CCR3(+) population produced higher eotaxin-2/CCL24 than the CCR3(-) population, indicating that differentiated eosinophils are capable of producing eotaxin-2/CCL24. During the ATRA-induced differentiation, expression of a transcriptional factor, GATA-1 was significantly increased. Introduction of siRNA against GATA-1 markedly reduced the ATRA-induced differentiation markers including CD11b and CCR3, as well as reduced eotaxin-2/CCL24 production. Finally, ATRA-induced differentiation and eotaxin-2/CCL24 production were greatly enhanced in the GATA-1-overexpressed clones. These results indicate that the ability to produce eotaxin-2/CCL24 is acquired during the differentiation into eosinophilic lineage which is dependent on GATA-1 expression.
We report a new case of transient myeloproliferative disorder (TMD) in a non Down syndrome neonate. The cytogenetic and molecular studies within from the blood blast cells identified a trisomy 21 and a partial deletion in exon 2 of the transcription factor GATA1. Spontaneous regression of the TMD was achieved at the age of 1 month as the clonal and molecular abnormalities. A survey by periodic cytological examinations of peripheral blood cells and GATA1 mutation analysis was instituted since three years and has not detected up to date acute leukaemia.
The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood.
To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription.
The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.
EWS/FLI-1 is a chimeric oncogene generated by chromosomal translocation in Ewing tumors, a family of poorly differentiated pediatric tumors arising predominantly in bone but also in soft tissue. The fusion gene combines sequences encoding a strong transactivating domain from the EWS protein with the DNA binding domain of FLI-1, an ETS transcription factor. A related fusion, TLS/ERG, has been found in myeloid leukemia. To determine EWS/FLI-1 function in vivo, we engineered mice with Cre-inducible expression of EWS/FLI-1 from the ubiquitous Rosa26 locus. When crossed with Mx1-cre mice, Cre-mediated activation of EWS/FLI-1 resulted in the rapid development of myeloid/erythroid leukemia characterized by expansion of primitive mononuclear cells causing hepatomegaly, splenomegaly, severe anemia, and death. The disease could be transplanted serially into naïve recipients. Gene expression profiles of primary and transplanted animals were highly similar, suggesting that activation of EWS/FLI-1 was the primary event leading to disease in this model. The Cre-inducible EWS/FLI-1 mouse provides a novel model system to study the contribution of this oncogene to malignant disease in vivo.
Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.
A previous investigation detailed the pathology of platelets in a family with the X-linked GATA-1 G208S mutation causing dyserythropoiesis and megathrombocytopenia. The present study has used ultrastructural immunocytochemistry, cytochemistry, and tannic acid staining to answer questions raised in the original investigation. Earlier studies, as well as ours, had shown that GATA-1 megathrombocytes are hypogranular, but did not definitively determine which organelles are decreased. Cytochemical localization of aryl sulfatase revealed that lysosomes were present in normal numbers, and the whole mount technique showed a normal frequency of dense bodies rich in arlenine nucleotides and serotonin. Thus alpha granules were the only organelles deficient in GATA-1 platelets. Tannic acid staining confirmed that the membranes wrapped around each other to form tubular inclusions come from elements of the dense tubular system. The unique tubular membrane inclusions in GATA-1 megathrombocytes, thought originally to derive from endoplasmic reticulum in the parent cell, were shown to be in direct continuity with elements of the surface connected open canalicular system (OCS), and to drive from the demarcation membrane system (DMS) of the megakaryocyte. Platelets in platelets and platelets in platelets in platelets were independent cells, and not derived by cytoplasmic sequestration in the enclosing macrothrombocytes. Fully spread GATA-1 platelets incubated with fibrinogen coated gold (Fgn/Au) particles before or after fixation bound as many Fgn/Au particles as normal spread platelets and moved the Fgn/Au- GPIIb/IIIa complexes from peripheral margins to cell centers and into channels of the OCS as efficiently. Exposure of spread normal platelets to bovine vWF resulted in coverage of the surface from edge to edge with multimers detected by anti-vWF antibody and protein A gold. Spread GATA-1 platelets bound very few vWF multimers, which were much smaller in size than those on normal spread cells, but were able to move then to cell centers. These findings support the concept that GATA-1 platelets are macrothrombocytes because they are not able to detach normally from each other during separation from megakaryocyte proplatelets. The marked decrease in the number and abnormal distribution of GPIb/IX receptors may play a role in GATA-1 megathrombocyte formation.
Runt-related transcription factor 1 (RUNX1) and GATA-1 are both transcription factors known to play essential roles in hematopoiesis. Genetic alterations of each are associated with abnormal platelet development, as well as predisposition to leukemia. In addition, in vitro and animal studies indicate that both factors are involved in megakaryopoiesis. We and others have previously shown that RUNX1 and GATA-1 physically interact and cooperate in the activation of megakaryocytic promoters such as alpha IIb integrin and glycoprotein Ibalpha. Moreover, transcriptional cooperation of RUNX1 with GATA-1 is conserved back to Drosophila in which RUNX1 and GATA-1 homologs cooperate in crystal cell development. In this article, we will review the molecular and functional significance of the transcriptional cross talk between RUNX1 and GATA-1. In particular, we will elaborate on recent data which suggest that GATA-1 targets RUNX1 for modification, in particular phosphorylation by cyclin-dependent kinases. Furthermore, targeting of RUNX1 by GATA-1 for phosphorylation may convert RUNX1 from a repressor to an activator. This is a potential mechanism of transcriptional cooperation and may be an essential step in megakaryocytic differentiation.
HOXA10 is a member of the HOX family of regulatory genes that are involved in hematopoiesis. Its role in megakaryopoiesis has been suggested by its expression in immature megakaryocytes and by the proliferation of megakaryocyte-primitive blast colonies upon HOXA10 overexpression. We sought to understand the role of HOXA10 in megakaryopoiesis better, by investigating its transcriptional regulation. Analysis of the 5' untranslated region and transfection of promoter/plasmids into human tissue culture cell lines identified transcriptionally active sequences that contain GATA-1 and Ets-1 sites and a putative binding site for its neighboring gene, HOXA11. Gel shift assays confirmed protein-DNA interactions at these sites. Mutation of the GATA-1 and the Ets-1 motifs amplified the expression of HOXA10 in HEL and K562 cells, confirming the importance of these cis-acting elements in regulating HOXA10 expression in megakaryocytic cells. Chromatin immunoprecipitation (ChIP) and chloramphenicol acetyl transferase (CAT) assays confirm that HOXA11 binds to the putative binding site, resulting in repression of HOXA10 expression. These data taken together give insight into the regulation of HOXA10 expression in megakaryocytic differentiation.
Activated mast cells produce Th2 cytokines that regulate allergic inflammation. We have previously shown that thymoquinone (TQ) attenuated airway inflammation in a mouse model of allergic airway inflammation. The present study investigated whether TQ affects Th2 cytokine response in vitro in lipopolysaccharide (LPS)-activated rat mast cells, RBL-2H3.
RBL-2H3 cells were stimulated for 12 h with 0.1 microg/ml LPS in the presence or absence of 10 microM TQ. Th2 cytokine production was measured in the culture supernatants by ELISA. The mRNA expression of IL-5, IL- 13 and GATA transcription factors was determined by RT-PCR. The expression of the transcription proteins c-Fos, c- Jun and phospho-c-Jun were determined by western blotting. The in vivo binding of GATA, AP-1 and NF-AT transcription factors to IL-5 promoter was assessed by chromatin immunoprecipitation analysis.
TQ significantly (p <0.05) inhibited LPS-induced IL-5 and IL-13 mRNA expression and protein production. However, TQ did not affect IL-10 production. GATA transcription factors are involved in the transcription of IL-5 and IL-13. TQ had no effect on the expression of AP-1 protein subunits, c-Jun and c-Fos, but markedly reduced the transcription of GATA-1 and -2 genes. Chromatin immunoprecipitation revealed that GATA, AP-1 and NF-AT binding to IL-5 promoter was induced by LPS stimulation and that TQ inhibited GATA binding at the IL-5 promoter but did not affect AP-1 and NF-AT binding.
These results suggest that TQ inhibits LPS-induced proinflammatory cytokine production in RBL-2H3 cells by blocking GATA transcription factor expression and promoter binding which demonstrates the anti-inflammatory effect of TQ.
The role of ischemia in collateral vessel development (arteriogenesis) is a contentious issue that cannot be addressed using mammalian models. To investigate this, we developed models of arteriogenesis using the zebrafish embryo, which gains sufficient oxygenation via diffusion to prevent ischemia in response to arterial occlusion.
We studied gridlock mutant embryos that suffer a permanently occluded aorta and show that these restore aortic blood flow by collateral vessels. We phenocopied gridlock mutants by laser-induced proximal aortic occlusion in transgenic Fli1:eGFP/GATA1:dsRED embryos. Serial imaging showed these restore aortic blood flow via collateral vessels by recruitment of preexisting endothelium in a manner similar to gridlocks. Collateral aortic blood flow in gridlock mutants was dependent on both nitric oxide and myeloid cells. Confocal microscopy of transgenic gridlock/Fli1:eGFP mutants demonstrated no aberrant angiogenic response to the aortic occlusion. qPCR of HIF1alpha expression confirmed the absence of hypoxia in this model system.
We conclude that NO and myeloid cell-dependent collateral vessel development is an evolutionarily ancient response to arterial occlusion and is able to proceed in the absence of ischemia.
In many developing tissues, signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Notch receptors are expressed on hematopoietic cells and cognate ligands on bone marrow stromal cells. Here, we investigate the role of mNotch1 signaling in the control of erythroid differentiation of multipotent progenitor cells.
Multipotent FDCP-mix cell lines engineered to permit the conditional induction of the constitutively active intracellular domain of mNotch1 (mN1(IC)) by the 4-hydroxytamoxifen (OHT)-inducible system were used to analyze the effects of activated mNotch1 on erythroid differentiation and on expression of Gata1, Fog1, Eklf, NF-E2, and beta-globin. Expression was analyzed by Northern blotting and real-time polymerase chain reaction. Enhancer activity of reporter constructs was determined with the dual luciferase system in transient transfection assays.
Induction of mN1(IC) by OHT resulted in increased and accelerated differentiation of FDCP-mix cells along the erythroid lineage. Erythroid maturation was induced by activated Notch1 also under conditions that normally promote self-renewal, but required the presence of erythropoietin for differentiation to proceed. While induction of Notch signaling rapidly upregulated Hes1 and Hey1 expression, the expression of Gata1, Fog1, Eklf, and NF-E2 remained unchanged. Concomitantly with erythroid differentiation, activated mNotch1 upregulated beta-globin RNA. Notch signaling transactivated a reporter construct harboring a conserved RBP-J (CBF1) binding site in the hypersensitive site 2 (HS2) of human beta-globin. Transactivation by activated Notch was completely abolished when this RBP-J site was mutated to prevent RBP-J binding.
Our results show that activation of mNotch1 induces erythroid differentiation in cooperation with erythropoietin and upregulates beta-globin expression.
Histone deacetylase inhibitors (HDAC inhibitors) are an emerging class of anticancer agents. To elucidate the mechanism of HDAC inhibitor-induced thrombocytopenia, we focused on the effects of HDAC inhibitors on megakaryocyte differentiation and performed Affymetrix GeneChip analysis of human megakaryocytic HEL cells treated with or without HDAC inhibitors. Here, we report that GATA-1 and 10 haematopoietic factors (SCL, NF-E2, EKLF, Pleckstrin, Thrombin-R, LMO2, PU.1, Fli-1, AML1, and TCF11) are transcriptionally repressed by HDAC inhibitors in a similar pattern (R>0.98), and putative GATA-1-binding sites are found in almost all promoters of these genes. In addition, luciferase reporter assays reveal that mutations of GATA-1-binding sites in the GATA-1 promoter abolish its sensitivity to HDAC inhibitor-mediated down-regulation in HEL cells. Further, this report also asserts that HDAC inhibitor increases megakaryocyte counts and inhibits GATA-1 gene expression in rat spleen. Together, these results suggest that HDAC inhibitors inhibit GATA-1 gene expression by decreasing the transactivation function of GATA-1 itself, and that this may in turn lead to a delay in megakaryocyte maturation and finally cause thrombocytopenia. Our findings may help our understanding of the molecular mechanism of HDAC inhibitor-mediated GATA-1 transcriptional repression and to reduce the risk of HDAC inhibitor-induced thrombocytopenia.
SET-CAN associated with the t(9;9) in acute undifferentiated leukemia encodes almost the entire sequence of SET and the C-terminal two-third portion of CAN, including the FG repeat region. To clarify a role(s) of SET-CAN in leukemogenesis, we developed transgenic mice expressing SET-CAN under the control of the Gata1 gene hematopoietic regulatory domain that is active in distinct sets of hematopoietic cells. SET-CAN transgenic mice showed anemia, thrombocytopenia, and splenomegaly. A significant number of transgenic mice started dying after 6 months post-birth, being in good agreement with the fact that red blood cells and platelets decreased. We found that a significant number of c-kit+ myeloid cells appeared in peripheral blood in transgenic mice. Characterization of the bone marrow cells of transgenic mice indicated impairment in hematopoietic differentiation of erythroid, megakaryocytic, and B cell lineages by SET-CAN. Transgenic mice, in particular, exhibited a high population of the c-kit+Sca-1+Lin- fraction in bone marrow cells compared with that of the control littermates. Our results demonstrate that SET-CAN blocks the hematopoietic differentiation program--one of the characteristics of acute myeloid leukemia.
For the proper development of vertebrate embryos as well as for survival of the adult organism, it is essential to form a functional vascular system. Molecules involved in this process are members of highly conserved families of proteins that exert conserved functions across species. Bone morphogenetic proteins (BMP) are extracellular factors that are regulated by extracellular modulators and bind to BMP receptors, which in turn activate intracellular signaling cascades. BMPs are necessary not only for induction of endothelial and hematopoietic lineages but also for further endothelial and hematopoietic cell differentiation. Previously, we identified BMPER (BMP endothelial cell precursor derived regulator) and demonstrated its spatiotemporal expression at sites of vasculogenesis and direct modulation of BMP activity. To directly investigate the role of BMPER in vascular development, we cloned the BMPER ortholog in zebrafish (zbmper). It is expressed at sites of high BMP activity, including vascular precursor cells located in the aortic arches and the intermediate cell mass during zebrafish embryonic development. Knockdown of zbmper results in a dorsalized phenotype, a reduced number of gata1 expressing hematopoietic precursor cells and of circulating blood cells as well as in a vascular phenotype. The generation of the caudal vein is compromised and the pattern guiding of the intersomitic vessels is disturbed, indicating that zbmper is required for early steps in vascular pattern formation and hematopoiesis in zebrafish.
To gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology.
Consistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFkappaBIA; a.k.a I-kappa-B-alpha, IKBalpha) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis.
This study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.
Capsaicin, the pungent component of chilli peppers, is known to induce mediators of hematopoiesis. We investigated the effect of capsaicin on hematopoiesis in mouse progenitor cells. Treatment of mouse bone marrow cells with capsaicin induced the formation of colony of burst-forming units-erythroid (BFU-E). We also found that the number of erythropoietin receptor (EpoR)-positive cells was increased by capsaicin. To clarify the effect of capsaicin on erythroid lineage, BFU-E colonies were separated from non-BFU-E colonies by colony-picking after in vitro culture of mouse bone marrow cells. Quantitative RT-PCR analysis revealed that capsaicin stimulated the expression of the erythroid-specific genes encoding EpoR, glycophorin A (GPA), beta-globin (Hbb-b1), GATA-1, PU.1, nuclear factor erythroid-derived 2 (NF-E2), and Krüppel-like factor 1 (KLF1) in the BFU-E colonies. Furthermore, capsaicin could effectively stimulate the transfected GATA-1 promoter in K562 cells. GATA-1 is known as an essential transcription factor for the development of erythroid cells. Our results show that development of the erythroid lineage from bone marrow cells can be induced by treatment with capsaicin, and that GATA-1 seems to play a role in this induced erythroid maturation.
A 34-year-old woman was referred for evaluation of a recent stillborn male fetus, gestational age 27 6/7 weeks, found to have congenital myeloid leukemia at autopsy. Autopsy findings included high weight for gestational age, hepatomegaly, and extensive intravascular leukemic infiltrates in the placenta, heart, liver, thymus, lung, kidneys, and brain. Genetic consultation and examination of photographs of the fetus revealed dysmorphic features.
Immunoperoxidase staining of placental tissue, fluorescence in situ hybridization of paraffin-embedded sections of the placenta using probes for t(12;21)(p13;q22), t(8;21)(q22;q22) and t/del(11q23), cytogenetic analysis of fetal tissue (tendon), sequence analysis of GATA1 in placental leukemic cells, and parental chromosome studies.
Down syndrome with in utero onset of GATA1 mutation-positive severe transient myeloproliferative disorder/acute megakaryoblastic leukemia.
Genetic counseling for the recurrence risk of Down syndrome on the basis of maternal age.
Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2'-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.
Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.
PGE(2) is involved in a wide variety of physiological and pathological processes; however, deciphering its role in early mammalian development has been difficult due to the maternal contribution of PGE(2). To overcome this limitation we have investigated the role of PGE(2) during T cell development in zebrafish. In this study, we show that zebrafish ep4a, a PGE(2) receptor isoform of EP4, is expressed at 26 h postfertilization in the dorsal aorta-posterior cardinal vein joint region, which has a high homology with the mammal aorta-gonad-mesonephros area and where definitive hemopoiesis arises. Furthermore, it is expressed in the presumptive thymus rudiment by 48 h postfertilization. Supplementation of PGE(2) results in a strong increase in rag1 levels and cell proliferation in the thymus. In contrast, the inhibition of PGE(2) production, as well as EP4 blockade, abrogates the expression of rag1 in the thymus and that of the lymphoid precursor marker ikaros, not only in the dorsal aorta-posterior cardinal vein joint region but also in the newly identified caudal hemopoietic tissue without affecting early hemopoietic (scl, gata2) and erythropoietic (gata1) markers. These results identify ep4a as the earliest thymus marker and define a novel role for the PGE(2)/EP4 pathway in controlling T cell precursor development in zebrafish.
Somatic mutations in the GATA1 gene are present in almost all cases of Down syndrome (DS)-associated acute megakaryoblastic leukemia (AMKL) and transient leukemia (TL). An in utero origin of the GATA1 mutation suggests it is an early leukemogenic event. To determine the detectable incidence and clinical relevance of GATA1 mutations in DS newborns, we screened Guthrie cards from 590 DS infants for mutations in the GATA1 gene. Twenty-two (3.8%) of 585 evaluable infants harbored a predicted functional GATA1 mutation; 2 were identified exclusively within intron 1. Hispanic newborns were 2.6 times more likely to have a mutated GATA1 gene than non-Hispanics (P = .02). Two newborns with a GATA1 mutation subsequently developed AMKL, and none of the infants without a functional GATA1 mutation were reported to have developed leukemia. In addition to screening for TL, a GATA1 mutation at birth might serve as a biomarker for an increased risk of DS-related AMKL.
GATA-1 is a transcription factor governing the production of erythroid and megakaryocytic cells. Unobstructed GATA-1 expression in early progenitor cells commits them to the myeloid lineage, channeling its differentiation towards erythrocytes and megakaryocytes. Myelodysplastic Syndromes (MDS) are clonal disorders of the hematopoietic stem cell frequently presenting dysplasia in erythroid and/or megakaryocytic lineage. We reasoned that measurement of GATA-1 expression levels in hematopoietic progenitor CD34(+) and the committed erythroid CD71(+) cells, from various MDS subcategories, could demonstrate GATA-1 involvement in the pathogenesis of the syndrome. In this study, MDS patients displayed significantly elevated GATA-1 mRNA expression, in bone marrow mononuclear cells (BMMCs), progenitor CD34(+) and erythroid CD71(+) cells in contrast to the control population (P < 0.001). Additionally, GATA-1 mRNA expression in MDS CD71(+) cells was positively correlated with their apoptotic levels (rho = 0.58, P = 0.03). Furthermore, GATA-1 expression levels were found to correlate with the disease progression. MDS patients in high/INT-2 IPSS risk group expressed significantly higher GATA-1 mRNA levels, in both CD34(+) and CD71(+) cells, as opposed to low/INT-1 patients (P < 0.001). Moreover, the former displayed increased apoptosis in the CD71(+) cells and significantly reduced neutrophil and platelet numbers and hemoglobin levels compared with the latter. We conclude that MDS patients display an increase of GATA-1 mRNA expression in BM cells, with high/INT-2 patients showing significantly higher levels. The higher level of GATA-1 mRNA in erythroid cells was positively correlated with their degree of apoptosis. These findings suggest that the up-regulation of GATA-1 may be responsible for the peripheral cytopenias in MDS.
GATA1 is one of the most fascinating transcription factors for biologists. It regulates many haematopoietic genes and interacts with a number of other transcription factors. Its functions are mostly conserved among vertebrates. Upon disruption of the GATA1 gene, mice show a drastic bloodless phenotype. GATA1 knockdown mice are predisposed to tumour development, the extent of which varies depending on the level of knockdown. When GATA1 is overexpressed, certain cells face an altered cell fate. These observations suggest the importance of regulation on the expression level and timing of GATA1. Deciphering such regulation is the key to understanding the commitment and differentiation of blood cells. There are so many splendid reviews describing GATA1 as a transcription factor. We therefore focus our topics on GATA1 gene regulation in this review.
Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2(617V>F)) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2a(UTR)-MO) and splice-site junction (jak2a(SS)-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2a(ca)). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1(+) (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, alphahe1 and betahe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2a(ca) significantly increased erythropoiesis and expression of gata1, alphahe1 and betahe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.
Various mutations in the X-linked transcription factor, GATA-1, may result in dyserythropoietic anemia, macrothrombocytopenia and/or erythropoietic porphyria. The present study has carried out detailed ultrastructural studies of abnormal platelet morphology in one, previously described family with a GATA-1 G208S mutation. The ultrastructural investigations revealed a large proportion of their circulating platelets were hypogranular macrothrombocytes, resembling cells from patients with the Gray Platelet Syndrome. However, most of their platelets contained some alpha granules and a small number contained as many as are present in normal platelets. GATA-1 platelets from family members also contained tubular inclusions formed from elements of the dense tubular system like those observed in the Medich Giant Platelets Disorder. The unique pathology of the GATA-1 family platelets found in this study involved features never observed previously in normal or abnormal platelets. Many of their cells contained unusual flat, tubular membrane sheets, often in parallel association and differing from all other membrane systems in normal platelets and megakaryocytes. In some macrothrombocytes the unusual membranes appeared to isolate areas of cytoplasm. The sequestered areas were platelets within platelets. On rare occasion there were two platelets within one platelet, or, even more rarely, a platelet within a platelet within a platelet. Another unique feature, probably related to platelets within platelets, was the frequent attachment of non-activated platelets to each other to form macrothrombocytes. GATA-1 platelets within platelets and attached to platelets, as well as giant platelets, suggest that proplatelet formation may be abnormal, or that GATA-1 platelets are unable to pinch off from megakaryocyte proplatelets in a normal manner.
Extra copies of chromosome 21 are often found in sporadic leukemias. Constitutional trisomy 21 of Down syndrome (DS) is associated with markedly increased risk for childhood leukemia. Thus the oncogenic role of trisomy 21 in the more common sporadic childhood leukemias may be revealed through the investigations of the relatively rare leukemias of DS. Recent studies of the megakaryoblastic leukemias of Down syndrome have uncovered a developmental leukemogenic mechanism characterized by a unique pre-natal collaboration between overexpressed genes from chromosome 21 and an acquired mutation in the transcription factor GATA1. The base of the markedly enhanced risk for acute lymphoblastic leukemia conferred by trisomy 21 is still unclear. Studies of the leukemias of DS are likely to contribute to the general understanding of the oncogenic mechanisms of chromosomal aneuploidies, the most common abnormalities in cancer.
Dendritic cells are key initiators and regulators of the immune response. Dendritic cell commitment and function require orchestrated regulation of transcription. Gata1 is a transcription factor expressed in several hematopoietic lineages. However, Gata1 function has not been explored in the monocytic or dendritic cell compartment. Here, we show that Gata1 is expressed in myeloid and plasmacytoid dendritic cells and that Gata1 ablation affects the survival of dendritic cells. Furthermore, lipopolysaccharide (LPS) stimulation of dendritic cells prompts Gata1 up-regulation, which is accompanied by increased levels of BclX and Ifng. Our findings show that Gata1 is a transcriptional regulator of dendritic cell differentiation and suggest that Gata1 is involved in the dendritic cell and macrophage lineage separation.
Myelofibrotic bone marrow displays abnormal angiogenesis but the pathogenic mechanisms of this are poorly understood. Since pericyte abnormalities are described on solid tumor vessels we studied whether vessel morphology and pericyte coverage in bone marrow samples from patients with myelofibrosis differed from that in samples from controls.
We assessed the microvascular density (MVD), vessel morphology and pericyte coverage in bone marrows from 19 myelofibrosis patients and nine controls. We also studied the same parameters in two mouse models of myelofibrosis, with genetic alterations affecting megakaryocyte differentiation (i.e. one model with low GATA-1 expression and the other with over-expression of thrombopoietin).
In myelofibrotic marrows, MVD was 3.8-fold greater than in controls (p<0.001) and vessels displayed 5.9-fold larger mean perimeters (p<0.001). MVD was 1.8-fold greater in JAK2 V617F-positive than in negative patients (p=0.026). Moreover, 92+/-11 % of vessels in patients with myelofibrosis were pericyte-coated but only 51+/-20 % of vessels in controls (p<0.001). In the two mouse models of myelofibrosis caused by targeting megakaryocytopoesis, wide, pericyte-coated and morphologically aberrant vessels were detected. MVD was significantly greater in bone marrow and spleen samples from animals with myelofibrosis than in wild-type mice.
We conclude that angiogenesis is similarly abnormal in human and murine myelofibrosis with intense pericyte coating, presumably related to abnormal megakaryocytopoiesis.
Mast cells are important elements of the body response to foreign antigens, being those represented either by small molecules (allergic response) or harbored by foreign microorganisms (response to parasite infection). These cells derive from hematopoietic stem/progenitor cells present in the marrow. However, in contrast with most of the other hematopoietic lineages, mast cells do not differentiate in the marrow but in highly vascularized extramedullary sites, such as the skin or the gut. Mast cell differentiation in the marrow is activated as part of the body response to parasites. We will review here the mast cell differentiation pathway and what is known of its major intrinsic and extrinsic control mechanisms. It will also be described that thrombopoietin, the ligand for the Mpl receptor, in addition to its pivotal rule in the control of thrombocytopoiesis and of hematopoietic stem/progenitor cell proliferation, exerts a regulatory function in mast cell differentiation. Some of the possible implications of this newly described biological activity of thrombopoietin will be discussed.
Identifying the transcription factor interactions that are responsible for cell-specific gene expression programs is key to understanding the regulation of cell behaviors, such as self-renewal, proliferation, differentiation, and death. The rapidly increasing availability of microarray-derived global gene expression data sets, coupled with genome sequence information from multiple species, has driven the development of computational methods to reverse engineer and dynamically model genetic regulatory networks. An understanding of the architecture and behavior of transcriptional networks should lend insight into how the huge number of potential gene expression programs is constrained and facilitates efforts to direct or redirect cell fate.
GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the beta-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.
The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.
Expression of the beta-globin genes proceeds from basal to exceptionally high levels during erythroid differentiation in vivo. High expression is dependent on the locus control region (LCR) and coincides with more frequent LCR-gene contacts. These contacts are established in the context of an active chromatin hub (ACH), a spatial chromatin configuration in which the LCR, together with other regulatory sequences, loops toward the active beta-globin-like genes. Here, we used recently established I/11 cells as a model system that faithfully recapitulates the in vivo erythroid differentiation program to study the molecular events that accompany and underlie ACH formation. Upon I/11 cell induction, histone modifications changed, the ACH was formed, and the beta-globin-like genes were transcribed at rates similar to those observed in vivo. The establishment of frequent LCR-gene contacts coincided with a more efficient loading of polymerase onto the beta-globin promoter. Binding of the transcription factors GATA-1 and EKLF to the locus, although previously shown to be required, was not sufficient for ACH formation. Moreover, we used knock-out mice to show that the erythroid transcription factor p45 NF-E2, which has been implicated in beta-globin gene regulation, is dispensable for beta-globin ACH formation.
The induction of Bcl-x(L) is critical for the survival of late proerythroblasts. The erythroid-specific transcriptional network that regulates Bcl-x(L) expression in erythropoiesis remains unclear. The activation of the central erythropoietic transcriptional factor, GATA-1, leads to the early, transient induction of a transcription repressor, Gfi-1B, followed by the late induction of Bcl-x(L) during erythroid maturation in G1ER cells. Chromatin immunoprecipitation assays demonstrated that a constant level of GATA-1 binds to the Bcl-x promoter throughout the entire induction period, while Gfi-1B is transiently associated with the promoter in the early phase. The sustained expression of Gfi-1B abolished GATA-1-induced Bcl-x(L) expression. Here, we present evidence that GATA-1 binds to the noncanonical GATT motif of the Bcl-x promoter for trans-activation. Gfi-1B expressed at increased levels is recruited to the Bcl-x promoter through its association with GATA-1, suppressing Bcl-x(L) transcription. Therefore, the down-regulation of Gfi-1B in the late phase of erythroid maturation is necessary for Bcl-x(L) induction. Furthermore, we show that the inhibition of Bcr-Abl kinase by treatment with imatinib caused the up-regulation of Gfi-1B in K562 cells, where Gfi-1B also cooperated with GATA-1 to repress Bcl-x(L) transcription. Gfi-1B knockdown by RNA interference diminished imatinib-induced apoptosis, while the overexpression of Gfi-1B sensitized K562 cells to arsenic-induced death. These findings illuminate the role of Gfi-1B in GATA-1-mediated transcription in the survival aspect of erythroid cells.
Lineage specification of multipotent progenitor cells is governed by a balance of lineage-affiliated transcription factors, such as GATA1 and PU.1, which regulate the choice between erythroid and myelomonocytic fates. But how ratios of lineage-determining transcription factors stabilize progenitor cells and resolve their indeterminacy to commit them to discrete, mutually exclusive fates remains unexplained. We used a simple model and experimental measurements to analyze the dynamics of a binary fate decision governed by a gene-circuit containing auto-stimulation and cross-inhibition, as embodied by the GATA1-PU.1 paradigm. This circuit generates stable attractors corresponding to erythroid and myelomonocytic fates, as well as an uncommitted metastable state characterized by coexpression of both regulators, explaining the phenomenon of "multilineage priming". GATA1 and PU.1 mRNA and transcriptome dynamics of differentiating progenitor cells confirm that commitment occurs in two stages, as suggested by the model: first, the progenitor state is destabilized in an almost symmetrical bifurcation event, resulting in a poised state at the boundary between the two lineage-specific attractors; second, the cell is driven to the respective, now accessible attractors. This minimal model captures fundamental features of binary cell fate decisions, uniting the concepts of stochastic (selective) and deterministic (instructive) regulation, and hence, may apply to a wider range of binary fate decision points.
Adhesion is one of the important biologic characteristics of leukemic cells. We previously reported a new megakaryocytic-erythroid cell line, JAS-R. In this study, JAS-R cells were segregated into two types by the differences of attachment to culture dishes. One type (designated as JAS-RAD cells) adhered to the substratum of the culture dishes, while the other (JAS-REN cells) grew as a single-cell suspension. Adhesion of JAS-RAD was inhibited by treatment with RGDS oligopeptide. Flow cytometric analysis revealed that JAS-RAD cells had high expression of CD41a and CD61 versus low CD235a expression, and JAS-REN showed low expression of CD41a, and CD61, and high CD235a. The two phenotypes were reciprocally exchangeable by selecting adherent or suspended cells from each type of culture. Microarray analysis and RT-PCR revealed that JAS-RAD cells expressed four major alpha-granule genes and JAS-REN cells expressed beta-globin. Interestingly, erythropoietin was only secreted by JAS-RAD cells. With regard to transcription factors, it was shown that GFI1, FLI1 and RUNX1 were strongly expressed in JAS-RAD cells while GATA1, FOG1 and NFE2 were equally expressed by both types. These findings indicate that adhesion via integrins is related to the phenotypic shift of JAS-R cells between megakaryocytic and erythroid lineages.
GATA-1 is the key transcription factor for the development of the erythroid, megakaryocytic, eosinophilic, and mast cell lineages. GATA-1 possesses the ability to self-associate, and this characteristic has been suggested to be important for GATA-1 function. To elucidate the roles self-associated GATA-1 plays during hematopoietic cell development in vivo, in this study we prepared GATA-1 mutants in which three lysine residues potentially contributing to the self-association (Lys-245, Lys-246, and Lys-312) are substituted in combination with alanines. Of the mutants, 3KA harboring alanine substitutions in all three lysines showed reduced self-association activity without considerable interference in the modification of GATA-1 by acetylation. We generated transgenic mouse lines that express these GATA-1 mutants utilizing the Gata1 hematopoietic regulatory domain, and crossed the mice to Gata1 knockdown (GATA-1.05) mutant mice. Although NKA (K245A and K246A) and CKA (K312A) mutants almost fully rescued the GATA-1.05 mice from anemia and embryonic lethality, the 3KA mutant only partially rescued the GATA-1.05 mutant mice. Even with the higher than endogenous level expression, GATA-1.05/Y::3KA embryos were prone to die at various stages in mid-to-late gestation. Live birth and an anemic phenotype were restored in some embryos depending on the expression level of the 3KA transgene. The expression of the transferrin receptor and heme biosynthesis enzymes was impaired in the yolk sac and liver of the 3KA-rescued embryos. Immature erythroid cells with insufficient expression of the transferrin receptor accumulated in the livers of 3KA-rescued embryos. These results provide the first convincing line of evidence that the self-association of GATA-1 is important for proper mammalian erythroid development in vivo.
The mechanism of osteosclerosis associated with myelofibrosis in megakaryocyte (MK)-related myeloproliferative disorders is largely unknown. However, growth factors released from the bone marrow cells, including from MKs, have been implicated in myelofibrosis, osteosclerosis, and angiogenesis. GATA-1 is a transcription factor required for normal MK development. GATA-1 deficiency in mice (GATA-1(low)) leads to increased megakaryocytic proliferation, followed by osteosclerosis and myelofibrosis. In this study we investigated the expression of bone morphogenetic proteins (BMPs) and BMP receptors and their possible role in the development of osteosclerosis in the MKs of 12-month-old GATA-1(low) mice by immunohistochemistry, cytomorphometry, and quantitative real-time PCR. Marrow MKs from both wild-type and GATA-1(low) mice showed moderate to intense staining for BMP-2, -4, and -6 and BMPR-IA and BMPR-II, whereas splenic MKs showed no BMP immunostaining. Presence of BMP protein in the bone marrow of GATA-1(low) mice was more than that seen in controls, owing to an increased number of MKs and osteoblasts. The osteosclerosis seen in GATA-1(low) mice appeared not to be due to a reduced number of functional osteoclasts because the number of tartrate-resistant acid phosphatase-positive osteoclasts was greater in GATA-1(low) mice than in controls. Our findings demonstrate the presence of significant amounts of BMP-2, -4, and -6 along with their receptors in bone marrow MKs of WT and GATA-1(low) mice. The increased levels of BMPs appear to be a result of increased numbers of MKs in GATA-1(low) mice and may, in part, account for the stimulation of osteoblastic activity and resulting osteosclerosis.
Ectopic expression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations or retroviral insertion, plays an important role in the onset of T-cell leukemias. Two transcripts of LMO2 gene (LMO2-a and LMO2-b) have been reported to encode a same 158-amino-acid protein. We have previously reported a novel transcript of human LMO2 gene (LMO2-c) encoding a 151-amino-acid protein, and defined its promoter region. In the present study, we investigated the regulation of the LMO2-c expression and the functions of LMO2-c. We found that LMO2-c expression is regulated by the cooperation of two essential hematopoietic transcription factors GATA-1 and PU.1 in various hematopoietic cell lines, suggesting an important functional role for LMO2-c in the hematopoietic system. More importantly, we demonstrated that LMO2-c acts as an antagonist of LMO2-a/b binding to its partners, therefore blocking the transactivation of LMO2-a/b on its target genes. These findings provide novel evidence to the functions of LMO2 gene in the hematopoietic system and leukemia.
GATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over approximately 100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells. Since it is unknown whether the individual complexes exert qualitatively distinct or identical functions to regulate Gata2 transcription in vivo, we compared the activity of the -3.9 and +9.5 kb sites of the Gata2 locus in transgenic mice. The +9.5 site functioned as an autonomous enhancer in the endothelium and fetal liver of embryonic day 11 embryos, whereas the -3.9 site lacked such activity. Mechanistic studies demonstrated critical requirements for a GATA motif and a neighboring E-box within the +9.5 site for enhancer activity in endothelial and hematopoietic cells. Surprisingly, whereas this GATA-E-box composite motif was sufficient for enhancer activity in an erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These results identify the first molecular determinant of Gata2 transcription in vascular endothelium, composed of a core enhancer module active in both endothelial and hematopoietic cells and regulatory modules preferentially required in endothelial cells.
The GATA-1-interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1-independent activation by GATA-1 has been described, all known examples of GATA-1-mediated repression are FOG-1 dependent. In the GATA-1-null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1-binding defective mutant of GATA-1 repressed several genes similarly upon activation with beta-estradiol. Repression also occurred in a FOG-1-null cell line expressing ER-GATA-1 and during ex vivo erythropoiesis. At the Lyl1 and Rgs18 loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1-mediated transcriptional regulation.
The nucleotide excision repair (NER) pathway and its leading gene excision-repair cross-complementary 1 (ERCC1) have been shown to be up-regulated in hepatocellular carcinomas even in the absence of treatment with chemotherapeutics. The aim of this study was to determine the mechanism involved in NER regulation during the liver cell growth observed in hepatocellular carcinoma. Both NER activity and ERCC1 expression were increased after exposure to the epidermal growth factor (EGF) in cultured normal and tumoral human hepatocytes. These increases correlated with the activation of the kinase signaling pathway mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK that is known to be a key regulator in the G(1) phase of the hepatocyte cell cycle. Moreover, EGF-mediated activation of ERCC1 was specifically inhibited by either the addition of U0126, a MEK/ERK inhibitor or small interfering RNA-mediated knockdown of ERK2. Basal expression of ERCC1 was decreased in the presence of the phosphoinositide-3-kinase (PI3K) inhibitor and small hairpin RNA (shRNA) against the PI3K pathway kinase FKBP12-rapamycin-associated protein or mammalian target of rapamycin. Transient transfection of human hepatocytes with constructs containing different sizes of the 5'-flanking region of the ERCC1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in EGF-treated cells, which correlated with the presence of the nuclear transcription factor GATA-1 recognition sequence. The recruitment of GATA-1 was confirmed by chromatin immunoprecipitation assay. In conclusion, these results represent the first demonstration of an up-regulation of NER and ERCC1 in EGF-stimulated proliferating hepatocytes. The transcription factor GATA-1 plays an essential role in the induction of ERCC1 through the mitogen-activated protein kinase (MAPK) pathway, whereas the PI3K signaling pathway contributes to ERCC1 basal expression.
Endomitosis is a unique form of cell cycle used by megakaryocytes, in which the latter stages of mitosis are bypassed so that the cell can increase its DNA content and size. Although several transcription factors, including GATA-1 and RUNX-1, have been implicated in this process, the link between transcription factors and polyploidization remains undefined. Here we show that GATA-1-deficient megakaryocytes, which display reduced size and polyploidization, express nearly 10-fold less cyclin D1 and 10-fold increased levels of p16 compared with their wild-type counterparts. We further demonstrate that cyclin D1 is a direct GATA-1 target in megakaryocytes, but not erythroid cells. Restoration of cyclin D1 expression, when accompanied by ectopic overexpression of its partner Cdk4, resulted in a dramatic increase in megakaryocyte size and DNA content. However, terminal differentiation was not rescued. Of note, polyploidization was only modestly reduced in cyclin D1-deficient mice, likely due to compensation by elevated cyclin D3 expression. Finally, consistent with an additional defect conferred by increased levels of p16, inhibition of cyclin D-Cdk4 complexes with a TAT-p16 fusion peptide significantly blocked polyploidization of wild-type megakaryocytes. Together, these data show that GATA-1 controls growth and polyploidization by regulating cyclin D-Cdk4 kinase activity.
Phospholipase C (PLC) gamma 1 has been shown to mediate signal transduction of tyrosine kinases and affect function of hematopoietic cells. However, its role in hematopoiesis during embryonic development is currently unclear. In this study, we examined this issue using morpholino (MO) gene knockdown in zebrafish embryos
MO targeting at the exon-1-intron-1 junction of zebrafish PLC-gamma1 was injected into embryos at the one- to four-cell stage (referred herein zPLC-gamma1(MO) embryos). Primitive hematopoiesis was examined quantitatively by flow cytometry in Tg(gata1:GFP) embryos and by real-time quantitative polymerase chain reaction at 18 hours-post-fertilization (hpf), before the onset of circulation. The embryos were also treated with receptor inhibitors of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor at 25, 1, and 30 micromol/L, respectively, from one cell until 48 hpf.
Erythropoiesis was reduced in zPLC-gamma1(MO) embryos, as shown by the reduction in gata1(+) cells (wild-type: 4.32% +/- 0.10% vs zPLC-gamma1(MO): 2.38% +/- 0.11%, p = 0.021) and gata1 and alpha-embryonic hemoglobin expression [0.47 +/- 0.06-fold (p = 0.013) and 0.46 +/- 0.04-fold (p = 0.013)]. Expression of scl, lmo-2 (early hematopoietic progenitors), pu.1, and l-plastin (myelomonocytic lineages) as well as fli1 (vascular progenitors) were unaffected. Fli1(+) cells in Tg(fli1:GFP) embryos were also unaffected by zPLC-gamma1(MO). When embryos were incubated with receptor inhibitors of vascular endothelial growth factor (VEGFRTKI), fibroblast growth factor (SU5402), or platelet-derived growth factor (AG1296), only VEGFRTKI reduced erythropoiesis [VEGFRTKI: 2.10% +/- 0.07% (p = 0.021) vs SU5402: 4.08% +/- 0.12% (p = 0.248) vs AG1296: 4.12% +/- 0.14% (p = 0.149)].
PLC-gamma1 is involved in the regulation of primitive hematopoiesis in zebrafish embryos, which is distinct from its later effect on vascular formation.
Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation.
We investigated the effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgene Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens.
Although the overall RNA yield (normalized to 1 x 10(7) leukocytes) was not different, the RNA preparation using unstabilized reference samples had an approximately 3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression (DeltaDeltaC(T) 0.16-0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure (DeltaDeltaC(T) 1.07 and 1.32).
The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.
We investigated the role of CCAAT enhancer-binding protein-alpha (C/EBPalpha) during zebrafish embryonic blood development.
Whole-mount mRNA in situ hybridization was performed to determine the spatio-temporal expression pattern of zebrafish cebpa in developing hematopoietic progenitors. A deletion mutation of cebpa (zD420), which mimics the human dominant-negative mutations of C/EBPalpha, was transfected into CV1 cell line to evaluate its transcriptional activity in vitro and injected into zebrafish embryos at the one- to two-cell stage to examine its effects on primitive hematopoiesis during early zebrafish development.
Zebrafish cebpa is expressed in the anterior and posterior lateral plate mesoderm at 12 hours postfertilization, along with scl, pu.1, and gata1 in developing hematopoietic progenitors. In vitro, the deletion mutation of cebpa (zD420) prevents expression of the full-length protein, allowing the expression of truncated isoforms from internal translational initiation sites. As in the human, the truncated zebrafish C/EBPalpha proteins did not activate the expression of known target granulocytic genes, and in fact suppressed transactivation that was induced in vitro by the full-length protein. Forced expression of the zD420 mRNA in zebrafish embryos led to an expansion of primitive erythropoiesis, without a discernible effect on granulopoiesis.
Expression of the truncated isoforms of cebpa alters the developmental pattern of hematopoietic progenitor cells during embryogenesis.
The GATA family of transcription factors appears to retain conserved roles in early germ layer patterning in most, if not all, animals; however, the number and structure of GATA factor genes varies substantially when different animal genomes are compared. Thus, the origin and relationships of invertebrate and vertebrate GATA factors, and their involvement in animal germ layer evolution, are unclear. We identified two highly conserved GATA factor genes in a marine annelid, the polychaete Platynereis dumerilii. A phylogenetic analysis indicates that the two Platynereis GATA factors are orthologous to the GATA1/2/3 and GATA4/5/6 subfamilies present in vertebrates. We also identified conserved motifs within each GATA class, and assigned the divergent Caenorhabditiselegans and Drosophila melanogaster GATA factor genes to the vertebrate classes. Similar to their vertebrate homologs, PdGATA123 mRNA expression was restricted to ectoderm, whereas PdGATA456 was detected only in endomesoderm. Finally, we identified in genome databases one GATA factor gene in each of two distantly related cnidarians that include motifs from both bilaterian GATA factor classes. Our results show that distinct orthologs of the two vertebrate GATA factor classes exist in a protostome invertebrate, suggesting that bilaterian GATA factors originated from GATA1/2/3 and 4/5/6 ancestral orthologs. Moreover, our results indicate that the GATA gene duplication and the functional divergence that led to these two ancestral GATA factor genes occurred after the split of the bilaterian stem group from the cnidarians.
Four years ago it was discovered that nearly all cases of transient myeloproliferative disorder and acute megakaryocytic leukemia in children with Down syndrome acquire mutations in the hematopoietic transcription factor gene GATA1. Studies within the past year, described within this review, have provided tremendous insights into the role of GATA1 mutations in these malignancies.
In the past year, our understanding of the molecular and cellular consequences of GATA1 mutations has been greatly enhanced. Most importantly, we have learned that these mutations, which result in the exclusive production of the short GATA1 isoform named GATA1s, have a distinct effect on fetal liver progenitors. In addition, multiple studies have shown that GATA1s can substitute for GATA1 in many aspects of megakaryocytic maturation. Finally, an important clinical study has revealed that GATA1 mutations alone are insufficient for leukemia.
Leukemia in children with Down syndrome requires at least three cooperating events--trisomy 21, a GATA1 mutation, and a third, as yet undefined, genetic alteration. Recent studies have provided tremendous insights into the GATA1 side of the story. Future experiments with human patient samples and mouse models will likely increase our awareness of the role of trisomy 21 in transient myeloproliferative disorder and acute megakaryocytic leukemia.
Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.
We identified a family with gray platelet syndrome (GPS) segregating as a sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia, and large agranular platelets characteristic of GPS, while obligate carrier females were asymptomatic but had dimorphic platelets on peripheral smear. Associated findings included mild erythrocyte abnormalities in affected males. Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578 and DXS6797, which segregated with the platelet phenotype and included the GATA1 gene. Sequencing of GATA1 revealed a G-to-A mutation at position 759 corresponding to amino acid change Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but not of gray platelet syndrome. Our findings suggest that XLTT is within a spectrum of disorders constituting the gray platelet syndrome, and we propose that GATA1 is an upstream regulator of the genes required for platelet alpha-granule biogenesis.
Runx1-deficient mice die around embryonic day 11.5 due to impaired hematopoiesis. This early death prevents the analysis of the role of Runx1 in the development of sensory ganglia. To overcome the early embryonic lethality, we adopted a new approach to utilize transgenic Runx1-deficient mice in which hematopoietic cells are selectively rescued by Runx1 expression under the control of GATA-1 promoter. In Runx1-deficient mice, the total number of dorsal root ganglion (DRG) neurons was increased, probably because of an increased proliferative activity of DRG progenitor cells and decreased apoptosis. In the mutant DRG, TrkA-positive neurons and peptidergic neurons were increased, while c-ret-positive neurons were decreased. Axonal projections were also altered, in that both central and peripheral projections of CGRP-positive axons were increased. In the dorsal horn of the spinal cord, projections of CGRP-positive axons expanded to the deeper layer, IIi, from the normal terminal area, I/IIo. Our results suggest that Runx1 is involved in the cell fate specification of cutaneous neurons, as well as their projections to central and peripheral targets.
Erythropoiesis is a complex multistage process for the differentiation of mature erythrocytes from hematopoietic stem cells. The function of several transcription factors has been reported in hematopoietic stem cell differentiation. However, the molecular basis governing its functional behavior is unclear. In this study, we characterized the role of Zfpm-1 during the erythropoietic differentiation of human hematopoietic stem cells. To verify the function of Zfpm-1 during erythropoietic differentiation, we established human CD34+ cell culture system by using human umbilical cord blood. At day 7 of the human CD34+ cell differentiation process to proerythocytes, Zfpm-1 was initially up-regulated and then dramatically down-regulated at day 9. The Zfpm-1 siRNA transfected HSCs contained 20% more GPA+ cells than the mock transfected cells, and showed repressed expression of the hematopoietic transcription factors, c-myc and c-myb, but increased expression of GATA-1. In contrast, the Zfpm-1 gain-of-function is the opposite of loss-of-function results above.
GATA-1 is a hematopoietic transcription factor expressed in erythroid, megakaryocytic, mast cell and eosinophil lineages. It is required for normal erythroid differentiation, the expression of erythroid-specific genes and for the establishment of an active chromatin structure throughout the beta-globin gene locus. GATA-1 is also necessary for the formation and function of the locus control region DNase I hypersensitive site (HS) core elements. To determine whether GATA-1 was sufficient to direct formation of the locus control region (LCR) and an erythroid pattern of gene expression, we expressed GATA-1 in the non-hematopoietic HeLa cell line that does not express other hematopoietic transcription factors but does express GATA-2, GATA-3, and GATA-6. We found that production of the GATA-1 protein resulted in the formation of LCR DNase I HSs 1-4 in their normal locations, and that histones became hyperacetylated within these regulatory elements. Transcription of several erythroid-specific genes was activated in HeLa cells expressing GATA-1, including those coding for alpha-globin, beta-globin, the erythropoietin receptor, the erythroid krüpple-like factor and p45 NF-E2. Despite increased expression of these genes at the mRNA level, their protein products were not detected. These results imply that GATA-1 is sufficient to direct chromatin structure reorganization within the beta-globin LCR and an erythroid pattern of gene expression in the absence of other hematopoietic transcription factors.
Mast cells are pivotal effector cells in IgE-mediated allergic reactions. GATA transcriptional factors such as GATA-1 and GATA-2 are expressed in mast cells, and recent studies have revealed that both GATA-1 and GATA-2 are required for mast cell development. However, the role of GATA transcriptional factors in differentiated mast cells has remained largely unknown. In this study, we repressed the activity of GATA-1 and GATA-2 by using three different approaches (inducible overexpression of a dominant-negative form of GATA, pharmacological inactivation, or small interfering RNA technology), and analyzed the molecular mechanisms of GATA transcriptional factors in the activation of mast cells. Surprisingly, the repression of GATA activity in differentiated mast cells led to the impairment of cell survival, IgE-induced degranulation, and cytokine production. Signal transduction and histone modification in the chromatin related to protein kinase Cbeta were defective in these cells. These results identify that GATA has a critical role in the activation of mast cell.
To determine the role of reactive oxygen species in erythroid differentiation, we investigated the effects of an antioxidant, N-acetyl-L-cysteine (NAC), on the differentiation of erythroid progenitors derived from mouse fetal liver. In response to erythropoietin (Epo), erythroid progenitors undergo differentiation in vitro and express erythroid-specific genes such as betamajor-globin, Alas2, MafK, p45, Eklf, and Gata1. Expression of these genes was decreased in the presence of NAC, whereas the expression of c-myb, which is downregulated during erythroid differentiation, remained constant. Moreover, NAC treatment inhibited an increase in the number of cells expressing high levels of erythroid-specific antigen TER119. Treatment with another antioxidant, pyrrolidine dithiocarbamate, also caused the attenuation of TER119 expression. These results suggest that reactive oxygen species are involved in Epo-mediated erythroid differentiation.
Caspase-3 is activated during both terminal differentiation and erythropoietin-starvation-induced apoptosis of human erythroid precursors. The transcription factor GATA-1, which performs an essential function in erythroid differentiation by positively regulating promoters of erythroid and anti-apoptotic genes, is cleaved by caspases in erythroid precursors undergoing cell death upon erythropoietin starvation or engagement of the death receptor Fas. In contrast, by an unknown mechanism, GATA-1 remains uncleaved when these cells undergo terminal differentiation upon stimulation with Epo. Here we show that during differentiation, but not during apoptosis, the chaperone protein Hsp70 protects GATA-1 from caspase-mediated proteolysis. At the onset of caspase activation, Hsp70 co-localizes and interacts with GATA-1 in the nucleus of erythroid precursors undergoing terminal differentiation. In contrast, erythropoietin starvation induces the nuclear export of Hsp70 and the cleavage of GATA-1. In an in vitro assay, Hsp70 protects GATA-1 from caspase-3-mediated proteolysis through its peptide-binding domain. The use of RNA-mediated interference to decrease the Hsp70 content of erythroid precursors cultured in the presence of erythropoietin leads to GATA-1 cleavage, a decrease in haemoglobin content, downregulation of the expression of the anti-apoptotic protein Bcl-X(L), and cell death by apoptosis. These effects are abrogated by the transduction of a caspase-resistant GATA-1 mutant. Thus, in erythroid precursors undergoing terminal differentiation, Hsp70 prevents active caspase-3 from cleaving GATA-1 and inducing apoptosis.
Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1(+) cells isolated from E7.0-7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1(+) cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1(+) hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo.
There is an increased risk of myeloid malignancy in individuals with Down's syndrome (DS), which is associated with a mutation in exon 2 of the transcription factor GATA-1. It is recognized that there is accelerated telomere shortening in blood cells of children with DS similar to that in conditions such as Fanconi anemia and dyskeratosis congenita. The latter conditions are associated with stem cell deficiency and clonal change, including acute myeloid leukemia. In this study we address the questions 1) whether the accelerated telomere shortening is associated with progenitor/stem cell deficiency in individuals with DS, predisposing to clonal change and 2) whether the occurrence of reduced numbers of stem/progenitor cells precede the incidence of mutations in exon 2 of GATA-1.
Peripheral blood from fetuses (23-35 weeks gestation) and/or bone marrow from children affected by DS and age-matched hematologically healthy controls were analyzed for telomere length, content of stem/progenitor cells, and mutations in exon 2 of GATA-1.
We found that hematopoietic stem/progenitor cell deficiency and telomere shortening occurs in individuals with DS in fetal life. Moreover, the presence of a low number of progenitor cells was not associated with mutations in exon 2 of GATA-1.
We propose that stem cell deficiency may be a primary predisposing event to DS leukemia development.
Congenital erythropoietic porphyria (CEP), an autosomal recessive disorder, is due to mutations of uroporphyrinogen III synthase (UROS). Deficiency of UROS results in excess uroporphyrin I, which causes photosensitization. We evaluated a 3-year-old boy with CEP. A hypochromic, microcytic anemia was present from birth, and platelet counts averaged 70 x 10(9)/L (70,000/microL). Erythrocyte UROS activity was 21% of controls. Red cell morphology and globin chain labeling studies were compatible with beta-thalassemia. Hb electrophoresis revealed 36.3% A, 2.4% A(2), 59.5% F, and 1.8% of an unidentified peak. No UROS or alpha- and beta-globin mutations were found in the child or the parents. The molecular basis of the phenotype proved to be a mutation of GATA1, an X-linked transcription factor common to globin genes and heme biosynthetic enzymes in erythrocytes. A mutation at codon 216 in the child and on one allele of his mother changed arginine to tryptophan (R216W). This is the first report of a human porphyria due to a mutation in a trans-acting factor and the first association of CEP with thalassemia and thrombocytopenia. The Hb F level of 59.5% suggests a role for GATA-1 in globin switching. A bone marrow allograft corrected both the porphyria and the thalassemia.
The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.
Platelets are small anucleate cells that travel near the vessel wall during laminar flow. In response to vascular injury, platelets undergo alterations in morphology which allow them to aggregate and cover the injured site. Platelets are produced by megakaryocytes in a process that involves the formation of platelet precursors called proplatelets and subsequent release of these proplatelets into the circulation. By forming a demarcation membrane system within the cytosol, megakaryocytes contain a membrane reservoir which allows for the production of thousands of platelets per mature megakaryocyte. Interestingly, the above process known as megakaryopoiesis is not yet fully understood. However, several groups have contributed evidence to unveil the role of thrombopoietin (TPO), the principal regulator of megakaryopoiesis in vivo. TPO is necessary for megakaryocyte maturation in that TPO deficient mice display greatly reduced megakaryocyte production as well as reduced numbers of mature megakaryocytes. Several transcription factors have also been implicated in megakaryopoiesis including, GATA-1, friend of GATA-1 (FOG-1), nuclear factor-erythroid 2 (NF-E2), and Fli-1. In fact, interactions among some of the transcription factors have been reported to produce synergistic effects. GATA-1 and Fli-1 interactions result in heightened GPIX and GPIb (2 components of von Willebrand Factor (vWF) receptor) expression, while GATA-1, RUNX1 and core-binding factor beta interactions result in improved alphaIIb promoter activity. Mutations in the vWF complex and alphaIIb beta3 have been linked to disorders such as Bernard-Soulier syndrome and Glazmann thrombasthenia respectively. Therefore, a more comprehensive understanding of the transcriptional control of megakaryopoiesis may lead to more effective treatments of platelet-related disorders.
Elongation factor 2 (EF-2) plays a key role in the essential process of protein synthesis by translocating tRNAs from the ribosomal A- and P-sites to the P- and E-sites. EF-2 regulates the outcome of protein synthesis in mammalian cells. This report demonstrates that chicken EF-2 protein levels are dependent on transcription in 8-bromo-cAMP, insulin and phorbol ester-treated cells. In order to delineate functional domains that control chicken EF-2 gene transcription, the 5'-flanking region of the chicken EF-2 promoter was analyzed. Deletion constructs from -550 and -86 had the same basal level promoter activity as the whole EF-2 promoter. The sequence between nucleotides -700 and -550 was determined to be a regulatory region for the chicken EF-2 basal promoter activity. The region between -700 and -550 has a negative regulatory region and two regulatory proteins (I, II). 8-bromo-cAMP increased chicken EF-2 promoter activity (-700/+102) in Rat 1 HIR fibroblast cells more than insulin and phorbol ester treatment. Binding of protein I and II were decreased by 8-bromo-cAMP but restored by a protein kinase A inhibitor (KT5720). GATA consensus sequence oligonucleotide and fragment -86/-50 prevented protein II binding of fragment -700/-550. This result suggested that protein II is a GATA-like protein. These observations provide a novel regulatory mechanism for the EF-2 promoter.
The Duffy blood group Ag (dfy) binds selective CXC and CC chemokines at high affinity and is expressed on erythrocytes and endothelial cells. However, it does not transmit a signal via G proteins, as occurs with other seven-transmembrane receptors. We hypothesized that dfy functions as a chemokine reservoir and regulates inflammation by altering soluble chemokine concentrations in the blood and tissue compartments. We determined whether Duffy Ag "loss-of-function" phenotypes (human and murine) are associated with alterations in plasma chemokine concentrations during the innate inflammatory response to LPS. Plasma CXCL8 and CCL2 concentrations from humans homozygous for the GATA-1 box polymorphism, a dfy polymorphism that abrogates erythrocyte chemokine binding, were higher than in heterozygotes following LPS stimulation of their whole blood in vitro. Similarly, dfy(-/-) mice showed higher plasma MIP-2 concentrations than dfy(+/+) mice following LPS stimulation of whole blood in vitro. We then determined the relative contributions of erythrocyte and endothelial Duffy Ag in modifying chemokine concentrations and neutrophil recruitment in the lungs following intratracheal LPS administration in dfy(-/-) and dfy(+/+) mice reconstituted with dfy(-/-) or dfy(+/+) marrow. Mice lacking endothelial dfy expression had higher MIP-2 and keratinocyte chemoattractant concentrations in the airspaces. Mice lacking erythrocyte dfy had higher MIP-2 and keratinocyte chemoattractant concentrations in the lung tissue vascular space, but lower plasma chemokine concentrations associated with attenuated neutrophil recruitment into the airspaces. These data indicate that dfy alters soluble chemokine concentrations in blood and local tissue compartments and enhances systemic bioavailability of chemokines produced during local tissue inflammation.
Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.
Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of gamma-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on gamma-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to beta-globin disorder, beta-thalassemia.
Newborns and children with Down Syndrome are predisposed to a range of blood disorders, which include acute lymphoblastic leukaemia and acute megakaryocytic leukaemia (AMKL). Over the last four years there has been considerable progress in our understanding of DS AMKL. Like other childhood leukaemias DS AMKL is initiated in utero and can present in the neonatal period as a clinically overt preleukaemic condition, transient myeloproliferative disorder (TMD). In addition to trisomy 21, fetal haemopoietic progenitors acquire N-terminal truncating mutations in the key megakaryocyte-erythroid transcription factor GATA1. These are the minimum required events for TMD to develop. In approximately 30% of TMD patients, additional as yet unidentified (epi)genetic mutations are required for progression to AMKL. Thus, DS TMD and AMKL provide a unique model of childhood leukaemia where the preleukaemic and leukaemic phases are ascertainable and separable allowing distinct steps in leukaemogenesis to be studied individually. These findings also have implications for the clinical management of DS TMD and AMKL specifically and also of childhood leukaemia more generally.
Hematologic abnormalities are common in individuals with Down syndrome (DS). Increased erythrocyte mean corpuscular volume (MCV) is frequently found among DS infants and remains elevated throughout life in two-thirds of patients, making interpretation of red cell indices for diagnosis of nutritional anemias or bone marrow failure disorders more challenging. Transient myeloproliferative disorder (TMD) associated with pancytopenia, hepatosplenomegaly, and circulating immature WBCs, is found almost exclusively in DS infants with an incidence of approximately 10%. In most cases, TMD regresses spontaneously within the first 3 months of life, but in some children, it can be life threatening or even fatal. Despite the high rate of spontaneous regression, TMD can be a preleukemic disorder in 20-30% of children with DS. The types of malignancy, response to therapy, and clinical outcome in children with DS are also unique. There is an increased risk of leukemia with an equal incidence of lymphoid and myeloid leukemia. Acute megakaryocytic leukemia (AMKL) subtype is the most common form of acute myeloid leukemia (AML) in this setting, and is uncommon in children without DS. Somatic mutations of the gene encoding the hematopoetic growth factor GATA1 have been shown to be specific for TMD and AMKL in children with DS. Myelodysplastic syndrome can precede AML. Children with DS and leukemia are more sensitive to some chemotherapeutic agents such as methotrexate than other children which requires careful monitoring for toxicity. Although the risk for leukemia is higher in individuals with DS, these patients have a lower risk of developing solid tumors, with the exception of germ cell tumors, and perhaps retinoblastoma and lymphoma.
Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration, and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate, or advanced differentiation and establish the value of this marker panel using mouse models of defective thrombopoiesis resulting from absence of GATA1, NF-E2, or tubulin beta1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stage-specific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis.
Changes in transcription factor levels and activities dictate developmental fate. Such a change might affect the full ensemble of target genes for a factor or only uniquely sensitive targets. We investigated the relationship among activity of the hematopoietic transcription factor GATA-1, chromatin occupancy, and target gene sensitivity. Graded activation of GATA-1 in GATA-1-null cells revealed high-, intermediate-, and low-sensitivity targets. GATA-1 activity requirements for occupancy and transcription often correlated. A GATA-1 amino-terminal deletion mutant severely deregulated the low-sensitivity gene Tac-2. Thus, cells expressing different levels of a cell type-specific activator can have qualitatively distinct target gene expression patterns, and factor mutations preferentially deregulate low-sensitivity genes. Unlike other target genes, GATA-1-mediated Tac-2 regulation was bimodal, with activation followed by repression, and the coregulator Friend of GATA-1 (FOG-1) selectively mediated repression. A GATA-1 mutant defective in FOG-1 binding occupied a Tac-2 regulatory region at levels higher than wild-type GATA-1, whereas FOG-1 facilitated chromatin occupancy at a distinct target site. These results indicate that FOG-1 is a determinant of GATA factor target gene sensitivity by either facilitating or opposing chromatin occupancy.
Myeloid leukemia of Down's syndrome (ML-DS) has characteristic biological features (e.g. expression of the truncated GATA1s), which are different from those of non-DS childhood acute myeloid leukemias (AML). The objective of this study was to investigate factors predisposing to the development of ML-DS.
We analyzed 134 bone marrow specimens from 64 children with ML-DS and non-DS AML during chemotherapy and 7 specimens from DS children with- out leukemia,who did not receive any chemotherapy,The specimens were analyzed by multiparameter flow cytometry and quantitative reverse transcriptase polymerase chain reaction for transcription factors involved in hematopoiesis.
Samples taken from children with ML-DS in complete remission during chemotherapy aberrantly expressed CD56 (NCAM) at the surface of monocytic and granulocytic cells. Compared to non-DS AML cases,children with ML-DS had a statistically significant higher proportion of CD56+ cells in the CD33+ fraction: 71%+/-6% vs. 4%+/-1% (p<0.00001). A significant decrease of the amount of CD33+/CD56+ cells was observed during and after maintenance therapy. An increased number of CD33+/CD56+ cells was also present (>85%) in children with DS who did not receive chemotherapy, but showed a left-shift (due to infection), compared with DS children without left-shift (<10% CD33+/CD56+ cells). Within the CD33+/CD56+ fraction, RUNX1 was overexpressed more than 5-fold (p<0.02) compared to CD33+/CD56- cells, whereas there were no differences regarding GATA1, SPI1, ERG or ETS-2 levels.
The combined overexpression of RUNX1 and NCAM during stress hematopoiesis in children with DS might be a key factor in the development of overt leukemia and/or in the growth advantage of the malignant GATA1s clone in ML- DS.
Multiple alignments of genome sequences are helpful guides to functional analysis, but predicting cis-regulatory modules (CRMs) accurately from such alignments remains an elusive goal. We predict CRMs for mammalian genes expressed in red blood cells by combining two properties gleaned from aligned, noncoding genome sequences: a positive regulatory potential (RP) score, which detects similarity to patterns in alignments distinctive for regulatory regions, and conservation of a binding site motif for the essential erythroid transcription factor GATA-1. Within eight target loci, we tested 75 noncoding segments by reporter gene assays in transiently transfected human K562 cells and/or after site-directed integration into murine erythroleukemia cells. Segments with a high RP score and a conserved exact match to the binding site consensus are validated at a good rate (50%-100%, with rates increasing at higher RP), whereas segments with lower RP scores or nonconsensus binding motifs tend to be inactive. Active DNA segments were shown to be occupied by GATA-1 protein by chromatin immunoprecipitation, whereas sites predicted to be inactive were not occupied. We verify four previously known erythroid CRMs and identify 28 novel ones. Thus, high RP in combination with another feature of a CRM, such as a conserved transcription factor binding site, is a good predictor of functional CRMs. Genome-wide predictions based on RP and a large set of well-defined transcription factor binding sites are available through servers at http://www.bx.psu.edu/.
Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. By single-cell cloning and sequential replating experiments of prospectively isolated progenitor cells, we demonstrate here that the hypomorphic Gata1low mutation increases the proliferation potential of a unique class of progenitor cells, similar in phenotype to adult common erythroid/megakaryocytic progenitors (MEPs), but with the "unique" capacity to generate erythroblasts, megakaryocytes, and mast cells in vitro. Conversely, progenitor cells phenotypically similar to mast cell progenitors (MCPs) are not detectable in the marrow from these mutants. At the single-cell level, about 11% of Gata1low progenitor cells, including MEPs, generate cells that will continue to proliferate in cultures for up to 4 months. In agreement with these results, trilineage (erythroid, megakaryocytic, and mastocytic) cell lines are consistently isolated from bone marrow and spleen cells of Gata1low mice. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which common myeloid progenitors become either MEPs or MCPs.
A Down syndrome male showed leukocytosis from birth and was diagnosed as transient myeloproliferative disorder (TMD). Eight months later, his condition had progressed to myelodysplastic syndrome after spontaneous resolution, and it then evolved to acute megakaryoblastic leukemia (AMKL) at the age of 20 months. Sequencing analysis showed that the predominant TMD and AMKL clones had different GATA1 mutations, although a minor TMD clone identical to the AMKL clone was present at birth. These observations suggest that a minor clone rather than the predominant clone at the time of TMD may give rise to AMKL later on.
p27KIP1 is known as a regulator of cellular differentiation and apoptosis in human cancer cells. We have previously reported that human chronic myeloid leukemia (CML) KU812 and K562 cells show inhibited cellular proliferation in response to treatment with activin A, a member of TGF-beta superfamily. Apoptosis and erythroid differentiation can be induced in KU812 and K562 cells, respectively. We report herein that activin A induced the expression of p27KIP1 in CML cells along with the induction of cellular differentiation and apoptosis. There are putative binding sequences of erythroid-related transcription factor GATA-1 in the promoter region of the human p27KIP1 gene. Expression of GATA-1 protein in activin A-treated KU812 and K562 cells showed dissimilar regulation in these two cell lines. Induction of p27KIP1 was commonly observed, but it did not correspond to the expression levels of GATA-1 in either line of activin A-treated CML cells. In addition, ERK protein was rapidly and transiently activated with activin A in both types of CML cells, suggesting that phosphorylation of ERK is required for activin A signaling in CML cells. These results indicate that both p27KIP1 induction and regulation of GATA-1 play essential roles in activin A-induced erythroid differentiation and apoptosis.
Transient leukemia of Down syndrome (DS-TL), also known as transient myeloproliferative disorder of Down syndrome (DS) and transient abnormal myelopoiesis of DS, occurs in approximately 10% of DS neonates and in phenotypically normal neonates with trisomy 21 mosaicism. In DS-TL, peripheral blood analysis shows variable numbers of blasts and, usually, thrombocytopenia; other cytopenias are uncommon. Bone marrow characteristics of DS-TL are, likewise, variable, though (in contrast to other leukemias) the bone marrow blast differential can be lower than the peripheral blood blast differential. The blasts of DS-TL typically show light microscopic, ultrastructural, and flow cytometric evidence of megakaryocyte differentiation. DS-TL neonates have a approximately 15% risk of developing potentially fatal liver disease and show <10% incidence of hydrops fetalis. Additional manifestations of DS-TL include cutaneous involvement, hyperviscosity, myelofibrosis, cardiopulmonary failure, splenomegaly, and spleen necrosis. Despite its typical transient nature, 20% to 30% of DS-TL patients develop overt (nontransient) acute leukemia, usually within 3 years and typically of the M7 phenotype (acute megakaryoblastic leukemia). The pathogenesis of DS-TL (and of subsequent acute leukemia) involves mutation of GATA1 (on chromosome X), which normally encodes a transcription factor integral to normal development of erythroid, megakaryocytic, and basophilic/mast cell lines. The pathogenetic role of trisomy 21 in DS-TL is unclear. Though indications for chemotherapy in DS-TL have not been firmly established, the blasts of DS-TL are sensitive to low-dose cytosine arabinoside.
Mouse gonadal development is regulated by a variety of transcription factors. Here we report the identification and characterization of a novel nuclear zinc finger protein called GATA like protein-1 (GLP-1), which is expressed at high levels in the somatic cells of the developing gonads, including Leydig cells in the testes and granulosa cells in the ovaries. Biochemical analysis of GLP-1 shows that it acts as a transcriptional repressor of GATA factor function. To determine the necessity of GLP-1 in gonadal development, a null allele in mice was generated by replacing all of the coding exons with the bacterial lacZ gene. GLP-1(lacZ) null mice are viable with no detectable defects in visceral organ development; however, both males and females are completely infertile. Loss of GLP-1 leads to defective sperm development in males with a marked reduction in mature spermatids observed as early as postnatal week 1. In females, loss of GLP-1 leads to a severe block in germ cell development as early as E17.5. Together, these data identify GLP-1 as a critical nuclear repressor in somatic cells of the gonad that is required for germ cell development, and highlight the importance of somatic-germ cell interactions in the regulation of this critical process.
The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.
We have generated mice from a N-ethyl-N-nitrosourea mutagenesis screen that carry a mutation in the translation initiation codon of Gata-1, termed Plt13, which is equivalent to mutations found in patients with acute megakaryoblastic leukemia and Down syndrome. The Gata-1 locus is present on the X chromosome in humans and in mice. Male mice hemizygous for the mutation (Gata-1Plt13/Y) failed to produce red blood cells and died during embryogenesis at a similar stage to Gata-1-null animals. Female mice that carry the Plt13 mutation are mosaic because of random inactivation of the X chromosome. Adult Gata-1Plt13/+ females were not anemic, but they were thrombocytopenic and accumulated abnormal megakaryocytes without a concomitant increase in megakaryocyte progenitor cells. Gata-1Plt13/+ mice contained large numbers of blast-like colony-forming cells, particularly in the fetal liver, but also in adult spleen and bone marrow, from which continuous mast cells lines were readily derived. Although the equivalent mutation to Gata-1Plt13 in humans results in production of GATA-1s, a short protein isoform initiated from a start codon downstream of the mutated initiation codon, Gata-1s was not detected in Gata-1Plt13/+ mice.
The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.
The development of mature blood cells from hematopoietic stem cells is regulated by transcription factors that control and coordinate the expression of lineage-specific genes. The GATA family consists of six transcription factors that function in hematopoietic and endodermal development. Among them, GATA-1 is expressed in erythroid, megakaryocytic, eosinophil and mast cell lineages, and GATA-2 is expressed in stem and progenitor cells, at more immature stage compared with GATA-1. Based on the characteristic phenotypes of GATA-1 and GATA-2 mutant mice, it has been suggested that mutations of these GATA genes in humans may result in the onset of certain clinical diseases. To date, mutations of GATA-1 gene have been found in inherited anemia and thrombocytopenia, and Down syndrome-related acute leukemia, which exhibits megakaryocytic phenotypes and frequently occurs in patients with Down syndrome. In contrast, no mutation of GATA-2 gene has been identified in hematological diseases; however, we found the expression level of GATA-2 is significantly decreased in CD34 positive cells in patients with aplastic anemia. Since GATA-2 functions in the proliferation of hematopoietic stem cells, the reduction of GATA-2 expression in CD34 positive cells may result in the decreased number of hematopoietic stem cells, which is the characteristic feature of aplastic anemia. Based on these lines of evidence, some types of hematological diseases may be defined as transcription factor diseases.
The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.
Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.
Vertebrate GATA transcription factors have been classified into two subgroups; GATA-1, GATA-2, and GATA-3 are expressed in hematopoietic cells, whereas GATA-4, GATA-5, and GATA-6 are expressed in mesoendoderm-derived tissues. We previously discovered that expression of GATA-2 or GATA-3 under the transcriptional control for the Gata1 gene eliminates lethal anemia in Gata1 germ line mutant mice (Gata1.05/Y). Here, we show that the GATA-4 expression by the same regulatory cassette prolongs the life span of Gata1.05/Y embryos from embryonic day 12.5 to 15.5 but fails to abrogate its embryonic lethality. Gata1.05/Y mice bearing the GATA-4 transgene showed impaired maturation of both primitive and definitive erythroid cells and defective erythroid cell expansion in fetal liver. Moreover, the incidence of apoptosis was observed prominently in primitive erythroid cells. In contrast, a GATA-4-GATA-1 chimeric protein prepared by linking the N-terminal region of GATA-4 to the C-terminal region of GATA-1 significantly promoted the differentiation and survival of primitive erythroid cells, although this protein is still insufficient for rescuing Gata1.05/Y embryos from lethal anemia. These data thus show a functional incompatibility between hematopoietic and endodermal GATA factors in vivo and provide evidence indicating specific roles of the C-terminal region of GATA-1 in primitive erythropoiesis.
Vascular endothelial (VE) cadherin, PECAM-1 (platelet endothelial cell adhesion molecule-1, CD31), Tie2, CD34, and endoglin are established markers for adult and embryonic endothelial cells (ECs). Here, we report that the expression of these EC markers is initiated in the extraembryonic region at the late-streak stage (nominal stage E6.75). Immunohistochemical analysis shows that EC marker-positive cells arise in a subset of Flk1 (VEGF-R2) mesodermal cells. In contrast, GATA1, a marker for primitive erythropoietic progenitors, is expressed in a more restricted subset of Flk1-positive cells. Using flow cytometry, we observed that the GATA1-positive cell population existed as a subset of the EC marker-positive cell. Consistent with this notion, we showed with the primitive hematopoietic colony assay that primitive erythropoietic progenitors are enriched in PECAM-1- and Tie2-positive cells. These results suggest that primitive hematopoietic cells arise from EC marker-positive cells. Thus, VE-cadherin, PECAM-1, CD34, endoglin, and Tie2 are expressed not only in adult and embryonic ECs but in extraembryonic Flk1-positive cells during gastrulation. The latter cell population includes progenitors that give rise to primitive hematopoietic cells, suggesting that primitive and definitive hematopoietic cells in the mouse embryo arise from EC marker-positive cells.
E-peptides and mature insulin-like growth factors (IGFs) are produced from pre-pro-IGFs during post-translational processing and co-secreted into the circulation. Previously, we reported that introduction of a transgene encoding the secreted form of rainbow trout (rt) Ea4-peptide or human (h) Eb-peptide into newly fertilized eggs of medaka (Oryzias latipes) and zebrafish (Danio rerio) resulted in developmental defects in heart, red blood cells and vasculature. In addition to vasculature and red blood cell developmental defects, multiple phenocopies of heart developmental defects categorized by developmental arrest at cardiomyocyte, heart tube and heart looping stages were also observed. These results raise a question of whether rtEa4- or hEb-peptide exerts pleiotropic inhibitory effects on heart, vasculature and red blood cell development in fish embryos. To answer this question, various amounts of recombinant rtEa4-peptide were microinjected into zebrafish eggs at 1.5, 2.5 and 5.5 h post-fertilization (hpf). Although a dose-dependent developmental defect in heart, vasculature and red blood cells was observed in embryos microinjected with rtEa4-peptide at 1.5 and 2.5 hpf, the heart development in all of the microinjected embryos was arrested at the cardiomyocyte stage. Furthermore, the mRNA levels of Nkx2.5, GATA5, VEGF, GATA1 and GATA2 genes in defective embryos were significantly reduced by rtEa4-peptide. These results confirm our previous findings that rtEa4- or hEb-peptide exhibits pleiotropic effects in inhibiting heart, vasculature and red blood cell development in zebrafish embryos.
Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes.
Hematopoiesis is controlled by multiple signaling molecules during embryonic and postnatal development. The function of the fibroblast growth factor (FGF) pathway in this process is unclear. Here we show that FGF plays a key role in the regulation of primitive hematopoiesis in chicks. Using hemoglobin mRNA expression as a sensitive marker, we demonstrate that timing of blood differentiation can be separated from that of initial mesoderm patterning and subsequent migration. High FGF activity inhibits primitive blood differentiation and promotes endothelial cell fate. Conversely, inhibition of FGFR activity leads to ectopic blood formation and down-regulation of endothelial markers. Expression and functional analyses indicate that FGFR2 is the key receptor mediating these effects. The FGF pathway regulates primitive hematopoiesis by modulating Gata1 expression level and activity. We propose that the FGF pathway mediates repression of globin gene expression and that its removal is essential before terminal differentiation can occur.
The 5'-HS4 chicken beta-globin insulator functions as a positional enhancer blocker on chromatinized episomes in human cells, blocking the HS2 enhancer of the human beta-globin locus control region from activating a downstream epsilon-globin gene. 5'-HS4 interrupted formation of a domain of histone H3 and H4 acetylation encompassing the 6-kb minilocus and inhibited transfer of RNA polymerase from the enhancer to the gene promoter. We found that the enhancer blocking phenotype was amplified when the insulated locus contained a weakened HS2 enhancer in which clustered point mutations eliminated interaction of the transcription factor GATA-1. The GATA-1 mutation compromised recruitment of histone acetyltransferases and RNA polymerase II to HS2. Enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator as revealed by micrococcal nuclease and DNase I digestion studies. Nucleosome depletion at 5'-HS4 was dependent on interaction of the insulator protein CCCTC-binding factor (CTCF) and was required for enhancer blocking. These findings provide evidence that a domain of active chromatin is formed by spreading from an enhancer to a target gene and can be blocked by a nucleosome-free gap in an insulator.
GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.
Regulation of transcription requires mechanisms to both activate and terminate transcription factor activity. GATA-1 is a key haemopoietic transcription factor whose activity is increased by acetylation. We show here that acetylated GATA-1 is targeted for degradation via the ubiquitin/proteasome pathway. Acetylation positively signals ubiquitination, suggesting that activation by acetylation simultaneously marks GATA-1 for degradation. Promoter-specific MAPK phosphorylation then cooperates with acetylation to execute protein loss. The requirement for both modifications is novel and suggests a way by which degradation of the active protein can be specifically regulated in response to external phosphorylation-mediated signalling. As many transcription factors are activated by acetylation, we suggest that this might be a general mechanism to control transcription factor activity.
Mutations in transcription factors constitute one means by which normal hematopoietic progenitors are converted to leukemic stem cells. Recently, acquired mutations in the megakaryocytic regulator GATA1 have been found in essentially all cases of acute megakaryoblastic leukemia (AMkL) in children with Down syndrome and in the closely related malignancy transient myeloproliferative disorder. In all cases, mutations in GATA1 lead to the expression of a shorter isoform of GATA-1, named GATA-1s. Because GATA-1s retains both DNA binding zinc fingers, but is missing the N-terminal transactivation domain, it has been predicted that the inability of GATA-1s to regulate its normal class of megakaryocytic target genes is the mechanism by which mutations in GATA1 contribute to the disease. Indeed, several recent reports have confirmed that GATA-1s fails to properly regulate the growth of megakaryocytic precursors, likely through aberrant transcriptional regulation. Although the specific target genes of GATA-1 mis-regulated by GATA-1s that drive this abnormal growth remain undefined, multiple candidate genes have been identified via gene array studies. Finally, the inability of GATA-1s to promote expression of important metabolic genes, such as cytadine deaminase, likely contributes to the remarkable hypersensitivity of AMkL blasts to cytosine arabinoside. Future studies to define the entire class of genes dysregulated by mutations in GATA1 will provide important insights into the etiology of these malignancies.
We have developed an animal model to investigate the mechanisms underlying an acute exacerbation of chronic asthma. Sensitized BALB/c mice were exposed to aerosolized ovalbumin, either as chronic low-level challenge (mass concentration approximately 3 mg/m(3)) for 4 wk, a single moderate-level challenge (approximately 30 mg/m(3)), or chronic low-level followed by single moderate-level challenge (the acute exacerbation group). Compared with animals receiving chronic challenge alone, mice in the acute exacerbation group exhibited a more marked inflammatory response, with involvement of intrapulmonary airways and lung parenchyma, and increased numbers of lymphocytes and eosinophils in bronchoalveolar lavage fluid. They also developed airway hyperreactivity (AHR) to methacholine, demonstrable as increased transpulmonary resistance and decreased compliance. This pattern of AHR was absent in chronically challenged animals, but was also present in animals given single moderate-level challenge. However, compared with animals receiving a single moderate-level challenge, inflammation and AHR were induced more rapidly in the acute exacerbation group. Eosinophil-deficient GATA1 Deltadbl mice exhibited undiminished AHR in the acute exacerbation model. We conclude that in mice with pre-existing airway lesions resembling mild chronic asthma, exposure to a moderately high concentration of inhaled antigen induces features of an acute exacerbation. The inflammatory response involves distal airways and is associated with a distinct pattern of AHR, which develops independent of the enhanced eosinophilic inflammation.
Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G --> C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.
The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.
Expression profiling is a powerful technique to sample cell state. This review shows how expression profiling is being applied to the study of erythroid differentiation.
Expression-based studies of multipotential hematopoietic progenitor cells has shown that these cells express lineage-restricted genes from multiple lineages at low levels, and that they are in effect 'primed' to develop into all hematopoietic cell types. Expression profiling of oligopotent and committed progenitor cells has further shown that commitment to the erythroid lineage is associated with a progressive decline in the number of expressed genes. Lineage commitment is regulated by lineage-restricted transcription factors, and studies show that the erythroid transcription factor GATA1, in addition to activating a subset of genes, has global repressive effects on gene expression. Terminal erythroid differentiation is associated with further reduction in the number of expressed genes. The erythroid program is defined by those genes that are still expressed, and their high-level expression depends on specific epigenetic modifications, recruitment of transcription factors, and posttranscriptional effects.
Expression profiling provides the means to identify novel targets for the therapy of erythrocytes disorders, and to obtain insights into the mechanisms of cellular differentiation.
Transient myeloproliferative disorder (TMD) occurs in 10% of infants with Down syndrome (DS). Down syndrome infants with resolved TMD may later develop acute megakaryocytic leukemia (AMKL). In these patients, AMKL is associated with somatic mutations in the X-linked transcription factor gene, GATA1. AMKL also has been described after TMD in children without DS. We report on a non-DS child identified with trisomy 21 mosaicism and a GATA1 mutation in the original blast cells who has been followed for 2 years without exhibiting AMKL. Currently, the risk for such infants developing acute leukemia is uncertain. We recommend that nondysmorphic infants with TMD undergo chromosome analysis for trisomy 21 and testing for GATA1 mutations to aid surveillance for leukemic transformation.
We describe a pathogenetic mechanism underlying a variant form of the inherited blood disorder alpha thalassemia. Association studies of affected individuals from Melanesia localized the disease trait to the telomeric region of human chromosome 16, which includes the alpha-globin gene cluster, but no molecular defects were detected by conventional approaches. After resequencing and using a combination of chromatin immunoprecipitation and expression analysis on a tiled oligonucleotide array, we identified a gain-of-function regulatory single-nucleotide polymorphism (rSNP) in a nongenic region between the alpha-globin genes and their upstream regulatory elements. The rSNP creates a new promoterlike element that interferes with normal activation of all downstream alpha-like globin genes. Thus, our work illustrates a strategy for distinguishing between neutral and functionally important rSNPs, and it also identifies a pathogenetic mechanism that could potentially underlie other genetic diseases.
The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.
Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied.
Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by chi2 goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm.
Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF < or = 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift.
The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases.
To evaluate the effects of Yisui Shengxue Granules on expressions of alpha-hemoglobin stabilizing protein (AHSP) and erythroid transcription factor GATA-1 mRNAs in bone marrow of patients with beta-thalassemia, and to explore its possible molecular mechanism.
Twelve patients with beta-thalassemia intermedia were treated with Yisui Shengxue Granules for three months. The blood indexes including hemoglobin (Hb), RBC, fetal hemoglobin (HbF) and reticulated corpuscles (Ret) were examined before and after treatment. Total RNA was extracted from bone marrow karyocyte in 8 patients selected from these 12 patients before and after treatment, and the expression levels of the AHSP and GATA-1 mRNAs were measured by real-time PCR.
Yisui Shengxue Granules could not only obviously improve the clinical symptoms of patients with beta-thalassemia intermedia, but also obviously increased the contents of Hb, RBC, HbF and Ret (P<0.05, or P<0.01). The expression levels of AHSP and GATA-1 mRNAs also significantly increased after treatment as compared with those before treatment (P<0.05, or P<0.01).
The results revealed that one of the possible molecular mechanism of the effects caused by Yisui Shengxue Granules is that it can up-regulate the expression levels of AHSP and erythroid transcription factor GATA-1 mRNAs, enhance the protein synthesis of AHSP which can bind the relative excess free alpha-globin, prevent the formation of alpha -globin-cytotoxic precipitates in red blood cells and decrease the hemolysis.
We observed that binding sites for the ubiquitously expressed transcription factor CP2 were present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), we demonstrated the functional importance of the adjacent CP2/GATA-1 sites. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impaired HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 were present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes.
Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C.
It has been reported that somatic mutations in the X-linked GATA1 gene are present in hematological clonal disorders in children with Down syndrome (DS). We analyzed retrospective samples of DS children with acute myeloid leukemia, transient leukemia (TL), and myelodysplastic syndrome (MDS) to test whether the specificity of GATA1 mutations can be helpful in distinguishing these hematopoietic disorders. A total of 49 samples were subjected to GATA1 mutation screening by direct sequencing and denaturing polyacrylamide gel electrophoresis (PAGE). Mutations in exon 2 of GATA1 were detected in six of eight DS-AML M7 samples and in four of six DS-TL; no mutation was detected in 13 children with acute lymphoblastic leukemia (DS-ALL), 6 with DS-AML (M0, M2, and M5), 6 with DS-MDS and in 8 DS infants without hematological disorders and 2 children with AML M7 without DS. Blast cells proportion in the sample represented a critical aspect on the sensitivity of mutation detection in GATA1, and a combination of sequence analysis and PAGE is necessary to detect mutations when blast percentage is low. The absence of detected mutations in any of the DS-MDS cases raises the question whether MDS in DS children is an intermediate stage between TL and AML M7, as previously suggested.
Vascular endothelial growth factor (VEGF) is known to play an essential role in vascular development. We have overexpressed VEGF122 or VEGF170, which are equivalent to mouse VEGF120 and VEGF164, in developing Xenopus embryos. Overexpression of VEGF170 but not VEGF122 demonstrated an absence of expression of hematopoietic markers alpha-globin and GATA-1 but only in the posterior portion of the blood island. Interestingly, strong signals of endothelial markers, msr, fli-1, and tie-2, were detectable in those regions, instead of hematopoietic markers. These results suggested both that injection of VEGF170 resulted in disturbance of vasculogenesis in the posterior portion of the blood island, with excessive production of endothelial cells at the expense of blood cells, and that the anterior and posterior portions of the VBI may have distinct characteristics.
When exposed to a pathogen, a naive CD4(+) T cell is forced to make a cell fate decision that leads to a polarized population of Th1 IFN-gamma- or Th2 IL-4- producing cells. Although IL-4 has traditionally been considered a factor that promotes Th2 cell differentiation, recent evidence has demonstrated that the site and timing of IL-4 expression in an immune response determines its ultimate effects on CD4(+) T cell fate. Using a mast cell (MC) reconstitution model, we demonstrate that MC-derived IL-4 promoted Th1 responses in vivo. Furthermore, MCs from genetically disparate mouse strains varied in their potential for IL-4 expression. Independent of the activation mode, MCs from Th1-prone C57BL/6 mice exhibited a more robust Il4 response than did the Th2-prone strain Balb/c. The hierarchy of IL-4 expression potential was directly associated with the degree of basal chromatin accessibility at cis-regulatory elements conserved noncoding sequence-1 and V(A) enhancer within the Th2 locus. GATA1/2 and Ikaros, factors with opposing roles in chromatin remodeling, acted at these sites. We propose that GATA and Ikaros proteins coordinately fine-tune accessibility at the Il4 locus during development to variably regulate IL-4 expression. These events likely contribute to the genetically determined heterogeneity in Th1 responses that underlie susceptibility to many diseases.
Hemangioblastomas are central nervous system (CNS) tumors of unknown histogenesis, which can occur sporadically or in von Hippel-Lindau disease. Hemangioblastomas are composed of neoplastic "stromal" cells of unknown origin, accompanied by intensive reactive angiogenesis. Failure to specify the cytologic origin of the stromal cell has precluded the development of nonsurgical therapies and limits understanding of its basic biology. We report that the stromal cells express proteins (Scl, brachyury, Csf-1R, Gata-1, Flk-1, and Tie-2) that characterize embryonic progenitor cells with hemangioblastic differentiation potential and conclude that embryonic progenitors with hemangioblast potential represent a possible cytologic equivalent of the stromal cell. We also identified a new autocrine/paracrine stimulatory loop between the receptor Tie-2 and the hypoxia-inducible factor target Ang-1, which, combined with previous observations, suggests that a variety of autocrine loops may be initiated in hemangioblastomas, depending on the differentiation status of the tumor cells and the extent of HIF downstream activation. Finally, the consistent identification of Scl in the stromal cells may help explain the unique and characteristic topographical distribution of hemangioblastomas within the CNS.
The lineage-determining transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is required for myeloid differentiation. Decreased function or expression of C/EBPalpha is often found in human acute myeloid leukemia. However, the precise impact of C/EBPalpha deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl-induced myeloproliferative disease. The expression of bcr/abl in C/EBPalphapos fetal liver cells led to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl-expressing C/EBPalpha-/- fetal liver cells failed to induce a myeloid disease in transplanted mice, but caused a fatal, transplantable erythroleukemia instead. Accordingly, increased expression of the transcription factors SCL and GATA-1 in hematopoietic precursor cells of C/EBPalpha-/-R01-EY-11298 ) fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl-positive cell line. Consistent with findings of the transplant model, expression of C/EBPalpha and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was identified as a critical direct target of C/EBPalpha. Down-regulation of Id1 by RNA interference impaired C/EBPalpha-induced granulocytic differentiation. Taken together, our study provides evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPalpha.
Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.
GATA-1, a zinc finger transcription factor, has been believed to be indispensable for the survival of proerythroblasts. However, we found that GATA-1-null proerythroblasts could survive and proliferate on OP9 stroma cells in the presence of erythropoietin. Furthermore, myeloid and mast cells were induced from the GATA-1-null proerythroblasts by the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), respectively, but lymphoid differentiation was not achieved by in vivo transfer. Thus, without activity of the transcription factor required for terminal differentiation, even relatively mature and committed cells proliferate continuously with the differentiation capacity to other lineages. Our data suggest that GATA-1 is a critical transcription factor to fix erythroid progenitors to the erythroid lineage.
Human acute myeloid leukemias with the t(8;21) translocation express the AML1-ETO fusion protein in the hematopoietic stem cell compartment and show impairment in erythroid differentiation. This clinical finding is reproduced in multiple murine and cell culture model systems in which AML1-ETO specifically interferes with erythroid maturation. Using purified normal human early hematopoietic progenitor cells, we find that AML1-ETO impedes the earliest discernable steps of erythroid lineage commitment. Correspondingly, GATA-1, a central transcriptional regulator of erythroid differentiation, undergoes repression by AML1-ETO in a nonconventional histone deacetylase-independent manner. In particular, GATA-1 acetylation by its transcriptional coactivator, p300/CBP, a critical regulatory step in programming erythroid development, is efficiently blocked by AML1-ETO. Fusion of a heterologous E1A coactivator recruitment module to GATA-1 overrides the inhibitory effects of AML1-ETO on GATA-1 acetylation and transactivation. Furthermore, the E1A-GATA-1 fusion, but not wild-type GATA-1, rescues erythroid lineage commitment in primary human progenitors expressing AML1-ETO. These results ascribe a novel repressive mechanism to AML1-ETO, blockade of GATA-1 acetylation, which correlates with its inhibitory effects on primary erythroid lineage commitment.
Erythroid progenitors have the potential to proliferate rapidly in response to environmental stimuli. This process is referred to as stress erythropoiesis, with erythropoietin (EPO) playing central roles in its promotion. In this study, we wanted to elucidate the molecular mechanisms governing the regulation of stress erythropoiesis and the maintenance of red-cell homeostasis. This was achieved by our development of a noninvasive real-time monitoring system for erythropoiesis using transgenic mouse lines expressing luciferase under the control of the mouse Gata1 hematopoietic regulatory domain (G1-HRD-luc) or human beta-globin locus control region (Hbb-LCR-luc). Optical bioluminescence images revealed that the luciferase was specifically expressed in spleen and bone marrow and was induced rapidly in response to anemia and hypoxia stimuli. The G1-HRD-luc activity tracked the emergence and disappearance of proerythroblast-stage progenitors, whereas the Hbb-LCR-luc activity tracked erythroblasts and later stage erythroid cells. Increased plasma EPO concentration preceded an increase in G1-HRD-luc, supporting our contention that EPO acts as the key upstream signal in stress erythropoiesis. Hence, we conclude that G1-HRD-luc and Hbb-LCR-luc reporters are differentially activated during stress erythropoiesis and that the transgenic mouse lines used serve as an important means for understanding the homeostatic regulation of erythropoiesis.
Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow-derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine.
A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR(-/-) rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR(-/-) rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR(-/-) rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR(-/-) rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR(-/-) rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR(-/-) rescued mice.
These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.
In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis.
In the zebrafish embryo, primitive hematopoiesis initiates in two spatially distinct regions. Rostrally, the cells of the anterior lateral plate mesoderm (ALPM) give rise exclusively to cells of the myeloid lineage in a pu.1-dependent manner. Caudally, in the posterior lateral plate mesoderm (PLPM), the expression of gata1 defines a precursor pool that gives rise predominantly to the embryonic erythrocytes. The transcription factor scl acts upstream of both gata1 and pu.1 in these precursor pools, activating a series of conserved transcription factors that cell-autonomously specify either myeloid or erythroid fates. However, the mechanisms underlying the spatial separation of the hematopoietic precursor pools and the induction of differential gene expression within these pools are not well understood. We show here that the Bmp receptor lost-a-fin/alk8 is required for rostral pu.1 expression and myelopoiesis, identifying an early genetic event that distinguishes between the induction of anterior and posterior hematopoiesis. Introducing a constitutively active version of the Alk8 receptor led to increased pu.1 expression, but the role of alk8 was independent of the scl-dependent cell-fate pathway. Furthermore, the role of Alk8 in myelopoiesis was genetically separable from its earlier role in dorsal-ventral embryonic patterning.
The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass, notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tal1 and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tal1, lmo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development.
Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.
Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias.
Hemoglobin consists of heme and globin proteins and is essential for oxygen transport in all vertebrates. Although biochemical features of heme synthesis enzymes have been well characterized, the function of these enzymes in early embryogenesis is not fully understood. We found that the sixth heme synthesis enzyme, coproporphyrinogen oxidase (CPO), is predominantly expressed in the intermediate cell mass (ICM) that is a major site of zebrafish primitive hematopoiesis. Knockdown of zebrafish CPO using anti-sense morpholinos (CPO-MO) leads to a significant suppression of hemoglobin production without apparent reduction of blood cells. Injection of human CPO RNA, but not a mutant CPO RNA that is similar to a mutant responsible for a hereditary coproporphyria (HCP), restores hemoglobin production in the CPO-MO-injected embryos. Furthermore, expression of CPO in the ICM is severely suppressed in both vlad tepes/gata1 mutants and in biklf-MO-injected embryos. In contrast, over-expression of biklf and gata1 significantly induces ectopic CPO expression. The function of CPO in heme biosynthesis is apparently conserved between zebrafish and human, suggesting that CPO-MO-injected zebrafish embryos might be a useful in vivo assay system to measure the biological activity of human CPO mutations.
Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. The effective concentration to cause 50% malformations (EC50) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang-1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.
Erythroid differentiation involves the transcription factor GATA-1 that positively regulates promoters of erythroid genes (including haemoglobin, glycophorin, erythropoietin receptor) and of erythropoietin. Terminal erythroid differentiation is characterized by major morphological changes that include chromatin condensation and cell size reduction. The morphological changes are partially similar at least to those observed during apoptosis. The production of red cells depends on the apoptosis rate of erythroid progenitors and precursors. Upon erythropoietin starvation or engagement of the death receptor Fas, caspases are activated in erythroid precursors and cleave GATA-1, thus inducing maturation arrest and apoptosis of immature erythroblasts. We have recently demonstrated that, upon erythropoietin stimulation, caspase-3 was also activated, an event required for human terminal erythroblast maturation. Proteins cleaved by caspases in erythroid cells undergoing terminal differentiation include Lamin B and Acinus, which are involved in chromatin condensation. In contrast, despite caspase-3 activation neither GATA-1 degradation nor apoptosis was observed. Thus, the fate of erythroid precursors is determined downstream of caspase activation by the pattern of cleaved targets. Therefore, there are some mechanisms underlying the selective protection of caspase-3 targets during erythropoiesis. This model in which caspases activation is required for differentiation may apply to other haematopoietic or non haematopoietic cellular systems which are described in this review.
In a mouse experimental asthma model, the administration of bacterial lipopolysaccharide (LPS), particularly at low doses, enhances the levels of ovalbumin (OVA)-induced eosinophilic airway inflammation. In an effort to clarify the cellular and molecular basis for the LPS effect, we demonstrate that the OVA-induced eosinophilic inflammation in the lung is dramatically increased by the administration of LPS in wild-type mice, whereas such increase was not observed in mast-cell-deficient mice or Toll-like receptor (TLR)4-deficient mice. Adoptive transfer of bone-marrow-derived mast cells (BMMCs) from wild-type, but not from TLR4-deficient, mice restored the increased eosinophilic inflammation in mast-cell-deficient mice. Wild-type BMMCs pretreated with LPS in vitro also reconstituted the eosinophilic inflammation. Moreover, in vitro analysis revealed that the treatment of BMMCs with LPS resulted in NF-kappaB activation, sustained up-regulation of GATA1 and -2 expression, and increased the capability to produce IL-5 and -13. Dramatic increases in the expression of IL-5 and -13 and Eotaxin 2 were detected in LPS-treated BMMCs after costimulation with LPS and IgE/Ag. Overexpression of GATA1, but not GATA2, in MC9 mast cells resulted in increased transcriptional activity of IL-4, -5, and -13. Furthermore, the levels of transcription of Th2 cytokines in BMMCs were decreased by the introduction of small interfering RNA for GATA1. Thus, mast cells appear to control allergic airway inflammation after their activation and modulation through TLR4-mediated induction of GATA1 and subsequent increase in Th2 cytokine production.
Hemgn (a gene symbol for hemogen in mouse, EDAG in human and RP59 in rat) encodes a nuclear protein that is highly expressed in hematopoietic tissues and acute leukemia. To characterize its regulatory mechanisms, we examined the activities of a Hemgn promoter containing 2975 bp of 5' flanking sequence and 196 bp of 5' untranslated region (5' UTR) sequence both in vitro and in vivo: this promoter is preferentially activated in a hematopoietic cell line, not in nonhematopoietic cell lines, and is sufficient to drive the transcription of a lacZ transgene in hematopoietic tissues in transgenic mice. Mutagenesis analyses showed that the 5' UTR including two highly conserved GATA boxes is critical for the promoter activity. GATA1, not GATA2, binds to the GATA binding sites and transactivates the Hemgn promoter in a dose-dependent manner. Furthermore, the expression of human hemogen (EDAG) transcripts were closely correlated with levels of GATA1 transcripts in primary acute myeloid leukemia specimens. This study suggests that the Hemgn promoter contains critical regulatory elements for its transcription in hematopoietic tissues and Hemgn is a direct target of GATA1 in leukemia cells.
Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector-based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter-driven transgenes or Pol III promoter-controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.
The transcriptional regulation of the human reduced folate carrier (hRFC), involved in cellular uptake of methotrexate and reduced folates, was studied in childhood acute lymphoblastic leukemia (ALL). The hRFC gene is regulated by six noncoding exons (A1/A2 and A to E) and multiple promoters. In ALL, hRFC-A1/A2 and hRFC-B are the major transcript forms.
RNAs from 18 ALL lymphoblast specimens and 10 nonobese diabetic/severe combined immunodeficient ALL xenografts were assayed by real-time reverse transcription-PCR for hRFC-A1/A2 and hRFC-B transcripts and for transcripts encoding USF1, GATA1, Sp1, and Ikaros transcription factors. For the xenografts, gel shift and chromatin immunoprecipitation assays assessed transcription factor binding to the hRFC-A1/A2 and hRFC-B promoters. CpG methylation density within a 334-bp region, including the core hRFC-B promoter, was established by bisulfite sequencing. hRFC-A1/A2 and hRFC-B promoter polymorphisms were assayed by DNA sequencing.
For the 28 ALLs, hRFC-A1/A2 and hRFC-B transcripts spanned a 546-fold range. By chromatin immunoprecipitation and gel shift assays, binding was confirmed for USF1 and GATA1 for hRFC-A1/A2, and for Sp1, USF1, and Ikaros for hRFC-B. hRFC transcript levels correlated with those for GATA1 and USF1 for hRFC-A1/A2 and with Sp1 and USF1 transcripts for hRFC-B. CpG methylation in ALL did not correlate with hRFC-B transcripts. In 40 ALL and 17 non-ALL specimens, 2 cosegregating high-frequency polymorphisms (T-1309/C-1217 and C-1309/T-1217; allelic frequencies of 36% and 64%, respectively) were detected in the A1/A2 promoter; none were detected in promoter B. The hRFC-A1/A2 polymorphisms only slightly affected promoter activity.
Our results show a complex regulation of hRFC in ALL involving the hRFC-A1/A2 and hRFC-B promoters and noncoding exons. Although Sp1, USF1, and GATA1 levels are critical determinants of hRFC transcription in ALL, neither DNA methylation nor promoter polymorphisms contribute to differences in hRFC expression.
The Myb proto-oncogene encodes a transcription factor (c-Myb) that is essential for normal hematopoiesis and is thought to regulate hematopoietic cell proliferation and differentiation by regulating expression of specific target genes. We identify the mouse erythroid-specific carbonic anhydrase I promoter (CAIe) as a target of c-Myb activity and demonstrate that Myb activity is critical for carbonic anhydrase I (CAI) expression in C19 MEL cells. CAI expression is downregulated when MEL cells differentiate in response to MEnT or treatment with N, N-hexamethylene bisacetamide (HMBA). Coexpression of GATA-1 with c-Myb results in synergistic activation of transcription from the CAIe promoter and both transcription factors interact with the CAIe promoter in vivo. We identify a novel 20 bp sequence in the CAIe promoter that is sufficient to mediate synergistic activation of the CAIe promoter by c-Myb and GATA-1. c-Myb and GATA-1 interact with this DNA sequence suggesting that c-Myb and GATA-1 may be contained in a complex that interacts with this region of the CAIe promoter. Forced expression of CAI delayed HMBA-induced differentiation of MEL cells and maintained them in a proliferating state. These data strongly suggest that CAI is a c-Myb target and is involved in regulating MEL cell proliferation and differentiation.
Sertoli cell number is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the seasonal breeding adult Djungarian hamster (Phodopus sungorus) model challenged this assertion by demonstrating a decrease in Sertoli cell number after gonadotropin depletion and a return to control levels following 7 days of FSH replacement. The present study aimed to determine whether adult Sertoli cells are terminally differentiated using known characteristics of cellular differentiation, including proliferation, junction protein localization, and expression of particular maturational markers, in the Djungarian hamster model. Adult long-day (LD) photoperiod (16L:8D) hamsters were exposed to short-day (SD) photoperiod (8L:16D) for 11 wk to suppress gonadotropins and then received exogenous FSH for up to 10 days. Sertoli cell proliferation was assessed by immunofluorescence by the colocalization of GATA4 and proliferating cell nuclear antigen and quantified by stereology. Markers of Sertoli cell maturation (immature, cytokeratin 18 [KRT18]; mature, GATA1) and junction proteins (actin, espin, claudin 11 [CLDN11], and tight junction protein 1 [TJP1, also known as ZO-1]) also were localized using confocal immunofluorescence. In response to FSH treatment, proliferation was upregulated within 2 days compared with SD controls (90% vs. 0.2%, P < 0.001) and declined gradually thereafter. In LD hamsters, junction proteins colocalized at the basal aspect of Sertoli cells, consistent with inter-Sertoli cell junctions, and were disordered within the Sertoli cell cytoplasm in SD animals. Exogenous FSH treatment promptly restored localization of these junction markers to the LD phenotype. Protein markers of maturity remain consistent with those of adult Sertoli cells. It is concluded that adult Sertoli cells are not terminally differentiated in the Djungarian hamster and that FSH plays an important role in governing the differentiation process. It is proposed that Sertoli cells can enter a transitional state, exhibiting features common to both undifferentiated and differentiated Sertoli cells.
Phosphorylation of transcription factors is important in posttranslational control of protein function. The indispensable zinc-finger transcription factor, Gata1, is phosphorylated constitutively at 6 serine residues (26, 49, 72, 142, 178, 187), and at a seventh (310) following induction of erythroid differentiation. However, the biologic consequences of phosphorylation with respect to function are unclear. To address this issue, we generated mice with serine-to-alanine mutations at the inducibly phosphorylated serine 310 alone or at conserved serine residues 72, 142, and 310 together. The peripheral blood parameters of the mice were normal, as was their response to acute erythropoietic stress. Analysis of hematopoietic progenitor populations during ontogeny and into adulthood showed a moderate decrease in erythroid burst-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) numbers only in the adult bone marrow of the triple mutant. Yet, later stage erythropoiesis was not perturbed. This suggests that any molecular consequences associated with loss of phosphorylation at residues 72, 142, and 310 can be compensated for in the in vivo environment.
RUNX1/AML1, located on chromosome 21, is a key factor in the generation and maintenance of hematopoietic stem cells and the gene most frequently implicated in human leukemias. Chromosome translocations and point mutations are well-documented genetic alterations in RUNX leukemia (also known as CBF leukemia). In addition, overdosage or overexpression of RUNX1 is suspected to be a third mode of RUNX1 involvement in leukemogenesis. The possibility that this mode might underlie Down syndrome-related leukemias caused by trisomy of chromosome 21 is discussed.
The chromosomal translocation t(8;21), generating the AML1-ETO fusion protein, is frequently associated with French-American-British (FAB) type M2 acute myeloid leukemia (AML). t(8;21) fuses the runt domain from the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. AML1-ETO inhibits normal definitive hematopoiesis and blocks erythroid differentiation. Several mechanistic models for the role of AML1-ETO in leukemia development have emerged over the last decade. Most of these models have emphasized the capacity of the fusion protein to redirect repressive cofactors, such as histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), to RUNX target genes, thereby reversing the hematopoietic transcriptional program activated by wild-type RUNX1a phenomenon referred to collectively in this review as the "classical" corepressor model. Because erythropoiesis occurs in a RUNX-independent manner, this dominant-negative "classical" model cannot explain the prominent repression of red-cell development by AML1-ETO. This review will consider the clinical and mechanistic significance of erythroid inhibition by AML1-ETO. Additional models to account for this mysterious oncogenic function are proposed.
Hemoglobin E (Hb E)-beta-thalassemia patients display a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. Given this clinical heterogeneity, additional genetic factors modifying disease severity remain to be discovered. Association studies are being conducted to elucidate the role of genetic polymorphisms as disease severity modifiers in Hb E-beta-thalassemia patients. Using strict scoring criteria, 1060 Hb E-beta-thalassemia patients were categorized into mild, moderate, and severe groups. Taking a candidate gene approach, we found no statistically significant differences between the mild and severe patients groups in allelic or genotypic frequencies for single nucleotide polymorphisms within five genes known to influence globin gene expression and erythropoiesis.
Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.
We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA-1 in erythroid cells. GATA-1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA-1 functions are mediated. Our work describes, for the first time, distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. We also provide evidence that distinct GATA-1 complexes are associated with specific GATA-1 functions in erythroid differentiation, for example, GATA-1/Gfi-1b with the suppression of cell proliferation and GATA-1/FOG-1/MeCP1 with the repression of other hematopoietic transcription programs. We next applied the biotinylation tag to Ldb-1, a known partner of GATA-1, and characterized a number of novel interaction partners that are essential in erythroid development, in particular, Eto-2, Lmo4, and CdK9. Last, we are in the process of applying the same technology to characterize the factors that are bound to the suppressed gamma-globin promoter in vivo.
During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp-morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.
Ets-1 is a widely expressed transcription factor implicated in development, tumorigenesis and hematopoiesis. We analyzed Ets-1 gene expression during human erythroid and megakaryocytic (MK) differentiation in unilineage cultures of CD34+ progenitor cells. During erythroid maturation, Ets-1 is downmodulated and exported from the nucleus into the cytoplasm through an active mechanism mediated by a leucine-rich nuclear export signal. In contrast, during megakaryocytopoiesis Ets-1 increases and remains localized in the nucleus up to terminal maturation. Overexpression of Ets-1 in erythroid cells blocks maturation at the polychromatophilic stage, increases GATA-2 and decreases both GATA-1 and erythropoietin receptor expression. Conversely, Ets-1 overexpressing megakaryocytes are characterized by enhanced differentiation and maturation, coupled with upmodulation of GATA-2 and megakaryocyte-specific genes. We show that Ets-1 binds to and activates the GATA-2 promoter, in vitro and in vivo, indicating that one of the pathways through which Ets-1 blocks erythroid and promotes MK differentiation is via upmodulation of GATA-2 expression.
Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.
Up-regulation of tumor necrosis factor alpha (TNFalpha) is linked to solid tumors as well as to hematologic disorders including different forms of anemia and multiple myeloma. This cytokine was shown to contribute to inhibition of erythroid maturation mechanisms which are characterized by the expression of specific genes regulated by GATA-1 and NF-E2 transcription factors. Here, we assessed the inhibiting effect of TNFalpha on erythroid differentiation using K562 cells which can be chemically induced to differentiate towards the erythroid pathway by aclacinomycin A, an anthracyclin. Results show that induced hemoglobinization of K562 cells as well as gamma-globin and erythropoietin receptor gene expression are decreased by TNFalpha via the inhibition of GATA-1 at its mRNA and protein expression level. Additionally, both constitutive and induced binding activity of GATA-1 is abolished and induced activation of a GATA-1 driven luciferase reporter construct is inhibited. Altogether, our results provide insight into the molecular mechanisms of inflammation-induced inhibition of erythroid differentiation.
The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (pro-neural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorso-central enhancer (DC) of ac/sc. We identify the LIM-homeo domain (LIM-HD) transcription factor Islet (Isl) by genetic screening and investigate its role in the thoracic pre-patterning. We show that isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) pro-neural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases pro-neural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC pro-neural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. We propose that sensory organ pre-patterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying pre-patterns within the different pro-neural clusters.
Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2 cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.
Transient myeloproliferative disorder (TMD) is found in 10% of newborns with Down syndrome (DS). Myeloid leukemia develops in 25% within the following 3 years. Little is known about markers predicting leukemia occurrence. We studied expression levels of the Wilms tumor gene (WT1) by real-time quantitative PCR (RQ-PCR) in peripheral blood of five infants with TMD. WT1 levels were elevated similar to findings in AML. Longitudinal studies showed normalization of the WT1 level in all patients except one who developed GATA1 mutated myeloid leukemia at 11 months of age. The lack of normalization of WT1 level may be a predictor of leukemia development and WT1 expression may be an attractive marker for monitoring of minimal residual disease.
Krüppel-like factor 6 (KLF6) is a member of a growing family of transcription factors that share a common 3 C2H2 zinc finger DNA binding domain and have broad activity in regulating proliferation and development. We have previously established that Klf6 is expressed in neuronal tissue, hindgut, heart, lung, kidney, and limb buds during midgestation. To explore the potential role of Klf6 in mouse development, we analyzed Klf6-/- mice and found that the homozygous mutation is embryonic lethal by embryonic day (E) 12.5 and associated with markedly reduced hematopoiesis and poorly organized yolk sac vascularization. Additionally, mRNA levels of Scl and Gata1 were reduced by approximately 80% in Klf6-/- yolk sacs. To further analyze this phenotype, we generated Klf6-/- embryonic stem (ES) cells by homologous recombination, and compared their capacity to differentiate into the hematopoietic lineage with that of either Klf6+/- or Klf6+/+ ES cells. Consistent with the phenotype in the early embryo, Klf6-/- ES cells displayed significant hematopoietic defects following differentiation into EBs. Prolongation of epiblast-like cells and delays in mesoderm induction were also observed in the Klf6-/- EBs, associated with delayed expression of Brachyury, Klf1, and Gata1. Forced expression of KLF6 using a tet-inducible system enhanced the hematopoietic potential of wild-type EBs. Collectively, these findings implicate Klf6 in ES-cell differentiation and hematopoiesis.
The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5' and 3' deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation.
We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.
Six cases of megakaryocytic leukemia (MKL) were identified and analyzed for morphology and molecular features. MKL were composed of megakaryocyte lineage cells ranging from immature to quite mature cells. VWF, GATA1 and RUNX1 were strongly expressed in megakaryocytes in both normal spleen and MKL as analyzed by immunohistochemistry (IHC). Altered expression of Meis1, Pbx1 and Psen2 and Lef1 in MKL detected with oligonucleotide microarrays was confirmed by qPCR and IHC. This is the first report of spontaneous MKL in mice, defining VWF as a biomarker for diagnosis and suggesting possible involvement of a series of genes in disease pathogenesis.
The stem cell leukemia (SCL) transcription factor is essential for vertebrate hematopoiesis. Using the powerful zebrafish model for embryonic analysis, we compared the effects of either reducing or ablating Scl using morpholino-modified antisense RNAs. Ablation of Scl resulted in the loss of primitive and definitive hematopoiesis, consistent with its essential role in these processes. Interestingly, in embryos with severely reduced Scl levels, erythroid progenitors expressing gata1 and embryonic globin developed. Erythroid maturation was deficient in these Scl hypomorphs, supporting that Scl was required both for the erythroid specification and for the maturation steps, with maturation requiring higher Scl levels than specification. Although all hematopoietic functions were rescued by wild-type Scl mRNA, an Scl DNA binding mutant rescued primitive and definitive hematopoiesis but did not rescue primitive erythroid maturation. Together, we showed that there is a distinct Scl hypomorphic phenotype and demonstrated that distinct functions are required for the roles of Scl in the specification and differentiation of primitive and definitive hematopoietic lineages. Our results revealed that Scl participates in multiple processes requiring different levels and functions. Further, we identified an Scl hypomorphic phenotype distinct from the null state.
Erythropoietin (Epo) stimulation of its receptor's downstream signaling pathways and optimum function of GATA-1 transcription factor are both essential for normal erythroid cell development. Epo-receptor (EpoR) signaling and GATA-1 regulate proliferation, survival, differentiation, and maturation of erythroid cells. Whether any signal that is generated by EpoR targets GATA-1 or affects GATA-1 transcriptional activity is not known. Here, we demonstrate that stimulation of EpoR results in phosphorylation of GATA-1 at serine 310 (S310) in primary fetal liver erythroid progenitors and in cultured erythroid cells. We show that phosphorylation of GATA-1 is important for Epo-induced maturation of fetal liver erythroid progenitor cells. The PI3-kinase/AKT signaling pathway is identified as a mediator of Epo-induced phosphorylation of GATA-1. AKT serine threonine kinase phosphorylates GATA-1S310 in vitro and in erythroid cells and enhances GATA-1 transcriptional activity. These data demonstrate that EpoR signaling phosphorylates GATA-1 and modulates its activity via the PI3-kinase/AKT signaling pathway.
The primary genetic lesion(s), as well as the biological processes responsible for the typical structural changes of the bone marrow microenvironment in idiopathic myelofibrosis, are still poorly understood, although a central role in disease pathogenesis has been attributed to the clonal proliferation and defective maturation of megakaryocytes. Two animal models of the disease have been described, that in the last few years significantly contributed to the elucidation of some of the pathogenetic steps of the human disease; these are represented by mice genetically modified to overexpress thrombopoietin and by knock-down mice with defective GATA-1 expression in megakaryocytes (GATA-1(low) mice). This review will outline these murine models, both characterized by extensive accumulation of megakaryocytes in hematopoietic tissues, and illustrate how they provided insights into the identification of some of the molecules and mechanisms responsible for the development of fibrosis and osteosclerosis that present major similarities with those observed in patients with idiopathic myelofibrosis.
The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.
To observe the differentiation-inducing properties of the novel erythroid differentiation related gene EDRF1, and demonstrate its functioning pathway involved in regulation of alpha- and gamma-globin gene expression.
EDRF1 sense and antisense constructs were transfected into HEL cells, then the expression of globin and erythropoietin receptor gene was identified by Northern blot analysis. RT-PCR and electrophoresis mobility shift assay (EMSA) were carried out to monitor the expression and DNA-binding activity of erythroid specific transcription factor GATA-1 and NF-E2.
In HEL cells with transfection of sense expression carrier, the experssion of EDRF1 mRNA was upregulated. In HEL cells with transfection of antisense expression carrier, the experession of EDRF1 mRNA was downregulated. In HEL cells with overexpression of EDRF1, production of alpha-globin was increased. In antisense EDRF1 overexpressed HEL cells, significant loss of alpha- and gamma-globin mRNA synthesis was observed. The expression of erythropoietin receptor was unchanged in HEL cells transfected with EDRF1 sense and antisense expression carriers. No significant change was found in expression of transcription factors GATA-1 and NF-E2 in HEL cells transfected with empty carrier, sense expression carrier, and antisense expression carrier. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression.
EDRF1 regulates alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor. Overexpression and underexpression of EDRF1 do not significantly regulate the expression of GATA-1 and NF-E2 mRNA. Monitoring the EPOR signal pathway is not the mechanism by which EDRF1 influences the differentiation of erythrocyte.
Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds and stabilizes alpha-hemoglobin during normal erythropoiesis and in pathological states of alpha-hemoglobin excess. AHSP has been proposed as a candidate gene in some Heinz body hemolytic anemias and as a modifier gene in the beta-thalassemia syndromes. To gain additional insight into the molecular mechanisms controlling the erythroid-specific expression of the AHSP gene and provide the necessary tools for further genetic studies of these disorders, we have initiated identification and characterization of the regulatory elements controlling the human AHSP gene. We mapped the 5'-end of the AHSP erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative AHSP gene promoter. In vitro studies using transfection of promoter/reporter plasmids in human tissue culture cell lines, DNase I footprinting analyses and gel mobility shift assays, identified an AHSP gene erythroid promoter with functionally important binding sites for GATA-1- and Oct-1-related proteins. In transgenic mice, a reporter gene directed by a minimal human AHSP promoter was expressed in bone marrow, spleen, and reticulocytes, but not in nonerythroid tissues. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of the promoter region and occupancy by GATA-1. The AHSP promoter is an excellent candidate region for mutations associated with decreased AHSP gene expression.
Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.
Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. Our laboratory has previously defined the human Gfi-1B promoter region and shown that GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, here we report that (i) Gfi-1B transcription is negatively regulated by its own gene product, (ii) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (iii) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, we propose that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells.
The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms.
The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.
GATA family transcription factors play essential roles in broad developmental settings. GATA-1, one of the hematopoietically expressed members, is required for normal erythroid and megakaryocytic differentiation. Over the past few years, mutations in the gene encoding GATA-1 have been linked to several human hematologic disorders, including X-linked dyserythropoietic anemia and thrombocytopenia, X-linked thrombocytopenia and beta-thalassemia, and Down syndrome acute megakaryoblastic leukemia. This review summarizes the role of GATA-1 during normal hematopoiesis and discusses how disease-associated mutations may affect its function.
Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.
Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc-kitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%-70% of the cells in BFU-E-derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%-84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.
Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 drives the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythromegakaryocytic differentiation. To investigate these functions further, we stimulated Gata1- murine embryonic stem-cell-derived hematopoietic cultures with thrombopoietin, a multistage cytokine. Initially, the cultures generated a wave of mutant megakaryocytes. However, these were rapidly overgrown by a unique population of thrombopoietin-dependent blasts that express immature markers and proliferate indefinitely. Importantly, on restoration of GATA-1 function, these cells differentiated into both erythroid and megakaryocytic lineages, suggesting that they represent bipotential progenitors. Identical cells are also present in vivo, as indicated by flow cytometry and culture analysis of fetal livers from Gata1- chimeric mice. Our findings indicate that loss of GATA-1 impairs the maturation of megakaryocyte-erythroid progenitors. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia, a clonal malignancy with features of both erythroid and megakaryocyte maturation.
Megakaryocytes are the hematopoietic precursors of platelets, which play an essential role in thrombosis and hemostasis. Platelet factor 4 (PF4) is expressed exclusively in megakaryocytes and platelets and serves as a lineage-specific marker of megakaryocytic differentiation. We previously characterized a number of upstream enhancer and repressor elements and demonstrated that GATA-1 and ETS-1 are important for PF4 gene expression. Recently, we have determined the novel regulatory element termed "TME" in the PF4 promoter and identified a group of binding proteins from megakaryocytic HEL cells. Here we review the function of these proteins in PF4 gene expression and discuss megakaryocyte-specific gene expression and megakaryocytepoiesis.
Aneuploidy is one of the hallmarks of cancer. Acquired additions of chromosome 21 are a common finding in leukemias, suggesting a contributory role to leukemogenesis. About 10% of patients with a germ line trisomy 21 (Down syndrome) are born with transient megakaryoblastic leukemia. We and others have shown acquired mutations in the X chromosome gene GATA1 in all these cases. The gene or genes on chromosome 21 whose overexpression promote the megakaryoblastic phenotype are presently unknown. We propose that ERG, an Ets transcription factor situated on chromosome 21, is one such candidate. We show that ERG is expressed in hematopoietic stem cells, megakaryoblastic cell lines, and in primary leukemic cells from Down syndrome patients. ERG expression is induced upon megakaryocytic differentiation of the erythroleukemia cell lines K562 and UT-7, and forced expression of ERG in K562 cells induces erythroid to megakaryoblastic phenotypic switch. We also show that ERG activates the gpIb megakaryocytic promoter and binds the gpIIb promoter in vivo. Furthermore, both ERG and ETS2 bind in vivo the hematopoietic enhancer of SCL/TAL1, a key regulator of hematopoietic stem cell and megakaryocytic development. We propose that trisomy 21 facilitates the occurrence of megakaryoblastic leukemias through a shift toward the megakaryoblastic lineage caused by the excess expression of ERG, and possibly by other chromosome 21 genes, such as RUNX1 and ETS2, in hematopoietic progenitor cells, coupled with a differentiation arrest caused by the acquisition of mutations in GATA1.
The abnormal megakaryocytopoiesis associated with idiopathic myelofibrosis (IM) plays a role in its pathogenesis. Because mice with defective expression of transcription factor GATA-1 (GATA-1(low) mutants) eventually develop myelofibrosis, we investigated the occurrence of GATA-1 abnormalities in IM patients. CD 34(+) cells were purified from 12 IM patients and 8 controls; erythroblasts and megakaryocytes were then obtained from unilineage cultures of CD 34(+) cells. Purified CD 61(+), GPA(+), and CD 34(+) cells from IM patients contained levels of GATA-1, GATA-2, and FOG-1 mRNA, as well as of GATA-2 protein, that were similar to controls. In contrast, CD 61(+) cells from IM patients contained significantly reduced GATA-1 protein. Furthermore, 45% of megakaryocytes in biopsies from IM patients did not stain with anti-GATA-1 antibody, as compared to controls (2%), essential thrombocythemia (4%), or polycythemia vera (11%) patients. Abnormalities in immunoreactivity for FOG-1 were not found, and no mutations in GATA-1 coding sequences were found. The presence of GATA-1(neg) megakaryocytes in bone marrow biopsies was independent of the Val 617 Phe JAK 2 mutation, making it unlikely that a downstream functional relationship exists. We conclude that megakaryocytes from IM patients have reduced GATA-1 content, possibly contributing to disease pathogenesis as in the GATA-1(low) mice and also representing a novel IM-associated marker.
Viruses rely on attachment to specific cell surface receptors to infect host cells. Selective expression of viral receptors has the potential to attenuate infection of susceptible tissues by redirecting virus to cells that cannot support viral replication. We propose that erythrocytes are an ideal instrument for this strategy, because they are present in vast numbers, permeate every organ, and cannot serve as hosts for viral propagation. To test this hypothesis, we generated a transgenic mouse, termed globin transcription factor 1 (GATA1)-coxsackie and adenovirus receptor (CAR), that expressed the CAR on erythrocytes. Coxsackievirus group B (CVB) adhered to the surface of CAR-expressing erythrocytes and was rendered noninfectious. Upon infection with CVB, GATA1-CAR mice had diminished viremia and reduced viral replication in heart, brain, and liver. Furthermore, when faced with a CVB challenge that was lethal to WT littermates, the survival of GATA1-CAR mice was prolonged, and their ultimate mortality was reduced. The GATA1-CAR mouse model presented here demonstrates that erythrocyte expression of CAR limits CVB pathogenesis. Erythrocytes also may be coated with a variety of receptors by nontransgenic methods, making this a very flexible model for the treatment of infectious diseases in humans.
All mice harboring the X-linked Gata1low mutation in a predominantly CD1 background are born anemic and thrombocytopenic. They recover from anemia at 1 month of age but remain thrombocytopenic all their life and develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, at 12 months. The effects of the genetic background on the myelofibrosis developed by Gata1low mice was assessed by introducing the mutation, by standard genetic approaches, in the C57BL/6 and DBA/2 backgrounds and by analyzing the phenotype of the different mutants at 12 to 13 (by histology) and 16 to 20 (by cytofluorimetry) months of age. Although all the Gata1low mice developed fibrosis at 12 to 13 months, variegations were observed in the severity of the phenotype expressed by mutants of different backgrounds. In C57BL/6 mice, the mutation was no longer inherited in a Mendelian fashion, and fibrosis was associated with massive osteosclerosis. Instead, DBA/2 mutants, although severely anemic, expressed limited fibrosis and osteosclerosis and did not present tear-drop poikilocytes in blood or extramedullary hemopoiesis in liver up to 20 months of age. We propose that the variegation in myelofibrosis expressed by Gata1low mutants of different strains might represent a model to study the variability of the clinical picture of the human disease.
The contribution of erythropoietin to the differentiation of the red blood cell lineage remains elusive, and the demonstration of a molecular link between erythropoietin and the transcription of genes associated with erythroid differentiation is lacking. In erythroid cells, expression of the tissue inhibitor of matrix metalloproteinase (TIMP-1) is strictly dependent on erythropoietin. We report here that erythropoietin regulates the transcription of the TIMP-1 gene upon binding to its receptor in erythroid cells by triggering the activation of phosphatidylinositol 3-kinase (PI3K)/Akt. We found that Akt directly phosphorylates the transcription factor GATA-1 at serine 310 and that this site-specific phosphorylation is required for the transcriptional activation of the TIMP-1 promoter. This chain of events can be recapitulated in nonerythroid cells by transfection of the implicated molecular partners, resulting in the expression of the normally silent endogenous TIMP-1 gene. Conversely, TIMP-1 secretion is profoundly decreased in erythroid cells from fetal livers of transgenic knock-in mice homozygous for a GATA(S310A) gene, which encodes a GATA-1 mutant that cannot be phosphorylated at Ser(310). Furthermore, retrovirus-mediated expression of GATA(S310A) into GATA-1(null)-derived embryonic stem cells decreases the rate of hemoglobinization by more than 50% compared to expressed wild-type GATA-1. These findings provide the first example of a chain of coupling mechanisms between the binding of erythropoietin to its receptor and GATA-1-dependent gene expression.
Hematopoiesis is a complex process regulated by nuclear proteins that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis has revealed critical roles for the X-linked transcription factor GATA-1 in erythrocyte and megakaryocyte differentiation. GATA-1 has two zinc fingers essential for normal function. The C-terminal finger is necessary for DNA binding. The N-terminal finger mediates interaction with FOG-1, a cofactor for GATA-1. Mutations in the N-terminal zinc finger of GATA-1 result in abnormal hematopoiesis. Here we report a family with a novel single base mutation that results in an amino acid substitution (Gly208Arg) within the highly conserved portion of the GATA-1 N-terminal finger domain, leading to dyserythropoietic anemia and macrothrombocytopenia. Another mutation described at the same codon (208) has been found to be associated with thrombocytopenia only. Our data support and extend the effect of the amino acid substitution at codon 208 on GATA-1 function not only regarding megakaryocyte but also regarding erythroid development.
PU.1, a hematopoietic Ets transcription factor, is required for development of the lymphoid and myeloid lineages. We have previously shown that PU.1 functions as both a transcriptional activator and repressor through complex formation with CBP/p300 and HDAC1/mSin3A/MeCP2, respectively. To determine whether modification of PU.1 is responsible for switching its association between co-activators and co-repressors, we examined whether acetylation regulates the physical and functional activities of PU.1. PU.1 was acetylated in vivo and its repressor activity was reduced when the putative acetylation motifs in the Ets domain were mutated. The mutant cooperated with CBP similar to wild type PU.1, but insufficiently with GATA-1 and mSin3A. Whereas overexpression of wild type PU.1 induced differentiation block, growth inhibition, and apoptotic cell death in MEL erythroleukemia cells as we reported previously, overexpression of the mutant-acetylation motif PU.1 did not. Taken together, our data suggest that acetylation might regulate the biological functions of PU.1 in erythroid cells.
GATA-1, the founding member of the GATA transcription factor family, is essential for cell maturation and differentiation within the erythroid and megakaryocytic lineages. GATA-1 regulates the expression of many genes within these lineages and its functionality depends upon its ability to bind both DNA and protein partners. Disruption of either of these functions causes severe hematopoietic dysfunction and results in blood disorders, such as thrombocytopenia and anemia. Within this review, we will focus on the structural aspects of GATA-1 with regard to interactions with its many partners and the identification of several mutations that disrupt these interactions.
Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.
We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.
Stem cell factor (SCF), erythropoietin (Epo), and GATA-1 play an essential role(s) in erythroid development. We examined how these proteins interact functionally in G1E cells, a GATA-1(-) erythroblast line that proliferates in an SCF-dependent fashion and, upon restoration of GATA-1 function, undergoes GATA-1 proliferation arrest and Epo-dependent terminal maturation. We show that SCF-induced cell cycle progression is mediated via activation of the Src kinase/c-Myc pathway. Restoration of GATA-1 activity induced G1 cell cycle arrest coincident with repression of c-Kit and its downstream effectors Vav1, Rac1, and Akt. Sustained expression of each of these individual signaling components inhibited GATA-1-induced cell cycle arrest to various degrees but had no effects on the expression of GATA-1-regulated erythroid maturation markers. Chromatin immunoprecipitation analysis revealed that GATA-1 occupies a defined Kit gene regulatory element in vivo, suggesting a direct mechanism for gene repression. Hence, in addition to its well-established function as an activator of erythroid genes, GATA-1 also participates in a distinct genetic program that inhibits cell proliferation by repressing the expression of multiple components of the c-Kit signaling axis. Our findings reveal a novel aspect of molecular cross talk between essential transcriptional and cytokine signaling components of hematopoietic development.
In the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life.
Fagaronine, a benzophenanthridine alkaloid from Fagara zanthoxyloides Lam. (Rutaceae), has been tested on the erythroleukemic cell line K562 in order to explain some previous results on cell differentiation. In this study we showed that fagaronine induces a significant hemoglobinization of the human erythroleukemic cell line K562. This hemoglobin synthesis was accompanied by a strong increase of erythroid mRNA expression such as gamma- and alpha-globin, and PBGD, an enzyme of heme synthesis. In addition, the Epo-R transcripts were also stimulated indicating that cells are engaged in a maturation process. Both transcription factors GATA-1 and NF-E2, which play an important role in the regulation of genes involved in the erythroid differentiation, were also transcriptionally up-regulated. To elucidate the possible role of GATA-1 in the FAG-induced differentiation of K562 cells, we transfected reporter constructs containing regulatory regions of erythroid genes encompassing GATA-1 binding sites. After 48 hours of treatment, FAG stimulated the EPO-R and gamma-globin promoters by 2- to 3-fold and the promoter/enhancer region of GATA-1 gene by 3.2-fold. A mutation within the GATA-1 binding sites strongly decreased the promoter activation induced by FAG. Taken together, our results represent a demonstration that FAG exerts its differentiating activity by a specific activation of the regulating GATA-1 regions of genes involved in the erythroid phenotype expression.
In the interleukin 3-dependent hematopoietic cell line Ba/F3, inhibition of mitogen-activated protein kinase, a member of the MAPK/c-Jun N-terminal kinase/stress-activated protein kinase kinase family that plays an important role in cell growth and death control, rapidly leads to severe apoptosis. However, most of the antiapoptotic substrates of MAPK remain to be identified. Here we report that, upon interleukin-3 stimulation of Ba/F3 cells, the transcription factor GATA-1 is strongly phosphorylated at residue serine 26 by a MAPK-dependent pathway. Phosphorylation of GATA-1 increases GATA-1-mediated transcription of the E4bp4 survival gene without significantly changing the DNA-binding affinity of GATA-1. Further characterization of GATA-1 phosphorylation site mutants revealed that the antiapoptotic function of GATA-1 is strongly dependent upon its phosphorylation at the Ser-26 position and is probably mediated through its up-regulation of Bcl-X(L) expression. Taken together, our data demonstrate that MAPK-dependent GATA-1 phosphorylation is important for its transactivation of the E4bp4 gene, Bcl-X(L) expression and cell survival. Therefore, GATA-1 may represent a novel MAPK substrate that plays an essential role in a cytokine-mediated antiapoptotic response.
GATA-1 exerts a concentration-dependent control on the differentiation of erythroid, megakaryocytic, mast, and eosinophilic cells. The concentration of GATA-1 is, in turn, regulated by specific sequences within the GATA-1 locus. On the basis of its levels of expression, the GATA-1 protein becomes associated with suitable partners forming transcription complexes that, by binding to lineage-specific enhancers, activate the expression of the corresponding target genes. Instrumental to our understanding of the role of GATA-1 in hemopoietic differentiation has been the generation of genetically engineered mutant mice and the discovery of naturally occurring mutations associated with either inherited or acquired human pathologies. We review our current understanding of the role of GATA-1 in normal and neoplastic hematopoiesis as emerging from these genetic approaches.
Eosinophil lineage-committed progenitors (EoPs) are phenotypically isolatable in the steady-state murine bone marrow. Purified granulocyte/monocyte progenitors (GMPs) gave rise to eosinophils as well as neutrophils and monocytes at the single cell level. Within the short-term culture of GMPs, the eosinophil potential was found exclusively in cells activating the transgenic reporter for GATA-1, a transcription factor capable of instructing eosinophil lineage commitment. These GATA-1-activating cells possessed an IL-5Ralpha(+)CD34(+)c-Kit(lo) phenotype. Normal bone marrow cells also contained IL-5Ralpha(+)CD34(+)c-Kit(lo) EoPs that gave rise exclusively to eosinophils. EoPs significantly increased in number in response to helminth infection, suggesting that the EoP stage is physiologically involved in eosinophil production in vivo. EoPs expressed eosinophil-related genes, such as the eosinophil peroxidase and the major basic protein, but did not express basophil/mast cell-related mast cell proteases. The enforced retroviral expression of IL-5Ralpha in GMPs did not enhance the frequency of eosinophil lineage read-outs, whereas IL-5Ralpha(+) GMPs displayed normal neutrophil/monocyte differentiation in the presence of IL-5 alone. Thus, IL-5Ralpha might be expressed specifically at the EoP stage as a result of commitment into the eosinophil lineage. The newly identified EoPs could be the cellular target in the treatment of a variety of disorders mediated by eosinophils.
The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.
Vanadium complexes are known to possess potent insulin-mimetic effects, high affinity for several enzymes and anticancer activity, which deserve increasing attention for application to biomedical sciences. Different vanadium complexes have been found to be more effective than the simple vanadium-(IV) and -(V) salts in experiments performed both in vitro and in vivo. Application of polyoxometalates as potential drugs against Herpes Simplex Virus and AIDS have also increased the interest to study the association between vanadium containing species and proteins. The aim of our research was to investigate the in vitro antiproliferative activity of a variety of vanadium-containing compounds, and study their ability to interfere with the molecular interactions between GATA-1 and NF-kappaB transcription factors and target DNA elements, employing electrophoretic mobility shift assays. All of the used vanadium compounds were found to exhibit antiproliferative activity, despite with differences in efficacy. Inhibition of K562 cell growth was not associated with differentiation, but with activation of apoptosis. Vanadium complexes with a +5 oxidation state and their discrete anionic units appear essential for the respective effects on K562 cells; a +4 oxidation state appears to be important in inhibiting transcription factors/DNA interactions.
Proteomics methods were used to characterize proteins that change their form or abundance in the nucleus of NRK49F rat kidney fibroblasts during prolonged hypoxia (1% O(2), 12 h). Of the 791 proteins that were monitored, about 20% showed detectable changes. The 51 most abundant proteins were identified by mass spectrometry. Changes in nuclear receptor transcription factors (THRalpha1, RORalpha4, HNF4alpha, NUR77), other transcription factors (GATA1, AP-2alpha, OCT1, ATF6alpha, ZFP161, ZNF354A, PDCD2), and transcription cofactors (PC4, PCAF, MTA1, TCEA1, JMY) are indicative of major, co-ordinated changes in transcription. Proteins involved in DNA repair/recombination, ribosomal RNA synthesis, RNA processing, nuclear transport, nuclear organization, protein translation, glycolysis, lipid metabolism, several protein kinases (PKCdelta, MAP3K4, GRK3), as well as proteins with no established functional role were also observed. The observed proteins suggest nuclear regulatory roles for proteins involved in cytosolic processes such as glycolysis and fatty acid metabolism, and roles in overall nuclear structure/organization for proteins previously associated with meiosis and/or spermatogenesis (synaptonemal complex proteins 1 and 2 (SYCP1, SYCP2), meiosis-specific nuclear structural protein 1 (MNS1), LMNC2, zinc finger protein 99 (ZFP99)). Proteins associated with cytoplasmic membrane functions (ACTN4, hyaluronan mediated motility receptor (RHAMM), VLDLR, GRK3) and/or endocytosis (DNM2) were also seen. For 30% of the identified proteins, new isoforms indicative of alternative transcription were detected (e.g., GATA1, ATF6alpha, MTA1, MLH1, MYO1C, UBF, SYCP2, EIF3S10, MAP3K4, ZFP99). Comparison with proteins involved in cell death, cancer, and testis/meiosis/spermatogenesis suggests commonalities, which may reflect fundamental mechanisms for down-regulation of cellular function.
Therapy-related myeloid leukemia (t-AML) is a distinctive clinical syndrome occurring after exposure to chemotherapy (CT) or radiotherapy (RT). We studied 306 consecutive patients referred to the University of Chicago with cytogenetic analyses. Since 1972, 141 males and 165 females with a median age of 51 years (range: 3-83 years) at primary diagnosis and 58 years (range: 6-86 years) at secondary diagnosis were analyzed. Patients had received various cytotoxic agents including alkylating agents (240 patients, 78%) and topoisomerase II inhibitors (115 patients, 39%). One hundred and twenty-one (40%) had received CT alone, 43 (14%) had received RT alone, and 139 (45%) had received both modalities. At diagnosis of t-AML, 282 (92%) had clonal abnormalities involving chromosome 5 (n=63), chromosome 7 (n=85), both chromosomes 5 and 7 (n=66), recurring balanced rearrangements (n=31), or other clonal abnormalities (n=39); 24 had a normal karyotype. Abnormalities of chromosomes 5 and/or 7 accounted for 76% of all cases with an abnormal karyotype. Seventeen patients had developed t-AML after autologous stem cell transplantation, but no unique pattern of cytogenetic abnormalities was observed. Patients presenting with acute leukemia were more likely to have a balanced rearrangement than those presenting with myelodysplasia (28% versus 4%, p<0.0001). Shorter latency was observed for patients with balanced rearrangements (median: 28 months versus 67 months; p<0.0001). Median survival after diagnosis of t-AML was 8 months; survival at 5 years was less than 10%. To gain insights into the molecular basis of this disease, we performed gene expression profiling of CD34+ hematopoietic progenitor cells from t-AML patients. We found distinct subtypes of t-AML that have characteristic gene expression patterns. Common to each of the subgroups are gene expression patterns typical of arrested differentiation in early progenitor cells. Leukemias with a -5/del(5q) have a higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), and loss of expression of the gene encoding interferon consensus sequence-binding protein (ICSBP). A second subgroup of t-AML is characterized by down-regulation of transcription factors involved in early hematopoiesis (TAL1, GATA1, and EKLF) and overexpression of proteins involved in signaling pathways in myeloid cells (FLT3) and cell survival (BCL2). Establishing the molecular pathways involved in t-AML may facilitate the identification of selectively expressed genes that can be exploited for the development of targeted therapies.
GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.
Transcription factor GATA-1 and its cofactor FOG-1 coordinate erythroid cell maturation by activating erythroid-specific genes and repressing genes associated with the undifferentiated state. Here we show that FOG-1 binds to the NuRD corepressor complex in vitro and in vivo. The interaction is mediated by a small conserved domain at the extreme N-terminus of FOG-1 that is necessary and sufficient for NuRD binding. This domain defines a novel repression module found in diverse transcriptional repressors. NuRD is present at GATA-1/FOG-1-repressed genes in erythroid cells in vivo. Point mutations near the N-terminus of FOG-1 that abrogate NuRD binding block gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing mutant forms of FOG-1 that are defective for NuRD binding. Together, these studies show that FOG-1 and likely other FOG-like proteins are corepressors that link GATA factors to histone deacetylation and nucleosome remodeling.
Acquired mutations in exon 2 of the GATA1 gene are detected in most Down syndrome (DS) patients with transient leukemia (TL) and acute megakaryoblastic leukemia (AMKL). We sought to determine if GATA1 mutations can be utilized as markers for minimal residual disease (MRD). GATA1 mutations were screened by SSCP analysis and sequenced. Using GATA1 mutation-specific primers, follow-up bone marrow samples from four patients were assayed by quantitative PCR. We show that molecular monitoring of GATA1 mutations is possible in Down syndrome patients with TL and AMKL, and GATA1 could be a stable marker for MRD monitoring.
The LIM-finger protein LMO2 forms a transcription factor complex with other hematopoietic regulator proteins, such as TAL1 (SCL), LDB1, GATA1, 2, and 3, in the promoters of several erythroid genes. To elucidate the functional role of two LIM domains in LMO2, we introduced deletion or mutation in each of the LIM domains and analyzed their phenotypic effects on the hematopoietic system when overexpressed in vivo or in vitro.
Protein interactions of LIM-modified LMO2 constructs with TAL1, LDB1, and GATAs were examined in an immunoprecipitation assay. In vivo hematopoiesis in transgenic mice with wild-type and LIM-modified Lmo2 was studied morphologically and by measuring the progenitor cells in fetal liver. Their effects on the erythroid differentiation of the dimethylsulfoxide (DMSO)-induced murine erythroleukemia (MEL) cells were evaluated.
Deletion of the LIM2 domain, but not of the LIM1 domain, abolished its binding of GATA proteins. Overexpression of wild-type LMO2 is known to have dominant negative inhibitory effects on erythropoietic development. Enforced expression of LMO2 constructs with mutant or absent LIM2 but with an intact LIM1 domain resulted in fetal death, small livers and hearts, and decreased hematopoiesis, as well as a hypoplastic thymus. DMSO-induced erythroid differentiation of the MEL cells was inhibited by the overexpressed LMO2 with mutant LIM2 but not by the LMO2 with modified LIM1.
Overexpression of the LMO2 with modified LIM2 inhibited hematopoiesis probably by interfering with the formation of the physiological complex or by replacing the functional LMO2 with mutants with reduced affinity to GATA proteins. In this experiment, no evident effect of the LMO2 with modified LIM1 could be observed.
Acquired mutations in the hematopoietic transcription factor GATA binding protein-1 (GATA1) are found in megakaryoblasts from nearly all individuals with Down syndrome with transient myeloproliferative disorder (TMD, also called transient leukemia) and the related acute megakaryoblastic leukemia (DS-AMKL, also called DS-AML M7). These mutations lead to production of a variant GATA1 protein (GATA1s) that is truncated at its N terminus. To understand the biological properties of GATA1s and its relation to DS-AMKL and TMD, we used gene targeting to generate Gata1 alleles that express GATA1s in mice. We show that the dominant action of GATA1s leads to hyperproliferation of a unique, previously unrecognized yolk sac and fetal liver progenitor, which we propose accounts for the transient nature of TMD and the restriction of DS-AMKL to infants. Our observations raise the possibility that the target cells in other leukemias of infancy and early childhood are distinct from those in adult leukemias and underscore the interplay between specific oncoproteins and potential target cells.
A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.
GATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1-deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1-deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1-deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s-expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.
Children with Down Syndrome (DS) have a 20-40 fold increased risk of developing acute myeloid leukemia (AML), mainly of the megakaryoblastic subtype (AMKL). Approximately 10 % of newborns with DS show transient myeloproliferative disease (TMD) which normally resolves spontaneously. The blast cells of both entities show megakaryoblastic/erythroblastic features (M7/M6) and cannot be distinguished by morphological characteristics.
Blast cells of 62 children were analyzed by four-color flow cytometry and dual color fluorescence microscopy.
The immunophenotype of blast cells from children with TMD and DS-AMKL is characterized by the expression of CD33 (+)/CD13 (+/-)/CD38 (+)/CD117 (+)/CD34 (+/-)/CD7 (+)/CD56 (+/-)/CD36 (+)/CD71 (+)/CD42b (+)/CD4dim (+)/TPO-R (+)/EPO-R (-)/IL-3-Ralpha (+)/IL-6-Ralpha (-). Non-DS children with morphologically related diseases, i. e. myelodysplastic syndrome (MDS), juvenile myelomonocytic leukemia (JMML), or AML-M6 and AML-M7, did not show this expression profile. CD34 expression was observed in 93 % of TMD, but only 50 % of DS-AMKL patients. The blast cells of all TMD and DS-AMKL cases were positive for TPO-R and IL-3R, whereas EPO-R and IL-6R were absent.
Immunophenotyping by the use of surface antigens and growth factor receptors is a useful tool to discriminate TMD and DS-AMKL from diseases with morphologically similar or identical blasts. The absence of EPO-R on the blast cells might be a sign of the high expression of the mutated -- and less active -- GATA1 in DS. The higher amount of CD34 co-expression in TMD may be interpreted to indicate that TMD is a slightly more immature disease than DS-AMKL.
The RUNX1/AML1 gene on chromosome 21 is most frequently inactivated in human leukemias. In addition, an increased dose of RUNX1 is suggested as a basis for several kinds of leukemias. Amplifications of chromosome 21 or the RUNX1 gene are shown to be associated with leukemias with lymphoid lineage, whereas its involvement in myeloid lineage remains unclear. In this study, we generated GATA-1 promoter-driven Runx1 transgenic (Tg) mice, which showed a transient mild increase of megakaryocyte marker-positive myeloid cells but no spontaneous leukemia. These mice were then crossed with BXH2 mice, which have a replication-competent retrovirus in the mouse and develop myeloid leukemia due to insertional mutagenesis by random integration of the virus. Overexpressed Runx1 transgene in BXH2 mice resulted in shortening of the latency of leukemia with increased frequency of megakaryoblastic leukemia, suggesting that increased Runx1 dosage is leukemogenic in myeloid lineage. Identifications of retroviral integration sites revealed the genetic alterations that may cooperate with Runx1 overdose in myeloid leukemogenesis. This mouse model may be useful for analysing the pathogenesis of myeloid leukemias with RUNX1 overdose, especially to examine whether an extra-copy of RUNX1 by trisomy 21 is causally related to Down's syndrome-related acute megakaryoblastic leukemia (DS-AMKL).
We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.
Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl(mo)). Distinct gene clusters were demarcated by stage-specific and mutant-specific gene regulation. These were found to correlate with the transcriptional program of hematopoietic progenitor cells, as well as of the erythroid, myeloid, and vascular lineages. Among these, several novel hematopoietic and vascular genes were detected, for instance, the erythroid transcription factors znfl2 and ncoa4. A specific regulation was found for myeloid genes, as they were more strongly expressed in vlt mutants compared with other erythroid mutants. A unique gene expression pattern of up-regulated isoprenoid synthesis genes was found in cloche and scl(mo), possibly in migrating cells. In conjunction with the high conservation of vertebrate hematopoiesis, the comparison of transcriptional profiles in zebrafish blood mutants represents a versatile and powerful tool to elucidate the genetic regulation of blood and blood vessel development.
Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16(INK4A) than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.
The zinc finger transcription factor GATA-2 plays a fundamental role in generating hematopoietic stem-cells in mammalian development. Less well defined is whether GATA-2 participates in adult stem-cell regulation, an issue we addressed using GATA-2 heterozygote mice that express reduced levels of GATA-2 in hematopoietic cells. While GATA-2+/- mice demonstrated decreases in some colony-forming progenitors, the most prominent changes were observed within the stem-cell compartment. Heterozygote bone marrow had a lower abundance of Lin(-)c-kit(+)Sca-1(+)CD34- cells and performed poorly in competitive transplantation and quantitative week-5 cobblestone area-forming cell (CAFC) assays. Furthermore, a stem-cell-enriched population from GATA1+/- marrow was more quiescent and exhibited a greater frequency of apoptotic cells associated with decreased expression of the anti-apoptotic gene Bcl-xL. Yet the self-renewal potential of the +/- stem-cell compartment, as judged by serial transplantations, was unchanged. These data indicate compromised primitive cell proliferation and survival in the setting of a lower GATA-2 gene dose without a change in the differentiation or self-renewal capacity of the stem-cells that remain. Thus, GATA-2 dose regulates adult stem-cell homeostasis by affecting select aspects of stem cell function.
CC Chemokine Receptor 3 (CCR3), the major chemokine receptor expressed on eosinophils, binds promiscuously to several ligands including eotaxins 1, 2, and 3. Even though the only cells that consistently accumulate following eotaxin administration in vivo are myeloid cells (primarily eosinophils), other cell types have recently been shown to express CCR3. It is therefore important to elucidate the molecular mechanisms regulating receptor expression.
In order to define regions responsible for CCR3 transcription, a DNAse hypersensitive site was identified in the vicinity of exon 1. Coupled with our previous data implicating exon 1 in CCR3 transcription, we hypothesized that transcription factors bind to exon-1. Electrophoretic mobility shift analysis revealed that nuclear proteins in eosinophilic cells bound to exon 1. Furthermore, antibody interference and mutation studies demonstrated GATA-1 binding to exon 1. In order to test the 1.6-kb CCR3 promoter element (that includes exon 1) for in vivo function, this region was used to generate transgenic mice that expressed a reporter protein. Strong transgene expression was achieved, with the pattern of expression suggesting a broad acting promoter.
The transcription factor GATA-1 binds to CCR3 exon 1. The 1.6-kb CCR3 promoter element, that includes exon 1, is a strong promoter in vivo.
The zebrafish cloche (clo) mutation affects the earliest known step in differentiation of blood and endothelial cells in vertebrates. We established clo/gata1-GFP transgenic line with erythroid-specific green fluorescent protein (GFP) expression, which allowed differentiation of clo and wild-type siblings at the midsomitogenesis stages before morphologically visible phenotypes appeared. To discover novel genes potentially involved in hematopoietic and vascular development, we performed microarray analysis of more than 15,000 zebrafish genes or expressed sequence tags (ESTs) in clo mutant embryos. We isolated the full-length sequences and determined the expression patterns for 8 novel cDNAs that were significantly down-regulated in clo-/- embryos. Dual specificity phosphatase 5 (dusp5), cadherin 5 (cdh5; VE-cadherin), aquaporin 8 (aqp8), adrenomedullin receptor (admr), complement receptor C1qR-like (crl), scavenger receptor class F, member 1 (scarf1), and ETS1-like protein (etsrp) were specifically expressed in the vascular endothelial cells, while retinol binding protein 4 (rbp4) was expressed in the yolk syncytial layer and the hypochord. Further functional studies of these novel genes should help to elucidate critical early steps leading to the formation of vertebrate blood vessels.
GATA proteins are transcription factors that bind GATA DNA elements through Cys4 structural zinc-binding domains and play critical regulatory roles in neurological and urogenital development and the development of cardiac disease. To evaluate GATA proteins as potential targets for lead, spectroscopically monitored metal-binding titrations were used to measure the affinity of Pb2+ for the C-terminal zinc-binding domain from chicken GATA-1 (CF) and the double-finger domain from human GATA-1 (DF). Using this method, Pb2+ coordinating to CF and DF was directly observed through the appearance of intense bands in the near-ultraviolet region of the spectrum (250-380 nm). Absorption data collected from these experiments were best fit to a 1:1 Pb2+ -CF model and a 2:1 Pb2+ -DF model. Competition experiments using Zn2+ were used to determine the absolute affinities of Pb2+ for these proteins. These studies reveal that Pb2+ forms tight complexes with cysteine residues in the zinc-binding sites in GATA proteins, beta1Pb = 6.4 (+/- 2.0) x 10(9) M(-1) for CF and beta2 = 6.3 (+/- 6.3) x 10(19) M(-2) for Pb(2+)2-DF, and within an order of magnitude of the affinity of Zn2+ for these proteins. Furthermore, Pb2+ was able to displace bound Zn2+ from CF and DF. Upon addition of Pb2+, GATA shows a decreased ability to bind to DNA and subsequently activate transcription. Therefore, the DNA binding and transcriptional activity of GATA proteins are most likely to be targeted by Pb2+ in cells and tissues that sequester Pb2+ in vivo, which include the brain and the heart.
Early erythroblasts from patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) show constitutive mitochondrial release of cytochrome c. Moreover, mature erythroblasts in RARS, but not in RA, display aberrant accumulation of mitochondrial ferritin (MtF). We analyzed cytochrome c release, MtF expression, and gene expression during erythroid differentiation in bone marrow cells from myelodysplastic syndrome (MDS) patients and healthy controls. Whereas none or few cultured erythroid cells from healthy individuals and RA patients expressed MtF, those from RARS patients showed MtF expression at an early stage, when cells were CD34+ and without morphologic signs of erythroid differentiation. The proportion of RARS erythroblasts that were MtF+ increased further upon in vitro maturation. Moreover, a significant overexpression of mRNA encoding cytochrome c, and proapoptotic Bid and Bax, was seen in freshly isolated cells from MDS patients. Genes involved in erythroid differentiation were also dysregulated in MDS cells. Importantly, GATA-1 expression increased during normal erythroid maturation, but remained low in MDS cultures, indicating a block of erythroid maturation at the transcriptional level. In conclusion, aberrant MtF expression in RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in this process.
In order to investigate expressions of transcription factor GATA-1 and GATA-2 genes in the bone marrow stromal cells (BMSCs) from patients with leukemia or normal controls, bone marrow stromal cells from 34 normal cases and 42 cases with leukemia were cultured long-term in vitro. Nonadherent cells (bone marrow hematopoietic cells) and amplified adherent cells (BMSC) were collected separately. Expressions of GATA-1 and GATA-2 genes were analyzed by using RT-PCR-ELISA; the semi-quantitative expression levels of GATA genes in the BMSCs from patients with leukemia were compared with normal controls. The results showed that expressions of GATA-1 and GATA-2 genes could be detected in the BMSCs and the bone marrow hematopoietic cells from both normal controls and the cases of leukemia. The expression ratio of GATA-1 in the BMSCs from acute lymphocytic leukemia (ALL) (85.7%) was similar to the normal controls (88.2%), whereas the expression ratios in BMSCs from acute myelocytic leukemia (AML) (55.6%) and chronic myelocytic leukemia (CML) (41.2%) were significant lower than the normal controls (P < 0.05). The rank of expression level of GATA-1 gene in the BMSCs was "ALL>AML>normal>CML". There was no difference in the expression level of GATA-2 gene within the BMSCs from normal controls and patients with leukemia. The ranks of expression levels of GATA-1 and GATA-2 genes in bone marrow hematopoietic cells were "AML>normal>ALL>CML" and "AML>CML>ALL>normal". The dominant expression of GATA-2 gene was found in the BMSCs from AML, CML or normal controls. It is inferred that the expressions of GATA-1 and GATA-2 genes in the BMSCs of normal controls and patients with leukemia may influence the regulation of hematopoiesis in the bone marrow stroma and it is worthy of further study to explore their roles in pathogenesis and development of leukemia.
Core binding factor (CBF) participates in specification of the hematopoietic stem cell and functions as a critical regulator of hematopoiesis. Translocation or point mutation of acute myeloid leukemia 1 (AML1)/RUNX1, which encodes the DNA-binding subunit of CBF, plays a central role in the pathogenesis of acute myeloid leukemia and myelodysplasia. We characterized the t(X;21)(p22.3;q22.1) in a patient with myelodysplasia that fuses AML1 in-frame to the novel partner gene FOG2/ZFPM2. The reciprocal gene fusions AML1-FOG2 and FOG2-AML1 are both expressed. AML1-FOG2, which fuses the DNA-binding domain of AML1 to most of FOG2, represses the transcriptional activity of both CBF and GATA1. AML1-FOG2 retains a motif that recruits the corepressor C-terminal binding protein (CtBP) and these proteins associate in a protein complex. These results suggest a central role for CtBP in AML1-FOG2 transcriptional repression and implicate coordinated disruption of the AML1 and GATAdevelopmental programs in the pathogenesis of myelodysplasia.
Defects in the X-linked DNA-binding megakaryocyte transcription factor GATA1 cause thrombocytopenia and abnormal platelet function. However, detailed studies of GATA1 function in platelet activation are lacking. Here, we studied platelets from GATA1-deficient mice and from a male patient (S14) with a bleeding diathesis attributed to a single amino acid substitution (R216Q) in the N-terminal GATA1 zinc finger that alters binding to DNA. In both cases there was inhibition of aggregation to collagen and decreased tyrosine phosphorylation of glycoprotein VI (GPVI)-signaling proteins. This effect was more marked in GATA1-deficient murine platelets, where it was associated with a significant reduction in surface GPVI expression. Moreover, both human and murine GATA1-mutant platelets showed reduced adhesion and aggregate formation on a collagen matrix at an intermediate rate of shear, although this could not be accounted solely by the thrombocytopenia and altered GPVI expression, indicating that GATA1 regulates additional factors important for platelet activation under shear. In contrast, there was no inhibition of responses to G protein-coupled receptor agonists in GATA1-perturbed platelets. Our results are consistent with GATA1 regulating some but not all pathways of platelet activation, leading to an impairment of aggregate formation under flow, which cannot be attributed solely to the thrombocytopenia.
Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.
Down syndrome children with acute megakaryocytic leukemia (AMkL) have higher cure rates than non-Down syndrome acute myeloid leukemia (AML) patients treated with cytosine arabinoside (ara-C). Megakaryoblasts from Down syndrome AML patients are more sensitive in vitro to ara-C than cells from non-Down syndrome AML patients. Somatic mutations in the GATA1 transcription factor have been detected exclusively and almost uniformly in Down syndrome AMkL patients, suggesting a potential linkage to the chemotherapy sensitivity of Down syndrome megakaryoblasts. Stable transfection of wild-type GATA1 cDNA into the Down syndrome AMkL cell line CMK resulted in decreased (8- to 17-fold) ara-C sensitivity and a threefold-lower generation of the active ara-C metabolite ara-CTP compared with that for mock-transfected CMK cells. High intracellular levels of uridine arabinoside (ara-U) (an inactive ara-C catabolite generated by cytidine deaminase) and cytidine deaminase transcripts were detected in GATA1-transfected CMK sublines, whereas no ara-U was detected in mock-transfected cells. Cytidine deaminase transcripts were a median 5.1-fold (P = .002) lower in Down syndrome megakaryoblasts (n = 16) than in blast cells from non-Down syndrome patients (n = 56). These results suggest that GATA1 transcriptionally upregulates cytidine deaminase and that the presence or absence of GATA1 mutations in AML blasts likely confers differences in ara-C sensitivities due to effects on cytidine deaminase gene expression, which, in turn, contributes to the high cure rate of Down syndrome AMkL patients.
Trisomy 21 [Down's syndrome (DS)] and mutations in transcription factor GATA1 predispose neonates to a transient myeloproliferative disorder (TMD) and/or acute megakaryocytic leukaemia (AMKL). The role of trisomy 21 in their pathogenesis is unclear. We previously reported two rare neonates without DS who had TMD, one of whom progressed to AMKL. Trisomy 21 was detected only in blood cells at presentation with TMD/AMKL and disappeared with disease resolution. We now show that the blood cells at presentation of TMD harboured GATA1 genomic DNA mutations, suggesting a requirement for trisomy 21 in haematopoietic cells, rather than other cell types, for development of TMD/AMKL.
Although GATA-1 and GATA-2 were shown to be essential for the development of hematopoietic cells by gene targeting experiments, they were also reported to inhibit the growth of hematopoietic cells. Therefore, in this study, we examined the effects of GATA-1 and GATA-2 on cytokine signals. A tamoxifen-inducible form of GATA-1 (GATA-1/ERT) showed a minor inhibitory effect on interleukin-3 (IL-3)-dependent growth of an IL-3-dependent cell line Ba/F3. On the other hand, it drastically inhibited TPO-dependent growth and gp130-mediated growth/survival of Ba/F3. Similarly, an estradiol-inducible form of GATA-2 (GATA-2/ER) disrupted thrombopoietin (TPO)-dependent growth and gp130-mediated growth/survival of Ba/F3. As for this mechanism, we found that both GATA-1 and GATA-2 directly bound to STAT3 both in vitro and in vivo and inhibited its DNA-binding activity in gel shift assays and chromatin immunoprecipitation assays, whereas they hardly affected STAT5 activity. In addition, endogenous GATA-1 was found to interact with STAT3 in normal megakaryocytes, suggesting that GATA-1 may inhibit STAT3 activity in normal hematopoietic cells. Furthermore, we found that GATA-1 suppressed STAT3 activity through its N-zinc finger domain. Together, these results suggest that, besides the roles as transcription factors, GATA family proteins modulate cytokine signals through protein-protein interactions, thereby regulating the growth and survival of hematopoietic cells.
Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (DeltaNF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the DeltaNF mutant, supporting our contention that DeltaNF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.
Idiopathic myelofibrosis (IM) is a disease characterized by marrow fibrosis, abnormal stem/progenitor cell trafficking, and extramedullary hematopoiesis frequently associated with alterations in megakaryocytes (Mks). Mice harboring genetic alterations in either the extrinsic (ectopic thrombopoietin expression, TPO(high) mice) or intrinsic (hypomorphic GATA-1 mutation, GATA-1(low) mice) control of Mk differentiation develop myelofibrosis, a syndrome similar to IM. The relationship, if any, between the pathobiologic mechanism leading to the development of myelofibrosis in the 2 animal models is not understood. Here we show that plasma from GATA-1(low) mice contained normal levels of TPO. On the other hand, Mks from TPO-treated wild-type animals (TPO(high) mice), as those from GATA-1(low) animals, had similar morphologic abnormalities and contained low GATA-1. In both animal models, development of myelofibrosis was associated with high transforming growth factor beta1 (TGF-beta1) content in extracellular fluids of marrow and spleen. Surprisingly, TPO treatment of GATA-1(low) mice restored the GATA-1 content in Mks and halted both defective thrombocytopoiesis and fibrosis. These data indicate that the TPO(high) and GATA-1(low) alterations are linked in an upstream-downstream relationship along a pathobiologic pathway leading to development of myelofibrosis in mice and, possibly, of IM in humans.
GATA-1 is the founding member of the GATA family of transcription factors. GATA-1 and GATA family member GATA-2 are expressed in erythroid and megakaryocytic lineages, in which they play a crucial role in cell maturation and differentiation. GATA-1 regulates the transcription of many specific and nonspecific erythroid genes by binding to DNA at the consensus sequence WGATAR, which is recognized by all of the GATA family of transcription factors. However, it was identified in eosinophilic cells and also in Sertoli cells in testis. Its activity depends on close cooperation with a functional network of cofactors, among them Friend of GATA, PU.1, and CBP/p300. The GATA-1 protein structure has been well described and includes two zinc fingers that are directly involved in the interaction with DNA and other proteins in vivo. GATA-1 mutations in the zinc fingers can cause deregulation of required interactions and lead to severe dysfunction in the hematopoietic system.
In the late 1980s, several research groups independently discovered the founding member of the GATA family of transcription factors, GATA-1. Each group had evidence that GATA-1 played an important role in erythroid gene expression, but little did they know that it would turn out to be a key regulator of development of not only red blood cells, but of several other hematopoietic cell types as well. Furthermore, few would have guessed that missense mutations in GATA1 would cause inherited blood disorders, while acquired mutations would be found associated with essentially all cases of acute megakaryoblastic leukemia (AMKL) in children with Down syndrome (DS). With respect to the latter disorder, the presence of a GATA1 mutation is now arguably the defining feature of this leukemia. In this review, I will summarize our current knowledge of the role of GATA-1 in normal development, and discuss how mutations in GATA1 lead to abnormal and malignant hematopoiesis.
The hierarchical gene regulatory network in hematopoiesis is highly complex, making elucidation of the processes of specification and differentiation of hematopoietic cells a challenging task. Recent discoveries have divulged the GATA factors as central to the genetic control of hematopoiesis. In particular, hematopoietic development is subject to extensive and precise regulation of GATA-1 and GATA-2 at the molecular level. We wish to emphasize the regulatory relationships between GATA-1 and GATA-2 implicated in cell development. An advanced experimental genetic approach has provided evidence that abnormalities in this network may result in a variety of blood disorders. The most striking new finding is the novel pathogenesis arising from GATA-1 dysfunction that leads to leukemia.
Transient myeloproliferative disorder is a form of self-limited leukemia that occurs almost exclusively in neonates with Down syndrome. The authors report an unusual case of a newborn without constitutional trisomy 21 who developed undifferentiated leukemia and subsequently achieved clinical and molecular remission without chemotherapy. Cytogenetics and molecular analysis have shown trisomy 21 and GATA1 mutation restricted to leukemic cells. G-to-T transversion was detected, which is predicted to result in a premature stop codon (c.119G>T; pGlu67X) in diagnosis samples. These findings emphasize that there must be a powerful interaction between GATA1 and trisomy 21 in leukemogenesis process.
Transient megakaryoblastic leukaemia is found in 10% of newborns with Down syndrome, characterized by constitutional trisomy 21. Although in most cases the leukaemic cells disappear spontaneously after the first months of life, irreversible acute megakaryoblastic leukaemia develops in 20% of these individuals within 4 years. The leukaemic cells typically harbour somatic mutations of the gene encoding GATA1, an essential transcriptional regulator of normal megakaryocytic differentiation. Leukaemia that specifically arises in the context of constitutional trisomy 21 and somatic GATA1 mutations is a unique biological model of the incremental process of leukaemic transformation.
The differentiation of hematopoietic progenitors into erythroid or myeloid cell lineages is thought to depend upon relative levels of the transcription factors gata1 and pu.1. While loss-of-function analysis shows that gata1 is necessary for terminal erythroid differentiation, no study has demonstrated that loss of gata1 alters myeloid differentiation during ontogeny. Here we provide in vivo evidence that loss of Gata1, but not Gata2, transforms primitive blood precursors into myeloid cells, resulting in a massive expansion of granulocytic neutrophils and macrophages at the expense of red blood cells. In addition to this fate change, expression of many erythroid genes was found to be differentially dependent on Gata1 alone, on both Gata1 and Gata2, or independent of both Gata factors, suggesting that multiple pathways regulate erythroid gene expression. Our studies establish a transcriptional hierarchy of Gata factor dependence during hematopoiesis and demonstrate that gata1 plays an integral role in directing myelo-erythroid lineage fate decisions during embryogenesis.
The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis, allowing for the critical in vivo analysis of cell lineage determination. In this study, we identify zebrafish myeloerythroid progenitor cells (MPCs) that are likely to represent the functional equivalent of mammalian common myeloid progenitors. Utilizing transgenic pu.1-GFP fish, real-time MPC differentiation was correlated with dynamic changes in cell motility, morphology, and gene expression. Unlike mammalian hematopoiesis, embryonic zebrafish myelopoiesis and erythropoiesis occur in anatomically separate locations. Gene knockdown experiments and transplantation assays demonstrated the reciprocal negative regulation of pu.1 and gata1 and their non-cell-autonomous regulation that determines myeloid versus erythroid MPC fate in the distinct blood-forming regions. Furthermore, forced expression of pu.1 in the bloodless mutant cloche resulted in myelopoietic rescue, providing intriguing evidence that this gene can function in the absence of some stem cell genes, such as scl, in governing myelopoiesis.
The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.
DYRKs are an emerging family of dual-specificity kinases that play key roles in cell proliferation, survival, and development. Up to seven mammalian DYRK isoforms have been reported, but only the DYRK1A gene (and its products) has been well characterized. Defined here are the genomic structures (and phylogenetics) of DYRK3, four additional murine and/or human DYRKs, and two related HIPK genes. For murine DYRK3, direct BAC sequences are provided, a basis for differential processing of murine versus human transcripts is defined, and tissue expression profiles plus a functional DYRK3 promoter are characterized. Unlike complex DYRKs 1A, 1B, and 4A, DYRK3 and DYRK2 possess simple 4-exon structures. For DYRK3, expression is strong in erythroid cells and testis, but is also detected in adult kidney and liver in situ. In addition, a 1930-bp DYRK3 promoter drives erythroid expression and transcript expression is inhibited sharply on GATA1-induced G1E cell differentiation.
Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements.
GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.
A proliferation marker, proliferating cell nuclear antigen (PCNA), a Sertoli cell specific transcription factor, GATA-1 and the male germ cell specific, RNA binding motif (RBM), were used to identify different cellular populations during postnatal development of the mouse testis.
Immunohistochemistry, RT-PCR and real-time quantitative RT-PCR (QRT-PCR) were used.
PCNA was expressed in pre-Sertoli and germ cells on the day of birth. Both pre-meiotic germ cells and spermatocytes expressed RBM throughout postnatal development. RBM-positive cell counts and QRT-PCR of RBM showed that average level of RBM per cell is highest in juvenile males between 14 and 21 days. From 42 days onward, there was a dramatic decrease in RBM expression in individual pre-meiotic and meiotic germ cells.
These markers were used to correlate cell proliferative capability, gene expression profile and anatomic location within the developing mouse testis. The majority of germ cells start active proliferation once they have migrated to the basement membrane or immediately before. RBM is more highly expressed during the first wave of spermatogenesis versus subsequent waves, suggesting that there may be a change in the activity of RBM.
In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn(-/-) mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn(-/-) erythroblasts, and the phenotype of Lyn(-/-) animals was remarkably similar to GATA-1(low) animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn(-/-) animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn(-/-) mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.
Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, chromatin immunoprecipitation analysis has revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the -2.8-kilobase (kb) region of the GATA-2 locus in the active state despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high resolution chromatin immunoprecipitation to show that GATA-1 and GATA-2 occupy two additional regions, -3.9 and -1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the -3.9- and -1.8-kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and GATA-2 displacement at the sites. In the transcriptionally active state, DNase I hypersensitive sites (HSs) were detected at the -3.9- and -1.8-kb regions, with a weak HS at the -2.8-kb region. Whereas ER-GATA-1-instigated repression abolished the -1.8-kb HS, the -3.9-kb HS persisted in the repressed state. Transient transfection analysis provided evidence that the -3.9-kb region functions distinctly from the -2.8- and -1.8-kb regions. We propose that GATA-2 transcription is regulated via the collective actions of complexes assembled at the -2.8- and -1.8-kb regions, which share similar properties, and through a qualitatively distinct activity of the -3.9-kb complex.
Blood group Duffy gene (FY) promoter in Duffy-negative individuals contains a point mutation in the GATA1 protein-binding motif, which was suggested to be responsible for erythroid suppression of FY. We developed two transgenic mouse lines with FY from both Duffy phenotypes. Transgenic mice with FY from Duffy-positive phenotype expressed Duffy protein both in red blood cells (RBCs) and non-erythroid tissues. Transgenic mice with FY from Duffy-negative phenotype did not express Duffy protein in RBCs, but it was expressed in non-erythroid tissues. This is the first in vivo experimental evidence showing the effect of -33T-->C promoter mutation on FY expression.
Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells.
Alpha-spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the alpha-spectrin gene. Previously, we demonstrated that the core promoter of the human alpha-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3' of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the alpha-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3' of the alpha-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased alpha-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.
Although physicians have known for many decades that children with Down syndrome are predisposed to developing transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL), many questions regarding these disorders remain unresolved. First, what is the relationship between TMD and AMKL? Second, what specific genetic alterations contribute to the leukemic process? Finally, what factors lead to the increased predisposition to these myeloid disorders? In this review I will summarize important new insights into the biology of TMD and AMKL gained from the recent discovery that GATA1, a gene that encodes an essential hematopoietic transcription factor, is mutated in the leukemic blasts from nearly all patients with these malignancies. In addition, I will discuss whether assaying for the presence of a GATA1 mutation can aid in the diagnosis of these and related megakaryoblastic leukemias. Future research aimed at defining the activity of mutant GATA-1 protein and identifying interacting factors encoded by chromosome 21 will likely lead to an even greater understanding of this intriguing leukemia.
It has been recognized that chromosomal abnormalities in childhood leukemia, are linked to both leukemogenesis and segregate patients into prognostic treatment groups. This is best exemplified in cases of children with Down syndrome (DS), who have significantly higher risks of developing leukemia compared to non-DS children and distinctive treatment outcomes, particularly in cases of acute myeloid leukemia (AML). The high event-free survival (EFS) rates of DS AML patients and in particular, patients with megakaryocytic leukemia (AMkL), at least in part reflects an increased sensitivity to cytosine arabinoside (ara-C) secondary to increased expression of the chromosome 21-localized gene, cystathionine-beta-synthase, and potentially global mechanisms which increase the susceptibility of cells to undergo apoptosis. Somatic mutations of the X-linked transcription factor gene, GATA1, have been detected uniformly and exclusively in DS AMkL cases, which may lead to altered expression of GATA1 target genes and alter the metabolism of drugs including ara-C. Hyperdiploid acute lymphoblastic leukemia (ALL) cells with extra copies of chromosome 21, generate higher levels of the active methotrexate (MTX) metabolite, MTX polyglutamates. This is on account of increased intracellular transport of MTX via the reduced folate carrier (RFC) whose gene is localized to chromosome 21 and may also account for the increased MTX-associated toxicity of DS ALL patients. Microarray technology should lead to the identification of additional gene targets linked to the treatment response of specific cytogenetic leukemia subgroups.
Infants with constitutional trisomy 21 are at increased risk of developing transient and acute megakaryoblastic leukemia (AMKL). Mutations in GATA1 have been identified in trisomy 21 patients with AMKL, and this lesion is thought to be an initial event by virtue of its presence during transient leukemia. Transient leukemia is also observed in phenotypically normal infants albeit much less commonly so. Almost all these infants are mosaic for trisomy 21, and the clinical course of transient leukemia recapitulates that observed in constitutional trisomy 21. We report a phenotypically normal infant with tetrasomy 21 transient leukemia, GATA1 mutation within exon 2, and trisomy 21 mosaicism restricted to the hematopoietic tissue. Two years after diagnosis, low levels of trisomy 21 persisted in the peripheral blood, which resolved 2.5 years after diagnosis. The GATA1 mutation was not detected at last follow-up. The literature review identified 32 phenotypically normal infants with transient leukemia. Ninety-one percent (29 of 32) were observed and three received chemotherapy at diagnosis of transient leukemia. Nineteen percent (6 of 32) developed acute leukemia, and four continued in remission (two died). Transient leukemia in trisomy 21 mosaicism recapitulates the condition observed in constitutional trisomy 21 at the biological and clinical levels. Infants should be followed for the development of acute leukemia.
Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.
The human beta-globin locus control region DNase I hypersensitive site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory indicate that positioned nucleosomes are present at the 5'- and 3'-flanking regions of HS-2. In addition, footprints were observed that indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 site exists in the region of the 3'-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds in vitro at the 3'-end of HS-2. An additional GATA-1 site was found to bind GATA-1 in vitro at a site positioned 40 bp upstream. At the 5'-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the core to the sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.
The amyloid beta-protein (Abeta) deposited in brains of Alzheimer's disease (AD) patients is proteolytically derived from a large Abeta precursor protein (APP). APP gene expression patterns in the AD brain region indicate that abnormalities of gene regulation may be important in AD pathology. To understand the contribution of different cell types to APP gene expression, we studied it at four levels: promoter activity (by reporter gene assay of transfected cells), DNA-nuclear protein interaction (by electrophoretic mobility shift assay), RNA message and protein (by northern and western blotting, respectively). APP mRNA and protein expression levels were greater in neuroblastoma and PC12 cells than in glial or cervix epithelial cells. Relative activity among 12 different promoter regions and within single regions varied according to cell type/cell line. An upstream regulatory region containing a GATA-1 site is necessary for activity in PC12 and glial cells but not in neuroblastoma cells. DNA-protein interactions were examined in three distal and one proximal promoter elements in nuclear extracts belonging to neuronal and non-neuronal cells. The proximal promoter region is important for cell line-specific APP gene expression. Characterization of the APP regulatory region's interaction with cell type-specific nuclear factor(s) is important to understand tissue-specific expression of APP seen in AD subjects.
Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1gamma (TIF1gamma) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1gamma mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1gamma mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1gamma functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1gamma protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.
Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.
GSTP1-1 gene expression mechanisms were investigated in hemin-induced erythroid differentiation of K562 cells. Hemoglobin production during differentiation was followed by a significant increase in GSTP1-1 mRNA (1.7-fold, P < 0.01) and protein (1.2-fold, P < 0.01) after 4 days of induction. This increase in mRNA production was not due to transcriptional up-regulation by GATA-1 previously shown to regulate GSTP1-1 during erythroid and megakaryocytic differentiation. Moreover, a drastic decrease in differentiation-specific GATA-1 mRNA expression was correlated to a reduction in GATA-1 promoter binding activity. Neither AP-1 nor NF-kappaB transcription factor binding activities could provide an explanation to the GSTP1-1 mRNA overexpression in hemin-treated cells. GSTP1-1 mRNA stability analysis using actinomycin D as an inhibitor of mRNA neosynthesis showed that mRNA half-life was doubled in hemin-induced erythroid differentiation of K562 cells. These results allow us to add stabilization of GSTP1-1 mRNA as a novel regulatory mechanism during hemin-mediated differentiation of K562 cells.
In this paper, we report new observations related to the mechanism of the negative regulation of the important adult beta-globin gene in the erythroid cells at the embryonic-fetal stage of their development. We focused on the role of the silencer II region located upstream of the beta-globin gene, which along with its cognate binding protein BP1, negatively regulates beta-globin transcription.
We prepared plasmid constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in the beta-globin promoter 690-bp insert, which in turn was linked to an enhanced green fluorescent protein (EGFP) reporter gene. A human erythroleukemia cell line (K562) with embryonic-fetal phenotype was transfected with these EGFP constructs.
Flow cytometry and fluorescence digital imaging showed about threefold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small interfering RNA (siRNA) complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous approximately 40% elevation of beta-globin promoter activity as well as an increase in beta-globin mRNA levels, as compared with controls. We detected no changes in the mRNA levels of positive regulators of hemoglobin transcription such as EKLF and GATA-1.
Our results support the involvement of BP1 in the mechanism of the negative regulation of beta-globin transcription. A better understanding of this mechanism may lay the groundwork for novel gene therapy approaches to inhibit the expression of abnormal structural variants of adult beta globin, such as sickle hemoglobin.
Transcription factor GATA-1 is required for erythropoiesis, yet its full actions are unknown. We performed transcriptome analysis of G1E-ER4 cells, a GATA-1-null erythroblast line that undergoes synchronous erythroid maturation when GATA-1 activity is restored. We interrogated more than 9000 transcripts at 6 time points representing the transition from late burst forming unit-erythroid (BFU-E) to basophilic erythroblast stages. Our findings illuminate several new aspects of GATA-1 function. First, the large number of genes responding quickly to restoration of GATA-1 extends the repertoire of its potential targets. Second, many transcripts were rapidly down-regulated, highlighting the importance of GATA-1 in gene repression. Third, up-regulation of some known GATA-1 targets was delayed, suggesting that auxiliary factors are required. For example, induction of the direct GATA-1 target gene beta major globin was late and, surprisingly, required new protein synthesis. In contrast, the gene encoding Fog1, which cooperates with GATA-1 in beta globin transcription, was rapidly induced independently of protein synthesis. Guided by bioinformatic analysis, we demonstrated that selected regions of the Fog1 gene exhibit enhancer activity and in vivo occupancy by GATA-1. These findings define a regulatory loop for beta globin expression and, more generally, demonstrate how transcriptome analysis can be used to generate testable hypotheses regarding transcriptional networks.
Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1(tm2Sho) or GATA-1(low) mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1(low) mutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1(low) mice. The block in maturation includes failure to properly organize alpha granules because von Willebrand factor is barely detectable in mutant MKs, and P-selectin, although normally expressed, is found frequently associated with the demarcation membrane system (DMS) instead of within granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in GATA-1(low) platelets. Mutant MKs are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process to establish contact with MKs by fusing their membrane with those of the DMS. As a result, 16% (in spleen) to 34% (in marrow) of GATA-1(low) MKs contain 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1(low) MKs have morphologic features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathologic neutrophil emperipolesis may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenvironment.
Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. We have determined the transcription start site of the human Gfi-1B gene and located its first non-coding exon approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, we have demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, our results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. Here we conclude that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter.
Cis-element identification is a prerequisite to understand transcriptional regulation of gene loci. From analysis of a limited number of conserved gene loci, sequence comparison has proved a robust and efficient way to locate cis-elements. Human and mouse GATA1 genes encode a critical hematopoietic transcription factor conserved in expression and function. Proper control of GATA1 transcription is critical in regulating myeloid lineage specification and maturation. Here, we compared sequence and systematically mapped position of DNase I hypersensitive sites, acetylation status of histone H3/H4, and in vivo binding of transcription factors over approximately 120 kilobases flanking the human GATA1 gene and the corresponding region in mice. Despite lying in approximately 10 megabase (Mb) conserved syntenic segment, the chromatin structures of the 2 homologous loci are strikingly different. The 2 previously unidentified hematopoietic cis-elements, one in each species, are not conserved in position and sequence and have enhancer activity in erythroid cells. In vivo, they both bind the transcription factors GATA1, SCL, LMO2, and Ldb1. More broadly, there are both species- and regulatory element-specific patterns of transcription factor binding. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. More generally, mouse human sequence comparison may fail to identify all cis-elements.
The human major histocompatibility complex (MHC) class Ib gene, HLA-E, codes for the major ligand of the inhibitory receptor NK-G-2A, which is present on most natural killer (NK) cells and some CD8(+) cytotoxic T lymphocytes. We have previously shown that gamma interferon (IFN-gamma) induction of HLA-E gene transcription is mediated through a distinct IFN-gamma-responsive element, the IFN response region (IRR), in all cell types studied. We have now identified and characterized a cell type-restricted enhancer of IFN-gamma-mediated induction of HLA-E gene transcription, designated the upstream interferon response region (UIRR), which is located immediately upstream of the IRR. The UIRR mediates a three- to eightfold enhancement of IFN-gamma induction of HLA-E transcription in some cell lines but not in others, and it functions only in the presence of an adjacent IRR. The UIRR contains a variant GATA binding site (AGATAC) that is critical to both IFN-gamma responsiveness and to the formation of a specific binding complex containing GATA-1 in K562 cell nuclear extracts. The binding of GATA-1 to this site in response to IFN-gamma was confirmed in vivo in a chromatin immunoprecipitation assay. Forced expression of GATA-1 in nonexpressing U937 cells resulted in a four- to fivefold enhancement of the IFN-gamma response from HLA-E promoter constructs containing a wild-type but not a GATA-1 mutant UIRR sequence and increased the IFN-gamma response of the endogenous HLA-E gene. Knockdown of GATA-1 expression in K562 cells resulted in a approximately 4-fold decrease in the IFN-gamma response of the endogenous HLA-E gene, consistent with loss of the increase in IFN-gamma response of HLA-E promoter-driven constructs containing the UIRR in wild-type K562 cells. Coexpression of wild-type and mutant adenovirus E1a proteins that sequester p300/CBP eliminated IFN-gamma-mediated enhancement through the UIRR, but only partially reduced induction through the IRR, implicating p300/CBP binding to Stat-1alpha at the IRR in the recruitment of GATA-1 to mediate the cooperation between the UIRR and IRR. We propose that the GATA-1 transcription factor represents a cell type-restricted mediator of IFN-gamma induction of the HLA-E gene.
The genetic lesion(s) underlying chronic idiopathic myelofibrosis, as well as the mechanisms leading to the typical fibro-osteosclerotic changes of the bone marrow microenvironment, are still undefined. Recently, animal models of the disease have been described. We will briefly review the characteristics of these models, the thrombopoietin-overexpressing mice and the GATA-1(low) mice (mice deficient for GATA-1 expression in megakaryocytes), and illustrate how they provided insights into pathogenetic mechanisms of myelofibrosis, with special regard to the role of abnormal megakaryocyte proliferation and maturation.
How does an emerging transcriptional programme regulate individual genes as stem cells undergo lineage commitment, differentiation and maturation? To answer this, we have analysed the dynamic protein/DNA interactions across 130 kb of chromatin containing the mouse alpha-globin cluster in cells representing all stages of differentiation from stem cells to mature erythroblasts. The alpha-gene cluster appears to be inert in pluripotent cells, but priming of expression begins in multipotent haemopoietic progenitors via GATA-2. In committed erythroid progenitors, GATA-2 is replaced by GATA-1 and binding is extended to additional sites including the alpha-globin promoters. Both GATA-1 and GATA-2 nucleate the binding of various protein complexes including SCL/LMO2/E2A/Ldb-1 and NF-E2. Changes in protein/DNA binding are accompanied by sequential alterations in long-range histone acetylation and methylation. The recruitment of polymerase II, which ultimately leads to a rapid increase in alpha-globin transcription, occurs late in maturation. These studies provide detailed evidence for the more general hypothesis that commitment and differentiation are primarily driven by the sequential appearance of key transcriptional factors, which bind chromatin at specific, high-affinity sites.
Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33 of Caenorhabditis elegans. Mutations of CRMP-2 result in abnormal axon termination. Recently, it was demonstrated that CRMP-2 binds to tubulin heterodimers to promote microtubule assembly that is critical for axonal differentiation and growth during development. Here we show that glial cell line-derived neurotrophic factor (GDNF) enhances CRMP-2 expression in TGW human neuroblastoma cells via activation of RET receptor tyrosine kinase. GDNF-mediated CRMP-2 expression was regulated mainly by the extracellular regulated kinase (ERK) pathway, but was independent of activation of phosphatidylinositol 3-kinase and Src family kinases. Analysis of the promoter region of the CRMP-2 gene revealed that the region 214-48 bp upstream of the transcriptional start site is important for CRMP-2 expression. The SP1, E2F, and GATA1/2 binding sites appeared to play some roles in regulation of CRMP-2 expression. As expected, the CRMP-2 protein accumulated in extended neurites of TGW cells treated with GDNF. However, neuritogenesis of TGW cells was mostly dependent on Src family kinase activity and not ERK activity, indicating that the increased expression of CRMP-2 alone was not sufficient for neuritogenesis.
Leukaemia is characterized by the accumulation of malignant haematopoietic precursors. Recent studies have revealed that acquired alterations in genes that regulate normal haematopoiesis are frequently detected in leukaemia. The progression to leukaemia depends on additional mutations that promote the survival of developmentally arrested cells. This review describes three examples of this general paradigm of leukaemogenesis: RUNX1 abnormalities in acute leukaemias, GATA1 mutations in the leukaemias of Down syndrome, and SCL and LMO2 ectopic expression in T cell acute lymphoblastic leukaemia.
To observe the role of Panax notoginosides (PNS) in up-regulation of GATA family transcription factors, and explore intracellular signal pathway of PNS in the proliferation of hematopoietic cells.
Human bone marrow cells were incubated with different concentrations of PNS for colony-forming assay. Human cell lines HL-60, K562, CHRF-288 and Meg-01 were incubated with PNS (10 mg/L) for 14 days. The cell nuclear proteins were extracted and analyzed by Western blot with antibodies against GATA-1, GATA-2. Electrophoretic mobility shift assay (EMSA) and antibody gel supershift assay was performed using (32)P labeled GATA consensus oligonucleotide which contains binding site for GATA transcription factors.
PNS could promote the proliferation of CFU-GM and CFU-E and induce the expression of GATA-1, GATA-2. The nuclear proteins of both GATA-1 and GATA-2 in K562, CHRF-288 and Meg-01 cells treated by PNS were increased by (1.5 - 2.8) and (2.0 - 3.1)-fold over untreated cells respectively. GATA binding activity initiated by PNS was apparently elevated to form higher density band of GATA-DNA complex. While there was no detectable change in HL-60 cells before and after PNS treatment. The predominant GATA binding complex was mainly attributable to both GATA-1 and GATA-2 proteins being in phosphorylated status.
PNS can induce the synthesis of transcription factors GATA-1 and GATA-2 and enhance their DNA binding activity, which could play a role in the up-regulation of the expression genes related to proliferation and differentiation in hematopoietic cells.
Transient myeloproliferative disorder (TMD) is a unique, spontaneously regressing neoplasia specific to Down's syndrome (DS), affecting up to 10% of DS neonates. In 20-30% of cases, it reoccurs as progressive acute megakaryoblastic leukaemia (AMKL) at 2-4 years of age. The TMD and AMKL blasts are morphologically and immuno-phenotypically identical, and have the same acquired mutations in GATA1. We performed transcript profiling of nine TMD patients comparing them with seven AMKL patients using Affymetrix HG-U133A microarrays. Similar overall transcript profiles were observed between the two conditions, which were only separable by supervised clustering. Taqman analysis on 10 TMD and 10 AMKL RNA samples verified the expression of selected differing genes, with statistical significance (P < 0.05) by Student's t-test. The Taqman differences were also reproduced on TMD and AMKL blasts sorted by a fluorescence-activated cell sorter. Among the significant differences, CDKN2C, the effector of GATA1-mediated cell cycle arrest, was increased in AMKL but not TMD, despite the similar level of GATA1. In contrast, MYCN (neuroblastoma-derived oncogene) was expressed in TMD at a significantly greater level than in AMKL. MYCN has not previously been described in leukaemogenesis. Finally, the tumour antigen PRAME was identified as a specific marker for AMKL blasts, with no expression in TMD. This study provides markers discriminating TMD from AMKL-M7 in DS. These markers have the potential as predictive, diagnostic and therapeutic targets. In addition, the study provides further clues into the pathomechanisms discerning self-regressive from the progressive phenotype.
Gata1 is a transcription factor essential for erythropoiesis. Erythroid cells lacking Gata1 undergo apoptosis, while overexpression of Gata1 results in a block in erythroid differentiation. However, erythroid cells overexpressing Gata1 differentiate normally in vivo when in the presence of wild-type cells. We have proposed a model, whereby a signal generated by wild-type cells (red cell differentiation signal; REDS) overcomes the intrinsic defect in Gata1-overexpressing erythroid cells. The simplest interpretation of this model is that wild-type erythroid cells generate REDS. To substantiate this notion, we have exploited a tissue specific Cre/loxP system and the process of X-inactivation to generate mice that overexpress Gata1 in half the erythroid cells and are Gata1 null in the other half. The results show that the cells supplying REDS are erythroid cells. This study demonstrates the importance of intercellular signalling in regulating Gata1 activity and that this homotypic signalling between erythroid cells is crucial to normal differentiation.
The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.
Gene-targeting experiments in transgenic mice have revealed an essential role for GATA-1 in the normal differentiation and development of erythroid cells. GATA-1 is phosphorylated in vivo on seven of its serine residues; the regulation and function of GATA-1 phosphorylation, however, is not understood. Here we demonstrate a role for MAP kinase (MAPK) signalling in the control of GATA-1 phosphorylation. We show that EGF-induced MAPK signalling results in the phosphorylation of ectopically expressed GATA-1 in COS cells. This phosphorylation can be positively or negatively regulated by genetic manipulation of the MAPK pathway through expression of constitutively activated, or dominant-negative, mutants of MAPK kinase (MAPKK), an upstream regulator of MAPK activity. In vitro phosphorylation experiments using purified MAPK and either recombinant GATA-1 or synthetic GATA-1 peptides suggest that GATA-1 is a MAPK substrate with MAPK phosphorylation occurring primarily on Ser26 and Ser178. We also show that GATA-1 is phosphorylated in factor-dependent haemopoietic progenitor cells in response to cytokine-induced signalling. Through the further use of a dominant-negative MAPKK mutant as well as chemical inhibitors of specific MAPKs, we identify ERK as an in vivo GATA-1 kinase. Finally, we demonstrate that mutation of serines 26 and 178 compromises the ability of GATA-1 to interact with the LIM-only protein LMO2 when both proteins are expressed in COS cells. These data implicate receptor-mediated signalling through the MAPK pathway as a control point in the regulation of transcription factor GATA-1.
Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210(BCR-ABL) oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210(BCR-ABL) versus those expressing an empty vector using cDNA macro- and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210(BCR-ABL)), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210(BCR-ABL) in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210(BCR-ABL) responsive element to a high affinity 5'(A/T)GATA(A/G)-3' "GATA" response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210(BCR-ABL)- and p185(BCR-ABL)-mediated trans-activation to an inert promoter. Short interfering RNA mediated "knockdown" of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein. We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.
Patients with 3q21q26 rearrangements seem to share similar clinicopathologic features and a common molecular mechanism, leading to myelodysplasia or acute myeloid leukemia (AML). The ectopic expression of EVI1 (3q26) has been implicated in the dysplasia that characterizes this subset of myeloid neoplasias. However, lack of EVI1 expression has been reported in several cases, and overexpression of EVI1 was detected in 9% of AML cases without 3q26 abnormalities. We report the molecular characterization of seven patients with inv(3)(q21q26), t(3;3)(q21;q26) or related abnormalities. EVI1 expression was detected in only one case, and thus ectopic expression of this gene failed to explain all of these cases. GATA2 (3q21) was found to be overexpressed in 5 of the 7 patients. GATA2 is highly expressed in stem cells, and its expression dramatically decreases when erythroid and megakaryocytic differentiation proceeds. No mutations in GATA1 were found in any patient, excluding loss of function of GATA1 as the cause of GATA2 overexpression. We report finding molecular heterogeneity in patients with 3q21q26 rearrangements in both breakpoints and in the expression pattern of the genes near these breakpoints. Our data suggest that a unique mechanism is not likely to be involved in 3q21q26 rearrangements.
Mutations in the GATA-1 gene have been identified in patients with familial macrothrombocytopenia and Down's syndrome patients with a transient myeloproliferative disorder and/or acute megakaryoblastic leukemia. We screened this gene in 46 patients with essential thrombocythemia and identified only a common single nucleotide polymorphism that is unlikely to be of pathological significance.
The GATA family of transcription factors establishes genetic networks that control developmental processes including hematopoiesis, vasculogenesis, and cardiogenesis. We found that GATA-1 strongly activates transcription of the Tac-2 gene, which encodes proneurokinin-B, a precursor of neurokinin-B (NK-B). Neurokinins function through G protein-coupled transmembrane receptors to mediate diverse physiological responses including pain perception and the control of vascular tone. Whereas an elevated level of NK-B was implicated in pregnancy-associated pre-eclampsia (Page, N. M., Woods, R. J., Gardiner, S. M., Lomthaisong, K., Gladwell, R. T., Butlin, D. J., Manyonda, I. T., and Lowry, P. J. (2000) Nature 405, 797-800), the regulation of NK-B synthesis and function are poorly understood. Tac-2 was expressed in normal murine erythroid cells and was induced upon ex vivo erythropoiesis. An estrogen receptor fusion to GATA-1 (ER-GATA-1) and endogenous GATA-1 both occupied a region of Tac-2 intron-7, which contains two conserved GATA motifs. Genetic complementation analysis in GATA-1-null G1E cells revealed that endogenous GATA-2 occupied the same region of intron-7, and expression of ER-GATA-1 displaced GATA-2 and activated Tac-2 transcription. Erythroid cells did not express neurokinin receptors, whereas aortic and yolk sac endothelial cells differentially expressed neurokinin receptor subtypes. Since NK-B induced cAMP accumulation in yolk sac endothelial cells, these results suggest a new mode of vascular regulation in which GATA-1 controls NK-B synthesis in erythroid cells.
Although platelets were already discovered light-microscopically in the 19th century, it became only clear in 1906 that they originate from megakaryocytes. The discovery of thrombopoietin in 1994 facilitated research on the origin of platelets, because this growth factor enabled expansion of megakaryocytes in culture. Thus, cell biological and molecular studies could be undertaken. Knowledge accumulated further by studying mouse models and by unravelling the defect in patients with hereditary thrombocytopenia. How a megakaryocyte originates from a hematopoietic stem cell and how this cell undergoes further maturation and differentiation is a complex process, controlled at different stages by several transcription factor. These transcription factors influence the expression of their own target genes, leading to megakaryocyte maturation and platelet release. One of the transcription factors that is most studied in this regard is GATA1, that forms a transcriptional complex with its cofactor FOG1. Families with hereditary thrombocytopenia have been described due to mutations in the GATA1 gene, and target genes were studied. This broadens our insight in normal megakaryocyte differentiation. The final release of platelets from the megakaryocyte is strongly dependent on the formation of so-called proplatelets. The formation of pseudopodia requires the presence of microtubuli. Beta 1-tubulin is a major part of these microtubuli and plays an important role not only in the genesis of platelets but also in the final discoid form of the platelet. Despite a renewed interest and expanding knowledge in this area, there are more questions than answers at this day.
Side population (SP) cells, a rare cell type identified by their ability to efflux the vital dye Hoechst 33342, are highly enriched for stem cell activity. Bone marrow (BM) SP cells uniformly express the pan-hematopoietic marker CD45, whereas tissue SP cells are heterogeneous in CD45 expression. In previous studies, we found that CD45 is expressed on 75% of lung SP cells. By performing whole BM transplantations, we determined that CD45-positive and CD45-negative lung SP cells are marrow derived. Transplantation of 200 highly purified BM SP cells indicated that both lung SP cell subtypes are derived from this marrow cell type. Morphologically, CD45-positive lung and BM SP cells possess similar features. They are small, round, and contain scant cytoplasm. CD45-negative lung SP cells are larger and contain abundant granular cytoplasm. Gene expression patterns for hematopoietic transcription factors GATA-1, GATA-2, and PU.1 further differentiated SP marrow and lung subtypes. By immunostaining for alpha-smooth muscle actin and cytokeratin, we found significant differences in the relative expression patterns of these markers in lung and marrow SP cell subtypes. In summary, these findings demonstrate that lung SP cells are derived from the BM and that CD45-positive and -negative subtypes can be distinguished by morphological differences and gene expression patterns.
Physical association between the transcription factor GATA-1 and the cofactor, Friend of GATA-1 (FOG-1), is essential for the differentiation of two blood cell types, erythroid cells and megakaryocytes. However, little is known regarding the mechanisms that modulate their interaction within cells. In the present study, we have identified TACC3 as a FOG-1-interacting protein. Transforming acidic coiled-coil protein 3 (TACC3), a protein that is highly expressed in hematopoietic cells, has been reported to have a critical role in the expansion of immature hematopoietic progenitors. We show that TACC3 affects FOG-1 nuclear localization, altering the interaction between GATA-1 and FOG-1. However, GATA-1 competes with TACC3 in the interaction with FOG-1. We observe that high levels of TACC3 inhibit the function of FOG-1 as a transcriptional cofactor of GATA-1. Furthermore, forced expression of TACC3 to levels similar to those found in progenitor cells delays terminal maturation of MEL and G1ER cells, two cell models of erythroid cell development. We suggest a role for TACC3 in regulating the cellular distribution of FOG-1 and thus the direct interaction of GATA-1 and FOG-1 as a mechanism to control the transition between expansion of multipotential progenitor cell populations and final stages of erythroid maturation.
Expression of the myeloid transcription factor GATA1 is required for early stages of eosinophil differentiation. Defining mechanisms regulating eosinophil GATA1 expression will be important to understand development of this lineage. However, the cis-elements required for eosinophil GATA1 expression are not fully characterized. Previous work identified HS 1 as a major GATA1 enhancer, but its role in eosinophil GATA1 expression is unclear. Here, we show that mouse HS 1 deletion leaves eosinophil GATA1 mRNA expression and eosinophil differentiation unaffected. Chromatin isolated from eosinophils and encompassing HS 1 is weakly enriched for acetylated histones H3/H4. HS 1 deletion does not alter eosinophil GATA1 locus histone acetylation. In eosinophils, GATA1 and CCAAT/enhancer binding protein epsilon (C/EBP epsilon) do not bind HS 1 but bind selectively a cis-element in the first GATA1 intron. Thus, HS 1 is not required for eosinophil GATA1 expression. Instead, this study suggests a previously unsuspected role for the GATA1 intron element for this function.
Mice deficient in GATA-1 or NF-E2 have a 200-300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice.
GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes.
Histomorphometry, microCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes.
A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact.
These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.
The multifunctional cytokine interleukin-1beta (IL-1beta) plays a central role in the body's immune and inflammatory responses. The mechanism of IL-1beta on thrombocytosis and megakaryocytopoiesis has remained controversial. In previous reports, we have demonstrated the expression of IL-1 receptors (IL-1RI and IL-1RII) and enhancing effects of IL-1beta on primary human megakaryocytic (MK) cells. In this study, we investigated the possible direct effects of IL-1beta on the expression of thrombopoietin (TPO) and transcription factors c-Jun, c-Fos, GATA-1, and p45 nuclear factor-E2 (NF-E2) in MK cell lines CHRF and Meg-01. Our results demonstrated that IL-1beta up-regulated messenger RNA (mRNA) and protein expressions of these transcription factors in a dose- and time-dependent manner. In CHRF cells, mRNA: c-Jun [3.4-fold, peaked at 15 minutes], c-Fos [4.2-fold, 15 minutes], GATA-1 [4.0-fold, 60 minutes], NF-E2 [3.2-fold, 120 minutes] and protein expression: c-Jun [3.0-fold, 30 minutes], c-Fos [1.7-fold, 30 minutes], GATA-1 [11.5-fold, 60 minutes], NF-E2 [12.5-fold, 120 minutes] were evidently enhanced after treatment with IL-1beta. The response to IL-1beta was consistent in the total cell and nuclear extracts and was significantly reduced by pretreatment with actinomycin D or cycloheximide. An IL-1-receptor antagonist (IL-1RA) inhibited the stimulatory effects of IL-1beta on these transcription factors by as much as 78%. TPO expression was increased by more than 9.9-fold on stimulation with IL-1beta. A TPO-neutralizing antibody did not significantly reduce the effects of IL-1beta. We conclude that IL-1beta up-regulates the expression of TPO, c-Jun, c-Fos, GATA-1, and NF-E2 in MK cells. The mechanism might be mediated by IL-1beta receptors and require transcription or protein synthesis. The direct involvement of IL-1beta in the MK lineage may provide an explanation for the phenomenon of thrombocytosis during inflammatory responses.
To study effects of chebulinic acid on erythroid and megakaryocytic differentiation in K562 cells.
The benzidine staining method was used to evaluate hemoglobin synthesis; the expression of erythroid specific glycophorin A (GPA) protein and megakaryocytic surface marker CD61 was determined by flow cytometry using fluorescence labeled antibodies; erythroid and megakaryocytic mRNA expression was analyzed by RT-PCR.
During erythroid differentiation induced by butyric acid (BA) or hemin, chebulinic acid not only inhibited the hemoglobin synthesis of BA- and hemin-treated K562 cells in concentration-dependent manner with IC50 of 4 micromol/L and 40 micromol/L respectively, but also inhibited another erythroid differentiation marker acetylcholinesterase at the concentration of 50 micromol/L in the cells either treated or untreated with each erythroid differentiation inducers, whereas chebulinic acid 50 micromol/L did not change GPA protein expression in these cells significantly. When K562 cells were treated with TPA 50 microg/L for 72 h to induce megakaryocytic differentiation, the presence of chebulinic acid 50 micromol/L slightly provoked the decrease of GPA protein expression induced by TPA. Chebulinic acid did not change the TPA-induced CD61 expression at the same concentration. Chebulinic acid also reduced the mRNA levels of erythroid relative genes including gamma-globin, PBGD, NF-E2, and GATA-1 genes in K562 cells either treated or untreated with BA, whereas chebulinic acid upregulated the mRNA levels of GATA-2 transcription factor in these cells.
Chebulinic acid had inhibitory effect on erythroid differentiation likely through changing transcriptional activation of differentiation relative genes, which suggests that chebulinic acid or other tannins might influence the efficiency of some anti-tumor drugs-induced differentiation or the hematopoiesis processes.
Paclitaxel (Px) is a cancer chemotherapeutic agent that causes bone marrow (BM) cytotoxicity by microtubule stabilization and by modifications in the expression of several genes. Hematopoietic progenitors show severe alterations following Px injury. Erythropoietic recovery should be accompanied by changes in the expression of transcription factors such as c-MYB, GATA-1, NF-E2, Bcl-x(L), and erythropoietin receptor (Epo-R). The aim of this work was to study the in vivo recovery of erythropoiesis and to correlate transcription factors, Bcl-x(L), and Epo-R expressions to apoptosis and changes in proliferation of murine erythroid progenitors following a single dose of Px (29 mg/kg, i.p.). BM total and differential cellularities, apoptosis (TdT-mediated dUTP Nick-End Labeling [TUNEL] assay), clonogenic assays, and immunoblots for transcription factors, Epo-R, and Bcl-x(L) were performed each day for 5 days post-injury. Apoptosis (24 +/- 0.81%, P < 0.01), inhibition of colony growth (burst-forming units-erythroid [BFU-E] and granulocyte-erythroid-macrophage [GEM]), and decrease in BM cellularities (28 +/- 4.2% of control) were maximal at 24 h following Px. The highest apoptosis was concomitant with the lowest BM cellularities. Apoptosis returned to normal values (3.08 +/- 0.61%) by day 3 post-Px. Up-regulation of c-MYB, GATA-1, Epo-R, and Bcl-x(L) expressions were observed between 24 and 48 h following Px. Correlations among c-MYB, GATA-1, Bcl-x(L), and Epo-R were extremely significant. Maximal expression of NF-E2 was observed on day 3 concomitant with the rise (threefold) of early erythroid precursors (BFU-E). Thus, cells that survive injury seem to be stimulated to produce early (24-48 h) erythroid-related and antiapoptotic proteins. Therefore, the results suggest an in vivo interplay between specific transcription factors and Bcl-x(L) during progenitor cell survival and proliferation; mechanisms triggered to restore size and composition of the erythroid compartment.
SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.
Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.
During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tal1 transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegf(lo/lo) embryos carrying a hypomorph allele, Flk1-positive cells reach the yolk sac at E8.5, but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development, different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegf(lo/lo) embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Vegf(lo) allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 and betaH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment.
The transcription factor GATA-1, together with its cofactor FOG-1, regulates erythropoiesis and megakaryocytopoiesis. Mutations in the DNA or FOG-1 binding sites of its N-terminal zinc finger result in different illnesses. Alterations of the FOG-1 face are responsible for dyserythropoietic anemia with thrombocytopenia while R216Q, the only mutation identified in the DNA face, induces X-linked thrombocytopenia with thalassemia (XLTT). The former disorder has been studied in detail whereas little is known about the latter since only one family has been investigated. We studied a second family with an R216Q, showing that XLTT and dyserythropoietic anemia with thrombocytopenia, even if different clinical entities, are closely related disorders. In both cases, patients present mild dyserythropoiesis, red cell hemolysis, severely defective maturation of megakaryocytes, macrothrombocytopenia with alpha-granule deficiency, and abnormalities of the cytoplasmic membrane system. However, a thalassemia minor phenotype has only been described in patients with XLTT whereas severe anemia and thrombocytopenia with evident defects of platelet composition and function may be observed only in dyserythropoietic anemia with thrombocytopenia.
The transcription factor GATA-1 plays a significant role in erythroid differentiation and association with CBP stimulates its activity by acetylation. It is possible that histone deacetylases (HDACs) repress the activity of GATA-1. In the present study, we investigated whether class I and class II HDACs interact with GATA-1 to regulate its function and indeed, GATA-1 is directly associated with HDAC3, HDAC4 and HDAC5. The expression profiling and our previous observation that GATA-2 interacts with members of the HDAC family prompted us to investigate further the biological relevance of the interaction between GATA-1 and HDAC5. Coexpression of HDAC5 suppressed the transcriptional potential of GATA-1. Our results demonstrated that GATA-1 and HDAC5 colocalized to the nucleus of murine erythroleukemia (MEL) cells. Furthermore, a portion of HDAC5 moved to the cytoplasm concomitant with MEL cell erythroid differentiation, which was induced by treatment with N,N'-hexamethylenebisacetamide. These observations support the suggestion that control of the HDAC5 nucleocytoplasmic distribution might be associated with MEL cell differentiation, possibly through regulated GATA-1 transactivation.
Members of the GATA and RUNX families of genes appear to have conserved functions during hematopoiesis from Drosophila to mammals. In Drosophila, the GATA factor Serpent (Srp) is required in blood cell progenitors for the formation of the two populations of blood cells (plasmatocytes and crystal cells), while the RUNX factor Lozenge (Lz) is specifically required for crystal cell development. Here we investigate the function and the mechanisms of action of Lz during hematopoiesis. Our results indicate that Lz can trigger crystal cell development. Interestingly, we show that Lz function is strictly dependent on the presence of functional Srp and that Srp and Lz cooperate to induce crystal cell differentiation in vivo. Furthermore, we show that Srp and Lz directly interact in vitro and that this interaction is conserved between Drosophila and mammals. Moreover, both Srp and mouse GATA1 synergize with mouse RUNX1 to activate transcription. We propose that interaction and cooperation between GATA and RUNX factors may play an important role in regulating blood cell formation from Drosophila to mammals.
Association of GATA-1 and its cofactor Friend of GATA-1 (FOG-1) is essential for erythroid and megakaryocyte development. To assess functions of GATA-1-FOG-1 association during mouse development, we used the GATA-1 hematopoietic regulatory domain to generate transgenic mouse lines expressing a mutant GATA-1, which contains a substitution of glycine 205 for valine (V205G) that abrogates its association with FOG-1. We examined whether the transgenic expression of mutant GATA-1 rescues GATA-1 germ line mutants from embryonic lethality. In high-expressor lines we observed that the GATA-1(V205G) rescues GATA-1-deficient mice from embryonic lethality at the expected frequency, revealing that excess GATA-1(V205G) can eliminate the lethal anemia that is due to GATA-1 deficiency. In contrast, transgene expression comparable to the endogenous GATA-1 level resulted in much lower frequency of rescue, indicating that the GATA-1-FOG-1 association is critical for normal embryonic hematopoiesis. Rescued mice in these analyses exhibit thrombocytopenia and display dysregulated proliferation and impaired cytoplasmic maturation of megakaryocytes. Although anemia is not observed under steady-state conditions, stress erythropoiesis is attenuated in the rescued mice. Our findings reveal an indispensable role for the association of GATA-1 and FOG-1 during late-stage megakaryopoiesis and provide a unique model for X-linked thrombocytopenia with inherited GATA-1 mutation.
Acquired mutations in megakaryocyte transcription factor GATA1 have recently been reported in Down syndrome (DS), transient myeloproliferative disorder (TMD), and acute megakaryoblastic leukemia (AMKL). To provide novel insight into GATA1 mutations in DS, genomic DNA was assayed from 12 AMKL and 4 TMD cases (including neonatal, prediagnosis samples in 4 of 16), neonatal blood spots from 21 DS children without clinically evident TMD or AMKL, and 62 non-DS cord blood samples, using techniques not previously employed with such samples. GATA1 mutations were present in all TMD and AMKL cases and at birth in 3 of 4 children without known clinical TMD, who later developed AMKL. They were present at birth in 2 of 21 DS neonates, who have not yet, but could still, develop AMKL (now 26 and 31 months). GATA1 mutations were not detected in 62 non-DS cord blood samples. In 4 AMKL patients multiple independent GATA1 mutations were observed. These data show GATA1 mutations occur in utero in most DS TMD and AMKL, that they may occur without clinical signs of disease, and that multiple separate GATA1 mutant clones can occur in an individual. The findings have implications for pathogenesis of DS TMD and AMKL and highlight parallels between DS AMKL and other childhood leukemias.
The early genetic pathway(s) triggering the pathogenesis of coronary artery disease (CAD) and myocardial infarction (MI) remain largely unknown. Here, we describe an autosomal dominant form of CAD/MI (adCAD1) that is caused by the deletion of seven amino acids in transcription factor MEF2A. The deletion disrupts nuclear localization of MEF2A, reduces MEF2A-mediated transcription activation, and abolishes synergistic activation by MEF2A and by the transcription factor GATA-1 through a dominant-negative mechanism. The MEF2A protein demonstrates strong expression in the endothelium of coronary arteries. These results identify a pathogenic gene for a familial vascular disease with features of CAD and implicate the MEF2A signaling pathway in the pathogenesis of CAD/MI.
Mutations in transcription factors often contribute to human leukemias by providing a block to normal differentiation. To determine whether mutations in the hematopoietic transcription factor GATA1 are associated with leukemia, we assayed for alterations in the GATA1 gene in bone marrow samples from patients with various subtypes of acute leukemia. Here we summarize our findings that GATA1 is mutated in the leukemic blasts of patients with Down syndrome acute megakaryoblastic leukemia (DS-AMKL). We did not find mutations in GATA1 in leukemic cells of DS patients with other types of acute leukemia, or in other patients with AMKL who did not have DS. Furthermore, we did not detect GATA1 mutations in DNAs from over 75 other patients with acute leukemia or from 21 healthy individuals. Since the GATA1 mutations were restricted to DS-AMKL, we also investigated whether GATA1 was altered in the "preleukemia" of DS, transient myeloproliferative disorder (TMD). TMD is a common myeloid disorder that affects 10% of DS newborns and evolves to AMKL in nearly 30% patients. We detected GATA1 mutations in TMD blasts from every infant examined. Together, these results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD and DS-AMKL, and that mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis. We hypothesize that disruption of normal GATA-1 function is an essential step in the initiation of megakaryoblastic leukemia in DS.
GATA transcription factors, together with Friend of GATA (FOG) cofactors, are required for the differentiation of diverse cell types. Multiple aspects of hematopoiesis are controlled by the interaction of FOG-1 with the GATA-1/2/3 subfamily. Likewise, FOG-2 is coexpressed with the GATA-4/5/6 subfamily at other sites, including the heart and gonads. FOG-2 and GATA-4 are required for cardiac development. Through transgenic rescue of hematopoietic defects of FOG-1-/- embryos we define an unsuspected role for FOG-1 in heart development. In particular, rescued FOG-1-/- mice die at embryonic day (E) 14.5 with cardiac defects that include double outlet right ventricle and a common atrioventricular valve. Using conditional inactivation of Fog-1 we assign the cell of origin in which FOG-1 function is required. Neural crest cells migrate properly into FOG-1-/- hearts and mice with FOG-1 conditionally excised from neural crest derivatives fail to develop cardiac abnormalities. In contrast, conditional inactivation of FOG-1 in endothelial-derived tissues by means of Tie-2-expressed Cre recapitulates the rescue-knockout defects. These findings establish a nonredundant requirement for FOG-1 in the outlet tract and atrioventricular valves of the heart that depend on expression in endothelial-derived tissue and presumably reflect cooperation with the GATA-4/5/6 subfamily.
The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.
The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. We show that this restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralising or posteriorising the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells.
Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3'-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.
The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and beta-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.
Oncogenic transformation usually inhibits normal cell differentiation processes. Certain chemical agents can force some tumor cells to resume their differentiation program and undergo cell cycle arrest, an approach termed differentiation therapy. Mouse erythroleukemia (MEL) cells represent an important cell culture model system for investigating the principles of differentiation therapy. MEL cells are malignant erythroblasts that are blocked from differentiating into mature erythroid cells because of inappropriate expression of the transcription factor PU.1, which binds to and represses GATA-1, a key transcriptional stimulator of red blood cell differentiation. We report here that the block to differentiation in MEL cells can be overcome by providing the cells with additional GATA-1. A conditionally active form of GATA-1 can trigger the cells to differentiate, undergo terminal cell division, and lose their tumorigenicity. We also show that the gene for the cell cycle inhibitor p21 is transcriptionally regulated by GATA-1 and is a likely downstream effector of GATA-1 that helps to promote differentiation and proliferation arrest.
Overexpression of the embryonic transcription factor, Wilms' tumour protein 1 (WT1), is common in acute myeloid leukaemias (AML). Mutations of Wilms' tumour gene 1 (WT1) in AML are rare and WT1 expression may be increased by other transcription factors. PAX2, PAX8 and GATA-1 are known physiological regulators of WT1. In the present study, we analysed either bone marrow or blood samples of 43 AML patients for the expression levels of WT1, PAX2, PAX8 and GATA-1 by real-time reverse transcription polymerase chain reaction (LightCycler). Bone marrow samples of patients without haematological malignancies and stem cell preparation samples from healthy donors and lymphoma patients served as controls. PAX2 expression was found in 11 of 43 AML samples, with a clear correlation of PAX2 with WT1 expression levels observed. PAX8 expression was found in two additional samples. GATA-1 expression was detectable in 41 of 43 AML samples and also in all control samples; no significant differences between these groups were observed and no correlation of GATA-1 expression with WT1 expression levels was apparent. In conclusion, PAX2, and possibly PAX8, appears to be a candidate for the upregulation of WT1 in a proportion of AML, whereas GATA-1 expression cannot be explained as an inducer of WT1. In two-thirds of leukaemias from our series, the basis of WT1 upregulation cannot be explained by the simple upregulation of the known WT1 activators.
GATA-1 is the founding member of a transcription factor family that regulates growth and maturation of a diverse set of tissues. GATA-1 is expressed primarily in hematopoietic cells and is essential for proper development of erythroid cells, megakaryocytes, eosinophils, and mast cells. Although loss of GATA-1 leads to differentiation arrest and apoptosis of erythroid progenitors, absence of GATA-1 promotes accumulation of immature megakaryocytes. Recently, we and others have reported that mutagenesis of GATA1 is an early event in Down syndrome (DS) leukemogenesis. Acquired mutations in GATA1 were detected in the vast majority of patients with acute megakaryoblastic leukemia (DS-AMKL) and in nearly every patient with transient myeloproliferative disorder (TMD), a "preleukemia" that may be present in as many as 10% of infants with DS. Although the precise pathway by which mutagenesis of GATA1 contributes to leukemia is unknown, these findings confirm that GATA1 plays an important role in both normal and malignant hematopoiesis. Future studies to define the mechanism that results in the high frequency of GATA1 mutations in DS and the role of altered GATA1 in TMD and DS-AMKL will shed light on the multistep pathway in human leukemia and may lead to an increased understanding of why children with DS are markedly predisposed to leukemia.
Transcription factors GATA-1 and GATA-2 are required for normal hematopoiesis. The loss of either leads to embryonic lethality in knockout mice because of the failure of erythroid maturation and the expansion of progenitors, respectively. As the expression of GATA-1 and GATA-2 overlaps within hematopoietic progenitors, the extent to which these factors functionally compensate for each other during embryogenesis is unknown. As shown here, we have analyzed double-knockout embryos at the yolk sac stage of development and have shown that the combined absence of these GATA factors virtually ablates primitive erythroid cell formation. Thus, the function of GATA-1 and GATA-2 overlaps at the yolk sac stage. Moreover, a GATA factor, either GATA-1 or GATA-2, is required to initiate blood formation in the embryo.
GATA-1 is an essential transcription factor for megakaryocyte and erythrocyte (MegE) development. Here we show that hematopoietic progenitors can be reprogrammed by the instructive action of GATA-1. Enforced expression of GATA-1 in hematopoietic stem cells led to loss of self-renewal activity and the exclusive generation of MegE lineages. Strikingly, ectopic GATA-1 reprogrammed common lymphoid progenitors as well as granulocyte/monocyte (GM) progenitors to differentiate into MegE lineages, while inhibiting normal lymphoid or GM differentiation. GATA-1 upregulated critical MegE-related transcription factors such as FOG-1 and GATA-2 in lymphoid and GM progenitors, and their MegE development did not require "permissive" erythropoietin signals. Furthermore, GATA-1 induced apoptosis of proB and myelomonocytic cells, which could not be prevented by enforced permissive Bcl-2 or myeloid cytokine signals. Thus, GATA-1 specifically instructs MegE commitment while excluding other fate outcomes in stem and progenitor cells, suggesting that regulation of GATA-1 is critical in maintaining multilineage homeostasis.
Mesenchymal stem cells (MSCs), precursors of diverse stromal cells, can support hematopoiesis in vitro and can promote the implantation of hematopoietic stem cells in vivo when co-transplanted with CD34(+) cells. The aim of this study was to investigate the potential effect of MSCs on the hematopoietic development of embryonic stem cells (ES cells) and the feasibility of a novel system in which ES cells will be co-cultured with MSCs. The murine bone marrow MSCs were isolated and cultured and then their phenotype and differentiation function were identified with FCM and histochemical technique. The CCE cells, murine ES cell line, were co-cultured with the isolated MSCs and the hematopoietic differentiation of CCE cells was observed with hematopoietic clonogenic assay and RT-PCR. The results showed that the morphology of MSCs became gradually homogeneous with the passage culture of cells. After passage 4, the marker of Sca-1, CD29, CD44 and CD105 were highly expressed, however, CD34 and CD45, the specific marker of hematopoietic and endothelial cells, could hardly be identified. The isolated MSCs differentiated into adipocytes and osteoblasts in specific induction culture system. After maintaining culture on mouse embryonic fibroblasts, CCE cells were plated in suspended culture system with only differentiation inductive agents and co-culture system in which MSCs were added. Compared with CCE cell suspended culture, the cells differentiated into embryoid body were obviously enhanced and there were no colony-forming cells in the co-culture system of ES cells and MSCs. In addition, transcription factor Oct-4 in co-cultured CCE cells was expressed and hematopoietic markers, Flk-1, GATA-1 and beta-H1, were negative. The ability of embryoid bodies derived from the co-culture system to produce hematopoietic colonies was markedly higher than that from the suspended culture system. It is concluded that MSCs inhibit the initial differentiation of ESC and enhance hematopoietic differentiation ability of the co-cultured ES cells.
The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning 'floxed' Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Surprisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse.
Smad7, an inhibitor of transforming growth factor beta superfamily signaling, is induced by bone morphogenetic protein (BMP) in an inhibitory feedback loop. Here, we identify multiple BMP response elements (BREs) in the Smad7 gene and demonstrate that they function differentially to interpret BMP signals in a cell type-specific manner. Two BREs (BRE-1 and -2) reside in the promoter region. One of these contains several conserved Smad1 and Smad4 binding sites that cooperate to mediate BMP-dependent induction, most likely in the absence of DNA binding partners. The third BRE (I-BRE) resides in the first intron and contains GATA factor binding sites. GATA-1, -5, or -6 is required for strong activation of I-BRE, and we show that they assemble with Smad1 on the I-BRE in living cells. Activation of the I-BRE is mediated by a specific region in GATA-5 and -6 but does not require direct physical interaction with Smad1. Comparison of I-BRE to BRE-1 showed that I-BRE is more responsive to low BMP concentrations. Moreover, analysis by chromatin immunoprecipitation experiments demonstrates that the endogenous I-BRE is occupied more robustly by endogenous Smad1 than is BRE-1. This correlates with regulation of the Smad7 gene, which is induced at lower BMP concentrations in GATA-expressing cell lines compared to non-GATA-expressing lines. These data thus define how cooperative and noncooperative Smad-dependent transcriptional regulation can function to interpret different BMP concentrations.
We found a novel polymorphism, -66T/C, in the promoter region of human FcepsilonRIalpha, the specific component of the high affinity receptor for IgE (FcepsilonRI), which is essential for the cell surface expression of FcepsilonRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at -66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (-80/-59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous -66T/C genotype, while most of allergic individuals have homozygous -66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcepsilonRIalpha polymorphism related to the allergic diseases.
Ets-family genes have been implicated in leukemia, as well as in normal hematopoiesis. ERF is an ets-related gene that represses transcription and is regulated by MAPK phosphorylation through its effect on ERF sub-cellular localization. Using pluripotent human cell lines, we studied the effect of ERF on erythroid differentiation. K562 and HEL cells expressing ERF expressed elevated levels of the erythroid-specific markers CD71 and Glycophorin A, as well as hemoglobin and GATA1. Treatment with the Erk kinase inhibitor PD98058 further enhanced the erythroid phenotype in ERF-expressing cells and cells expressing a non-phosphorylatable ERF mutant exhibited an even more enhanced phenotype. These results are consistent with the fact that ERF function is regulated by MAPK, and suggest that the effect of the MAPK pathway in erythroid differentiation is partially mediated by ERF. The effect of ERF is similar to the one shown for ETS1 and opposite to the FLI1 function in these cells, suggesting that several ets genes may play key roles in hematopoietic differentiation.
Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues.
SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis.
Interplay among GATA transcription factors is an important determinant of cell fate during hematopoiesis. Although GATA-2 regulates hematopoietic stem cell function, mechanisms controlling GATA-2 expression are undefined. Of particular interest is the repression of GATA-2, because sustained GATA-2 expression in hematopoietic stem and progenitor cells alters hematopoiesis. GATA-2 transcription is derepressed in erythroid precursors lacking GATA-1, but the underlying mechanisms are unknown. Using chromatin immunoprecipitation analysis, we show that GATA-1 binds a highly restricted upstream region of the approximately 70-kb GATA-2 domain, despite >80 GATA sites throughout the domain. GATA-2 also binds this region in the absence of GATA-1. Genetic complementation studies in GATA-1-null cells showed that GATA-1 rapidly displaces GATA-2, which is coupled to transcriptional repression. GATA-1 also displaces CREB-binding protein (CBP), despite the fact that GATA-1 binds CBP in other contexts. Repression correlates with reduced histone acetylation domain-wide, but not altered methylation of histone H3 at lysine 4. The GATA factor-binding region exhibited cell-type-specific enhancer activity in transient transfection assays. We propose that GATA-1 instigates GATA-2 repression by means of disruption of positive autoregulation, followed by establishment of a domain-wide repressive chromatin structure. Such a mechanism is predicted to be critical for the control of hematopoiesis.
The erythroid transcription factor GATA-1 is also expressed in Sertoli cells of the testis. The testicular expression of GATA-1 is regulated in a developmental and spermatogenic stage-specific manner. To further clarify the regulatory mechanisms of testicular GATA-1 gene expression, we carried out transgenic reporter gene expression analyses.
We found that GATA-1 expression in Sertoli cells is markedly decreased concomitant with the emergence of elongated spermatids in the seminiferous tubules. Transgenic reporter mouse analyses revealed that a 15 kb GATA-1 genomic region is sufficient to recapitulate the gene expression profile in Sertoli cells. While the GATA-1 haematopoietic enhancer and the proximal first exon are included within the 15 kb genomic region, these regulatory elements are not essential for GATA-1 expression in Sertoli cells. Further analyses using deletion constructs revealed that a 1.5 kb region 5' to the GATA-1 haematopoietic enhancer is essential for gene expression in Sertoli cells and this region is referred to as the GATA-1 testis activation region.
These results thus demonstrated that the GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene. The 15 kb genomic region is applicable and useful for the expression vector system specific for adult Sertoli cells in stage VII to IX.
Oncogenes involved in the development of hematological malignancies were first discovered through the study of experimental leukemias induced in animals by retroviruses. The discovery that some of these genes were located at the breakpoints of chromosome rearrangements in human malignancies, such as the MYC gene in Burkitt's lymphoma and the ABL gene in chronic myeloid leukemia (CML) has suggested that chromosome abnormalities were causally implicated in the pathogenesis of human diseases. Numerous nonrandom somatically acquired chromosomal translocations or inversions have been identified in human leukemias. The molecular cloning of the genes located at the breakpoints of these rearrangements allowed to identify more than 100 new oncogenes, the products of which affect normal programs of cell proliferation, differentiation and survival. Chromosome translocations can lead to the deregulated expression of a normal gene product, but in most cases of leukemia, chromosome rearrangements result in the expression of a chimeric fusion protein. Oncogene products associated with acute leukemias are often transcription factors while tyrosine kinases and antiapoptotic proteins are more commonly activated or overexpressed in chronic leukemias and in lymphomas. Recent data indicated that gene rearrangements were not the sole gene alterations occurring in human leukemia since point mutations could also affect the function of transcription factors playing a key role in hematopoiesis such as C/EBP alpha, GATA1 and AML1. But the most exciting finding was the discovery of activating point mutations in tyrosine kinase receptors such as FLT3 and c-KIT in acute leukemia. Treatment of leukemia could therefore benefit from new therapeutic approaches targeting the function of specific oncogene products as already demonstrated for CML and acute promyelocytic leukemia.
Transient myeloproliferative disorder (TMD) is a leukemoid reaction occurring occasionally in Down syndrome newborn infants. Acute megakaryocytic leukemia (AMKL) develops in approximately 20% to 30% of the cases with TMD. Recently, acquired mutations in the N-terminal activation domain of the GATA-1 gene, encoding the erythroid/megakaryocytic transcription factor GATA-1, have been reported in Down syndrome-related AMKL (DS-AMKL). To understand the multistep leukemogenesis in Down syndrome, GATA-1 mutations were investigated in patients with TMD. We show here that mutations in the GATA-1 gene were detected in 21 of 22 cases with TMD. Most of the mutations in TMD were located in the regions including exon 2 and were essentially identical to those observed in DS-AMKL. In the DS-AMKL cell line, MGS, which itself expresses only a truncated mutant of GATA-1, expression of full-length GATA-1 induced the differentiation toward the erythroid lineage. However, expression of the short form of GATA-1 did not induce erythroid differentiation. These results indicate that expression of GATA-1 with a defective N-terminal activation domain contributes to the expansion of TMD blast cells and that other genetic changes contribute to the development of AMKL in Down syndrome.
The developmentally regulated mammalian beta-globin genes are activated by a distant locus control region/enhancer. To understand the role of chromatin remodeling complexes in this activation, we used stably replicated chromatin templates, in which transcription activation of the human embryonic epsilon-globin gene depends on the tandem Maf-recognition elements (MAREs) within the beta-globin locus control region HS2 enhancer, to which the erythroid factor NF-E2 binds. The HS2 MAREs are required for nucleosome mobilization and histone hyperacetylation at the distant promoter. Nucleosome mobilization also requires the promoter TATA box, and is independent of histone hyperacetylation. In contrast, promoter hyperacetylation requires the promoter GATA-1, and CACC-factor activator motifs, as well as the TATA box. ChIP analysis reveals that NF-E2 is associated with the active epsilon-globin promoter, which lacks an NF-E2 binding sequence, in a TATA box and HS2/MARE-dependent fashion. NF-E2 association with the epsilon-globin promoter coincides with that of RNA polymerase II at both regulatory sites. The results emphasize MARE-TATA box interactions in the recruitment of complexes modifying promoter chromatin for transcription activation and imply close physical interaction between widely separated regulatory sequences mediated through these sites.
As most transcription factors, GATA-1 activities are mediated by interactions with multiple proteins. Those identified so far associate with the zinc-finger domain and/or surrounding sequences. In contrast, no proteins interacting with the N-terminal domain have been identified although several evidences suggest its involvement in the control of hematopoiesis. In an attempt to identify proteins that interact with the N-terminal transactivation domain of GATA-1, a random phage peptide library was screened with recombinant GATA-1 protein and the sequence of a selected peptide was used for database protein sequence retrieval. We selected a set of peptides sharing the core sequence phi-B((2-3))-nu((2-4)) (where phi, B, and nu represent hydrophobic, basic, and neutral residues, respectively). Using the sequence of the most represented peptide (pep5) as query, we retrieved the HIV accessory protein Nef. We show that Nef binds GATA-1 and GATA-3 in vitro in virtue of its sequence homology with pep5.
Asthma is characterized by an increased production of eosinophil-active C-C chemokines by the airway epithelium. Recent studies have identified the presence of important quantities of labile zinc in the conducting airways. We hypothesized that modulation of this labile zinc could influence the production of proinflammatory chemokines in respiratory epithelial cells. The zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the heavy metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS) were used to reduce the labile zinc content of A549, BEAS-2B, and HFL-1 cells. Northern blot analysis and RNase protection assay were used to study the effects of the zinc chelators on mRNA expression. DMPS and TPEN specifically inhibited the production of eotaxin, regulated on activation, normal T-cell expressed, and presumably secreted, and monocyte chemotactic protein-1 in TNF-alpha-stimulated respiratory epithelial cells and fibroblasts through labile zinc chelation. The inhibitory effects of DMPS and TPEN were associated with the decreased binding of the zinc-finger transcription factor GATA-1, whereas no change in NF-kappaB activation was observed. Together these results demonstrate that modulation of the labile pool of zinc can regulate gene expression and protein synthesis of C-C chemokines in lung epithelial cells and fibroblasts.
Transient myeloid disorder is a unique self-regressing neoplasia specific to Down's syndrome. The transcription factor GATA1 is needed for normal growth and maturation of erythroid cells and megakaryocytes. Mutations in GATA1 have been reported in acute megakaryoblastic leukaemia in Down's syndrome. We aimed to investigate changes in GATA1 in patients with Down's syndrome and either transient myeloid disorder (n=10) or acute megakaryoblastic leukaemia (n=6). We recorded mutations eliminating exon 2 from GATA1 in all patients with transient myeloid disorder (age 0-24 days) and in all with acute megakaryoblastic leukaemia (age 14-38 months). The range of mutations did not differ between patients with each disorder. Patients with transient myeloid disorder with mutations in GATA1 can regress spontaneously to complete remission, and mutations do not necessarily predict later acute megakaryoblastic leukaemia.
Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbalpha genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.
YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6% identical to that of mouse YY1 and 97.8% identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114-193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56-113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1-78/100-106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.
32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response to erythropoietin (EPO). In order to clarify the mechanisms underlying such restriction, we compared 32D and 32D Ro cells for their capacity to express erythroid-specific transcription factors (Gata1, Gata2, Scl, Nef2, Eklf, and Id) and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate erythroid differentiation potential in 32D Ro cells. By Northern analysis, the two cell lines expressed similar levels of all these genes. However, after being treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent clones (1 clone/10(4) cells) which gave rise to EPO-dependent cell lines. All the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D Ro cells had erythroid morphology and expressed high levels of alpha- and beta-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage colony-stimulating factor-dependent clones concurrently isolated, as a control, showed erythroid properties. Therefore, 5-AzaC treatment reactivates the potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive erythroid clones suggesting that gene methylation played an important role in the G-CSF-mediated restriction/activation of the differentiation potential of these cells.
GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.
Platelet-derived growth factor (PDGF) is a platelet alpha-granule protein. In previous reports, we demonstrated the expression of PDGF receptors on platelets and megakaryocytic cells and that PDGF enhanced the proliferation of megakaryocytic progenitor cells. In this study, we investigated the effects of PDGF on mRNA and protein expressions of megakaryocyte-associated transcription factors, c-Fos, GATA-1, NF-E2 and PU.1, in two human megakaryocytic cell lines CHRF-288-11 and DAMI. RT-PCR/Southern blot analysis and Real-time PCR demonstrated that PDGF increased the mRNA expression of c-Fos, GATA-1 and NF-E2, but not PU.1 in a dose- and time-dependent manner. The activation was confirmed at the protein level by Western blot analysis of both total cell and nuclear lysates. The addition of increasing concentrations of Tyrphostin AG1295, an inhibitor of PDGF receptor kinase, blocked the stimulatory effect of PDGF on the mRNA and protein expressions of these transcription factors. The up-regulation of c-Fos, GATA-1 and NF-E2 protein by PDGF was inhibited by actinomycin D and cycloheximide, suggesting that mRNA and protein synthesis might be involved in the mechanism. Our data suggest a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions and we speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis.
Patients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.
GATA-1 is a transcription factor essential for erythroid cell development, and knockdown of GATA-1 gene results in maturation arrest and transformation of erythroblasts. To clarify the mechanism that gives rise to this abnormal phenotype, genes that are aberrantly expressed in the spleen of heterozygous GATA-1 knockdown mutant mouse (referred to as GATA-1 mutant mouse) were identified by using cDNA array. One of these genes, YB-1, was found to be highly expressed in the spleen of GATA-1 mutant mouse. Reporter and electrophoretic mobility shift assays revealed that the proximal GATA element in 5'-UTR region of YB-1 gene functions positively in K562 cells. Furthermore, both GATA-1 and GATA-2, which were transiently expressed in COS-7 cells, bound to this element and activated the YB-1 promoter through this element. These results suggest that YB-1 functions under the regulation of GATA factors in erythroid differentiation and aberrant expression of YB-1 gene may result in dyserythropoiesis.
We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from the cytoplasm to the nucleus in a c-Myc-dependent manner. AMY-1 works as an inducer of human K562 cell differentiation upon induction of AraC. To characterize the expression or functional importance of AMY-1, the genomic DNA of mouse AMY-1 was cloned and characterized. Both mouse and human genomic DNAs, the latter of which was retrieved from a human DNA database, comprise five exons spanning about 11 kb. To characterize the promoter of the mouse AMY-1 gene, a series of deletion constructs of the region upstream of the first ATG was linked to the luciferase gene, and their luciferase activities were measured in human HeLa and K562 cells. The results showed that Sp1 was essential for AMY-1 expression in both cell lines and that GATA-1 is also necessary in K562 cells. Sp1 in both cell lines and GATA-1 only in K562 cells were identified as proteins binding to these sites by a mobility shift assay. Furthermore, it was found that GATA-1 stimulated AMY-1 expression synergistically with Sp1 in ectopically expressed insect cells and that both proteins were associated in K562 cells.
To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression.
By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2.
It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression.
The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.
We have cloned the genomic DNA encoding the human NeuAc alpha2,3Gal beta1,3GalNAc alpha2,6-sialyltransferase (hST6GalNAc IV) and analysed its structure. The hST6GalNAc IV gene was found to span about 9 kb and to be composed of six exons. The 5'-RACE (rapid amplification of cDNA ends) results indicated that mRNA isoform of the hST6GalNAc IV was generated by alternative splicing in the 5'-untranslated region. The expression of this gene was highly restricted in human fetal tissues. The potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5'-flanking region of this gene lacked canonical TATA and CAAT boxes, but contained several putative binding sites for transcription factors SP1, MZF1, GATA1, LMO2COM, NFAT, HFH8 and USF, etc. Functional analysis of the 5'-flanking region by transient expression method revealed a high transcriptional activity in both HepG2 cells and Molt4 cells in a cell type-dependent manner, but not in SK-N-MC cells. These results suggest cell type-specific regulation of the basal hST6GalNAc IV promoter activity.
The platelet marginal band consists of a single peripheral microtubule (MT) that is wound in 8 to 12 coils and maintains discoid cell shape. About 90% of beta-tubulin in the marginal band is of the divergent, megakaryocyte (MK)/platelet-restricted beta1 isoform. beta1-tubulin-null mice show reduced proplatelet formation, thrombocytopenia, and platelet spherocytosis. Here, we show that structural abnormalities in resting beta1-tubulin-/- platelets include frequent kinks and breaks in the marginal band. Platelets derived from mice lacking the transcription factor GATA1 show similar defects, probably as a direct consequence of absent beta1-tubulin. beta1-tubulin+/- platelets have normal ratios of beta-tubulin isotypes but the marginal band is half the normal thickness, which is sufficient to maintain elliptical cell shape. Thus, a threshold 50% or less of the normal amount of beta1-tubulin is required to preserve marginal band integrity and cell shape. beta1-tubulin-/- platelets have normal size and contents and show no defects in serotonin release or aggregation. Accordingly, the apparently isolated spherocytosis allows investigation of the role of discoid platelet shape in hemostasis. On agonist stimulation, the disorganized MTs in beta1-tubulin-/- platelets fail to condense into central rings and instead are dispersed in short bundles and linear arrays. Nevertheless, intravital microscopy and flow chamber studies demonstrate full functionality of these spherocytic platelets under physiologic shear conditions. Together, these findings highlight the essential requirements of the MK/platelet-restricted beta1-tubulin isoform in platelet structure and suggest that spherocytosis does not impair many aspects of platelet function.
Children with constitutional trisomy 21 (Down syndrome) have an approximately 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia. Unique to newborn infants with Down syndrome is a transient leukemia (TL), also referred to as transient myeloproliferative syndrome, that undergoes spontaneous remission in the majority of cases but in approximately 20% is followed by AMKL later in life. Recently mutations of the gene encoding the hematopoietic transcription factor GATA1 were shown to be specific for AMKL of Down syndrome. Here, we demonstrate that GATA1 mutations are present in blasts of TL and show the identical GATA1 mutation in sequential samples collected from a patient during TL and subsequent AMKL. These findings suggest a model of malignant transformation in Down syndrome AMKL in which GATA1 mutations are an early event and AMKL arises from latent TL clones following initial apparent remission.
To investigate the action of Ginsenosides (GS) in inducing transcription factor c-fos and GATA-1 to explore the mechanism of GS in hematopoietic cells.
The proliferation effects of GS on granulocytic (HL-60), monocytic (U937), erythrocytic (K562) and megaryocytic (Meg-01) cell lines were observed by using proliferation test of MTT and colony formation of progenitor cells. The combining reaction of transcription factors c-fos and GATA-1 with nuclear protein antigen were analyzed by Western Blot after being treated by GS.
(1) GS (10 micrograms/ml) could stimulate and promote proliferation of 3 cell lines with significant difference between GS and non-GS control (P < 0.05 in all) in both MTT test and colony assay. (2) After treatment with GS, c-fos protein in HL-60, K562 and Meg-01 cell lines was increased by 1.5, 2.0 and 2.5 fold respectively, while U937 cell did not express c-fos. (3) Except that U937 cell hadn't expressed GATA-1, the other cell lines after the treatment by GS, the GATA-1 protein level was elevated to 1.5, 2.1 and 1.3 fold of that before treatment.
The proliferation of three lines initiated by GS was involved in transcription factor c-fos or GATA-1, which could pay the role in the GS induced up-regulation correlated with proliferation and differentiation of hematopoiesis.
Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor beta (CBFbeta), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFbeta up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFbeta, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins alphaIIb and alpha2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).
Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neo Delta HS or GATA-1(low) mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue(+) mast cells and apoptotic metachromatic(-) mast cell precursors in connective tissues and peritoneal lavage and numerous (60-70% of all the progenitors) "unique" trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.
As many as 10% of infants with Down syndrome (DS) present with transient myeloproliferative disorder (TMD) at or shortly after birth. TMD is characterized by an abundance of blasts within the peripheral blood and liver, and notably undergoes spontaneous remission in the majority of cases. TMD may be a precursor to acute megakaryoblastic leukemia (AMKL), with an estimated 30% of TMD patients developing AMKL within 3 years. We recently reported that mutations in the transcription factor GATA1 are associated with DS-AMKL. To determine whether the acquisition of GATA1 mutations is a late event restricted to acute leukemia, we analyzed GATA1 in DNA from TMD patients. Here we report that GATA1 is mutated in the TMD blasts from every infant examined. These results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD, and mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis.
FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the beta-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on beta-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.
One function of lineage-restricted transcription factors may be to control the formation of tissue-specific chromatin domains. In erythroid cells, the beta-globin gene cluster undergoes developmentally regulated hyperacetylation of histones at the active globin genes and the locus control region (LCR). However, it is unknown which transcription factor(s) governs the establishment of this erythroid-specific chromatin domain. We measured histone acetylation at the beta-globin locus in the erythroid cell line G1E, which is deficient for the essential hematopoietic transcription factor GATA-1. Restoration of GATA-1 activity in G1E cells led to a substantial increase in acetylation of histones H3 and H4 at the beta-globin promoter and the LCR. Time course experiments showed that histone acetylation occurred rapidly after GATA-1 activation and coincided with globin gene expression, indicating that the effects of GATA-1 are direct. Moreover, increases in histone acetylation correlated with occupancy of GATA-1 and the acetyltransferase CBP at the locus in vivo. Together, these results suggest that GATA-1 and its cofactor CBP are essential for the formation of an erythroid-specific acetylation pattern that is permissive for high levels of gene expression.
HEL cells, a human erythroleukemia cell line, expressing mainly the gamma-globin genes, small amount of epsilon-globin gene, but not beta-globin gene. Our previous studies demonstrated that beta-globin gene could be expressed in HEL cells induced by hydroxyurea. However, the molecular mechenism is still unknown. Here the binding patterns of GATA factors (GATA-1 and GATA-2) to the regulatory elements of human beta-globin gene were examined with the nuclear extracts from hydroxyurea-induced and uninduced HEL cells. Our results showed in EMSA assay that GATA factors could bind to the core sequence of HS2(-10681 to -10971 bp), the 3' flanking sequence of HS2 core(-10323 to -10680 bp) and the promoter of human beta-globin gene(+20 to -112 bp). However, the binding patterns between hydroxyurea-induced and uninduced HEL cells were different. Furthermore, by using Western-blot analysis, our data showed that the amount of GATA-2 was decreased in hydroxyurea-induced HEL cells. In contrast to GATA-2, the amount of GATA-1 was increased in hydroxyurea-induced HEL cells. These results showed that the different members of GATA family might play different roles during the differentiation of erythrocytes. GATA-1 may stimulate the differentiation of HEL cells, while GATA-2 can probably inhibit the differentiation of HEL cells.
In the bone marrow of C/EBP epsilon(-/-) mice, expression of neutrophil secondary and tertiary granule mRNAs is absent for lactoferrin (LF), neutrophil gelatinase (NG), murine cathelin-like protein (MCLP), and the cathelin B9; it is severely reduced for neutrophil collagenase (NC) and neutrophil gelatinase-associated lipocalin (NGAL). In addition, the expression of eosinophil granule genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBP alpha, C/EBP beta, and C/EBP delta in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo. Stable inducible expression of C/EBP epsilon and C/EBP alpha in the murine fibroblast cell line NIH 3T3 activated expression of mRNAs for B9, MCLP, NC, and NGAL but not for LF. In transient transfections of C/EBP epsilon and C/EBP alpha, B9 was strongly induced with weaker induction of the other genes. C/EBP beta and C/EBP delta proteins weakly induced B9 expression, but C/EBP delta induced NC expression more efficiently than the other C/EBPs. The expression of MBP was inefficiently induced by C/EBP epsilon alone and weakly induced with C/EBP epsilon and GATA-1, but the addition of PU.1 resulted in a striking cooperative induction of MBP in NIH 3T3 cells. Mutation of a predicted PU.1 site in the human MBP promoter-luciferase reporter construct abrogated the response to PU.1. Gel-shift analysis demonstrated binding of PU.1 to this site. MBP and EPX mRNAs were absent in a PU.1-null myeloid cell line established from the embryonic liver of PU.1(-/-) mice. Restitution of PU.1 protein expression restored MBP and EPX protein expression. This study demonstrates that C/EBP epsilon is essential and sufficient for the expression of a particular subset of neutrophil secondary granule genes. Furthermore, it indicates the importance of PU.1 in the cooperative activation of eosinophil granule genes.
WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.
The FcR beta-chain, a subunit of two related multisubunit receptor complexes, the FcepsilonRI and FcgammaRIII, amplifies the mast cell response and is necessary for the cell surface expression of FcepsilonRI in mouse. The transient reporter assay indicated that -69/+4 region is required for cell type-specific transcriptional regulation of mouse beta-chain gene. EMSA using Abs against transcription factors or competitive oligonucleotides demonstrated that -58/-40 region (containing overlapping three GATA-1 sites, -53/-48, -46/-51, and -42/-47) and -31/-26 region (containing one GATA-1 site) are recognized by GATA-1. The promoter activity of beta-chain was decreased by nucleotide replacements of the GATA-1 sites in mouse mast cell line PT18. Furthermore, exogenously produced GATA-1 up-regulated the promoter activity in CV-1 cells, which are negative in the beta-chain production and the up-regulation was apparently suppressed by GATA-1 site mutations. These results indicate that cell type-specific transcription of mouse beta-chain gene is regulated by GATA-1.
Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects.
Anthracycline antitumor drugs induce erythroid differentiation of the K562 erythroleukemic cell line at subtoxic concentrations. Aclacinomycin (ACM) stimulates this process by activating the erythroid transcription factor GATA-1, that controls genes involved in hemoglobin biosynthesis. To investigate the implication of GATA-1 in this process, we used a specific anti-GATA-1 polyclonal antibody that we produced in our laboratory. The GATA-1 transcription factor was then monitored during erythroid differentiation induced by aclacinomycin. Here we show that a cellular redistribution and a modification of the phosphorylation state of this transcription factor occurred during ACM-mediated cell differentiation. It suggests that anthracyclines can induce the erythroid differentiation of neoplastic cells by activating the transcription factor GATA-1, probably via its clustering into nuclear foci.
The GATA-1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo, indicating that additional regulatory sequences are necessary.
We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis-acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality.
A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo.
Erythroid and megakaryocytic lineage differentiation and maturation are regulated via cooperation between transcription factor GATA1 and its essential cofactor friend-of-GATA1 (FOG1). The interaction between these two murine proteins is well studied in vitro and depends on the binding of Fog1 to the N-terminal zinc finger (N-finger) of Gata1. We identified the human FOG1 gene on chromosome 16q24 and found expression mainly in hematopoietic cells and also in several other tissues. Sequencing of FOG1 cDNA revealed a 1006 amino-acid protein that contained nine zinc fingers, highly homologous to murine Fog1 fingers. The amino acid sequence and the GATA1-binding capacity of the human and murine finger 5 are however different. Ex vivo binding studies demonstrated that FOG1 interacts with both GATA1 and GATA2. We and others have described patients with mutations in the GATA1 N-finger (V205 M, D218G, D218Y, or G208S), who suffer from macrothrombocytopenia and erythrocyte abnormalities. We now show ex vivo that the interaction between GATA1 and FOG1 is indeed disturbed in platelets and erythrocytes of those patients carrying D218 GATA1 mutations. The identification of the human FOG1 gene will enable the genetic screening of patients with non X-linked thrombocytopenia and dyserythropoiesis.
Hydroxyurea (HU) has been shown to increase the proportion of fetal haemoglobin (HbF) in most sickle cell patients. A low-dosage regimen increased total haemoglobin (Hb) levels in some thalassaemia intermedia patients by preferentially increasing beta-globin biosynthesis. To further characterize these apparent dose-dependent effects of HU, we examined erythroid cells exposed to HU (5-100 micro mol/l) in two-phase liquid culture. Low doses (from 5 to 25 micro mol/l) increased Hb levels by up to 2.7-fold, and a high dose (100 micro mol/l) increased Hb levels when added at d 3-6 of phase II, with no significant changes in response to HU during the late stage of phase II culture (> or = 9 d). HU exposure during d 0-3 of phase II culture increased the number of erythroid colonies to a maximum of fivefold at 5 micro mol/l HU. GATA-1 mRNA was downregulated at a high dose and GATA-2 was dose dependently upregulated over a lower dosage range. Treatment with 100 micro mol/l HU dramatically upregulated the death receptor DR-5, caspase 3, as determined by cDNA microarray analysis. In contrast, 10 micro mol/l HU modestly upregulated mRNA levels of the early growth response gene. Our results suggest that HU exerts concentration-dependent effects on HbF production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.
Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.
The transcription factors GATA-1 and GATA-2 play key roles in gene regulation during erythropoiesis. Gene ablation studies in mouse revealed that GATA-2 is crucial for the maintenance and proliferation of immature hematopoietic progenitors, whereas GATA-1 is essential for the survival of erythroid progenitors as well as the terminal differentiation of erythroid cells. Both GATA-1 and GATA-2 are regulated in a cell-type-specific manner, their expression being strictly controlled during the development and differentiation of erythroid cells. Closer examination revealed a cross-regulatory mechanism by which GATA-1 can control the expression of GATA-2 and vice versa, possibly via essential GATA binding sites in their cis-acting elements. In addition, recent studies identified several human inherited hematopoietic disorders that are caused by mutations in cis-acting GATA binding motifs or mutations in GATA-1 itself.
A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx gene, but their N-terminal peptides are encoded by alternative first exons. The expression of the two enzymes is differentially regulated in various cells, but little is known about the regulatory mechanisms. To explore the tissue-specific regulation of expression of the two isoenzymes, we first investigated their tissue distribution. Whereas phGPx is expressed at low levels in many organs, snGPx was only detected in testis, kidney, and in the human embryonic kidney cell line HEK293. Subcellular fractionation studies and immunoelectron microscopy revealed a cytosolic localization. To explore the mechanistic reasons for the differential expression pattern, we first tested the activity of the putative phGPx and snGPx promoters. The 5'-flanking region of the joint ph/snGPx gene exhibits strong promoter activity. In contrast, the putative snGPx promoter, which comprises 334 bp of intronic sequences, lacks major promoter activity. However, it strongly suppresses the activity of the ph/snGPx promoter. These data suggest negative regulatory elements in the first intron of the ph/snGPx gene, and DNase protection assays revealed the existence of several protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1, GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1 and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for the first time somatic expression of the snGPx and provide evidence for the existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our failure to detect an alternative snGPx promoter suggests that transcription of the ph/snGPx gene may be regulated by a joint basic promoter. The decision, which GPx isoform is expressed in a given cell, appears to be made by alternative splicing of a joint primary transcript.
One of the most serious consequences of cytotoxic cancer therapy is the development of therapy-related acute myeloid leukemia (t-AML), a neoplastic disorder arising from a multipotential hematopoietic stem cell. To gain insights into the molecular basis of this disease, we performed gene expression profiling of CD34(+) hematopoietic progenitor cells from t-AML patients. Our analysis revealed that there are distinct subtypes of t-AML that have a characteristic gene expression pattern. Common to each of the subgroups are gene expression patterns typical of arrested differentiation in early progenitor cells. Leukemias with a -5/del(5q) have a higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), and loss of expression of the gene encoding IFN consensus sequence-binding protein (ICSBP). A second subgroup of t-AML is characterized by down-regulation of transcription factors involved in early hematopoiesis (TAL1, GATA1, and EKLF) and overexpression of proteins involved in signaling pathways in myeloid cells (FLT3) and cell survival (BCL2). Establishing the molecular pathways involved in t-AML may facilitate the identification of selectively expressed genes that can be exploited for the development of urgently needed targeted therapies.
The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.
Hematopoiesis initiates in the extraembryonic yolk sac. To isolate various types of precursor cells from this blood cell-forming tissue, yolk sac cells were immortalized by retroviral-mediated expression of the HOX11 homeobox-containing gene. Among the cell lines derived, some were able to spontaneously generate adherent stromal-like cells capable of taking up acetylated low-density lipoprotein, and they could be induced to form tubelike structures when cultured on Matrigel. Although these cell lines were negative for hematopoietic cell surface markers, they gave rise to hematopoietic colonies--containing cells belonging to the monocytic, megakaryocytic, and definitive erythroid lineages--when plated in methylcellulose medium supplemented with hematopoietic growth factors. Low amounts of Flk-1 mRNA could be detected in these cells, and they showed significant responsiveness to vascular endothelial growth factor, stem cell factor, basic fibroblast growth factor, and interleukin 6. They also expressed the transcription factors SCL, GATA2, GATA1, PU.1, and c-myb. These yolk sac-derived cell lines may represent a transitional stage of early hematopoietic development.
Human beta-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in beta-globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 beta/gamma-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P <.05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P <.05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 beta/gamma-globin transgene expression and should be retained in LCRbeta-globin expression cassettes designed for gene therapy.
Vertebrate hematopoiesis is regulated by distinct cell-specific transcription factors such as GATA-1 and SCL. Mammalian p45-NFE2 was characterized for its ability to bind the hypersensitive sites of the globin locus control region. NFE2 is a member of a cap'n'collar (CNC) and basic zipper (BZIP) superfamily that regulates gene transcription. It has been implicated in diverse processes such as globin gene expression, oxidative stress, and platelet lineage differentiation. Here, we have isolated the zebrafish ortholog of NFE2. The gene is highly homologous, particularly in the DNA-binding domain. Mapping the zebrafish NFE2 to linkage group 23 establishes a region of chromosomal synteny with human chromosome 12, further suggesting evolutionary conservation. During embryogenesis, the zebrafish gene is expressed specifically in erythroid cells and also in the developing ear. NFE2 expression is lacking in zebrafish mutants that have no hematopoietic cells. An analysis of the sauternes mutant, which carries a mutation in the ALAS-2 gene and thus has defective heme synthesis, demonstrates higher levels of NFE2 expression than normal. This further establishes the block to erythroid differentiation in the sauternes mutant. Our studies demonstrate conservation of the vertebrate genetic program for the erythroid lineage.
Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown. We tested whether the hematopoietic-specific activators NF-E2 and GATA-1, which mediate transactivation of the beta-globin genes, induce both histone acetylation and H3-meK4. Through the use of NF-E2- and GATA-1-null cell lines, we show that both activators induce H3 acetylation at the promoter upon transcriptional activation. However, analysis of H3-mek4 revealed that NF-E2 and GATA-1 differentially regulate chromatin modifications at the betamajor promoter. NF-E2, but not GATA-1, induces H3-meK4 at the promoter. Thus, under conditions in which NF-E2 and GATA-1 activate the transcription of an endogenous gene at least 570-fold, these activators differ in their capacity to induce H3-meK4. Despite strong H3-meK4 at hypersensitive site 2 of the upstream locus control region, neither factor was required to establish H3-meK4 at this site. These results support a model in which multiple tissue-specific activators collectively function to assemble a composite histone modification pattern, consisting of overlapping histone acetylation and methylation. As GATA-1 induced H3 acetylation, but not H3-meK4, at the promoter, H3 acetylation and H3-meK4 components of a composite histone modification pattern can be established independently.
Platelet glycoprotein VI is a collagen receptor belonging to the immunoglobulin-like protein family that is essential for platelet interactions with collagen and is exclusively expressed in the megakaryocytic lineage. The objective of this study was to characterize the human glycoprotein VI gene (GP6) 5' regulatory and promoter regions.
We first used 5' RACE to establish experimentally that the major transcription start site lies 28 bp upstream from the start codon. We next subcloned the 5' regulatory region of GP6 into pGL3-basic [pGL3(-1576)] and used deletion mutagenesis to identify important regulatory regions, comparing the activity of transiently expressed promoter-luciferase constructs in Dami and HeLa cells. We found that megakaryocyte lineage-specific transcription is largely controlled within the segment -191/-39. By site-directed mutagenesis, we confirmed that a GATA-1 site at -176 and an Ets-1 site at -45 play important roles in the regulation of GP6 transcriptional activity.
We have determined that the GP6 sequence -191 to -39 represents the core promoter and that transcription is driven largely by GATA-1 (-176) and c-Ets-1 (-45) sites within this segment.
The exposure of collagen fibers at sites of vascular injury results in the adherence of platelets and their subsequent activation. The platelet collagen receptor glycoprotein (GP)(1) VI plays a crucial role in platelet activation and thrombus formation and decreased levels or defective GPVI may lead to excessive bleeding. In addition, elevated levels of collagen receptors may predispose individuals to coronary heart disease or strokes. GPVI expression is restricted to platelets and their precursor cell, the megakaryocyte. In this study we investigate the regulation of GPVI expression and show that thrombopoietin induces its expression in the megakaryocytic cell line UT-7/TPO. A 5'-region flanking the transcription start point of the GPVI gene was cloned (-694 to +29) and we report that this putative GPVI promoter bestows megakaryocye-specific expression. Deletion analyses and site-directed mutagenesis identified Sp1(227), GATA(177), and Ets(48) sites as essential for GPVI expression. We show that transcription factors GATA-1, Fli-1, and Sp1 can bind to and activate this promoter. Finally, GPVI mRNA was detected only in megakaryocytic cell lines expressing both Fli-1 and GATA-1, and we show that overexpression of Fli-1 in a stable cell line (which expresses endogenous GATA-1 and Sp1) results in expression of the endogenous GPVI gene.
The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. Here, we examine the molecular basis for the megakaryocyte-restricted activation of the alphaIIb gene. FOG-1 stimulates GATA-1-dependent alphaIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the alphaIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. Our studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes.
We investigated the expression of the PLZF gene in purified human hematopoietic progenitors induced to unilineage erythroid, granulocytic or megakaryocytic differentiation and maturation in serum-free culture. PLZF is expressed in quiescent progenitors: the expression level progressively rises through megakaryocytic development, whereas it gradually declines in erythroid and granulopoietic culture. To investigate the role of PLZF in megakaryopoiesis, we transduced the PLZF gene into the erythro-megakaryocytic TF1 cell line. PLZF overexpression upmodulates the megakaryocytic specific markers (CD42a, CD42b, CD61, PF4) and induces the thrombopoietin receptor (TpoR). The proximal promoter of the TpoR gene is activated in PLZF-expressing TF1 cells: in this promoter region, a PLZF DNA-binding site was identified by deletion constructs studies. Interestingly, PLZF and GATA1 proteins coimmunoprecipitate in PLZF-expressing TF1 cells: enforced expression of both PLZF and GATA1 in TF1 cells results in increased upregulation of megakaryocytic markers, as compared to exogenous PLZF or GATA1 alone, suggesting a functional role for the PLZF/GATA1 complex. Our data indicate that PLZF plays a significant stimulatory role in megakaryocytic development, seemingly mediated in part by induction of TpoR expression at transcriptional level. This stimulatory effect is potentiated by physical interaction of PLZF and GATA1, which are possibly assembled in a multiprotein transcriptional complex.
The transcription factors GATA-1 and GATA-4 have been implicated in the regulation of testicular development and function. Their cofactors FOG-1 and FOG-2 are expressed in the gonads, but their cell-specific and developmental expression in the testis remains unresolved. Therefore, we analyzed GATA-1, GATA-4, FOG-1 and FOG-2 expression in detail, from undifferentiated male urogenital ridge to adult testis.
Immunohistochemistry and in situ hybridization were applied on mouse testicular samples.
GATA-4 and FOG-2, but not GATA-1 or FOG-1, were expressed as early as in the male urogenital ridge. FOG-2 expression was localized in the Sertoli cells at embryonal day 12.5 (E12.5), but it diminished with advancing fetal testicular development. In E17.5 testis, FOG-2 was present only in the testicular capsule and a subset of fetal Leydig cells. FOG-1 was expressed from E15.5 Sertoli cells onwards, whereas GATA-1 was not detected during the fetal period at all. In the postnatal testis, FOG-2 was abundantly expressed immediately after birth, but in adult testis its expression was predominantly restricted to stage VII-XII seminiferous tubules. Stage specificity was also found for FOG-1, which, similarly to GATA-1, was abundantly expressed in stage VII-XII tubules during adulthood.
Our results indicate that FOG-2, in addition to GATA-4, has a role in early gonadal development and sexual differentiation, and FOG-1 at later fetal stages, while GATA-1 executes its action postnatally. The findings suggest that, in contrast to the hematopoietic system and the heart, GATA-1 and GATA-4 do not use FOG-1 and FOG-2 respectively as their only cofactors during the early stages of testicular development.
GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPepsilon isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPepsilon(27) isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPepsilon(27) are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPepsilon(32/30) and epsilon(27) physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPepsilon isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPepsilon(27).
Transcription factor GATA-1 is essential for the development of erythroid cells and megakaryocytes. Each of its 2 zinc fingers is critical for normal function. The C-terminal finger is necessary for DNA binding. The N finger mediates interaction with FOG-1, a cofactor for GATA-1, and also modulates DNA-binding affinity, notably at complex or palindromic GATA sites. Residues of the N finger-mediating interaction with FOG-1 lie on the surface of the N finger facing away from DNA. Strong sequence conservation of residues facing DNA suggests that this other surface may also have an important role. We report here that a syndrome of X-linked thrombocytopenia with thalassemia in humans is caused by a missense mutation (Arg216Gln) in the GATA-1 N finger. To investigate the functional consequences of this substitution, we used site-directed mutagenesis to alter the corresponding residue in GATA-1. Compared with wild-type GATA-1, Arg216Gln GATA-1 shows comparable affinity to single GATA sites but decreased affinity to palindromic sites. Arg216Gln GATA-1 interacts with FOG-1 similarly with wild-type GATA-1. Arg216Gln GATA-1 supports erythroid maturation of GATA-1 erythroid cells, albeit at reduced efficiency compared with wild-type GATA-1. Together, these findings suggest that residues of the N finger of GATA-1-facing DNA contribute to GATA-1 function apart from interaction with the cofactor FOG-1. This is also the first example of beta-thalassemia in humans caused by a mutation in an erythroid transcription factor.
alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.
Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.
The hematopoietic transcription factor GATA-1 regulates erythropoiesis and beta-globin expression. Although consensus GATA-1 binding sites exist throughout the murine beta-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the beta-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner.
Children with Down syndrome have a 10-20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein, the genetic alterations that promote Down syndrome-related AMKL (DS-AMKL) have remained elusive. Here we show that leukemic cells from every individual with DS-AMKL that we examined contain mutations in GATA1, encoding the essential hematopoietic transcription factor GATA1 (GATA binding protein 1 or globin transcription factor 1). Each mutation results in the introduction of a premature stop codon in the gene sequence that encodes the amino-terminal activation domain. These mutations prevent synthesis of full-length GATA1, but not synthesis of a shorter variant that is initiated downstream. We show that the shorter GATA1 protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor Friend of GATA1 (FOG1; encoded by ZFPM1) to the same extent as does full-length GATA1, but has a reduced transactivation potential. Although some reports suggest that the activation domain is dispensable in cell-culture models of hematopoiesis, one study has shown that it is required for normal development in vivo. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.
The phenotype induced by the GATA-1(low) (neodeltaHS) mutation is here further characterized by analyzing the hemopoietic system during the aging (up to 20 months) of a GATA-1(low) colony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct = 45.9 +/- 4.0) until 12 months but became anemic from 15 months on (Hct = 30.9 +/- 3.9; P <.05). Anemia was associated with several markers of myelofibrosis such as the presence of tear-drop poikilocytes and progenitor cells in the blood, collagen fibers in the marrow and in the spleen, and hemopoietic foci in the liver. Semiquantitative reverse transcription-polymerase chain reaction showed that growth factor genes implicated in the development of myelofibrosis (such as osteocalcin, transforming growth factor-beta1, platelet-derived growth factor, and vascular endothelial growth factor) were all expressed in the marrow from the mutants at higher levels than in corresponding normal tissues. The GATA-1(low) mutants experienced a slow progression of the disease because the final exitus was not observed until at least 15 months with a probability of survival more favorable than that of W/Wv mice concurrently kept in the animal facility (P <.001, by Kaplan-Meier analysis). In conclusion, impaired GATA-1 expression may contribute to the development of myelofibrosis, and the GATA-1(low) mutants may represent a suitable animal model for the human disease that may shed light on its pathogenesis.
Previous studies have established that protein kinase C-zeta (PKC-zeta) is critical for neuronal cell differentiation. However, the role of PKC-zeta in haematopoietic cell differentiation is less clear. In this study, we have investigated the influence of PKC-zeta overexpression on the phenotype of the human monocytic U937 leukaemic cells. In two PKC-zeta-overexpressing clones (U937 zetaJ and U937 zetaB), PKC-zeta expression levels and activity were three to fourfold higher, and the enzyme accumulated both in the cytoplasm and in the nucleus compared with U937 control cells. PKC-zeta-overexpressing U937 cells exhibited an erythroid phenotype characterized by high levels of glycophorin A, cell haemoglobinization, increased GATA-1 transcripts and protein expression, compared with controls. Immunoprecipitation studies revealed that GATA-1 protein was constitutively phosphorylated in PKC-zeta-overexpressing cells. Moreover, GATA-1 did not interact with PKC-zeta but interacted with ERK1, which was constitutively activated and accumulated in the nucleus of U937 zetaJ. However, ERK1 phosphorylation inhibition by PD098059 did not influence either GATA-1 phosphorylation or GATA-1/ERK1 interaction. Collectively, these results suggest a model in which PKC-zeta induces MEK-dependent ERK1 activation, ERK1 translocation to the nucleus, GATA-1/ERK1 interaction and ERK1-independent GATA-1 phosphorylation resulting in GATA-1 accumulation. To conclude, this study provides evidence for the role of PKC-zeta in erythroid gene regulation.
The development of red blood cells (erythrocytes) is distinguished by high-level production of the oxygen carrier, haemoglobin A (HbA), a heterotetramer of alpha- and beta-haemoglobin subunits. HbA synthesis is coordinated to minimize the accumulation of free subunits that form cytotoxic precipitates. Molecular chaperones that regulate globin subunit stability, folding or assembly have been proposed to exist but have never been identified. Here we identify a protein stabilizing free alpha-haemoglobin by using a screen for genes induced by the essential erythroid transcription factor GATA-1 (refs 4, 5). Alpha Haemoglobin Stabilizing Protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free alpha-haemoglobin but not with beta-haemoglobin or haemoglobin A (alpha(2)beta(2)). Moreover, AHSP specifically protects free alpha-haemoglobin from precipitation in solution and in live cells. AHSP-gene-ablated mice exhibit reticulocytosis and abnormal erythrocyte morphology with intracellular inclusion bodies that stain positively for denatured haemoglobins. Hence, AHSP is required for normal erythropoiesis, probably acting to block the deleterious effects of free alpha-haemoglobin precipitation. Accordingly, AHSP gene dosage is predicted to modulate pathological states of alpha-haemoglobin excess, such as beta-thalassaemia.
Transcription factor GATA-1 reprograms immature myeloid cells to three different hematopoietic lineages-erythroid cells, megakaryocytes, and eosinophils. GATA-1 is essential for maturation of erythroid and megakaryocytic precursors, as revealed by gene targeting in mice. Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss of the eosinophil lineage. These findings suggest that GATA-1 is required for specification of this lineage during hematopoietic development. Mice lacking the ability to produce eosinophils should prove useful in ascertaining the role of eosinophils in a variety of inflammatory or allergic disorders.
GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1-deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.
Previous work has demonstrated that lineage-specific transcription factors play essential roles in red blood cell development. More recent studies have shown that these factors participate in critical protein-protein interactions in addition to binding DNA. The zinc finger transcription factor GATA-1, a central mediator of erythroid gene expression, interacts with multiple proteins including FOG-1, EKLF, SP1, CBP/p300 and PU.1. The mechanisms by which these interactions influence GATA-1 function, as well as any possible relationships between these seemingly disparate complexes, remain incompletely understood. However, several new findings have provided further insight into the functional significance of some of these interactions. Studies involving point mutants of GATA-1 have shown that a direct physical interaction between GATA-1 and FOG-1 is essential for normal human erythroid and megakaryocyte maturation in vivo. In addition, evidence has emerged that physical interaction between GATA-1 and the myeloid/lymphoid specific factor PU.1, an oncogene implicated in murine erythroleukemia, acts to functionally cross-antagonize one another. This provides a possible mechanism by which dysregulated expression of hematopoietic transcription factors leads to lineage maturation arrest in leukemias.
FOG family zinc finger proteins play essential roles in development through physical interaction with GATA factors. FOG-1, like its interacting partner GATA-1, is required for normal differentiation of erythroid and megakaryocytic cells. Here, we have developed a functional assay for FOG-1 based on its ability to rescue erythroid and megakaryocytic maturation of a genetically engineered FOG-1(-/-) cell line. We demonstrate that interaction through only one of FOG-1's four GATA-binding zinc fingers is sufficient for rescue, providing evidence against a model in which FOG-1 acts to bridge multiple GATA-binding DNA elements. Importantly, we find that distinct regions of FOG-1 differentially influence erythroid versus megakaryocyte maturation. As such, we propose that FOG-1 may modulate the fate of a bipotential erythroid/megakaryocytic precursor cell.
E4bp4, a member of the basic region/leucine zipper transcription factor superfamily, is up-regulated by the interleukin-3 (IL-3) signaling pathway and plays an important role in the anti-apoptotic response of IL-3. In this study, we demonstrated that E4bp4 is regulated by IL-3 mainly at the transcriptional level. Promoter analysis revealed that a GATA motif downstream of a major transcription initiation site is essential for E4bp4 expression in the IL-3-dependent Ba/F3 cell line. Gel shift assays demonstrated that both GATA-1 and GATA-2 proteins bind to the E4bp4 GATA site in vitro, and the chromatin immunoprecipitation assay further confirmed the in vivo binding of GATA-1 to the E4bp4 promoter. Overexpression of GATA-1 alone transactivates the E4bp4 reporter, whereas transactivation of the E4bp4 reporter by GATA-2 is dependent on the stimulation of IL-3. Last, we demonstrated that alteration of GATA-1 binding to the GATA site by stably overexpressing GATA-1 or a GATA-1 mutant containing only the DNA-binding domain not only modulates the expression of the E4bp4 gene but also influences apoptosis induced by IL-3 removal. Taken together, our results suggest that the GATA factors play an important role in transducing the survival signal of IL-3, and one of their cellular targets is E4bp4.
Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.
Aberrant expression of PU.1 inhibits erythroid cell differentiation and contributes to the formation of murine erythroleukemias (MEL). The molecular mechanism by which this occurs is poorly understood. Here we show that PU.1 specifically and efficiently inhibits CBP-mediated acetylation of several nuclear proteins, including the hematopoietic transcription factors GATA-1, NF-E2, and erythroid Krüppel-like factor. In addition, PU.1 blocks acetylation of histones and interferes with acetylation-dependent transcriptional events. CBP acetyltransferase activity increases during MEL cell differentiation as PU.1 levels decline and is inhibited by sustained PU.1 expression. Finally, PU.1 inhibits the differentiation-associated increase in histone acetylation at an erythroid-specific gene locus in vivo. Together, these findings suggest that aberrant expression of PU.1 and possibly other members of the Ets family of oncoproteins subverts normal cellular differentiation in part by inhibiting the acetylation of critical nuclear factors involved in balancing cellular proliferation and maturation.
Transcriptional regulation of the gene-encoding human Fc epsilon RI alpha-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The alpha-chain promoter activity was up-regulated approximately 2-fold by exogenously expressed YY1 or PU.1 and approximately 7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter approximately 41- or approximately 27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the alpha-chain gene expression. Transcriptional regulation of the alpha-chain gene through four transcriptional factors is discussed.
The signal transducer and activator of transcription 3 (Stat3), a member of the Stat family of proteins, is commonly activated by thrombopoietic cytokines including thrombopoietin (TPO), interleukin (IL)-6, and interleukin-11. This finding strongly suggested that Stat3 has an important role in megakaryopoiesis and thrombopoiesis. To clarify the functional role of Stat3 in in vivo megakaryopoiesis and thrombopoiesis, we generated transgenic mice overexpressing a dominant-negative Stat3, Stat3F, to suppress the function of endogenous Stat3. To accomplish the selective expression of Stat3F in megakaryocytic lineage cells, we used the regulatory gene region of GATA-1 transcription factor selectively expressed in megakaryocytic and erythroid lineage cells. Two independent transgenic (Tg) mice lines were established. It was confirmed by Western blotting analysis that Stat3F proteins were highly expressed in the platelets from the Tg mice. In addition, it was found that Stat3 activation induced by TPO stimulation was drastically suppressed in these Tg mice compared with littermates. These findings indicate that Stat3F works well in the Tg mice. Platelet counts were within the normal range in steady-state conditions and were recovered normally from transient thrombocytopenia induced by antiplatelet serum injection. Interestingly, the platelet recovery from myelosuppression after 5-fluorouracil treatment was significantly delayed in the Tg mice. Collectively, our results strongly suggest that Stat3 plays an important role in the early stage of megakaryopoiesis, presumably through the expansion of megakaryocytic progenitor cells.
Vlad tepes (vlt(m651)) is one of only five "bloodless" zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in vlt(m651). Embryos homozygous for the vlt(m651) mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C --> T (Arg-339 --> Stop), in vlt(m651). The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in vlt(m651). These results show that the vlt(m651) mutation is a previously uncharacterized gata1 allele in the zebrafish. The vlt(m651) mutation sheds new light on Gata1 structure and function in vivo, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.
Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [DeltaLNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34+ hematopoietic stem-progenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position-effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.
The expression of the beta-like globin genes is intricately regulated by a series of both general and tissue-restricted transcription factors. The hemapoietic lineage-specific transcription factor GATA-1 is important for erythroid differentiation and has been implicated in regulating the expression of the erythroid-specific genes including the genes of the beta-globin locus. In the human erythroleukemic K562 cell line, only one DNA region has been identified previously as a putative site of GATA-1 interaction by in vivo footprinting studies. We mapped GATA-1 binding throughout the beta-globin locus by using chIp-chip analysis of K562 cells. We found that GATA-1 binds in a region encompassing the HS2 core element, as was previously identified, and an additional region of GATA-1 binding upstream of the gammaG gene. This approach will be of general utility for mapping transcription factor binding sites within the beta-globin locus and throughout the genome.
The distal locus control region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the beta-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1-5) mark the LCR. Sequence conservation and direct experimental evidence have implicated sequences within and between the HS cores in function of the LCR. In this report we confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences.
We and others demonstrated that the mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis, and in isolated rat Sertoli cells and testicular tumor cell lines derived from Leydig and Sertoli cells. In this study, we investigated the possible effects of gonadotropins and cAMP on the expression of GATA-binding protein genes in testicular cells. Unexpectedly, FSH negatively regulated GATA-1 (but not GATA-4) mRNA in a dose-dependent manner in primary cultures of rat Sertoli cells isolated from 21-d-old animals. GATA-1 mRNA was also negatively regulated by cAMP in a dose- and time-dependent manner in MA-10, a mouse Leydig tumor cell line. When 0.3 mM cAMP was administered to MA-10 cell cultures for 4 h, more than 95% of the GATA-1 mRNA and protein was abolished. The reduction of GATA-1 mRNA by cAMP can be mimicked by treatment with forskolin, which elevates intracellular cAMP levels. The inhibitory effect of cAMP was specific to the GATA-1 gene, given that GATA-4 and alpha-tubulin mRNA levels were not changed by any of the cAMP treatments. Inhibin alpha-subunit mRNA, on the other hand, was evidently increased by cAMP treatment in both MA-10 and Sertoli cells. However, inhibin alpha-subunit mRNA levels were elevated at 60-90 min before the suppression of GATA-1 mRNA detected. The inhibitory effect of cAMP on GATA-1 mRNA and protein was shown to be specific to testicular cells. The GATA-1 mRNA expressed in MEL, a mouse erythroid leukemia cell line, was not affected by cAMP. The reduction of GATA-1 mRNA by cAMP can be prevented when a translational inhibitor, cycloheximide, is added. In summary, we demonstrated that gonadotropins via cAMP negatively regulate the mRNA and protein levels of GATA-1, but not GATA-4, in testicular cells. The inhibitory effect on GATA-1 gene expression was specific to testicular cells and was not observed in erythroid cells.
Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.
Multiple myeloma (MM) is associated with severe normochromic/normocytic anemia. This study demonstrates that the abnormal up-regulation of apoptogenic receptors, including both Fas ligand (L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), by highly malignant myeloma cells is involved in the pathogenesis of the ineffective erythropoiesis and chronic exhaustion of the erythroid matrix. By measuring Fas-L and TRAIL in plasma cells and the content of glycophorin A (GpA) in erythroblasts from a cohort of 28 untreated, newly diagnosed patients with MM and 7 with monoclonal gammopathy of undetermined significance (MGUS), selected in relation to their peripheral hemoglobin values, results showed that both receptors occurred at high levels in 15 severely anemic MM patients. Their marrow erythropoietic component was low and included predominantly immature GpA(+dim) erythroblasts, in contrast with the higher relative numbers of mature GpA(+bright) erythroid cells observed in the nonanemic patients and those with MGUS. In cocultures with autologous Fas-L(+)/TRAIL(+) myeloma cells, the expanded GpA(+dim) erythroid population underwent prompt apoptosis after direct exposure to malignant plasma cells, whereas erythroblasts from nonanemic patients were scarcely affected. The evidence that Fas-L(+)/TRAIL(+) malignant plasma cells prime erythroblast apoptosis by direct cytotoxicity was also supported by the increase of FLICE in fresh immature GpA(+dim) erythroid cells, whereas ICE and caspase-10 increased in subsequent maturative forms. In addition, GATA-1, a survival factor for erythroid precursors, was remarkably down-regulated in fresh erythroblasts from the severely anemic patients. These results indicate that progressive destruction of the erythroid matrix in aggressive MM is due to cytotoxic mechanisms based on the up-regulation in myeloma cells of Fas-L, TRAIL, or both. It is conceivable that the altered regulation of these receptors defines a peculiar cytotoxic phenotype that drives the progression of aggressive MM.
GATA-1, an important hematopoietic transcription factor, plays a critical role in differentiation and maturation of erythroid and megakaryocytic cell lines. GATA-1 appears to serve as a factor for virtually all characterized erythroid and megakaryocytic-expressed genes. Thus, defining the mechanisms by which the GATA-1 gene and protein are regulated should provide important clues regarding the establishment of erythroid and megakaryocytic programs of gene expression in committed cells and their maintenance thereafter in maturing precursors. This review focuses on the regulation of GATA-1 expression and elucidates the regulation of GATA-1 at the gene and protein levels. Such research is expected to provide insights into the mechanisms involved in hematopoietic commitment.
GATA1 is the X-linked transcriptional activator required for megakaryocyte and erythrocyte differentiation. Missense mutations in the N-terminal zinc finger (Nf) of GATA1 result in abnormal hematopoiesis, as documented in four families: the mutation V205M leads to both severe macrothrombocytopenia and dyserythropoietic anemia, D218G to macrothrombocytopenia and mild dyserythropoiesis without anemia, G208S to macrothrombocytopenia and R216Q to macrothrombocytopenia with beta-thalassemia. The three first GATA1 mutants display a disturbed binding to their essential transcription cofactor FOG1, whereas the fourth mutant shows an abnormal direct DNA binding. In this study, we describe a new family with deep macrothrombocytopenia, marked anemia and early mortality, if untreated, due to a different GATA1 mutation (D218Y) in the same residue 218 also implicated in the above mentioned milder phenotype. Zinc finger interaction studies revealed a stronger loss of affinity of D218Y-GATA1 than of D218G-GATA1 for FOG1 and a disturbed GATA1 self-association. Comparison of the phenotypic characteristics of patients from both families revealed that platelet and erythrocyte morphology as well as expression levels of the platelet GATA1-target gene products were more profoundly disturbed for the hemizygote D218Y mutation. The D218Y allele (as opposed to the D218G allele) was not expressed in the platelets of a female carrier while her leukocytes showed a skewed X-inactivation pattern. We conclude that the nature of the amino acid substitution at position 218 of the Nf of GATA1 is of crucial importance in determining the severity of the phenotype in X-linked macrothrombocytopenia patients and possibly also in inducing skewed X inactivation.
Human parvovirus B19 (B19) infection during pregnancy is associated with the adverse foetal outcome known as non-immune hydrops fetalis (NIHF). Although B19 is known to infect erythroid-lineage cells in vivo as well as in vitro, the mechanism leading to the occurrence of NIHF is not clear. To investigate the possible involvement of the B19 non-structural protein NS1 in NIHF, three independent lines of transgenic mice were generated that expressed NS1 under the control of the Cre-loxP system and the GATA1 promoter. Two of the three lines expressed NS1 in erythroid-lineage cells. Most of the transgenic mice died at the embryonic stage, some of which developed hydropic changes caused by severe anaemia at embryonic day 15.5 (E15.5). Histological examination of embryos at E15.5 showed significantly fewer erythropoietic islands in the liver parenchyma, whereas their hearts showed no abnormal signs, such as cardiomegaly and apoptotic cells. The NS1-transgenic mouse lines established here provide an animal model for human NIHF and suggest that NS1 plays a crucial role in the adverse outcome associated with intrauterine B19 infection in humans.
Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated beta-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.
Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of receptor-bound Janus kinase (Jak2). Jak2 then mediates the phosphorylation of receptor tyrosine sites and the recruitment of 25 or more Src homology 2 domain-encoding proteins and associated factors. Previous studies have shown that an EPO receptor form containing Jak2-binding domains plus a single phosphotyrosine(343) (PY(343))-STAT5-binding site provides all signals needed for erythroid cell development. However, roles for PY(343) and STAT5 remain controversial, and findings regarding PY-null receptor activities and erythropoiesis in STAT5-deficient mice are disparate. To study activities of a PY-null EPO receptor in primary cells while avoiding compensatory mechanisms, a form retaining domains for Jak2 binding and activation, but lacking all cytoplasmic tyrosine sites, was expressed in transgenic mice from a GATA1 gene-derived vector as a human epidermal growth factor receptor- murine EPO receptor chimera (EE-T-Y343F). The bio-signaling capacities of this receptor form were investigated in CFUe from thiamphenicol-treated mice. Interestingly, this PY-null EPO receptor form supported CFUe development (in the absence of detectable STAT5 activation) at efficiencies within 3-fold of those levels mediated by either an EE-T-Y343 form or the endogenous EPO receptor. However, EE-T-Y343F-dependent Ter119(+) erythroblast maturation was attenuated. In tests of cosignaling with c-Kit, EE-T-Y343F nonetheless retained full capacity to synergize with c-Kit in promoting erythroid progenitor cell proliferation. Thus, EPO receptor PY-dependent events can assist late erythropoiesis but may be nonessential for EPO receptor-c-Kit synergy.
HEL cells, a human erythroleukemia cell line, mainly express the fetal (gamma) globin gene and trace amount of the embryonic (epsilon) globin gene, but not adult (beta) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (beta) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human beta-globin gene, including the proximal regulatory element (the beta-promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU-induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human beta-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human beta-globin gene in HU-induced HEL cells.
The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct cis elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.
Although erythropoietin (EPO) and its receptor (EPOR) are crucial for the proliferation, survival, and terminal differentiation of erythroid progenitors, it remains to be elucidated whether EPOR-unique signaling is required for erythropoiesis. To address this issue, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR)-transgenic mice and heterozygous EPOR mutant mice were crossed by in vitro fertilization. In methylcellulose clonal culture of fetal liver (FL) cells of generated hGMR-expressing EPOR(-/-) embryos at embryonic day (E) 12.5 of gestation, hGM-CSF stimulated erythroid colony formation under serum-containing and serum-free conditions. Analysis of globin expression in individual erythrocyte-containing colonies formed from E12.5 FL cells showed that hGM-CSF supports primitive and definitive erythropoiesis even in EPOR(-/-) embryos. In comparison of activities between hGM-CSF and EPO in hGMR-expressing EPOR(+/+) embryos, the 2 substances supported the formation of similar numbers of erythroid colonies in clonal culture of E12.5 FL cells; enhanced adult, but not embryonic, globin synthesis; and induced increase of GATA-1 expression and decrease of erythroid Kruppel-like factor and cMyb expression in the FL cells. On the other hand, in E8.0 yolk sac erythropoiesis, both substances had a similar effect on erythroid colony formation, but hGM-CSF induced an increase of beta-major globin expression, while EPO did not. All together, the results of the present study demonstrated that hGM-CSF can stimulate the proliferation and differentiation of primitive and definitive erythroid cells independently of EPOR signal if they express hGMR, and the activity is comparable to that of EPO in definitive, but not primitive, erythropoiesis.
The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage.
We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells.
These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.
GATA-1 is a key regulator of terminal erythroid differentiation in mammals and birds. The structural and biochemical studies of human GATA-1 (hGATA-1) are limited by the difficulty of its purification in a sufficient amount. Here we describe the procedure for obtaining pure bacterial recombinant hGATA-1 in an active functional state. We demonstrate that this protein may be successfully used for preparing an affinity column, producing GATA-1-specific rabbit polyclonal antibodies, and studying DNA-protein and protein-protein interactions.
A family with recessive X-linked thrombocytopenia affecting 4 males in 2 generations, characterized by macrothrombocytopenia, profound bleeding, and mild dyserythropoiesis, is described. Microsatellite linkage analysis identified a region of the X chromosome including the GATA-1 gene, which encodes a critical transcription factor involved in erythrocyte and megakaryocyte development. By sequencing the entire coding region of GATA-1, a 2-base mutation was detected that results in a single amino acid substitution (glycine 208 to serine) within a highly conserved portion of the N-terminal zinc finger domain. Restriction fragment length polymorphism confirmed that this novel mutation segregated with the affected males and female carrier. Although not required for DNA binding, Gly208 of GATA-1 is involved in direct interaction with Friend of GATA-1 (FOG), a cofactor required for normal megakaryocytic and erythroid development. These results demonstrate that the GATA-1-FOG interaction is partially disrupted by the mutation and that the greatest effect involves contact with the FOG zinc finger 9. These findings help describe a novel mutation of GATA-1 in humans as a cause of X-linked thrombocytopenia, and they confirm the vital role played by this transcription factor during in vivo megakaryocyte development.
Vascular endothelial growth factor (VEGF, VEGF-A), a selective mitogen for endothelial cells is a critical factor for vascular development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(121), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both endothelial and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both vascular and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate endothelial cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice, the Cre/loxP system was used, and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However, double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation, causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes, mast cells, and eosinophils, but not in neutrophils, lymphocytes, or nonhematopoietic tissues. In summary, GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time, mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells.
GATA-1 is a transcription factor essential for erythroid/megakaryocytic cell differentiation. To investigate the contribution of individual domains of GATA-1 to its activity, transgenic mice expressing either an N-terminus, or an N- or C-terminal zinc finger deletion of GATA-1 (Delta NT, Delta NF or Delta CF, respectively) were generated and crossed to GATA-1 germline mutant (GATA-1.05) mice. Since the GATA-1 gene is located on the X-chromosome, male GATA-1 mutants die by embryonic day 12.5. Both Delta NF and Delta CF transgenes failed to rescue the GATA-1.05/Y pups. However, transgenic mice expressing Delta NT, but not the Delta NF protein, were able to rescue definitive hematopoiesis. In embryos, while neither the Delta CF protein nor a mutant missing both N-terminal domains (Delta NTNF) was able to support primitive erythropoiesis, the two independent Delta NT and Delta NF mutants could support primitive erythropoiesis. Thus, lineage-specific transgenic rescue of the GATA-1 mutant mouse revealed novel properties that are conferred by specific domains of GATA-1 during primitive and definitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA-1.
Megakaryocytes, among the rarest of hematopoietic cells, serve the essential function of producing numerous platelets. Genetic studies have recently provided rich insights into the molecular and transcriptional regulation of megakaryocyte differentiation and thrombopoiesis. Three transcription factors, GATA-1, FOG-1, and NF-E2, are essential regulators of distinct stages in megakaryocyte differentiation, extending from the birth of early committed progenitors to the final step of platelet release; a fourth factor, Fli-1, likely also plays an important role. The putative transcriptional targets of these regulators, including the NF-E2-dependent hematopoietic-specific beta-tubulin isoform beta1, deepen our understanding of molecular mechanisms in platelet biogenesis. The study of rare syndromes of inherited thrombocytopenia in mice and man has also refined the emerging picture of megakaryocyte maturation. Synthesis of platelet-specific organelles is mediated by a variety of regulators of intracellular vesicle membrane fusion, and platelet release is coordinated through extensive and dynamic reorganization of the actin and microtubule cytoskeletons. As in other aspects of hematopoiesis, characterization of recurrent chromosomal translocations in human leukemias provides an added dimension to the molecular underpinnings of megakaryocyte differentiation. Long regarded as a mysterious cell, the megakaryocyte is thus yielding many of its secrets, and mechanisms of thrombopoiesis are becoming clearer. Although this review focuses on transcriptional control mechanisms, it also discusses recent advances in broader consideration of the birth of platelets.
Hematopoietic cells arise from ventral mesoderm in different vertebrates, but the mechanisms through which various factors contribute to the hematopoietic processes, including erythrogenesis, remain incompletely understood. The Krüppel-like transcription factor Biklf is preferentially expressed in blood islands throughout zebrafish embryogenesis, marking the region of future erythropoiesis [1]. In this paper, we show that expression of biklf is significantly suppressed in the blood-less mutants vampire and m683 in which primitive hematopoiesis is impaired. Knockdown of biklf using morpholino-based antisense oligonucleotides (biklf-MO) led to a potent reduction in the number of circulating blood cells and deficiency in hemoglobin production. Consistently, we found that the expression of beta(e3)globin is strongly suppressed in biklf-MO-injected embryos, while gata1 expression is partly inhibited at the 10-somite stage. In addition, analysis of reporter constructs driven by the GATA1 and beta-globin promoters showed that Biklf can positively regulate both genes. These results indicate that Biklf is required for erythroid cell differentiation in zebrafish.
Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.
Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation.
Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.
The follicle-stimulating hormone receptor (FSHR) gene is expressed in Sertoli cells in males and in granulosa cells in females. Cis-acting sequences and associated binding factors responsible for the transcription of the TATA-less FSHR gene in Sertoli cells were analyzed with dimethylsulfate (DMS) footprinting assays and electrophoretic mobility shift assay (EMSA). In vivo footprints in the core promoter using nuclear proteins from Sertoli cells identified several protected sequences, including an inverted GATA (TATC, -88/-85), and an E2F (TTTCGCG, -45/-39) motif. EMSA showed the presence of one or more sequence-specific proteins interacting with these potential regulatory elements. Antibody-supershift assays as well as competition assays further revealed that testis-specific GATA-1 recognized the inverted GATA element. The functional role of the potential cis-acting elements was analyzed by transient transfection assays with and without mutations of the putative elements. The mutational analysis indicated that the GATA and E2F elements were each required for optimal promoter activity. The effects of each of the promoter elements was examined in transfections in which mutations were made in each of the known regulatory sites, including the E box, GATA, and E2F sites in various combinations. All of these sites contribute to the maximum promoter activity such that mutations of the E box, GATA, and E2F sites eliminated nearly all promoter activity.
The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.
A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for beta-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbbeta and GPIX but also a significantly lower expression of the nondirectly GATA1-regulated Gsalpha gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbalpha, GPIbbeta, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion, GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
A multipotent immature myeloid cell population was produced from a basic fibroblast growth factor (bFGF)-dependent hematopoietic stem cell line, A-6, when cultured with stem cell factor (SCF) replacing bFGF. Those cells were positive for stem cell markers, c-kit and CD34, and a myeloid cell marker, F4/80. Some cell fractions were also positive for Mac-1, a macrophage marker or Gr-1, a granulocytic maker, but negative for an erythroid marker TER119. They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1. Among various cytokines, interleukin-3 (IL-3) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and beta-major globin. The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3. SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line.
The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.
Cathepsin E is an intracellular aspartic proteinase that is considered to have a number of physiological roles including antigen processing. Quantitation of procathepsin E mRNA by LightCyclertrade mark technology indicated that the gene was transcribed in lung but not in kidney of both human and mouse origin. In contrast, the transcript was present in mouse spleen and alveolar macrophages but not in the counterpart tissue/cells from humans. Regulation of human and mouse procathepsin E gene expression was shown not to be influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in each promoter region by transcription factors such as GATA1, PU1 and YY1, as revealed by functional analysis using a series of promoter/luciferase reporter gene fusion constructs. Thus the extent to which the procathepsin E gene is expressed in a particular cell type may depend on the balance between the effects produced by positive-acting, cell-specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous YY1 factor. In this way, the relative abundance and influence of general and cell-specific transcription factors can govern the production of cathepsin E and thereby account for the sporadic cell and tissue distribution of this enzyme in different species.
In this study, megakaryocytopoiesis was investigated in the recessive mutant mouse, jumonji, obtained by a gene-trap strategy. We investigated the number of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygous embryos by in vitro colony forming assay and monitored colony formation from single megakaryocyte progenitors. We also investigated the differentiation of jumonji-deficient megakaryocytes in terms of the expression of megakaryocyte differentiation markers PF4, CD62P, and GATA-1, proplatelet formation, cytoplasmic maturation, and endomitosis. We found that the population of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygotes increased. A fraction of megakaryocyte progenitors derived from the fetal liver of jumonji homozygotes formed larger colonies in vitro when compared with controls. This abnormality is caused by delayed growth arrest in the progeny. Immature megakaryocyte progenitors showed this abnormality. The megakaryocytes of jumonji homozygotes expressed PF4, CD62P, and GATA-1, obtained cytoplasmic maturation, extended proplatelet-like processes, and underwent endomitosis. The loss of the jumonji gene causes an increase in the number of megakaryocyte lineage cells. Our data suggest that the jumonji gene regulates proliferation but not differentiation of megakaryocyte lineage cells.
Although normal megakaryocytic development has been shown to require the presence of functional GATA-1 and NF-E2 transcription factors in vivo, the roles of other members of the GATA binding factors and NF-E2 family during megakaryocytic differentiation are unclear. the present study, the expression of GATA family members, GATA-1 and GATA-2, a GATA-binding factor, EVI-1, the large subunit of NF-E2 factor, p45 and the related factors, Nrf1, Nrf2, Nrf3, BACH1, BACH2, and the small subunit of NF-E2, MAFK and MAFG has been examined in human megakaryocytic and erythroid cells by reverse transcriptase-polymerase chain reaction. CD34+ cells isolated from human cord blood were induced to unilineage megakaryocytic or erythroid differentiation in liquid suspension culture in the presense of thrombopoietin or erythropoietin, respectively. Each lineage was identified by monoclonal antibody against GPIIb/IIIa or glycophorin A. In megakaryocytic culture, p45, Nrf1, Nrf2, BACH1, MAFK and MAFG mRNAs were induced similarly to erythroid culture. Nrf3 mRNA was barely detected in both cultures. BACH2 was induced only in megakaryocytic culture, although the level of expression was low. Furthermore, the profiles of transcription factors involved in hematopoiesis, EVI-1 and Ets-1 mRNAs were induced only in megakaryocytic culture. Megakaryocytic and erythroid differentiation pathways are closely related to each other, and these two lineage cells share a number of lineage-specific transcription factors. However, the results showed that the profile of the expression of these transcription factors in megakaryocytic cells is distinct from that of erythroid lineage. The dynamic changes in the levels of different transcription factors that occur during primary megakaryocytic differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.
Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.
GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human alpha- and beta-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both beta-and alpha clusters and in proximal regulatory regions of the zeta-, epsilon- and gamma-globin but not the alpha, delta or beta-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of epsilon-globin mRNA but had no effect on the levels of expression of gamma, zeta or alpha-globin genes. Increasing GATA-2 levels increased epsilon-globin and gamma-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.
The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1-mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function. (Blood. 2000;95:2543-2551)
Using a combination of mouse bacterial artificial chromosome (BAC) genomic library screening, long-range polymerase chain reaction (PCR) amplification, genomic walking and DNA sequencing, we have characterized the intron/exon boundaries, the sizes of each intron and 5' flanking region of the mouse PFK-C gene. The gene spans approximately 55 kb and comprises 22 exons separated by 21 introns. All intron/exon splice junctions conform to the GT/AG rule. The mouse PFK-C gene organization is similar to that of the human and rabbit PFK-A and human and mouse PFK-B genes. However, PFK-C has much larger intronic sequences throughout the gene. Anchored PCR was performed to amplify about 1.0 kb of genomic DNA upstream of the translational start site. Sequence analysis of the PFK-C 5' flanking region revealed that it is devoid of TATA and CAAT boxes at the usual positions, but it contained several putative binding sites for transcription factors AP1, GATA1, NKX2.5 and STAT. The 5' flanking region was not enriched in GC dinucleotides and lacked CpG islands and putative binding sites for SP1. Four transcription initiation sites have been identified by full-length RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) between -61 and -32 bp from the translation initiation codon. Reverse transcription-PCR analysis revealed that PFK-A, PFK-B and PFK-C genes were expressed, in all mouse tissues tested, at varying levels. PFK-A mRNA was more abundantly expressed in all tissues than were the PFK-B and PFK-C genes. Based on the mouse PFK-C signal normalized to 18S rRNA, the PFK-C mRNA was expressed at the highest levels in the brain, heart, thymus and testicles, whereas low levels were observed in the kidney, liver, muscle, and lung.
The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation. CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity. Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity. Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones. Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP. Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA. In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBPalpha, strongly stimulated nucleosomal HAT activity. In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity. These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.
We have previously reported that CDV (carnitine deficiency-associated gene expressed in ventricle)-1 was a downregulated gene in the hypertrophied ventricle of carnitine-deficient juvenile visceral steatosis mice and that the related gene (CDV-1R) showed no tissue specificity and no sensitivity to carnitine deficiency. In the present paper, the CDV-1/1R gene was isolated from a mouse genomic BAC library, and the genomic structure was characterized. We found that the CDV-1/1R gene consisted of at least 19 exons and encompassed approximately 48 kb. The splice sites conformed to the GT-AG rule, and the CDV-1R mRNA containing 19 exons was processed. CDV-1 mRNA containing 5 exons was constructed from the 3' half of CDV-1R. The first exon of CDV-1 consisted of the 3' side (116 bp) of intron 14 and exon 15 (87 bp) of CDV-1R. The presumed promoter sequence for CDV-1 located in the intron 14 of CDV-1R contained the common TATA box and consensus binding sites for various transcription factors (Nkx-2.5, Spl, C/EBP, SRF, YY1, and CREB), which seem to play roles in the heart-specific expression and carnitine deficiency-associated suppression of CDV-1. In the upstream region of the CDV-1 promoter, we found two VNTRs, 13 repeats of GATA1, and 16 copies of STRE involved in yeast stress response. The CDV-1/1R gene was located close to DSMIT68 on mouse Chromosome (Chr) 5, corresponding to human Chr 12q24. All these data revealed that two mRNA species, CDV-1 and CDV-1R, are expressed tissue-specifically by using promoters peculiar to each transcript in a single gene.
Human FcgammaRIIA, expressed on platelets, neutrophils, and macrophages, plays a major role in platelet activation and immune clearance. Clinical observations indicate that regulation of expression of this receptor is an important factor influencing the course of immune thrombocytopenia. We used both transient transfection with FcgammaRIIA promoter constructs and electrophoretic mobility shift assays (EMSA) to study the regulation of FcgammaRIIA transcription. In HEL (erythromegakaryocytic) cells, the 200 bp immediately 5' of the ATG start codon accounted for the majority of the activity of a 3.6-kb promoter fragment. Putative GATA (-161) and NF-Y (-119) sites are present. EMSA analyses demonstrate specific binding of both GATA-1 and GATA-2 to labeled oligonucleotides containing the putative GATA site with HEL but not U937 (myelomonocytic) nuclear extracts. Antibodies to NF-Y supershift the specific -119 NF-Y complex with HEL, U937, Jurkat (T-lymphocytic), and HeLa (nonhematopoietic) nuclear extracts. Comparison of the activity of GATA and NF-Y mutant constructs in HEL and U937 demonstrates that while either GATA or NF-Y mutation results in a large decrease in the promoter activity (2.2- and 2.3-fold, respectively) in HEL cells, neither mutation is effective in reducing activity in U937 cells. This is the first example of a promoter active in the megakaryocyte lineage in which NF-Y cooperates additively with GATA factors to regulate transcription. Identification of other factors that must be operational for FcgammaRIIA transcription in myelomonocytic cells which lack GATA factors will bolster our ongoing efforts to dissect the function of these Fc receptors in megakaryocytic and myelomonocytic cells in vivo.
Uroporphyrinogen-III (URO) synthase is the heme biosynthetic enzyme defective in congenital erythropoietic porphyria. The approximately 34-kb human URO-synthase gene (UROS) was isolated, and its organization and tissue-specific expression were determined. The gene had two promoters that generated housekeeping and erythroid-specific transcripts with unique 5'-untranslated sequences (exons 1 and 2A) followed by nine common coding exons (2B to 10). Expression arrays revealed that the housekeeping transcript was present in all tissues, while the erythroid transcript was only in erythropoietic tissues. The housekeeping promoter lacked TATA and SP1 sites, consistent with the observed low level expression in most cells, whereas the erythroid promoter contained GATA1 and NF-E2 sites for erythroid specificity. Luciferase reporter assays demonstrated that the housekeeping promoter was active in both erythroid K562 and HeLa cells, while the erythroid promoter was active only in erythroid cells and its activity was increased during hemin-induced erythroid differentiation. Thus, human URO-synthase expression is regulated during erythropoiesis by an erythroid-specific alternative promoter.
Mammalian CBFB encodes a transcription factor (CBF beta) that in combination with CBF alpha 2 binds to specific DNA sequences and regulates expression of a number of hematopoietic genes. CBFB is associated with human leukemias through a chromosome 16 inversion and is essential for definitive hematopoiesis during mouse embryo development. We have isolated a zebrafish cbfb complementary DNA (cDNA) clone from a zebrafish kidney cDNA library. This cbfb is highly homologous to human and mouse CBFB/Cbfb genes at both the DNA and protein level. In biochemical analyses, cbfbeta binds to human CBF alpha 2 and enhances its DNA binding. During zebrafish development, cbfb is expressed in the lateral plate mesoderm at tail bud stage and in the intermediate cell mass (ICM, the location of embryonic hematopoiesis) between the 21- to 26-somite stages. The cbfb is also expressed in Rohon-Beard cells, cranial nerve ganglia, hindbrain, retina, branchial arches, jaw, and fin buds. Expression of cbfb is decreased or absent in the ICM and Rohon-Beard cells in some hematopoietic mutants and is unaffected in others. We have also analyzed the expression of scl and gata-1 in the same hematopoietic mutants to ascertain the relative order of these transcription factors to cbfb in zebrafish hematopoiesis. Our results indicate that cbfb is expressed in early hematopoietic progenitors and that its expression pattern in the hematopoietic mutants is similar to that of scl. (Blood. 2000;96:4178-4184)
Megakaryocytes lacking transcription factor GATA-1 fail to complete maturation in vivo and hyperproliferate. To define how GATA-1 regulates megakaryocyte cell growth we searched for mRNA transcripts expressed in primary wild-type, but not GATA-1(-), megakaryocytes. One differentially expressed transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and also has lesser activity against soluble analogues of this lipid, inositol 3, 4-bisphosphate and inositol 1,3,4-triphosphate. Reintroduction of 4-Ptase I into both primary GATA-1(-) and wild-type megakaryocytes significantly retards cell growth, suggesting that absence of 4-Ptase I may contribute to the hyperproliferative phenotype of GATA-1(-) megakaryocytes. Overexpression of 4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken together, these data indicate that 4-Ptase I is a regulator of cell proliferation.
Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1.
We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity.
Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.
The c-myb proto-oncogene product (c-Myb) is a sequence-specific DNA-binding protein that functions as a transcriptional activator. The transcriptional coactivator CREB-binding protein (CBP) binds via its KIX domain to the activation domain of c-Myb and mediates c-Myb-dependent transcriptional activation. CBP possesses intrinsic histone acetyltransferase activity, and can acetylate not only histones but also certain transcriptional factors such as GATA1 and p53. Here we demonstrate that the C/H2 domain of CBP, which is critical for the acetyltransferase activity, also directly interacts with the negative regulatory domain (NRD) of c-Myb. Consistent with this observation, CBP acetylated c-Myb in vitro at Lys(438) and Lys(441) within the NRD. In addition, CBP acetylated c-Myb in vivo not only at the sites found in this study but also at the p300-induced acetylation sites reported recently. Replacement of lysine by arginine at all of these sites dramatically decreased the trans-activating capacity of c-Myb. The results of transcriptional activation assays with c-Myb acetylation site mutants suggested that acetylation of c-Myb at each of these five sites synergistically enhances c-Myb activity. Mutations of these acetylation sites reduced the strength of the interaction between c-Myb and CBP. Thus, acetylation of c-Myb by CBP increases the trans-activating capacity of c-Myb by enhancing its association with CBP. These results demonstrate a novel molecular mechanism of regulation of c-Myb activity.
The CCAAT boxes of the beta-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPgamma, and address its role in globin gene regulation by erythroid overexpression of C/EBPgamma, and a dominant-negative form C/EBPgammaDeltaB, in mice. Elevated levels of C/EBPgamma, but not C/EBPgammaDeltaB, increase expression of the (fetal) gamma-globin relative to the (adult) beta-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPgamma and C/EBPgammaDeltaB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.
Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.
Optimal production of red cells in vivo requires collaboration between c-Kit, erythropoietin receptor (Epo-R), and GATA-1. However, the mechanism(s) of collaboration remain unclear. Utilizing an embryonic stem cell-derived erythroid progenitor cell line from mice deficient in GATA-1, we have examined the role of c-Kit and Epo-R in erythroid cell proliferation, survival, and differentiation. In the absence of GATA-1, we demonstrate an essential role for c-Kit in survival and proliferation of erythroid progenitors via the regulation of Bcl-2 expression. In addition, we demonstrate that Epo-R and Stat5 are regulated by a second, novel mechanism. We demonstrate that c-Kit stimulation by stem cell factor is essential for the maintenance of Epo-R and Stat5 protein expression, which results in significantly enhanced Bcl-x(L) induction and survival of erythroid progenitors in response to Epo stimulation. Restoration of GATA-1 function results in terminal erythroid maturation and up-regulation of Epo-R and Bcl-x(L) expression, leading also to significantly enhanced survival of terminally differentiating erythroid progenitors in the presence of only Epo. These results demonstrate that c-Kit and Epo-R have unique role(s) during distinct phases of erythroid maturation, and both stem cell factor and Epo contribute to the regulation of the Epo-R-Stat5-Bcl-x(L) pathway to ensure optimal survival, proliferation, and differentiation of erythroid progenitors.
The HEL cells, a human erythroleukemia cell line, express mainly the fetal globin gene and small amount of the embryonic (epsilon) globin gene, but not the adult (beta) globin gene. Hydroxyurea, a small organic compound, has been successfully used to treat sickle cell anemia and beta-thalessaemia. Our data demonstrated that the growth rate of HEL cell proliferation was inhibited by different doses of hydroxyurea (from 50 mumol/L to 200 mumol/L). Using both routine RT-PCR and quantitative PCR analyses, we revealed that the expression of beta-globin gene was sharply activated and alpha-globin gene was almost completely silenced when HEL cells were induced for 3 or 5 days. Meanwhile, gamma-globin gene was expressed with no much difference between induced and uninduced HEL cells. We also demonstrated that the expression of GATA-1 and NF-E 2, which were two of the most important transcription factors in erythrocyte development, was activated about 3 folds in the induced cells. We suggested that the induction of GATA-1 and NF-E 2 expression by hydroxyurea might lead to activation of the adult beta-globin gene through some pathways of signal transduction, therefore, hydroxyurea might play a role to induce HEL cells to terminal differentiation.
The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1-estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1-mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein-protein interactions. These findings contribute to understanding how protein-protein interactions participate in hematopoietic differentiation and leukemogenesis. (Blood. 2000;96:2641-2648)
Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-epsilon. In transfection studies, the isolated variable domain of PKC-epsilon selectively blocked exogenous activation of the megakaryocyte-specific alpha IIb promoter. Constitutively active mutants of PKC-epsilon, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the alpha IIb promoter. The functional cooperation between GATA-1 and PKC-epsilon displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-epsilon specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters.
Targeted gene disruption of two distinct lineage-restricted hematopoietic transcription factors has provided useful insights into the transcriptional control of platelet production. Absence of either the basic leucine-zipper protein NF-E2 or of the zinc-finger protein GATA-1 in vivo results in severe thrombocytopenia secondary to distinct patterns of arrested megakaryocyte cytoplasmic maturation; in addition, megakaryocyte-selective loss of GATA-1 expression leads to dysregulated proliferation of progenitor cells. The ultrastructure of the defective megakaryocytes suggests that absence of the respective transcription factors impairs biogenesis of platelet-specific granules and proper development and organization of demarcation membranes. In particular, transcriptional targets of NF-E2 may be implicated in the very final stages of megakaryocyte differentiation, which involve the organization and release of platelets. Preliminary characterization of genes that are downregulated in NF-E2-/- megakaryocytes is in progress and is likely to lead to mechanistic insights into thrombocytopoiesis.
The transcription factor GATA-1 is specifically expressed in hematopoietic lineages. Prior gene knockout experiments established an essential role for GATA-1 in red blood cell production, but could not provide direct evidence with respect to a requirement in megakaryopoiesis. We summarize here recent lineage-selective gene targeting in mice that establishes critical functions for GATA-1 in controlling megakaryocyte growth and maturation, and platelet production. GATA-1 megakaryocytes are delayed in their cellular maturation, exhibit marked hyperproliferation and generate fewer than normal, yet enlarged, platelets in vivo. Thus GATA-1 is a central regulator in both the erythroid and megakaryocytic lineages.
During embryogenesis, endothelial and blood precursors are hypothesized to arise from a common progenitor, the hemangioblast. Several genes that affect the differentiation of, or are expressed early in, both the endothelial and blood lineages may in fact function at the level of the hemangioblast. For example, the zebrafish cloche mutation disrupts the differentiation of both endothelial and blood cells. The transcription factor gene scl is expressed in both endothelial and blood lineages from an early stage and can regulate their differentiation. Here we report that in zebrafish the homeobox gene hhex (previously called hex) is also expressed in endothelial and blood lineages from an early stage. We find that hhex expression in these lineages is significantly reduced in cloche mutant embryos, indicating that hhex functions downstream of cloche to regulate endothelial and blood differentiation. Ectopic expression of hhex through injection of a DNA construct leads to the premature and ectopic expression of early endothelial and blood differentiation genes such as fli1, flk1 and gata1, indicating that Hhex can positively regulate endothelial and blood differentiation. However, analysis of a hhex deficiency allele shows that hhex is not essential for early endothelial and blood differentiation, suggesting that another gene, perhaps scl, compensates for the absence of Hhex function. Furthermore, we find that hhex and scl can induce each other's expression, suggesting that these two genes cross-regulate each other during early endothelial and blood differentiation. Together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation.
Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-ras(G12V) evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-ras(G12V). In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-ras(G12V), respectively. In GATA-1-transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb-transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.
Induction of specific gene expression may provide an alternative or a support to conventional cytotoxic chemotherapy of cancer, as well as to therapy for sickle cell diseases. In this respect, pharmacological induction of expression of the endogenous gamma-globin gene is a realistic approach to therapy of beta-globin disorders. Erythroid differentiation and inhibition of proliferation of the human CML K562 cell line was induced by guanosine 5'-triphosphate (GTP). The hemoglobin production in cells was correlated to an increase in alpha- and gamma-globin mRNA expression. At the transcriptional level, we showed that both the expression of the major erythroid transcription factor GATA-1 (protein and mRNA) and its binding capacity to the gamma-globin gene promoter was transiently increased. Moreover, GTP moderately stimulated the gamma-globin gene promoter after 48 h of treatment. At the post-transcriptional level, GTP treatment led to a drastic increase of the gamma-globin mRNA half-life. This stabilizing effect of GTP was mediated via the 3'-untranslated region (3'-UTR) of the gamma-globin mRNA. In conclusion, mechanism of GTP-mediated differentiation of K562 cells is linked to an early activation of gamma-globin gene transcription followed by a stabilization of its mRNA.
GATA-1 is a tissue-specific transcription factor that is essential for the production of red blood cells. Here we show that overexpression of GATA-1 in erythroid cells inhibits their differentiation, leading to a lethal anaemia. Using chromosome-X-inactivation of a GATA-1 transgene and chimaeric animals, we show that this defect is intrinsic to erythroid cells, but nevertheless cell nonautonomous. Usually, cell nonautonomy is thought to reflect aberrant gene function in cells other than those that exhibit the phenotype. On the basis of our data, we propose an alternative mechanism in which a signal originating from wild-type erythroid cells restores normal differentiation to cells overexpressing GATA-1 in vivo. The existence of such a signalling mechanism indicates that previous interpretations of cell-nonautonomous defects may be erroneous in some cases and may in fact assign gene function to incorrect cell types.
The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)
A 40-bp DNA, consisting of seven tandem GATA repeats, is located near the HS5 site in the 5' boundary area of the locus control region (LCR) of human beta-globin gene. This (GATA)(7) motif, named 5a, exhibits silencer activity in erythroid cells. In transfected, recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the activity of the cis-linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis-linked HS2 enhancer regardless of whether the CAT gene was driven by the pgk or the epsilon-globin promoter. Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer activity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a. Band shift experiments show that the GATA-1 protein binds to both 5a and the mutant 5a and forms a large protein complex. Together, the results suggest that GATA-1 bound at 5a is a strong, proximal repressor of HS2 enhancer activity.
We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.
The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.
GATA-1 and other vertebrate GATA factors contain a DNA binding domain composed of two adjacent homologous zinc fingers. Whereas only the C-terminal finger of GATA-1 is capable of independent binding to the GATA recognition sequence, double GATA sites that require both fingers for high affinity interaction are found in several genes. We propose a mechanism whereby adjacent zinc fingers interact to influence the binding and transactivation properties of GATA-1 at a subset of DNA-binding sites. By using two such double GATA sites we demonstrate that the N-terminal finger and adjacent linker region can alter the binding specificity of the C-terminal finger sufficiently to prevent it from recognizing some consensus GATA sequences. Therefore, the two zinc fingers form a composite binding domain having a different DNA binding specificity from that shown by the constituent single C-terminal finger. Furthermore, we compare two of these double sites and show that high affinity binding of GATA-1 to a reporter gene does not necessarily induce transactivation, namely the sequence of the DNA-binding site can alter the ability of GATA-1 to stimulate transcription.
Transcription factor GATA-1 is essential for normal erythropoiesis. GATA-binding sites are consistently found in promoters or enhancers of genes expressed selectively in erythroid cells. To discover novel GATA-1-regulated genes, we searched for GATA-1-activated transcripts in G1E cells, an erythroid line derived from GATA-1(-) embryonic stem cells. By subtractive analysis, we identified a new ATP-binding cassette (ABC) transporter that is strongly and rapidly induced by GATA-1. This protein, named ABC-me (for ABC-mitochondrial erythroid), localizes to the mitochondrial inner membrane and is expressed at particularly high levels in erythroid tissues of embryos and adults. ABC-me is induced during erythroid maturation in cell lines and primary hematopoietic cells, and its overexpression enhances hemoglobin synthesis in erythroleukemia cells. The ABC proteins participate in diverse physiological processes by coupling ATP hydrolysis to the transport of a variety of substrates across cell membranes. We speculate that ABC-me, a newly identified erythroid-expressed ABC superfamily member, may mediate critical mitochondrial transport functions related to heme biosynthesis.
Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is one of the earliest and best-known markers of Sertoli cell differentiation and is expressed until around puberty. The present study is aimed at the better understanding of the molecular pathways involved in testicular development and establishment of adult functions with regards to AMH regulation. We found, within the mouse AMH promoter, putative GATA motifs (A/T)GATA(A/G), known to be specifically bound by members of the GATA transcription factor family. We then carried out RNase protection assays and immunohistochemical techniques aimed at comparing precisely the chronological expression patterns of AMH and GATA-1, this latter being expressed in the testis after birth. Using both approaches we found an inverse and close relationship between AMH and GATA-1 mRNA and protein expression during the pre-pubertal period. These results allowed us to define a transitory 4-5-day period, starting from 3 dpp when both proteins are heterogeneously expressed in Sertoli cells and showed that the appearance of GATA-1 is associated with the decrease of AMH expression in these cells. Furthermore DNA-protein interaction in in vitro studies showed first that GATA-1 binds with various affinities on sites found in the AMH promoter and second that the proximity of the two strongest affinity sites leads to a synergistic binding effect. Altogether, the present study suggests that GATA-1 participates in AMH gene repression during the pre-pubertal period.
The zinc finger protein GATA-1 functions in a concentration-dependent fashion to activate the transcription of erythroid and megakaryocytic genes. Less is understood, however, regarding factors that regulate the GATA-1 gene. Presently elements within intron 1 are shown to markedly affect its erythroid-restricted transcription. Within a full-length 6. 8-kilobase GATA-1 gene construct (G6.8-Luc) the deletion of a central subdomain of intron 1 inhibited transcription >/=10-fold in transiently transfected erythroid SKT6 cells, and likewise inhibited high-level transcription in erythroid FDCW2ER-GATA1 cells. In parental myeloid FDCER cells, however, low-level transcription was largely unaffected by intron 1 deletions. Within intron 1, repeated GATA and Ap1 consensus elements in a central region are described which when linked directly to reporter cassettes promote transcription in erythroid SKT6 and FDCER-GATA1 cells at high rates. Moreover, GATA-1 activated transcription from this subdomain in 293 cells, and in SKT6 cells this subdomain footprinted in vivo. For stably integrated GFP reporter constructs in erythroid SKT6 cells, corroborating results were obtained. Deletion of intronic GATA and Ap1 motifs abrogated the activity of G6.8-pEGFP; activity was decreased by 43 and 56%, respectively, by the deletion of either motif; and the above 1800-base pair region of intron 1 per se was transcribed at rates uniformly greater than G6.8-pEGFP. Also described is the differential utilization of exons 1a and 1b among primary erythromegakaryocytic and myeloid cells.
GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 we performed a functional analysis of the FOG-2 protein. The results demonstrated that 1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins, 2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and 3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, we have identified a novel N-terminal domain of FOG-2 (amino acids 1-247) that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development.
The Notch signal transduction pathway is a highly conserved regulatory system that controls multiple developmental processes. We have established an erythroleukemia cell model to study how Notch regulates cell fate and erythroleukemic cell differentiation. K562 and HEL cells expressed the Notch-1 receptor and the Notch ligand Jagged-1. The stable expression of the constitutively active intracellular domain of Notch-1 (NIC-1) in K562 cells inhibited erythroid without affecting megakaryocytic maturation. Expression of antisense Notch-1 induced spontaneous erythroid maturation. Suppression of erythroid maturation by NIC-1 did not result from down-regulation of GATA-1 and TAL-1, transcription factors necessary for erythroid differentiation. Microarray gene expression analysis identified genes activated during erythroid maturation, and NIC-1 disrupted the maturation-dependent changes in the expression of these genes. These results show that NIC-1 alters the pattern of gene expression in K562 cells leading to a block in erythroid maturation and therefore suggest that Notch signaling may control the developmental potential of normal and malignant erythroid progenitor cells.
The erythroid-enriched transcription factor NF-E2 is composed of two subunits, p45 and p18, the former of which is mainly expressed in the hematopoietic system. We have isolated and characterized the mouse p45 NF-E2 gene; we show here that, similar to the human gene, the mouse gene has two alternative promoters, which are differentially active during development and in different hematopoietic cells. Transcripts from the distal promoter are present in both erythroid and myeloid cells; however, transcripts from an alternative proximal 1b promoter, lying in the first intron, are abundant in erythroid cells, but barely detectable in myeloid cells. During development, both transcripts are detectable in yolk sac, fetal liver, and bone marrow. Transfection experiments show that proximal promoter 1b has a strong activity in erythroid cells, which is completely dependent on the integrity of a palindromic GATA-1 binding site. In contrast, the distal promoter 1a is not active in this assay. When the promoter 1b is placed 3' to the promoter 1a and reporter gene, in an arrangement that resembles the natural one, it acts as an enhancer to stimulate the activity of the upstream promoter la.
The glycoprotein gp91(phox) is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91(phox) gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91(phox) promoter in various types of gp91(phox)-expressing cells. A 10-base pair (bp) region from bp -105 to -96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp -101 to -96 (-98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the -98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the -105/+12 construct in a -98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91(phox) gene in human eosinophils.
Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs 9-12). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.
The erythroid Krüppel-like factor (EKLF) is a key regulatory protein in globin gene expression. This zinc finger transcription factor is required for expression of the adult beta globin gene, and it has been suggested that it plays an important role in the developmental switch from fetal gamma to adult beta globin gene expression. We have previously described a sequence element in the distal promoter region of the mouse EKLF gene that is critical for the expression of this transcription factor. The element consists of an E box motif flanked by 2 GATA-1 binding sites. Here we demonstrate that mutation of the E box or the GATA-1 consensus sequences eliminates expression from the EKLF promoter in transgenic mice. These results confirm the importance of this activator element for in vivo expression of the EKLF gene. (Blood. 2000;95:1652-1655)
Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19(INK4D) expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19(INK4D) induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2. (Blood. 2000;95:1264-1273)
3'-Azido-3'-deoxythymidine (AZT) treatment in HIV-infected patients is limited by bone marrow suppression including neutropenia and anemia. Previous studies had shown a direct effect of high concentrations of this drug on globin gene expression in K-562 erythroleukemia cells. To better define the mechanism(s) of AZT-induced bone marrow toxicity, the present study evaluates these effects in more relevant human erythroid progenitor liquid cultures, because AZT is 100 times more toxic to human bone marrow cells than K-562 cells. At a clinically relevant concentration of 1 microM, AZT inhibited specifically erythroid cell growth by approximately 58% as compared with untreated cells. The percentage of cells synthesizing hemoglobin was decreased also by 47% in AZT-treated cells with beta-globin mRNA levels accounting for 0.27 pmol in treated cells as compared with 1.44 under control conditions while beta-actin levels remained unchanged. Under the same conditions, AZT inhibited the beta-globin chain synthesis by approximately 60% as compared with the control. Consistent with the data described above was the finding that a concentration as low as 0.1 microM of AZT decreased by almost 40% the binding level of the erythroid-specific transcription factor GATA-1. These findings demonstrate that AZT, at clinical relevant concentrations, specifically inhibits beta-globin gene expression in human erythroid progenitor liquid cell culture.
Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.
Gene targeting experiments have revealed that transcription factors such as c-Myb and GATA-1 play crucial roles during hematopoietic differentiation. c-Myb is necessary in the immature cells of almost every hematopoietic lineage and GATA-1 is essential for the development of the erythroid lineage. In addition, CREB-binding protein (CBP) acts as a transcriptional adapter for various transcription factors, including c-Myb and GATA-1. In this paper, we show that the transcription factors c-Myb and GATA-1 each inhibit the transcriptional activity of the other and that any possible bipartite complexes c-Myb, GATA-1, and CBP could be formed, but the tripartite complex was hardly formed. The exclusive binding of GATA-1 and c-Myb to CBP is probably the molecular basis for the mutual inhibition of their transcriptional activity. Our data suggest that cross-talk between these three factors might be important for hematopoietic differentiation and that CBP functions as a key molecule during the process.
A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.
A systemic approach is proposed, which makes it possible to increase the accuracy of recognition of functional sites in arbitrary DNA sequences. The approach is based on the Central limit theorem and consists in the averaging of a large number of recognitions of a particular site. To obtain a rather large number of recognitions within the framework of conventional methods of recognition, consensus, and frequency matrix, 20 novel oligonucleotide alphabets were used. The approach was used to study the binding sites of GATA-1 and C/EBP transcription factors. It was found that the averaged recognition of these sites is more precise than each of specific recognitions, which just follows from the Central limit theorem.
The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid lineage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remains unknown. To gain insights into its function, mouse HS5 was cloned and sequenced. Comparison of the HS5 sequences of mouse, human, and galago revealed two extensively conserved regions, designated HS5A and HS5B. DNase I hypersensitivity mapping revealed that two hypersensitive sites are located within the HS5A region (designated HS5A(major) and HS5A(minor)), and two are located within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, suggesting that these protein binding sites are implicated in the formation of HS5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific. HS5 was detected in murine brain but not in murine kidney or adult liver, suggesting that this site is not ubiquitous. The presence of GATA-1 and NF-E2 motifs (which are common features of the DNase I hypersensitive sites of the LCR) suggests that the HS5 is organized in a manner similar to that of the other HSs. Taken together, our results suggest that HS5 is an inherent component of the beta-globin locus control region.
Fli-1 is a proto-oncogene which is rearranged in tumors induced by three different retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identified several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from different cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/EBS element was bound by several factors that seemed Ets-related, one of which was found to be specifically expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identified to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.
We investigated the requirements for enhancer-promoter communication by using the human beta-globin locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the epsilon-globin gene in chromatinized minichromosomes in erythroid cells. Activation of globin genes during development is accompanied by localized alterations of chromatin structure, and CACCC binding factors and GATA-1, which interact with both globin promoters and the LCR, are believed to be critical for globin gene transcription activation. We found that an HS2 element mutated in its GATA motif failed to remodel the epsilon-globin promoter or activate transcription yet HS2 nuclease accessibility did not change. Accessibility and transcription were reduced at promoters with mutated GATA-1 or CACCC sites. Strikingly, these mutations also resulted in reduced accessibility at HS2. In the absence of a globin gene, HS2 is similarly resistant to nuclease digestion. In contrast to observations in Saccharomyces cerevisiae, HS2-dependent promoter remodeling was diminished when we mutated the TATA box, crippling transcription. This mutation also reduced HS2 accessibility. The results indicate that the epsilon-globin promoter and HS2 interact both structurally and functionally and that both upstream activators and the basal transcription apparatus contribute to the interaction. Further, at least in this instance, transcription activation and promoter remodeling by a distant enhancer are not separable.
The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.
Gata1 is expressed from either one of two alternative promoters, the erythroid (proximal to the AUG) and the testis (distal to the AUG) promoter, both used by hemopoietic cells. To clarify the role of the distal and proximal Gata1 transcripts in erythroid differentiation, we determined by specific reverse transcriptase-polymerase chain reactions their relative levels of expression during the differentiation of erythroid precursors purified from the spleen of mice treated with phenylhydrazine (PHZ) or infected with the anemia-inducing strain of the Friend virus (FVA cells). PHZ cells are erythroid precursors that progress in vivo to erythroblasts in 3 days. Both PHZ and FVA cells synchronously proliferate and differentiate in vitro in the presence of erythropoietin (EPO). The levels of total and of distal, but not of proximal, Gata1 transcripts increased by five- to eightfold during in vivo and in vitro differentiation of FVA and PHZ cells. The increase in expression was temporally associated with an increase in the expression of Eklf, Scl, and Nfe2, three genes required for erythroid differentiation, and preceded by 24 h the repression of Gata2 and Myb expression. The day 1 PHZ cells that survived 18 h in the absence of EPO do not express globin genes and express detectable levels of distal but not of proximal Gata1 transcripts. These cells activate the expression of the globin genes within 2 h when exposed to EPO. Therefore, during erythroid differentiation of primary cells, increased expression of distal Gata1 transcripts underlies the increase in the expression of total Gata1 associated with the establishment of the erythroid differentiation program.
The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is functionally implicated in their survival and proliferation. We have used estrogen and tamoxifen-inducible forms of GATA-2 to modulate the levels of GATA-2 in the IL-3-dependent multipotential hematopoietic progenitor cell model FDCP mix. Ligand-dependent induction of exogenous GATA-2 activity did not rescue cells deprived of IL-3 from apoptosis. However, induction of GATA-2 activity in cells cultured in IL-3 blocked factor-dependent self-renewal but not factor-dependent survival: Cells undergo cell cycle arrest and cease proliferating but do not apoptose. This was accompanied by differentiation down the monocytic and granulocytic pathways. Differentiation occurred in the presence of IL-3 and did not require addition of exogenous differentiation growth factors such as G-CSF or GM-CSF normally required to induce granulomonocytic differentiation of FDCP-mix cells. Conversely, EPO-dependent erythroid differentiation was inhibited by GATA-2 activation. These biological effects were obtained with levels of exogenous GATA-2 representing less than twofold increases over endogenous GATA-2 levels and were not observed in cells overexpressing GATA-1/ER. Similar effects on proliferation and differentiation were also observed in primary progenitor cells, freshly isolated from murine bone marrow and transduced with a GATA-2/ER-containing retrovirus. Taken together, these data suggest that threshold activities of GATA-2 in hematopoietic progenitor cells are a critical determinant in influencing self-renewal versus differentiation outcomes.
The process through which multipotential hematopoietic cells commit to distinct lineages involves the induction of specific transcription factors. PU.1 (also known as Spi-1) and GATA-1 are transcription factors essential for the development of myeloid and erythroid lineages, respectively. Overexpression of PU.1 and GATA-1 can block differentiation in lineages in which they normally are down-regulated, indicating that not only positive but negative regulation of these factors plays a role in normal hematopoietic lineage development. Here we demonstrate that a region of the PU.1 Ets domain (the winged helix-turn-helix wing) interacts with the conserved carboxyl-terminal zinc finger of GATA-1 and GATA-2 and that GATA proteins inhibit PU.1 transactivation of critical myeloid target genes. We demonstrate further that GATA inhibits binding of PU.1 to c-Jun, a critical coactivator of PU.1 transactivation of myeloid promoters. Finally, PU.1 protein can inhibit both GATA-1 and GATA-2 transactivation function. Our results suggest that interactions between PU.1 and GATA proteins play a critical role in the decision of stem cells to commit to erythroid vs. myeloid lineages.
Protein 4.2 (P4.2) is an important component in the erythrocyte membrane skeletal network that regulates the stability and flexibility of erythrocytes. Recently, we provided the evidence for specific P4.2 expression in erythroid cells during development (L. Zhu et al., 1998, Blood 91, 695-705). Using dimethyl sulfoxide (DMSO)-induced differentiation of murine erythroleukemia (MEL) cells as a model, transcription of the P4.2 gene was found to be induced during erythroid differentiation. To examine the mechanism for this induction, we isolated the mouse P4.2 genomic DNA containing the 5' flanking sequence and defined the location of the P4.2 promoter. Transcription of the mouse P4.2 gene initiates at multiple sites, with the major initiation site mapped at 174 nucleotides upstream of the ATG start codon. The mouse P4.2 promoter is TATA-less and contains multiple potential binding sites for erythroid transcription factors GATA-1, NF-E2, EKLF, and tal-1/SCL. Transient transfection experiments demonstrated that a 1.7-kb mouse P4.2 promoter fused with the luciferase coding regions was induced in DMSO-treated MEL cells. Deletion analysis showed that a 259-bp P4.2 promoter DNA (nucleotide position -88 to +171 relative to the major transcription initiation site designated +1), containing a GATA-binding site at position -29 to -24, could still respond to the induction in differentiated MEL cells. Importantly, mutations in the -29/-24 GATA motif rendered the promoter unresponsive to DMSO induction. Electrophoretic mobility shift assay revealed that GATA-1 could bind to the -29/-24 GATA motif and this was confirmed by the observation that the nuclear protein bound to the motif was supershifted by an anti-GATA-1 monoclonal antibody. Taken together, these results suggest that the erythroid transcription factor GATA-1 plays an important role in the induction of P4.2 gene expression during erythroid cell differentiation.
Key regulatory regions necessary for the expression of the gene encoding FcepsilonRI alpha-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, -74/-69 and -55/-47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the alpha-chain promoter. Both the regulatory elements enhanced the promoter activity only in alpha-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the alpha-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcepsilonRI alpha-chain.
The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals.
Various strategies led to the identification of transcription factors that take part to the control of different steps during the formation of new blood vessels: the description of the expression pattern of genes encoding these factors during embryonic development for ETS-1, ERG and FLI, SCL/TAL, GATA1 and 2, the description of the phenotype of embryos obtained after gene inactivation by homologous recombination for ARNT or LKLF, and the study of transcriptional regulation in cultured endothelial cells for EGR1 or HOX-D3. Altogether, these results showed that there is no transcription factor specific for endothelial cells or for one step in the formation of blood vessels. Rather, factors controlling gene expression induced by hypoxia, shear-stress or growth factors take part in the morphogenesis of the vascular tree. The study of these factors may allow to identify potential therapeutic targets for treatments aimed at inhibiting or stimulating the development of new blood vessels.
The analysis of functional DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. The organization of erythroid LCRs is complex, consisting of multiple, interdigested cis elements. As in situ binding to these sites is determined by the accessibility of these regulatory regions in native chromatin and the availability of relevent cell-specific and ubiquitous factors, in vivo footprinting was used to define protein DNA interactions in human globin LCRs. To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine resides, rather than exclusively at guanines. This modification, termed GA-LMPCR in vivo footprinting, was essential for the analysis of GATA-1 motifs in the alpha-LCR and HS-3 of the beta-LCR. Moreover, GA-LMPCR in vivo footprinting provided high-resolution analysis of AP-1/NF-E2 elements and revealed protein contacts at sequences that are not coincident with previously described regulatory motifs. A comprehensive discussion of this modification and sample illustrations from our studies have been presented to demonstrate the enhanced detection and resolution obtained with this procedure.
The Wilms' tumor protein, WT1, represses transcription from several growth factor genes. WT1 transcription is regulated in erythroid and myeloid lineages by the transcription factor GATA-1. Using a sensitive, isotopic duplex RT-PCR procedure amplifying WT1 or GATA-1 together with beta-actin as the internal control in a single reaction mix, we quantitated the expression of WT1 and GATA-1 mRNA of 16 patients with myelodysplastic syndrome (MDS), 56 with acute myeloid leukemia (AML) and 22 with acute lymphoblastic leukemia (ALL). K562 was used as reference positive control for this cell line expresses both WT1 and GATA-1. Among MDS patients, increased WT1 expression was found in refractory anemia with excess blast (RAEB) and RAEB in transformation (RAEB-T) subtypes compared to the normal controls, whereas WT1 expression in refractory anemia (RA) was not different from the normal control level. All of AML cases of subtypes M0, M1, M2 and M3 expressed WT1 more than three times the normal WT1 level. Subtypes M4 to M7 showed significantly lower WT1 levels than M1 to M3 and AML cases with CD14+ expressed less WT1 than CD14-. Higher than normal WT1 levels were also expressed in cases of ALL.
The chromatin structure of the human beta-globin gene locus assumes a transcriptionally-active conformation in erythroid cells. One feature of this chromatin reorganization is the formation of DNase 1 hypersensitive sites in the regions of active globin gene promoters. This reorganization requires the globin locus control region and is associated with normal expression of the beta-like globin genes. To determine whether it is possible to artificially enhance the opening of the chromatin structure of a minimal beta-globin promoter, we placed a 101bp, erythroid-specific DNase 1 hypersensitive site-forming element (HSFE) immediately upstream of the beta-globin promoter and gene. This element includes binding sites for NF-E2, AP-1, GATA-1 and Sp-1. Constructs were stably transfected into murine erythroleukemia cells and promoter chromatin structure and gene expression were analyzed. The HSFE induced an area of enhanced DNase 1 hypersensitivity extending from the transcriptional start site to -300bp of the artificial promoter and significantly increased the proportion of beta-globin promoters in an open chromatin configuration. This remodeling of promoter chromatin structure resulted in 3-fold increases in beta-globin gene transcription and induction, and inhibited long-term beta-globin gene silencing. These results indicate that a relatively small cis-acting element is able to enhance remodeling of promoter chromatin structure resulting in increased beta-globin gene expression.
The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.
Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.
Friend of GATA-1 (FOG-1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA-binding protein GATA-1 and modulate its transcriptional activity. Recently, two new members of the FOG family have been identified: a mammalian protein, FOG-2, that also associates with GATA-1 and other mammalian GATA factors; and U-shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier. FOG proteins contain multiple zinc fingers and it has been shown previously that the sixth finger of FOG-1 interacts specifically with the N-finger but not the C-finger of GATA-1. Here we show that fingers 1, 5 and 9 of FOG-1 also interact with the N-finger of GATA-1 and that FOG-2 and U-shaped also contain multiple GATA-interacting fingers. We define the key contact residues and show that these residues are highly conserved in GATA-interacting fingers. We examine the effect of selectively mutating the four interacting fingers of FOG-1 and show that each contributes to FOG-1's ability to modulate GATA-1 activity. Finally, we show that FOG-1 can repress GATA-1-mediated activation and present evidence that this ability involves the recently described CtBP co-repressor proteins that recognize all known FOG proteins.
Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.
In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1-deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIbalpha, GPIbbeta, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1-deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.
Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.
GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.
We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.
Butyric acid (BA) is known to induce overexpression of fetal hemoglobin and then erythroid differentiation. Therefore, BA is currently under clinical investigation as a potential therapy for the treatment of sickle cell disease and cancer. Nevertheless, the molecular mechanisms involved in BA-induced differentiation remain largely unknown. Previous reports have shown that BA-induced overexpression of erythroid genes occurred at the transcriptional level, suggesting the involvement of erythroid transcription factors. Here, we intend to demonstrate the requirement of GATA-1 and NF-E2 transcription factors in the BA-induced erythroid differentiation of human leukemic K562 cells. Time-course experiments showed that nuclear levels of GATA-1 and p45 NF-E2 proteins increased during BA treatment. Moreover, antisense oligodeoxynucleotides targeting either GATA-1 or p45 NF-E2 proteins inhibited both protein expression and BA-induced differentiation. In contrast, BA-induced cell growth inhibition was not affected. These results provide the first direct evidence for the requirement of GATA-1 and NF-E2 in BA-induced differentiation process.
GATA-1 is a transcription factor required for development of erythroid cells. The expression of GATA-1 is tightly restricted to the hematopoietic lineage. Using transgene constructs containing zebrafish GATA-1 genomic sequences and the green fluorescent protein (GFP) reporter gene, we previously showed that a 5.6-kb enhancer/promoter fragment is sufficient to direct erythroid-specific expression of the GFP. In this study, we used enhancer/promoter fragments containing various deletion and point mutations to further characterize the cis-acting elements controlling tissue-specific GATA-1 expression. We report here the identification of distinct cis-acting elements that cooperate to confer on GATA-1 its hematopoietic expression pattern. A CACCC box, located 142 bp upstream of the translation start codon, is critical for the initiation of GATA-1 expression. A distal double GATA element is required for maintaining and enhancing the hematopoietic expression of GATA-1. The erythroid-specific activity of the GATA-1 promoter is also enhanced by a 49-bp sequence element located 218 bp upstream of the CACCC element and a CCAAT box adjacent to the double GATA motif. Finally, the hematopoietic specificity of the GATA-1 promoter is secured by a negative cis-acting element that inhibits expression in the notochord.
In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions -880, -75 and -135, -30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either -75Ets, -135GATA, or -30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel-retardation assays revealed that Ets-1 and GATA-1 proteins from HEL and MEG-01 cells bound to the Ets motifs and GATA motifs, respectively. Co-transfection experiments showed that the overexpression of Ets-1 and/or GATA-1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets-1 and GATA-1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets-1 increased 10-fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets-1 and GATA-1 play key roles in the positive regulation of PF4 gene expression.
A unique A/T-rich sequence (5'-AAAAAGTAAAAA-GTAAAAAAGTAAAAAG-3), referred to as the AGTA repeat, is found in the silencer region of the pumpkin ascorbate oxidase gene. A cDNA for protein (AOBP) that binds to the AGTA repeat was isolated from pumpkin by the southwestern method. The AOBP protein has a new class of zinc/DNA-binding domain named Dof/MOA domain that is highly conserved in many plant proteins and is significantly related to those of steroid hormone receptors and GATA1. Gel retardation analysis indicated that AOBP bound to the AGTA repeat through the Dof/MOA domain. Metal chelators, 1,10-phenanthroline and EDTA, specifically inhibited the DNA binding of AOBP, indicating that metal coordination plays an important role in DNA binding of AOBP. Thus, the Dof/MOA domain acts as a zinc/DNA-binding domain in AOBP. Gel retardation analysis with mutated oligonucleotides suggested that the Dof/MOA domain recognized the AGTA core sequence. AOBP mRNA was expressed in mature tissues of pumpkin, but was expressed only in small amounts or was not expressed in growing tissues. Furthermore, the expression was auxin-independent. The expression pattern of AOBP and that of ascorbate oxidase did not show a positive correlation.
Modification of histones, DNA-binding proteins found in chromatin, by addition of acetyl groups occurs to a greater degree when the histones are associated with transcriptionally active DNA. A breakthrough in understanding how this acetylation is mediated was the discovery that various transcriptional co-activator proteins have intrinsic histone acetyltransferase activity (for example, Gcn5p, PCAF, TAF(II)250 and p300/CBP. These acetyltransferases also modify certain transcription factors (TFIIEbeta, TFIIF, EKLF and p53). GATA-1 is an important transcription factor in the haematopoietic lineage and is essential for terminal differentiation of erythrocytes and megakaryocytes. It is associated in vivo with the acetyltransferase p300/CBP. Here we report that GATA-1 is acetylated in vitro by p300. This significantly increases the amount of GATA-1 bound to DNA and alters the mobility of GATA-1-DNA complexes, suggestive of a conformational change in GATA-1. GATA-1 is also acetylated in vivo and acetylation directly stimulates GATA-1-dependent transcription. Mutagenesis of important acetylated residues shows that there is a relationship between the acetylation and in vivo function of GATA-1. We propose that acetylation of transcription factors can alter interactions between these factors and DNA and among different transcription factors, and is an integral part of transcription and differentiation processes.
Blood formation (hematopoiesis) entails the generation of hematopoietic stem cells (HSCs) within the embryo and subsequent commitment of multipotential progenitors to differentiation along single lineages. These processes are controlled in large part by cell-restricted transcription factors which cooperate with more widely expressed factors to direct lineage-specific gene expression. Candidate hematopoietic transcriptional regulators have been identified by characterizing factors mediating cell-specific gene transcription and by defining genes involved in chromosomal rearrangements in leukemia. The application of transgenic and embryonic stem cell methods have provided insights into their in vivo functions and suggested mechanisms by which lineage selection may be achieved. One of the first, and best, characterized hematopoietic transcription factors is GATA-1. Herein studies of GATA-1 are reviewed to illustrate how manipulations of its locus in the mouse have contributed to current understanding in unique and unexpected ways.
Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.
Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of epsilony-globin mRNA, the blastlike cells accumulated in the definitive stage showed beta-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that the GATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.
Previously we have shown that overexpression of PU.1, an Ets family transcription factor, in murine erythroleukemia (MEL) cells results in apoptotic cell death in the presence of the differentiation-inducing reagent dimethyl sulfoxide (DMSO). In this study, we examined the dynamics of GATA-1 and NF-E2 hematopoietic transcription factors during the induction of apoptosis, because GATA-1 has been shown to be implicated in survival of erythroid cells. Formation of the GATA-1-DNA complex as judged by EMSA was markedly reduced when apoptosis was induced, although subcellular localization of the GATA-1 protein and expression levels of the GATA-1 mRNA and protein were not changed during the apoptotic process. Complex formation was not reduced when apoptosis was avoided by adding 30% serum in culture medium and when mutant PU.1 proteins with the deletion of the DNA-binding (Ets) or transactivation domain were expressed. Complex formation in nuclear extracts of parental MEL cells was reduced when they were mixed with those of apoptotic cells, suggesting that apoptotic cells may contain a factor(s) preventing GATA-1 from binding to DNA. In contrast to GATA-1, formation of the NF-E2-DNA complex was not changed during the process of apoptosis, although the expression level of the NF-E2 p45 gene was reduced in the process. These results suggest that reduction of the DNA-binding activity of GATA-1 may partly account for PU.1-mediated apoptosis in MEL cells.
We have isolated and characterized a human glutathione transferase A4 (hGSTA4) subunit gene from a yeast artificial chromosome containing several other glutathione transferase alpha genes and pseudogenes. The homodimeric protein hGSTA4-4, is involved in the detoxification of 4-hydroxynonenal and other reactive electrophiles produced by oxidative metabolism, and may have a significant role in protecting intracellular components from oxidative damage. The hGSTA4 gene spans nearly 18 kb, contains seven exons, maps onto chromosome 6p12, and lies in close proximity to the 7SK small nuclear RNA gene in a head-to-tail orientation. The intron/exon borders conform to the standard rules, an open reading frame is present beginning at position 154 in exon 2, and the stop codon is at position 822 in exon 7. The transcription initiation site has been determined by primer extension analysis and is located 135 bp upstream of intron 1. Isolation and sequencing of the hGSTA4 gene 5'-flanking region revealed it to be devoid of TATA or CCAAT boxes but it does contain an initiator element overlapping the transcription start site, a GC box and putative binding sites for transcription factors AP1, STAT, GATA1 and NF-kappaB. Reverse transcription-PCR analysis revealed that hGSTA4 mRNA was present in all the tissues tested, although in low amounts, suggesting that this subunit may be ubiquitously expressed.
We examined expression of the erythroid-associated genes GATA-1 and erythropoietin receptor (EPOR) in primary leukemia using the reverse transcriptase-polymerase chain reaction (RT-PCR). GATA-1 and EPOR mRNAs were detectable in all cases of erythroleukemia (French-American-British classification: M6) or early erythroblastic leukemia. In all other leukemia cases, including M2 through M5, stem cell leukemia, and adult T-cell leukemia, these gene transcripts were undetectable. GATA-2 was detectable in all the cases of primary leukemias examined in this study, except one case of M5. In one case, the phenotype switched from myeloid (M2) to erythroid (M6) and then back to myeloid. Northern blotting and RT-PCR revealed that GATA-1 and EPOR mRNAs were significantly upregulated at the M6 stage compared with the M2 stage. GATA-1 may be involved in the expression of an erythroid phenotype in acute leukemia. We generated HL-60 transfectants exogenously expressing GATA-1. The majority of HL-60 cells expressing GATA-1 lacked azurophilic granules, and electron microscopic analysis revealed that myeloperoxidase activity was negative. Platelet peroxidase activity, which was detectable in both megakaryoblasts and erythroid progenitors, was positive. However, EPOR and glycophorin A mRNAs were undetectable by RT-PCR. These findings suggest that besides GATA-1, a third factor may be required for the expression of mature erythroid phenotypes. In addition, our results indicate that GATA-1 is involved in inactivation of myeloperoxidase and activation of the platelet peroxidase.
Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.
Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.
Mechanisms of platelet production and release by mammalian megakaryocytes are poorly understood. We used thrombocytopenic knockout mice to better understand these processes. Proplatelets are filamentous extensions of terminally differentiated megakaryocytes that are thought to represent one mechanism of platelet release; however, these structures have largely been recognized in cultured cells and there has been no correlation between thrombocytopoiesis in vivo and proplatelet formation. Mice lacking transcription factor NF-E2 have a late arrest in megakaryocyte maturation, resulting in profound thrombocytopenia. In contrast to normal megakaryocytes, which generate abundant proplatelets, cells from these mice never produce proplatelets, even after prolonged stimulation with c-Mpl ligand. Similarly, megakaryocytes from thrombocytopenic mice with lineage-selective loss of transcription factor GATA-1 produce proplatelets very rarely. These findings establish a significant correlation between thrombocytopoiesis and proplatelet formation and suggest that the latter represents a physiologic mechanism of platelet release. We further show that proplatelet formation by normal megakaryocytes and its absence in cells lacking NF-E2 are independent of interactions with adherent (stromal) cells. Similarly, thrombocytopenia in NF-E2(-/-) mice reflects intrinsic defects in the megakaryocyte lineage. These observations improve our understanding of platelet production and validate the study of proplatelets in probing the underlying mechanisms.
As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.
The murine delayed-early serum-responsive gene T1 encodes glycoproteins of the interleukin-1 receptor family. Transcriptional initiation in fibroblasts is regulated by c-Fos and gives rise to a rare 5-kb mRNA and an abundant 2.7-kb mRNA. These transcripts are translated into a receptor-like membrane-anchored protein and a secreted protein consisting only of the ectodomain. In mast cells, T1 gene transcription is initiated 10.5 kb further upstream than in fibroblasts and gives rise predominantly to the 5-kb transcript under normal growth conditions. Here we demonstrate that calcium ionophore stimulation of mast cells resulted in an upregulation of T1 gene expression and a switch from the long to the short T1 transcript. This was paralleled by the disappearance of the receptor-type T1 protein on the mast cell surface and the secretion of large amounts of the truncated T1 protein. c-Fos and a T1 enhancer, which have previously been identified to be essential for T1 expression in fibroblasts, were not required for calcium ionophore-mediated T1 gene upregulation. Overexpression of the transcription factor GATA-1 in mast cells caused elevated T1 synthesis. Three GATA elements were identified in the minimal GATA-responsive mast cell promoter. Mutational analysis revealed that all three GATA elements are involved in T1 gene expression. Point mutations within the middle GATA element eliminated promoter activity completely, while mutations of the distal and proximal GATA binding sites reduced promoter strength by factors of 2 and 5, respectively. Exogenous expression of GATA-1 was not sufficient to activate the mast cell-specific promoter in NIH 3T3 fibroblasts.
Human TR2 orphan receptor, isolated from the testis and prostate, is a member of the steroid/thyroid hormone receptor superfamily. With the screening of a human genomic library and the combination of primer walking and PCR sequencing, we found that the entire TR2 orphan receptor gene coding region and 5'-untranslated region feature 13 introns and 14 exons, and that the consensus splice sequences (GT-AG) are present in all intron-exon boundaries. Within the region that codes for the DNA binding domain, TR2 orphan receptor gene has a distinct intron-exon junction. Whereas all other known steroid receptors have one splice site that separates their first and second zinc fingers in the DNA binding domain, TR2 orphan receptor has a rare splice site located in the middle of its first zinc finger. The identification of specific junction sequences for potential alternative splicing sites helps to explain the existence of multiple forms of TR2 orphan receptor cDNA (TR2-5, 7, 9, 11). The S1 nuclease protection assay for TR2 message revealed that there are multiple transcription initiations, and that the major cap site surrounded by an initiator-like sequence is located at the 104th nucleotide upstream from the translation start codon. Sequence analysis of a 2.7-kb DNA fragment upstream of the TR2 orphan receptor translation start codon unveiled several potential cis-acting elements, such as AP-1, HNF-5, GATA1 binding sites, and GC boxes. Using fluorescence in situ hybridization combined with a high-resolution G-banding technique, we found that the TR2 orphan receptor gene was mapped to human chromosome 12 at band q22, whereas the structurally closely related TR4 orphan receptor gene was mapped to human chromosome 3 at band q24.3.
The TAL1 gene is disrupted by translocation or deletion (tal(d)) in up to 30% of T-cell acute lymphoblastic leukaemia (T-ALL), leading to aberrant transcriptional activation, as a SIL-TAL1 fused transcript in tal(d). It has been suggested that TAL1 transcription occurs in approximately 50% of a T-ALLs without apparent rearrangement. SIL-TAL1 was positive in 15/60 (25%) of T-ALL, whereas wild-type TAL1 transcripts were detected in all 13 SIL-TAL1 and in 19/43 (44%) T-ALL without SIL-TAL1. To investigate the cellular origin of TAL1 we exploited the fact that GATA1 and TAL1 are co-ordinately expressed in non-lymphoid haemopoietic cells, whereas only the latter is found in T-ALL. GATA1 was detected in 10/23 (43%) TAL1-negative T-ALLs but in 17/19 (89%) 'unexplained' TAL1-positive cases, suggesting a common non-lymphoid cellular origin. Immunocytochemical analysis with a TAL1-specific monoclonal antibody showed nuclear expression in the blasts of 10/34 (29%) cases, including 8/10 SIL-TAL1+ and two RT-PCR TAL1+, SIL-TAL1- cases. In the remaining cases TAL1 expression was restricted to a minor population (< 5%) of larger, strongly TAL1-positive cells which comprised erythroid cells, CD34+ CD3- precursors and an unidentified TAL1+ CD45- population which morphologically resembled monocytes/macrophages. We therefore suggest that appropriate diagnostic evaluation of T-ALL should include molecular detection of SIL-TAL1 transcripts and in situ immunocytochemical detection of TAL1 protein expression by leukaemic blasts. This approach will enable accurate analysis of the prognostic significance of TAL1 deregulation in T-ALL.
Little is known about the transcription factors that mediate lineage commitment of multipotent hematopoietic precursors. One candidate is the Ets family transcription factor PU.1, which is expressed in myeloid and B cells and is required for the development of both these lineages. We show here that the factor specifically instructs transformed multipotent hematopoietic progenitors to differentiate along the myeloid lineage. This involves not only the up-regulation of myeloid-specific cell surface antigens and the acquisition of myeloid growth-factor dependence but also the down-regulation of progenitor/thrombocyte-specific cell-surface markers and GATA-1. Both effects require an intact PU.1 transactivation domain. Whereas sustained activation of an inducible form of the factor leads to myeloid lineage commitment, short-term activation leads to the formation of immature eosinophils, indicating the existence of a bilineage intermediate. Our results suggest that PU.1 induces myeloid lineage commitment by the suppression of a master regulator of nonmyeloid genes (such as GATA-1) and the concomitant activation of multiple myeloid genes.
AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.
The GATA-1 gene encodes a transcription factor expressed in early multipotent haemopoietic progenitors, in more mature cells of the erythroid, megakaryocytic and other lineages, but not in late myeloid precursors; its function is essential for the normal development of the erythroid and megakaryocytic system. To define regulatory elements of the mouse GATA-1 gene, we mapped DNaseI-hypersensitive sites in nuclei of erythroid and haemopoietic progenitor cells. Five sites were detected. The two upstream sites, site 1 and site 2, represent a new and a previously defined erythroid enhancer respectively. The site 1 enhancer activity depends both on a GATA-binding site (also footprinted in vivo) and on several sites capable of binding relatively ubiquitous factors. A DNA fragment encompassing site 1, placed upstream of a GATA-1 minimal promoter, is able to drive expression of a simian virus 40 (SV40) T-antigen in the yolk sac, but not bone-marrow cells, obtained from mice transgenic for this construct, allowing in vitro establishment of immortalized yolk-sac cells. A similar construct including site 2, instead of site 1, and previously shown to be able to immortalize adult marrow cells is not significantly active in yolk-sac cells. Sites 4 and 5, located in the first large intron, have no enhancer activity; they include a long array of potential Ets-binding sites. MnlI restriction sites, overlapping some of the Ets sites, are highly accessible, in intact nuclei, to MnlI. Although these sites are present in all GATA-1-expressing cells studied, they are the only strong sites detectable in FDCP-mix multipotent progenitor cells, most of which do not yet express GATA-1. The data indicate that appropriate GATA-1 regulation may require the co-operation of different regulatory elements acting at different stages of development and cell differentiation.
A transgenic mouse line (Tg.AC) carrying an activated v-Ha-ras oncogene fused to the embryonic zeta-globin promoter develops an array of spontaneous epithelial and mesenchymal neoplasms. In this report we describe the morphological, immunophenotypic, and molecular features of a unique hematopoietic neoplasm in these mice. The cardinal lesion of this disease is marked hepatomegaly due to leukemic proliferation and infiltration. In the peripheral blood, there is a marked increase in the number of metarubricytes and other less differentiated erythroid progenitor cells. Leukemic cells stain positively with an erythroid-associated nuclear transcription factor (GATA-1). Using a reverse transcription polymerase chain reaction assay, co-expression of GATA-1 and endogenous zeta-globin genes is detected in hematopoietic tissues of nonleukemic transgenic and nontransgenic mice. ras transgene expression is, however, detected only in normal bone marrow and leukemic tissues of transgenic mice, and 5' mapping experiments using S1 protection analysis of total RNA from leukemic tissue indicates that transcription of the transgene mRNA is initiated from the natural zeta-globin promoter start site, supporting the belief that the zeta-globin promoter directs v-Ha-ras expression in erythroid progenitor cells, ultimately leading to leukemic transformation.
To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem 272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to as GATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.
The EOS47 antigen is an early and specific marker of eosinophil differentiation in the chicken haematopoietic system. To elucidate the transciptional events controlling commitment to the eosinophil lineage, we studied the regulation of the eosinophil-specific EOS47 promoter. This promoter is TATA-less, and binds trancription factors of the Ets, C/EBP, GATA and Myb families. These sites are contained within a 309 bp promoter fragment which is sufficient for specific high level transcription in an eosinophil cell line. Co-transfection experiments in Q2bn fibroblasts showed cooperative activation of the EOS47 proximal promoter by c-Myb, Ets-1/Fli-1, GATA-1 and C/EBPalpha. The Ets-1/Fli-1 and C/EBPalpha proteins were the most potent activators, and acted with high synergy through juxtaposed binding sites located approximately 60 bp upstream of the transcription start site. The Ets-1 and C/EBPalpha proteins were found to associate physically via their DNA-binding domains and to bind their combined binding site cooperatively. GATA-1 showed biphasic regulation of the EOS47 promoter, activating at low and repressing at high protein concentrations. These results demonstrate combinatorial activation of an eosinophil-specific promoter by ubiquitous and lineage-restricted haematopoietic transcription factors. They also indicate that direct interactions between C/EBPs and specific Ets family members, together with GATA-1, are important for eosinophil lineage determination.
Transcription factors regulating the process of megakaryocyte development remain largely unclarified. To clarify them further, we used a human megakaryoblastic cell line, Meg-J, which showed prominant polyploidization and augmented platelet glycoprotein (GP) Ib expression after incubation with thrombopoietin (TPO, c-mpl ligand) and K252a (an indolocarbasole derivative). Under these conditions, we analyzed the expression of the transcription factors and observed that the expression of NF-E2 p45, but not those of GATA-1, GATA-2, Tal-1/SCL, Evi-1, and MafK, was increased after TPO and K252a stimulation. Gel-shift assay confirmed the enhanced binding activity to the NF-E2 site. The abolishment of NF-E2 p45 with NF-E2 antisense oligomers inhibited TPO plus K252a-induced polyploidization. These findings suggest that NF-E2 p45 is essential for the polyploidization of megakaryocytic cells.
Erythrocyte development has previously been shown to depend upon the expression of the lineage-restricted trans-acting factor GATA-1. Despite predicted roles for this factor during early development, GATA-1-deficient cells in chimeric mice and embryonic stem cell cultures mature to a late proerythroblast stage and express at least certain genes that normally are thought to be regulated by GATA-1 (including erythroid Krüppel-like factor [EKLF] and the erythropoietin [Epo] receptor). Opportunities to test roles for GATA-1 in erythroid gene activation in these systems therefore are limited. In the present study, in an alternate approach to test the function of GATA-1, GATA-1 has been expressed together with the Epo receptor in myeloid FDCW2 cells and the resulting effects on cytokine-dependent proliferation and erythroid gene expression have been assessed. GATA-1 expression at low levels delayed FDCW2ER cell cycle progression at the G1 phase specifically during Epo-induced mitogenesis. Upon expression of GATA-1 at increased levels, proliferation in response to Epo, interleukin-3 (IL-3), and stem cell factor was attenuated and endogenous GATA-1, EKLF and betamaj-globin gene expression was activated. Friend of GATA-1 (FOG) transcript levels also were enhanced, and ets-1 and c-mpl but not Epo receptor gene expression was induced. Finally, in FDCW2 cells expressing increased levels of GATA-1 and a carboxyl-terminally truncated Epo receptor, Epo (with respect to IL-3 as a control) was shown to markedly promote globin transcript expression. Thus, novel evidence for select hierarchical roles for GATA-1 and Epo in erythroid lineage specification is provided.
The solution structure of a complex between the DNA binding domain of a fungal GATA factor and a 13 base-pair oligonucleotide containing its physiologically relevant CGATAG target sequence has been determined by multidimensional nuclear magnetic resonance spectroscopy. The AREA DNA binding domain, from Aspergillus nidulans, possesses a single Cys2-Cys2 zinc finger module and a basic C-terminal tail, which recognize the CGATAG element via an extensive network of hydrophobic interactions with the bases in the major groove and numerous non-specific contacts along the sugar-phosphate backbone. The zinc finger core of the AREA DNA binding domain has the same global fold as that of the C-terminal DNA binding domain of chicken GATA-1. In contrast to the complex with the DNA binding domain of GATA-1 in which the basic C-terminal tail wraps around the DNA and lies in the minor groove, the structure of complex with the AREA DNA binding domain reveals that the C-terminal tail of the fungal domain runs parallel with the sugar phosphate backbone along the edge of the minor groove. This difference is principally attributed to amino acid substitutions at two positions of the AREA DNA binding domain (Val55, Asn62) relative to that of GATA-1 (Gly55, Lys62). The impact of the different C-terminal tail binding modes on the affinity and specificity of GATA factors is discussed.
Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify the cis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5' deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp -117 to -67 were deleted. To identify transcription factors that bind to and transactivate through the bp -117 to -67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.
While mast cells have been previously shown to express both GATA-1 and GATA-2 mRNAs, individual functions for these related factors during their course of differentiation within the mast cell lineage have not yet been defined. To address this question, the expression of GATA-1 and GATA-2 mRNAs and proteins were examined in three mouse mast cell progenitor lines as well as in mast cells isolated from both wild-type and GATA-1-deficient mice.
Both mast cell progenitor lines, as well as primary mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells (PMCs) were examined by RNA blotting and immunological analyses. GATA-2 protein was abundantly expressed in all three mast cell lines and in BMMCs, but only weakly in some of PMCs. In contrast, GATA-1 protein was expressed in PMCs and BMMCs after culture in the presence of IL3 and SCF. We also found the presence of Alcian blue staining-positive but berberine staining-negative mast cells in the skin of mice heterozygous to GATA-1 knock-down allele.
These results suggest that the expression of GATA factor-dependent genes is regulated by GATA-2 during mast cell development and that GATA-1 is required for the specification of differentiated mast cell phenotypes.
The chemokine receptor CCR5 is a cofactor for cellular entry of macrophage-tropic strains of HIV-1. Expression of CCR5 is restricted to T cells, macrophages, and certain cell lines; however, the mechanisms controlling its expression remain largely unknown. To delineate these mechanisms, approximately 1.0 kb of DNA from the immediate 5' upstream region of CCR5 was cloned and characterized. CCR5 promoter activity was up-regulated by PMA, and a region spanning -417 to +61 relative to the transcription start site was sufficient for the basal and induced activity. DNase I footprinting assays demonstrated several protected areas within this region, and gel shift assays determined binding sites for transcriptional factors Oct-1, Oct-2, T cell factor 1alpha, and GATA1. CCR5 promoter activity was also induced by IL-2 or anti-CD3 Ab, while stimulation with anti-CD28 Ab markedly reduced CD3-mediated up-regulation of the CCR5 promoter. Flow cytometry confirmed the findings at the level of cell surface expression. Further delineation of the regulation of the CCR5 promoter will be important for a more comprehensive understanding of the pathogenesis of HIV disease.
The transcription factor GATA-1 coordinates multiple events during terminal erythroid cell maturation. GATA-1 participates in the transcription of virtually all erythroid-specific genes, blocks apoptosis of precursor cells, and controls the balance between proliferation and cell cycle arrest. Prior studies suggest that the function of GATA-1 is mediated in part through association with transcriptional cofactors. CREB-binding protein (CBP) and its close relative p300 serve as coactivators for a variety of transcription factors involved in growth control and differentiation. We report here that CBP markedly stimulates GATA-1's transcriptional activity in transient transfection experiments in nonhematopoietic cells. GATA-1 and CBP also coimmunoprecipitate from nuclear extracts of erythroid cells. Interaction mapping pinpoints contact sites to the zinc finger region of GATA-1 and to the E1A-binding region of CBP. Expression of a conditional form of adenovirus E1A in murine erythroleukemia cells blocks differentiation and expression of endogenous GATA-1 target genes, whereas mutant forms of E1A unable to bind CBP/p300 have no effect. Our findings add GATA-1, and very likely other members of the GATA family, to the growing list of molecules implicated in the complex regulatory network surrounding CBP/p300.
The collagenase B type IV (Col4B) gene is highly expressed in the osteoclast, the primary bone-resorbing cell. However, factors that regulate expression of the Col4B gene are not well characterized. A murine P1 genomic clone containing a 94 kb sequence insert which contains the Col4B gene was isolated. A 4 kb EcoR1 DNA fragment containing the 5' flanking sequence of the gene was further subcloned and restriction mapped. Putative transcription factors such as SRY, Lyf-1, and GATA1 and 2, binding motifs were identified by sequence analysis in this promoter region. Enhancer and suppressor regions were mapped by transient expression of Col4B gene promoter deletion mutant-luciferase reporter gene constructs in HepG2 cells. Col4B mRNA expression in different murine tissues was analyzed by reverse transcription-polymerase chain reaction and demonstrated high levels of expression in bone, clavaria, spleen and thymus. This promoter provides a valuable tool for targeting gene expression to the osteoclast.
We have previously demonstrated that the basal transcription of rat inhibin alpha-subunit gene in a mouse testicular Leydig tumor cell line, MA-10, depends upon a 67-bp DNA fragment at the position of -163 to -97. Within this promoter region two GATA motifs were observed. In this study, we investigated the possible role of GATA-binding proteins in the regulation of inhibin alpha-subunit gene transcription in testicular cells. Northern blot and RT-PCR analyses showed that mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis and in MA-10 and rat Sertoli cells. Testis-specific GATA-1 mRNA, which is transcribed from a promoter 8 kb upstream to the erythroid exon I of mouse GATA-1 gene, was also identified in MA-10 cells. Mutations of GATA sequences in alpha-subunit promoter markedly decreased the transcriptional activity of alpha-subunit gene when measured by their ability of transient expression of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), in MA-10 cells. Cotransfection of alphaCAT chimeric construct with cDNA expression plasmid coding for mouse GATA-1 or GATA-4 protein revealed that GATA-1 but not GATA-4 can transactivate alpha-subunit promoter in a dose-dependent manner. The transactivation by GATA-1 was inhibited if GATA sequences in alpha-subunit promoter were mutated. Furthermore, electrophoretic mobility shift assay demonstrated that GATA-binding proteins present in nuclear extracts of MA-10 cells and rat testis interacted with the GATA motifs in alpha-subunit promoter, and the GATA-1 in these nuclear extracts formed a supershifted immunocomplex with antibody raised against mouse GATA-1 protein. We therefore concluded that the basal transcription of inhibin alpha-subunit gene in testicular MA-10 cells is up-regulated by testicular GATA-1 but not GATA-4 through its interaction with the GATA motifs in alpha-subunit promoter. In summary, we have provided the first evidence of the functional role of a GATA-binding protein in the regulation of testicular gene expression.
The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes, RCHE and RHD. Rh50 glyco-protein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression of RH50 gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5' flanking region of RH50 gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence of RH genes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter of RH50 gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Krüppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms for RH vs RH50 genes.
The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.
The "zinc-finger" transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and beta-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.
In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.
The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor which is expressed in a tissue-specific manner. Our studies indicate that in addition to the promoter, other regulatory elements are required for tissue-specific expression of this gene. A 258-base pair hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8-10-fold in K562 and HL60 cells. Sequence analysis revealed both a GATA and a c-Myb motif in the enhancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that the GATA-1 protein in K562 nuclear extracts binds to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 expression vector showed that GATA-1 transactivated this enhancer, increasing the CAT reporter activity 10-15-fold. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has also been mapped in the 258-base pair enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo.
Butyric acid (BA) was shown to induce hemoglobinization of K562 cells in a dose- and time-dependent manner. The maximal differentiation (54% of hemoglobinized cells) was obtained with the 0.5 mM concentration, which induced a 60% inhibition of cell growth at day 3 without cytotoxicity. Parallel to the kinetics of hemoglobinization, a rapid increase in gamma-globin and porphobilinogen deaminase (PBGD) mRNAs was observed in BA-treated cells. This increase was time-dependent and higher for gamma-globin than for PBGD (six- and two-fold at day 3, respectively). In contrast, erythropoietin receptor mRNAs were not affected by BA treatment. Analysis of erythroid transcription factor mRNA levels during the time course of BA treatment showed, for the first time, an early and marked (up to three-fold) increase in p45 NF-E2 mRNA, contrasting with that of GATA-1 mRNA (<1.5-fold). Taken together, these results showed the rapid differentiating effect of BA and suggest the involvement of the NF-E2 transcription factor.
The Wilms'-tumor gene WT1 may have a different function from a tumor-suppressor gene in some leukemias. Using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia system, we examined whether WT1 expression was involved during leukemogenesis, since this model enabled us to analyze cells altered by DMBA at various stages of leukemogenesis. By the semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) method, WT1 expression was detected in 15 (71%) of 21 DMBA-induced erythroblastic leukemias. Among 15 WT1-expressing leukemias, GATA-1, which is an erythroid-specific transcription factor and might regulate WT1 expression, was also expressed in 13 cases (p < 0.05). On the other hand, WT1 expression was not detected in any normal or early pre-leukemic rats and was detected in 1 of 8 rats in late pre-leukemic stages. These results showed that cells with a high expression level of WT1 tended to develop into leukemia and that WT1 contributed to leukemogenesis in the late stage, suggesting that the expression of WT1 plays an important role in cell proliferation and in maintaining the viability of some leukemia cells.
The transcription factor GATA-1, which is expressed in several hematopoietic lineages and multipotential progenitors, is required for the development of red blood cells and platelets. To identify control elements of the mouse GATA-1 gene, we analyzed DNase I hypersensitivity of the locus in erythroid chromatin and the expression of GATA-1/Escherichia coli beta-galactosidase (lacZ) transgenes in mice. Transgenes with 2.7 kb of promoter sequences are expressed infrequently and only within adult (definitive) erythroid cells. We show that inclusion of an upstream hypersensitive site (HS I) markedly enhances the frequency of expressing transgenic lines and activates expression in primitive erythroid cells. This pattern recapitulates the proper pattern of GATA-1 expression during development. By breeding a GATA-1/lacZ transgene into a GATA-1(-) background, we also have shown that the activation or maintenance of GATA-1 expression does not require the presence of GATA-1 itself, thereby excluding simple models of positive autoregulation. The transgene cassette reported here should be useful in directing expression of foreign sequences at the onset of hematopoiesis in the embryo and may assist in the identification of upstream regulators of the GATA-1 gene.
The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.
Transcription factor GATA-1 is essential for red blood cell maturation and, therefore, for survival of developing mouse embryos. GATA-1 is also expressed in megakaryocytes, mast cells, eosinophils, multipotential hematopoietic progenitors and Sertoli cells of the testis, where its functions have been elusive. Indeed, interpretation of gene function in conventional knockout mice is often limited by embryonic lethality or absence of mature cells of interest, creating the need for alternate methods to assess gene function in selected cell lineages. Emerging strategies for conditional gene inactivation through site-specific recombinases rely on the availability of mouse strains with high fidelity of transgene expression and efficient, tissue-restricted DNA excision. In an alternate approach, we modified sequences upstream of the GATA-1 locus in embryonic stem cells, including a DNase I-hypersensitive region. This resulted in generation of mice with selective loss of megakaryocyte GATA-1 expression, yet sufficient erythroid cell levels to avoid lethal anemia. The mutant mice have markedly reduced platelet numbers, associated with deregulated megakaryocyte proliferation and severely impaired cytoplasmic maturation. These findings reveal a critical role for GATA-1 in megakaryocyte growth regulation and platelet biogenesis, and illustrate how targeted mutation of cis-elements can generate lineage-specific knockout mice.
The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoDeltaHS) or lacking the selection cassette (DeltaneoDeltaHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoDeltaHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the DeltaneoDeltaHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the "knockdown" effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.
We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3), or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the alpha and beta subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and alpha- and beta-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (> or = 17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the alpha subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The beta subunits of the IL-3 and GM-CSF receptor, the alpha subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, alpha- and beta-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the alpha subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor, in particular, is activated both in erythroid (alpha- and beta-globin positive and in myeloid (alpha- and beta-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis.
To elucidate the in vivo function of GATA-1 during hematopoiesis, we specifically disrupted the erythroid promoter of the GATA-1 gene in embryonic stem cells and generated germ line chimeras. Male offspring of chimeras bearing the targeted mutation were found to die by 12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia to normal erythropoiesis. When female heterozygotes were crossed with transgenic males carrying a reporter gene, which specifically marks primitive erythroid progenitors, massive accumulation of undifferentiated erythroid cells were observed in the yolk sacs of the GATA-1-mutant embryos, demonstrating that GATA-1 is required for the terminal differentiation of primitive erythroid cells in vivo.
Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally controlled in vivo, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice. We found that a transcriptional control element located between -3.9 and -2.6 kb 5' to the erythroid first exon serves as an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells. The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5' element displays properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1 first intron specifically augments this activity in definitive erythroid cells.
Coproporphyrinogen oxidase (CPO; EC 1.3.3.3), the sixth enzyme of heme biosynthesis, transcribed from a single promoter is markedly induced during erythroid differentiation. CPO is ubiquitously expressed in all cells. To determine cis-acting elements of the human CPO gene, the promoter region of the gene was isolated, and three potential GATA-1 motifs and four GC boxes were found within this fragment. In a functional analysis of various deletion mutants, we found that the GATA-1 binding site at -143 to -138 was essential for basic and inducible expressions of the CPO gene in mouse erythroleukemia (MEL) cells. Gel mobility shift assay revealed that GATA-1 bound to the region is required for the expression and this was confirmed by observations that the nuclear protein bound to the GATA-1 motif was supershifted with anti GATA-1 antibody, by gel mobility shift assay. Furthermore, co-expression of mouse GATA-1 in MEL cells led to an increase in the promoter activity, which was markedly increased by dimethyl sulfoxide-treatment. These results indicate that GATA-1 plays an important role in regulation of transcription of the CPO gene in erythroid cells.
The molecular events which underlie lineage commitment and differentiation in hematopoietic cells are still incompletely understood. Microcell fusion is a versatile technique which has been utilized in characterizing and mapping genes involved in tumor suppression, cell senescence, and certain aspects of differentiation. Microcell fusion has the potential to contribute to the understanding of hematopoietic differentiation; however, application of this technique is limited by the need to use adherent cells as microcell donors, by the need to tag candidate chromosomes with a selectable marker, and by the need for prolonged selection of fused cells prior to characterization of their phenotype. We developed a modified technique of microcell fusion using square wave electroporation, which allows higher efficiency fusion than polyethylene glycol fusion. By using cross-species fusion and species-specific PCR primers, we were able to detect new gene induction events 48 h after microcell fusion. To study erythroid gene expression, we fused microcells from human erythroid K562 cells to murine B-lymphoid SP-2 cells. We found that microcell fusion induced the nonerythroid recipient cells to express alpha-globin mRNA in a dose-dependent manner. They also expressed RNA for beta-globin, GATA-1, and NF-E2. In contrast, there was no expression of heart- or liver-specific genes. We conclude that microcells from erythroid cells contain all the information necessary to induce expression of multiple erythroid genes. Analysis of the components of the microcells responsible for this new gene induction may allow the characterization of cellular factors responsible for erythroid-specific gene expression.
The LIM-only protein Lmo2, originally identified as an oncogenic protein in human T cell leukemia, is essential for erythropoiesis. A possible role for Lmo2 in transcription during erythropoiesis has been investigated. Direct interaction of Lmo2 was observed in vitro and in vivo with the zinc finger transcription factor GATA-1, as well as with the basic helix-loop-helix (bHLH) transcription factor Tall. By using mammalian two-hybrid analysis, E47/Tall/Lmo2/GATA-1 protein complex could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis. This data suggest that variations in amounts of complexes involving Lmo2, Tall, and GATA-1 could be important for erythroid differentiation.
The DLX gene family is a family of divergent homeobox genes which are related to the Drosophila distal-less (Dll) gene and has been reported to be expressed primarily in the forebrain and craniofacial structures. We have previously identified a new member of this family, DLX-7. We now report that this gene is expressed in normal hematopoietic cells and leukemia cell lines with erythroid characteristics. We used an antisense oligonucleotide targeted against the translation start site of DLX-7 mRNA to inhibit its expression in a human erythroleukemia cell line K562, which expresses DLX-7 at a high level. The antisense oligonucleotide efficiently reduced the DLX-7 mRNA, while control oligonucleotides, including a mutant oligonucleotide identical to the antisense sequence except for four nucleotide mismatches, had no effect on DLX-7 mRNA level. Inhibition of DLX-7 expression decreased the plating efficiency by approximately 70% compared with control. The antisense treatment caused apoptosis, as shown by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) method. Down-regulation of DLX-7 expression by antisense treatment was associated with a reduction in GATA-1 and c-myc mRNA levels. Thus, we conclude that DLX-7 is expressed in hematopoietic cells and that the inhibition of its expression results in the decreased levels of GATA-1 and c-myc genes, with an accompanying induction of apoptosis.
To test whether human GATA-1 (hGATA-1) is involved in the transcriptional control of globin gene switching, we produced transgenic mice overexpressing hGATA-1, crossed them with mice carrying a human beta-globin locus yeast artificial chromosome (beta YAC), and analyzed globin gene expression in their progeny. Mice carrying both the hGATA-1 and the beta YAC transgenes had normal levels of gamma- and beta-globin mRNA and no distortion in the rate or in the timing of gamma-to-beta switch, indicating that hGATA-1 is not involved in the developmental control of gamma- and beta-globin genes. In contrast, mice carrying the hGATA-1 and the beta YAC transgenes had 5- to 6-fold lower expression of the human epsilon globin gene compared with beta YAC mice lacking the hGATA-1 transgene. These results provide direct in vivo evidence that hGATA-1 is a specific repressor of human epsilon gene expression. Our findings also suggest that binary transgenic mouse systems based on overexpression of transcriptional factors can be used to investigate the trans control of human globin gene switching. Systems as the one we describe here should be useful in the study of any developmentally controlled human gene for which transgenic mice are available.
EVI1, located at chromosome band 3q26, encodes a 1051 amino acid zinc finger protein inappropriately expressed in the leukemic cells of 2-5% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. The activation of EVI1 often follows a chromosomal rearrangement involving band 3q26, and the two most frequent rearrangements are the t(3;3)(q21;q26) and the inv(3)(q21q26). EVI1 exists also as a longer protein that includes 188 additional amino acids at the N-terminus, named MDS1/EVI1. Both genes are expressed at very low levels in the normal bone marrow. The genomic region between the first coding exon of MDS1/EVI1 and the first coding exon of EVI1 is 150-300 kb. The majority of the chromosomal breakpoints at the 5' end of EVI1 in the t(3;3) resulting in EVI1 activation have been mapped in this region. As a consequence of the t(3;3), the cell would be unable to express MDS1/EVI1, although it would express EVI1. We have compared the transcriptional activity of MDS1/EVI1 and EVI1, and we show that MDS1/EVI1 is a strong activator of promoters containing the AGATA motif, whereas EVI1 is a repressor. In addition, whereas EVI1 represses activation by the GATA-1 erythroid factor, MDS1/EVI1 does not, and is itself repressed by EVI1. By gene fusion to the DNA-binding domain of Gal4, we further show that the activation properties of MDS1/EVI1 are restricted to an acidic segment encoded by the second and third exons in the 5' untranslated region of EVI1. We have also examined the relative expression of the two genes in normal bone marrow and in the bone marrow of leukemia patients with 3q26 rearrangements. Our results indicate that the rearrangements at 3q26 affect expression of EVI1, but not of MDS1/EVI1. We propose that rearrangements at 3q26 involving EVI1 could result in leukemia by a two-step process involving first transcriptional disruption of MDS1/EVI1, and next by inappropriately activating expression of EVI1.
Patients with myelodysplastic syndrome (MDS) have ineffective in vivo and in vitro erythropoiesis, characterized by an impaired response to erythropoietin (Epo). We examined proliferation and maturation of MDS marrow cells in response to Epo in more detail. Epo-dependent DNA synthesis as well as induction of GATA-1 binding activity in marrow cells from 15 MDS cases were severely reduced as compared with normal bone marrow (NBM). Additionally, the appearance of morphologically identifiable erythroid cells was decreased in MDS cell cultures. These data indicate that both the Epo-dependent proliferation as well as the differentiation induction by Epo is suppressed. To study more upstream events of the Epo signal transduction route we investigated activation of the signal transducer and activator of transcription (STAT) 5. In all 15 MDS samples tested, STAT5 activation was absent or greatly suppressed in response to Epo. In contrast, interleukin-3 induced a normal STAT5 response in MDS cells. Further, in MDS the subset of CD71+ BM cells that is phenotypically similar to Epo-responsive cells in normal marrow, was present. We conclude that the Epo response in MDS is disturbed at an early point in the Epo-receptor (EpoR) signal transduction pathway.
The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at -24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position -24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and transacting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved.
To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.
Hematopoiesis in the mouse conceptus begins in the visceral yolk (VYS), with primitive erythroblasts first evident in blood islands at the headfold stage (E8.0). VYS erythropoiesis is decreased or abrogated by targeted disruption of the hematopoietic transcription factors tal-1, rbtn2, GATA-1, and GATA-2. To better understand the potential roles of these genes, and to trace the initial temporal and spatial development of mammalian embryonic hematopoiesis, we examined their expression patterns, and that of betaH1-globin, in normal mouse conceptuses by means of in situ hybridization. Attention was focused on the 36-hour period from mid-primitive streak to early somite stages (E7.25 to E8.5), when the conceptus undergoes rapid morphologic changes with formation of the yolk sac and blood islands. Each of these genes was expressed in extraembryonic mesoderm, from which blood islands are derived. This VYS expression occurred in a defined temporal sequence: tal-1 and rbtn2 transcripts were detected earlier than the others, followed by GATA-2 and GATA-1, and then by betaH1-globin. Transcripts for all of these genes were present in VYS mesoderm cell masses at the neural plate stage (E7.5), indicating commitment of these cells to the erythroid lineage before the appearance of morphologically recognizable erythroblasts. By early somite stages (E8.5), GATA-2 mRNA expression is downregulated in VYS blood islands as terminal primitive erythroid differentiation proceeds. We conclude that primitive mammalian erythropoiesis arises during gastrulation through the ordered temporal expression of tal-1, rbtn2, GATA2, and GATA-1 in a subset of extraembryonic mesoderm cells. During the stages analyzed, tal-1 and rbtn2 expression was also present in posterior embryonic mesoderm, while GATA-1 and GATA-2 expression was evident in extraembryonic tissues of ectodermal origin.
GATA-1 is a transcription factor expressed both in the hematopoietic system and in the Sertoli cells of the testis, and is essential for correct erythropoiesis. Hematopoietic and Sertoli cells transcribe GATA-1 from two different promoters: the proximal (erythroid) is active in hematopoietic cells; the distal (testis) is active in Sertoli cells. We investigated by RT-PCR the possibility that GATA-1 might be transcribed from the testis promoter also in hematopoietic cells. Testis promoter-derived transcripts are present at low levels in vivo at all stages of hematopoietic development. Purified multipotent progenitors, fractionated into populations expressing low or high levels of GATA-1, do not contain any "testis" transcripts. However, when grown in vitro, they rapidly express GATA-1 from the testis promoter in the presence of Erythropoietin (Epo) but not in that of other growth factors. This result reflects an Epo-dependent differentiation event, rather than a direct effect of Epo. Indeed, immortalized progenitor cell lines which respond to both Epo and SCF, continue to express testis-derived transcripts when switched from Epo to SCF.
We have utilized Aspergillus nidulans as a model system for the characterization of the major vertebrate transcription factor GATA-1. This has been achieved both by analysing the function of murine GATA-1 directly and by using direct gene replacement to introduce chimaeric areA::GATA-1 derivatives at the areA locus, which encodes a GATA factor involved in regulating nitrogen metabolism in A. nidulans. Although GATA-1 shows only limited function when expressed in A. nidulans, the C-terminal GATA DNA-binding domain can replace the native GATA domain of AREA and retain near wild-type function. Surprisingly, inclusion of the N-terminal DNA-binding domain of GATA-1 has a major role in determining the function of areA::GATA constructs in vivo, leading to a general loss of activation. This negative function is partially dominant and is dependent on both the fidelity of the zinc-chelating structure and a second factor encoded by A. nidulans. The presence of two GATA domains also disrupts modulation of AREA activity. The ability of duplicate GATA domains to disrupt normal signal transduction is not dependent on the relative position of the domains or on the fidelity of the zinc-chelating structure. This demonstrates the utility of nitrogen metabolism's regulation in A. nidulans as a model system for the molecular and genetic characterization of heterologous GATA factors while also providing insights into native Aspergillus regulatory components.
GATA-1 is a tissue-specific DNA-binding protein containing two zinc-finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non-transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D-dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E-dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus.
All mature blood lineages in the peripheral circulation are derived from pluripotent haematopoietic stem cell. Progressive lineage-restriction of this stem cell is executed, in part, by the interplay and cross-talk between a host of lineage-restricted as well as ubiquitous transcription factors. To elucidate the regulatory mechanisms underlying the erythroid gene regulation, it is essential to understand how individual transcription factors contribute to the regulation of specific target genes, and how these erythroid transcription factor genes are regulated in turn. These key issues of mammalian development have been addressed by examining the activities controlling the prototype transcription factor, GATA-1. The transcriptional regulation of GATA-1 has been intensively investigated, thereby leading to the identification of its developmental stage-specific regulatory sequences. Loss-of-function mutant animals, combined with specific marking of the primitive and definitive erythroid lineages have also shed new insight into how GATA-1 activity is required in vivo at specific developmental stages. Procedures have also been developed for ascertaining whether or not the GATA-1 protein actually binds in vivo to regulatory GATA motifs in candidate target genes. Application of a similar multifaceted approach should enable investigators to examine the physiological roles that any transcription factor might play in vivo during the differentiation of any well defined cell lineage.
Transcription factor GATA-1 was first identified in erythroid cells, but was later shown to also be expressed in Sertoli cells of the mouse testis. GATA-1 transcription in testis initiates from a different first exon (exon IT) than the erythroid mRNA (transcribed from exon IE). To begin to address the question of how expression of GATA-1 might be differentially regulated in Sertoli and erythroid cells, we have cloned and determined the structure of the IT promoters of both the rat and mouse GATA-1 genes. The transcription regulatory mechanism(s) controlling the synthesis of exon IT-derived mRNA was investigated by transfection of wild-type and mutant reporter genes, with and without co-transfected GATA factor expression plasmids, into either fibroblasts or Sertoli cell lines. Two GATA binding sites in the IT promoter were found to be required for GATA factor-mediated activation in fibroblasts: GATA-IT-directed reporter gene expression was activated only after co-transfection with GATA-1, implying that transcriptional activation of GATA-1 in the testis might be at least partially mediated through these GATA regulatory elements. We also found that the endogenous GATA-1 gene was silent in primary culture and two different Sertoli cell lines, and that the repression of co-transfected GATA-1 reporter genes could not be relieved by forced expression of GATA-1 in Sertoli cells. Thus the GATA-IT promoter may be under the control of a regulatory network in Sertoli cells which involves both positive and negative regulation of transcription, and conserved GATA motifs found in the IT promoter may be required for transducing these effects.
We have set out to test a model for tissue-specific gene expression that relies on the early replication of expressed genes to sequester limiting activating transcription factors. Using an erythroid cell line, we have tested the changes in the DNA binding activity of the lineage-restricted transcription factor GATA-1 through the cell cycle. We find that GATA-1 activity is low in G1, peaks in mid-S phase, and then decreases in G2/M. In contrast, the binding activities of two ubiquitous transcription factors, Oct1 and Sp1, remain high in G2/M. GATA-1 protein and mRNA vary in a similar manner through the cell cycle, suggesting that the expression of the gene or the stability of its message is regulated. Although a number of transcription factors involved in the control of the cell cycle or DNA replication have been shown to peak in S phase, this is the first example of a lineage-restricted transcription factor displaying S phase-specific DNA binding activity. One interpretation of these data leads to a model in which the peak in GATA-1 DNA binding amplifies the effect of early replication on the activation of erythroid-specific genes at the same time as preventing activation of non-erythroid genes containing GATA-responsive elements. These results may also relate to recent data implicating GATA-1 function in apoptosis and cell cycle progression.
Interaction of erythropoietin with its type 1 receptor is essential to the development of late erythroid progenitor cells. Through the ectopic expression of receptor mutants in lymphoid and myeloid cell lines, insight has been gained regarding effectors that regulate Epo-induced proliferation. In contrast, effectors that regulate Epo-induced differentiation events (e.g. globin gene expression) are largely undefined. For in vitro studies of this pathway, erythroleukemic SKT6 cell sublines have been isolated which stably and efficiently hemoglobinize in response to Epo. Epo rapidly activated Jak2, STAT5 and detectably STATs 1 and 3, while no effects on GATA-1, EKLF or STAT5 expression were observed. Finally, efficient hemoglobinization of SKT6 cells was shown to be mediated by chimeric receptors comprised of the EGF receptor extracellular domain and truncated cytoplasmic subdomains of either the Epo receptor or the prolactin Nb2 receptor. This work further establishes SKT6 cells as an important model for studies of Epo-stimulated differentiation, and shows that this signaling pathway is promoted by a limited set of membrane-proximal receptor domains and effectors.
During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.
Phenotype of P815 mouse mast cells changes markedly during culture in the peritoneal cavity of syngenic BDF1 mice. The cells, cultured for 1 week in the peritoneal cavity of syngenic BDF1 mice, proliferate and express high levels of L-histidine decarboxylase (HDC) and mouse mast cell protease (MMCP)-6 mRNAs, indicating the ability of P815 cells to differentiate toward mature connective tissue mast cells. Peritoneal fluid aspirated from P815-inoculated BDF1 mouse and added to cultured P815 cells in vitro was also found to induce HDC mRNA expression, suggesting that at least some of the humoral factors in the peritoneal fluid induce HDC mRNA transcription. Among the erythroid transcription factors, P815 cells expressed GATA-2 but not GATA-1 mRNA before and after the intraperitoneal incubation. In contrast, the expression of NF-E2 subunit p45 disappeared, while expression of subunit mafK was markedly reduced after incubation. Cotransfection assays using HDC-luciferase reporter and p45 and/or mafK expression constructs showed that NF-E2 affects the transactivation of HDC gene. These results suggest that NF-E2 is also an important transcription factor in mast cell differentiation.
Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages.
Transcription factors play a key role in controlling the cellular differentiation of hematopoietic cells. Among the known transcription factors, both GATA-1 and SCL play roles in the cellular differentiation of erythrocytic and megakaryocytic lineages, while GATA-2 is thought to maintain and promote the proliferation of early hematopoietic progenitors. In this review, the clinical implications of expression of the GATA family, SCL, and EVI1 gene in various types of human leukemia are discussed. De novo acute myeloid leukemia (AML) patients may be subdivided into three categories depending on the expression pattern of transcription factors, i.e., GATA-1(+)SCL(+), GATA-1(+)SCL(-), and GATA-1(-)SCL(-). AML patients with both GATA-1 and SCL expression have a poor prognosis and have some characteristic clinical and hematologic features. The EVI1 gene may be expressed through at least two pathways in hematologic malignancies; one is related to chromosomal changes at 3q26, while the other is related to myelodysplasia regardless of chromosomal changes at 3q26 region. These findings suggest that the pattern of expression in transcription factors in abnormal hematopoietic cells is reflected in the malignant phenotype and play a role in the pathogenesis of the disease.
Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.
The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.
The X-linked mouse mutant phenotype, tattered (Td), is associated with prenatal lethality of males and has been mapped previously to the proximal region of the mouse X chromosome. We report here a refined position for Td and demonstrate that it lies in the approximately 0.9-cM interval between DXMit55 and Xkh. This enables us to predict that the human homologue lies either between CLCN5 and the evolutionary breakpoint that lies between GATA1 and PFC or distal to XK and proximal to the evolutionary breakpoint that lies between XK and DMD. Histological analysis of dorsal skin taken from 5-day-old heterozygous animals revealed that the mutation was associated with patches of hyperkeratinzation in the epidermis and in the hair follicles, accompanied by a mild inflammatory infiltrate in the underlying dermis.
Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic level in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.
The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.
Bernard-Soulier Syndrome (BSS) is a rare congenital bleeding disorder due to absent or decreased expression of the glycoprotein Ib-IX-V (GpIb-IX-V) receptor complex on the platelet surface. To date, only mutations in GpIbalpha or GpIX have been reported in patients with BSS. GpIbbeta differs from the other proteins in this receptor in that the gene is more complex, and an alternative form is expressed in cells of non-megakaryocytic lineage, including endothelial cells. It appears that the megakaryocytic and endothelial cell mRNA species are transcribed from different start sites and have different proximal promoter regions. We have identified a patient with BSS who has a deletion on one chromosome 22, resulting in velocardiofacial syndrome. The GpIbbeta gene has been mapped to this deleted (22q11.2) region of chromosome 22. The patient has greatly reduced levels of GpIbbeta mRNA and no detectable platelet GpIbbeta protein, suggesting that his BSS results from a mutation in his remaining GpIbbeta allele. Sequence analysis revealed that the coding region of GpIbbeta is normal, but the 5'-upstream region contains a C to G transversion at base -133 from the transcription start site used in megakaryocytes. The mutation changes a GATA consensus binding site, disrupts GATA-1 binding to the mutated site, and decreases promoter activity by 84%. Thus, in this patient, Bernard-Soulier syndrome results from a deletion of one copy of GpIbbeta and a mutated GATA binding site in the promoter of the remaining allele, resulting in decreased promoter function and GpIbbeta gene transcription.
The myb-ets-containing ME26 virus causes erythroleukemia in mice by a novel mechanism involving the inappropriate activation of erythroid-specific genes in hematopoietic precursor cells. We have previously shown that the ME26 viral protein can transactivate the GATA-1 promoter in transient transactivation assays carried out in mouse fibroblasts. The mouse GATA-1 promoter, whose activity is regulated by the GATA-1 protein itself, contains a double GATA consensus sequence at its 5' end and two CACCC elements at its 3' end, both of which are crucial for promoter activity in erythroid cells, as well as a nonconsensus GATA sequence and several putative c-myb and c-ets binding sites. In order to determine which sequences in the GATA-1 promoter are crucial for activation by the ME26 viral protein, we made deletions of the promoter, cloned them into a luciferase expression vector and tested their activity in mouse fibroblasts, which do not express GATA-1. Our results indicate that sequences in the 3' end of the GATA-1 promoter, which include two CACCC elements, are essential for transactivation by ME26 virus, while other upstream sites contribute to full activation by the virus. Mutation of the CACCC sites abolishes ME26 viral transactivation. The interaction of cell extracts containing ME26 viral protein and the GATA-1 promoter fragment containing the two CACCC elements was examined by electrophoretic mobility shift analysis (EMSA) and the results showed no direct interaction between the two. However, we could detect the ubiquitous transcription factor Sp1 bound to this sequence. These data demonstrate that the CACCC element is necessary for GATA-1 promoter transactivation by ME26 virus and that the viral protein may indirectly transactivate the promoter by binding to Sp1.
In an effort to understand how the heme biosynthetic pathway is uniquely regulated in erythroid cells, we examined the structure of the gene encoding murine delta-aminolevulinate dehydratase (ALAD; EC4.2.1.24), which is the second enzyme of the pathway. The gene contains two first exons, named 1A and 1B, which are alternatively spliced to exon 2, where the coding region begins. Each first exon has its own promoter. The promoter driving exon 1A expression is TATA-less and contains many GC boxes. In contrast, the exon 1B promoter bears regulatory sequences similar to those found for beta-globin and other erythroid-specific genes. Tissue distribution studies reveal that ALAD mRNA containing axon 1A is ubiquitous, whereas mRNA containing axon 1B is found only in erythroid tissues. This finding, together with our further observation that GATA-1 mRNA levels increase 3-fold during maturation of murine erythroid progenitor cells, may help explain simultaneous 3-fold increases in exon 1B expression. The unexpected result that axon 1A expression also increases 3-fold during CFU-E maturation may be attributable to the action of NF-E2, since there is a potential binding site in a position analogous to the NF-E2 site in the locus control region of the beta-globin gene cluster.
WT1 is a tumor suppressor gene that can repress transcription of many growth-factor and growth-factor receptor genes. We quantitated WT1 expression levels in 62 acute myelogenous leukemia (AML) samples and found that 82% strongly expressed WT1. WT1 expression levels are highest in the undifferentiated and granulocytic French-American-British (FAB) subclasses and lower in the monocytic subclasses. WT1 was strongly expressed in normal CD34+ bone marrow (BM) stem cells but only weakly or not expressed in normal mature blood cells. This suggests that WT1 gene expression is associated with immature cells, which have high proliferative capacities. Previous studies of WT1 gene regulation showed that GATA-1 may regulate WT1 expression. To understand the relationship between WT1 and GATA-1 expression in leukemia, we examined the expression pattern of GATA-1 in the cells described above. Overall, AML samples expressed significant amounts of both WT1 and GATA-1. However, AML samples with 16q22 abnormalities, presumably interrupting the core binding factor (CBF) beta gene expressed lower than normal levels of GATA-1 but high levels of WT1. Our data suggest that the transcription factor CBF beta may be important for GATA-1 gene regulation. Thus, WT1 expression varied in different FAB subclasses, and GATA-1 expression was strongly affected by the presence of chromosome 16q22 abnormalities.
Previous comparisons of gene location in the three major groups of mammals (eutherians, marsupials, and monotremes) have suggested that the long arm of the human X represents the ancestral mammalian X chromosome, whereas the short arm represents an autosomal region(s) recently added to the eutherian X chromosome. To identify the fusion point of this ancient X-autosome rearrangement, we have mapped four genes, three of which map near the centromere of the human Xp, in marsupials and in a monotreme. We found that ARAF1, ALAS2, and GATA1 are located on the X chromosome in marsupials, and ALAS2 and GATA1 are also located on the X in the platypus. This implies that the proximal short arm of the human X chromosome, including the centromere, was part of the ancestral mammalian X chromosome. The fusion point between the conserved region and the recently added regions therefore maps to human Xp11.23, although gene order on the human X indicates that there has been some rearrangement of this region.
To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.
The far upstream region (-1.2-0.9 kilobase pairs) of the mouse glycophorin gene contains the locus control region (LCR)-like region, which acts as an erythroid-specific enhancer dependent on chromosomal integration in murine erythroleukemia (MEL) cells. In the present study, we demonstrated that this region binds six nuclear factors. The binding of GATA-1 to corresponding sites did not show any change before or after induction with dimethyl sulfoxide, but the binding of Spi-1/PU.l and an unidentified factor called glycophorin regulatory element binding factor (GRBF) showed a change during induction. While binding activity of Spi-l/PU.l dropped soon after induction, the GRBF activity increased after induction when expression of the glycophorin gene began. After identification of the consensus binding site of GRBF, we cloned cDNA for that factor by Southwestern method, and it was identified as a previously reported transcription factor, delta, a murine form of YY-l which is a versatile transcription factor. Mutation analysis in the delta/YY-1 binding site within the LCR-like region indicated that delta/YY-1 acts as a regulatory protein in combination with the E-box-binding protein that binds to the neighboring sequence.
The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans-acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.
UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.
A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23-p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internal Alu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter-ZNF21-DXS7465E (MG66)-DXS7927E (MG81)-WASP, DXS1011E, DXS7467E (MG21)-DXS- 7466E (MG44)-GATA1-DXS7469E (Xp664)-TFE3-SYP (DXS1007E)-Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.
GATA-1, a transcription factor essential for the development of the erythroid lineage, contains two adjacent highly conserved zinc finger motifs. The carboxy-terminal finger is necessary and sufficient for specific binding to the consensus GATA recognition sequence: mutant proteins containing only the amino-terminal finger do not bind. Here we identify a DNA sequence (GATApal) for which the GATA-1 amino-terminal finger makes a critical contribution to the strength of binding. The site occurs in the GATA-1 gene promoters of chickens, mice, and humans but occurs very infrequently in other vertebrate genes known to be regulated by GATA proteins. GATApal is a palindromic site composed of one complete [(A/T)GATA(A/G)] and one partial (GAT) canonical motif. Deletion of the partial motif changes the site to a normal GATA site and also reduces by as much as eightfold the activity of the GATA-1 promoter in an erythroid precursor cell. We propose that GATApal is important for positive regulation of GATA-1 expression in erythroid cells.
GATA-1, mainly expressed during erythroid differentiation, has been shown to regulate the genes specifically expressed in the late stages of erythropoiesis and to protect erythroid cells from apoptosis, suggesting that it might interfere with the cell cycle. By expressing the retrovirally transduced human GATA-1 cDNA in NIH3T3 fibroblasts, we have shown that GATA-1 alone was unable to transactivate its erythroid-specific target genes in these nonerythroid cells. However, GATA-1 expression had a dramatic effect on the proliferation of these fibroblasts. The cloning efficiency of the GATA-1-expressing fibroblasts was maintained but their S phase was greatly elongated and their G1 and G2/M phases were reduced, impairing substantially their proliferation. When cultured at low serum concentrations for 48 hours, GATA-1-expressing fibroblasts failed to accumulate in the G0/G1 phases but did not become serum independent. GATA-1-expressing fibroblasts expressed D1, A, and B1 cyclin mRNAs under conditions of serum starvation or at confluence, whereas these cyclin mRNAs were downregulated in the parental NIH3T3 cells cultured under the same conditions. Moreover, these effects of GATA-1 expression on proliferation were not limited to NIH3T3 cells, since different clones of hGATA-1 virus-infected FDCP-1 cells, a murine interleukin-3-dependent hematopoietic cell line, had a slower growth rate than control cells. Based on these data, we hypothesize that GATA-1 plays a role in the regulation of the cell cycle during terminal erythroid differentiation.
Steroid hormones regulate diverse biological functions, including programmed cell death (apoptosis). Although steroid receptors have been studied extensively, relatively little is known regarding the cellular targets through which apoptosis is triggered. We show here that the ligand-activated estrogen receptor (ER) induces apoptosis in an erythroid cell line by binding to, and consequently inhibiting the activity of, GATA-1, an erythroid transcription factor essential for the survival and maturation of erythroid precursor cells. GATA-1 inhibition is reflected in the downregulation of presumptive GATA-1 target genes. Constitutive overexpression of a GATA-binding protein resistant to the effects of the ER partially rescues ER-induced apoptosis. Induction of apoptosis by a mutant ER defective in binding to the estrogen response element but active in GATA-1 inhibition suggests that ER-mediated inhibition of GATA-1 is direct and does not require estrogen response element-dependent transcriptional activation. Thus, a lineage-restricted transcription factor, such as GATA-1, constitutes one cellular target through which steroid hormones may control apoptosis. As GATA-binding proteins are evolutionarily conserved, we speculate that members of the steroid receptor family may exert some of their diverse biological functions in different cellular contexts through interference with the function of GATA-binding proteins.
Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.
To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5' flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.
GATA and CACC elements commonly are codistributed within the regulatory domains of a variety of erythroid genes. Using Drosophila S2 cells, the actions of GATA1, Sp1, and erythroid Kruppel-like factor (EKLF) at these elements within model erythroid promoters have been assessed. For each promoter studied (erythroid pyruvate kinase, glycophorin B, and a murine betamaj globin-derived construct, GCT) Sp1 and EKLF each activated transcription despite differences in CACC element sequence, orientation, and positioning. However, GATA1 acted in apparent cooperativity with Sp1 at the pyruvate kinase promoter; with EKLF at the betamaj globin-derived GCT promoter; and with either Sp1 or EKLF at the glycophorin B promoter. Thus, GATA1 may functionally interact with each of these Krüppel-like factors depending on promoter context; and at the GCT promoter, transcriptional activation by GATA1 and EKLF was > or = 10-fold higher than levels attributable to additive effects. The possibility that interactions between these activators may be direct was supported by the specific binding of baculoviral-expressed EKLF to GATA1. This report underlines the likelihood that discrete roles exist for Sp1 and EKLF in erythroid gene activation, and supports a mechanism of direct cooperativity for EKLF and GATA1 as coregulators.
Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.
The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. In order to understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the regulatory elements of this gene. Two regions are hypersensitive to DNase I in nuclei of cells that express MPL: the promoter and a portion of intron 6. The latter behaves as a chromatin-dependent enhancer. A 200 bp fragment of the promoter is sufficient for high-level specific expression. This fragment can bind several transacting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in human erythroleukemia (HEL) cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. This study indicates a molecular basis for the coregulation of markers of megakaryocyte differentiation. Finally, we describe other nuclear factor binding sites that may be involved in the cell-type-specific expression of MPL.
To understand the molecular mechanisms of erythroid differentiation, we analyzed by semiquantitative RT-PCR the expression of the transcription factor GATA1, the erythropoietin receptor (EpoR), and erythroid (beta-globin) differentiation markers in purified hematopoietic stem cells (HSCs) after in-vitro-induced differentiation. Whether GATA1 transcription was from the proximal (with respect to the AUG, also known as erythroid) or the distal (also known as testis) promoter was analyzed as well. Low-density marrow cells which bind to wheat germ agglutinin, but not to the antibody 15.1.1, and which either do or do not retain the dye rhodamine-123 (Rho-bright and Rho-dull, respectively), were purified. Rho-dull, but not Rho-bright cells permanently reconstitute lymphomyelopoiesis in W/Wv and severe-combined-immunodeficiency mice and, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give rise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricted or myeloid-restricted differentiation is observed when the cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF + IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, respectively. Rho-dull cells have barely detectable reconstitution potential at day 5 of culture. None of the genes examined were expressed in purified Rho-bright or Rho-dull cells. The only exception was GATA1 which was expressed at maximal levels in Rho-bright cells at the onset of culture. Rho-dull cells did not express GATA1 before day 3 of culture (maximal expression at days 10-15). Activation of GATA1 and EpoR was observed in all growth of mRNA for the two genes expressed by the cells. In contrast, beta-globin mRNA was detected only in the presence of Epo. The transcription of GATA1 was exclusively from the proximal promoter in the absence of Epo but both proximal and distal transcripts were observed in its presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1 mRNA) coincided with maximal globin mRNA levels (day 5 or day 15 for Rho-bright and Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSCs and pluripotent progenitor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.
The present study examines genetic mechanism(s) possibly involved in the observed 3'-azido-3'-deoxythymidine (AZT)-induced inhibition of globin gene transcription by evaluating the direct phenotypic erythroid effects of AZT on erythroid-specific transcription factors which regulate globin gene promoters. In vitro binding of GATA-1 or NFE-2 to its consensus sequence was decreased in the presence of AZT reaching a maximum inhibition as early as 24 h after AZT treatment. Nuclear extracts from butyric acid-induced K562 cells treated with an IC50 concentration of AZT exhibited a decrease in GATA-1 and NFE-2 binding by approximately 30% and 35%. In contrast, 2',3'-dideoxycytidine which inhibits cell growth without affecting hemoglobin synthesis, had no effect on binding of GATA-1 and NFE-2 factors. Northern blot analysis revealed a 25% decrease by AZT in GATA-1 mRNA steady-state levels at 24 h and this inhibitory effect was maintained until 72 h after drug addition. A similar decrease in NFE-2 mRNA steady-state levels was observed at 72 h after AZT treatment. This study suggests that AZT inhibition of erythroid differentiation is subsequent to a decrease of nuclear factors gene expression which affect their DNA binding.
GATA-1 is a zinc-finger transcription factor that plays a critical role in the normal development of hematopoietic cell lineages. In human and murine erythroid cells a previously undescribed 40-kDa protein is detected with GATA-1-specific antibodies. We show that the 40-kDa GATA-1 (GATA-1s) is produced by the use of an internal AUG initiation codon in the GATA-1 transcript. The GATA-1 proteins share identical binding activity and form heterodimers in erythroleukemic cells but differ in their transactivation potential and in their expression in developing mouse embryos.
Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.
Using cDNA selection with a YAC from the Xp11.2 region, we have identified a novel gene (RBM3) that encodes a polypeptide with high sequence similarity to a group of proteins that bind to RNA. On a YAC contig map, RBM3 is located between OATL1 and GATA1/TFE3 in sub-band Xp11.23, and gives rise to alternatively spliced transcripts in a variety of human tissues. The longest open reading frame encodes a 157 amino acid protein with a predicted molecular weight of 17 kDa. Its putative RNA-binding domain most closely resembles that of two previously characterized human RNA-binding proteins, YRRM, the gene for which has been implicated in azoospermia, and hnRNP G, a glycoprotein, also identified as an auto-antigen. The homology of RMB3 in both sequence and size to an RNA binding protein from maize, AAIP , suggests that it functions in a fundamental pathway conserved from plants to mammals.
Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks posttransplant, animals that received cells transduced by the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) count than control animals that received cells transduced with a virus containing a neor gene. Erythropoiesis was stimulated in GATA-1 and control animals by phlebotomy. GATA-1 animals required more extensive phlebotomy to reach a hematocrit less than 25 and their hematocrit returned to normal levels sooner than control animals. The effect of twice-daily injections of 10 U recombinant erythropoietin (epo) was also examined. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data suggest that dysregulated expression of GATA-1 in primitive hematopoietic cells enlarges the pool of epo-responsive erythroid progenitor cells.
Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.
Phenotypic characteristics of blasts were studied in a Down's infant with transient abnormal myelopoiesis (TAM). Two major subpopulations were identified: (1) CD33+CD42b+ cells with platelet peroxidase activity, the commitment of which to megakaryocytic lineage was supported by an increased expression of GATA-1 mRNA; (2) CD33+CD34+CD7+CD4+ cells with immature ultrastructure, which could be either immature megakaryocytic or myeloid cells with aberrant differentiation. Mixed colonies containing megakaryocytes and monocyte/macrophages in the peripheral blood suggested the presence of progenitors common to these subpopulations. These results may indicate that subpopulations of blasts with phenotypic diversity could be derived from aberrant common progenitors to megakaryocytic and myeloid lineages in this patient.
To understand the clinical implications of transcription factors and their biologic roles during cellular differentiation in the hematopoietic system, we examined the expression of GATA-1, GATA-2, and stem cell leukemia (SCL) gene in human leukemia cell lines and various leukemia patients using the reverse transcriptase-polymerase chain reaction. Cell lines exhibiting megakaryocytic or erythrocytic phenotypes had GATA-1, GATA-2, and SCL gene transcripts, while monocytic cell lines had no detectable GATA-1, GATA-2, or SCL gene mRNA. In some myeloid cell lines, GATA-1 expression, but not SCL gene expression, was detected; GATA-1 expression in HL-60 cells was downregulated during the process of monocytic differentiation. We next examined GATA-1, GATA-2, and SCL gene expression in 110 leukemia samples obtained from 76 patients with acute myeloid leukemia (AML), 19 with acute lymphoblastic leukemia (ALL), and 15 with chronic myeloid leukemia in blast crisis (CML-BC). SCL gene expression was usually accompanied by GATA-1 expression and was preferentially detected in patients with leukemia exhibiting megakaryocytic or erythrocytic phenotypes, while patients with monocytic leukemia were clustered in the group with no detectable GATA-1 expression. None of the patients with ALL or CML-lymphoid-BC expressed SCL. De novo AML patients with SCL gene expression had a lower complete remission (CR) rate and had a significantly poorer prognosis. Among the patients with AML not expressing SCL, a high percentage of patients with CD7+ AML and CD19+ AML had detectable GATA-1, while patients with GATA-1-negative AML had the best CR rate (87.5%). Our results suggest that the expression pattern of transcription factors reflects the lineage potential of leukemia cells, and GATA-1 and SCL gene expression may have prognostic value for the outcome of patients with AML.
The transcription factor GATA-1 recognizes a consensus motif present in regulatory regions of numerous erythroid-expressed genes. Mouse embryonic stem cells lacking GATA-1 cannot form mature red blood cells in vivo. In vitro differentiation of GATA-1- embryonic stem cells gives rise to a population of committed erythroid precursors that exhibit developmental arrest and death. We show here that the demise of GATA-1- erythroid cells is accompanied by several features characteristics of apoptosis. This process occurs despite normal expression of all known GATA target genes examined, including the erythropoietin receptor, and independent of detectable accumulation of the tumor suppressor protein p53. Thus, in addition to its established role in regulating genes that define the erythroid phenotype, GATA-1 also supports the viability of red cell precursors by suppressing apoptosis. These results illustrate the multifunctional nature of GATA-1 and suggest a mechanism by which other hematopoietic transcription factors may ensure the development of specific lineages.
The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.
We have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression.
We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized. One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma. A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis. We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis. The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255146- Xcen, agrees with genomic pulsed-field maps of the region. In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CPG islands. These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases.
The mouse WASP gene, the homolog of the gene mutated in Wiskott-Aldrich syndrome, has been isolated and sequenced. the predicted amino acid sequence is 86% identical to the human WASP sequence. A distinct feature of the mouse gene is an expanded polymorphic GGA trinucleotide repeat that codes for polyglycine and varies from 15 to 17 triplets in different Mus musculus strains. The genomic structure of the mouse WASP gene is expressed as an approximately 2.4-kb mRNA in thymus and spleen. Chromosomal mapping in an interspecific M. Musculus/M. spretus backcross placed the Wasp locus near the centromere of the mouse X chromosome, inseparable from Gata1, Tcfe3, and scurfy (sf). This localization makes Wasp a candidate for involvement in scurfy, a T cell-mediated fatal lymphoreticular disease of mice that has previously been proposed as a mouse homolog of Wiskott-Aldrich syndrome. Northern analysis of sf tissue samples indicated the presence of WASP mRNA in liver and skin, presumably as a consequence of lymphocytic infiltration, but non abnormalities in the amount or size of mRNA present.
Thrombopoietin (Tpo) is a cytokine that specifically regulates megakaryocyte maturation and platelet production. Little is known about the molecular and cellular mechanism of the Tpo-induced megakaryocyte maturation process including polyploidization and platelet release. To study Tpo-induced megakaryocyte differentiation, a mouse cell line FD-TPO, which responds and grows with Tpo, was established from a interleukin-3-dependent hematopoietic progenitor cell line FDC-P2. The FD-TPO cells, expressing endogenous Tpo receptor, grew with Tpo in a dose-dependent manner. Further, Tpo stimulation dramatically induced expression of megakaryocyte/erythroid-specific transcription factors GATA-1 and NF-E2 in FD-TPO cells. Flow cytometry analysis demonstrated that expression of platelet-specific cell surface antigens including CD61 (GPIIIa) dramatically increased in Tpo-stimulated FD-TPO cells and that expression of myeloid-specific antigens, Gr-1 and Mac-1, decreased. Therefore, we concluded that the binding of Tpo to FD-TPO cells induces not only cell growth but also differentiation into mature megakaryocyte-like cells, and thus this cell line was found to be useful for the study of Tpo receptor-mediated growth and differentiation signals.
The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators.
Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression.
The murine homologues of the loci for McLeod syndrome (XK), Dent's disease (CICN5), and synaptophysin (SYP) have been mapped to the proximal region of the mouse X chromosome and positioned with respect to other conserved loci in this region using a total of 948 progeny from two separate Mus musculus x Mus spretus backcrosses. In the mouse, the order of loci and evolutionary breakpoints (EB) has been established as centromere-(DXWas70, DXHXF34h)-EB-Clcn5-(Syp, DXMit55, DXMit26)-Tfe3-Gata1-EB-Xk-Cybb-telomere. In the proximal region of the human X chromosome short arm, the position of evolutionary breakpoints with respect to key loci has been established as DMD-EB-XK-PFC-EB-GATA1-C1CN5-EB-DXS1272E-ALAS2-E B-DXF34-centromere. These data have enabled us to construct a high-resolution genetic map for the approximately 3-cM interval between DXWas70 and Cybb on the mouse X chromosome, which encompasses 10 loci. This detailed map demonstrates the power of high-resolution genetic mapping in the mouse as a means of determining locus order in a small chromosomal region and of providing an accurate framework for the construction of physical maps.
The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.
Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. Thus studies on the transcriptional regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation of this lineage. In previous experiments, the promoter of GPIIb gene was isolated and we have shown that a fragment extending 643 bp upstream the transcription start site was able to control the cell specificity of a reporter gene in transfection experiments of different permanent cell lines. Most of the transcriptional activity is contained in an enhancer containing binding sites for members of the GATA and ets transcription factors families. The transcription factor GATA1 is not only a major regulator of the transcription of erythroid genes, but it also regulates the expression of GPIIb and other megakaryocytic genes. We suggest that the lineage specificity and the temporal activation of GPIIb gene during hematopoiesis rely on the activity of a repressor that has been identified on the promoter. To test this hypothesis, we have developed a cell model allowing the study of the megakaryocytes differentiation from very immature progenitors to fully differentiated cells. This model is based on the differentiation of mouse embryonic stem cells. We have obtained megakaryocytes together with erythrocytic and granulo-macrophagic cells. The transfection in these ES cells of GPIIb promoter constructs mutated or not on different regions, including the repressor element will provide important information on the mechanisms controlling gene activation or repression during megakaryocyte differentiation.
Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.
Acute megakaryoblastic leukaemia (M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. To clarify properties of the blast cells in M7 and TMD cases, we examined erythroid markers expression in blasts from six cases with M7 and seven cases with TMD in this study. Erythroid-specific mRNAs encoding gamma-globin and erythroid delta-aminolevulinate synthase were found to be expressed in blasts from most of these cases, indicating that majorities of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. We also found that mRNAs encoding GATA-1 and GATA-2 are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells.
We investigated expression of the human ecotropic virus integration site-1 (EVI1) gene in patients with leukemia and myelodysplastic syndrome (MDS) using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The EVI1 transcripts were detected in 5 (10.0%) of 50 patients with de novo acute myeloid leukemia (AML), including two AML patients with trilineage myelodysplasia, and in 8 (34.8%) of 23 patients with post-myelodysplastic syndrome AML (post-MDS AML). EVI1 expression was also detected in 6 (35.3%) of 17 MDS patients and three of six patients with chronic myeloid leukemia (CML) in myelomegakaryoblast crisis. No EVI1 transcripts were detected in patients with acute lymphoid leukemia (n = 15) or CML in lymphoid blast crisis (n = 4). Chromosomal abnormalities at the 3q26 region, where the EVI1 gene is located, were found in one patient with MDS and two patients with CML myelomegakaryoblast crisis who had EVI1 expression. Our results showed that EVI1 expression was frequent in patients with post-MDS AML and AML with trilineage myelodysplasia, regardless of the presence or absence of 3q26 abnormalities. EVI1 expression was accompanied by expression of GATA-1 and GATA-2, and often by stem cell leukemia (SCL) gene expression. In patients with post-MDS AML, EVI1 expression was not always associated with a 3q26 abnormality, whereas EVI1 expression in CML myelomegakaryoblast crisis was often linked to a 3q26 abnormality. Our results suggest that the leukemogenic role of EVI1 expression may differ between post-MDS AML and leukemia, with EVI1 expression associated with a 3q26 abnormality.
High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.
The mRNA for the Duffy blood group antigen, the erythrocyte receptor for the Plasmodium vivax malaria parasite, has recently been cloned and shown to encode a widely expressed chemokine receptor. Here, we show that the Duffy antigen/chemokine receptor gene (DARC) is composed of a single exon and that most Duffy-negative blacks carry a silent FY*B allele with a single T to C substitution at nucleotide -46. This mutation impairs the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor. With the recent characterization of the FY*A and FY*B alleles, these findings provide the molecular basis of the Duffy blood group system and an explanation for the erythroid-specific repression of the DARC gene in Duffy-negative individuals.
We have studied gene expression of GATA-1, GATA-2, and SCL, which are known as cell-specific transcription factors, in 110 various leukemias consisted of 76 patients with acute myeloid leukemia (AML), 19 with acute lymphoblastic leukemia (ALL), and 15 with chronic myeloid leukemia (CML) in blast crisis by the revearse transcription-polymerase chain reaction assay. Accordingly, we divided into three groups. Group I (GATA-1+SCL+): patients with AML exhibiting phenotypic characteristics of erythroid or megakaryocytic lineage and most of CML myeloid blast crisis were included. Group II (GATA-1+, SCL-): Not only CD7-positive and CD19-positive AML, but also a part of Ph+ALL demonstrated this pattern. Leukemia in this group is considered to have a capability to differentiate into myeloid and lymphoid lineages. Group III (GATA-1-, SCL-): patients in this group consisted of leukemias which are differentiated into specific cell-lineages, either myeloid or lymphoid, when compared to groups I or II. Our data suggest that the expression pattern of transcription factors reflects lineage potential in leukemia cells, leading to classification of leukemias.
The expression of the major erythroid DNA-binding protein GATA-1 was studied during erythropoietin and chemically induced erythroid differentiation of J2E and murine erythroleukemia (MEL) cells. An increase in GATA-1 mRNA levels was observed shortly after hormonal stimulation of J2E cells, due to increased transcription and not stabilization of the short-lived transcripts. Concomitantly, an increase in protein capable of binding to the GATA motif was detected. Premature removal of erythropoietin from culture resulted in submaximal GATA-1, globin, and hemoglobin production. In contrast, differentiation of J2E cells initiated by sodium butyrate resulted in a sudden depletion of GATA-1 transcripts. Similarly, dimethyl sulphoxide induction of MEL cells produced a transient decrease in GATA-1 mRNA. Surprisingly, these decreases in mRNA were not reflected in alterations to GATA-1 protein content, or the ability of the transcription factor to bind DNA. We concluded from this study that the sequence of events initiated by erythropoietin was not followed during chemical stimulation of erythroid differentiation.
Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.
An unresolved aspect of current understanding of erythroid cell-specific gene expression relates to how a limited number of transcriptional factors cooperate to direct high-level expression mediated by cis-regulatory elements separated over large distances within globin loci. In this report, we provide evidence that GATA-1, the major erythroid transcription factor, activates transcription in a synergistic fashion with two Krüppel family factors, the ubiquitous protein Sp1 and the erythroid-restricted factor EKLF (erythroid Krüppel-like factor), which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Krüppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. We have shown that GATA-1 interacts physically with Sp1 and EKLF and that interactions are mediated through their respective DNA-binding domains. Moreover, we show that GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. Finally, the formation of GATA-1-SP1 complexes was demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops between locus control regions and globin promoters (or enhancers) might be formed or stabilized.
We have explored the expression of the transcription factors GATA-1, GATA-2, and NF-E2 in purified early hematopoietic progenitor cells (HPCs) induced to gradual unilineage erythroid or granulocytic differentiation by growth factor stimulus. GATA-2 mRNA and protein, already expressed in quiescent HPCs, is rapidly induced as early as 3 h after growth factor stimulus, but then declines in advanced erythroid and granulocytic differentiation and maturation. NF-E2 and GATA-1 mRNAs and proteins, though not detected in quiescent HPCs, are gradually induced at 24-48 h in both erythroid and granulocytic culture. Beginning at late differentiation/early maturation stage, both transcription factors are further accumulated in the erythroid pathway, whereas they are suppressed in the granulopoietic series. Similarly, the erythropoietin receptor (EpR) is induced and sustainedly expressed during erythroid differentiation, although beginning at later times (i.e., day 5), whereas it is barely expressed in the granulopoietic pathway. In the first series of functional studies, HPCs were treated with antisense oligomers targeted to transcription factor mRNA: inhibition of GATA-2 expression caused a decreased number of both erythroid and granulocyte-monocytic clones, whereas inhibition of NF-E2 or GATA-1 expression induced a selective impairment of erythroid colony formation. In a second series of functional studies, HPCs treated with retinoic acid were induced to shift from erythroid to granulocytic differentiation (Labbaye et al. 1994. Blood. 83:651-656); this was coupled with abrogation of GATA-1, NF-E2, and EpR expression and conversely enhanced GATA-2 levels. These results indicate the expression and key role of GATA-2 in the early stages of HPC proliferation/differentiation. Conversely, NF-E2 and GATA-1 expression and function are apparently restricted to erythroid differentiation and maturation: their expression precedes that of the EpR, and their function may be in part mediated via the EpR.
The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.
To study oncogenesis in the erythroid lineage, we have generated transgenic mice carrying the human c-MYC proto-oncogene under the control of mouse GATA-1 regulatory sequences. Six transgenic lines expressed the transgene and displayed a clear oncogenic phenotype. Of these, five developed an early onset, rapidly progressive erythroleukemia that resulted in death of the founder animals 30-50 d after birth. Transgenic progeny of the sixth founder, while also expressing the transgene, remained asymptomatic for more than 8 mo, whereupon members of this line began to develop late onset erythroleukemia. The primary leukemic cells were transplantable into nude mice and syngeneic hosts. Cell lines were established from five of the six leukemic animals and these lines, designated erythroleukemia/c-MYC (EMY), displayed proerythroblast morphology and expressed markers characteristic of the erythroid lineage, including the erythropoietin receptor and beta-globin. Moreover, they also manifested a limited potential to differentiate in response to erythropoietin. Studies in the surviving transgenic line indicated that, contrary to our expectations, the transgene was not expressed in the mast cell lineage. That, coupled with the exclusive occurrence of erythroleukemia in all the transgenic lines, suggests that the GATA-1 promoter construct we have used includes regulatory sequences necessary for in vivo erythroid expression only. Additional sequences would appear to be required for expression in mast cells. Further, our results show that c-MYC can efficiently transform erythroid precursors if expressed at a vulnerable stage of their development.
This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.
Tattered (Td) is an X-linked dominant mouse mutation that causes prenatal lethality in affected males. To map the locus, we analyzed 199 normal male and affected female progeny from a backcross of Td and Mus castaneus. Pedigree analysis of these animals suggests a gene order of cen-DXWas70-(Td, DXMit26, Gata1, Tcfe3)-(Cybb, Otc)-tel, where Tcfe3 is a transcription factor homologous to a gene involved in the murine microphthalmia (mi) mutation [Hodgkinson et al. Cell 74, 395-404, 1993]. To evaluate Tcfe3 as a candidate for Td, heterozygous tattered females were crossed to xid males to obtain females in which > 95% of B cells expressed genes solely from the Td X Chromosome (Chr). Fluorescent activated cell sorting (FACS) analysis and Western blotting of isolated splenocytes from Td/xid double heterozygotes rule out Tcfe3 as a likely candidate for the Td mutation.
Using J2E cells and the murine beta maj-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta maj promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGg-TAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT (-75) and CACCC (-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA]4-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]4-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me2SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.
The vegetative and reproductive (flowering) phases of Arabidopsis development are clearly separated. The onset of flowering is promoted by long photoperiods, but the constans (co) mutant flowers later than wild type under these conditions. The CO gene was isolated, and two zinc fingers that show a similar spacing of cysteines, but little direct homology, to members of the GATA1 family were identified in the amino acid sequence. co mutations were shown to affect amino acids that are conserved in both fingers. Some transgenic plants containing extra copies of CO flowered earlier than wild type, suggesting that CO activity is limiting on flowering time. Double mutants were constructed containing co and mutations affecting gibberellic acid responses, meristem identity, or phytochrome function, and their phenotypes suggested a model for the role of CO in promoting flowering.
The Wilms' tumor gene, WT1, is believed to play a role in hematopoiesis as it is expressed in the spleen and in immature leukemias in addition to the developing genitourinary system. WT1 is down-regulated in differentiated leukemia cells both in vivo and in vitro and is up-regulated in fetal spleen and immature leukemia cells. The modulation of WT1 expression was examined in many cell types, and a hematopoietic-specific enhancer element has been identified. Here we describe the transcriptional response of this enhancer to hematopoietic-specific transcription factors. We found co-expression of WT1 and GATA-1 mRNA in K562 cells and in mouse spleen, suggesting potential interactions between these two transcription factors. We find that the activity of the 3' WT1 enhancer is positively correlated with the expression of GATA-1. Gel shift competition experiments and transactivation studies revealed that this functional activity is mediated via binding at a GATA-binding site in the WT1 enhancer. The transactivation of the WT1 enhancer by GATA-1 implies that GATA-1 plays a role in the regulation of WT1 during hematopoiesis.
The human HOXB2 gene is a member of the vertebrate Hox gene family that contains genes coding for specific developmental stage DNA-binding proteins. Remarkably, within the hematopoietic compartment, genes of the HOXB complex are expressed specifically in erythromegakaryocytic cell lines and, for some of them, in hematopoietic progenitors. Here, we report the study of HOXB2 gene transcriptional regulation in hematopoietic cells, an initial step in understanding the lineage-specific expression of the whole HOXB complex in these cells. We have isolated the HOXB2 5'-flanking sequence and have characterized a promoter fragment extending 323 base pairs upstream from the transcriptional start site, which, in transfection experiments, was sufficient to direct the tissue-specific expression of HOXB2 in the erythroid cell line K562. In this fragment, we have identified a potential GATA-binding site that is essential to the promoter activity as demonstrated by point mutation experiments. Gel shift analysis revealed the formation of a specific complex in both erythroleukemic lines K562 and HEL that could be prevented by the addition of a specific antiserum raised against GATA-1 protein. These findings suggest a regulatory hierarchy in which GATA-1 is upstream of the HOXB2 gene in erythroid cells.
Understanding the mechanism of developmental regulation of hemoglobin switching has scientific as well as clinical relevance because of the influence of fetal hemoglobin (HbF) production in adulthood on the clinical manifestation of thalassemia and sickle cell anemia. We have previously found that the normal developmental patterns of globin gene expression are recapitulated in an experimental system of primary cultures that support differentiation of erythroid progenitors. We further found that high activities of the transcriptional activators, GATA-1 and SP1, are associated with normal adult erythroid differentiation.
In the present work, we have studied, the activities of GATA-1 and SP1 during differentiation of cultured erythroid progenitors derived from cord blood and from fetal livers, as well as from beta zero-thalassemia patients.
The results showed high GATA-1 binding activity and very low SP1 activity in the fetal liver cultures. This pattern was in contrast to cultures derived from normal adult peripheral blood, in which both GATA-1 and SP1 activities were high. Cord blood cultures showed an additive combination of "adult" and "fetal" patterns. The progenitors derived from a beta zero-thalassemia patient with high HbF production showed "fetal" pattern. On the other hand, in cultures of 2 beta zero-thalassemia patients without high HbF, "adult" pattern was observed.
In the present work, we show that human fetal and adult erythroid progenitors are distinct in their transcription factors, and that the commitment to fetal or adult program occurs at a very early differentiation stage. Our studies also demonstrate that under anemic stress, recruitment of fetal progenitors may occur in adulthood.
The proacrosin gene is transcribed in diploid spermatogenic cells and translated in haploid round spermatids. In order to evaluate sequences which are involved in proacrosin gene transcription, DNA-protein interactions were analyzed in 1.2 kb of the 5'flanking region of the rat gene. 13 protein binding sites were identified by DNase I footprinting using nuclear extracts from rat testis and brain, respectively. Five footprints (F1, F3, F7, TS2, TS3) which suggest an interaction with testis specific nuclear factors were further examined by gel retardation assays. Three testis specific binding sites (F1, F7, TS2, located 472bp, 697bp and 1004bp upstream of ATG, respectively) could be identified with both methods. The binding site F1 contains a motif which is similar to a testis specific footprint found in mouse protamine 1 gene. The nucleotide sequence of F7 contains the recognition motif of an isoform of the transcription factor GATA1, which is expressed in testis. Furthermore F1 and F7 are located in that part of the 5'flanking region of the proacrosin gene, which can direct proacrosin gene expression in germ cells of male transgenic mice.
The human epsilon-globin gene is transcribed in erythroid cells only during the embryonic stages of development. Expression of epsilon-globin gene, however, can be maintained in adult transgenic mice following removal of DNA positioned between -467 and -182 bp upstream of the epsilon-globin cap site. We have identified three protein binding regions within this silencer; a CCACC motif around -379, two overlapping motifs for YY1 and GATA around -269 and a GATA motif around -208 and we have analyzed their function during development by studying several mutants in transgenic mice. Mutation of the -208 GATA motif allows high epsilon-globin transgene expression in the adult suggesting that, in addition to its positive effects on transcription, GATA-1 also plays a negative role in the regulation of globin gene expression during development. Repression of epsilon gene expression in the adult also requires a functional YY1 binding site at position -269. Finally, mutation of the -379 CCACC site results in a small but detectable level of epsilon expression in adult erythroid cells. Thus, multiple proteins, including GATA-1, participate in the formation of the epsilon gene repressor complex that may disrupt the interaction between the proximal epsilon-promoter and the locus control region (LCR) in definitive erythroid cells.
The GATA-1 and GATA-2 transcription factors, which each contain two homologous zinc fingers, are important hematopoietic regulators expressed within the erythroid, mast cell, and megakaryocytic lineages. Enforced expression of either factor in the primitive myeloid line 416B induces megakaryocytic differentiation. The features of their structure required for this activity have been explored. The ability of 12 GATA-1 mutants to promote 416B maturation was compared with their DNA-binding activity and transactivation potential. Differentiation did not require any of the seven serine residues that are phosphorylated in vivo, an N-terminal region bearing the major transactivation domain, or a C-terminal segment beyond the fingers. Removal of a consensus nuclear localization signal following the second finger did not block differentiation or nuclear translocation. The N-terminal finger was also dispensable, although its removal attenuated differentiation. In contrast, the C-terminal finger was essential, underscoring its distinct function. Remarkably, only 69 residues spanning the C-terminal finger were required to induce limited megakaryocytic differentiation. Analysis of three GATA-2 mutants led to the same conclusion. Endogenous GATA-1 mRNA was induced by most mutants and may contribute to differentiation. Because the GATA-1 C-terminal finger could bind its target site but not transactivate a minimal reporter, it may direct megakaryocytic maturation by derepressing specific genes and/or by interacting with another protein which provides the transactivation function.
The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure.
GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-1 in hematopoietic cell differentiation, we have tested the ability of embryonic stem cells, carrying a targeted mutation in the X-linked GATA-1 gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-1- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this factor at some point in the erythroid lineage (L. Pevny et al., (1991) Nature 349, 257-260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-1 in other lineages. We found that the development of erythroid progenitors in embryonic yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation.
A t(X;1)(p11.2;q21.2) has been reported in cases of papillary renal cell tumors arising in males. In this study two cell lines derived from this tumor type have been used to indicate the breakpoint region on the X chromosome. Both cell lines have the translocation in addition to other rearrangements and one is derived from the first female case to be reported with the t(X;1)(p11.2;q21.2). Fluorescence in situ hybridization (FISH) has been used to position YACs belonging to contigs in the Xp11.2 region relative to the breakpoint. When considered together with detailed mapping information from the Xp11.2 region the position of the breakpoint in both cell lines was suggested as follows: Xpter-->Xp11.23-OATL1-GATA1-WAS-TFE3-SY P-t(X;1)-DXS255-CLCN5-DXS146-OATL2- Xp11.22-->Xcen. The breakpoint was determined to lie in an uncloned region between SYP and a YAC called FTDM/1 which extends 1 Mb distal to DXS255. These results are contrary to the conclusion from previous FISH studies that the breakpoint was near the OATL2 locus, but are consistent with, and considerably refine, the position that had been established by molecular analysis.
In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions.
We have isolated and characterized the 5'-flanking region of the gene for human ferrochelatase (HFC), the last enzyme of the heme biosynthetic pathway. The proximal promoter of the gene is contained within a region that structurally resembles a CpG island and is devoid of general cis elements such as TATA and CAAT boxes. Recognition sites for the ubiquitous Sp1 family of transcription factors, as well as for the erythroid-specific trans-acting factors NF-E2 and GATA-1 were found, and binding of regulatory proteins to these elements was analyzed by in vitro DNase I protection assays. The contribution of the various cis elements to both ubiquitous and erythroid preferential expression of the HFC gene was assessed by using transient transfection assays. These showed that a minimal Sp1-driven promoter devoid of the upstream erythroid-specific elements was sufficient for erythroid preferential expression of the HFC gene. However, elimination of a repressor sequence lying between the minimal promoter and the erythroid-specific elements resulted in high levels of expression in human erythroleukemic K562 cells only when the cis elements recognized by GATA-1 and NF-E2 were present, suggesting that the activity of these factors is regulated by a downstream repressor in erythroid cells.
GATA-1 is a cys-2/cys-2 zinc finger transcriptional activator that is required for erythrocyte development in chimeric mice and contributes to the expression of all erythroid genes studied to date, including the erythropoietin receptor, glycophorin B, and porphobilinogen deaminase genes. Transactivation by GATA-1 is mediated by either an amino-terminal acidic domain, R1, or an independent adjacent domain, R2, and may involve the coordinate action of cofactors (NF-E2, EKLF, and Sp1) which bind adjacent cis-elements. To directly assess mechanisms of transactivation, we have developed an efficient cell-free transcription system using recombinant human GATA-1 (rhGATA-1) expressed in SF9 cells. Levels of baculoviral expression of GATA-1 were > or = 200-fold higher than endogenous levels in erythroid K562 cells. Factors from each source were essentially equivalent in molecular weight and DNA binding properties, and highly similar in phosphotryptic peptide composition. Notably, DNA binding was inhibited following treatment with alkaline phosphatase. In both SF9 and K562 cells, GATA-1 occurred largely as heterogeneous multimers, thus complicating its isolation by standard procedures. However, significant purification of this factor (> or = 100-fold; > or = 75% purity) was accomplished via DNA affinity chromatography. In cell-free assays, this rhGATA-1 was shown to be remarkably active in transactivating model erythroid promoters. This work establishes an efficient in vitro system for direct analyses of mechanisms, cofactors, and functional domains of GATA-1 which regulate transcription at defined proximal promoters.
The DNA-binding domain of the erythroid transcription factor GATA-1 consists of two closely related, but distinct zinc-fingers which are highly conserved among the members of the growing family of GATA-like factors. The DNA-binding domain of the human GATA-1 (F1F2) was expressed as a histidine-tagged fusion protein in Escherichia coli. The denaturated protein was purified by Ni(2+)-chelate affinity chromatography and renaturated in situ. The active recombinant protein was purified by DNA affinity chromatography. F1F2 displayed GATA-1 specific binding activity toward its DNA recognition sequences within the hypersensitive site 3 of the human locus control region and the human gamma-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F1F2 bound also the CCAAT-box region of the human gamma-globin promoter.
M-TAT is a cytokine-dependent cell line with the potential to differentiate along the erythroid and megakaryocytic lineages. We cultured M-TAT cells long term (> 1 year) in the continuous presence of erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF). These long term cultures are referred to as M-TAT/EPO, M-TAT/GM-CSF, and M-TAT/SCF cells, respectively. Hemoglobin concentration and gamma-globin and erythroid delta-aminolevulinate synthase mRNA levels were significantly higher in M-TAT/EPO cells than in M-TAT/GM-CSF cells. When the supplemented cytokine was switched from GM-CSF to EPO, hemoglobin synthesis in M-TAT/GM-CSF cells increased rapidly (within 5 h), and the level of GATA-1 mRNA increased. In contrast, the addition of GM-CSF to the M-TAT/EPO cell culture decreased the amount of hemoglobin, even in the presence of EPO, indicating that the EPO signal for erythroid differentiation is suppressed by GM-CSF. Thus, erythroid development of M-TAT cells is promoted by EPO and suppressed by GM-CSF. These results support the hypothesis that EPO actively influences the programming of gene expression required for erythroid progenitor cell differentiation.
Recently, we demonstrated that a DNA-binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA-binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA-1. Rat GATA-1 expressed as a GST-fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA-1 and GATA-4 mRNAs are expressed in fetal hepatocytes.
In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster.
The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.
We have established a hemopoietic cell line, BL3, that possesses a rearranged retroviral genome, which we used as a genetic tag for their engraftment into lethally irradiated mice. Analysis of recipients up to 7 months after engraftment indicates that the marker was present in differentiated cells of various hemopoietic organs, in colony-forming cells, pre-CFU-S-forming cells, and in organs of secondary recipients from bone marrow cells of primary recipients. BL3 cells are Thy-1+, Sca-1+, B220-, Mac-1-, and Gr-1-. They express GATA-1 and are able to develop "cobblestones" with stromal cells. They do not express and respond to several hemopoietic growth factors known to facilitate marrow recovery. However, they are negatively regulated by TGF beta and can be stimulated by mitogen-stimulated spleen cell-conditioned medium. We conclude that BL3 cells possess properties of hemopoietic stem cells, including their capability to contribute to long-term repopulation.
The tal-1 gene, which is frequently activated in human T cell acute leukemias (T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and potentially a transcription factor. In human and murine hematopoiesis tal-1 is expressed during the differentiation of the erythroid, megakaryocytic and mastocytic cell lineages. The expression of tal-1 appears to be comodulated with that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein may regulate the tal-1 gene activity in these hematopoietic lineages. To get further insights into the molecular mechanisms that control tal-1 expression, we have isolated 5' sequences of the murine gene and compared them to their human counterparts. The 5' flanking sequences from the two genes show several regions of high homology. The alignment of both sequences enabled us to predict that similarly, to the human, the mouse gene contains two alternative first exons (Ia and Ib). Remarkably, in both species, the proximal region of the tissue-specific exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33) and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate that GATA proteins are able to interact with both GATA-motifs in a sequence specific fashion, but with different efficiencies. Moreover transfection studies show that the GATA-1 protein directly mediates tal-1 transcription by interacting with the -122/+1 fragment, defined as a minimal promoter in erythroid cells. Mutagenesis of the promoter establishes that the -33 GATA-binding site present in this fragment is critical for tal-1 expression in erythroid cells, but by itself does not lead to full promoter activity. Indeed, further mutations show that the second -65 GATA-binding site and the binding motif for SP1 (-59) significantly contribute to the overall activity of the proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1 cooperates with the transcription factor SP1 to mediate the erythroid-specific expression of the tal-1 gene.
Interleukin 4 (IL-4), a critical immunoregulatory cytokine, is produced by a subset of T lymphocytes and cells of the mast cell/basophil lineage. There are cell-specific differences in the regulatory elements that control IL-4 transcription in these two cell types. A 683-bp Bgl II fragment, located within the second intron of the murine IL-4 gene, was previously shown to exhibit mast cell-specific enhancer activity. To define critical cis-acting elements that regulate this enhancer, a series of deletions from the 5' and 3' ends of the Bgl II fragment were generated. Their effect on enhancer activity was assessed in IL-4-producing mast cell lines in transient transfection assays. Two functionally independent subregions, E1 and E2, were defined in this analysis. Both are required for full enhancer activity. Sequences identical to previously defined DNA-binding sites for SP1 and GATA are present within E1, and an ets binding site is located within E2. Although mutation of the SP1 sites had no effect on enhancer function, alteration of either the GATA or ets site reduced enhancer activity by 50-60%. Proteins that associate with the IL-4 intronic GATA and ets sites were detected in mast cell nuclear extracts by mobility-shift assays. Specific antibodies identified these factors as GATA-1 and GATA-2 and the ets family member PU.1. GATA-1, GATA-2, and PU.1 exhibit cell-specific expression, suggesting that these proteins play a critical role in the lineage-restricted activity of the IL-4 intronic enhancer in mast cells.
In order to elucidate the role of c-myb gene in erythroid differentiation of K562 cell induced by hemin (Hm) and erythropoietin (Epo), we constructed recombinant plasmid that could produce antisense myb RNA after induction with dexamethasone. During treatment with Hm, K562 cells constitutively expressed c-myb mRNA, and 50% of them began to synthesize hemoglobin (Hb). Expression of antisense myb RNA reduced the amount of c-myb mRNA, and the percentage of Hb-synthesizing cells was decreased to 20%. In the presence of Epo, c-myb mRNA declined and 20% of K562 cells synthesized Hb regardless of antisense myb RNA expression. It is suggested that constitutive expression of c-myb mRNA is necessary for Hm-induced differentiation, and that a decrease in the amount of c-myb mRNA induced by antisense myb RNA expression suppresses Hm-induced differentiation. The amount of c-myb mRNA in K562 cells was reduced during the differentiation induced by Epo. Expression of GATA-1 mRNA was almost constant during Hm-induced differentiation, but increased during Epo treatment. It is supposed that the mechanism of Hm-induced differentiation is distinguished from that of Epo-induced differentiation in K562 cells.
Transcription factors play an important role in the normal developmental process of hematopoietic cells. However, expression of transcription factors and its implication in various human leukemias is not well understood. We have studied GATA-1, GATA-2, and stem cell leukemia (SCL) gene expression in 30 patients with acute myeloid leukemia (AML) by the reverse transcription-polymerase chain reaction assay. In AML both GATA-1 and SCL genes were commonly expressed in M6 and M7 leukemias, and also in leukemias bearing the platelet-associated antigen. We found some AML patients with GATA-1, but not SCL expression. Most CD7+ AML and t(8;21)(q22;q22)-AML were included in this group, which often demonstrated immunoglobulin heavy chain and/or T-cell receptor gene rearrangements. Consequently, GATA-1+ SCL- AML may originate from early myeloid progenitors. Moreover, most AML patients of the M3, M4, or M5 groups were GATA-1- SCL-. Our data suggest that the expression pattern of transcription factors may help to define distinct phenotypes of AML cells.
GATA-1 is an essential factor for the transcriptional activation of erythroid-specific genes, and is also abundantly expressed in a discrete subset of cells bordering the seminiferous epithelium in tubules of the murine testis. In examining normal and germ-line defective mutant mice, we show here that GATA-1 is expressed only in the Sertoli cell lineage in mouse testis. GATA-1 expression in Sertoli cells is induced concomitantly with the first wave of spermatogenesis, and GATA-1-positive cells are uniformly distributed among all tubules during prepubertal testis development. However, the number of GATA-1-positive cells declines thereafter and were found only in the peripheral zone of seminiferous tubules in stages VII, VIII and IX of spermatogenesis in the adult mouse testis. In contrast, virtually every Sertoli cell in mutant W/Wv, jsd/jsd or cryptorchid mice (all of which lack significant numbers of germ cells) expresses GATA-1, thus showing that the expression of this transcription factor is negatively controlled by the maturing germ cells. These observations suggest that transcription factor GATA-1 is a developmental stage- and spermatogenic cycle-specific regulator of gene expression in Sertoli cells.
GATA-1 is a zinc finger DNA-binding protein thought to be involved in the expression of the vast majority of erythroid specific genes. We have examined the phosphorylation of GATA-1 in murine erythroleukemia (MEL) cells and have mapped the sites of phosphorylation by overexpression of GATA-1 in monkey kidney COS cells. We show that GATA-1 is phosphorylated on 6 serines within its amino terminus in uninduced MEL cells and that a 7th site, serine 310, becomes phosphorylated after MEL cells are induced to differentiate by exposure to dimethyl sulfoxide. This site lies near the carboxyl boundary of the DNA-binding domain in a conserved region of the protein believed to be involved in DNA bending. Detailed analyses indicate, however, that phosphorylation at this site, or the other sites identified, does not significantly influence DNA-binding affinity or specificity, DNA bending, or transcriptional transactivation by GATA-1.
Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that binds a CACCC motif found in the human beta-globin gene promoter. We have studied the promoter of the EKLF gene and identified binding sites for the transcription factors GATA-1 and CCAAT-binding Protein 1 (CP1). We show that both types of binding sites are required for full activity, and that the GATA motif at -60 is essential. The EKLF promoter can be directly activated in nonerythroid cells in cotransfection experiments by forced expression of GATA-1. These results suggest that EKLF is dependent on GATA-1 for its expression and lies downstream of, or coincident with, GATA-1 in a regulatory hierarchy in erythroid development.
Expression of the transcription factor GATA-1, which regulates several erythroid specific genes and possibly also some megakaryocytic genes, has been previously detected in normal erythroblasts, megakaryocytes, and basophils, and in some myeloid cell lines. It has been suggested that GATA-1 may be first expressed in a common progenitor and then further activated during erythroid-megakaryocytic and basophilic differentiation and repressed during myeloid maturation. We investigated GATA-1 mRNA expression in highly purified leukemic blasts representing different lineages and stages of myeloid differentiation and in a recently established leukemic cell line, GF-D8, which exhibits morphological, cytochemical and immunophenotypic characteristics of early myeloid progenitor cells. We found GATA-1 expression in five of five myeloid and in one megakaryocytic blast crisis of CML, in four of six cases of myelomonocytic leukemias (M4 according to FAB classification), in one case of erythroleukemia (M6), whereas lymphoid blast crisis of CML and all other FAB groups were completely negative. In addition, a low level of GATA-1 mRNA was also expressed by the GF-D8 cell line. These data further support the hypothesis that GATA-1 expression may occur not only in erythroid and megakaryocytic progenitors, but also in early myeloid progenitors, and then be further regulated during lineage-specific maturation.
The control of the expression of the alpha- and beta-globin gene clusters is effected by sequences called locus control regions (LCRs). Detailed analysis of these elements is therefore essential to the understanding of the complex mechanisms of globin gene regulation. In this paper we describe the characterization of the mouse alpha-globin LCR. This element is localized 26 kb upstream of the mouse embryonic globin gene (Hba-x) and is highly conserved at the protein binding sites. However, three of four CACC boxes and one GATA-1 binding site, identified in the human alpha-LCR, are not conserved in the mouse. Interestingly, we identified a highly conserved putative transcription factor binding sequence (AAAGG) that may play an important role in LCR function. The identification of the mouse alpha-LCR makes it now possible to analyze this element in its natural environment and will certainly contribute to the understanding of the alpha-globin gene expression.
The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.
The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.
In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.
Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5' flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid-specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5' flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.
The recent development of a whole panoply of multidimensional heteronuclear-edited and -filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the 10-kDa limit of conventional 2-dimensional NMR to systems up to potentially 35-40 kDa. The basic strategy for solving 3-dimensional structures of larger proteins and protein-ligand complexes in solution using 3- and 4-dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin-1 beta, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA-1 with its cognate DNA target site.
All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.
Previous studies have demonstrated the expression of GATA-1 (a DNA-binding nuclear protein) in erythrocytes, megakaryocytes, eosinophils, basophils, mast cells, early marrow progenitor cells and in mouse and human erythroid leukaemia cell lines. We studied 31 bone marrow specimens from patients with acute myeloid leukaemia (AML) for GATA-1 expression by reverse transcriptase/polymerase chain reaction (RT/PCR) analysis. GATA-1 expression was detected in all of the patients with erythroleukaemia, and in one of nine patients with megakaryoblastic leukaemia, but absent from 17 patients with French-American-British (FAB) M1-5 leukaemia. In AML, GATA-1 expression is indicative of differentiation to the erythroid and possibly megakaryocytic lineages, analogous to its expression in normal haemopoiesis.
Two conserved loci, DXHX674h and DXHX679h, which map to Xp11.22-Xp11.21 on the human X chromosome short arm, have been positioned between the loci for proteolipid protein (Plp) and the E1a subunit of pyruvate dehydrogenase (Pdha1) in the distal region of the mouse X chromosome using Mus musculus x Mus spretus interspecific backcrosses. These data, together with previous comparative mapping studies on another conserved locus (DXF34) and the locus that encodes the erythroid transcription factor (GATA1), reveal that loci that map to the proximal region of the human X chromosome short arm lie in four different regions of the mouse X chromosome and that the human and mouse X chromosomes contain a minimum of eight conserved segments.
The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.
Erythropoietin (Epo) is a cytokine which specifically regulates differentiation and proliferation of erythroid progenitor cells. We report here that Epo receptor expressed in interleukin 3-dependent lymphoid Ba/F3 cells transmits both differentiation and growth signals. Epo stimulation of these cells leads to activation of transcription and/or translation of the erythroid-specific transcription factors GATA-1 and SCL, followed by the accumulation of both alpha- and beta-globin chains. These results suggest that expression and activation of the Epo receptor regulates erythroid-specific gene expression and might play a role in determining a cell lineage in vivo and that GATA-1 and SCL may exert their effects after Epo binds to its receptor. It was further found that chimeric receptors composed of extracellular domains of Epo receptor and cytoplasmic domains of interleukin 2 or interleukin 3 receptors could also induce erythroid-specific gene expression in Ba/F3 cells. Taking these data together with previous observations, we conclude that interaction of the extracellular domains of the Epo receptor with other membrane components is essential for transmission of both the erythroid differentiation and the growth signals.
The GATA-1 transcription factor has been shown to be important in the regulation of globin and non-globin genes in erythroid, megakaryocytic and mast cell lineages. It is a member of a family of GATA proteins which both overlap in their expression patterns and bind the motif (A/T)GATA(A/G). The GATA family of proteins are also members of the superfamily of zinc finger-like domain proteins and have two similar domains of the type Cys-X2-Cys-X17-Cys-X2-Cys which direct the DNA binding of the protein. A random oligonucleotide selection procedure has been employed to further elucidate the mechanism of GATA-1-DNA recognition. The resulting oligonucleotides were tested for binding activity to both wild-type and mutant GATA-1 proteins. Two classes of GATA-1-DNA interaction have been defined, the first requiring only the carboxy finger of GATA-1 to bind and having the motif GAT(A/T), the second requiring both finger domains to bind and having the core motif (T/C)AAG. By using sequence comparison and depurination analysis it is concluded that the two finger-like domains of GATA-1 have different DNA binding recognition motifs. Binding of GATA-1 to GAT(A/T) motifs is associated with transcriptional activation of linked genes. The only known (T/C)AAG motif is in the distal CAAT-box promoter region of the human A gamma-globin gene where the binding of GATA-1 appears to regulate the correct developmental suppression of gamma-globin expression.
Four distinct factors in extracts from murine erythroleukemia (MEL) cells interacted with the human beta-globin gene promoter CAAT box: CP1, GATA-1, and two novel factors, denoted a and b, one of which is highly inducible in the MEL system. GATA-1 binding to the CAAT element was very unstable (half-life < 1 min), whereas bindings of a, b, and CP1 were comparatively stable, with half-lives of 18, 19, and 3.5 min, respectively. Stable transfections of MEL cells showed that in the presence of the beta-globin locus control region (LCR), the wild-type CAAT box, a mutant which bound to GATA-1 with increased stability over the normal sequences, and a mutant which bound a, b, and CP1 specifically could all stimulate transcription greater than ninefold over that induced by a null CAAT mutation in both uninduced and terminally differentiated MEL cells. A mutant which bound the a and b factors specifically gave only a twofold stimulation of promoter activity, and this lower activity correlated with a decrease in the stability of binding of the b protein. On the other hand, CP1 binding alone did not stimulate transcription. Taken together, these results suggest that in the context of the wild-type beta-globin CAAT element the b factor stimulates transcription directed by the LCR in MEL cells, although the LCR can also function through more stable GATA-1-binding sequences. However, in K562 cells, the wild-type beta-globin CAAT box alone was unable to stimulate gene expression directed by the LCR and high levels of transcription were obtained only upon inclusion of more upstream beta-globin promoter sequences. In contrast, a construct containing only the A gamma-globin CAAT box region did give high expression levels in K562 cells. Thus, there is a fundamental difference in the way the LCR functions in these two model systems in terms of its requirements at the promoter level.
Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. We have previously shown that a fragment extending 643 base pairs upstream the transcription start site of the human GPIIb promoter was able to control the tissue-specific expression of the CAT gene in transfection experiments. Four potential GATA-binding sites, located at positions -463, -376, -243, and -54 are present within this fragment. Gel shift analysis revealed that nuclear extracts from the erythroleukemic cell line HEL contain a DNA-binding protein that recognizes these GATA sites. Using an antiserum raised to an hydrophilic region of the transcription factor GATA-1, the HEL GATA-binding protein was found to be GATA-1. Point mutations of the different GATA sites indicated that they did not equally contribute to GPIIb promoter activity. The -463 GATA motif located in an enhancer region is essential for full transcription activity and was found to be dominant upon the other GATA motifs. When this site is mutated, the -54 GATA site appears to be essential for the remaining CAT activity. These results indicate that the transcription factor GATA-1 plays an important role in the regulation of the transcription of the megakaryocyte specific GPIIb gene.
The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut-NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single-copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.
GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation.
Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.
Inappropriate expression of the Evi-1 zinc-finger gene in hematopoietic cells has been associated with acute myelogenous leukemia and myelodysplastic syndromes in murine models and in humans. Consistent with this, previous studies have shown that aberrant expression of the Evi-1 gene in a myeloid progenitor cell line blocks granulocytic differentiation. Here we demonstrate that the aberrant expression of the Evi-1 gene impairs the normal response of erythroid cells or bone-marrow progenitors to erythropoietin. Erythroid differentiation has been shown to require the GATA-1 transcription factor that binds to a sequence contained within the consensus binding sequence identified for Evi-1. In the studies presented here we also show that Evi-1 can repress GATA-1-dependent transactivation in transient chloramphenicol acetyltransferase assays. Together the data support the hypothesis that inappropriate expression of the Evi-1 gene blocks erythropoiesis by repressing the transcription of a subset of GATA-1 target genes.
Ets family proteins activate transcription via binding to the GGAA core sequence located in the promoter/enhancer elements of many cellular and viral genes. GATA-1 is an erythroid-specific transcription factor. The promoter of the chicken GATA-1 gene contains multiple ets binding sites (EBS), two of them are present in palindromic form. The GATA-1 promoter has been shown to be activated by the E26 virus. In this study, we have analysed whether the palidromic EBS of the chicken GATA-1 promoter is a target for binding and activation by members of the cellular ets gene family products. The results herein indicate that both EBS in the palindrome are required for DNA-binding because mutations in either site reduces the activity by at least 95%. Moreover, DNA binding of ETS1 to the EBS palindrome is dramatically stabilized in the presence of a specific monoclonal antibody whose epitope maps between amino acid positions 240-260. Although each of the single sites bind, the efficiency of binding is extremely low. Furthermore, for efficient binding the two sites must be in an inverted configuration because of the fact that the oligonucleotide containing the left and right EBS in the same orientation binds 10-fold less than the oligonucleotide containing the EBS palindrome. Additionally, we show that the transcription of a reporter gene (CAT) either linked to the GATA-1 EBS palindrome or GATA-1 promoter can be activated by cotransfection with ETS1, alternatively-spliced ETS1, ETS2 or ERGB/Hu-FLI-1 expression vectors.
Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma-globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3).(ABSTRACT TRUNCATED AT 400 WORDS)
Treatment of mouse erythroleukemia (MEL) cells with hexamethylene bisacetamide induces a program of erythrodifferentiation, as judged by an increase in the synthesis of globins and other erythroid-specific products. This induction can be inhibited by glucocorticoids, e.g. dexamethasone. All globin and other erythroid-specific genes tested contain GATA response elements (GATA-RE) and can be transactivated by GATA-1, a transcription factor. GATA-1 is highly expressed in erythroid cells, including MEL cells. We noted a glucocorticoid receptor (GR) response element motif near a GATA-RE motif in the promoter region of the mouse beta-major and beta-minor globin genes and about 130 bases away from a GATA-RE in the alpha 1-globin gene promoter and, therefore, investigated the possibility that the dexamethasone-induced inhibition of induced MEL cell differentiation may involve effects of the GR on GATA-1 activity. Evidence obtained from transfection assays and DNA electrophoretic mobility shift assays indicates that the GR binds GATA-1 and interferes with its function before any interaction with DNA, but that the presence of a glucocorticoid response element near a GATA-RE augments the GR effect. The N-terminal 106-amino acid domain of the GR was found to be essential for the effect, possibly by binding to GATA-1. Since GATA-1 is autoregulatory, i.e. it has been shown by others to bind to its own promoter and up-regulate its own transcription, the finding that activated GR can interfere with GATA-1 function may provide an explanation for the inhibition by glucocorticoids of the entire program of erythroid differentiation in MEL cells. That is, by interfering with GATA-1 function, the GR inhibits not only the expression of erythroid structural genes, but may also inhibit the expression of a primary erythroid regulatory gene, GATA-1. It was also shown that the GATA-RE in each of the beta-globin promoters responds to mouse GATA-1 in a functional transfection assay.
The sequence-specific DNA-binding protein factor F6, which binds upstream of the cluster of the chicken alpha-globin genes, has previously been found to interact with a DNA fragment containing a replication origin and a nuclear matrix binding site. This protein has been partially characterized. Based on its molecular weight and binding affinity, F6 belongs to a family of GATA proteins, the chicken equivalent of transcription factor NFE-1. An oligonucleotide including the binding site for F6 competes for binding of the above-mentioned DNA fragment to the nuclear matrix. This indicates an involvement of this protein in the interaction between DNA and the nuclear matrix.
The transcription factor GATA-1 is a fundamental regulator of genes in haematopoietic cell lineages and belongs to a family of factors that bind to the consensus sequence WGATAR. The GATA motif was originally identified in cis-regulatory regions of globin and other erythroid-specific genes, but the range of genes controlled by the GATA factors has since expanded. Members of the GATA transcription factor family share a conserved zinc-finger DNA-binding domain, but the expression profile of each GATA factor is distinct. Here we show that a testis form of murine (m)GATA-1 messenger RNA is transcribed from a promoter located 5' to the erythroid first exon, and the remaining exons (which encode the mGATA-1 protein) are used in common by both testis and erythroid transcripts. We use an anti-mGATA-1 monoclonal antibody to show that the factor expressed in erythroid cells is the same as that found in the seminiferous tubules of murine testis. The GATA-1-expressing cells in 10-week-old testis were found only in contact with the basement membrane of seminiferous tubules, suggesting that GATA-1 regulates genes during the earliest stages of spermatogenesis.
We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.
Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.
Previous studies have shown that a -112 to +78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid-specific activity. This PBGD-(-112 to +78) promoter contains a CCACC binding site (position -100), a GATA binding site (position -70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA-1 trans-activates the PBGD-(-112 to +78) promoter in non-erythroid cells. We show that, if trans-activation is abolished by mutations that destroy either the -100 CCACC binding or the -70 GATA binding sites, replacement of the -100 CCACC binding site by a simian-virus-40-protein-1 (Sp1) binding site maintains both the erythroid-specific activity of this promoter and the human GATA-1 trans-activation. Thus, human GATA-1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD-(-112 to +78) promoter is activated 20-fold by a cis-linked 5' hypersensitive site 2 (5'HS-2) of the human beta-globin locus control region. This activation depends on the -70 GATA and -100 CCACC or Sp1 binding sites. When a longer -714 to +78 fragment of the PBGD promoter is used, the -70 GATA mutant still displays erythroid-specific activity and is cis-activated by the 5'HS-2 enhancer, while the -100 CCACC mutant is completely inactive in the absence or in the presence of the 5'HS-2 enhancer. Thus, the -100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5'HS-2 enhancer, whereas the -70 GATA binding site can functionally be replaced by upstream cis-acting elements.
Recently, many investigators have been interested in the study on eosinophil biology since genes association with eosinophils such as interleukin-5 or eosinophil granule proteins (EPO, ECP, EDN, MBP, and CLC), were isolated. However, the molecular basis for the commitment of progenitors to the eosinophil lineage has not been determined. The mechanism by which eosinophil-specific genes encoding primary and secondary granule proteins (e.g. ECP, EDN, EPO, MBP, and CLC) are expressed and regulated during eosinophilopoiesis is also unknown. In this paper, I described the characterization of genes encoding eosinophil granule proteins and the mRNA expression of GATA-1 binding transcription factor during eosinophil differentiation.
Proper expression of the human beta-like globin genes is completely dependent on the presence of the locus control region or LCR, a region containing four DNase hypersensitive sites (HS1-4) situated 5' to the structural genes. Linkage of the LCR to a transgene results in copy number-dependent transcription, independent of the site of integration in the host genome. We have analysed a small region of the LCR (HS3) in transgenic animals to determine the minimal interactions that are required for this property. The results show that a specific combination of a G-rich sequence flanked on each side by one binding site for the transcription factor GATA1 is essential to obtain position-independent expression of a linked beta globin gene in erythroid cells. The overall transcriptional activity of HS3 is achieved through synergy with other combinations of similar binding sites.
Anthracycline antitumor drugs, particularly aclacinomycin (ACM) have been shown to be potent inducers of erythroid differentiation in human leukemic K562 cells. Here we report that such an event is associated with an overexpression of the erythroid-specific transcription factors GATA-1 and NFE-2. Using the electrophoretic mobility shift assay, during differentiation over 3 days of culture, we have observed an increase in the binding either of GATA-1 to the promoter of the gamma-globin gene (region -201 to -156) or NFE-2 to the promotor of the porphobilinogen deaminase gene (region -170 to -142). Both events were paralleled by a recruitment of hemoglobinized cells and a stimulation of heme synthesis. Enhanced binding capacity of GATA-1 was confirmed by an increase in its mRNAs. Moreover, GATA-1 and NFE-2 overexpression has been shown to be specific of the differentiating effect of the drug and not of its growth inhibitory effect. In contrast, no change was observed in the binding of the ubiquitous factors OTF-1 and AP-1, except on day 3, where AP-1 decreased. Although ACM is a DNA-intercalating agent, it did not directly affect transcription factors binding to their cis-sequences as assessed by the preincubation of the oligonucleotides probes with increasing concentrations of ACM. Taken together, these results strongly suggest that ACM could exert their erythroid-differentiating activity by modulating the expression of transcription factors which specifically regulate the transcription of erythroid genes.
Erythroid differentiation leads to the production of red blood cells that contain a high level of hemoglobin. This level is mainly regulated by globin gene transcription during development and differentiation. Although numerous cis-acting sequences are involved in transcriptional activity of globin genes, combinations of three motifs, CCACC, SP1 and GATA represent the core elements of their regulatory sequences. These combinations are also found in promoters and/or enhancers of non-globin genes specifically expressed in the late stages of erythroid differentiation. The CCACC and SP1 sequences bind proteins that do not display erythrocytic specificity, and the GATA sequences bind a family of transacting factors recently cloned. The GATA family members are distinctive for a highly homologous DNA binding domain that exists in two zinc fingers reminiscent of those of the glucocorticoid receptor. None of the GATA family members displays only erythroid specificity, but gene disruption followed by rescue indicates that GATA-1 is necessary for terminal erythroid differentiation throughout development. The GATA/SP1 and GATA/CCACC associations are present in positive, negative or inducible regulatory sequences suggesting that other elements control the fine tuning of erythroid gene expression. NF-E2, which is a major transcriptional activator, members of the ets family which are implicated in the early stages of erythropoiesis and finally c-erbA which directly regulates a set of erythroid-specific genes are proteins that bind these latter regulatory motifs.
The developmental control of expression of the human epsilon-globin gene appears to be mediated, at least in part, by a transcriptional silencer in the DNA 5' to the cap site of this gene. We have used site-directed mutagenesis and DNA-protein binding assays to define the active motifs of this epsilon-globin silencer. DNase I foot-printing of the silencer region with K562 cell nuclear extracts defined a sequence, which we designate as the epsilon-globin silencer motif or epsilon GSM (epsilon -278 to -258 base pairs (bp)) containing a region (epsilon -270 to -258) with 90% homology to the yeast mating type silencer, ABF-1 (autonomous replicating sequence binding factor one) and which also overlaps at (epsilon -269 to -262) with the human YY1 consensus sequence, an element which mediates transcription repression and activation of viral, mouse, and human genes. The DNase I footprint extended 5' in the silencer region to include an inverted repeat of a six-nucleotide motif (epsilon -267 to -278 bp) which shares 5 of 6 bases with the GATA-1 consensus sequence. In gel mobility shift assays, two specific proteins (A and B) in nuclear extracts from erythroleukemia K562 cells bound to the DNase I-footprinted region. Protein B, associated with epsilon-globin silencer activity in vitro, required an intact epsilon GSM sequence for binding. Mutation of 5 bases within the epsilon GSM in an epsilon-globin promoter-containing fragment extending upstream to 1400 bp in transient transfection assays increased activity by 3.0-fold compared with the native sequence, suggesting that the silencer activity was mediated by the epsilon GSM sequence. We found that protein A could be displaced by a competitor containing the GATA-1 consensus sequence, suggesting that protein A is a GATA-like protein. The region from -267 to -271 within the epsilon GSM and GATA-1 homology region was important for binding of both proteins A and B. These data suggest that protein binding to the epsilon GSM and GATA motifs mediate the negative effect of the silencer on transcription, possibly via direct competition for binding to this DNA region. Recombinant yeast ABF-1 and human YY1 bound to the epsilon GSM. Mutating three bases (epsilon -259, -262, -264) in the epsilon GSM decreased the binding affinity of protein B and recombinant human YY1 but increased the binding affinity of recombinant yeast ABF-1. Furthermore, competitor containing the YY1 consensus sequence competed for protein B binding, whereas competitor containing a perfect yeast ABF-1 consensus sequence did not.(ABSTRACT TRUNCATED AT 400 WORDS)
In summary, we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level. We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis. By ablating the expression of the endogenous GATA-1 gene, we are in a position to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences. We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment, experiments that have not been possible heretofore. Although the assay involves the dramatic loss of red cell production, it should be possible to define important regulatory domains that can then be assayed using less stringent systems, such as cell-free extracts for in vitro transcription. The ideal situation would be analyses conducted in GATA-1- erythroid cells. However, these cells have been impossible to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability (C. Simon and S. Orkin, unpublished observations). It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene. If GATA-1- red cells were available, the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes. Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied. However, the ES cell system can be used as an alternative until targeted erythroleukemia cells become available. Other applications involve the introduction of other GATA-binding protein family members to determine whether they rescue the mutation. If they cannot, chimeric proteins can be tested to identify which amino acids distinguish the different family members. We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny. How does GATA-1 regulate red cell genes like globin or the Epo receptor? Once we identify the functional domains of the GATA-binding proteins, we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin. Is GATA-1 necessary for globin gene switching? GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters. We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression.
We analyzed epsilon-globin transcription in erythroid cells and in erythroid extracts to determine the requirements for enhancer-dependent expression of this gene. Mutations that abolished GATA-1 binding at a single position in the promoter prevented interaction with enhancers, whereas elimination of a second more distal promoter GATA-1 site had no effect. Deletion or mutation of the GATA-1 sites in either the human beta-globin locus control region DNase-hypersensitive site II enhancer or the chicken beta A/epsilon-globin enhancer did not diminish the ability of the enhancers to interact with the promoter. In contrast, mutation of the AP-1/NF-E2 sites in these enhancers resulted in elimination of enhancement. In vitro transcription of these constructs was promoter dependent and was not sensitive to abolition of GATA-1 binding in the promoter, consistent with the role of GATA-1 solely as a mediator of the enhancer effect. Thus, GATA-1 regulates the response of the epsilon-globin gene to enhancers through a specific site in the promoter and requires enhancer AP-1/NF-E2 binding to transduce the enhancer effect on transcription.
Tal-1 rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the tal-1 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the GATA-1 gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied GATA-1 expression in these purified hematopoietic cells. We found that GATA-1 and tal-1 genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, tal-1 and GATA-1 genes are coexpressed in committed progenitors cells (CD34+/CD38(2+)), whereas they are not detectable in the most primitive cells (CD34(2+)/CD38-). In contrast, GATA-2 is strongly expressed in both most primitive and committed progenitors cells, whereas GATA-3 is mostly detected in most primitive ones. Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages.
The locus for the erthyroid transcription factor, GATA1, has been positioned in the small interval between DXS255 and TIMP on the proximal short arm of the human X Chromosome (Chr) by use of a partial human cDNA clone and a well-characterized somatic cell hybrid panel. Analysis of selected recombinants from 108 Mus musculus x Mus spretus backcross progeny with the same clone confirmed that the homologous murine locus (Gf-1) lies between Otc and the centromere of the mouse X Chr. These data imply that a partial inversion of gene order has occurred within the conserved segment that represents Xp21.1-Xp11.23 in human (CYBB-GATA1) and the proximal 6 cM of the mouse X Chr (Gf-1-Timp). Furthermore, they indicate that the mouse mutant scurfy and the human genetic disorder Wiskott-Aldrich syndrome, which have been mapped to the same regions as GATA1/Gf-1 in both species, may indeed be homologous disorders.
We have investigated interactions between the erythroid transcription factor GATA-1 and factors binding two cis-acting elements commonly linked to GATA sites in erythroid control elements. GATA-1 is present at all stages of erythroid differentiation, is necessary for erythropoiesis, and binds sites in all erythroid control elements. However, minimal promoters containing GATA-1 sites are inactive when tested in erythroid cells. Based on this observation, two erythroid cis elements, here termed CACCC and AP-1/NFE-2, were linked to GATA sites in minimal promoters. None of the elements linked only to a TATA box created an active promoter, but GATA sites linked to either CACCC or AP-1/NFE-2 elements formed strong erythroid promoters. A mutation of T to C at position -175 in the gamma-globin promoter GATA site, associated with hereditary persistence of fetal hemoglobin (HPFH), increased expression of these promoters in both fetal and adult cells. A construct bearing the beta-globin CACCC element was more active in adult and less active in fetal erythroid cells, when compared with the gamma-globin CACCC element. These studies suggest that erythroid control elements are formed by the interactions of at least three transcription factors, none of which functions alone.
We have performed a detailed analysis of the cis-acting sequences involved in the erythroid-specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, -1 to -60, contains a GATA binding sequence around -37 and an SP1 binding sequence around -50. This region is active in erythroid and non-erythroid cells. The distal region, -60 to -95, contains two overlapping protein binding sites around -75, one for hGATA-1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non-erythroid cells and defines the -95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the -37 GATA and the -50 SP1 binding sites are necessary for efficient activity of the -95 GPB construct. Mutations that impair the -75 GATA-1 binding result in extinction of the -95 GPB construct activity if the -75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the -75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA-1 can efficiently activate transcription of the -95 GPB construct in non-erythroid cells. This transactivation is abolished by mutations that impair either the -37 GATA-1 or the -50 SP1 binding. Mutations that impair the -75 GATA-1 binding and still allow the -75 ubiquitous binding also abolish the transactivation of the -95 GPB construct, indicating that hGATA-1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
The SCL (tal-1, TCL5) gene is a member of the basic domain, helix-loop-helix (bHLH) class of putative transcription factors. We found that (i) the SCL promoter for exon Ia contains a potential recognition site for GATA-binding transcription factors, (ii) SCL mRNA is expressed in all erythroid tissues and cell lines examined, and (iii) SCL mRNA increases upon induced differentiation of murine erythroleukemia (MEL) cells, and inferred that SCL may play a physiologic role in erythroid differentiation. We used gel shift and transfection assays to demonstrate that the GATA motif in the SCL promoter binds GATA-1 (and GATA-2), and also mediates transcriptional transactivation. To identify a role for SCL in erythroid differentiation, we generated stable transfectants of MEL and K562 (a human chronic myelogenous leukemia cell line that can differentiate along the erythroid pathway) cells overexpressing wild-type, antisense or mutant SCL cDNA. Increasing the level of SCL expression in two independent MEL lines (F4-6 and C19, a 745 derivative) and K562 cells increased the rate of spontaneous (i.e. in the absence of inducer) erythroid differentiation. Conversely, induced differentiation was inhibited in MEL transfectants expressing either antisense SCL cDNA or a mutant SCL lacking the basic domain. Our experiments suggest that the SCL gene can be a target for the erythroid transcription factor GATA-1 and that the SCL gene product serves as a positive regulator of erythroid differentiation.
Delta-thalassemia is a complex group of inherited disorders of globin genes characterized by impaired synthesis of the delta-globin chain. The T-C substitution was detected at position -77 of the delta-globin gene isolated from three independent Japanese individuals who were homozygotes for delta-thalassemia. To elucidate the significance of the mutation in delta-globin gene expression, we investigated the genotype of three delta-thalassemia homozygotes and 58 normal individuals using dot blot hybridization of the polymerase chain reaction (PCR)-amplified DNA. The mutation was observed in six alleles of three homozygotes, while no mutation was detected in 116 alleles of normal individuals, thereby indicating the close association of this mutation with the thalassemia phenotype. Since the mutation (TTATCT-TCATCT) is located within the inverted binding motif of GATA-1 (T or A-G-A-T-A-G or A), an erythroid cell-specific transcription factor, we did gel retardation assays using nuclear extracts from the erythroid cells. We found that GATA-1 binds the oligonucleotide spanning positions -61 to -90, but does not bind to the oligonucleotide with the mutation at position -77. Competition gel retardation assays showed that GATA-1 binding can be competed out by the fragment with the GATA-1 motif, but not with the mutant oligonucleotide. Analysis of the transient expression of the CAT gene linked to the delta-globin gene promoter region demonstrated that the construct with the mutant promoter region was expressed about 20-fold less compared with the normal one. Thus, the mutation at position -77 impairs delta-globin gene expression by abolishing GATA-1 binding to the AGATAA sequence of the promoter region of the delta-globin gene. This provides a good example of involvement of tissue-specific transacting factors in the molecular pathogenesis of hereditary diseases.
We performed a systematic functional analysis of the human gamma-globin promoter to identify its activator domains. We used a panel of truncation and scanning mutants as well as transfection in human K562 fetal erythroid cells. The various mutations produced relatively small changes in promoter function in both transient and stable transfection assays. The CACCC region and the region containing the binding sites for protein GATA-1 behaved as activator domains. We also obtained evidence for a minor activator site in the - 200 to - 190 region. The results are consistent with the interpretation that gamma-globin gene regulation may occur in part through multiple small effects of promoter elements.
The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (i) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors.
The gene coding for glycoprotein IIb (GPIIb), the alpha subunit of platelet integrin GPIIb/IIIa is an early and specific marker of the megakaryocytic lineage. Thus, studies on the regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation in this lineage. The promoter region of this gene was isolated and analyzed to understand its tissue-specific transcriptional activity. A region extending from nucleotides -414 to -554 was found to be extremely important for the promoter function. Deletion of this region results in a 70% decrease of the promoter activity, as measured in CAT assays. This region has the properties of an enhancer. It is able to activate a heterologous promoter, in a distance- and orientation-independent manner, in both megakaryocytic and erythroid cells. This enhancer contains binding sites for nuclear factors and mutation of these sites, individually or together, abolish the enhancer activity. These nuclear factors are present in megakaryocytic and erythroid cell lineages, but they are absent in the other tested cells. One of the sites, named domain D, contains a TTATC motif that may interact with the transcription factor GATA1, active in erythroid and megakaryocytic cells. These results indicate that the promoter of a megakaryocytic gene contains a tissue specific enhancer, active in both the erythroid and the megakaryocytic lineages, and may implicate the erythroid factor GATA1.
ME26 virus is a recombinant mouse retrovirus construct homologous to the avian E26 virus. Both encode a 135-kDa gag-myb-ets fusion protein which is localized in the nucleus. We have recently shown that ME26 virus can induce erythropoietin (Epo) responsiveness in hematopoietic cells. Mice infected with ME26 virus develop a hyperplasia of Epo-dependent hematopoietic precursor cells from which permanent cell lines can be established. In vitro, ME26 virus specifically induces Epo responsiveness in the interleukin-3-dependent myeloid cell line FDC-P2 by enhancing expression of the Epo receptor (EpoR). In the present study we demonstrate that ME26 virus infection of FDC-P2 cells also results in enhanced expression of beta-globin and the erythroid-specific transcription factor GATA-1, a protein which can transactivate both the EpoR promoter and globin genes. In addition, these cells exhibit a down-regulation of c-myb expression similar to that seen in differentiating erythroid cells. To determine the molecular basis for activation of erythroid genes in ME26 virus-infected cells, we carried out transient expression assays with DNA constructs of either the EpoR promoter of the GATA-1 promoter linked to reporter genes. Our results indicate that while ME26 virus did not directly enhance expression from the EpoR promoter, both it and its avian parent, E26, transactivated the GATA-1 promoter. Furthermore, ME26 virus cooperates with the GATA-1 protein to enhance expression of the EpoR gene. We propose that the mechanism by which ME26 virus induces erythroleukemia involves transactivation of the GATA-1 gene, thus positively regulating the expression of the EpoR and leading to the proliferation of a unique population of Epo-responsive cells. By specifically inducing Epo responsiveness in hematopoietic cells via transactivation of a transcription factor, ME26 virus utilizes a novel mechanism for retrovirus pathogenesis.
Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.
The SCL gene encodes a putative transcription factor with a basic helix-loop-helix (B-HLH) motif and is known to be predominantly expressed in erythroid cells. Here we also demonstrate expression of SCL mRNA in normal mast cells, mast cell lines and megakaryocytic cell lines. SCL is therefore expressed in the same three lineages as GATA-1, a well-recognized hemopoietic transcription factor. SCL and GATA-1 mRNA were also co-expressed in interleukin 3-dependent primitive myeloid lines. In murine erythroleukemia (MEL) cells SCL and GATA-1 underwent coordinated biphasic modulation during hexamethylene bisacetamide (HMBA)-induced erythroid differentiation. The kinetics of SCL and GATA-1 mRNA expression was inversely correlated with changes in ID, a negative regulator of B-HLH proteins, and was distinct from changes in MYC, MYB and erythropoietin receptor transcripts. During myeloid differentiation of K562 cells, SCL and GATA-1 mRNA levels also underwent biphasic modulation. Thus SCL and GATA-1 are coordinately expressed in multiple hemopoietic lineages and coordinately regulated during induced erythroid and myeloid differentiation. In nonhemopoietic tissues SCL was only detected in adult and developing brain where GATA-1 is reportedly not expressed. In day 14.5 embryos analysed by in situ hybridization, SCL transcripts were detected in post-mitotic neurons in the metencephalon and roof of the mesencephalon. This suggests a previously unexpected role for SCL in neural differentiation.
The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or followed by dramatic increases in EpoR transcription. To determine whether gene activity could be correlated with chromatin alterations, DNase-hypersensitive sites (HSS) were mapped. Two major HSS located in the promoter region and within the first intron of the EpoR gene are present in all embryonal stem and hematopoietic cells tested, the intensities of which correlate well with EpoR expression levels. In addition, a third major HSS also located within the first intron of the EpoR gene is uniquely present in erythroid cells that express high levels of EpoR. Transfection assays show that sequences surrounding this major HSS impart erythroid cell-specific enhancer activity to a heterologous promoter and that this activity is at least in part mediated by GATA-1. These data, together with concordant expression levels of GATA-1 and EpoR in both early multipotent hematopoietic and committed erythroid cells, support a regulatory role of the erythroid cell-specific transcription factor GATA-1 in EpoR transcription in these cells. However, the lack of significant levels of GATA-1 expression in embryonal stem cells implies an alternative regulatory mechanism of EpoR transcription in cells not committed to the hematopoietic lineage.
We have previously described a family of Northern Sardinian descent in which the propositus was affected by thalassemia major resulting from compound heterozygosity for codon 39 nonsense mutation and the beta +IVS II nt 745 mutation and in which all heterozygotes for the beta +IVS II nt 745 mutation had normal hemoglobin (Hb) A2 levels. To define the reasons for normal HbA2 levels in otherwise typical beta-thalassemia heterozygotes, we cloned and sequenced the delta-thalassemia gene in cis to the beta +IVS II nt 745 mutation. The sequence analysis showed a single nucleotide substitution (G----A) at position 69 nts (delta +69) downstream to the polyA addition site. Dot blot analysis with an oligonucleotide probe complementary to the delta +69 mutation detected this mutation in several heterozygotes for the beta +IVS II nt 745 mutation from the proband's family, but failed to show it either in a group of normal individuals of the same origin or in nonrelated heterozygotes for the beta +IVS II nt 745 mutation of the same or different descent from the proband. The delta +69 (G----A) mutation may be responsible for the low delta-globin output from the beta +IVS II nt 745 chromosome or could be a silent polymorphism not affecting the function of the delta-globin gene. The normal G at position 69 is part of a sequence very similar to the core DNA (A/T)GATA(A/G) motif (GATA box) that is a binding site for the GATA-1 protein. Gel-retardation assay has shown that a DNA fragment containing the GATA motif with the G----A at position +69 has increased binding affinity for erythroid-specific DNA binding protein(s) as compared with the wild-type sequence. These findings may suggest that the delta +69 mutation is responsible for the deficient function of the in cis delta-globin gene.
Some of the elements involved in the erythroid-specific transcriptional regulation of the human gamma- and beta-globin genes and located inside or in the immediate proximity of these genes have been identified as sequences which bind erythroid-specific factors. In the present study, we found two regions located within 1 kb in 5' to the alpha 2- and in 3' to the alpha 1-globin genes which contribute to the induction of human alpha-globin genes following erythroid differentiation in stable MEL transformants. By DNAse I footprinting and gel mobility shift assays, we identified several GATA-1 and one AP-1/NF-E2-binding sites located inside these regions. These results strengthen the idea that, like for all other globin genes, flanking regions contribute in vivo to the regulation of human alpha-globin gene expression.
Regulation of the expression of the erythropoietin (Epo) receptor (EpoR) gene is under the control of transcriptional regulatory factor GATA-1. GATA-1 is expressed widely among the nonerythroid, factor-dependent subclones of the interleukin 3-dependent mouse cell line 32D. Consequently, to determine whether GATA-1 and EpoR gene expression are linked even in nonerythroid cells, we have studied the correlation of GATA-1 expression with expression and function of EpoR in these cell lines. EpoR mRNA (by RNase protection analysis) and EpoR protein (by specific antibody immunoprecipitation of metabolically labeled EpoR protein) were detectable not only in 32D and 32D Epo (an Epo-dependent subclone) but also in 32D GM, a subclone dependent for growth on granulocyte/macrophage colony-stimulating factor. EpoR mRNA also was detectable by PCR in 32D G, a subclone dependent for growth on granulocyte colony-stimulating factor. However, only 32D Epo cells bound 125I-labeled Epo and expressed EpoR protein on the cell surface, as determined by immunoprecipitation of surface-labeled proteins. These results indicate that, in these factor-dependent cell lines, the major regulatory step determining the erythroid-specific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Mechanisms that could affect lineage-specific translocation are the presence of a chaperone protein, erythroid-specific editing of EpoR mRNA, or altered processing of the EpoR protein to the cell surface. In this model, lineage-restricted responses to growth factors such as Epo are determined not by expression of the genes for growth factor receptors but, rather, by appropriate processing of the receptor protein.
Erythropoietin, a glycoprotein produced by the kidneys in response to anemia and hypoxia, is a major growth factor for cells of the erythroid lineage. Erythropoietin interacts with high-affinity cell surface receptors (EpoR) present on developing progenitors and is required for their survival. Previously we characterized the gene for EpoR and demonstrated that its promoter acts in a cell-specific manner. Here we show that the hematopoietic-specific transcription factor GATA-1 is necessary, and indeed is sufficient as the sole cell-restricted regulator, for activation of the EpoR promoter in fibroblast transfection assays. Hence, GATA-1, which participates in transcriptional control of the majority of erythroid-expressed genes, also acts on the promoter of an essential lineage-restricted receptor (EpoR). This central contribution of GATA-1 to EpoR promoter function provides a mechanism whereby a cell-restricted regulator may ensure the viability and subsequent maturation of progenitor cells during hematopoietic differentiation.
The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.
The 5' DNase I-hypersensitive site 2 (5' HS-2) is an erythroid-specific enhancer located 11 kilobases (kb) upstream of the human beta-globin gene cluster. Presence in cis of 5' HS-2 confers a high level of erythroid cell-specific and developmentally regulated promoter activities of human globin genes in transfected cell cultures and in transgenic mice. Combining the use of the methylation protection assay and polymerase chain reaction, we have studied nuclear factor-DNA interactions of the 5' HS-2 enhancer in vitro and in vivo. The data from analyses of three different sequence motifs within 5' HS-2 represent three different modes of protein-DNA interaction with respect to cell-type specificities and in vivo vs. in vitro differences. First, a GATA-1 motif was found to bind nuclear factor(s), presumably the GATA-1 factor, present in K-562 cell extracts and in living K-562 cells. No such binding was seen in nonerythroid HeLa cells or extract. A second motif, NF-E2/AP1 (nuclear factor-erythroid 2/activator protein 1), consists of tandemly arranged dimers of AP1 binding consensus. The presence of either HeLa extract or K-562 extract protects the NF-E2/AP1 motif from methylation, but the footprints are different. This is most likely due to different protein-DNA contacts of the AP1-DNA complex formed in HeLa extract and the NF-E2-DNA complex in K-562 extract. In vivo methylation protection patterns of this motif parallel those observed in vitro, suggesting that it is also bound by NF-E2 in K-562 cells and by AP1 in HeLa cells. Finally, a GT-I motif binds apparently to one or more similar factors in both types of nuclear extracts, but the in vivo methylation protection patterns are not identical between living HeLa and K-562 cells. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the 5' HS-2 enhancer in erythroid cells. The detection of conformationally different nuclear factor-DNA complexes at the same sequence motifs in HeLa and Raji cell lines also raises interesting questions regarding the origin and function of these complexes in nonerythroid cells.
Erythropoietin is a cytokine which specifically regulates the proliferation and differentiation of erythroid progenitor cells. The expression of erythropoietin receptor on the cell membrane of the progenitor cells is a critical event during the erythroid differentiation process. In order to clarify the tissue-specific and differentiation stage-specific expression of the erythropoietin receptor gene, its transcriptional regulation was examined by transient expression assay, gel mobility shift assay and DNase I footprinting. The results clearly showed that GATA-1 transactivates the gene expression through a single GATA motif located around -200 bp upstream from the ATG codon in a dose dependent manner. Furthermore, Northern blot analysis revealed that erythropoietin receptor-mediated signals strongly enhanced GATA-1 gene expression in accordance with the appearance of hemoglobin-positive cells. Taken together with other observations, these results suggested the following scheme of erythroid differentiation: 1)GATA-1 is expressed in the early stage of blood cell development; 2) GATA-1 transactivates the erythropoietin receptor gene; 3) erythropoietin binds its receptor and the receptor-mediated signals enhance GATA-1 gene expression in erythroid progenitor cells; and 4) GATA-1 finally transactivates hemoglobin synthesis-related genes and globin genes in relatively matured erythroid cells.
5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway. cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library. It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd, and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd. The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd, respectively. The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two-thirds of their proteins. An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site. An iron-responsive element (IRE) motif has been identified in the 5'-untranslated region of the human erythroid ALAS mRNA, but is not present in the housekeeping ALAS mRNA. Gel retardation experiments established that this IRE motif formed a protein - RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs. A transcript of the ALAS IRE, mutated in the conserved loop of the IRE, did not readily form this protein - RNA complex. These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis.
The human GATA1, hGATA1 (previously called NF-E1, GF-1 or Eryf-1), a major sequence-specific DNA-binding protein of the erythrocytic lineage, is a member of a zinc-finger family of DNA-binding proteins. We report here the cloning of a human cDNA for a new member of this family. This member, called hGATA3, has 85% amino acid homology with hGATA1 in the DNA-binding domain and no homology elsewhere in the protein. Unlike hGATA1, hGATA3 is not localized on the X chromosome and we map it to the 10p15 band of the human genome. Northern blot analysis indicates that this factor is a T-cell specific transcription factor, present before activation and up-regulated during T-cell activation. The encoded hGATA3 protein, made in an in vitro transcription-translation assay, binds the WGATAR motif present in the human T-cell receptor (TCR) delta gene enhancer and, by transfection in HeLa cells, we show that hGATA3 can transactivate this TCR delta gene enhancer. Interestingly this enhancer binds and is also transactivated by hGATA1. Conversely, the promoter of the human glycophorin B (GPB), which is erythroid-specific and contains two WGATAR motifs, binds and is transactivated by hGATA1 and, to a lesser extent, by hGATA3. These results indicate that the activation of specific genes by hGATA1 or hGATA3 is partly governed by the lineage expression of these two factors during haematopoiesis and that, in the T-cell lineage, hGATA3 binds the human TCR delta gene enhancer and is involved in its expression.
Transcription of erythroid-expressed genes and normal erythroid development in vivo are dependent on a regulatory protein (GATA-1) that recognizes a consensus GATA motif. GATA-1 expression is itself restricted to erythroid progenitors and to two related hematopoietic lineages, megakaryocytes and mast cells. During cellular maturation the levels of GATA-1 RNA and protein increase progressively. In an effort to delineate mechanisms by which this pivotal transcription factor is itself regulated we have characterized the mouse GATA-1 gene and cis-elements within its promoter. We find that the isolated promoter retains cell specificity exhibited by the intact gene. Full promoter activity requires the presence of proximal CACCC box sequences and an upstream, double GATA motif that binds a single GATA-1 molecule in an asymmetric fashion. Using in vivo footprinting of mouse erythroleukemic cells we detect protein binding in vivo to both cis-elements. On the basis of these findings we propose that a positive feedback loop mediated through GATA-1 serves two complementary functions: maintenance of the differentiated state by locking the promoter into an "on" state, and programming the progressive increase in protein content throughout cellular maturation.
The epsilon-globin gene is the first of the human beta-like globin genes to be expressed during development. We have analyzed protein-DNA interactions in the epsilon-globin promoter region by DNase I footprinting and electrophoretic mobility shift experiments using nuclear extracts from K562 human erythroid cells and from nonerythroid HeLa cells. A restricted set of ubiquitous proteins, including Sp1, bound to regions of the promoter including the CACCC and CCAAT sites. Three interactions, at positions -213, -165, and +3 relative to the transcription start site, were erythroid specific and corresponded to binding of GATA-1, a transcription factor highly restricted to the erythroid lineage. Interestingly, the GATA-1 site at -165 has been conserved in the promoters of 10 mammalian embryonic globin genes. Point mutations demonstrate that GATA-1 binding to this site is necessary for interaction with an erythroid-specific enhancer but that in the absence of an enhancer, GATA-1 does not increase transcription.
In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA-3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA-3 contains a zinc finger domain that is highly related to the DNA-binding domain of the erythroid-specific transcription factor, GATA-1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA-3 is expressed exclusively in T cells. Overexpression of hGATA-3 in HeLa cells or human B cells specifically activated transcription from a co-transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA-3 is a novel lineage-specific hematopoietic transcription factor that appears to play an important role in regulating the T cell-specific expression of the TCR alpha gene.
The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.
The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.
The transcriptional binding protein NFE-1 (also called GF-1 and Ery-f1) is thought to play a necessary, but not sufficient, role in the regulation of differentiation-related gene expression in a subset of hematopoietic lineages (erythroid, megakaryocytic, and basophil-mast cell). In order to clarify the mechanism which underlies the lineage-specificity of the NFE-1 expression, as well as the relationship between the expression of this factor and growth factor responsiveness, we have evaluated the capacity of erythropoietin (Epo)-, granulomonocytic (GM)-colony stimulating factor (CSF)-, and granulocyte (G)-CSF-dependent subclones derived from the interleukin 3 (IL-3)-dependent cell line 32D, to express 1) NFE-1 mRNA, 2) NFE-1-related nuclear proteins, and 3) chloramphenicol acetyl transferase (CAT) activity when transfected with a CAT gene under the control of NFE-1 cognate sequences. NFE-1 mRNA was found to be expressed not only in cells with mast cell (IL-3-dependent 32D) and erythroid (Epo-dependent 32D Epo1) phenotypes, but also in cells with predominantly granulocyte/macrophage properties, such as the GM-CSF- (early myelomonocytic) and G-CSF- (myelocytic) dependent subclones of 32D. However, a gradient of expression, correlating with the lineage, the stage of differentiation, and the growth factor responsiveness of the cell lines, was found among the different subclones: Epo greater than or equal to IL-3 greater than GM-CSF greater than G-CSF. Binding experiments demonstrated NFE-1 activity in all cell lines except the G-CSF-dependent line. Function of the NFE-1 protein was assessed by the expression of the CAT gene linked to the SV40 promoter and a mutant (-175 T----C) HPFH gamma-globin promoter. High level CAT expression was seen only in the Epo1 cells although low level expression was also seen in the parent 32D. These results demonstrate that the specificity of the expression of NFE-1 for the erythroid--megakaryocytic--mast cell lineages is obtained by progressive inactivation of its expression in alternative lineages.
Human glycophorin C (GPC) is an integral membrane protein found in many cell types but which exhibits increased expression in red cells. We have investigated the in vitro binding of nuclear protein factors to the GPC gene 5'-flanking region which was transcriptionally active when transfected into erythroid and non-erythroid cells. We found that in addition to known ubiquitous or erythroid trans-acting factors, a presently uncharacterized erythroid/megakaryocyte-specific protein binds the GPC promoter. This factor, that we called NFE-6, is present in embryonic, fetal, or adult-like human or mouse erythroleukemic cell lines, in a megakaryoblastic cell line, but not in lymphoid or non-hematopoietic cell lines. These data suggest that NFE-6, like the well characterized NFE-1 factor, is tissue- but not stage-specific and is conserved across species. It is proposed that NFE-6 might be involved in the quantitative change in the expression of the house-keeping GPC gene related to red cell specialization.
We have used DNAase I footprinting and the gel mobility shift assay to study proteins which bind to promoter elements located between -140 and -382 upstream of the human A gamma globin gene. Footprints are found with both erythroid and nonerythroid nuclear extracts at three sites: from -294 to -264, -242 to -227, and -189 to -172 from the transcription initiation site. An erythroid-specific footprint is identified from -194 to -189. We demonstrate that two known transcription factors, the ubiquitous octamer-binding protein OTF-1 and the erythroid regulatory factor NFE-1, bind to the -194 to -172 region and that their footprints overlap. Binding of OTF-1 to this region is reduced by a mutation at -175 associated with a form of non-deletion hereditary persistence of fetal hemoglobin. We conclude that OTF-1 may compete with NFE-1 for the -175 binding site, possibly functioning as a repressor of gamma globin transcription.
Human parturition is associated with many pro-inflammatory mediators which are regulated by the nuclear factor-kappaB (NF-κB) family of transcription factors. In the present study, we employed a ChIP-on-chip approach to define genomic loci within chromatin of PHM1-31 myometrial cells that were occupied by RelA-containing NF-κB dimers in response to a TNF stimulation of 1 h. In TNF-stimulated PHM1-31 cells, anti-RelA serum enriched 13 300 chromatin regions; importantly, 11 110 regions were also enriched by anti-RelA antibodies in the absence of TNF. DNA sequences in these regions, from both unstimulated or TNF-stimulated PHM1-31 cultures, were associated with genic regions including IκBα, COX-2, IL6RN, Jun and KCNMB3. TNF-induced binding events at a consensus κB site numbered 1667; these were represented by 112 different instances of the consensus κB motif. Of the 1667 consensus κB motif occurrences, 770 (46.2%) were identified within intronic regions. In unstimulated PHM1-31 cells, anti-RelA-serum-enriched regions were associated with sequences corresponding to open reading frames of ion channel subunit genes including CACNB3 and KCNB1. Moreover, in unstimulated cells, the consensus κB site was identified 2116 times, being defined by 103 different sequence instances of this motif. Of these 2116 consensus κB motifs, 1089 (51.5%) were identified within intronic regions. Parallel expression array analyses in PHM1-31 cultures demonstrated that TNF stimulated a >2-fold induction in 51 genes and a fold repression of >1.5 in 18 others. We identified 14 anti-RelA-serum-enriched genomic regions that correlated with 17 TNF-inducible genes, such as COX2, Egr-1, Jun, IκBα and IL6, as well as five regions associated with TNF-mediated gene repression, including Col1A2.
Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV)-1 and tumor necrosis factor (TNF)-α-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic aggressive tumors. However, little is known about its role in astrocyte responses during HIV-1 central nervous system (CNS) infection and whether it contributes toward the development of HIV-associated neurocognitive disorders (HAND). Therefore, in this study, we investigated changes in AEG-1 CNS expression in HIV-1-infected brain tissues and elucidated a potential mechanism of AEG-1-mediated regulation of HAND. Immunoblotting and immunohistochemical analyses of HIV-1 seropositive and HIV-1 encephalitic human brain tissues revealed significantly elevated levels of AEG-1 protein. Immunohistochemical analyses of HIV-1 Tat transgenic mouse brain tissues also showed a marked increase in AEG-1 staining. Similar to in vivo observations, cultured astrocytes expressing HIV-1 Tat also revealed AEG-1 and cytokine up-regulation. Astrocytes treated with HAND-relevant stimuli, TNF-α, interleukin (IL)-1β, and HIV-1, also significantly induced AEG-1 expression and nuclear translocation via activation of the nuclear factor (NF)-κB pathway. Co-immunoprecipitation studies demonstrated IL-1β- or TNF-α-induced AEG-1 interaction with NF-κB p65 subunit. AEG-1 knockdown decreased NF-κB activation, nuclear translocation, and transcriptional output in TNF-α-treated astrocytes. Moreover, IL-1β treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2, increased expression of excitatory amino acid transporter 2 repressor ying yang 1, and reduced glutamate clearance, a major transducer of excitotoxic neuronal damage. Findings from this study identify a novel transcriptional co-factor function of AEG-1 and further implicate AEG-1 in HAND-associated neuroinflammation.
Peripheral endothelin-1 (ET-1) levels are increased in chronic systemic disorders such as congestive cardiac failure, diabetes and chronic renal failure. Bone infections are also associated with poor prognoses in these conditions. In the present study, we examined the alterations in Toll-like receptor 2 (TLR2) signaling induced by ET-1 in an in vitro osteoblast cell model. The TLR2-positive murine osteoblast cell line MC3T3-E1 was treated with heat-killed Listeria monocytogenes (HKLM), a TLR2 ligand, in the presence or absence of ET-1. We examined TLR2 expression, intranuclear NF-κB phosphorylation and interleukin 6 (IL-6) production. ET-1 suppressed cell surface expression of TLR2, NF-κB phosphorylation and IL-6 production. As TLR2 represents an important mechanism by which osteoblasts recognize bacterial pathogens, a continuously elevated ET-1 status may impair pathogenic recognition by osteoblasts and consequently affect bone metabolism during infections.
Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.
Multiple Myeloma (MM) is an incurable plasma cell cancer that is caused by several chromosomal translocations and gene deletions. Although deregulation of several signaling pathways including the Nuclear Factor-Kappa B (NF-κB) pathway has been reported in MM, the molecular requirement and the crosstalk between NF-κB and its target genes in MM cell survival has been largely unclear. Here, we report that Yin Yang1 (YY1), a target gene for NF-κB, is hyperexpressed in most MM tumor cells obtained from human patients, exhibits constitutive nuclear localization, and is essential for survival of MM cells. Mechanistically, we report a novel YY1-RelA complex formation, which is essential to transcriptionally repress a proapoptotic gene Bim. In line with this, depletion of YY1 or RelA resulted in elevated levels of Bim and apoptosis. Moreover, both YY1 and RelA are recruited to the Bim promoter and are required to repress the Bim promoter. Importantly, depletion of YY1 or RelA almost completely impaired the colony forming ability of MM progenitor cells suggesting that both RelA and YY1 are essential for the survival and growth of MM progenitor cells. Moreover, depletion of either YY1 or RelA completely inhibited MM tumor growth in xenograft models for human myeloma. Thus, a novel RelA-YY1 transcriptional repression complex is an attractive drug target in MM.
Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene which encodes the tumor necrosis factor (TNF) receptor, TNFR1. We investigated the effect of three high penetrance and three low penetrance TNFRSF1A mutations upon NF-κB transcription factor family subunit activity, and the resulting impact upon secretion of 25 different cytokines. Whilst certain mutations resulted in elevated NF-κB p65 subunit activity, others instead resulted in elevated c-Rel subunit activity. Interestingly, high p65 activity was associated with elevated IL-8 secretion, whereas high c-Rel activity increased IL-1β and IL-12 secretion. In conclusion, while all six TNFRSF1A mutations showed enhanced NF-κB activity, different mutations stimulated distinct NF-κB family subunit activities, and this in turn resulted in the generation of unique cytokine secretory profiles.
Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level.
β-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-κB p65 and inhibitor of κB (IκB) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas.
STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and β-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of β-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase) in the pancreas, a decreased NF-κB, and increased IκB pancreatic protein expression. In the fat-1-treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E₂ and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A₄ was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased.
Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ-induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology-inflammation, β-cell damage-through cytokine response and lipid mediator production.
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease.
Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor alpha (TNFalpha) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFalpha inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFalpha had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-kappaB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-kappaB by the overexpression of the dominant negative IkappaBalpha. Although TNFalpha failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFalpha suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-kappaB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFalpha inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB.
Copper is a persistent environmental contaminant, and exposure to elevated levels of this transition metal can result in a variety of pathologies. Copper affects the transcription of multiple defense and repair genes to protect against metal-induced pathologies. HepG2 cells were treated with copper under multiple conditions and microarray analyses were previously performed to better understand the mechanisms by which copper affects the transcription of stress-responsive genes. Analysis of the microarray data indicated that copper modulates multiple signal transduction pathways, including those mediated by NF-kappaB. Luciferase assays, quantitative reverse transcription real-time PCR, and chemical inhibition in HepG2 cells validated the microarray results and confirmed that NF-kappaB was activated by stress-inducible concentrations of copper. In addition, two novel NF-kappaB-regulated genes, SRXN1 (sulfiredoxin 1 homolog) and ZFAND2A (zinc-finger, AN1-type domain 2A), were identified. Our results indicate that the activation of NF-kappaB may be important for survival under elevated concentrations of copper.
In the past few decades, the use of natural compounds, such as flavonoids, as anti-inflammatory agents has gained much attention. Our current study focuses on the preventive effects of quercetin, apigenin, and luteolin on cytokine-induced beta-cell damage.
Pancreatic beta-cells or islets were treated with cytokine mixtures in the presence or absence of flavonoids and the inhibitory effect of flavonoids against cytokine toxicity was determined.
Treatment of RINm5F (RIN) rat insulinoma cells with interleukin 1beta (IL-1beta) and interferon gamma (IFN-gamma) induced cell damage. Quercetin, apigenin, and luteolin completely protected against IL-1beta- and IFN-gamma-mediated cytotoxicity in RIN cells. Incubation with quercetin, apigenin, and luteolin resulted in a significant reduction in IL-1beta- and IFN-gamma-induced nitric oxide production, a finding that correlated well with reduced levels of the inducible form of NO synthase messenger RNA and protein. The molecular mechanism by which quercetin, apigenin, and luteolin inhibited inducible NO synthase gene expression appeared to involve the inhibition of nuclear factor kappaB (NF-kappaB) activation. The IL-1beta- and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity, p50 and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared with unstimulated cells. Quercetin, apigenin, and luteolin also prevented IL-1beta- and IFN-gamma-mediated inhibition of insulin secretion.
Quercetin, apigenin, and luteolin inhibited cytotoxicity in RIN cells and attenuated the decrease of glucose-stimulated insulin secretion in islets by IL-1beta and IFN-gamma.
OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40 mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-kappaB (p50 and p65) also binds to the OX40 promoter region, but the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipitation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-kappaB binds to the OX40 promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-kappaB might be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4(+)CD25(+) regulatory T (Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg cell. In CD4(+)CD25(+) Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence of in vitro activation.
NF-kappaB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-kappaB activity. Interestingly, even in proliferating myoblasts, the absence of NF-kappaB caused the pronounced induction of several myofibrillar genes, suggesting that NF-kappaB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-kappaB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-kappaB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-kappaB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-kappaB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-kappaB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-kappaB activity. Based on these results, we propose that NF-kappaB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-kappaB functions in myoblasts to modulate skeletal muscle differentiation.
Activation of the transcriptional factor NF-kappaB is triggered by signal-dependent degradation of its inhibitor protein IkappaB through the ubiquitin (Ub)-proteasome pathway. We found here that a phosphorylated IkappaBalpha immunoprecipitated (IP-pIkappaBalpha) from the crude extract of HeLa cells which had been treated with tumor necrosis factor-alpha (TNFalpha) caused a dramatic ubiquitination of itself, termed autoubiquitination, when incubated with ATP, Ub, and E1-activating and E2-conjugating enzymes. IP-pIkappaBalpha also catalyzed ubiquitination of an in vitro synthesized 35S-IkappaBalpha previously phosphorylated by IkappaB-kinase (IKK) which is referred to as transubiquitination. No appreciable activity of auto- and transubiquitination was observed in an unphosphorylated IP-IkappaBalpha. Moreover, the putative IkappaBalpha-Ub ligase (IkappaBalpha-E3) present in HeLa cell cytosol associated in vitro with an IKK-phosphorylated recombinant IkappaBalpha, a process independent of NF-kappaB binding to IkappaBalpha or TNFalpha stimulation. Replacement of the two Ser residues at positions 32 and 36 corresponding to IKK phosphorylation sites by Ala resulted in almost complete prevention of binding of an IkappaBalpha-E3 to IkappaBalpha. These results indicate that phosphorylation of IkappaBalpha is necessary and sufficient for recruitment of this IkappaBalpha-E3 to associate with IkappaBalpha.
Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks.
Genome-wide association studies (GWAS) have prioritized a transcription factor, nuclear receptor 2 family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15 bp deletion in exon 2 leading to a five-amino-acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2(mutant) rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein-protein interaction is examined. Interaction of Nr2f2(mutant) protein with Fog2 is greater than that with the wild-type Nr2f2, indicating that the extent of interaction between these two transcription factors critically influences BP.
The mammalian thalamus is an essential diencephalic derivative that plays unique roles in processing and relaying sensory and motor information to and from the cerebral cortex. The profile of transcription factors and lineage tracing experiments revealed a spatiotemporal relationship between diencephalic progenitor domains and discrete differentiated neurons contributing to thalamic nuclei. However, the precise molecular mechanisms by which heterogeneous thalamic neurons become specified and assemble into distinct thalamic nuclei are still poorly understood. Here, we show that a combinatorial interaction between the bHLH transcription factors Ascl1 and Helt is required for acquiring thalamic progenitor identity. Surprisingly, in the combined absence of Ascl1 and Helt, rostral thalamic progenitors (TH-R) adopt a molecular profile of a more rostral diencephalic derivative, the prethalamus. Furthermore, we show that the prethalamic factors Dlxs upregulated by Ascl1/Helt deficiency play unique roles in regulating thalamic progenitor specification, and that derepression of Dlx2 and Dlx5 suppress generation of TH-R neurons. Taken together, our results suggest a model whereby the combined activity of two distinct bHLH factors plays a key role in the development of discrete classes of thalamic interneurons.
Transcriptional regulation is normally based on the recognition by a transcription factor of a defined base sequence in a process of direct read-out. However, the nucleic acid secondary and tertiary structure can also act as a recognition site for the transcription factor in a process known as indirect read-out, although this is much less understood. We have previously identified such a transcriptional control mechanism in early Xenopus development where the interaction of the transcription factor ilf3 and the gata2 promoter requires the presence of both an unusual A-form DNA structure and a CCAAT sequence. Rapid identification of such promoters elsewhere in the Xenopus and other genomes would provide insight into a less studied area of gene regulation, although currently there are few tools to analyse genomes in such ways.
In this paper we report the implementation of a novel bioinformatics approach that has identified 86 such putative promoters in the Xenopus genome. We have shown that five of these sites are A-form in solution, bind to transcription factors and fully validated one of these newly identified promoters as interacting with the ilf3 containing complex CBTF. This interaction regulates the transcription of a previously uncharacterised downstream gene that is active in early development.
A Perl program (APTE) has located a number of potential A-form DNA promotor elements in the Xenopus genome, five of these putative targets have been experimentally validated as A-form and as targets for specific DNA binding proteins; one has also been shown to interact with the A-form binding transcription factor ilf3. APTE is available from http://www.port.ac.uk/research/cmd/software/ under the terms of the GNU General Public License.
In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX[1] (insulin, dexamethasone and 3-isobutyl-1-methylxanthine), oleate, and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of peroxisome proliferator-activated receptor-γ (PPARγ) and adipocyte fatty acid-binding protein (AFABP), but decreasing level of GATA binding protein 2 (GATA2). In contrast, for cells cultured in basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes.
Lymphedema is caused by dysfunction of lymphatic vessels, leading to disabling swelling that occurs mostly on the extremities. Lymphedema can be either primary (congenital) or secondary (acquired). Familial primary lymphedema commonly segregates in an autosomal dominant or recessive manner. It can also occur in combination with other clinical features. Nine mutated genes have been identified in different isolated or syndromic forms of lymphedema. However, the prevalence of primary lymphedema that can be explained by these genetic alterations is unknown. In this study, we investigated 7 of these putative genes. We screened 78 index patients from families with inherited lymphedema for mutations in FLT4, GJC2, FOXC2, SOX18, GATA2, CCBE1, and PTPN14. Altogether, we discovered 28 mutations explaining 36% of the cases. Additionally, 149 patients with sporadic primary lymphedema were screened for FLT4, FOXC2, SOX18, CCBE1, and PTPN14. Twelve mutations were found that explain 8% of the cases. Still unidentified is the genetic cause of primary lymphedema in 64% of patients with a family history and 92% of sporadic cases. Identification of those genes is important for understanding of etiopathogenesis, stratification of treatments and generation of disease models. Interestingly, most of the proteins that are encoded by the genes mutated in primary lymphedema seem to act in a single functional pathway involving VEGFR3 signaling. This underscores the important role this pathway plays in lymphatic development and function and suggests that the unknown genes also have a role.
The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.
Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine-paracrine factor in this tissue. GNRH has been shown to increase pituitary Adcyap1 gene expression through the interaction of CREB and jun/fos with CRE/AP1 cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognate cis-elements which regulate Adcyap1 gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulate Adcyap1 promoter activity via a GATA cis-element located at position -191 in the rat Adcyap1 gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation of Adcyap1 gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.
The first haematopoietic stem cells share a common origin with the dorsal aorta and derive from putative adult haemangioblasts in the dorsal lateral plate (DLP) mesoderm. Here we show that the transcription factor (TF) stem cell leukaemia (Scl/Tal1) is crucial for development of these adult haemangioblasts in Xenopus and establish the regulatory cascade controlling its expression. We show that VEGFA produced in the somites is required to initiate adult haemangioblast programming in the adjacent DLP by establishing endogenous VEGFA signalling. This response depends on expression of the VEGF receptor Flk1, driven by Fli1 and Gata2. Scl activation requires synergy between this VEGFA-controlled pathway and a VEGFA-independent pathway controlled by Fli1, Gata2 and Etv2/Etsrp/ER71, which also drives expression of the Scl partner Lmo2. Thus, the two ETS factors Fli1 and Etv6, which drives the VEGFA expression in both somites and the DLP, sit at the top of the adult haemangioblast gene regulatory network (GRN). Furthermore, Gata2 is initially activated by Fli1 but later maintained by another ETS factor, Etv2. We also establish that Flk1 and Etv2 act independently in the two pathways to Scl activation. Thus, detailed temporal, epistatic measurements of key TFs and VEGFA plus its receptor have enabled us to build a Xenopus adult haemangioblast GRN.
The first axis to be specified during vertebrate development is that between the site where gastrulation will begin and the opposite pole of the embryo (dorsoventral axis in amphibians and fish, anteroposterior in amniotes). This relies on Nodal activity, but different vertebrates differ in how this activity is positioned. In chick, the earliest known asymmetry is posterior expression of the TGFβ-related factor Vg1, close to the future Nodal expression domain. Here we show that the transcription factor Gata2 is expressed anteriorly before this stage. Gata2 influences the site of primitive streak formation and its role is independent from, and upstream of, Vg1 and Wnt. However, although Vg1 is required for streak formation, Gata2 does not act as an absolute anterior specifier, but provides an anterior bias. These findings point to previously unsuspected global determinants of polarity of the early amniote embryo.
Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.
Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study were: 1) to verify if GATA and AP-1 sites could bind GATA2 and c-Jun/c-Fos factors, respectively; 2) to investigate whether such sites modulate the enhancer activity of the SIRT3-VNTR alleles. DAPA assay proved that GATA2 and c-Jun/c-Fos factors are able to bind the corresponding sites. Moreover, co-transfection experiments showed that the over-expression of GATA2 and c-Jun/c-Fos factors boosts the VNTR enhancer activity in an allelic-specific way. Furthermore, we established that GATA2 and c-Jun/c-Fos act additively in modulating the SIRT3-VNTR enhancer function. Therefore, GATA2 and AP-1 are functional sites and the T S> C transition of the second VNTR repeat affects their activity.
Certain beta(2)-adrenoceptor agonists, such as clenbuterol, are known to elicit a muscle-specific anabolism or hypertrophy in both normal and catabolic muscle in a wide variety of species. However, the underlying mechanism(s) of the beta(2)-agonist-induced anabolism remains unclear. This study aimed to determine the effects of clenbuterol administration in utero on skeletal muscle and to examine the underlying molecular mechanisms. Pregnant rats were fed clenbuterol (2 mg/kg diet) from Day 4 of gestation (4 dg) until weanling and fetal samples were taken from 13.5, 15.5, 17.5, and 19.5 dg and from 1d neonatal pups. Muscles were analyzed for total DNA, RNA and protein and sections examined morphologically for changes in muscle development. Western and immunohistochemical analyses were performed to identify changes in known myogenic signaling proteins. Clenbuterol increased the size of both fast and slow fibers in utero which was associated with a decreased DNA:protein ratio (28%) and an increased RNA:DNA ratio (36%). Additionally, drug treatment in utero induced a decrease in the fast:slow fiber ratio (38%). These myogenic changes were correlated with an increase in the GATA-2 hypertrophic transcription factor at both 17.5 dg (by 250%) and 19.5 dg (by 40%) in fetuses from clenbuterol treated dams. In addition, drug treatment resulted in increased membrane association of PKC-micro at 17.5 dg (325%) and increased PKC-alpha cytosolic abundance (40%) and PKC-theta membrane abundance at 19.5 dg (250%). These results are the first demonstration that beta(2)-agonists such as clenbuterol may act through upregulating the GATA-2 transcription factor and implicate certain PKC isoforms in the drug-induced regulation of skeletal muscle development.
Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHbeta transcripts, suggesting a specific function for TSHbeta transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHbeta promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHbeta promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHbeta promoter. Thus, the TSHbeta gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.
The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.
Beyond enumeration, circulating tumor cells (CTCs) can provide genetic information from metastatic cancer that may facilitate a greater understanding of tumor biology and enable a precision medicine approach.
CTCs and paired leukocytes from men with metastatic castration-resistant prostate cancer (mCRPC) were isolated from blood through red cell lysis, CD45 depletion, and flow sorting based on EpCAM/CD45 expression. We next performed whole genomic copy number analysis of CTCs and matched patient leukocytes (germline) using array-based comparative genomic hybridization (aCGH) from 16 men with mCRPC, including longitudinal and sequential CTCs aCGH analyses in the context of enzalutamide therapy.
All patients had mCRPC and primary or acquired resistance to abiraterone acetate or enzalutamide. We compiled copy gains and losses, with a particular focus on those genes highly implicated in mCRPC progression and previously validated as being aberrant in metastatic tissue samples and genomic studies of reference mCRPC datasets. Genomic gains in >25% of CTCs were observed in AR, FOXA1, ABL1, MET, ERG, CDK12, BRD4, and ZFHX3, while common genomic losses involved PTEN, ZFHX3, PDE4DIP, RAF1, and GATA2. Analysis of aCGH in a sample with sequential enzalutamide resistant visceral progression showed acquired loss of AR amplification concurrent with gain of MYCN, consistent with evolution toward a neuroendocrine-like, AR-independent clone.
Genomic analysis of pooled CTCs in men with mCRPC suggests a reproducible, but highly complex molecular profile that includes common aberrations in AR, ERG, c-MET, and PI3K signaling during mCRPC progression, which may be useful for predictive biomarker development.
Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCβ specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCβ level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCβ. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCβ inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCβ inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCβ-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.
The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshβ), glycoprotein subunit α (gsuα), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshβ and gsuα expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slβ, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshβ and gsuα expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshβ and gsuα expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhβ and fshβ) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary.
Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression.
Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up- or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSHβ) by liganded thyroid hormone receptor (TR).
In an effort to better understand the mechanism that drives TSHβ down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSHβ promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSHβ promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner.
Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.
Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.
Transcriptional repression of the TSH-specific beta subunit (TSHbeta) gene has been regarded to be specific to thyroid hormone (tri-iodothyronine, T(3)) and its receptors (TRs) in physiological conditions. However, TSHbeta mRNA levels in the pituitary were reported to decrease in the administration of pharmacologic doses of estrogen (17-beta-estradiol, E(2)) and increase in E(2) receptor (ER)-alpha null mice. Here, we investigated the molecular mechanism of inhibition of the TSHbeta gene expression by E(2)-bound E(2)-estrogen receptor 1 (E(2)-ERalpha). In kidney-derived CV1 cells, transcriptional activity of the TSHbeta promoter was stimulated by GATA2 and suppressed by THRBs and ERalpha in a ligand-dependent fashion. Overexpression of PIT1 diminished the E(2)-ERalpha-induced inhibition, suggesting that PIT1 may protect GATA2 from E(2)-ERalpha targeting by forming a stable complex with GATA2. Interacting surfaces between ERalpha and GATA2 were mapped to the DNA-binding domain (DBD) of ERalpha and the Zn finger domain of GATA2. E(2)-dependent inhibition requires the ERalpha amino-terminal domain but not the tertiary structure of the second Zn finger motif in E(2)-ERalpha-DBD. In the thyrotroph cell line, TalphaT1, E(2) treatment reduced TSHbeta mRNA levels measured by the reverse transcription PCR. In the human study, despite similar free thyroxine levels, the serum TSH level was small but significantly higher in post- than premenopausal women who possessed no anti-thyroid antibodies (1.90 microU/ml+/-0.13 S.E.M. vs 1.47 microU/ml+/-0.12 S.E.M., P<0.05). Our findings indicate redundancy between T(3)-TR and E(2)-ERalpha signaling exists in negative regulation of the TSHbeta gene.
Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.
Acute erythroid leukemia (AEL), characterized by a predominant erythroid proliferation, is a subtype of acute myelogenous leukemia. The genetic basis of AEL remains poorly defined. Through whole-exome sequencing, we identified high frequencies of mutations in CEBPA (32.7%), GATA2 (22.4%), NPM1 (15.5%), SETBP1 (12.1%) and U2AF1 (12.1%). Structure prediction analysis revealed that most of the GATA2 mutations were located at the DNA-binding N-terminal zinc-finger near the DNA-binding interface, suggesting that mutations could result in at least partial inactivation of GATA2 protein. On co-transfection of a GATA-responsive reporter construct together with plasmids expressing either GATA2 wild-type or GATA2 ZF1 mutants (P304H, L321P and R330X) in 293T cells, we found a reduced transcriptional activation in cells transfected with GATA2 mutants. To determine whether reduced GATA2 function is involved in leukemogenesis of AEL, we transfected 32D cells with GATA2 mutants and evaluated the impact of GATA2 mutations on erythroid differentiation. Our data revealed an increased expression of erythroid-related antigens Ter-119, β-globin and βh1-globin, as well as increased hemoglobin positivity in 32D cells transfected with GATA2 mutants compared with control cells. Our results suggest that the decline of GATA2 resulting from mutations contributes to the erythroid commitment, differentiation and the development of AEL.Leukemia advance online publication, 8 July 2016; doi:10.1038/leu.2016.162.
GATA2 gene encodes a member of the GATA family of zinc-finger transcription factors that play a pivotal role during the transition of primitive blood forming cells into white blood cells. Mutation in GATA2 results in the loss of function or even gain of function, including abnormal proliferation of white blood cells that may predispose to acute myeloid leukemia. Our results showed that the codon usage in GATA2 has been influenced by GC mutation bias where nature has highly favored fourteen most over represented codons but disfavored the ATA codon across five mammals. Purifying natural selection has affected GATA2 gene in human and other mammals to maintain its protein function during the period of evolution. Our findings report an insight into the codon usage patterns in gaining the clues for codon optimization to alter the translational efficiency as well as for the functional conservation of gene expression and the significance of nucleotide composition in GATA2 gene within mammals.
Gata2 is a zinc finger transcription factor that is important in hematopoiesis and neuronal development. However, the roles of Gata2 in the mesenchymal lineages are poorly understood. In vitro studies suggest that Gata2 modulates adipocyte differentiation and mesenchymal stem cell (MSC) proliferation. To systematically determine the in vivo functions of Gata2 in the MSC lineage commitment and development, we have generated three mouse models in which Gata2 is specifically deleted in MSCs, adipocytes, or osteoblasts. During the MSC expansion stage, Gata2 promotes proliferation and attenuates differentiation; thereby Gata2 loss in MSCs results in enhanced differentiation of both adipocytes and osteoblasts. During the differentiation stage, Gata2 also plays MSC-independent roles to impede lineage commitment; hence, Gata2 loss in adipocyte or osteoblast lineages also augments adipogenesis and osteoblastogenesis, respectively. These findings reveal Gata2 as a crucial rheostat of MSC fate to control osteoblast and adipocyte lineage development.
Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.
Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.
The transcriptional regulation of the gene encoding α-synuclein (SNCA) is thought to play a critical role in the pathogenesis of Parkinson's disease (PD), as common genetic variability in this gene is associated with an elevated risk of developing PD. However, the relevant mechanisms are still poorly understood. So far, only few proteins have been identified as transcription factors (TFs) of SNCA in cellular models. Here we show that two of these TFs bind to the DNA in human brain tissue: the zinc finger protein ZSCAN21 occupies a region within SNCA intron 1, as described before, while GATA2 occupies a specific region within intron 2, where we have identified a new binding site within the complex structure of the 5'-promoter region of SNCA. Electrophoretic mobility shift assays confirmed these binding sites. Genetic investigations revealed no polymorphisms or mutations within these sites. A better understanding of TF-DNA interactions within SNCA may allow to develop novel therapies designed to reduce α-synuclein levels.
Heterozygous familial or sporadic GATA2 mutations cause a multifaceted disorder, encompassing susceptibility to infection, pulmonary dysfunction, autoimmunity, lymphoedema and malignancy. Although often healthy in childhood, carriers of defective GATA2 alleles develop progressive loss of mononuclear cells (dendritic cells, monocytes, B and Natural Killer lymphocytes), elevated FLT3 ligand, and a 90% risk of clinical complications, including progression to myelodysplastic syndrome (MDS) by 60 years of age. Premature death may occur from childhood due to infection, pulmonary dysfunction, solid malignancy and MDS/acute myeloid leukaemia. GATA2 mutations include frameshifts, amino acid substitutions, insertions and deletions scattered throughout the gene but concentrated in the region encoding the two zinc finger domains. Mutations appear to cause haplo-insufficiency, which is known to impair haematopoietic stem cell survival in animal models. Management includes genetic counselling, prevention of infection, cancer surveillance, haematopoietic monitoring and, ultimately, stem cell transplantation upon the development of MDS or another life-threatening complication.
Increased local immune and inflammatory responses in obese individuals with periodontitis may explain the aggressive clinical presentation and altered treatment response when compared to that of normal weight subjects. Our goal was to identify any differences in microRNA (miRNA) expression profiles of gingival tissue in periodontitis when obesity is present, which may suggest novel molecular pathways that this miRNA network may affect. Total RNA was extracted from gingival tissue biopsies collected from normal weight and obese individuals with periodontitis; miRNA expression profiling was performed with Affymetrix GeneChip miRNA 3.0 arrays; and results were validated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). In silico identification of previously confirmed miRNA gene targets was conducted through miRTarBase and miRWalk databases, and pathway enrichment analysis identified enriched miRNA gene sets. Expression of selected genes in the same biopsy samples was tested with qRT-PCR. The gingival tissue miRNA profile of obese patients, compared to that of normal weight patients, showed 13 upregulated and 22 downregulated miRNAs, among which miR-200b was validated by qRT-PCR to be significantly increased in obesity. Functional analysis of 51 experimentally validated miR-200b gene targets identified enrichment of genes involved in cell motility, differentiation, DNA binding, response to stimulus, and vasculature development pathways not previously identified in the obesity-specific disease profile. Furthermore, the expression of the miR-200b gene targets ZEB1/2, GATA2, and KDR was confirmed by qRT-PCR as being lower in obese patients with periodontitis versus normal weight patients, suggesting a role of miR-200b in regulation of a set of gene targets and biological pathways relevant to wound healing and angiogenesis. Functional studies to explore the role of miR-200b in the above processes may offer new insights on putative therapeutic targets for this group of patients.
The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.
GATA2 deficiency is a germline disease that causes a wide spectrum of phenotypes including viral and bacterial infections, cytopenias, myelodysplasia, myeloid leukemias, pulmonary alveolar proteinosis and lymphedema. The age of clinical presentation ranges from early childhood to late adulthood, with most occurring in adolescence to early adulthood. We review the expanding GATA2-deficient phenotype, molecular genetics of disease and developments in treatment.
GATA2 mutations have been found in up to 10% of those with congenital neutropenia and/or aplastic anemia. Heterozygous mutations appear to cause haploinsufficiency due to either protein dysfunction or uniallelic reduced transcription. Disease-associated mutations in intronic regulatory elements or variations within the 5' leader exons indicate that regulation of GATA2 is critical. Those with GATA2 mutations are at high risk for myelodysplasia, cytogenetic abnormalities, acute myeloid leukemia or chronic myelomonocytic leukemia. Bone marrow transplantation has been successful for both hematopoietic and pulmonary alveolar proteinosis repair.
GATA2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis as well as lymphatic angiogenesis. GATA2 deficiency is caused by a variety of mutations in the GATA2 gene and can have variable presentation, onset and outcome. Patients are susceptible to mycobacterial, viral and fungal infections and can develop myelodysplasia, acute or chronic leukemias, lymphedema and pulmonary alveolar proteinosis. Hematopoietic stem cell transplantation reverses most of the clinical phenotype with good long-term outcomes.
Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1 % of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.
A 50-year-old woman was diagnosed with acute myeloid leukemia (AML). She has history of thrombocytopenia for 25 years and a significant family history of thrombocytopenia, affecting her mother, siblings and their children, as well as her own children. Both her mother and maternal aunt died from myelodysplastic syndrome (MDS). Additional genetic analysis was performed and identified two heterozygous missence mutations in the second zinc finger domain of GATA2 gene (p.Thr358Lys, and p.Leu359Val), occurring in cis on the same allele. Given the patient's family history and clinical manifestation, this was interpreted as an acute myeloid leukemia with heritable GATA2 mutations associated with familial AML-MDS. Germline GATA2 mutations are involved in a group of complex syndromes with overlapping clinical features of immune deficiency, lymphedema and propensity to acute myeloid leukemia or myelodysplastic syndrome (AML-MDS). Here we reported a case of familial AML-MDS with two novel GATA2 mutations. This case illustrates the importance of recognizing the clinical features for this rare category of AML-MDS and performing the appropriate molecular testing. The diagnosis of heritable gene mutations associated familial AML-MDS has significant clinical implication for the patients and affected families. Clinical trials are available to further investigate the role of allogeneic hematopoietic stem cell transplant in managing these patients.
The GATA2 gene encodes a zinc-finger transcription factor that acts as a master regulator of normal hematopoiesis. Mutations in GATA2 have been implicated in the development of myelodysplastic syndrome and acute myeloid leukemia (AML). Using RNA sequencing we now report that GATA2 is either mutated with a functional consequence, or expressed at low levels in the majority of normal karyotype AML (NK-AML). We also show that low-GATA2-expressing specimens (GATA2(low)) exhibit allele-specific expression (ASE) (skewing) in more than half of AML patients examined. We demonstrate that the hypermethylation of the silenced allele can be reversed by exposure to demethylating agents, which also restores biallelic expression of GATA2. We show that GATA2(low) AML lack the prototypical R882 mutation in DNMT3A frequently observed in NK-AML patients and that The Cancer Genome Atlas AML specimens with DNMT3A R882 mutations are characterized by CpG hypomethylation of GATA2. Finally, we validate that several known missense single-nucleotide polymorphisms in GATA2 are actually loss-of-function variants, which, when combined with ASE, represent the equivalent of homozygous GATA2 mutations. From a broader perspective, this work suggests for the first time that determinants of ASE likely have a key role in human leukemia.
GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway.
We identified two novel GATA2 mutations in acute myeloid leukemia (AML). One mutation (p.R308P-GATA2) was a R308P substitution within the zinc finger (ZF)-1 domain, and the other (p.A350_N351ins8-GATA2) was an eight-amino-acid insertion between A350 and N351 residues within the ZF-2 domain. p.R308P-GATA2 did not affect DNA-binding and transcriptional activities, while p.A350_N351ins8-GATA2 reduced them, and impaired G-CSF-induced granulocytic differentiation of 32D cells. Although p.A350_N351ins8-GATA2 did not show a dominant-negative effect over wild-type (Wt)-GATA2 by the reporter assay, it might be involved in the pathophysiology of AML by impairing myeloid differentiation because of little Wt-GATA2 expression in primary AML cells harboring the p.A350_N351ins8 mutation.
On July 21, 2016, the Roundtable on Health Literacy of the National Academies of Sciences, Engineering, and Medicine convened a workshop on health literacy and health insurance literacy in the context of health reform in the United States. Since 2010, the year the Patient Protection and Affordable Care Act (ACA) became law, the roundtable has convened two workshops related to this topic as well as commissioned two papers, and several members have prepared individually written perspectives. The roundtable's attention to the topic of health insurance is appropriate because health reform has created major changes in the health care system in this country. It has brought millions of people into the system that previously did not have access. In addition, many of these individuals have limited experience with health care and health insurance and are from populations that traditionally have high rates of low health literacy. The workshop focused on the challenges and opportunities in helping consumers obtain, understand, and use health insurance.
Pregnant women should be tested with immune tests for tuberculosis when they undergo TB screening according to the German Infectious Diseases Protection Act. Interferon Gamma Release Assays (IGRAs) should be the priority tests used for pregnant refugees and asylum seekers, especially when migrating from high incidence countries (> 20 cases/100.000 population according to the ECDC) or when they fled from crisis regions; positive tuberculin skin tests should be veryfied with IGRA. Once immunological tests are suggesting a latent tuberculosis infection it should be considered that pregnant women have an increased risk of developing active tuberculosis with potential health threats for the unborn child. Therefore, in case of a justified clinical suspicion of active tuberculosis or when a positive immunological test is present, pregnant women should be considered for chest X-ray examination, in order to rule out active pulmonary TB. Further examinations (such as ultrasound) to rule out extrapulmonary TB should be undertaken, once active pulmonary TB has been ruled out. A treatment indication is given for pregnant women during pregnancy, once active TB has been veryfied.
Idiopathic central precocious puberty (ICPP) is the premature activation of the hypothalamic-pituitary-gonadal axis in the absence of organic disease. Up to now, just gain-of-function mutations of KISS1/KISS1R and loss-of-function mutations of the maternally imprinted gene MKRN3 are the known genetic causes of ICPP. Our intention is to evaluate variants present in genes related to the pubertal onset pathway that could act as disease-causing or predisposing variants.
We studied the clinical exome of 20 patients diagnosed with ICPP using the Illumina platform. The bioinformatics analysis was performed using 2 different programs, and the variants were filtered according to a list of genes related to the gonadotropin-releasing hormone pathway.
In a "sporadic case," we found a missense variant in MKRN3 NM_005664.3: c.203G>A, causing the protein change NP_005655.1:p.Arg68His, predicted as pathogenic by 2 informatics tools. The proband carrying this variant was diagnosed with ICPP at 7.75 years of age. We did not find any pathogenic variants in KISS1, KISS1R, LIN28, GNRH, GNRHR, TACR3, and TAC3.
MKRN3 is the most frequent genetic cause of familial ICPP, so it is wise to screen for MKRN3 mutations in all patients with familial ICPP and in patients with an unclear paternal pubertal history.
Because cannabis use is a major public health concern and cannabis is known to act on central neurotransmission, studying the retinal ganglion cells in individuals who regularly use cannabis is of interest.
To determine whether the regular use of cannabis could alter the function of retinal ganglion cells in humans.
For this case-control study, individuals who regularly use cannabis, as well as healthy controls, were recruited, and data were collected from February 11 to October 28, 2014. Retinal function was used as a direct marker of brain neurotransmission abnormalities in complex mental phenomena.
Amplitude and implicit time of the N95 wave on results of pattern electroretinography.
Twenty-eight of the 52 participants were regular cannabis users (24 men and 4 women; median age, 22 years [95% CI, 21-24 years]), and the remaining 24 were controls (20 men and 4 women; median age, 24 years [95% CI, 23-27 years]). There was no difference between groups in terms of age (P = .13) or sex (P = .81). After adjustment for the number of years of education and alcohol use, there was a significant increase for cannabis users of the N95 implicit time on results of pattern electroretinography (median, 98.6 milliseconds [95% CI, 93.4-99.5]) compared with controls (median, 88.4 milliseconds [95% CI, 85.0-91.1]), with 8.4 milliseconds as the median of the differences (95% CI, 4.9-11.5; P < .001, Wald logistic regression). A receiver operating characteristic curve analysis (area under the curve, 0.84 [95% CI, 0.73-0.95]; P < .001) revealed, for a cutoff value of 91.13 milliseconds, a sensitivity of 78.6% (95% CI, 60.5%-89.8%) and a specificity of 75.0% (95% CI, 55.1%-88.0%) for correctly classifying both cannabis users and controls in their corresponding group. The positive predictive value was 78.6% (95% CI, 60.5%-89.8%), and the negative predictive value was 75.0% (95% CI, 55.1%-88.0%).
Our results demonstrate a delay in transmission of action potentials by the ganglion cells in regular cannabis users, which could support alterations in vision. Our findings may be important from a public health perspective since they could highlight the neurotoxic effects of cannabis use on the central nervous system as a result of how it affects retinal processing.
Electronic cigarette (e-cigarette) simulates the act of tobacco smoking by vaporizing a mixture of propylene glycol, nicotine, and flavoring agents. e-cigarette has been proposed as a product able to aid to stop smoking. The aim of the study is to verify the clinical variations of periodontal health induced by e-cigarettes use and, moreover, to investigate about the awareness of the e-smokers about their health variations and about their hypothetical need to turn back to smoke combustible cigarettes.This clinical observational pilot study involved 110 out of 350 smokers, who switched to e-cigarette. Patients were subjected to oral examinations. A questionnaire to self-assess the variations of some parameters of general health, and to self-assess the need to smoke combustible cigarettes, was distributed to such subjects involved in the study.At the end of the study, we registered a progressive improvement in the periodontal indexes, as well as in the general health perception. Finally, many patients reported an interesting reduction in the need to smoke.In the light of this pilot study, the e-cigarette can be considered as a valuable alternative to tobacco cigarettes, but with a positive impact on periodontal and general health status.
Dendritic cells (DCs) and monocytes develop from a series of bone-marrow-resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box-binding protein associated with epithelial-mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6C(hi) monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals.
A number of hormones work together to control plant cell growth. Rapid Alkalinization Factor 1 (RALF1), a plant-derived small regulatory peptide, inhibits cell elongation through suppression of rhizosphere acidification in plants. Although a receptor-like kinase, FERONIA (FER), has been shown to act as a receptor for RALF1, the signaling mechanism remains unknown. In this study, we identified a receptor-like cytoplasmic kinase (RPM1-induced protein kinase, RIPK), a plasma membrane-associated member of the RLCK-VII subfamily, that is recruited to the receptor complex through interacting with FER in response to RALF1. RALF1 triggers the phosphorylation of both FER and RIPK in a mutually dependent manner. Genetic analysis of the fer-4 and ripk mutants reveals RIPK, as well as FER, to be required for RALF1 response in roots. The RALF1-FER-RIPK interactions may thus represent a mechanism for peptide signaling in plants.
Transposable elements (TEs) are repeated DNA sequences that can constitute a substantial part of genomes. Studying TEs' activity, interactions, and accumulation dynamics is thus of major interest to understand genome evolution. Here, we describe the transposition dynamics of cut-and-paste mariner elements during experimental (short- and longer-term) evolution in Drosophila melanogaster Flies with autonomous and nonautonomous mariner copies were introduced in populations containing no active mariner, and TE accumulation was tracked by quantitative PCR for up to 100 generations. Our results demonstrate that (i) active mariner elements are highly invasive and characterized by an elevated transposition rate, confirming their capacity to spread in populations, as predicted by the "selfish-DNA" mechanism; (ii) nonautonomous copies act as parasites of autonomous mariner elements by hijacking the transposition machinery produced by active mariner, which can be considered as a case of hyperparasitism; (iii) this behavior resulted in a failure of active copies to amplify which systematically drove the whole family to extinction in less than 100 generations. This study nicely illustrates how the presence of transposition-competitive variants can deeply impair TE dynamics and gives clues to the extraordinary diversity of TE evolutionary histories observed in genomes.
The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.
Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely diminishes antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity.
Staff in pediatric intensive care units (PICU) are inherently exposed to potentially traumatic events. Posttraumatic growth (PTG) is the occurrence of positive changes after experiencing a traumatic event. This study aims (a) to evaluate the prevalence of PTG in PICU staff, and whether their scores are different from those reported by professionals working in other pediatric units, (b) to explore the role of resilience and coping strategies in predicting PTG, and (c) to explore the relation of demographic and work-related variables with PTG.
Participants of this multicentric, cross sectional study were 298 PICU workers and 189 professionals working in noncritical pediatric units. They completed the Brief Resilience Scale, a Coping Strategies Questionnaire, the Posttraumatic Growth Inventory (PTGI), and provided demographic and work-related information.
Of PICU staff, 68.8% experienced growth to a "great" or "very great" degree in at least one of the PTGI's dimensions. Higher PTG was reported following the death of a child or after a recent conflict with a work colleague. PICU workers and noncritical pediatric staff showed equivalent PTG levels. Multigroup path analysis with latent variables showed that emotion-focused coping was related to PTG only in PICU staff, whereas problem-focused coping was related to PTG in both groups. The relation between resilience and PTG was not significant.
Work-related trauma can act as a catalyst for positive posttrauma changes. Modifying coping strategies may be a way to foster PTG in health care providers. (PsycINFO Database Record
Convective cloud formation and evolution strongly depend on environmental temperature and humidity profiles. The forming clouds change the profiles that created them by redistributing heat and moisture. Here we show that the evolution of the field's thermodynamic properties depends heavily on the concentration of aerosol, liquid or solid particles suspended in the atmosphere. Under polluted conditions, rain formation is suppressed and the non-precipitating clouds act to warm the lower part of the cloudy layer (where there is net condensation) and cool and moisten the upper part of the cloudy layer (where there is net evaporation), thereby destabilizing the layer. Under clean conditions, precipitation causes net warming of the cloudy layer and net cooling of the sub-cloud layer (driven by rain evaporation), which together act to stabilize the atmosphere with time. Previous studies have examined different aspects of the effects of clouds on their environment. Here, we offer a complete analysis of the cloudy atmosphere, spanning the aerosol effect from instability-consumption to enhancement, below, inside and above warm clouds, showing the temporal evolution of the effects. We propose a direct measure for the magnitude and sign of the aerosol effect on thermodynamic instability.
Many cohort studies have shown that consumption of diets containing a higher composition of foods derived from plants reduces mortality from coronary heart disease (CHD). Here, we examined the active components of a plant-based diet and the underlying mechanisms that reduce the risk of CHD using three rat models and a quantitative proteomics approach. In a short-term myocardial infarction (MI) model, intake of wheat extract (WE), the representative cardioprotectant identified by screening approximately 4,000 samples, reduced myocardial injury by inhibiting apoptosis, enhancing ATP production, and maintaining protein homeostasis. In long-term post-MI models, this myocardial protection resulted in ameliorating adverse left-ventricular remodelling, which is a predictor of heart failure. Among the wheat components, arabinose and xylose were identified as active components responsible for the observed efficacy of WE, which was administered via ingestion and tail-vein injections. Finally, the food components of plant-based diets that contained cell wall polysaccharides rich in arabinose, xylose, and possibly fucose were found to confer protection against myocardial injury. These results show for the first time that specific monosaccharides found in the cell wall polysaccharides in plant-based diets can act as active ingredients that reduce CHD by inhibiting postocclusion steps, including MI and heart failure.
The NET (for NocA, Nlz, Elbow, TLP-1) protein family is a group of conserved zinc finger proteins linked to embryonic development and recently associated with breast cancer. The members of this family act as transcriptional repressors interacting with both class I histone deacetylases and Groucho/TLE co-repressors. In Drosophila, the NET family members Elbow and NocA are vital for the development of tracheae, eyes, wings and legs, whereas in vertebrates ZNF703 and ZNF503 are important for the development of the nervous system, eyes and limbs. Despite the relevance of this protein family in embryogenesis and cancer, many aspects of its origin and evolution remain unknown. Here, we show that NET family members are present and expressed in multiple metazoan lineages, from cnidarians to vertebrates. We identified several protein domains conserved in all metazoan species or in specific taxonomic groups. Our phylogenetic analysis suggests that the NET family emerged in the last common ancestor of cnidarians and bilaterians and that several rounds of independent events of gene duplication occurred throughout evolution. Overall, we provide novel data on the expression and evolutionary history of the NET family that can be relevant to understanding its biological role in both normal conditions and disease.
In sarcoidosis, the proinflammatory cytokines interferon gamma, tumour necrosis factor and interleukin-6 are released by monocyte-derived macrophages and lymphocytes in the lungs and other affected tissues. Regulatory receptors expressed on monocytes and macrophages act to suppress cytokine production, and reduced expression of regulatory receptors may thus promote tissue inflammation. The aim of this study was to characterise the role of regulatory receptors on blood monocytes in patients with sarcoidosis. Cytokine release in response to stimulation of whole blood was measured in healthy controls and Caucasian non-smoking patients with sarcoidosis who were not taking disease modifying therapy. Expression of the regulatory molecules IL-10R, SIRP-α/β, CD47, CD200R, and CD200L was measured by flow cytometry, and functional activity was assessed using blocking antibodies. Stimulated whole blood and monocytes from patients with sarcoidosis produced more TNF and IL-6 compared with healthy controls. 52.9% of sarcoidosis patients had monocytes characterised by low expression of CD200R, compared with 11.7% of controls (p < 0.0001). Patients with low monocyte CD200R expression produced higher levels of proinflammatory cytokines. In functional studies, blocking the CD200 axis increased production of TNF and IL-6. Reduced expression of CD200R on monocytes may be a mechanism contributing to monocyte and macrophage hyper-activation in sarcoidosis.
Axonal injury is a common feature of central nervous system insults that culminates with the death of the affected neurons, and an irreversible loss of function. Inflammation is an important component of the neurodegenerative process, where the microglia plays an important role by releasing proinflammatory factors as well as clearing the death neurons by phagocytosis. Here we have identified the purinergic signaling through the P2X7 receptor as an important component for the neuronal death in a model of optic nerve axotomy. We have found that in P2X7 receptor deficient mice there is a delayed loss of retinal ganglion cells and a decrease of phagocytic microglia at early times points after axotomy. In contralateral to the axotomy retinas, P2X7 receptor controlled the numbers of phagocytic microglia, suggesting that extracellular ATP could act as a danger signal activating the P2X7 receptor in mediating the loss of neurons in contralateral retinas. Finally, we show that intravitreal administration of the selective P2X7 receptor antagonist A438079 also delays axotomy-induced retinal ganglion cell death in retinas from wild type mice. Thus, our work demonstrates that P2X7 receptor signaling is involved in neuronal cell death after axonal injury, being P2X7 receptor antagonism a potential therapeutic strategy.
In everyday social interactions, people's facial expressions sometimes reflect genuine emotion (e.g., anger in response to a misbehaving child) and sometimes do not (e.g., smiling for a school photo). There is increasing theoretical interest in this distinction, but little is known about perceived emotion genuineness for existing facial expression databases. We present a new method for rating perceived genuineness using a neutral-midpoint scale (-7 = completely fake; 0 = don't know; +7 = completely genuine) that, unlike previous methods, provides data on both relative and absolute perceptions. Normative ratings from typically developing adults for five emotions (anger, disgust, fear, sadness, and happiness) provide three key contributions. First, the widely used Pictures of Facial Affect (PoFA; i.e., "the Ekman faces") and the Radboud Faces Database (RaFD) are typically perceived as not showing genuine emotion. Also, in the only published set for which the actual emotional states of the displayers are known (via self-report; the McLellan faces), percepts of emotion genuineness often do not match actual emotion genuineness. Second, we provide genuine/fake norms for 558 faces from several sources (PoFA, RaFD, KDEF, Gur, FacePlace, McLellan, News media), including a list of 143 stimuli that are event-elicited (rather than posed) and, congruently, perceived as reflecting genuine emotion. Third, using the norms we develop sets of perceived-as-genuine (from event-elicited sources) and perceived-as-fake (from posed sources) stimuli, matched on sex, viewpoint, eye-gaze direction, and rated intensity. We also outline the many types of research questions that these norms and stimulus sets could be used to answer.
Typical dicots possess equal-sized cotyledons and leaf-bearing shoots topped with a shoot apical meristem (SAM), the source of lateral organs, and where KNOX1 homeobox genes act as key regulators. New World Gesneriaceae show typical cotyledons, whereas Old World Gesneriaceae show anisocotyly, the unequal post-germination growth of cotyledons, and include unifoliate (one-leaf) plants. One-leaf plants show an extremely reduced body plan: the adult above-ground photosynthetic tissue consisting of a single cotyledon, a macrocotyledon enlarged by the basal meristem (BM), but lacking a SAM. To investigate the origin and evolution of the BM and one-leaf plants, the meristem activity and KNOX1 SHOOTMERISTEMLESS (STM) expression in cotyledons and leaves were systematically studied by RT-PCR and in situ hybridization across the family Gesneriaceae, Jovellana in Calceolariaceae (sister family to Gesneriaceae), and Antirrhinum in Plantaginaceae, all families of order Lamiales (asterids), in comparison to Arabidopsis (Brassicales, rosids). In all examined Lamiales samples, unlike Arabidopsis, BM activity accompanied by STM expression was found in both cotyledons in early stages. Foliage leaves of Gesneriaceae and Jovellana also showed the correlation of BM and STM expression. An extension of BM activity was found following a phylogenetic trajectory towards one-leaf plants where it is active throughout the lifetime of the macrocotyledon. Our results suggest that KNOX1 involvement in early cotyledon expansion originated early on in the diversification of Lamiales and is proposed as the prerequisite for the evolution of vegetative diversity in Gesneriaceae. Step-wise morphological shifts, driven by transfers of meristematic activity, as evidenced by shifts in KNOX1 expression, may be one mechanism by which morphological diversity evolves in plants.
Cardioprotective strategies aim to salvage myocardium from ischemia/reperfusion injury and to reduce infarct size and its consequences. Different stimuli, acting at sites remote from the heart (remote conditioning), activate molecular self-defense mechanisms at the target organ heart as well as in other parenchymal organs. Remote conditioning of the heart has been established in many experimental studies and successfully translated to patients. Remote ischemic conditioning by short repetitive cycles of ischemia/reperfusion on an extremity reduces infarct size and improves the prognosis of patients with reperfused myocardial infarction. The present review focuses on three levels of remote conditioning and its resulting cardioprotection: I) at the stimulus level, electrical stimulation, chemical/pharmacological substances, mechanical trauma and cycles of ischemia/reperfusion act at sites remote from the heart, II) at the transfer level, neuronal and humoral mediators transfer the protective signal from the periphery to the heart, and III) at the target level, receptor activation and intracellular signal transduction ultimately affect protection of the myocardium and other organs, as established in different animal models and humans/patients. Remote conditioning is obviously a systemic response. Further mechanistic understanding is mandatory to translate the protection by remote conditioning more successfully to patients with cardiovascular disease.
We studied the predator-prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.
Unsettled knowledge as to whether scrapie transmits prenatally in sheep and goats and transmits by semen and preimplantation embryos has a potential to compromise measures for controlling, preventing and eliminating the disease. The remedy may be analysis according to a systematic review, allowing comprehensive and accessible treatment of evidence and reasoning, clarifying the issue and specifying the uncertainties. Systematic reviews have clearly formulated questions, can identify relevant studies and appraise their quality and can summarise evidence and reasoning with an explicit methodology. The present venture lays a foundation for a possible systematic review and applies three lines of evidence and reasoning to two questions. The first question is whether scrapie transmits prenatally in sheep and goats. It leads to the second question, which concerns the sanitary safety of artificial breeding technologies, and is whether scrapie transmits in sheep and goats by means of semen and washed or unwashed in vivo derived embryos. The three lines of evidence derive from epidemiological, field and clinical studies, experimentation, and causal reasoning, where inferences are made from the body of scientific knowledge and an understanding of animal structure and function. Evidence from epidemiological studies allow a conclusion that scrapie transmits prenatally and that semen and embryos are presumptive hazards for the transmission of scrapie. Evidence from experimentation confirms that semen and washed or unwashed in vivo derived embryos are hazards for the transmission of scrapie. Evidence from causal reasoning, including experience from other prion diseases, shows that mechanisms exist for prenatal transmission and transmission by semen and embryos in both sheep and goats.
Objective: Suicide attempt may follow a process right from the inception of the first information about ‎suicide until the act itself. This study was conducted to determine the relationship between ‎perception of suicide prevention with the process of suicide attempt and demographic ‎variables following a suicidal attempt.‎ Method: In this hospital based cross-sectional study, 168 consecutive admitted participants with a ‎suicide attempt were screened, and 109 who met the study criteria were recruited to ‎participate in this study before discharge. They were assessed using the socio-demographic ‎and clinical proforma designed for this study as well as by the Pierce Suicide Intent Scale. To ‎assess the process of suicide attempt and perception of suicide prevention, a 17- item ‎questionnaire was developed and used after rigorous literature search. The Cronbach's ‎alpha coefficient value of this questionnaire found to be 0.84 in the reliability analysis.‎ Results: Media was the first source of information, and the majority had short duration of ‎preoccupation and interval between making the decision and the actual attempt and the ‎control of emotion during the attempt. A significant positive correlation was observed ‎between the source of the first information and age (p<01), reason for the method used and ‎economic status (p<01), duration since the first information and family history of suicide (p<01). Psychiatric diagnosis had a statistically significant association with the method used (p<01), duration of preoccupation (p<01), preparedness (p<01) and emotional state during ‎the attempt (p<01). A statistically significant negative correlation was found between the ‎source of the first information and education (p<01), any psychiatric diagnosis and duration ‎since the first death wish (p<01). On the score of perception about suicide prevention, a ‎significant group difference was observed for marital status, occupation, medical diagnosis, ‎opinion about an attempt, duration since the decision to attempt, and emotional control ‎during the attempt.‎ Conclusion: Based on the findings, it can be concluded that perception of suicide prevention may vary ‎with the process of suicide attempts and demographic characteristics.‎.
Metastasis is a multi-step process that ultimately depends on the ability of disseminating cancer cells to establish favorable communications with their microenvironment. The tumor microenvironment consists of multiple and continuously changing cellular and molecular components. One of the factors regulating the tumor microenvironment is TNF-α, a pleiotropic cytokine that plays key roles in apoptosis, angiogenesis, inflammation and immunity. TNF-α can have both pro- and anti-tumoral effects and these are transmitted via two major receptors, the 55 kDa TNFR1 and the 75 kDa TNFR2 that have distinct, as well as overlapping functions. TNFR1 is ubiquitously expressed while the expression of TNFR2 is more restricted, mainly to immune cells. While TNFR1 can transmit pro-apoptotic or pro-survival signals through a complex network of downstream mediators, the role of TNFR2 is less well understood. One of its main functions is to act as a survival factor and moderate the pro-apoptotic effects of TNFR1, particularly in immune cells. In this review, we summarize the evidence for the involvement of the TNF system in the progression of the metastatic process from its contribution to the early steps of tumor cell invasion to its role in the colonization of distant sites, particularly the liver. We show how the TNF receptors each contribute to these processes by regulating and shaping the tumor microenvironment. Current evidence and concepts on the potential use of TNF targeting agents for cancer prevention and therapy are discussed.
Viruses are under relentless selective pressure from host immune defenses. To study how poxviruses adapt to innate immune detection pathways, we performed serial infections of vaccinia virus in primary human cells. Independent courses of experimental evolution with a recombinant strain lacking E3L revealed several high frequency point mutations in conserved poxvirus genes, suggesting important roles for essential poxvirus proteins in innate immune subversion. Two distinct mutations were identified in the viral RNA polymerase gene A24R, which seem to act through different mechanisms to increase virus replication. Specifically, a Leu18Phe substitution in A24R conferred fitness tradeoffs, including increased activation of the antiviral factor Protein kinase R (PKR). Intriguingly, this A24R variant underwent a drastic selective sweep during passaging, despite enhanced PKR activity. We show that the sweep of this variant can be accelerated by the presence of copy number variation (CNV) at the K3L locus, which with multiple copies strongly reduces PKR activation. Therefore, adaptive cases of CNV can also facilitate the accumulation of point mutations separate from the expanded locus. This study reveals how rapid bouts of gene copy number amplification during accrual of distant point mutations can potently facilitate poxvirus adaptation to host defenses.
Viruses can quickly evolve to defeat host immune functions. For poxviruses, little is known about how multiple adaptive mutations emerge in populations at the same time. In this study, we uncovered a means of vaccinia virus adaptation involving the accumulation of distinct genetic variants within a single population. We identified adaptive point mutations in the viral RNA polymerase gene A24R, and surprisingly, found that one of these mutations activates the nucleic acid sensing factor PKR. We also found that gene copy number variation (CNV) can provide dual benefits to evolving virus populations, including evidence that CNV facilitates the accumulation of a point mutation distant from the expanded locus. Our data suggest that transient CNV can accelerate the fixation of mutations conferring modest benefits, or even fitness tradeoffs, and highlight how structural variation might aid poxvirus adaptation through both direct and indirect action.
Plant growth, development and interaction with the environment involve the action of multiple phytohormones. Transcription factors (TFs) of diverse families play essential roles in the signalling cascades triggered by the perception of a particular hormone. TFs may act alone or in a combinatorial fashion with other TFs, and may act specifically in a single hormonal signalling cascade or as signalling hubs for multiple hormones. In the signalling cascades triggered by the phytohormone jasmonate (JA), which modulates a diverse, but specific, range of aspects of plant growth, development and defence, the TFs of the basic helix-loop-helix (bHLH) family play an essential and often conserved role in the plant kingdom. Here, we first discuss the bHLH TFs involved in all kinds of JA-modulated processes in the model plant Arabidopsis thaliana Secondly, we elaborate on the identity and role of bHLH TFs in the conserved JA-mediated elicitation of specialized metabolism of medicinal and crop species. Finally, we discuss which directions future fundamental research on the functioning of bHLH TFs in JA signalling may head for and how this research can be translated from model plants into crop and medicinal plant species to engineer traits of agronomical and industrial interest.
Anthocyanins, a class of flavonoids, are responsible for the orange to blue coloration of flowers and act as visual attractors to aid pollination and seed dispersal. Malonyl-CoA is the precursor for the formation of flavonoids and anthocyanins. Previous studies have suggested that malonyl-CoA is formed almost exclusively by acetyl-CoA carboxylase, which catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. In the present study, the full-length cDNA of Petunia hybrida acyl-activating enzyme 13 (PhAAE13), a member of clade VII of the AAE superfamily that encodes malonyl-CoA synthetase, was isolated. The expression of PhAAE13 was highest in corollas and was down-regulated by ethylene. Virus-induced gene silencing of petunia PhAAE13 significantly reduced anthocyanin accumulation, fatty acid content, and cuticular wax components content, and increased malonic acid content in flowers. The silencing of PhAAE3 and PhAAE14, the other two genes in clade VII of the AAE superfamily, did not change the anthocyanin content in petunia flowers. This study provides strong evidence indicating that PhAAE13, among clade VII of the AAE superfamily, is specifically involved in anthocyanin biosynthesis in petunia flowers.
Social interventions targeted at people with severe mental illness (SMI) often include volunteers. Volunteers' perspectives are important for these interventions to work. This article investigates the experiences of volunteer families who befriend a person with SMI.
Qualitative interviews with members of volunteer families.
The families were motivated by helping a vulnerable person and by engaging in a rewarding relationship. However, the families often doubted their personal judgement and relied on mental health workers to act as safety net.
The volunteer involvement is meaningful but also challenging. The families value professional support.
Disorders of mast cell activation can be classified as primary (mastocytosis), secondary (reactive) or idiopathic. This article discusses how to recognise and approach the diagnosis of patients suspected to have symptoms of abnormal mast cell activation. Given the highly varied and often complex symptomatology of such patients, we advocate applying a logical step-wise approach to investigating these patients to ensure the correct diagnosis is made. Treatments of mast cell activation disorders are discussed, dividing them into those that ameliorate the effects of mast cell mediators and those that act to stabilise the mast cell.
Mitochondria play a central role in pancreatic β-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic β-cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester-induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC-dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester-stimulated INS-1E cells maintained under resting (2.5mM) glucose revealed a large number of phosphorylation sites that were altered during short-term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C-Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12-myristate 13-acetate-induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non-nutrient secretagogues act, in part, via PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to β-cell activation.-Santo-Domingo, J., Chareyron, I., Dayon, L., Galindo, A. N., Cominetti, O., Giménez, M. P. G., De Marchi, U., Canto, C., Kussmann, M., Wiederkehr, A. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β-cells.
Retrospective review of prospectively maintained database.
To review myelomeningocele patients with severe kyphosis undergoing kyphectomy surgery in terms of complications and clinical and radiographic outcomes.
Because of posterior element abnormality in myelomeningocele, the extensor muscles act as perverted flexors, driving progressive kyphosis that resulted in sitting, respiratory, and skin breakdown problems.
Clinical case notes and x-rays of seven myelomeningocele patients undergoing kyphectomy surgery were reviewed with a minimum follow-up of 24 months. They consisted of four males and three females with an average age of 9.5 years at surgery. Surgery was performed in three despite open pressure ulcers that failed to heal. These wounds were all closed primarily at initial operation, and no flaps were required. Pedicle screw and sublaminar wire constructs were utilized with iliac screws for distal control.
The median surgical time was 245 minutes (165-285), with an estimated blood loss of 700 mL (500-2,550). The preoperative kyphosis of 142 degrees (90-180) was corrected to 15 degrees (5-45) representing a 92% correction. All experienced improved sitting. There were no early complications but 2 patients with preoperative pressure ulcers returned at 13 months with recurrent sepsis and wound breakdown. Their osteotomy had fused, and the infection settled after instrumentation removal and antibiotic administration.
Although an infrequent presentation today, severe kyphosis in myelomeningocele patients causes not only a major functional impairment but threat to their life with apical pressure sores. Kyphectomy and posterior instrumented spinal fusion can be performed safely, even in the face of an open sore with excellent kyphotic correction and resultant improved functionality and ability to sit. These open sores can be closed primarily without the requirement of plastic surgery as a result of the shortening and extension of the spine.
Australian abattoir workers, farmers, veterinarians and people handling animal birthing products or slaughtering animals continue to be at high risk of Q fever despite an effective vaccine being available. National Notifiable Diseases Surveillance System data were analysed for the period 1991-2014, along with enhanced risk factor data from notified cases in the states of New South Wales and Queensland, to examine changes in the epidemiology of Q fever in Australia. The national Q fever notification rate reduced by 20% [incident rate ratio (IRR) 0·82] following the end of the National Q fever Management Program in 2006, and has increased since 2009 (IRR 1·01-1·34). Highest rates were in males aged 40-59 years (5·9/100 000) and 87% of Q fever cases occurred in New South Wales and Queensland. The age of Q fever cases and proportion of females increased over the study period. Based on the enhanced risk factor data, the most frequently listed occupation for Q fever cases involved contact with livestock, followed by 'no known risk' occupations. More complete and comparable enhanced risk factor data, at the State/Territory and national levels, would aid in further understanding of the epidemiology of Q fever.
Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a major wheat disease which is mainly controlled through the release of resistant cultivars containing one or several resistance genes. Considerable effort has been put into the discovery of new resistance genes, but knowledge of their mechanisms of action is often lacking. In this study, the mechanism of resistance conferred by a recently discovered stem rust resistance locus on wheat chromosome 7AL was investigated through microscopic observations and RNA-sequencing, using the susceptible line Columbus and the independent, backcrossed, resistant lines Columbus-NS765 and Columbus-NS766.
Microscopic observations of infected leaves revealed that the resistance conferred by the 7AL resistance locus was initiated 2 days post-inoculation, upon the fungus entry into the plant through the stoma. Resistance was manifested by death of guard and epidermal cells adjacent to an infection site. Occasionally, similar observations were made in the susceptible line, suggesting that the resistance response was the same in all genotypes, but enhanced in the resistant lines. Transcriptomic analysis, combined with assignment of genes to wheat chromosomes, revealed a disproportionately high number of differentially expressed genes were located on chromosomes 7AL and 6A. A number of genes annotated as cysteine-rich receptor-like kinases were located on chromosome 7AL. Closer investigation indicated that the encoded proteins were in fact putative receptor-like cytoplasmic kinases. One of the putative RLCK genes contained a SNP marker previously shown to co-segregate with the 7AL resistance locus. The results also indicated the presence of a large introgression on chromosome 6A in both resistant lines, but whether it has any role in the resistance response is unclear.
This study represents the first investigation on the resistance mechanism conferred by the wheat 7AL stem rust resistance locus. The resistance response was associated with pre-haustorial cell death, and the transcriptome analysis suggested putative receptor-like cytoplasmic kinases as candidate resistance genes for further investigation.
Health and fitness applications (apps) have gained popularity in interventions to improve diet, physical activity and sedentary behaviours but their efficacy is unclear. This systematic review examined the efficacy of interventions that use apps to improve diet, physical activity and sedentary behaviour in children and adults.
Systematic literature searches were conducted in five databases to identify papers published between 2006 and 2016. Studies were included if they used a smartphone app in an intervention to improve diet, physical activity and/or sedentary behaviour for prevention. Interventions could be stand-alone interventions using an app only, or multi-component interventions including an app as one of several intervention components. Outcomes measured were changes in the health behaviours and related health outcomes (i.e., fitness, body weight, blood pressure, glucose, cholesterol, quality of life). Study inclusion and methodological quality were independently assessed by two reviewers.
Twenty-seven studies were included, most were randomised controlled trials (n = 19; 70%). Twenty-three studies targeted adults (17 showed significant health improvements) and four studies targeted children (two demonstrated significant health improvements). Twenty-one studies targeted physical activity (14 showed significant health improvements), 13 studies targeted diet (seven showed significant health improvements) and five studies targeted sedentary behaviour (two showed significant health improvements). More studies (n = 12; 63%) of those reporting significant effects detected between-group improvements in the health behaviour or related health outcomes, whilst fewer studies (n = 8; 42%) reported significant within-group improvements. A larger proportion of multi-component interventions (8 out of 13; 62%) showed significant between-group improvements compared to stand-alone app interventions (5 out of 14; 36%). Eleven studies reported app usage statistics, and three of them demonstrated that higher app usage was associated with improved health outcomes.
This review provided modest evidence that app-based interventions to improve diet, physical activity and sedentary behaviours can be effective. Multi-component interventions appear to be more effective than stand-alone app interventions, however, this remains to be confirmed in controlled trials. Future research is needed on the optimal number and combination of app features, behaviour change techniques, and level of participant contact needed to maximise user engagement and intervention efficacy.
Eclipta alba (Linn) Hassk. (Asteraceae) has been reported to be a nerve tonic and has been used to treat epilepsy in folk medicine.
The present study isolates and characterizes luteolin from E. alba and evaluates its antiepileptic potential in chemically induced acute and chronic models in mice.
The methanol extract (16.85% w/w) of E. alba leaves was subjected to fractionation for isolation of luteolin. In acute pentylenetetrazole (PTZ) model, luteolin (5, 10, 20 mg/kg, i.p.) was administered 30 min prior to PTZ injection (100 mg/kg) in Swiss albino mice. Kindling was induced by chronic administration of PTZ (35 mg/kg) on every alternate day (48 days). Luteolin was investigated on the course of kindling development and oxidative stress markers [reduced glutathione (GSH) and malondialdehyde (MDA)] in kindled mice.
Single-dose pretreatment with luteolin (10 and 20 mg/kg, i.p.) was found to be effective in an acute PTZ model (100% protection from mortality) and it did not exhibit any effect on motor coordination at the same doses. PTZ-induced kindling was significantly (p < 0.001) prevented by luteolin (5, 10, 20 mg/kg, i.p.) in a dose-dependent manner. Luteolin restored levels of reduced GSH (p < 0.001) and decreased the level of MDA (p < 0.001), a marker of lipid peroxidation.
The results of the present study demonstrated that luteolin had an anticonvulsant effect in an acute PTZ model. Luteolin exhibited and inhibitory effect on the course of kindling and associated oxidative stress and hence could be a potential molecule in the treatment of epilepsy.
NURSES COULD face legal risks under the Human Rights Act by using 'pain compliance' techniques to restrain patients, a leading researcher has warned.
Nurses do not need to have responsibilities delegated; they must be able to carry out assessments and provide care and treatment in their own right. This will work better provided that nurses recognise and act on the importance of 'real' professional skills development.
Last january the Guardian reported that 'the NHS is in an impossible situation which endless reforms have failed to address'. It sounds familiar and is possibly true, with staffing and social problems aggravated by economic reforms and demographic change. But a _ group of nurses in the West Midlands are showing that £ some of the issues raised by the Community Care Act can be managed and even turned to advantage.
This book gives a detailed insight into the Human Rights Act, placing it in the context of health care and discussing the many challenges the Act could create. It explores the impact the Act will have on key areas such as health law, ethics and patients' rights.
Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like C.elegans protein DAF-28. DAF-28(R37C) is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype-deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C) animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations.
Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals.
In developing epithelia, the core planar polarity proteins physically interact with each other and localize asymmetrically at opposite cell ends, forming intercellular complexes that link the polarity of neighboring cells. Using quantitative imaging to examine the composition of the core protein complex in vivo, we find that complex composition is unexpectedly plastic. The transmembrane proteins Frizzled and Flamingo form a stoichiometric nucleus in the complex, while the relative levels of the other four core proteins can vary independently. Exploring the functional consequences of this, we show that robust cell polarization is achieved over a range of complex stoichiometries but is dependent on maintaining appropriate levels of the components Frizzled and Strabismus. We propose that the core proteins assemble into signalosome-like structures, where stable association is not dependent on one-to-one interactions with binding partners, and signaling functions can act over a wide range of complex compositions.
Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca(2+) channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca(2+) channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca(2+) level, defective Ca(2+)-induced stomatal closure, and Ca(2+)-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca(2+) channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca(2+) channels are distinct from previously characterized ROS-activated Ca(2+) channels and act upstream of ROS, providing Ca(2+) transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca(2+) channels responsible for maintaining basal [Ca(2+)]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.
The United States is increasingly racially diverse. Racial disparities in maternal-child health persist. Despite national calls for workforce diversification, more than 90% of certified nurse-midwives are white. This systematic review examines how racism and midwifery's lack of racial diversity impact both midwives and their patients.
Databases were searched in January 2016 for studies that explored 1) racially concordant or racially discordant maternity care provided, at least in part, by midwives; 2) women of color's experience of race and discrimination in maternity care provided, at least in part, by midwives; and 3) midwives of color's experience of race and discrimination in clinical, educational, and/or professional settings. Studies were excluded if they were conducted outside the United States, focused on recent immigrant populations, or didn't have an English-language abstract. Selected studies were each reviewed by 2 independent reviewers, and data from the studies were entered into literature tables and synthesized for discussion.
A total of 7 studies was retained for review-3 on the experience of patients and 4 on the experience of providers. The studies show racism is common in midwifery education, professional organizations, and clinical practices. Racism and midwifery's lack of racial diversity act as a barrier to people of color completing midwifery education programs and fully participating in midwifery professional organizations. Both patients and midwives of color identified midwives of color as uniquely positioned to provide high-quality care for communities of color.
The midwifery profession and its patients stand to substantially benefit from diversification of the field, which requires addressing racism within the profession. Structural competency is a new theory that offers an effective framework to guide these efforts.
A retrospective cohort study of prospectively collected data.
As an initial effort to address readmissions after lumbar discectomy, reasons for hospital readmission are identified and discussed.
Lumbar discectomy is a commonly performed procedure. The Affordable Care Act codifies penalties for hospital readmissions. New quality-based reimbursements tied to readmissions call for a better understanding of the causes of readmission after procedures such as lumbar discectomy.
Lumbar discectomies performed in 2012-2014 were identified in the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. Patient Demographics, surgical variables, and reasons for readmissions within 30 days were recorded. Pearson's Chi Square was used to compare rates of demographics and surgical variables between readmitted and non-readmitted patients. Multivariate regression was used to identify risk factors for readmission.
Of 20,376 lumbar discectomies, 533 patients (2.62%) were readmitted within 30 days of surgery. The most common reasons for readmission were surgical site infections (n = 130, 0.64% of all discectomies, 24.4% of all readmissions), followed by pain issues (n = 89, 0.44%, 16.7%), and thromboembolic events (43, 0.21%, 8.1%). Overall time to readmission was 13.0 days ± 8.0 days (mean ± standard deviation). Factors most associated with readmission after lumbar discectomy were higher ASA class (Relative risk [RR] = 1.49, p < 0.001) and prolonged operative time (RR = 1.41, p = 0.002).
Surgical site infection, postoperative pain, and thromboembolic events were the most common reasons for readmission after lumbar discectomy. These findings identify potential areas for quality improvement initiatives.
Environmental stress, such as oxidative or heat stress, induces the activation of the heat shock response (HSR) and leads to an increase in the heat shock proteins (HSPs) level. These HSPs act as molecular chaperones to maintain cellular proteostasis. Controlled by highly intricate regulatory mechanisms, having stress-induced activation and feedback regulations with multiple partners, the HSR is still incompletely understood. In this context, we propose a minimal molecular model for the gene regulatory network of the HSR that reproduces quantitatively different heat shock experiments both on heat shock factor 1 (HSF1) and HSPs activities. This model, which is based on chemical kinetics laws, is kept with a low dimensionality without altering the biological interpretation of the model dynamics. This simplistic model highlights the titration of HSF1 by chaperones as the guiding line of the network. Moreover, by a steady states analysis of the network, three different temperature stress regimes appear: normal, acute, and chronic, where normal stress corresponds to pseudo thermal adaption. The protein triage that governs the fate of damaged proteins or the different stress regimes are consequences of the titration mechanism. The simplicity of the present model is of interest in order to study detailed modelling of cross regulation between the HSR and other major genetic networks like the cell cycle or the circadian clock.
Germ line-specific genes are activated in somatic cells during tumorigenesis, and are accordingly referred to as cancer germline genes. Such genes that act on piRNA (Piwi-interacting RNA) processing play an important role in the progression of cancer cells. Here, we show that the spermatogenic transposon silencer maelstrom (Mael), a piRNA-processing factor, is required for malignant transformation and survival of cancer cells. A specific Mael isoform was distinctively overexpressed in diverse human cancer cell lines and its depletion resulted in cancer-specific cell death, characterized by apoptosis and senescence, accompanied by an increase in reactive oxygen-species and DNA damage. These biochemical changes and death phenotypes induced by Mael depletion were dependent on ATM. Interestingly Mael was essential for Myc/Ras-induced transformation, and its overexpression inhibited Ras-induced senescence. In addition, Mael repressed retrotransposon activity in cancer cells. These results suggest that Mael depletion induces ATM-dependent DNA damage, consequently leading to cell death specifically in cancer cells. Moreover, Mael possesses oncogenic potential that can protect against genetic instability.
Over the last couple of decades, a body of theories has emerged that explains when and why people are motivated to act. Multiple disciplines have investigated the origins and consequences of motivated behavior, and have done so largely in parallel. Only recently have different disciplines, like psychology and economics, begun to consolidate their knowledge, attempting to integrate findings. The following chapter presents and discusses the most prominent approaches to motivation in the disciplines of biology, psychology, and economics. Particularly, we describe the specific role of incentives, both monetary and alternative, in various motivational theories. Though monetary incentives are pivotal in traditional economic theory, biological and psychological theories ascribe less significance to monetary incentives and suggest alternative drivers for motivation.
To evaluate the effect of precompression on power Doppler visualization of blood flow in breast masses.
This Institutional Review Board-approved and Health Insurance Portability and Accountability Act-compliant study evaluated 30 patients with breast masses (16 benign and 14 malignant) undergoing ultrasound-guided breast biopsy. A computational mathematics program was used to calculate the number of color pixels in a region of interest at various degrees of compression of the breast by the transducer. The amount of precompression was calculated as previously described. The percentage of color pixels compared to minimal compression was plotted against the percentage of precompression. The amount of precompression needed to decrease the number of color pixels by 50% and 100% was calculated. The differences between benign and malignant lesions were compared.
The mean percentages of precompression ± SD needed to decrease the number of color voxels by 50% in were 15.9% ± 6.43% (range, 8%-30%) for benign lesions and 14.0% ± 4.17% (range, 8%-20%) for malignant lesions (P = .35). The percentages of precompression needed to decrease the number of color pixels by 100% in were 34.7% ± 12.33% (range, 23%-62%) for benign lesions and for malignant lesions 26.7% ± 3.89% (range, 18%-31%), which were statistically significant (P = .027).
The amount of precompression normally used when obtaining B-mode images can substantially decrease the number of color voxels on power Doppler sonography. When performing quantitative work on Doppler evaluation of breast lesions, precompression needs to be controlled.
The role of formic acid together with the effect of the solvent type and their synergic interactions with a NiMo catalyst were studied for the conversion of lignin into bio-oil in an alcohol/formic acid media. Replacing formic acid with molecular hydrogen or isopropanol decreased the oil yield to a considerable degree, increased the solid yield and altered the nature of the bio-oil. The differences induced by the presence of molecular hydrogen were comparable to the ones observed in the isopropanol system, suggesting similar lignin conversion mechanisms for both systems. Additional semi-batch experiments confirmed that formic acid does not act merely as an in situ hydrogen source or hydrogen donor molecule. On the contrary, formic acid seems to react with lignin through a formylation-elimination-hydrogenolysis mechanism that leads to the de-polymerization of the biopolymer. This reaction competes with the formic acid decomposition giving mainly H2 and CO2, forming a complex reaction system. To the best of our knowledge, this is the first time that the distinctive role/mechanism of formic acid has been observed in the conversion of real lignin feedstock. In addition, the solvent, and especially ethanol, seems also to play a vital role in the stabilization of the de-polymerized monomers, and in the elimination/deformylation step.
Dual process models, such as the Prototype Willingness Model (PWM), propose to account for both intentional and reactive drinking behaviour. Current methods of measuring constructs in the PWM rely on self-report, thus require a level of conscious deliberation. Implicit measures of attitudes may overcome this limitation and contribute to our understanding of how prototypes and willingness influence alcohol consumption in young people. This study aimed to explore whether implicit alcohol attitudes were related to PWM constructs and whether they would add to the prediction of risky drinking.
The study involved a cross-sectional design. The sample included 501 participants from the United Kingdom (Mage 18.92; range 11-51; 63% female); 230 school pupils and 271 university students.
Participants completed explicit measures of alcohol prototype perceptions, willingness, drunkenness, harms, and intentions. They also completed an implicit measure of alcohol attitudes, using the Implicit Association Test.
Implicit alcohol attitudes were only weakly related to the explicit measures. When looking at the whole sample, implicit alcohol attitudes did not add to the prediction of willingness over and above prototype perceptions. However, for university students implicit attitudes added to the prediction of behaviour, over and above intentions and willingness. For school pupils, willingness was a stronger predictor of behaviour than intentions or implicit attitudes.
Adding implicit measures to the PWM may contribute to our understanding of the development of alcohol behaviours in young people. Further research could explore how implicit attitudes develop alongside the shift from reactive to planned behaviour. Statement of contribution What is already known on this subject? Young people's drinking tends to occur in social situations and is driven in part by social reactions within these contexts. The Prototype Willingness Model (PWM) attempts to explain such reactive behaviour as the result of social comparison to risk prototypes, which influence willingness to drink, and subsequent behaviour. Evidence also suggests that risky drinking in young people may be influenced by implicit attitudes towards alcohol, which develop with repeated exposure to alcohol over time. One criticism of the PWM is that prototypes and willingness are usually measured using explicit measures which may not adequately capture young people's spontaneous evaluations of prototypes, or their propensity to act without forethought in a social context. What does this study add? This study is novel in exploring the addition of implicit alcohol attitudes to the social reaction pathway in the model in order to understand more about these reactive constructs. Implicit alcohol attitudes added to the prediction of behaviour, over and above intentions and willingness for university students. For school pupils, willingness was a stronger predictor of behaviour than intentions or implicit attitudes. Findings suggest that adding implicit alcohol attitudes into the PWM might be able to explain the shift from reactive to intentional drinking behaviours with age and experience.
Genes encoding cellular membrane trafficking components, namely RAB7L1 and RAB39B, are more recently recognized factors associated with Parkinson's disease (PD). Encoded by a gene within the PARK16 locus, RAB7L1 interacts with Leucine-rich repeat kinase 2 (LRRK2) to act in intracellular transport processes that are likely important in neuronal survival and function. LRRK2 also directly phosphorylates a number of other Rab proteins. On the other hand, nonsense and missense mutations of the X-chromosome localized RAB39B, were shown to underlie X-linked intellectual disability (ID) in male patients with early-onset PD. The cellular or neuronal functions of RAB39B are not yet known with certainty, but it has recently been shown to play a role in glutamate receptor trafficking. Importantly, RAB39B is also functionally connected to components of autophagy regulation, which affects α-synuclein processing and clearance. In this review, we discuss the association of Rabs with PD pathology, and potential etiological mechanisms whereby defects or deficiencies in certain Rab proteins could lead to PD susceptibility. This article is protected by copyright. All rights reserved.
The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction via intravenous antigen administration such that it could be used to either: improve efficiency of inhibition of MS or to inhibit secondary autoimmunities that may occur following alemtuzumab use in multiple sclerosis. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses assessed clinically. A dose-response of CD4 mAb depletion indicated that 60-70% functional CD4 T cell depletion achieved in perceived failed trials in MS, was perhaps too low to even stop disease in animals. However, more marked (~75-90%) physical depletion CD4 T cells via CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness via a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rational for the rapid B cell hyper-repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T cell regulatory mechanisms that promote immune tolerance may account for the secondary B cell autoimmunities, which occur following alemtuzumab treatment of multiple sclerosis. This article is protected by copyright. All rights reserved.
Toxoplasma gondii is an intracellular protozoan with worldwide distribution and dogs act as sentinels of human infection. This search aimed to determine the occurrence of antibodies against T. gondii in dogs of the communities on the Cuiabá River, Mato Grosso and variables associated with infection. The dogs of the riverside communities in Cuiabá River, which includes Barranco Alto, Praia do Poço, Engenho Velho, Varginha, Bom Sucesso, Passagem da Conceição and São Gonçalo Beira Rio, were evaluated for the presence of T. gondii antibodies by indirect immunofluorescence antibody test (IFAT). The prevalence and factors associated with infection were calculated by chi-squared test (χ2) or Fisher's exact test, and univariate and multiple analysis. Of the 248 dogs surveyed, 107 (43.1%) were seropositive for T. gondii. The seroprevalence ranged from 25.6% to 64.3%. There was no statistically significant difference between the communities studied (p > 0.05). As for the associated factors, the only statistically significant factor was that of dogs living with cats (p = 0.02), with approximately twice the risk of acquiring infection. In conclusion, the seroprevalence in dogs of riverside communities in the Baixada Cuiabana demonstrated that high rates of infection, being the factor associated with infection, contact with domestic cats.
Neighborhood dogs may act as reservoirs for several zoonotic protozoan infections, particularly in urban areas, thus constituting a potential public health threat. Accordingly, the aim of the present study was to evaluate the exposure of neighborhood dogs to four protozoan pathogens in public areas with high levels of human movement in Curitiba, southern Brazil. Blood samples from 26 neighborhood dogs were screened by means of the indirect immunofluorescent antibody test (IFAT) for Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum, and a questionnaire was answered by the respective keeper. A total of 8/26 dogs (30.7%) seroreactive to T. gondii, 3/26 (11.5%) to N. caninum and 2/26 (7.7%) to both were identified. All the samples were seronegative for T. cruzi and Leishmania spp. Pathogen seroreactivity was not associated with the daily human movements or other epidemiological variables investigated (p > 0.05). In conclusion, the low seroprevalence for T. gondii and N. caninum indicated low environmental and food risk for animal infection and the seronegativity for Leishmania spp. and T. cruzi may reflect the absence of these pathogens in urban areas of Curitiba. Moreover, neighborhood dogs may be used as environmental sentinels for the presence of protozoan pathogens and their vectors.
The homeodomain transcription factor distal-less homeobox 3 gene (DLX3) is required for hair, tooth and skeletal development. DLX3 mutations have been found to be responsible for Tricho-Dento-Osseous (TDO) syndrome, characterized by kinky hair, thin-pitted enamel and increased bone density. Here we show that the DLX3 mutation (c.533 A>G; Q178R) attenuates osteogenic potential and senescence of bone mesenchymal stem cells (BMSCs) isolated from a TDO patient, providing a molecular explanation for abnormal increased bone density. Both DLX3 mutations (c.533 A>G and c.571_574delGGGG) delayed cellular senescence when they were introduced into pre-osteoblastic cells MC3T3-E1. Furthermore, the attenuated skeletal aging and bone loss in DLX3 (Q178R) transgenic mice not only reconfirmed that DLX3 mutation (Q178R) delayed cellular senescence, but also prevented aging-mediated bone loss. Taken together, these results indicate that DLX3 mutations act as a loss of function in senescence. The delayed senescence of BMSCs leads to increased bone formation by compensating decreased osteogenic potentials with more generations and extended functional lifespan. Our findings in the rare human genetic disease unravel a novel mechanism of DLX3 involving the senescence regulation of bone formation.
Because torque teno virus (TTV) has been implicated in tumorigenesis as a cocarcinogen, we studied TTV prevalence in saliva and biopsy samples from head and neck cancer (HNCC) patients, patients with premalignant lesions of oral cancer, and controls. We also wished to determine the TTV genotypes in HNCC patients.
A seminested polymerase chain reaction (PCR) amplifying the N22 region of the TTV genome, as well as direct sequencing of PCR fragments, was used.
TTV prevalence was higher in HNCC patients (saliva: 27/71, 38%; tumor biopsy: 22/74, 30%) than in controls (saliva: 8/56, 14%; oral mucosa: 1/19, 5%). TTV prevalence was also high in patients with premalignant lesions of oral carcinoma (saliva: 9/18, 50%; biopsy: 5/21, 24%). By phylogenetic analysis, TTV belonging mostly to genotypes 1 and 2 was found in HNCC patients. In most of the cases, identical TTV strains were present in the biopsy and salivary sample of the same HNCC patient. In addition, the same TTV strain was detected in 2 laryngeal carcinoma biopsies obtained from 2 independent patients.
Our data are compatible with the idea that TTV might act as a cocarcinogen in certain cases of HNCC. Alternatively, HNCC may facilitate either TTV replication or TTV entry into the saliva.
Empathy can be defined as the ability to understand the other's thoughts and feelings. It contains both cognitive and emotional components.
The aim of this study was to investigate the empathy ability of patients with alcohol dependency in association with cognitive and emotional functions, after acute detoxification and during long-term abstinence.
Thirty-three alcohol dependent inpatients that completed a detoxification process and stayed abstinent throughout the study, and 33 healthy comparison subjects that matched the patients for age, gender, and education level were included in the study. All the participants were administered the Facial Emotion Identification Test (FEIT), Facial Emotion Discrimination Test (FEDT), Trail Making Test (TMT), Digit Span Test (DST), Auditory Consonant Trigram Test (ACT), and Empathy Quotient Scale (EQS). All the tests were repeated after 3 months of abstinence.
At the first evaluation conducted after detoxification, patients performed significantly worse than healthy comparisons in almost all tests. At the second evaluation, which was conducted after 3 months of abstinence, the patients improved significantly in all measures, and no significant differences were detected between the patient and comparison groups. There were significant correlations between the test scores and EQS score.
Alcohol dependency has deleterious effects on empathy ability, and cognitive and emotional functions. Those impairments can improve with abstinence. Empathy ability has strong relationships with cognitive and emotional functions.
The past three decades have witnessed an enormous progress in the elucidation of the ERK/MAPK signaling pathway and its involvement in various cellular processes. Because of its importance and complex wiring, the ERK pathway has been an intensive subject for mathematical modeling, which facilitates the unraveling of key dynamic properties and behaviors of the pathway. Recently, however, it became evident that the pathway does not act in isolation but closely interacts with many other pathways to coordinate various cellular outcomes under different pathophysiological contexts. This has led to an increasing number of integrated, large-scale models that link the ERK pathway to other functionally important pathways. In this chapter, we first discuss the essential steps in model development and notable models of the ERK pathway. We then use three examples of integrated, multipathway models to investigate how crosstalk of ERK signaling with other pathways regulates cell-fate decision-making in various physiological and disease contexts. Specifically, we focus on ERK interactions with the phosphoinositide-3 kinase (PI3K), c-Jun N-terminal kinase (JNK), and β-adrenergic receptor (β-AR) signaling pathways. We conclude that integrated modeling in combination with wet-lab experimentation have been and will be instrumental in gaining an in-depth understanding of ERK signaling in multiple biological contexts.
The ubiquitous role of microRNAs (miRNAs) in a number of pathological processes has suggested that they could act as potential drug targets. RNA-binding small molecules offer an attractive means for modulating miRNA function. The availability of bioassay data sets for a variety of biological assays and molecules in public domain provides a new opportunity toward utilizing them to create models and further utilize them for in silico virtual screening approaches to prioritize or assign potential functions for small molecules. Here, we describe a computational strategy based on machine learning for creation of predictive models from high-throughput biological screens for virtual screening of small molecules with the potential to inhibit microRNAs. Such models could be potentially used for computational prioritization of small molecules before performing high-throughput biological assay.
With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at ~36 °C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the "injectability", i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo.
An engineer who becomes an educator in a school of software engineering has the mission to teach how to design and construct software systems, therein applying his or her knowledge and expertise. However, due to their engineering background, engineers may forget that educating a person is not the same as designing a machine, since a machine has a well-defined goal, whilst a person is capable to self-propose his or her own objectives. The ethical implications are clear: educating a free person must leave space for creativity and self-determination in his or her own discovery of the way towards personal and professional fulfillment, which cannot consist only in achieving goals selected by others. We present here an argument that is applicable to most fields of engineering. However, the dis-analogy between educating students and programming robots may have a particular appeal to software engineers and computer scientists. We think the consideration of three different stages in the educational process may be useful to engineers when they act as educators. We claim that the three stages (instructing, training and mentoring) are essential to engineering education. In particular, education is incomplete if the third stage is not reached. Moreover, mentoring (the third stage aimed at developing creativity and self-determination) is incompatible with an educational assessment framework that considers the goals of the engineer are always given by others. In our view, then, an integral education is not only beyond programming the behavior of students, but also beyond having them reach those given goals.
The increasing demand which requires ascertaining the legal age of undocumented individuals who reach the various countries of the European Community means that new lines of research must be developed which help respond to questions posed by the Justice Administration. For this reason, this study has been designed on the basis of fusion times of the proximal humeral epiphysis. Moreover, the ultrasound scan has been used as the diagnostic method. It is a non-invasive technique, unlike the radiograph, which is used under current standards for the forensic diagnosis of age. Used as a study sample were the ultrasound images of the proximal humeral epiphysis among 221 individuals belonging to the Spanish population, of both genders, of ages ranging from 5 to 30 years. All of the images were classified into 6 stages of fusion based on the morphology of each. The results display differences among the six age groups proposed for each of the stages of fusion and are of great interest from the perspective of enforcing the Spanish Criminal Law Act on Minors, because Stage 4 would mean that the person being studied is under the age of 16 years in the case of males and 15 years in the case of females. These results, coupled with the use of ultrasound as a non-invasive diagnostic technique, make this study a very useful method when the use of radiographs is not possible.
Sesquiterpenes are an abundant group belonging to the terpenoid family, with a C15 structure comprise of three isoprene units. Many sesquiterpenes are volatile compounds and it act as chemical messenger in plant signalling, particularly in the defense mechanism against biotic and abiotic stresses. Panax ginseng Meyer is important medicinal herbs with various reported pharmacological efficacies in which its triterpenoid saponins, called ginsenosides, were mostly studied. However, there have been few studies on volatile sesquiterpenes compounds regulation on P. ginseng. As slow-growing perennial plant, P. ginseng received many kind of stresses during its cultivation. The pathogen attack is one of the most devastated perturbation for ginseng yield. Thus, we aimed to analyze P. ginseng STS gene (PgSTS) expressions in ginseng organs as well as mono-, sesquiterpenes contents from ginseng seedlings treated with elicitors. qRT-PCR and GC-MS analysis showed that two elicitors- salicylic acid (SA) and methyl jasmonate (MeJA) triggered PgSTS expression at different time points and significantly induced mono-, sesquiterpene yield. Overexpression of PgSTS in Arabidopsis also induced high terpene content and conferred tolerance against Pseudomonas syringae pv. tomato infection. These results suggested that PgSTS transcripts are involved in terpenoid biosynthesis in response to environmental stress mediated by MeJA and SA elicitors; thus, generate tolerance against pathogen attack.
In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.
The Tobacco Control Act requires public disclosure of information about toxic constituents in cigarette smoke. To inform these efforts, we studied public understanding of cigarette smoke constituents.
We conducted phone surveys with national probability samples of adolescents (n=1125) and adults (n=5014) and an internet survey with a convenience sample of adults (n=4137), all in the USA. We assessed understanding of cigarette smoke constituents in general and of 24 specific constituents.
Respondents commonly and incorrectly believed that harmful chemicals in cigarette smoke mostly originate in additives introduced by cigarette manufacturers (43-72%). Almost all participants had heard that nicotine is in cigarette smoke, and many had also heard about carbon monoxide, ammonia, arsenic and formaldehyde. Less than one-quarter had heard of most other listed constituents being in cigarette smoke. Constituents most likely to discourage respondents from wanting to smoke were ammonia, arsenic, formaldehyde, hydrogen cyanide, lead and uranium. Respondents more often reported being discouraged by constituents that they had heard are in cigarette smoke (all p<0.05). Constituents with names that started with a number or ended in 'ene' or 'ine' were less likely to discourage people from wanting to smoke (all p<0.05).
Many people were unaware that burning the cigarette is the primary source of toxic constituents in cigarette smoke. Constituents that may most discourage cigarette smoking have familiar names, like arsenic and formaldehyde and do not start with a number or end in ene/ine. Our findings may help campaign designers develop constituent messages that discourage smoking.
The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1's G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43's cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1's C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as 'doorkeepers' and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly.
MYD88 mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of Activated B-Cell like (ABC) Diffuse Large B Cell Lymphoma (DLBCL), leading to constitutive NFkB pathway activation. The aim of this study was to examine the distinct genomic profiles of MYD88 mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants.
A cohort of 361 DLBCL cases (94 MYD88 mutant and 267 MYD88 wild-type) was submitted to next generation sequencing (NGS) focusing on 34 genes in order to analyze associated mutations and copy number variations, as well as gene expression profiling, and clinical and prognostic analyses.
Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis also segregated subgroups according to associated genetic alterations among patients with the same MYD88 mutation. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFkB pathway activation, although the majority of MYD88 L265P mutant cases harbor downstream NFkB alterations, which can predict BTK inhibitor resistance. Finally, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated CD79B mutations significantly improved the survival of MYD88 L265P mutant ABC DLBCL in our cohort.
This study highlights the relative heterogeneity of MYD88 mutant DLBCBL, adding to the field's knowledge of the theranostic importance of MYD88 mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy.
The modulation exerted by glucocorticoids in physiological responses to stressors is essential for maintaining short-term homeostasis. However, highly frequent and/or prolonged activation of the hypothalamic-pituitary-adrenal/interrenal axis may inhibit processes that are important to long-term fitness and health, including reproduction and immunocompetence. The present study evaluates the response to adrenocorticotropic hormone (ACTH) injection in the adult male tree frog, Hypsiboas faber, as indicated by levels of plasma corticosterone (CORT), plasma testosterone (T), ocular melatonin (MEL), hematocrit and immune functioning (total leukocyte count and bacterial killing ability against Escherichia coli). All levels were measured 1, 3 and 6h after treatment. ACTH increased CORT levels whilst decreasing T and MEL levels at 1h post-treatment. Six hours after ACTH injection, hematocrit and MEL levels increased. ACTH treatment did not significantly modulate the immune measures over the time-range sampled. The hormonal changes observed in response to ACTH treatment suggest that stressors could act as inhibitors of reproductive activity, as well as differentially modulating melatonin levels at different time-points.
MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, about 1 molecule of flavin nucleotide is sufficient to reduce 100 molecules of the mitoNEET [2Fe-2S] clusters in 4minutes under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane α-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10Å, which may facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells.
Toxicological studies of defined chemical mixtures assist human health risk assessment by establishing how chemicals interact with one another to induce an effect. This paper reviews how antiandrogenic chemical mixtures can alter reproductive tract development in rats with a focus on the reproductive toxicant phthalates. The reviewed studies compare observed mixture data to mathematical mixture model predictions based on dose addition or response addition to determine how the individual chemicals in a mixture interact (e.g., additive, greater, or less than additive). Phthalate mixtures were observed to act in a dose additive manner based on the relative potency of the individual phthalates to suppress fetal testosterone production. Similar dose additive effects have been reported for mixtures of phthalates with antiandrogenic pesticides of differing mechanisms of action. Overall, data from these phthalate experiments in rats can be used in conjunction with human biomonitoring data to determine individual hazard indices, and recent cumulative risk assessments in humans indicate an excess risk to antiandrogenic chemical mixtures that include phthalates only or phthalates in combination with other antiandrogenic chemicals.
We previously demonstrated the presence of sonic hedgehog (Shh) in nasal mucus in normal subjects and in patients with smell loss (hyposmia). Nasal mucus Shh levels were found significantly diminished in untreated hyposmic patients of multiple etiologies. Since treatment with oral theophylline has been previously associated with improvement in smell function we wished to study if such treatment increased nasal mucus Shh as well as improved smell function in patients with hyposmia.
Forty-four patients with hyposmia of several etiologies were evaluated for changes in hyposmia by subjective measurements of smell, taste and flavor perception and by olfactometry. Measurements of nasal mucus Shh were made in relationship to each set of sensory measurements. Patients were treated with oral theophylline at doses of 200-800mg for periods of 2-10months with sensory function, nasal mucus Shh and serum theophylline levels evaluated at these time intervals. Nasal mucus Shh measurements were made with a sensitive spectrophotometric ELISA assay and theophylline with a fluorometric assay.
There was consistent, significant improvement in subjective responses in smell, taste and flavor perception and in olfactometry associated with increased nasal mucus Shh and serum theophylline after theophylline treatment.
Improvement in smell function and in nasal mucus Shh was positively correlated in a dose-response relationship after treatment with oral theophylline. Results are consistent with a successful role for theophylline in improvement of smell function in hyposmic patients of multiple etiologies associated with increased nasal mucus Shh which can act as a biochemical marker for smell function.
In the Netherlands in 2006 a major health care reform was introduced, aimed at reinforcing regulated competition in the health care sector. Health insurers were provided with strong incentives to compete and more room to negotiate and selectively contract with health care providers. Nevertheless, the bargaining position of health insurers vis-à-vis both GPs and hospitals is still relatively weak. GPs are very well organized in a powerful national interest association (LHV) and effectively exploit the long-standing trust relationship with their patients. They have been very successful in mobilizing public support against unfavorable contracting practices of health insurers and enforcement of the competition act. The rapid establishment of multidisciplinary care groups to coordinate care for patients with chronic diseases further strengthened their position. Due to ongoing horizontal consolidation, hospital markets in the Netherlands have become highly concentrated. Only recently the Dutch competition authority prohibited the first hospital merger. Despite the highly concentrated health insurance market, it is unclear whether insurers will have sufficient countervailing buyer power vis-à-vis GPs and hospitals to effectively fulfill their role as prudent buyer of care, as envisioned in the reform. To prevent further consolidation and anticompetitive coordination, strict enforcement of competition policy is crucially important for safeguarding the potential for effective insurer-provider negotiations about quality and price.
MicroRNAs (miRNAs) are small non-coding RNA comprising approximately 19-25 nucleotides. miRNAs can act as tumour suppressors or oncogenes, and aberrant expression of miRNAs has been reported in several human cancers and has been associated with cancer initiation and progression. Recent evidence suggests that miRNAs play a major role in thyroid carcinogenesis. In this review, we summarize the role of miRNAs in thyroid cancer and describe the oncogenic or tumour suppressor function of miRNAs as well as their clinical utility as prognostic or diagnostic markers in thyroid cancer.
Decision Support for the Therapy Planning for Young Refugees and Asylum-Seekers with Posttraumatic Disorders Due to the Convention on the Rights of the Child and § 6 of the Asylum Seekers' Benefit Act, there are legal and ethical obligations for the care of minor refugees suffering from trauma-related disorders. In Germany, psychotherapeutic care of adolescent refugees is provided by specialized treatment centers and Child and Adolescent psychiatries with specialized consultation-hours for refugees. Treatment of minor refugees is impeded by various legal and organizational barriers. Many therapists have reservations and uncertainties regarding an appropriate therapy for refugees due to a lack of experience. This means that only a fraction of the young refugees with trauma-related disorders find an ambulatory therapist. In a review of international literature, empirical findings on (interpreter-aided) diagnostics and therapy of young refugees were presented. Practical experiences on therapeutic work with traumatized young refugees were summarized in a decision tree for therapy planning in the ambulatory setting. The decision tree was developed to support therapists in private practices by structuring the therapy process.
Intended to be a brief guide to making Mental Health Act assessments, this updated edition includes an overview of the relevant legal frameworks, and how to set up and manage assessments. It also describes how to make and implement decisions about, for example, hospital admission.
Significance Redox signaling is one of the key elements involved in cardiovascular diseases. Two important molecules are the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) and the oxidoreductase Thioredoxin-1 (Trx-1). Recent Advances During the last years, a lot of studies investigated Nrf2 and Trx-1 as protective proteins in cardiovascular disorders. Moreover, post-translational modifications of those molecules were identified which play an important role in the cardiovascular system. This review will summarize changes in the vasculature in atherosclerosis and ischemia reperfusion injury of the heart and the newest findings achieved with Nrf2 and Trx-1 therein. Interestingly, Nrf2 and Trx-1 can act together as well as independently of each other in protection against atherosclerosis and ischemia and reperfusion injury. Critical Issues In principle, pharmacological activation of a transcription factor like Nrf2 can be dangerous, since a transcription regulator has multiple targets and the pleiotropic effects of such activation should not be ignored. Moreover, over activation of Nrf2 as well as long term treatment with Trx-1 could be deleterious for the cardiovascular system. Future Directions Therefore, length of treatment with Nrf2 activators and/or Trx-1 has first to be studied in more detail in cardiovascular disorders. Moreover, a combination of Nrf2 activators and Trx-1 should be investigated and taken into consideration.
The humidifier disinfectant disaster (HDD) was not a simple poisoning accident by biocides, but a singular disaster in history created by chemicals in household products. This disaster was a result of the failure of a system for the management of chemical and product safety. Since the management authority for chemical usage safety is different from those for chemical safety in products, many blind areas for chemical safety management in products still remain. The 'Act on the Registration and Evaluation, etc. of Chemical Substances (ARECS)' or the new 'Biocidal Product Act' must not only address the blind areas in the management system for chemical and product safety, but also prevent a second HDD. To prevent another HDD, an integrated registration, evaluation, and management system for chemicals and consumer products must be incorporated into the 'ARECS' as an essential part for chemical safety in consumer products.
The government is preparing to make meaningful changes to its lambasted plans for a new Mental Health Act - at least that is the message coming from civil servants and ministers. If true, it is the result of the fantastic response generated by members and supporters of the more than 50 organisations brought together by the Mental Health Alliance.
Like everyone who cares, we search for an answer to the reason why a fellow human could carry out such a senseless act as the Soho bombing. We are horrified because, as Paul Boateng, the home minister said, we have a right to be different in a pluralistic society.
MINISTERS SHOULD urge the European Union to overturn a decision that will allow 'underqualified' Polish nurses the automatic right to practise in the UK, nursing organisations have said.
So, it's official, even unto the wife of the prime minister - a lifestyle guru has become an essential prerequisite to happiness. Having someone in your life to help you look right, speak right and act right is now as necessary as that not so subtly hidden designer label or the particular holiday destination that is 'in' this year.
In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.
The Americans With Disabilities Act requires that nursing programs not discriminate against students with disabilities. This article describes a qualitative study of RNs who had disabilities while in nursing school. As students, participants tried to hide their disabilities, experienced fear and anger from faculty, were frequently told they could never be nurses, wanted to be treated like everyone else, had to work harder than others to prove themselves worthy, and learned to advocate for themselves.
The Mental Capacity Act (MCA) is statutory legislation introduced in 2007 to protect and empower people to make decisions for themselves and those who were deemed as unable to make choices would have decisions made on their behalf, often by health professionals. All health professionals must follow the guiding principles of this legislation. Yet a scrutiny report by the House of Lords concluded this legislation was under-used with a lack of knowledge among professionals regarding applying the legislation in practice. A review of the literature also supports these findings. A mixed method study was carried out among adult community nurses to explore if these findings were applicable within a health trust in the North West region. An electronic questionnaire (n=60), focus group (n=7) and a paired interview (n=2) were utilised to collect data regarding community nurse's experience and confidence of using the MCA in practice. Analysis identified key themes: training, knowledge and confidence among staff, working together with the wider multidisciplinary team, empowerment and improving documentation. While findings suggest clear examples of self-appraised confidence and excewllent patient care, there is also scope for development regarding the knowledge and utilisation of the MCA in the clinical practice of community nurses.
The optical density of pristine graphene is high and broad in the near infrared region of the electromagnetic spectrum positioning this material as a highly efficient photothermal agent for in vivo applications. In this study, surfactant assisted exfoliated graphene was incorporated within bulk lipid samples of varying lipid types: glyceryl monoether, glyceryl monooleate and phytantriol. The pristine graphene sheets did not disrupt the packing of the liquid crystals while being in sufficiently intimate contact to provide localized heating and induce phase transitions. The phase progressions induced through heating using NIR irradiation of the entrained graphene particles within the bulk liquid crystal were studied using SAXS and confirmed using polarized optical microscopy. Increases in apparent temperature experienced by the matrix of up to 50 °C were observed by establishing a SAXS versus bulk temperature calibration curve allowing in situ measurements. The studies demonstrate the potential for use of graphene as a photothermal actuator across a range of lipid based systems of interest in controlled drug delivery.
A recent study makes the surprising observation that autophagosomes can still form in the absence of the core conjugation machinery. Furthermore, while such autophagosomes can fuse with lysosomes, their degradation is delayed, and this is associated with delayed destruction of the inner autophagosomal double membrane, highlighting a new role for proteins thought to act exclusively in the formation of autophagosomes in late stages of the autophagic itinerary within autolysosomes.
Patients and their family members often ask about genetic testing for asymptomatic individuals who are at risk for developing a genetic disorder. Ordering a genetic test is a complex process involving consideration of many basic ethical principles including autonomy, beneficence, and nonmaleficence, as well as the physician's duty to act in the patient's best interest. Physicians have many choices regarding what tests to order, and they must develop the knowledge and skills to best discuss genetic testing with their patients. Integration of core ethical principles into these processes will permit physicians to best serve their patients when obtaining informed consent, considering advantages and harms of potential results, disclosing those results, and providing follow-up.
To review the legal issues concerning family members' access to information when patients are in the ICU.
U.S. Code, U.S. Code of Federal Regulations, and state legislative codes.
Relevant legal statutes and regulations were identified and reviewed by the two attorney authors (L. F., M. A. V.).
Not applicable.
Review by all coauthors.
The Health Insurance Portability and Accountability Act and related laws should not be viewed as barriers to clinicians sharing information with ICU patients and their loved ones. Generally, under Health Insurance Portability and Accountability Act, personal representatives have the same authority to receive information that patients would otherwise have. Persons involved in the patient's care also may be given information relevant to the episode of care unless the patient objects. ICUs should develop policies for handling the issues we identify about such information sharing, including policies for responding to telephone inquiries and methods for giving patients the opportunity to object to sharing information with individuals involved in their care. ICU clinicians also should be knowledgeable of their state's laws about how to identify patients' personal representatives and the authority of those representatives. Finally, ICU clinicians should be aware of any special restrictions their state places on medical information. In aggregate, these strategies should help ICU managers and clinicians facilitate robust communication with patients and their loved ones.
Computerised cognitive behaviour therapy (cCBT) is an efficient form of therapy potentially improving access to psychological care. Indirect evidence suggests that the uptake and effectiveness of cCBT can be increased if facilitated by telephone, but this is not routinely offered in the NHS.
To compare the clinical effectiveness and cost-effectiveness of telephone-facilitated free-to-use cCBT [e.g. MoodGYM (National Institute for Mental Health Research, Australian National University, Canberra, ACT, Australia)] with minimally supported cCBT.
This study was a multisite, pragmatic, open, two-arm, parallel-group randomised controlled trial with a concurrent economic evaluation.
Participants were recruited from GP practices in Bristol, Manchester, Sheffield, Hull and the north-east of England.
Potential participants were eligible to participate in the trial if they were adults with depression scoring ≥ 10 on the Patient Health Questionnaire-9 (PHQ-9).
Participants were randomised using a computer-generated random number sequence to receive minimally supported cCBT or telephone-facilitated cCBT. Participants continued with usual general practitioner care.
The primary outcome was self-reported symptoms of depression, as assessed by the PHQ-9 at 4 months post randomisation.
Secondary outcomes were depression at 12 months and anxiety, somatoform complaints, health utility (as assessed by the European Quality of Life-5 Dimensions questionnaire) and resource use at 4 and 12 months.
Clinical effectiveness: 182 participants were randomised to minimally supported cCBT and 187 participants to telephone-facilitated cCBT. There was a difference in the severity of depression at 4 and 12 months, with lower levels in the telephone-facilitated group. The odds of no longer being depressed (defined as a PHQ-9 score of < 10) at 4 months were twice as high in the telephone-facilitated cCBT group [odds ratio (OR) 2.05, 95% confidence interval (CI) 1.23 to 3.42]. The benefit of telephone-facilitated cCBT was no longer significant at 12 months (OR 1.63, 95% CI 0.98 to 2.71). At 4 months the between-group difference in PHQ-9 scores was 1.9 (95% CI 0.5 to 3.3). At 12 months the results still favoured telephone-facilitated cCBT but were no longer statistically significant, with a difference in PHQ-9 score of 0.9 (95% CI -0.5 to 2.3). When considering the whole follow-up period, telephone-facilitated cCBT was asssociated with significantly lower PHQ-9 scores than minimally supported cCBT (mean difference -1.41, 95% CI -2.63 to -0.17; p = 0.025). There was a significant improvement in anxiety scores over the trial period (between-group difference 1.1, 95% CI 0.1 to 2.3; p = 0.037). In the case of somatic complaints (assessed using the Patient Health Questionnaire-15), there was a borderline statistically significant difference over the trial period (between-group difference 1.1, 95% CI 0.0 to 1.8; p = 0.051). There were gains in quality-adjusted life-years at reduced cost when telephone facilitation was added to MoodGYM. However, the results were subject to uncertainty.
The results showed short-term benefits from the addition of telephone facilitation to cCBT. The effect was small to moderate and comparable with that of other primary care psychological interventions. Telephone facilitation should be considered when offering cCBT for depression.
Participants' depression was assessed with the PHQ-9, cCBT use was quite low and there was a slightly greater than anticipated loss to follow-up.
Improve the acceptability of cCBT and its capacity to address coexisting disorders. Large-scale pragmatic trials of cCBT with bibliotherapy and telephone-based interventions are required.
Current Controlled Trials ISRCTN55310481.
This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 89. See the NIHR Journals Library website for further project information.
Class IIa HDACs show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as "Readers" of acetyl marks, whereas enzymatically active HDACs from class I or IIb are called "Erasers" to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small molecule ligands of class IIa histone deacetylases (HDACs) gained tremendous attention during the last decade and were suggested as pharmaceutical target in several indication areas such as cancer, Huntington's disease or muscular atrophy. Up to now only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes with simple mix-and-measure procedure and extraordinary robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The assay principle is generic and can also be transferred to class I HDAC8.
There are two ways to synchronize oscillators: by coupling and by common forcing, which can be pure noise. By virtue of the Ott-Antonsen ansatz for sine-coupled phase oscillators, we obtain analytically tractable equations for the case where both coupling and common noise are present. While noise always tends to synchronize the phase oscillators, the repulsive coupling can act against synchrony, and we focus on this nontrivial situation. For identical oscillators, the fully synchronous state remains stable for small repulsive coupling; moreover it is an absorbing state which always wins over the asynchronous regime. For oscillators with a distribution of natural frequencies, we report on a counter-intuitive effect of dispersion (instead of usual convergence) of the oscillators frequencies at synchrony; the latter effect disappears if noise vanishes.
We report the unprecedented Lapita exploitation and subsequent extinction of large megafauna tortoises (?Meiolania damelipi) on tropical islands during the late Holocene over a 281,000 km(2) region of the southwest Pacific spanning from the Vanuatu archipelago to Viti Levu in Fiji. Zooarchaeological analyses have identified seven early archaeological sites with the remains of this distinctive hornless tortoise, unlike the Gondwanan horned meiolaniid radiation to the southwest. These large tortoise radiations in the Pacific may have contributed to the rapid dispersal of early mobile Neolithic hunters throughout southwest Melanesia and on to western Polynesia. Subsequent rapid extinctions of these terrestrial herbivorous megafauna are likely to have led to significant changes in ecosystems that help explain changes in current archaeological patterns from Post-Lapita contexts in the region.
We study the enhanced dewetting of ultrathin Polystyrene (PS)/Poly (methyl methacrylate) (PMMA) blend films in a mixed solution, and reveal the dewetting can act as a simple and effective method to fabricate large-area surface-enhanced Raman scattering (SERS) substrate. A bilayer structure consisting of under PMMA layer and upper PS layer forms due to vertical phase separation of immiscible PS/PMMA during the spin-coating process. The thicker layer of the bilayer structure dominates the dewetting structures of PS/PMMA blend films. The diameter and diameter distribution of droplets, and the average separation spacing between the droplets can be precisely controlled via the change of blend ratio and film thickness. The dewetting structure of 8 nm PS/PMMA (1:1 wt%) blend film is proved to successfully fabricate large-area (3.5 cm × 3.5 cm) universal SERS substrate via deposited a silver layer on the dewetting structure. The SERS substrate shows good SERS-signal reproducibility (RSD < 7.2%) and high enhancement factor (2.5 × 10(7)). The enhanced dewetting of polymer blend films broadens the application of dewetting of polymer films, especially in the nanotechnology, and may open a new approach for the fabrication of large-area SERS substrate to promote the application of SERS substrate in the rapid sensitive detection of trace molecules.
The anti-cancer activity of the benzo[h]quinolines was evaluated on cultured human skin cancer (G361), lung cancer (H460), breast cancer (MCF7) and colon cancer (HCT116) cell lines. The inhibitory effect of these compounds on the cell growth was determined by the MTT assay. The compounds 3e, 3f, 3h and 3j showed potential cytotoxicity against these human cancer cell lines. Effect of active compounds on DNA oxidation and expression of apoptosis related gene was studied. We also developed a quantitative method to measure the activity of cyclin-dependent kinases-2 (CDK2) by western blotting in the presence of active compound. In addition, molecular docking revealed that benzo[h]quinolines can correctly dock into the hydrophobic pocket of the targets receptor protein aromatase and CDK2, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. These findings reveal that benzo[h]quinolines act as anti-cancer agents by inducing oxidative stress-mediated DNA damage.
Physical exercise can reduce adverse conditions during aging, while both exercise and aging act as metabolism modifiers. The present study investigates rat fecal and cecal metabolome alterations derived from exercise during rats' lifespan.
Groups of rats trained life-long or for a specific period of time were under study. The training protocol consisted of swimming, 15-18 min per day, 3-5 days per week, with load of 4-0% of rat's weight. Fecal samples and cecal extracts were analyzed by targeted and untargeted metabolic profiling methods (GC-MS and LC-MS/MS). Effects of exercise and aging on the rats' fecal and cecal metabolome were observed.
Fecal and cecal metabolomics are a promising field to investigate exercise biochemistry and age-related alterations.
The present generation of women of childbearing age more frequently suffer from overweight, obesity, initial as well as fully established metabolic syndrome, which together with postponing motherhood until the third decade in life plays an important role in the increasing incidence of gestational diabetes (GDM) that currently affects about 1/5 of pregnant women. However the causal link between diabetes during pregnancy and metabolic diseases in the whole population is mutual. By way of epigenetic changes, maternal diabetes unfavourably programmes metabolism of the offspring, who tend to transfer the disorder to the next generations. Gestational diabetes is therefore an important link fitting into the accumulation curve of the incidence of overweight, obesity, metabolic syndrome and consequently also T2DM among the whole population. Genetic as well as epigenetic factors play a great role in the GDM pathogenesis, which is shown by the fact that this complication also affects women with normal BMI. When it comes to diagnosing GDM, we will need to manage also in future with establishing fasting glycemia and glycemia following glucose challenge (OGTT) that may include a considerable degree of measurement inaccuracy. It is therefore necessary to observe pre-analytical and analytical conditions of measurements in order to obtain a reliable result. It is a positive sign that the Czech professional associations have adopted new international criteria for diagnosing GDM which, as opposed to those valid earlier, better reflect the risk of pregnancy-related and perinatal complications.The care for gestational patients with diabetes at a low risk (due to satisfactory glycemic control through a diet or small pharmacotherapeutic doses, with an eutrophic fetus and without associated complications) is provided by an outpatient gynecologist and a diabetes specialist, they can give birth in standard maternity hospitals. The care for gestational patients with diabetes at a higher risk is taken over by specialist centres. The early and appropriate treatment of gestational diabetes demonstrably reduces the risk of complications. The base for therapy is formed by regimen-related measures: the therapeutic diet and increased physical activity. The best results of the dietary therapy are achieved with foods low on glycemic index and glycemic load that can also act as efficient prevention of GDM and subsequent development of T2DM. A small number of cases require adding of pharmacological therapy: insulin and newly also metformin. Metformin is the drug of choice primarily in obese patients, however in almost half of the cases insulin must be added. Medication, in particular with insulin, must be introduced carefully, following re-education and elimination of dietary mistakes. The aim of the treatment is not only to achieve normoglycemia, but also to improve, or at least to not further worsen insulin resistance. Insulin resistance alone without diabetes, e.g. due to obesity or a great weight gain, may lead to macrosomia and epigenetic changes. In this regard, the prevention within the whole population of pregnant women needs to be improved and the vicious circle of the causation of metabolic disorders among the population needs to be broken.Key words: recommended procedure - epigenetic changes - gestation diabetes mellitus - macrosomia - screening.
Polyamines (PAs) and nitric oxide (NO) are vital signals in modulating plant response to abiotic stress. However, to our knowledge, studies on the relationship between NO and PAs in response to cold stress in tomato are limited. Accordingly, in this study, we investigated the effects of putrescine (Put) and spermidine (Spd) on NO generation and the function of Spd-induced NO in the tolerance of tomato seedling under chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the seedlings, whereas Put had no such effect. Moreover, H2O2 might act as an upstream signal to stimulate NO production. Both exogenous NO donor (sodium nitroprusside (SNP)) and Spd enhanced chilling tolerance in tomato, thereby protecting the photosynthetic system from damage. Compared to chilling treatment alone, Spd enhanced the gene expressions of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and their enzyme activities in tomato leaves. However, a scavenger or inhibitor of NO abolished Spd-induced chilling tolerance and blocked the increased expression and activity due to Spd of these antioxidant enzymes in tomato leaves under chilling stress. The results showed that NO induced by Spd plays a crucial role in tomato's response to chilling stress.
Short endogenous peptides represent one of the most important constituents of the mammalian body's general regulatory system. Some synthesized analogs and modified natural peptides (eg, corticotropins) also show high biological activity. Nevertheless, the mechanism of action of regulatory peptides remains unclear. To explain the effects of peptides of intermolecular processes, the hypothesis that a synactonal mechanism underlies the action of regulatory peptides, exemplified by the heptapeptide Semax, has been proposed. Thus, in the total pool of Semax metabolites, which includes the cleavage products of the parental molecule, we can distinguish the functional core, represented by the major metabolic products-peptides HFPGP and PGP. These peptides have their own binding sites with similar although differing characteristics. Together with Semax, they constitute a single complex of bioregulators acting in a certain sequence and in interaction, ie, synacton. It can be assumed that the diverse clinically significant effects of the drug Semax are determined by its synacton. Specific interactions between some tritium-labeled peptides (basic constituents of the Semax synacton) and plasma membranes of neurons have been characterized. Only a few peptides of the Semax synacton showed competitive activity for the Semax binding sites. Fragments comprising 5 amino acid residues (EHFPG and HFPGP) showed the highest competitive activity. We also characterized the processes of specific ligand-receptor interactions of some tritium-labeled corticotropins ([(3) H-Pro]MEHFPGP, [(3) H-Pro]HFPGP, and [(3) H-Pro]PGP) by applying mathematical discriminative models (Scatchard, Hill, Bjerrum, and Lineweaver-Burk plots). So the intermolecular interactions of these peptides with plasma membranes of neuronal brain targets are probably not limited by specific binding at orthosteric sites. The effect of peptides that act in the synacton considerably extends the regulatory potential of the initial molecule.
The case here reported involves a schizophrenic 19-year-old girl under treatment with clotiapine, which was well tolerated except for a moderate dry mouth. The woman ingested a whole sole (Solea solea), which caused a very rapid death by choking. A complete autopsy was performed 24 h later, as well as histological and toxicological analysis. At autopsy, the sole was wedged in the esophagus causing a choking ab extrinseco. The fish had a length of 18 cm and a maximum width of 6 cm, weighing 188.7 g. Toxicological analysis detected 0.57 mg/L of clotiapine in blood, which falls within the therapeutic range. The peculiarity of this case is represented by two factors: one is the choking by fish and the second was the adverse affect caused by clotiapine, which induced a condition of dry mouth making the act of swallowing even more difficult, thereby contributing to a very rapid mechanical asphyxia and the death of the young woman.
Recent studies have identified Engrailed-2 (EN-2), a homeobox-containing transcription factor, as a candidate oncogene in prostate cancer (PC). Therapeutic targeting on EN-2, however, is limited because the mechanism underlying EN-2 overexpression in prostatic cancer cells is unknown. This study was to investigate the potential regulatory role of miR-33a on EN-2 expression and explore this signaling axis in ability of prostate cancer survival and metastasis.
The relative expression of miR-33a and EN-2 in paired prostate cancer tissue and adjacent normal tissue as well as in prostate cancer cell lines, PC3 and DU145, was determined using quantitative real-time PCR or western blot, respectively. Cells survival, migration and invasion were evaluated by assays of MTT, TUNEL and Boyden chamber assays, respectively. Direct regulation of EN-2 by miR-33a was examined by luciferase reporter assay.
The data showed that miR-33a was upregulated and EN-2 was downregulated in both prostate cancer tissue and prostate cancer cells. miR-33a overexpression suppresses prostate cancer cell survival and metastasis. miR-33a can directly act on EN-2 expression by binding to 3'UTR of its mRNA. Also, miR-33a negatively regulated EN-2 mRNA and protein expression. In pcDNA-EN-2 and miR-33a mimic co-transfected PC3 and DU145 cells, EN-2 overexpression reverses the anti-cell survival and metastasis actions of miR-33a overexpression. The pivotal role of miR-33a in inhibiting prostate tumor growth was confirmed in xenograft models of prostate cancer.
Our data suggest that the functional interaction of miR-33a and EN-2 is involved in tumorigenesis of prostate cancer. Also in this process EN-2 serves as a negative responder for miR-33a.
Volitional action and self-control-feelings of acting according to one's own intentions and in being control of one's own actions-are fundamental aspects of human conscious experience. However, it is unknown whether high-level cognitive control mechanisms are affected by socially salient but nonconscious emotional cues. In this study, we manipulated free choice decisions to act or withhold an action by subliminally presenting emotional faces: In a novel version of the Go/NoGo paradigm, participants made speeded button-press responses to Go targets, withheld responses to NoGo targets, and made spontaneous, free choices to execute or withhold the response for Choice targets. Before each target, we presented emotional faces, backwards masked to render them nonconscious. In Intentional trials, subliminal angry faces made participants more likely to voluntarily withhold the action, whereas fearful and happy faces had no effects. In a second experiment, the faces were made supraliminal, which eliminated the effects of angry faces on volitional choices. A third experiment measured neural correlates of the effects of subliminal angry faces on intentional choice using EEG. After replicating the behavioural results found in Experiment 1, we identified a frontal-midline theta component-associated with cognitive control processes-which is present for volitional decisions, and is modulated by subliminal angry faces. This suggests a mechanism whereby subliminally presented "threat" stimuli affect conscious control processes. In summary, nonconscious perception of angry faces increases choices to inhibit, and subliminal influences on volitional action are deep seated and ecologically embedded.
Behavior analysts who supervise staff are responsible for establishing a healthy supervisory relationship and for teaching basic behavior analytic skills (e.g., verbal repertoires, technical repertoires, clinical decision-making). In addition, supervisors should prepare their supervisees to succeed in their subsequent professional activities by developing their interpersonal skills and professionalism repertoires. Difficulties in the supervisor relationship and problematic personal and professional skills often become the focus of targeted supervision efforts after the effects of deficits (e.g., avoidance of supervision, complaints from consumers, persistent tardiness) are detected. The primary purpose of this paper is to provide guidance to the supervisor's effort to identify and address barriers to successful supervision related to a damaged supervisory relationship and persistent interpersonal and professional skills of the supervisee. A secondary purpose of this paper is to act as a general call to supervisors to continually and thoughtfully reflect on their own history, repertoires, and behavior, such that they may continue professional growth as supervisors.
Rationale: Current first-line treatments for stress-related disorders such as major depressive disorder (MDD) act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted "α5-PAM"), a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites, has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS) and treated with α5-PAM acutely (30 min prior to assessing behavior) or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as "behavioral emotionality") across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in the hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusion: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.
A 50-year-old male with advanced non-small-cell lung cancer was unable to have standard-of-care molecular testing performed at diagnosis as a result of inadequacy of the available tissue. A subsequently performed commercial liquid tumor biopsy (Foundation ACT(®)) revealed an epidermal growth factor receptor exon 19 deletion, but due to the progression of the tumor and rapid deterioration in the patient's performance status, a meaningful attempt at therapy directed to this recognized therapeutic target was not possible. This case provides important support for the relevance of liquid tumor biopsies in documenting highly clinically relevant molecular targets, particularly in the setting where limited solid tumor tissue is available for analysis.
Astronauts and vestibular patients face analogous challenges to orientation function due to adaptive exogenous (weightlessness-induced) or endogenous (pathology-induced) alterations in the processing of acceleration stimuli. Given some neurovestibular similarities between these challenges, both affected groups may benefit from shared research approaches and adaptation measurement/improvement strategies. This article reviews various past strategies and introduces two plausible ground-based approaches, the first of which is a method for eliciting and assessing vestibular adaptation-induced imbalance. Second, we review a strategy for mitigating imbalance associated with vestibular pathology and fostering readaptation. In discussing the first strategy (for imbalance assessment), we review a pilot study wherein imbalance was elicited (among healthy subjects) via an adaptive challenge that caused a temporary/reversible disruption. The surrogate vestibular deficit was caused by a brief period of movement-induced adaptation to an altered (rotating) gravitoinertial frame of reference. This elicited adaptation and caused imbalance when head movements were made after reentry into the normal (non-rotating) frame of reference. We also review a strategy for fall mitigation, viz., a prototype tactile sway feedback device for aiding balance/recovery after disruptions caused by vestibular pathology. We introduce the device and review a preliminary exploration of its effectiveness in aiding clinical balance rehabilitation (discussing the implications for healthy astronauts). Both strategies reviewed in this article represent cross-disciplinary research spin-offs: the ground-based vestibular challenge and tactile cueing display were derived from aeromedical research to benefit military aviators suffering from flight simulator-relevant aftereffects or inflight spatial disorientation, respectively. These strategies merit further evaluation using clinical and astronaut populations.
Thyroid cancer is the most frequent malignancies of the endocrine system, and it has became the fastest growing type of cancer worldwide. Much still remains unknown about the molecular mechanisms of thyroid cancer. Studies have found that some certain relationship between ARAP3 and human cancer. However, the role of ARAP3 in thyroid cancer has not been well explained. This study aimed to investigate the role of ARAP3 gene in papillary thyroid carcinoma.
Whole exon sequence and whole genome sequence of primary papillary thyroid carcinoma (PTC) samples and matched adjacent normal thyroid tissue samples were performed and then bioinformatics analysis was carried out. PTC cell lines (TPC1, BCPAP, and KTC-1) with transfection of small interfering RNA were used to investigate the functions of ARAP3 gene, including cell proliferation assay, colony formation assay, migration assay, and invasion assay.
Using next-generation sequence and bioinformatics analysis, we found ARAP3 genes may play an important role in thyroid cancer. Downregulation of ARAP3 significantly suppressed PTC cell lines (TPC1, BCPAP, and KTC-1), cell proliferation, colony formation, migration, and invasion.
This study indicated that ARAP3 genes have important biological implications and may act as a potentially drugable target in PTC.
The Affordable Care Act requires state Medicaid programs to cover substance use disorder treatment for their Medicaid expansion population but allows states to decide which individual services are reimbursable. To examine how states have defined substance use disorder benefit packages, we used data from 2013-14 that we collected as part of an ongoing nationwide survey of state Medicaid programs. Our findings highlight important state-level differences in coverage for substance use disorder treatment and opioid use disorder medications across the United States. Many states did not cover all levels of care required for effective substance use disorder treatment or medications required for effective opioid use disorder treatment as defined by American Society of Addiction Medicine criteria, which could result in lack of access to needed services for low-income populations.
This study provides a forward-thinking assessment of the factors likely to affect future trends in dental care in the United States. We developed a forecasting model based on historical data from the Medical Expenditure Panel Survey to determine how demographic trends and recent health care policies will affect dental care use in the future. Our forecasts suggest that the medical and dental insurance reforms instituted under the Affordable Care Act will increase rates of dental care use and the number of dental visits, with utilization rates reaching 47 percent in 2026 and the number of visits reaching 334 million, under optimistic assumptions about take-up of pediatric dental coverage. Our forecasting model also indicates that visits for preventive dental care will increase in the future, while visits for dental treatment will decline. Our forecasts can be used to infer future need for different types of dental care providers and to provide policy makers with the information needed to consider the expansion of mandates for dental benefit coverage to adult populations covered by Medicaid, Medicare, and individual and small-group plans sold on health insurance exchanges.
The Affordable Care Act is improving access to and the affordability of a wide range of health care services. While dental care for children is part of the law's essential health benefits and state Medicaid programs must cover it, coverage of dental care for adults is not guaranteed. As a result, even with the recent health insurance expansion, many Americans face financial barriers to receiving dental care that lead to unmet oral health needs. Using data from the 2014 National Health Interview Survey, we analyzed financial barriers to a wide range of health care services. We found that irrespective of age, income level, and type of insurance, more people reported financial barriers to receiving dental care, compared to any other type of health care. We discuss policy options to address financial barriers to dental care, particularly for adults.
Oligomerization of the mixed lineage kinase domain-like protein (MLKL) is essential for its cation channel function in necroptosis. Here we show that the MLKL channel is an octamer comprising of two previously identified tetramers most likely in their side-by-side position. Inter-molecule disulfide bonds are present in the tetramer but are not required for the octamer assembly and necroptosis. MLKL forms oligomers in the necrosome, and is then released from the necrosome before or during its membrane translocation. We identified two MLKL mutants that could not oligomerize into octamers although they formed tetramer and also one MLKL mutant that could spontaneously form a disulfide bond-linked octamer. Subsequent analysis revealed that the tetramers fail to translocate to the plasma membrane, and that the MLKL octamer formation depends on α-helices 4 and 5. While MLKL could be detected from outside of the cells, its N- and C-terminal ends could not be detected, indicating that the MLKL octamer spans across the plasma membrane, leaving its N- and C-terminal inside the cell. These data allowed us to propose a 180° symmetry model of the MLKL octamer and concluded that the fully assembled MLKL octamers, but not the previously described tetramers act as effectors of necroptosis.
Enterohaemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally-encoded 365 kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonisation of cattle by EHEC serogroup O5, O111 and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill-defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells, and to a lesser extent proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, 8, WC-1 and γδ-TCR) and cytokines examined (IL-2, -4, -10, -17A and IFN-γ), and rendered T cells refractory to mitogen for a least 18 hours after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavilin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB, L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.
The mammalian brain is supplied with blood by specialized vasculature that is structurally and functionally distinct from that of the periphery. A defining feature of this vasculature is a physical blood-brain barrier (BBB). The BBB separates blood components from the brain microenvironment, regulating the entry and exit of ions, nutrients, macromolecules, and energy metabolites. Over the last two decades, physiological studies of cerebral blood flow dynamics have demonstrated that substantial intercellular communication occurs between cells of the vasculature and the neurons and glia that abut the vasculature. These findings suggest that the BBB does not function independently, but as a module within the greater context of a multicellular neurovascular unit (NVU) that includes neurons, astrocytes, pericytes, and microglia as well as the blood vessels themselves. Here, we describe the roles of these NVU components as well as how they act in concert to modify cerebrovascular function and permeability in health and select diseases.
WHO recently issued new guidance on the prevention of sexual transmission of Zika virus. The updated guidance states that '[c]ountry health programmes should ensure that… [i]n order to prevent adverse pregnancy and fetal outcomes, men and women of reproductive age, living in areas where local transmission of Zika virus is known to occur, be correctly informed and oriented to consider delaying pregnancy'. While the media has reported this advice as WHO telling couples in Zika-affected regions to avoid pregnancy, WHO states that they are not doing that. In an interview with the New York Times, a spokesperson from WHO stated, 'it's important to understand that this is not WHO saying, "Hey everybody, don't get pregnant." It's that they should be advised about this, so they themselves can make the final decision'. In this statement, the WHO's spokesperson distinguishes between actively directing individuals to delay pregnancy and advising them, which is portrayed as a merely informative act that facilitates but does not direct an individual's final decision. This paper proposes that advising should not be understood as a purely informational and non-directive act. The choices that agencies make in what advice to offer and to whom to offer the advice are ethical choices with practical implications. We will thus lay out a framework for considering the ethical issues that arise in the context of advising and demonstrate how it can be used to evaluate the WHO guidance.
Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLK) are essential for plant growth, development and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as co-regulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as co-regulatory and physical partners of EMS1. The SERK1/2 gene functions in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation (BiFC), Förster resonance energy transfer (FRET), and co-immunoprecipitation (co-IP) approaches revealed that SERK1 biochemically interacted with EMS1. Trans-phosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among twelve in vitro auto- and trans-phosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 auto-phosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence on the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.
In monkeys, motor outputs from premotor cortex (PM) involve cortico-cortical connections with primary motor cortex (M1). However, in humans, the functional organization of PM and its relationship with the corticospinal tract (CST) is still uncertain. This study was carried out in 21 patients undergoing intraoperative brain mapping prior to tumor resection. The left ventrolateral premotor cortex (vlPM-BA6) was identified preoperatively by functional magnetic resonance imaging, and then investigated intraoperatively using high frequency direct electrical stimulation (HF-DES) of the convexity of M1 and vlPM-BA6, with simultaneous recording of motor-evoked potentials (MEPs) from oro-facial, hand and arm muscles. The somatotopy, organization of evoked responses, latency of MEPs, and cortical excitability of vlPM-BA6 were compared with reference data from M1. vlPM-BA6 was found to be less excitable, with significantly longer MEP latencies than M1. In addition to the pure oro-facial and hand-arm muscle representation, a "transition oro-hand zone" was identified in vlPM-BA6. The longer latency of vlPM-BA6 MEPs suggests that human vlPM could act on spinal motoneurons either directly through more slowly conducting CST fibers or via less direct pathways through M1, brainstem, or spinal mechanisms. The results help in disclosing the very different roles of vlPM and M1 in motor control.
Even though splicing repression by hnRNP complexes bound to exonic sequences is well-documented, the responsible effector domains of hnRNP proteins have been described for only a select number of hnRNP constituents. Thus, there is only limited information available for possible varying silencer activities amongst different hnRNP proteins and composition changes within possible hnRNP complex assemblies. In this study, we identified the glycine-rich domain (GRD) of hnRNP proteins as a unifying feature in splice site repression. We also show that all four hnRNP D isoforms can act as genuine splicing repressors when bound to exonic positions. The presence of an extended GRD, however, seemed to potentiate the hnRNP D silencer activity of isoforms p42 and p45. Moreover, we demonstrate that hnRNP D proteins associate with the HIV-1 ESSV silencer complex, probably through direct recognition of "UUAG" sequences overlapping with the previously described "UAGG" motifs bound by hnRNP A1. Consequently, this spatial proximity seems to cause mutual interference between hnRNP A1 and hnRNP D. This interplay between hnRNP A1 and D facilitates a dynamic regulation of the repressive state of HIV-1 exon 3 which manifests as fluctuating relative levels of spliced vpr- and unspliced gag/pol-mRNAs.
Dextransucrase (EC2.4.5.1) from strain Leuconostoc mesenteroides 0326, which synthesizes dextran and oligosaccharides, which act as prebiotics, are popularly used in such industries as food and medicine. A novel dextransucrase efficient in synthesizing oligosaccharides was designed. We constructed the truncation mutant DSR-S1-ΔA (residues 1-3087bp) by deleting the 1494bp fragment of the C-terminal. The novel enzyme (MW: 110kDa) loss activity, when sucrose was used as only substrate. After adding an acceptor, DSR-S1-ΔA was fully activated but with heavily impaired polysaccharide synthesis ability. Instead, the enzyme produced a large amount of oligosaccharides. DSR-S1-ΔA showed transglycosylation for synthesizing more oligosaccharides of lower degree of polymerization (DP) with different acceptors, and it also improved the selection range of dextransucrase acceptor response to acceptors. The enzyme developed in this study can be applied in glycodiversifcation studies.
Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu(+2) and Cd(+2) ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.
Programmed cell clearance is a highly regulated physiological process of elimination of dying cells that occurs rapidly and efficiently in healthy organisms. It thus ensures proper development as well as homeostasis. Recent studies have disclosed a considerable degree of conservation of cell clearance pathways between nematodes and higher organisms. The externalization of the anionic phospholipid phosphatidylserine (PS) has emerged as an important "eat-me" signal for phagocytes and its exposition on apoptotic cells is controlled by phospholipid translocases and scramblases. However, there is mounting evidence that PS exposure occurs not only in apoptosis, but may also be actively expressed on the surface of cells undergoing other forms of cell death including necrosis; PS is also expressed on the surface of engulfing cells. Additionally, PS may act as a "save-me" signal during axonal regeneration. Here we discuss mechanisms of PS exposure and its recognition by phagocytes as well as the consequences of PS signaling in nematodes and in mammals.
Hensen's node is the "organizer" of the avian and mammalian early embryo. It has many functions, including neural induction and patterning of the ectoderm and mesoderm. Some of the signals responsible for these activities are known but these do not explain the full complexity of organizer activity. Here we undertake a functional screen to discover new secreted factors expressed by the node at this time of development. Using a Signal Sequence Trap in yeast, we identify several candidates. Here we focus on Calreticulin. We show that in addition to its known functions in intracellular Calcium regulation and protein folding, Calreticulin is secreted, it can bind to BMP4 and act as a BMP antagonist in vivo and in vitro. Calreticulin is not sufficient to account for all organizer functions but may contribute to the complexity of its activity.
Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae.
Critical hand ischemia (CHI) can be devastating and may result in amputation. Distal vessel calcification has been shown to be a major factor in causing CHI. Atherectomy in the upper extremities is not typically considered due to the small anatomy; however, the Diamondback 360° Peripheral Orbital Atherectomy System (OAS) (Cardiovascular Systems, Inc.) can access treatment areas with a reference vessel diameter of 1.5mm.
A retrospective, observational, single center (Merit Health Center, Jackson, MS) analysis of 11 CHI patients with calcific disease of the radial artery treated with orbital atherectomy (OAS) was completed. Demographics and procedural to 30-day outcomes were assessed.
All patients had good blood flow to the hand after intervention and none experienced complications during or immediately post-procedure. At 30-days the freedom from revascularization and amputation was 100%, and all the wounds were healed. The following important principles were followed during the use of OAS for CHI: (1) ACT was therapeutic (~250s); (2) Gentle wire manipulation; (3) Utilization of a small OAS crown (1.25mm); (4) Aggressive vasodilator use-given through the exchange catheter; (5) Angioplasty balloon was matched to the size of the vessel and long and low pressure inflations were completed.
Critical hand ischemia can be treated with endovascular techniques. Obtaining good outflow to the fingers is critical for wound healing and preventing amputation. Orbital atherectomy is a useful tool in preparing vessels for balloon angioplasty; particularly in cases where calcification is present.
The magnitude of the clinical benefits produced by inhibitors of the renin-angiotensin system in heart failure has been modest, possibly because of the ability of renin-angiotensin activity to escape from suppression during long-term treatment. Efforts to intensify pharmacological blockade by use of dual inhibitors that interfere with the renin-angiotensin system at multiple sites have not yielded consistent incremental clinical benefits, but have been associated with serious adverse reactions. By contrast, potentiation of endogenous compensatory vasoactive peptides can act to enhance the survival effects of inhibitors of the renin-angiotensin system, as evidenced by trials that have compared angiotensin-converting enzyme inhibitors with drugs that inhibit both the renin-angiotensin system and neprilysin. Several endogenous vasoactive peptides act as adaptive mechanisms, and their augmentation could help to broaden the benefits of renin-angiotensin system inhibitors for patients with heart failure.
Many factors enter into the decision by the Food and Drug Administration (FDA) to approve a new drug for use by physicians and other health care providers in treating diseases. Initially, the FDA authority was restricted to issues of safety and only later did the documentation of efficacy become part of the review process required for approval. However, all drugs have the potential for causing harm at some dose level to all and at lower doses in certain patients with vulnerability to the particular pharmacology of the agent. As new drugs have been designed to manage disorders that are uncommon, but of significant consequence, they may have adverse effects that are acceptable only because they are so uniquely beneficial to these specific conditions. The risk of these adverse effects may be acceptable since the benefit can outweigh the harm in most patients and the adversity can be predicted and managed. The approval of this category of drugs has grown rapidly since definition of a mechanism of action to manage and modify the risk has been provided by a process known as known as Risk Evaluation and Mitigation Strategy or "REMS." In 2007, the Food and Drug Administration Amendments Act (FDAAA) allowed the FDA to require postmarketing studies and the authority to mandate the implementation of a REMS for drugs with efficacy but documented potential for harm. Two relatively new drugs useful in the management of severe elevations of low-density lipoprotein cholesterol have been approved under a requirement for a REMS. These are lomitapide, an inhibitor of microsomal triglyceride transfer protein and mipomersen, an antisense oligonucleotide which reduces the synthesis of apolipoprotein B.
Fatigue is common in patients with JIA and affects daily life negatively. We assessed the presence and severity of fatigue in patients with JIA, including factors presumed associated with fatigue (e.g., disease activity, disability, pain, physical activity, exercise capacity, and self-efficacy), and whether fatigue is related to participation in physical education classes, school attendance, and sports frequency.
The current study used baseline data of 80 patients with JIA (age 8-13) who participated in an intervention aimed at promoting physical activity. Primary outcome measurements were fatigue, assessed using the Pediatric-Quality-of-Life-Inventory (PedsQl)-Fatigue-scale and energy level assessed using a VAS scale. Other outcome measurements were disease activity (VAS Physician Global Assessment Scale), disability (Childhood Health Assessment Questionnaire), physical activity (accelerometer), exercise capacity (Bruce treadmill test), self-efficacy (Childhood Arthritis Self-Efficacy Scale), and participation (self-report).
Sixty percent of patients with JIA suffered from daily low-energy levels; 27% suffered from very low-energy levels more than half the week. Low energy levels were best predicted by disability and low physical activity. Fatigue measured with the PEDsQL was higher compared to the control-population. Disability and low self-efficacy were main predictors of fatigue. Self-efficacy was a predictor of fatigue but did not act as moderator. Fatigue was a predictor for sports frequency but not for school attendance.
Fatigue is a significant problem for JIA patients. Interventions aimed at reducing perceived disability, stimulating physical activity, and enhancing self-efficacy might reduce fatigue and thereby enhance participation.
Trial number ISRCTN92733069.
The implementation of nutrition policies in schools has been recommended as a strategy to improve child dietary intake. Internationally, research suggests that the majority of schools do not implement these policies. In New South Wales (NSW), Australia, the NSW Healthy School Canteen Policy requires that school canteens prohibit the sale of 'red' foods (i.e. foods that are typically nutrient poor and high in energy, such as confectionary and deep-fried foods) and 'banned'drinks (i.e. soft drinks); and that the majority of items on the menu are 'green' (i.e. foods that are good sources of nutrients, such fruits, vegetables and lean meats). This study examined the impact of a multicomponent audit and feedback intervention on schools' implementation of the NSW Healthy School Canteen Policy. A secondary aim was to assess the impact of the intervention on menu composition.
This study was a parallel group randomised controlled trial with 72 rural and remote primary schools (36 interventions, 36 controls) located in one region within NSW, Australia. Intervention schools received an initial face to face contact and up to four cycles of audit and feedback (consisting of a menu audit, written feedback report and telephone feedback) over a 12-month period. The primary trial outcomes were the proportion of schools with a canteen menu that had: i) no 'red' foods or 'banned' drinks; and ii) >50% 'green' items, as assessed via standardised menu audits undertaken by trained dietitians. For each primary outcome, between-group differences were assessed using Fisher's exact test under an intention to treat approach.
There was insufficient evidence to conclude the intervention had a positive impact on the proportion of intervention schools with no 'red' or 'banned' items on their menu (RR = 2.8; 95% CI: 0.9 to 8.9; p = 0.0895), or on the proportion of intervention schools with more than 50% 'green' items (RR = 1.5; 95% CI: 0.7 to 3.2; p = 0.2568). These findings remained non-significant in the multiple imputation analyses. Intervention schools were significantly more likely to have a lower percentage of 'red' items (p-value: 0.007) and a higher percentage of 'green' items on the menu (p-value: 0.014). This remained statistically significant in the multiple imputation analyses for 'red items' (p-value: 0.0081) but not for 'green' items (p-value: 0.0910).
While there was insufficient statistical evidence to suggest that this multicomponent audit and feedback intervention was effective in improving primary schools' compliance with a healthy canteen policy, the intervention demonstrated some positive impact in reducing the availability of 'red' items on the menu.
This trial was prospectively registered with the Australian New Zealand Clinical Trials Registry ( ACTRN12613000543785 ). Registered 15th May 2013.
The recent finding of asbestos fibres in drinking water (up to 700.000 fibres/litres) in Tuscany (Central Italy) leads to concerns about health risks in exposed communities. Exposure to asbestos has been linked with cancer at several levels of the gastrointestinal tract, and it has been documented, in an animal model, a direct cytotoxic effect of asbestos fibres on the ileum. It has been recently described a possible link between asbestos and intrahepatic cholangiocarcinoma, and asbestos fibres have been detected in humans in histological samples from colon cancer and in gallbladder bile. Taken together, these findings suggest the possibility of an enterohepatic translocation of asbestos fibres, alternative to lymphatic translocation from lungs. In animal models, asbestos fibres ingested with drinking water act as a co-carcinogen in the presence of benzo(a) pyrene and, according to the International Agency for Research on Cancer (IARC ), there is evidence pointing to a causal effect of ingested asbestos on gastric and colorectal cancer. The risk seems to be proportional to the concentration of ingested fibres, to the extent of individual water consumption, to exposure timing, and to the possible exposure to other toxics (i.e., benzo(a)pyrene). Furthermore, the exposure to asbestos by ingestion could explain the epidemiological finding of mesothelioma in subjects certainly unexposed by inhalation. In conclusion, several findings suggest that health risks from asbestos could not exclusively derive from inhalation of fibres. Health hazards might also be present after ingestion, mainly after daily ingestion of drinking water for long periods. In Italy, a systemic assessment of the presence of asbestos fibres in drinking water is still lacking, although asbestos-coated pipelines are widely diffused and still operating. Despite the fact that the existence of a threshold level for health risks linked to the presence of asbestos in drinking water is still under debate, the precautionary principle should impose all possible efforts in order to revise health policies concerning this topic, and a systematic monitoring of drinking water to quantify the presence of asbestos is certainly needed in all regions. Further epidemiological studies aimed to the identification of exposed communities and to an adequate health risk assessment in their specific geographical areas are urgently needed.
Health professionals face serious legal risks if they act as go-betweens for researchers seeking participants for clinical trials, the RCN Research Society's conference was told. A study has shown that patients.
Two recent reports offer an interesting contrast regarding provision of mental health services. One supports the idea of legislation to improve services, whereas the other calls upon services to get their act together.
Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins.
The purpose of this study was to investigate the effect of knee extension/flexion and fatigue on muscle co-activation of the Rectus Femoris (RF) and Biceps Femoris (BF) during the eggbeater kick. Ten national level male water polo players executed eggbeater kicks at maximum effort for the duration of the test. The eggbeater kick cycle was divided into four phases (FLX1, FLX2, EXT1, EXT2). Surface electromyographs were recorded from RF and BF. EMG activity normalized to the maximum voluntary isometric contraction, muscle co-activation (CCI) and angular velocity (AV) of the right and left knee were calculated. Highest levels of RCCI and LCCI were observed during final phase of flexion (FLX2) and initial phase of extension (EXT1) (p<0.05). FLX2 and final phase of extension (EXT2) revealed the highest AV during the cycle. A decrease in CCI was observed with fatigue for FLX2 while AV was reduced for all phases. During the cycle RF and BF act as agonist/antagonist to accelerate and decelerate knee flexion/extension. The high AV and low CCI levels observed for EXT2 might increase joint instability and consequent risk of injury. This knowledge provides a better understanding of the mechanisms involved in stabilizing and controlling the knee during underwater movement.
After sudden traumatic brain injuries, secondary injuries may occur during the following days or weeks, which leads to the accumulation of reactive oxygen species (ROS). Since ROS exacerbate brain damage, it is important to protect neurons against their activity. Zinc finger protein 179 (Znf179) was shown to act as a neuroprotective factor, but the regulation of gene expression under oxidative stress remains unknown. In this study, we demonstrated an increase in Znf179 protein levels in both in vitro model of hydrogen peroxide (H2O2)-induced ROS accumulation and animal models of traumatic brain injury. Additionally, we examined the sub-cellular localization of Znf179, and demonstrated that oxidative stress increases Znf179 nuclear shuttling and its interaction with specificity protein 1 (Sp1). Subsequently, the positive autoregulation of Znf179 expression, which is Sp1-dependent, was further demonstrated using luciferase reporter assay and green fluorescent protein (GFP)-Znf179-expressing cells and transgenic mice. The upregulation of Sp1 transcriptional activity induced by the treatment with nerve growth factor (NGF) led to an increase in Znf179 levels, which further protected cells against H2O2-induced damage. However, Sp1 inhibitor, mithramycin A, was shown to inhibit NGF effects, leading to a decrease in Znf179 expression and lower cellular protection. In conclusion, the results obtained in this study show that Znf179 autoregulation through Sp1-dependent mechanism plays an important role in neuroprotection, and NGF-induced Sp1 signaling may help attenuate more extensive (ROS-induced) damage following brain injury.
Mosquitoes act as vectors of key pathogens and parasites. Plant essential oils have been recognized as important sources of biopesticides, which do not induce resistance and have limited toxic effects on human health and non-target organisms. In this research, we evaluated the larvicidal and oviposition deterrence activity of Hedychium larsenii essential oil (EO) and its major compounds ar-curcumene and epi-β-bisabolol. Both molecules showed high toxicity against early third instars of Anopheles stephensi (LC50=10.45 and 14.68µg/ml), Aedes aegypti (LC50=11.24 and 15.83µg/ml) and Culex quinquefasciatus (LC50=12.24 and 17.27µg/ml). In addition, low doses of ar-curcumene and epi-β-bisabolol were effective as oviposition deterrents against the three tested mosquito species. Notably, the acute toxicity of H. larsenii oil and its major compounds against the mosquito biocontrol agent Poecilia reticulata was low, with LC50 higher than 1500ppm. Overall, the results from this study revealed that ar-curcumene and epi-β-bisabolol from the H. larsenii oil can be considered for the development of novel and effective mosquito larvicides.
Voltage-gated inward Ca(2+) currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca(2+) concentration. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of CaV1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla).
In this study, we generated stable CaV1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis.
The knockdown of CaV1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules.
Our results suggest that CaV1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs.
Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase (mTcTXNPx) which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mTcTXNPx was analyzed by the Western blotting technique using polyclonal antibody anti mTcTXNPx obtained from a rabbit immunized with the mTcTXNPx recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5kDa polypeptide of mTcTXNPx did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5kDa mTcTXNPx is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.
The present study examined the effects of a 6-week whole body vibration training (WBVT) regimen on heart rate variability (HRV) and body composition in obese Hispanic postmenopausal women. Participants were randomly assigned to either WBVT (n=13) or non-exercising control group (n=14). HRV and body composition were measured before and after 6 weeks. There was a significant group x time interaction (P<0.05) for heart rate, sympathovagal balance and body fat percentage (BF%) such that all significantly decreased (P<0.05); and R-R intervals which significant increased (P<0.05) following WBVT compared to no changes after control. The changes in sympathovagal balance were correlated with changes in BF% (r=0.63, P<0.05). Our findings indicate that WBVT improves HRV and BF% in obese Hispanic postmenopausal women. The improvement in BF% partially explained the decrease in sympathovagal balance. Since obese and older individuals are at increased risk of developing cardiovascular diseases, they could potentially benefit from WBVT.
Physical inactivity is common in persons with multiple sclerosis (MS), but there is very little known about the pattern and predictors of changes in physical activity over time.
This study examined changes in moderate-to-vigorous physical activity (MVPA) over a 30-month time period and the demographic and clinical predictors of such changes in relapsing-remitting MS (RRMS).
269 persons with MS wore an accelerometer for a 7-day period and completed a demographic/clinical scale every 6 months over a 30-month period. Data were analyzed using latent class growth modeling (LCGM).
LCGM identified a two-class model for changes in levels of MVPA over time. Class 1 involved higher initial levels of MVPA and linear decreases in MVPA over time, whereas Class 2 involved lower initial levels of MVPA and linear increases in MVPA over time. LCGM further indicated that males were more likely (OR=5.8, p<.05) and those with higher disability status were less likely (OR=0.51, p<.05) to belong to Class 1 than Class 2.
Levels of MVPA change over time in persons with RRMS and the pattern of change suggests that behavioral physical activity interventions for persons with MS might target men and those with lower disability.
We investigated the differences and over time changes in recommended physical activity among foreign-born (FB) from English speaking countries (ESC) and non-English speaking countries (NESC) relative to native-born (NB) Australians, and whether the association between nativity and duration of residence (DoR) and physical activity is mediated by English language proficiency, socio-economic status and social engagement/membership.
This study applies multilevel group-mean-centred mixed (hybrid) logistic regression models to twelve waves of longitudinal data (12,634 individuals) from the Household, Income and Labour Dynamics in Australia survey with engagement in physical activities for more than three times a week as the outcome variable.
Immigrants from ESC had higher odds of physical activity, while immigrants from NESC had significantly lower odds of physical activity than NB Australians, after adjusting for covariates. There was no evidence that these differences changed by DoR amongst immigrants from NESC, whereas ESC immigrants had higher odds of physical activity when their DoR was more than 20 years. We also found a mediating role of English language proficiency on immigrants physical activities.
Appropriate health promotion interventions should be implemented to foster physical activities among NESC immigrants, considering English language proficiency as an important factor in designing interventions.
Approximately 17.4% of people in Mexico self-report physical activity levels below the World Health Organization's guidelines and an average sedentary time of 16 hours per day.1 Low physical activity has been associated with non-communicable disease risk factors and previous research suggests that urbanicity might be an important determinant of physical activity. The aim of this study was to measure urbanicity in Mexico and assess if it is associated with physical activity and sitting time.
A sample of 2,880 men and 4,211 women aged 20-69 was taken from the 2012 Mexico National Health and Nutrition Survey and multivariable linear regression models were used to examine the association between physical activity, sitting time and urbanicity; adjusting for sex, education level, socioeconomic status and Body Mass Index. The urbanicity score and the seven urbanicity sub-scores were estimated from the CENSUS 2010.
The sub-scores of demographic, economic activity, diversity and communication were negatively associated with physical activity. Sitting time was positively associated with the overall urbanicity, and the demographic and health sub-scores.
There was evidence of associations between urbanicity and physical activity in Mexico.
To date, there is no physical activity (PA) questionnaire with convergent and construct validity for the oldest-old. The aim of the present study was to investigate the validity of questionnaire-assessed PA in comparison with objective measures determined by uniaxial and triaxial accelerometers and physical performance measures in the oldest-old.
Participants were 155 elderly (mean age 90 years) who were examined at the university and agreed to wear an accelerometer for 7 days in the 3-year-follow-up survey of the Tokyo Oldest-Old Survey of Total Health. Fifty-nine participants wore a uniaxial and triaxial accelerometer simultaneously. Self-rated walking, exercise, and household PA were measured using a modified Zutphen PA Questionnaire (PAQ). Several physical performance tests were done, and the associations among PAQ, accelerometer-assessed PA, and physical performances were compared by Spearman's correlation coefficients.
Significant, low to moderate correlations between PA measures were seen on questionnaire and accelerometer assessments (rho = 0.19 to 0.34). Questionnaire-assessed PA measure were correlated with a range of lower extremity performance (rho = 0.21 to 0.29).
This PAQ demonstrated convergent and construct validity. Our findings suggest that the PAQ can reasonably be used in this oldest-old population to rank their PA level.
To examine associations between physical activity (PA) and depressive symptoms among adults with type 2 diabetes mellitus (Type 2 DM), and whether associations varied according to weight status.
Diabetes MILES - Australia is a national survey of adults with diabetes, focused on behavioral and psychosocial issues. Data from 705 respondents with Type 2 DM were analyzed, including: demographic and clinical characteristics, PA (IPAQ-SF), depressive symptoms (PHQ-9), and BMI (self-reported height and weight). Data analysis was performed using ANCOVA.
Respondents were aged 59±8 years; 50% women. PA was negatively associated with depressive symptoms for the overall sample (ηp(2)= 0.04, p<0.001) and all weight categories separately: healthy (ηp(2)0.11, p=0.041, overweight (ηp(2)= 0.04, p =0.025) and obese (ηp(2)=0.03, p=0.007,). For people who were healthy (BMI 18.5-24.9) or overweight (BMI 25-29.9), high amounts of PA were significantly associated with fewer depressive symptoms; for adults who were obese (BMI ≥ 30) however, both moderate and high amounts were associated with fewer depressive symptoms.
PA is associated with fewer depressive symptoms among adults with Type 2DM, however the amount of PA associated with fewer depressive symptoms varies according to weight status. Lower amounts of PA might be required for people who are obese to achieve meaningful reductions in depressive symptoms compared to those who are healthy weight or overweight. Further research is needed to establish the direction of the relationship between PA and depressive symptoms.
Sedentary behavior (SB) increases throughout adolescence, and is associated with adverse health outcomes.
Examine psychosocial and friend influences on SB and screen time in adolescents using a mixed-methods design.
108 middle and high school students wore accelerometers to measure objective SB, completed screen time and psychosocial questionnaires, and nominated friends to complete activity questionnaires. Focus groups centered around influences on SB behavior. Regression analyses and NVivo software analyzed quantitative and qualitative data.
Screen time was associated with greater screen time enjoyment, lower self-efficacy, and friends' screen time (r(2)=.21, p<.0001). Friends influenced whether adolescents engaged in screen time behaviors, with active friends encouraging less screen time.
Active friends influenced adolescents to engage in less SB. Interventions should place an emphasis on encouraging less screen time, and providing opportunities for adolescents and their friends to engage in activities that promote physical activity rather than SB.
This study examined associations between sedentary time, physical activity (PA), and executive function among youth participating in the Study of Early Child Care and Youth Development.
>: Sedentary time and PA (light, moderate, vigorous, and moderate-to-vigorous (MVPA)) were objectively assessed at 9 and 15 yrs, while executive function (inhibition, working memory, and fluid intelligence) were assessed at 15 yrs. Regression models were used to examine associations.
Sedentary time at 9 yrs predicted fluid intelligence at 15 yrs (B = 0.031), whereas increased sedentary time from 9 to 15 yrs predicted higher inhibition (B = 0.003), working memory (B = 0.074), and fluid intelligence (B = 0.029). Relatively lower levels of working memory at 15 yrs were predicted from increased levels of light PA, moderate PA, and MVPA from 9 to 15 yrs (B = -0.075, -0.293, and -0.173, respectively). At 15 yrs, inhibition, working memory, and fluid intelligence were significantly associated with sedentary time (B = 0.003, 0.055, and 0.045, respectively).
Childhood sedentary time and PA may affect executive function at 15 yrs; however, prospective studies are needed to examine the concurrent change in both sedentary time and PA with executive function.
Few studies have examined the associations among social cohesion, physical activity, and obesity in older adults. This study explored the influences of social cohesion and leisure-time physical activity on obesity in older adults, and tested whether these relationships varied by race/ethnicity and income level.
A cross-sectional analysis of adults in the 2013 National Health Interview Study (NHIS) who were over 65 years of age (N=7714) was used. Logistic regressions were performed to examine the impacts of social cohesion and physical activity on obesity, and the relative risks (RR) were reported.
The median age was 73 years old, and 59.8% of respondents were female. 23.8% met the recommended level of moderate physical activity. Neighborhood social cohesion was not associated with obesity for older adults. Meeting the recommended level of vigorous physical activity was related to a lower probability of obesity only for older Hispanic adults (RR=0.41, 95% CI: 0.31-0.50), while older adults were less likely to be obese if they met the recommended level of moderate physical activity.
Increasing the level of physical activity may profoundly reduce the probability of obesity for older adults. Moreover, the results implied the need for future physical activity interventions for minorities.
This study evaluated the feasibility and efficacy of the Aging(Plus) intervention program. Aging(Plus) is an 8-week multi-component motivational program which promotes increased physical activity by targeting adults' negative views on aging (NVOA) and perceptions of control, two known psychological barriers to physical exercise. 62 adults, ages 50-82 years, participated in this feasibility study. We assessed NVOA, perceptions of control, and physical activity level at baseline (Week 0), immediate posttest (Week 4), and delayed posttest (Week 12). High attendance rates, low attrition, and positive participant feedback indicated that the program had high acceptability. Repeated measures multivariate analyses of variance (RM-MANOVA) showed statistically significant and substantively meaningful improvements in NVOA, control beliefs, and physical activity from pretest to immediate and delayed posttest. The program effects did not differ between those younger or older than age 65. These findings provide promising support for the feasibility and efficacy of the Aging(Plus) program.
There are several well-known risk factor monitoring systems, but few examples of comprehensive surveillance systems designed specifically to inform physical activity (PA) policy. This paper examines the utility of Canada`s Physical Activity and Sport Monitoring System in guiding policy and practice.
Indicators were determined in conjunction with government, non-governmental associations and academics. Serial measures were collected from representative population (telephone interviews, n=4,000-11,000) and setting-based (postal surveys, n=1,425-4,304) surveys.
Adult PA was higher in 2014 (47%) than 1998 (37%). The prevalence of knowledge about sufficient PA to meet national guidelines increased (31% to 57%). Most adults (66%) reported having many safe places to walk locally. Having policies to encourage walking and cycling when redeveloping communities increased by community size (5% to 37%). PA promotion was available in 10-15% of workplaces. Most parents (64%) provided transportation to support their child's PA. The prevalence of policies mandating daily PE increased 2001-2011 (36% to 55%), as did having no policy to hire qualified PE teachers (25% to 34%).
Canada's surveillance system has provided information for guiding policy planning, resource allocation, setting and tracking national goals, assessing changes in PA determinants, and evaluating national campaigns, naturally occurring experiments, and innovative policies.
Physical education (PE) is mandated in most states, but few studies of PE in private schools exist.
We assessed selected PE policies and practices in private secondary schools (grades 6-12) in California using a 15-item questionnaire related to school characteristics and their PE programs.
Responding schools (n=450; response rate, 33.8%) were from 37 counties. Most were coeducational (91.3%) and had a religious affiliation (83%). Secular schools had more PE lessons, weekly PE min, and smaller class sizes. Most schools met guidelines for class size, but few met national recommendations for weekly PE minutes (13.7%), not permitting substitutions for PE (35.6%), and programs being taught entirely by PE specialists (29.3%).
Private schools, which serve about five million US children and adolescents, may be falling short in providing quality PE. School stakeholders should encourage adoption and implementation of policies and practices that abide by professional guidelines and state statutes.
This study examined associations between physical activity (recreational, non-recreational) and sleep duration among a nationally representative diverse sample of U.S. adults.
We used cross-sectional data from 9,205 National Health and Nutrition Examination Survey 2007-2012 participants aged 20-65 years who identified as White, Black, or Hispanic. Activity (i.e., recreation, occupation, and transportation activity) was categorized into quartiles. Sleep duration was categorized as short (≤6 hours/night) or normal (>6 to ≤9 hours/night). Logistic regression was used to estimate associations of activity with sleep duration.
Recommended levels of recreation activity and moderate levels of transportation activity were associated with normal sleep duration (Odds Ratio (OR): = 1.33, 95% Confidence Interval (CI) = 1.08, 1.65; OR= 1.28, 95% CI= 1.02, 1.62, respectively). High occupation physical activity was associated with shorter sleep duration (OR = 0.59, 95% CI = 0.49, 0.71). Differences were observed by race/ethnicity in associations of recreation and occupation activity with sleep duration.
White individuals who engaged in some recreation activity, relative to being inactive, had more favorable sleep duration; whereas, high levels of occupation activity were associated with worse sleep duration among White and Black individuals. Physical activity was not associated with sleep duration among Hispanics.
Physical activity during pregnancy has numerous benefits, but the influence on the duration of labor is unclear. We investigated the influence of habitual physical activity during late pregnancy on the duration of labor, with consideration of previous delivery experience and the stage of labor.
This prospective study included 103 women (48 nulliparous, 55 multiparous) in late pregnancy. Habitual physical activity was evaluated using the Baecke physical activity questionnaire (BQ). Women were divided into a high activity group (HA) and a low activity group (LA) based on their median total BQ score. Data pertaining to the duration of labor were obtained from the birth records after delivery.
In multiparous women, the duration of the second stage of labor was significantly shorter in the HA group than in the LA group [median (range): HA, 11 min (1-102 min); LA, 20 min (4-175 min); p < 0.05]. The significant difference persisted after adjusting for confounding variables (standardized β = -0.34; p = 0.01). In nulliparous women, there were no significant differences in duration of labor between groups.
Higher physical activity in multiparous women during late pregnancy might positively influence the duration of the second stage of labor.
Carbon nanodots (CNDs)@BaSO4 hybrid phosphors are fabricated in an easy and low-cost process by sequentially assembling Ba(2+) and SO4(2-) ions onto the surface of carbon nanodots through electrostatic attraction. CNDs act as the nucleus to attract these reactive ions and provide the luminescent centers in the hybrid phosphors. This strategy is versatile for a variety of negatively charged CNDs with different emission colors. The advantage of the resultant hybrid phosphors is that their luminescence exhibits excellent thermal and photostability, as well as remarkable resistance to strong acid/alkali and common organic solvents. These merits allow for the fabrication of CNDs-based light-emitting diodes using the CNDs@BaSO4 hybrid phosphors as a color conversion layer.
The first example of nickel-catalyzed hydroamination of allenes is reported. The new cationic [(3-iminophosphine)nickel(allyl)]+ catalysts have been fully characterized and act regioselectively in the catalytic hydroamination of allenes with secondary amines at room temperature.
HPV positive patients suffering from head and neck cancer benefit from intensified radiotherapy when applied as a primary as well as an adjuvant treatment strategy. However, HPV negative patients treated with surgery and adjuvant radiotherapy lack validated prognostic biomarkers. It is therefore important to define prognostic biomarkers in this particular patient population. Especially, ´high-risk groups´ need to be defined in order to adapt treatment protocols. Since dysregulation of the sonic hedgehog pathway plays an important role in carcinogenesis, we aimed to assess whether members of the sonic hedgehog-signaling pathway may act as prognostic factors in patients with HPV negative head and neck squamous cell carcinoma.
In this prospective study, pretreatment tumor biopsies of patients with head and neck squamous cell carcinoma were taken during panendoscopy (2005 to 2008). All patients were treated with surgery and postoperative radiotherapy. After assessment of HPV and p16 status, protein expression profiles of the Sonic hedgehog-signaling pathway were determined by immunohistochemistry and tissue microarray analyses in 36 HPV negative tumor biopsies. Expression profiles of Sonic hedgehog, Indian hedgehog, Patched, Smoothened, Gli-1, Gli-2 and Gli-3 were correlated with patients´ clinical data, local-control rate, disease-free as well as overall survival. Data from The Cancer Genome Atlas databank were used for external validation of our results.
Gli-1 (p = 0.04) and Gli-2 (p = 0.02) overexpression was significantly linked to improved overall survival of HPV negative patients. Gli-2 (p = 0.04) overexpression correlated significantly with prolonged disease-free survival. Cox-multivariate analysis showed that overexpression of Gli-2 correlated independently (HR 0.40, 95% CI 0.16-0.95, p = 0.03) with increased overall survival.
Gli-1 and Gli-2 overexpression represents a substantial prognostic factor for overall and disease-free survival in patients with locally advanced HPV negative head and neck cancer undergoing surgery and postoperative radiotherapy.
N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.
Indoor air pollution is an increasing health concern, especially in enclosed environments such as underground subway stations because of increased global usage by urban populations. This study measured the indoor air quality of underground platforms at 10 metro stations of the Taipei Rapid Transit system (TRTS) in Taiwan, including humidity, temperature, carbon monoxide (CO), carbon dioxide (CO₂), formaldehyde (HCHO), total volatile organic compounds (TVOCs), ozone (O₃), airborne particulate matter (PM10 and PM2.5), bacteria and fungi. Results showed that the CO₂, CO and HCHO levels met the stipulated standards as regulated by Taiwan's Indoor Air Quality Management Act (TIAQMA). However, elevated PM10 and PM2.5 levels were measured at most stations. TVOCs and bacterial concentrations at some stations measured in summer were higher than the regulated standards stipulated by Taiwan's Environmental Protection Administration. Further studies should be conducted to reduce particulate matters, TVOCs and bacteria in the air of subway stations.
Implementation of the Affordable Care Act (2010) enabled more than 30 million people to have new access to primary care services. On the basis of current utilization patterns, demand for primary care providers is expected to grow more rapidly than physician supply. This imbalance is expected to worsen, as the aging population requires more health care resources. In addition, more patients are requiring critical care services and physician numbers are not keeping with this growing need. Restrictions on resident physician practice hours have impacted inpatient care as well. Revisiting outdated state practice laws, and considering Full Practice Authority (FPA) for nurse practitioners (NP), is needed for improving access to care while creating greater flexibility for development of patient-centered health care homes and other emerging models of care delivery. Currently, 21 states and the District of Columbia have adopted FPA for NPs, with 15 more states planning legislation in 2016. Allowing FPA and Prescriptive Authority (PA) enables NPs to become more efficient and effective patient care team members. However, physician resistance to FPA and PA presents barriers to implementation.
The healthcare provider landscape is rapidly changing. Given the imminent retirement of baby boomer physicians, implementation of the Affordable Care Act, and the increased utilization of health care services by an ever-aging population, the supply of providers cannot keep pace with the demand for services. This has led to an increased utilization of advanced clinical practitioners (ACPs). This article shows how one large highly-matrixed health care system approached identifying this workforce, and how thought leaders worked collaboratively with physicians, administrators, and ACPs to meet a growing demand for providers. Carolinas HealthCare System developed a 3-pronged approach to this opportunity. The development of a Center for Advanced Practice was explored and implemented. This Center serves as a 2-way conduit of information and ideas between system administrators and providers. It also serves as a central source of regulatory and practice information for administrators and providers. The growing number of open ACP positions, along with the reluctance to employ novice and new graduate ACPs, led to the development of a postgraduate transition to practice fellowship program. This program's clinical tracks and curriculum are described. Finally, a collaborative effort between the health care system and a local university resulted in the local offering of an acute care nurse practitioner program, which allowed system nurses to continue their education without the need for relocation. Higher satisfaction and engagement, lower turnover, better career opportunities, more satisfied administrators, and physicians all contributed to the overwhelming success of this initiative.
Nursing executives are continually challenged to address a rapidly evolving health care system. Every aspect of care delivery is being scrutinized, yet the constant requirement remains assurance of high-quality, highly reliable care that is cost-effective and easily accessible. The nursing workforce remains visible in traditional roles as well as in new and innovative capacities, whereas nurse executives experience expanded realms of focus and responsibility. As health systems continue to adapt to the changing environment, the ability to leverage the role and scope of the advanced practice registered nurse (APRN) as a health care provider is emerging as an essential business and care strategy. While the APRN role is more commonly accepted in primary care, ambiguity persists in how to best use the role along the continuum of care to achieve the cost-effective solutions required in today's health care environment. Successful health care systems of today and tomorrow are those that act boldly in a strategic direction. Data-driven decision making is key to meaningful change, whether clinical or operational. The need to develop high-functioning, integrated provider teams is apparent and immediate. Nurse executives and their nursing leadership colleagues must lead with evidence in hand to create infrastructures that support true integration, optimization, and engagement of all members of the provider team to better serve our patients and communities.
Polymorphism is the ability of a solid material to exist in more than one form or crystal structure and this is of interest in the fields of crystal engineering and solid-state chemistry. 2,2'-(Disulfanediyl)dibenzoic acid (also called 2,2'-dithiosalicylic acid, DTSA) is able to form different hydrogen bonds using its carboxyl groups. The central bridging S atoms allow the two terminal arene rings to rotate freely to generate various hydrogen-bonded linking modes. DTSA can act as a potential host molecule with suitable guest molecules to develop new inclusion compounds. We report here the crystal structures of three new polymorphs of the inclusion compound of DTSA and trimethylamine, namely trimethylazanium 2-[(2-carboxyphenyl)disulfanyl]benzoate 2,2'-(disulfanediyl)dibenzoic acid monosolvate, C3H10N(+)·C14H9O4S2(-)·C14H10O4S2, (1), tetrakis(trimethylazanium) bis{2-[(2-carboxyphenyl)disulfanyl]benzoate} 2,2'-(disulfanediyl)dibenzoate 2,2'-(disulfanediyl)dibenzoic acid monosolvate, 4C3H10N(+)·2C14H9O4S2(-)·C14H8O4S2(2-)·C14H10O4S2, (2), and trimethylazanium 2-[(2-carboxyphenyl)disulfanyl]benzoate, C3H10N(+)·C14H9O4S2(-), (3). In the three polymorphs, DTSA utilizes its carboxyl groups to form conventional O-H...O hydrogen bonds to generate different host lattices. The central N atoms of the guest amine molecules accept H atoms from DTSA molecules to give the corresponding cations, which act as counter-ions to produce the stable crystal structures via N-H...O hydrogen bonding between the host acid and the guest molecule. It is noticeable that although these three compounds are composed of the same components, the final crystal structures are totally different due to the various configurations of the host acid, the number of guest molecules and the inducer (i.e. ancillary experimental acid).
Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M1 and M3, and (3) memory retrieval is more impaired by inactivation of multiple M1-M4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach.
The sensory properties of a reward-paired cue (a conditioned stimulus; CS) may impact the motivational value attributed to the cue, and in turn influence the form of the conditioned response (CR) that develops. A cue with multiple sensory qualities, such as a moving lever-CS, may activate numerous neural pathways that process auditory and visual information, resulting in CRs that vary both within and between individuals. For example, CRs include approach to the lever-CS itself (rats that "sign-track"; ST), approach to the location of reward delivery (rats that "goal-track"; GT), or an "intermediate" combination of these behaviors. We found that the multimodal sensory features of the lever-CS were important to the development and expression of sign-tracking. When the lever-CS was covered, and thus could only be heard moving, STs not only continued to approach the lever location but also started to approach the food cup during the CS period. While still predictive of reward, the auditory component of the lever-CS was a much weaker conditioned reinforcer than the visible lever-CS. This plasticity in behavioral responding observed in STs closely resembled behaviors normally seen in rats classified as "intermediates." Furthermore, the ability of both the lever-CS and the reward-delivery to evoke dopamine release in the nucleus accumbens was also altered by covering the lever-dopamine signaling in STs resembled neurotransmission observed in rats that normally only GT. These data suggest that while the visible lever-CS was attractive, wanted, and had incentive value for STs, when presented in isolation, the auditory component of the cue was simply predictive of reward, lacking incentive salience. Therefore, the specific sensory features of cues may differentially contribute to responding and ensure behavioral flexibility.
The growth of social media and user-created content on online sites provides unique opportunities to study models of human declarative memory. By framing the task of choosing a hashtag for a tweet and tagging a post on Stack Overflow as a declarative memory retrieval problem, 2 cognitively plausible declarative memory models were applied to millions of posts and tweets and evaluated on how accurately they predict a user's chosen tags. An ACT-R based Bayesian model and a random permutation vector-based model were tested on the large data sets. The results show that past user behavior of tag use is a strong predictor of future behavior. Furthermore, past behavior was successfully incorporated into the random permutation model that previously used only context. Also, ACT-R's attentional weight term was linked to an entropy-weighting natural language processing method used to attenuate high-frequency words (e.g., articles and prepositions). Word order was not found to be a strong predictor of tag use, and the random permutation model performed comparably to the Bayesian model without including word order. This shows that the strength of the random permutation model is not in the ability to represent word order, but rather in the way in which context information is successfully compressed. The results of the large-scale exploration show how the architecture of the 2 memory models can be modified to significantly improve accuracy, and may suggest task-independent general modifications that can help improve model fit to human data in a much wider range of domains. (PsycINFO Database Record
Bacterial spoilage of food products is regulated by density dependent communication system called quorum sensing (QS). QS control biofilm formation in numerous food pathogens and Biofilms formed on food surfaces act as carriers of bacterial contamination leading to spoilage of food and health hazards. Agents inhibiting or interfering with bacterial QS and biofilm are gaining importance as a novel class of next-generation food preservatives/packaging material. In the present study, Zinc nanostructures were synthesised using Nigella sativa seed extract (NS-ZnNPs). Synthesized nanostructures were characterized hexagonal wurtzite structure of size ~24 nm by UV-visible, XRD, FTIR and TEM. NS-ZnNPs demonstrated broad-spectrum QS inhibition in C. violaceum and P. aeruginosa biosensor strains. Synthesized nanostructures inhibited QS regulated functions of C. violaceum CVO26 (violacein) and elastase, protease, pyocyanin and alginate production in PAO1 significantly. NS-ZnNPs at sub-inhibitory concentrations inhibited the biofilm formation of four-food pathogens viz. C. violaceum 12472, PAO1, L. monocytogenes, E. coli. Moreover, NS-ZnNPs was found effective in inhibiting pre-formed mature biofilms of the four pathogens. Therefore, the broad-spectrum inhibition of QS and biofilm by biogenic Zinc oxide nanoparticles and it is envisaged that these nontoxic bioactive nanostructures can be used as food packaging material and/or as food preservative.
The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure-function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure of Clostridium thermocellum arabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported. CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure of CtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes. CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis of CtAbf43A along with the other member of GH43 subfamily 16 with known structure, the Bacillus subtilis arabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.
Temperature sensitivity is found in all multicelleular organisms, as well as in most primitive life forms. The ubiquity of this temperature sensitivity is an indicator of its effects at the multicellular, cellular and molecular levels [1]. Previous studies have shown that temperature-based regulation is present in the transcriptional process [2]. RNA Thermometers, temperature-sensitive sequences, have been shown to act on heat-shock genes to regulate temperature-dependant systems in many organisms [3,4]. The goal of this study was to characterize the shifts in the functioning of these RNA Thermometers at various temperatures. In addition, using the principle of transcriptional thermoregulation, an automated temperature-responsive system stimulating inverse endothermic and exothermic enzymatic reactions for heat stabilization was proposed.
The endothermic enzymatic reaction was designated as the breakdown of urea, reflecting the function of urease, and the exothermic reaction was designated as the breakdown of hydrogen peroxide, reflecting the function of catalase [5].
The proposed system was built upon the translation of urease and the inhibition of catalase translation at higher temperatures, and the inverse at lower temperatures. As RNA Thermometers can be used only to drive transcription at higher temperatures, the installation of a lac-regulated 2-way system was suggested. This system would also provide a synthetic solution to thermoregulation and the current systems employed today. This system could be applied where the current thermoregulatory systems prove insufficient and could be further developed and optimized to replace them in the future.
With millions of adolescents becoming infected with HIV globally, it is essential that barriers to much-needed interventions are reduced for at-risk adolescents. In this article we review the legal and policy framework in South Africa for adolescent access to male circumcision. We are of the view that the framework does confer protection for adolescent boys while enabling access to male circumcision; however, we identify ambiguities and tensions that exist between the Children's Act, regulations and national guidelines. We recommend reform to further enable access by this vulnerable group to this prevention modality.
Exploration of the clinical uptake of a novel conversation partner training (CPT) programme in aphasia in 10 Dutch rehabilitation facilities and identification of its perceived facilitators and barriers in service providers, and the evaluation of the implementation methods used.
Ten rehabilitation centres took part in a multifaceted implementation of CPT over 13 months. Each centre selected two speech and language therapists to act as knowledge brokers whose role was to raise awareness of CPT in the team and to facilitate getting partners of people with aphasia into the programme. The implementation was evaluated using analysis of recruitment data and questionnaires, supplemented by consensus data and scrutiny of implementation plans.
Successful implementation was described as (1) four dyads included during the intervention period, (2) two more dyads included after the intervention period, before the end of the study, and (3) inclusion of the Partners of Aphasic clients Conversation Training (PACT) programme in a description of the logistics of local stroke care (stroke care pathway). Seven centres were successful in reaching the target inclusion of six dyads in total. Only one centre had care pathways in place. From a recruitment pool of 504 dyads, 41 dyads were recruited and 34 partners completed the implementation study of the PACT. Observed facilitators included the motivation to engage partners in the rehabilitation process and the perceived added value of PACT. The perceived barriers focused on time limitations within current systems to discuss the consequences of PACT with relevant professionals and to establish allocated time for this training within existing care routines.
The motivation of professionals to involve partners in the rehabilitation process assisted with the introduction of PACT in practice. The main barrier was time, linked to the requirement to think through integration of this innovation within existing care. Longer term evaluation would ascertain how centres sustain uptake without support. Implications for Rehabilitation The integration of a new treatment method that reaches beyond the boundaries of one group of professionals needs to be facilitated by providing time to all team members involved to discuss and think through the consequences of that approach for clinical decision making within the care trajectory of a client and his/her significant other. Partners of people with aphasia need to be properly informed about the collaborative nature of communication with a view to the longer term adjustment to living with someone with aphasia.
As part of the Healthy, Hunger-Free Kids Act, snacks, and desserts sold in K-12 schools as of the 2014-2015 school year are required to meet the "Smart Snacks" nutritional guidelines. Although studies exist in tracking progress in local and national efforts, the proportion of snack food procured by school districts compliant with the Smart Snacks standard prior to its full implementation is unknown.
We repurposed a previously untapped database, Interflex, of public bid records to examine the nutritional quality of snacks and desserts procured by school districts. We selected 8 school districts with at least 90% complete data each year during 2011-2012, 2012-2013, and 2013-2014 school years and at locations across different regions of the United States. We quantified the amount of calories and sugar of each product contained in the won bids based on available online sources and determined whether the produce complied with Smart Snack guidelines.
In all 8 districts (snack expenditure analyzed ranging from $152,000 to $4.4 million), at least 50% of snack bids were compliant with the US Department of Agriculture Smart Snacks standard during the 2013-2014 school year. Across sampled districts, we observed a general trend in lower caloric density (kcal per product) and sugar density (grams of sugar per product) over a 3-year period.
Many districts across the country have made headway in complying with the Smart Snack guidelines, though gaps remain.
Human subjects protection in healthcare contexts rests on the premise that a principled boundary distinguishes clinical research and clinical practice. However, growing use of evidence-based clinical practices by health systems makes it increasingly difficult to disentangle research from a wide range of clinical activities that are sometimes called "research on medical practice" (ROMP), including quality improvement activities and comparative effectiveness research. The recent growth of ROMP activities has created an ethical and regulatory gray zone with significant implications for the oversight of human subjects research.
We conducted six semi-structured, open-ended focus group discussions with IRB members to understand their experiences and perspectives on ethical oversight of ROMP, including randomization of patients to standard treatments.
Our study revealed that IRB members are unclear or divided on the central questions at stake in the current policy debate over ethical oversight of ROMP: IRB members struggle to make a clear distinction between clinical research and medical practice improvement, lack consensus on when ROMP requires IRB review and oversight, and are uncertain about what constitutes incremental risk when patients are randomized to different treatments, any of which may be offered in usual care. They characterized the central challenge as a balancing act, between, on the one hand, making information fully transparent to patients and providing adequate oversight, and on the other hand, avoiding a chilling effect on the research process or harming the physician-patient relationship.
Evidence-based guidance that supports IRB members in providing adequate and effective oversight of ROMP without impeding the research process or harming the physician-patient relationship is necessary to realize the full benefits of the learning health system.
Macroautophagy (hereafter referred to as autophagy) is a housekeeping process constitutively executed at basal level in all cells to promote cellular homeostasis by regulating organelle and protein turnover. However, autophagy deregulation caused by several stress factors, such as hypoxia, is prevalent in many cancers. It is now well established that autophagy can act as tumor suppressor or tumor promoter depending on tumor type, stage, and genetic context. In developed tumors, autophagy promotes the survival of cancer cells and therefore operates as a cell resistance mechanism. Emerging evidence point to the prominent role of autophagy in disabling the antitumor immune response by multiple overlapping mechanisms leading to tumor escape from immune cell attack mediated by both natural killer cells and cytotoxic T-lymphocytes. Such a role has inspired significant interest in applying anti-autophagy therapies as an entirely new approach to overcome tumor escape from immune surveillance, which constitutes so far a major challenge in developing more effective cancer immunotherapies. In this review, we will summarize recent reports describing how tumor cells, by activating autophagy, manage to hijack the immune system. In particular, we will focus on the emerging role of hypoxia-induced autophagy in shaping the antitumor immune response and in allowing tumor cells to outmaneuver an effective immune response and escape immunosurveillance. In keeping with this, we strongly believe that autophagy represents an attractive future therapeutic target to develop innovative and effective cancer immunotherapeutic approaches.
It has been demonstrated that microRNAs (miRNAs or miRs) can act as prognostic and diagnostic markers, and potential therapeutic targets. miR-95-3p has been reported to be downregulated in osteosarcoma tissues, but its potential as a serum biomarker has not been assessed in human osteosarcoma. The purpose of the present study was to examine the expression levels of miR-95-3p in serum of patients with osteosarcoma and to investigate the diagnostic and prognostic value of miR-95-3p. The serum levels of miR-95-3p in osteosarcoma patients were detected by a real-time quantitative reverse transcription-polymerase chain reaction assay. Associations between miR-95-3p expression and various clinicopathological characteristics were analyzed using Chi square test. Differences in patient survival were determined using the Kaplan-Meier method and a log-rank test. A Cox proportional hazards regression analysis was used for multivariate analyses of prognostic values. Compared to healthy controls, the expression levels of miR-95-3p in serum of osteosarcoma patients were significantly decreased (P < 0.0001). Low miR-95-3p expression had significant association with clinical stage (P < 0.001) and metastasis (P < 0.001). The Kaplan-Meier curve showed that patients with high miR-95-3p expression survived significantly longer than patients with low miR-95-3p expression (P = 0.017). Multivariate analysis demonstrated that miR-95-3p expression level (P = 0.014) was an independent prognostic biomarker for overall survival. Our findings suggested that down-expression of serum miR-95-3p might be associated with poor prognosis of osteosarcoma patients, suggesting that decreased expression of serum miR-95-3p may serve as a valuable diagnostic/prognostic marker for osteosarcoma patients.
Feldspar minerals are the most common rock formers in Earth's crust. As such they play an important role in subjects ranging from geology to climate science. An atomistic understanding of the feldspar structure and its interaction with water is therefore desirable, not least because feldspar has been shown to dominate ice nucleation by mineral dusts in Earth's atmosphere. The complexity of the ice/feldspar interface arising from the numerous chemical motifs expressed on the surface makes it a challenging system. Here we report a comprehensive study of this challenging system with ab initio density functional theory calculations. We show that the distribution of Al atoms, which is crucial for the dissolution kinetics of tectosilicate minerals, differs significantly between the bulk environment and on the surface. Furthermore, we demonstrate that water does not form ice-like overlayers in the contact layer on the most easily cleaved (001) surface of K-feldspar. We do, however, identify contact layer structures of water that induce ice-like ordering in the second overlayer. This suggests that even substrates without an apparent match with the ice structure may still act as excellent ice nucleating agents.
Plant growth promoting rhizobacteria produce chemical compounds with different benefits for the plant. Among them, HCN is recognized as a biocontrol agent, based on its ascribed toxicity against plant pathogens. Based on several past studies questioning the validity of this hypothesis, we have re-addressed the issue by designing a new set of in vitro experiments, to test if HCN-producing rhizobacteria could inhibit the growth of phytopathogens. The level of HCN produced by the rhizobacteria in vitro does not correlate with the observed biocontrol effects, thus disproving the biocontrol hypothesis. We developed a new concept, in which HCN does not act as a biocontrol agent, but rather is involved in geochemical processes in the substrate (e.g., chelation of metals), indirectly increasing the availability of phosphate. Since this scenario can be important for the pioneer plants living in oligotrophic alpine environments, we inoculated HCN producing bacteria into sterile mineral sand together with germinating plants and showed that the growth of the pioneer plant French sorrel was increased on granite-based substrate. No such effect could be observed for maize, where plantlets depend on the nutrients stored in the endosperm. To support our concept, we used KCN and mineral sand and showed that mineral mobilization and phosphate release could be caused by cyanide in vitro. We propose that in oligotrophic alpine environments, and possibly elsewhere, the main contribution of HCN is in the sequestration of metals and the consequential indirect increase of nutrient availability, which is beneficial for the rhizobacteria and their plant hosts.
In the burgeoning field of e-mental health interventions, avatars are increasingly being utilized to facilitate online communication between clients and therapists, and among peers. Avatars are digital self-representations, which enable individuals to interact with each other in computer-based virtual environments. In this narrative review, we examine the psychotherapeutic applications of avatars that have been investigated and trialed to date. Five key applications were identified (1) in the formation of online peer support communities; (2) replicating traditional modes of psychotherapy by using avatars as a vehicle to communicate within a wholly virtual environment; (3) using avatar technology to facilitate or augment face-to-face treatment; (4) as part of serious games; and (5) communication with an autonomous virtual therapist. Across these applications, avatars appeared to serve several functions conducive to treatment engagement by (1) facilitating the development of a virtual therapeutic alliance; (2) reducing communication barriers; (3) promoting treatment-seeking through anonymity; (4) promoting expression and exploration of client identity; and (5) enabling therapists to control and manipulate treatment stimuli. Further research into the feasibility and ethical implementation of avatar-based psychotherapies is required.
The purpose of this study is to examine what factors contributing to the variability in chronic obstructive pulmonary disorder (COPD) and asthma hospitalization rates when the influence of patient characteristics is being simultaneously considered by applying a risk adjustment method. A longitudinal analysis of COPD and asthma hospitalization of rural Medicare beneficiaries in 427 rural health clinics (RHCs) was conducted utilizing administrative data and inpatient and outpatient claims from Region 4. The repeated measures of risk-adjusted COPD and asthma admission rate were analyzed by growth curve modeling. A generalized estimating equation (GEE) method was used to identify the relevance of selected predictors in accounting for the variability in risk-adjusted admission rates for COPD and asthma. Both adjusted and unadjusted rates of COPD admission showed a slight decline from 2010 to 2013. The growth curve modeling showed the annual rates of change were gradually accentuated through time. GEE revealed that a moderate amount of variance (marginal R(2) = 0.66) in the risk-adjusted hospital admission rates for COPD and asthma was accounted for by contextual, ecological, and organizational variables. The contextual, ecological, and organizational factors are those associated with RHCs, not hospitals. We cannot infer how the variability in hospital practices in RHC service areas may have contributed to the disparities in admissions. Identification of RHCs with substantially higher rates than an average rate can portray the need for further enhancement of needed ambulatory or primary care services for the specific groups of RHCs. Because the risk-adjusted rates of hospitalization do not very by classification of rural area, future research should address the variation in a specific COPD and asthma condition of RHC patients. Risk-adjusted admission rates for COPD and asthma are influenced by the synergism of multiple contextual, ecological, and organizational factors instead of a single factor.
The {Cu(NO₃)(H₂O)}(HTae)(4,4'-Bpy) (H₂Tae = 1,1,2,2-tetraacetylethane, 4,4'-Bpy = 4,4'-Dipyridyl) 1D coordination polymer has been obtained by slow evaporation. The crystal structure consists of parallel and oblique {Cu(HTae)(4,4'-Bpy)} zig-zag metal-organic chains stacked along the [100] crystallographic direction. Copper(II) ions are in octahedral coordination environment linked to two nitrogen atoms of two bridging 4,4'-Bpy and to two oxygen atoms of one HTae molecule in the equatorial plane. The occupation of the axial positions varies from one copper atom to another, with different combinations of water molecules and nitrate anions, giving rise to a commensurate super-structure. By means of the thermal removal of water molecules, copper coordinatively unsaturated centres are obtained. These open metal sites could act as Lewis acid active sites in several heterogeneous catalytic reactions. The dehydrated compound, CuHTaeBpy_HT, has been tested as a heterogeneous recoverable catalyst for Knoevenagel condensation reactions. The catalyst is active and heterogeneous for the condensation of aldehydes with malononitrile at 60 °C using a molar ratio catalyst:substrate of 3 % and toluene as solvent. The catalyst suffers a partial loss of activity when reusing it, but can be reused at least four times.
Foul-smelling environmental pollution was a major concern following a chemical workplace explosion. Malodorous pollution has previously been associated with aggravated physical and psychological health, and in persons affected by a trauma, an incidence-related odour can act as a traumatic reminder. Olfaction may even be of significance in the development and persistence of post-traumatic stress symptoms (PTSS). The present longitudinal study assessed whether perceived smell related to malodorous environmental pollution in the aftermath of the explosion was a determinant of subjective health complaints (SHC) and PTSS among gainfully employed adults, when the malodorous pollution was present, and after pollution clean-up. Questionnaire data from validated instruments were analysed using mixed effects models. Individual odour scores were computed, and the participants (n=486) were divided into high and low odour score groups, respectively. Participants in the high odour score group (n=233) reported more SHC and PTSS than those in the low odour score group (n=253), before and even after the pollution was eliminated. These associations lasted for at least three years after the pollution was removed, and might indicate that prompt clean-up is important to avoid persistent health effects after malodorous chemical spills.
The intergeniculate leaflet (IGL) is a flat thalamic nucleus implicated in the modulation of circadian rhythmicity. In rat, two main GABAergic subpopulations can be distinguished in the IGL: neurons synthesizing neuropeptide Y (NPY), which directly innervates the suprachiasmatic nuclei, and enkephalinergic cells, which connect contralaterally located leaflets. The aim of this study was to evaluate possible effects of inner IGL neurotransmitters on the spontaneous and synaptic activity of IGL neurons. The data presented in this article provide evidence that enkephalin, and not NPY, could act upon the majority of IGL neurons. Moreover, we investigated the type of opioid receptor activated by enkephalin and showed that the μ-receptor is functionally predominant in the IGL. The application of met-enkephalin not only robustly hyperpolarized IGL neurons (both putatively NPY-synthesizing and putatively enkephalinergic neurons), but it also was able to inhibit GABAergic and glutamatergic synaptic transmission. Based on this and previous studies, we hypothesize that IGL enkephalinergic neurons may act as powerful interneurons that inhibit themselves and NPY-synthesizing neurons, also in the contralaterally located IGL.
In the present work, the release mechanisms of active pharmaceutical ingredients (APIs) enclosed in self-pore forming regenerated cellulose (RC) two-piece hard shell capsules are described. The RC capsules were fabricated using a modified dip-coating approach, which yielded an assembled dosage form that was equivalent in size and shape to a conventional gelatin two-piece hard shell capsule. Drug release characteristics from RC capsules were evaluated using potassium chloride, diphenhydramine hydrochloride, tramadol hydrochloride, niacinamide, acetaminophen and ketoprofen as model APIs. The RC capsules act as a barrier coated reservoir device that releases the enclosed API at a zero order release rate. When comparing all the API's release behavior from RC capsules, a power-law relationship was observed between their zero-order release rates and their respective aqueous solubilities. Osmotic as well as diffusive mechanisms are involved in the release of the enclosed API. The osmotic mechanism's contribution to zero order release rate increases as the aqueous solubility of the tested APIs inside the capsule increases. The osmotic mediated flux and the apparent diffusivity of the APIs through the capsule wall is a competitive process and the osmotic mediated flux of the enclosed API begins to override its diffusivity through the capsule wall as the API solubility increases. This behavior is attributed to the wide range of pore sizes observed in RC membranes, from our prior analysis. The fluid permeability analysis shows that the RC capsules presented in this work may be better suited for osmotic drug delivery applications than conventional encapsulated systems described in the literature.
Botanical-mediated synthesis of nanomaterials is currently emerging as a cheap and eco-friendly nanotechnology, since it does not involve the use of toxic chemicals. In the present study, we focused on the synthesis of gold nanoparticles using the aqueous peel extract of Musa paradisiaca (MPPE-AuNPs) by green approach following a facile and cheap fabrication process. The green synthesized MPPE-AuNPs were bio-physically characterized by UV-Vis spectroscopy, FTIR, XRD, TEM, Zeta potential and EDX. MPPE-AuNPs were crystalline in nature, spherical to triangular in shape, with particle size ranged within 50 nm. The biofilm inhibition activity of MPPE-AuNPs was high against multiple antibiotic resistant (MARS) Gram-positive Enterococcus faecalis. Light and confocal laser scanning microscopic observation evidenced that the MPPE-AuNPs effectively inhibited the biofilm of E. faecalis when tested at 100 μg mL(-1). Cytotoxicity studies demonstrated that MPPE-AuNPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 100 μg ml(-1). The morphological changes in the MPPE-AuNPs treated A549 lung cancer cells were visualized under phase-contrast microscope. Furthermore, the ecotoxicity of MPPE-AuNPs on the freshwater micro crustacean Ceriodaphnia cornuta were evaluated. Notably, no mortality was recorded in MPPE-AuNPs treated C. cornuta at 250 μg mL(-1). This study concludes that MPPE-AuNPs are non-toxic, eco-friendly and act as potential biomaterial for biomedical applications.
Epidemiological studies indicate that hyperuricaemia is an independent risk factor for cardiovascular disease. Alongside uric acid formation, increased xanthine oxidase activity also results in the formation of oxidative free radicals and superoxide particles. Oxidative stress significantly contributes to the development of cardiovascular disease, including endothelial cell dysfunction, atherosclerosis, vascular calcification and impaired myocardial energetics. Allopurinol, a competitive xanthine oxidase inhibitor, in addition to reducing serum uric acid levels, can act as a free radical scavenger. Although traditionally used for the management of gout, there has been renewed interest in the role of allopurinol in the management of cardiovascular disease. In this review, we summarise the role of the xanthine oxidase pathway in the generation of oxidative stress and evaluate the current body of evidence assessing the clinical effects of allopurinol in patients with cardiovascular disease. A number of small clinical studies have shown a beneficial effect of allopurinol in reducing ischemia-reperfusion injury in the setting of bypass surgery and coronary angioplasty. Additionally, studies in heart failure indicate a potential favourable effect of allopurinol on endothelial dysfunction, LV function and haemodynamic indices, particularly in those with raised serum uric acid levels. Whilst this cheap and readily available pharmacological option may offer a very cost effective therapeutic option, large-scale prospective studies are required to better delineate its role in reducing hard clinical end-points.
Radiologists frequently image women with the sole complaint of mastalgia (breast pain). We hypothesized that whereas the vast majority of women ultimately have no imaging explanation for their breast pain, a small percentage of patients may have a correlative imaging finding and confirm the current American College of Radiology Appropriateness Criteria recommendations.
In this Health Insurance Portability and Accountability Act (HIPAA)-compliant, institutional review board-approved retrospective review, we evaluated 236 women between the ages of 18 and 83 years who presented to our Breast Care Center in 2013 with the sole complaint of breast pain or tenderness. Patients' clinical presentation, diagnostic imaging work-up, and clinical and radiographic follow-up were documented. Outcomes of the diagnostic work-up were compared with the American College of Radiology Appropriateness Criteria recommendations.
Of the 236 patients, 10 women had cyclical breast pain, 116 had noncyclical, nonfocal breast pain, and 110 had noncyclical, focal breast pain. No imaging correlates were discovered to explain the etiology of cyclical pain, supporting the American College of Radiology Appropriateness Criteria rating values. A definitive imaging correlate for breast pain was identified in seven women (3%) with noncyclical, focal pain, one of which was a cancer diagnosis (0.4%), which correlates with the American College of Radiology Appropriateness Criteria ratings. No imaging correlates were found in women with noncyclical, nonfocal pain, supporting the American College of Radiology Appropriateness Criteria ratings.
There was no radiological imaging finding to explain the etiology of mastalgia in most women. Diagnostic imaging may be an appropriate diagnostic evaluation in patients with noncyclical, focal breast pain, supporting the American College of Radiology Appropriateness Criteria recommendations.
The Patient Protection and Affordable Care Act established health insurance marketplaces to allow consumers to make educated decisions about their health care coverage. During the first open enrollment period in 2013, the federally facilitated marketplace in Pima County, Arizona listed 119 plans, making it one of the most competitive markets in the country. This study compares these plans based on differences in consumer cost sharing, including deductibles, co-pays and premiums. Consumer costs were reviewed using specific cases including a normal delivery pregnancy, the management of Type II Diabetes, and the utilization of specialty drugs to treat Hepatitis C. Total cost of care was calculated as the cost of managing the condition or event plus the cost of monthly premiums, evaluated as a single individual age 27. Evaluating a plan on premium alone is not sufficient as cost sharing can dramatically raise the cost of care. A rating system and better cost transparency tools could provider easier access to pertinent information for consumers.
Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-β (IFNβ) in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNβ significantly induced markers of immunogenic cell death. In situ gene therapy with IFNβ, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1β, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNβ acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.
To assess gender utilization of the Family and Medical Leave Act (FMLA) in radiology practices across the United States.
The Practice of Radiology Environment Database was utilized to identify U.S. practice leaders, who were asked to complete an electronic survey developed by the ACR Human Resources (HR) Commission. In 2016, new survey questions asked about number of radiologists in each practice who took FMLA, the reasons why, the average number of weeks taken, and how such absences were covered.
Thirty-two percent (579/1815) of practice group leaders responded to the survey and of these, 73% (432/579) answered FMLA questions, with 15% of those (64/432) answering affirmatively that a radiologist in their practice had taken FMLA leave. Reasons for this in 2015 included to care for a newborn/adopted child (49%), because of a personal serious health condition (42%), to care for an immediate family member (8%), or for active military duty (1%). Women took a greater number of weeks of FMLA leave than men for all reasons (care of newborn/adopted child: 10.7 versus 4.7; personal serious health condition: 10.3 versus 8.0; care of immediate family member: 9.7 versus 8.7) except for military duty (24 weeks taken, all by men). At least 69% of leave time was paid, irrespective of reason for leave or gender of person taking it. Most practices (82%) made no workforce changes to cover FMLA leave.
Both genders of radiologists needed absences from work for FMLA-sanctioned reasons.
Canonical growth factors act indirectly via receptor-mediated signal transduction pathways. Here, we report on an autonomous pathway in which a growth factor is internalized, has its localization regulated by phosphorylation, and ultimately uses intrinsic catalytic activity to effect epigenetic change. Angiogenin (ANG), a secreted vertebrate ribonuclease, is known to promote cell proliferation, leading to neovascularization as well as neuroprotection in mammals. Upon entering cells, ANG encounters the cytosolic ribonuclease inhibitor protein, which binds with femtomolar affinity. We find that protein kinase C and cyclin-dependent kinase phosphorylate ANG, enabling ANG to evade its inhibitor and enter the nucleus. After migrating to the nucleolus, ANG cleaves promoter-associated RNA, which prevents the recruitment of the nucleolar remodeling complex to the ribosomal DNA promoter. The ensuing derepression of rDNA transcription promotes cell proliferation. The biochemical basis for this unprecedented mechanism of signal transduction suggests new modalities for the treatment of cancers and neurological disorders.
The lipidome comprises a large array of molecules with diverse physicochemical properties. Lipids are structural components of cells, act as a source of energy, and function as signaling mediators. Alterations in lipid metabolism are involved in the onset and progression of a variety of diseases, including metabolic syndrome and cancer. Because of this, interest in lipidomics, the comprehensive characterization of the lipidome by mass spectrometry, has intensified in recent years. However, obtaining a truly complete overview of all lipids in a sample has remained very challenging due to their enormous structural diversity. Here, we provide an overview of the collection of analytical approaches used to study various lipid classes, emphasizing innovations in sample preparation and liquid chromatography-mass spectrometry (LC-MS). Additionally, we provide practical suggestions for increasing the coverage of the lipidome.
Cognitive behavioral therapy (CBT) and acceptance and commitment therapy (ACT) have both garnered empirical support for the effective treatment of social anxiety disorder. However, not every patient benefits equally from either treatment. Identifying moderators of treatment outcome can help to better understand which treatment is best suited for a particular patient.
Forty-nine individuals who met criteria for social anxiety disorder were assessed as part of a randomized controlled trial comparing 12 weeks of CBT and ACT. Pre-treatment avoidance of social situations (measured via a public speaking task and clinician rating) was investigated as a moderator of post-treatment, 6-month follow-up, and 12-month follow-up social anxiety symptoms, stress reactivity, and quality of life.
Public speaking avoidance was found to be a robust moderator of outcome measures, with more avoidant individuals generally benefitting more from CBT than ACT by 12-month follow-up. In contrast, clinician-rated social avoidance was not found to be a significant moderator of any outcome measure.
Results were found only at 12-month follow-up. More comprehensive measures of avoidance would be useful for the field moving forward.
Findings inform personalized medicine, suggesting that social avoidance measured behaviorally via a public speaking task may be a more robust factor in treatment prescription compared to clinician-rated social avoidance.
Losing weight is a goal for many people, but it is hard to pursue. However, dieting cues in the environment hold promise for improving individuals' eating behavior. For example, exposure to thin, human-like sculptures by the artist Alberto Giacometti has been found to promote healthy snack choices at a vending machine. Whether health- or weight-related processes drive such effects has not yet been determined. However, a detailed understanding of the content-related drivers of environmental cues' effects provides the first indications regarding a cue's possible use. Therefore, two laboratory studies were conducted to examine the Giacometti sculptures' effects on unhealthy and healthy food intake (Study 1) and on the completion of weight- and health-related fragmented words (Study 2). Study 1 indicated that the sculptures are weight-related by showing that they reduced food intake independent of food healthiness. Furthermore, the "Giacometti effect" was moderated by restrained eating. Restrained eaters, who are known for their weight-control goal, ate less after having been exposed to the thin sculptures. The results of Study 2 pointed in the same direction. Restrained eaters completed more weight-related words after being exposed to the sculptures. Overall, these studies suggest that the thin sculptures are primarily weight-related cues and particularly helpful for restrained eaters. Environmental weight-control cues such as the Giacometti sculptures could act as a counterforce to our obesogenic environment and help restrained eaters pursue their weight-control goal. In this way, they could nudge food decisions in a healthier direction.
Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins' mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD50 concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.
Actein (ACT), isolated from the rthizomes of Cimicifuga foetida, is a triterpene glycoside, showing inhibitory role in breast cancer cells. However, the effects of ACT treatment on gastric cancer have little been known. Thus, the study is conducted to explore the in vitro and in vivo role of ACT in gastric cancer. And the interactions between ACT and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were investigated in gastric cancer cells. A synergistic effect of ACT and TRAIL combination on apoptosis induction in gastric cancer cells was observed. The cancer cells were insensitive to TRAIL single therapy. However, gastric cancer cells receiving ACT were sensitive to TRAIL-triggered apoptotic response by enhancing Caspases cleavage, due to elevation of decoy receptor 1 and 2 (DcR1 and DcR2) dependent on p53. Bcl-2 family members of Bcl-2 and Mcl-1, belonging to anti-apoptosis, were decreased, whereas Bad and Bak, as pro-apoptotic members, were increased for ACT and TRAIL combined treatment. Additionally, the mouse xenograft model suggested that ACT and TRAIL in combination markedly inhibited gastric cancer growth in comparison to ACT or TRAIL monotherapy without toxicity. The present study revealed a dramatically therapeutic strategy for promoting TRAIL-induced anti-cancer effects on gastric cancer cells via ACT combination.
The US Food and Drug Administration (FDA) Amendments Act of 2007 granted the FDA new authorities to enhance drug safety by requiring application holders to submit a proposed Risk Evaluation and Mitigation Strategy (REMS). A REMS is a required risk management plan that uses tools beyond the package insert. REMS elements may include a medication guide and patient package insert for patients and a communication plan focused on health care professionals. Elements to assure safe use (ETASUs) are put in place to mitigate a specific known serious risk when other less restrictive elements of a REMS are not sufficient to mitigate such risk. An implementation system is required for an REMS that includes the ETASUs. With approximately eight years of experience with REMS programs, many health care settings have created systems to manage REMS and also to integrate REMS into their practice settings. At the same time, there are issues associated with the development and implementation of REMS. In 2011, FDA created the REMS Integration Initiative to develop guidance on how to apply statutory criteria to determine when a REMS is required, to improve standardization and assessment of REMS, and to improve integration of REMS into the existing healthcare system. A key component of the REMS Integration Initiative is stakeholder outreach to better understand how existing REMS programs are working and to identify opportunities for improvement. This review attempts to share our company's experience with the REMS program, and to provide updates on FDA's efforts to improve REMS communication, to standardize REMS process, to reduce REMS program burdens and to build a common REMS platform.
The purpose of this study was to examine the validity of the 1971-2010 United States Department of Agriculture's (USDA's) loss-adjusted food availability (LAFA) per capita caloric consumption estimates. Estimated total daily energy expenditure (TEE) was calculated for nationally representative samples of US adults, 20-74 years, using the Institute of Medicine's predictive equations with "low-active" (TEE L-ACT) and "sedentary" (TEE SED) physical activity values. TEE estimates were subtracted from LAFA estimates to create disparity values (kcal/d). A validated mathematical model was applied to calculate expected weight change in reference individuals resulting from the disparity. From 1971-2010, the disparity between LAFA and TEE L-ACT varied by 394kcal/d-(P < 0.001), from -205kcal/d (95% CI: -214, -196) to +189kcal/d (95% CI: 168, 209). The disparity between LAFA and TEE SED varied by 412kcal/d (P < 0.001), from -84kcal/d (95% CI: -93, -76) to +328kcal/d (95% CI: 309, 348). Our model suggests that if LAFA estimates were actually consumed, reference individuals would have lost ~1-4kg/y from 1971-1980 (an accumulated loss of ~12 to ~36kg), and gained ~3-7kg/y from 1988-2010 (an accumulated gain of ~42 to ~98kg). These estimates differed from the actual measured increments of 10kg and 9kg in reference men and women, respectively, over the 39-year period. The USDA LAFA data provided inconsistent, divergent estimates of per capita caloric consumption over its 39-year history. The large, variable misestimation suggests that the USDA LAFA per capita caloric intake estimates lack validity and should not be used to inform public policy.
Individuals routinely receive information about their risk of coronary heart disease (CHD) based on traditional risk factors as part of their primary care. We are also able to calculate individual's risk of CHD based on their genetic information and at present genetic testing for common diseases is available to the public. Due to the limitations in previous studies further understanding is needed about the impact of the risk information on individual's well-being and health-behaviour. We aimed to explore the short term response to receiving different forms of CHD risk information and lifestyle advice for risk reduction.
We conducted fourty-one face-to-face interviews and two focus groups across England with participants from the INFORM trial who received a combination of individualised phenotypic and genotypic CHD risk scores and web-based lifestyle advice. Risk scores were presented in different formats, e.g. absolute 10 year risk was presented as a thermometer and expressed as a percentage, natural frequency and 'heart age'. Interviews and focus groups explored participants' understanding and reaction to the risk scores and attempts to change lifestyle during the intervention. We tape-recorded and transcribed the interviews and focus groups and analysed them using thematic analysis.
Three main themes were identified: limitations of risk scores to generate concern about CHD risk; the advantages of the 'heart age' format of risk score presentation in communicating a message of sub-optimal lifestyle; and intentions and attempts to make moderate lifestyle changes which were prompted by the web-based lifestyle advice.
There are a number of limitations to the use of risk scores to communicate a message about the need for a lifestyle change. Of the formats used, the 'heart age', if noticed, appears to convey the most powerful message about how far from optimal risk an individual person is. An interactive, user friendly, goal setting based lifestyle website can act as a trigger to initiate moderate lifestyle changes, regardless of concerns about risk scores.
Current Controlled Trials ISRCTN17721237 . Registered 12 January 2015.
Global constitutionalism is a way of looking at the world, at global rules and how they are made, as if there was a global constitution, empowering global institutions to act as a global government, setting rules which bind all states and people.
This essay employs global constitutionalism to examine how and why global health governance, as currently structured, has struggled to advance the right to health, a fundamental human rights obligation enshrined in the International Covenant on Economic, Social and Cultural Rights. It first examines the core structure of the global health governance architecture, and its evolution since the Second World War. Second, it identifies the main constitutionalist principles that are relevant for a global constitutionalism assessment of the core structure of the global health governance architecture. Finally, it applies these constitutionalist principles to assess the core structure of the global health governance architecture.
Leading global health institutions are structurally skewed to preserve high incomes countries' disproportionate influence on transnational rule-making authority, and tend to prioritise infectious disease control over the comprehensive realisation of the right to health.
A Framework Convention on Global Health could create a classic division of powers in global health governance, with WHO as the law-making power in global health governance, a global fund for health as the executive power, and the International Court of Justice as the judiciary power.
Improving assessment guidance and feedback for students has become an international priority within higher education. Podcasts have been proposed as a tool for enhancing teaching, learning and assessment. However, a stronger theory-based rationale for using podcasts, particularly as a means of facilitating assessment guidance and feedback, is required.
To explore students' experiences of using podcasts for assessment guidance and feedback. To consider how these podcasts shaped beliefs about their ability to successfully engage with, and act on, assessment guidance and feedback Design Exploratory qualitative study. Setting Higher education institution in North-East Scotland. Participants Eighteen third year undergraduate nursing students who had utilised podcasts for assessment guidance and feedback within their current programme of study.
Participants took part in one of four focus groups, conducted between July and September 2013. Purposive sampling was utilised to recruit participants of different ages, gender, levels of self-assessed information technology skills and levels of academic achievement. Data analysis was guided by the framework approach.
Thematic analysis highlighted similarities and differences in terms of students' experiences of using podcasts for assessment guidance and feedback. Further analysis revealed that Self-Efficacy Theory provided deeper theoretical insights into how the content, structure and delivery of podcasts can be shaped to promote more successful engagement with assessment guidance and feedback from students. The structured, logical approach of assessment guidance podcasts appeared to strengthen self-efficacy by providing readily accessible support and by helping students convert intentions into action. Students with high self-efficacy in relation to tasks associated with assessment were more likely to engage with feedback, whereas those with low self-efficacy tended to overlook opportunities to access feedback due to feelings of helplessness and futility.
Adopting well-structured podcasts as an educational tool, based around the four major sources of information (performance accomplishments, vicarious experience, social persuasion, and physiological and emotional states), has potential to promote self efficacy for individuals, as well as groups of students, in terms of assessment guidance and feedback.
Accurate segmentation of anatomical structures in medical images is important in recent imaging based studies. In the past years, multi-atlas patch-based label fusion methods have achieved a great success in medical image segmentation. In these methods, the appearance of each input image patch is first represented by an atlas patch dictionary (in the image domain), and then the latent label of the input image patch is predicted by applying the estimated representation coefficients to the corresponding anatomical labels of the atlas patches in the atlas label dictionary (in the label domain). However, due to the generally large gap between the patch appearance in the image domain and the patch structure in the label domain, the estimated (patch) representation coefficients from the image domain may not be optimal for the final label fusion, thus reducing the labeling accuracy. To address this issue, we propose a novel label fusion framework to seek for the suitable label fusion weights by progressively constructing a dynamic dictionary in a layer-by-layer manner, where the intermediate dictionaries act as a sequence of guidance to steer the transition of (patch) representation coefficients from the image domain to the label domain. Our proposed multi-layer label fusion framework is flexible enough to be applied to the existing labeling methods for improving their label fusion performance, i.e., by extending their single-layer static dictionary to the multi-layer dynamic dictionary. The experimental results show that our proposed progressive label fusion method achieves more accurate hippocampal segmentation results for the ADNI dataset, compared to the counterpart methods using only the single-layer static dictionary.
Cytokine-mediated immunity plays a dominant role in the pathogenesis of various immune diseases, including asthma. The recent identification of the family interleukin (IL)-1-related cytokine IL-18 now contributes to our understanding of the fine-tuning of cellular immunity. IL-18 can act as a cofactor for Th2 cell development and IgE production and also plays an important role in the differentiation of Th1 cells. Recent work identified an IL-18 association with the pathogenesis of asthma, wherein increased IL-18 expression was found in the serum of patients. Furthermore, IL-18 polymorphisms with susceptibility to asthma were reported, suggesting that IL-18 may be therapeutically relevant to asthma. In this review, we discuss the role of IL-18 in the pathogenesis of asthma and its therapeutic potential based on current research.
Latent growth curve modelling was used to contrast the developmental trajectories of hyperactivity-inattention (H-I) problems across childhood for children with a language difficulty at the start of school and those with typical language and to examine if the presence of a language difficulty moderates the associations of child, parent and peer predictors with these trajectories. Unconditional and language-status conditional latent growth curves of H-I problems were estimated for a large nationally representative cohort of children, comprising 1627 boys (280 - language difficulty) and 1609 girls (159 - language difficulty) measured at age 4 to 5, 6 to 7, 8 to 9 and 10 to 11. Multiple regression tested interaction between language status and predictors of the level and slope of the trajectory of H-I problems. On average, boy's H-I behaviours showed temporal stability while for girls H-I decreased over time with a slower rate of decrease with age. For both boys and girls, the levels of H-I problems were persistently elevated for those with a language difficulty compared to their peers. Neither the shape nor rate of change of H-I problems were associated with language status. Child sociability predicted the rate of growth in H-I for boys with a language difficulty but not for other boys. Child prosocial behaviours and parental psychological distress predicted the rate of growth in H-I for girls with a language difficulty but not for other girls. Parental hostility was associated with the rate of growth only for boys with typical language. The findings indicate that having a language difficulty at school entry is associated with persistently higher levels of H-I problems across childhood and moderates the rate of their growth in some circumstances.
The direct oral anticoagulants (DOACs) have emerged as a good alternative for the treatment of thromboembolic diseases, and their use in clinical practice is increasing rapidly. The DOACs act by blocking the activity of one single step in the coagulation cascade. These drugs act downstream in the common pathway of the coagulation cascade by directly antagonising the action of thrombin or factor Xa. The development of DOACs represents a paradigm shift from the oral vitamin K antagonists such as warfarin. This article aims to describe the properties of the currently available DOACs including pharmacology and dosing. We also address the strategies for periprocedural management and reversal of anticoagulation of patients treated with these agents.
The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5' end of the mRNA and scans the 5' untranslated region (5'UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5'UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N (6)-methyladenosine (m(6)A) residues in their 5'UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.
Successful fertilization in flowering plants depends on the precise directional growth control of pollen tube through the female pistil tissue, towards the female gametophyte contained in the ovule for delivery of non-motile sperm cells. Cysteine-rich peptides LUREs secreted from the synergid cells on either side of the egg cell act as ovular attractants of pollen tubes. Competency control by the pistil is crucial for the response of pollen tubes to these ovular attractants. We recently reported that ovular 4-O-methyl-glucuronosyl arabinogalactan AMOR induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. The β isomer of the terminal disaccharide 4-O-methyl-glucuronosyl galcatose was essential and sufficient for the competency induction. However, critical and non-critical structures in the disaccharide have not been dissected deeply. Herein, we report the synthesis of new AMOR analogs and the structure-activity relationships (SAR) for AMOR activity in the presence of these synthesized analogs. Removal of 4-O-methyl group or -COOH from the glucuronosyl residue of the disaccharide dramatically reduces AMOR activity. The pyranose backbone of the second sugar of disaccharide is essential for the activity but not hydroxy groups. The role of β isomer of the disaccharide 4-Me-GlcA-β(1,6)-Gal is very specific for competency control as there was no difference in effect among the sugar analogs tested for pollen germination. This study represents the first SAR study of sugar molecule involved in plant reproduction, which opens a way for modification of the molecule without loss of activity.
Successful male gametogenesis involves orchestration of sequential gene regulation for somatic differentiation in pre-meiotic anthers. We report here the cloning of Male Sterile23 (Ms23), encoding an anther-specific predicted basic helix-loop-helix (bHLH) transcription factor required for tapetal differentiation; transcripts localize initially to the precursor secondary parietal cells then predominantly to daughter tapetal cells. In knockout ms23-ref mutant anthers, five instead of the normal four wall layers are observed. Microarray transcript profiling demonstrates a more severe developmental disruption in ms23-ref than in ms32 anthers, which possess a different bHLH defect. RNA-seq and proteomics data together with yeast two-hybrid assays suggest that MS23 along with MS32, bHLH122, and bHLH51 act sequentially as either homo- or heterodimers to choreograph tapetal development. Among them, MS23 is the earliest-acting factor, upstream of bHLH51 and bHLH122, controlling tapetal specification and maturation. In contrast, MS32 is constitutive and independently regulated and is required later than MS23 in tapetal differentiation.
Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino-acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences non-specific chemical acetylations.
The objective is to summarise recent findings from the 2010 Australian Survey of High Impact Psychosis (SHIP) and examine their implications for future policy and planning to improve mental health, physical health and other circumstances of people with a psychotic disorder.
Survey of High Impact Psychosis collected nationally representative data on 1825 people with psychotic illness. Over 60 papers have been published covering key challenges reported by participants: financial problems, loneliness and social isolation, unemployment, poor physical health, uncontrolled symptoms of mental illness, and lack of stable, suitable housing. Findings are summarised under the rubric of participant-ranked top challenges.
The main income source for the majority (85%) of participants was a government benefit. Only one-third was employed, and the most appropriate employment services for this group were under-utilised. High rates of loneliness and social isolation impacted mental and physical health. The rate of cardiometabolic disease was well above the general population rate, and associated risk factors were present from a very young age. Childhood abuse (30.6%), adult violent victimisation (16.4%) and alcohol and substance abuse/dependence (lifetime rates of 50.5% and 54.5%, respectively) complicated the clinical profile. Treatment with medication was suboptimal, with physical health conditions undertreated, a high rate of psychotropic polypharmacy and underutilisation of clozapine in chronic persistent psychotic illness. Only 38.6% received evidence-based psychosocial therapies. In the previous year, 27.4% had changed housing and 12.8% had been homeless, on average for 155 days.
Money, social engagement and employment are the most important challenges for people with psychotic illness, as well as good physical and mental health. An integrated approach to recovery is needed to optimise service delivery and augment evidence-based clinical practice with measures to improve physical health and social circumstances. Meeting these challenges has the potential to reduce costs to government and society, as well as promote recovery.
The canonical view about the effect of thyroid hormones (THs) on thermogenesis assumes that the hypothalamus acts merely as a modulator of the sympathetic outflow on brown adipose tissue (BAT). Recent data have challenged that vision by demonstrating that THs act on the ventromedial nucleus of the hypothalamus (VMH) to inhibit AMP-activated protein kinase (AMPK), which regulates the thermogenic program in BAT, leading to increased thermogenesis and weight loss. Current data have shown that in addition to activation of brown fat, the browning of white adipose tissue (WAT) might be also an important thermogenic mechanism. However, the possible central effects of THs on the browning of white fat remain unclear. Here, we show that 3,3',5,5' tetraiodothyroxyne (T4)-induced hyperthyroidism promotes a marked browning of WAT. Of note, central or VMH-specific administration of 3,3',5-triiodothyronine (T3) recapitulate that effect. The specific genetic activation of hypothalamic AMPK in the VMH reversed the central effect of T3 on browning. Finally, we also showed that the expression of browning genes in human WAT correlates with serum T4. Overall, these data indicate that THs induce browning of WAT and that this mechanism is mediated via the central effects of THs on energy balance.
Total nominal US health care spending increased 5.8 percent and reached $3.2 trillion in 2015. On a per person basis, spending on health care increased 5.0 percent, reaching $9,990. The share of gross domestic product devoted to health care spending was 17.8 percent in 2015, up from 17.4 percent in 2014. Coverage expansions that began in 2014 as a result of the Affordable Care Act continued to affect health spending growth in 2015. In that year, the faster growth in total health care spending was primarily due to accelerated growth in spending for private health insurance (growth of 7.2 percent), hospital care (5.6 percent), and physician and clinical services (6.3 percent). Continued strong growth in Medicaid (9.7 percent) and retail prescription drug spending (9.0 percent), albeit at a slower rate than in 2014, contributed to overall health care spending growth in 2015.
A new efficient adaptive optimal control approach is presented in this paper based on the indirect model reference adaptive control (MRAC) architecture for improvement of adaptation and tracking performance of the uncertain system. The system accounts here for both matched and unmatched unknown uncertainties that can act as plant as well as input effectiveness failures or damages. For adaptation of the unknown parameters of these uncertainties, the frequency selective learning approach is used. Its idea is to compute a filtered expression of the system uncertainty using multiple filters based on online instantaneous information, which is used for augmentation of the update law. It is capable of adjusting a sudden change in system dynamics without depending on high adaptation gains and can satisfy exponential parameter error convergence under certain conditions in the presence of structured matched and unmatched uncertainties as well. Additionally, the controller of the MRAC system is designed using a new optimal control method. This method is a new linear quadratic regulator-based optimal control formulation for both output regulation and command tracking problems. It provides a closed-form control solution. The proposed overall approach is applied in a control of lateral dynamics of an unmanned aircraft problem to show its effectiveness.
Cascade regression is a popular face alignment approach, and it has achieved good performances on the wild databases. However, it depends heavily on local features in estimating reliable landmark locations and therefore suffers from corrupted images, such as images with occlusion, which often exists in real-world face images. In this paper, we present a new adaptive cascade regression model for robust face alignment. In each iteration, the shape-indexed appearance is introduced to estimate the occlusion level of each landmark, and each landmark is then weighted according to its estimated occlusion level. Also, the occlusion levels of the landmarks act as adaptive weights on the shape-indexed features to decrease the noise on the shape-indexed features. At the same time, an exemplarbased shape prior is designed to suppress the influence of local image corruption. Extensive experiments are conducted on the challenging benchmarks, and the experimental results demonstrate that the proposed method achieves better results than state-of-the-art methods for facial landmark localization and occlusion detection.
Histone deacetylases (HDACs)(2) play important roles in the epigenetic regulation of gene expression in cells and are emerging therapeutic targets for treating a wide range of diseases. HDAC inhibitors (HDACi)(3) that act on multiple HDAC enzymes have been used clinically to treat a number of solid and hematological malignancies. HDACi are currently being studied also for their efficacy in non-malignant diseases, including pathologic bone loss, but this has necessitated a better understanding of the roles of individual HDAC enzymes, particularly the eleven zinc-containing isozymes. Selective isozyme-specific inhibitors currently being developed against class I HDACs (1, 2, 3 and 8) and class II HDACs (4, 5, 6, 7, 9 and 10) will be valuable tools for elucidating the roles played by individual HDACs in different physiological and pathological settings. Isozyme-specific HDACi promise to have greater efficacy and reduced side effects, as required for treating chronic disease over extended periods of time. This article reviews the current understanding of roles for individual HDAC isozymes and effects of HDACi on bone cells, (osteoblasts, osteoclasts and osteocytes), in relation to bone remodelling in conditions characterised by pathological bone loss, including periodontitis, rheumatoid arthritis and myeloma bone disease.
Numerous animal species display behavioral changes in response to changes in social status or territory possession. For example, in male European starlings only males that acquire nesting sites display high rates of sexual and agonistic behavior. Past studies show that mu and delta opioid receptors regulate behaviors associated with social ascension or defeat. Opioids also act at kappa receptors, with dynorphin binding with the highest affinity; however, the role of these opioids in social behavior has not been well studied. We observed flocks of male starlings during the breeding season and ran quantitative real-time polymerase chain reaction (qPCR) to measure expression of kappa opioid receptors (OPRK1) and prodynorphin (PDYN) in brain regions involved in social behavior and motivation (ventral tegmental area [VTA], medial preoptic nucleus [mPOA]) and vocal behavior (Area X). Males with nesting territories displayed more sexual/agonistic behavior than males without nesting territories. They also had lower OPRK1 expression in VTA and mPOA. OPRK1 expression in VTA correlated negatively with sexual/agonistic behaviors, consistent with past studies showing kappa receptors in VTA to inhibit sociosexual behaviors. PDYN in mPOA correlated negatively with a measure of nesting behavior that may also reflect sexual motivation. PDYN in Area X related positively to song. Distinct patterns of OPRK1 and PDYN expression in VTA, mPOA, and Area X related to gonad volume, suggesting that breeding condition may modify (or be modified by) OPRK1 and PDYN expression. Studies are now needed to further characterize the role of OPRK1 and PDYN in status-appropriate social behaviors.
The immune system plays an important role in enhancing an individual's ability to survive in a world inhabited by pathogens and parasites. The innate immune system is regulated by processes encoded in an individual's genome, providing an avenue for selection to act on this system, as well as the phenotypic relationships generated between this system and other traits of interest. While relationships between innate immunity and endocrine traits (e.g. testosterone) have been reported often in the literature, these relationships are complex and may differ under varying environmental conditions. To better understand the relative contribution of innate immunity (or an endocrine or behavioral trait) to a phenotypic correlation with another trait, an estimation of the underlying heritable genetic variation of the trait of interest is needed. An upper level estimate of the heritability of such traits can be obtained from calculating its repeatability. We conducted a literature review to determine how often repeated samples of measures of innate immune function were conducted and repeatability estimates obtained. This review revealed a very limited number of repeatability estimates, with a large range (0.0-0.9); estimates were exclusively from livestock that have undergone strong artificial selection. This observation of the present literature suggests more work is needed in non-domesticated and free-living animals to begin to understand the underlying genetic contribution of innate immune function to phenotypic correlations of interest (e.g. testosterone and immunity) to behavioral ecologists, evolutionary physiologists and ecoimmunologists.
Multiple sclerosis (MS) is a very common disease of vital importance. In the MS treatment, some drugs such as fingolimod which help to protect nerves from damage are used. The main goal of the drug therapy in MS is to take control of the inflammation which leads to the destruction of myelin and axons in nerve cell and thus prevent and stop the progression of the disease. Fingolimod (FTY720) is an orally active immunomodulatory drug that has been used for the treatment of relapsing-remitting multiple sclerosis. It is a sphingosine-1-phosphate receptor modulator which prevents lymphocytes from contributing to an autoimmune reaction by inhibiting egress of lymphocytes them from lymph nodes. In this study, we have computer designed, synthesized and characterized two novel derivatives of FTY720, F1-12h and F2-9, and have determined their underlying mechanism of their beneficial effect in SH-SY5Y, SK-N-SH, and U-118 MG cell lines. For this purpose, we first determined the regulation of the cAMP response element (CRE) activity and cAMP concentration by F1-12h and F2-9 together with FTY720 using pGL4.29 luciferase reporter assay and cAMP immunoassay, respectively. Then, we have determined their effect on MS- and GPCR-related gene expression profiles using custom arrays along with FTY720 treatment at non-toxic doses (EC10). It was found that both derivatives significantly activate CRE and increase cAMP concentration in all three cell lines, indicating that they activate cAMP pathway through cell surface receptors as FTY720 does. Furthermore, F1-12h and F2-9 modulate the expression of the pathway related genes that are important in inflammatory signaling, cAMP signaling pathway, cell migration as well as diverse receptor and transcription factors. Expression of the genes involved in myelination was also increased by the treatment with F1-12h and F2-9. In summary, our data demonstrate that the two novel FTY720 derivatives act as anti-inflammatory ultimately by influencing the gene expression via the cAMP and downstream transcription factor CRE pathway. In conclusion, F1-12h and F2-9 might contribute future therapies for autoimmune diseases such as multiple sclerosis.
Despite the benefits of improving transitions across care, literature is very limited on inpatient "Communication about Medicines" (ComMed) by staff across United States (U.S.) hospitals.
To evaluate ComMed quality variations by hospital characteristics.
In a cross-sectional, retrospective study of publicly available U.S. Medicare's Hospital Consumer Assessment of Health Care Plans Survey (HCAHPS) data (January 2013-September 2014), ComMed quality (high = above average/excellent vs. low = average/below average/poor star ratings) of 3125 hospitals were compared across region, rural-urban location, and health information technology (HIT) infrastructure giving providers access to patients' electronic medical records. Multivariate logistic regression analysis was conducted with adjusting for confounders (hospital - bed size, ownership, type, ED services, the number of completed HCAHPS surveys).
After adjusting for other characteristics, Midwest versus Western region hospitals (OR = 1.55, 95% CI: 1.21-1.98, p=<0.0001), hospitals with HIT infrastructure (OR = 1.29, 95% CI: 1.05-1.59, p = 0.02) were more likely while Northeast vs. Western region hospitals (OR = 0.67, 95% CI: 0.50-0.89, p=<0.0001) and hospitals in metropolitan areas with 1 million or more population vs. Nonmetro area with less than 2500 population were less likely (OR = 0.68, 95% CI: 0.48-0.95, p=<0.0001), to be associated with high ComMed quality. Hospitals' small bed-size, physician/non-profit ownership, critical-access type, absent ED services, and 100-299 HCAHPS completed surveys were more likely to be associated with high ComMed quality.
One of the first national studies found significant variations in ComMed quality across U.S. hospitals by location (high in Midwest and low in Northeast regions and urban areas) and by access to HIT infrastructure (high) after controlling for other hospital characteristics. With this baseline data, hospital providers and policymakers can design, implement, and evaluate service programs with pharmacists and HIT to enhance ComMed quality in the future delivery of patient-centered care.
The study was carried out to address a method for separation of terrestrial and marine biogenic silica (BSi) in estuaries based on BSi compositions and δ(13)C values in BSi associated organic matter (δ(13)CBSi). We used two world-class major rivers - the Changjiang (Yangtze) and Huanghe (Yellow) Rivers as examples to illustrate our approach. Our results for these rivers indicate that riverine BSi is comprised mainly of phytoliths and diatoms. River BSi concentrations vary with terrestrial inputs and in-stream primary production. Although the fluvial BSi sources are complex, the terrestrial δ(13)CBSi signals are quite unique (-24.7±0.8), significantly lower than the marine δ(13)CBSi values (-21.3±0.07, central Yellow Sea) (p<0.01). Thus, the variation of δ(13)C within BSi organic matter can provide terrestrial source information on the biogeochemistry of silicon in estuaries and the adjacent shelf. The δ(13)CBSi combination could potentially act as an efficient tool to study environmental change in coastal areas on decadal time-scales since this index may respond to variable terrestrial fluxes from land, as well as to changed phytoplankton assemblages in the coastal ocean.
Critical care patients are at increased risk of infection. Near-patient surfaces act as reservoirs of microbial soil, which may contain pathogens.
To correlate soil levels with hand-touch frequency of near-patient sites in an intensive care unit (ICU).
Five sites around each bed in a 10-bed ICU were screened for total microbial soil (cfu/cm(2)) and Staphylococcus aureus every month for 10 months. Selected sites were infusion pump and cardiac monitor, left and right bedrails, and bed table. Ten 1 h covert audits of hand-touch frequency of these sites were performed in order to provide an average hand-touch count, which was modelled against soil levels obtained from microbiological screening.
Seven of 10 staphylococci were found in conjunction with gross contamination of a specific site (P=0.005) and the same proportion from three most frequently touched sites (bedrails and bed table). There was a linear association between four sites demonstrating gross microbial contamination (>12 cfu/cm(2)) and mean number of hand-touch counts (P=0.08). The bed table was handled most but was not the most contaminated site. We suspected that customary placement of alcohol gel containers on bed tables may have reduced microbiological yield. Removing the gel container from one table confirmed its inhibitory effect on microbial contamination after rescreening (19% vs 50% >12 cfu/cm(2): P=0.007).
Surface bioburden at near-patient sites in ICU is associated with hand-contact frequencies by staff and visitors. This supports the need for targeted hygienic cleaning in a high-risk healthcare environment.
Essential oil from Cananga odorata (ylang-ylang essential oil, YYO) is usually used in reducing blood pressure, improving cognitive functioning in aromatherapy in human. Few reports showed its effect on anxiety behaviors.
To investigate the anxiolytic effects of YYO exposure on anxiety animal models, determine the major active constituents and investigate the change of neurotransmitters after odor exposure.
ICR mice were subjected to three anxiety models including open field, elevated plus maze and light-dark box tests after acute and chronic YYO exposure. Main constituents of YYO were defined using GC/MS. These compounds were then tested on the male mice separately on three anxiety models. The monoamines neurotransmitters and their metabolites were analyzed after acute odor exposure and elevated plus maze test.
YYO exposure only showed significant anxiolytic effect on the male mice. It increased the time that mice visited open arms and light box area in elevated plus maze and light-dark box tests after acute and chronic YYO exposures. Three main constituents of YYO, benzyl benzoate, linalool and benzyl alcohol showed anxiolytic effect on the male mice individually. YYO exposure brought changes of neurotransmitters on the male mice more significantly than the female mice. It decreased the dopamine (DA) concentration in the striatum and increased the 5-hydroxytryptamine (5-HT) concentration in the hippocampus of the male mice. The major constituent benzyl benzoate changed neurotransmitters concentration in accordance with the YYO. Moreover, it decreased the ratio of 5-HIAA/5-HT in the hippocampus.
Both acute and chronic YYO exposure showed anxiolytic effect on the male mice. YYO and its major constituent benzyl benzoate might act on the 5-HTnergic and DAnergic pathways.
Knowledge of the microbial quality of irrigation waters is extremely limited. For this reason, the US FDA has promulgated the Produce Rule, mandating the testing of irrigation water sources for many farms. The rule requires the collection and analysis of at least 20 water samples over two to four years to adequately evaluate the quality of water intended for produce irrigation. The objective of this work was to evaluate the effect of interannual weather variability on surface water microbial quality. We used the Soil and Water Assessment Tool model to simulate E. coli concentrations in the Little Cove Creek; this is a perennial creek located in an agricultural watershed in south-eastern Pennsylvania. The model performance was evaluated using the US FDA regulatory microbial water quality metrics of geometric mean (GM) and the statistical threshold value (STV). Using the 90-year time series of weather observations, we simulated and randomly sampled the time series of E. coli concentrations. We found that weather conditions of a specific year may strongly affect the evaluation of microbial quality and that the long-term assessment of microbial water quality may be quite different from the evaluation based on short-term observations. The variations in microbial concentrations and water quality metrics were affected by location, wetness of the hydrological years, and seasonality, with 15.7-70.1% of samples exceeding the regulatory threshold. The results of this work demonstrate the value of using modeling to design and evaluate monitoring protocols to assess the microbial quality of water used for produce irrigation.
Worldwide, ecosystems are increasingly dominated by exotic plant species, a shift hypothesized to result from numerous ecological factors. Two of these, increased resource availability and enemy release, may act in concert to increase exotic success in plant communities (Resource-Enemy Release Hypothesis, R-ERH). To test this, we manipulated the availability of soil nutrients and access of vertebrate herbivores, insect herbivores, and fungal pathogens to intact grassland communities containing both native and exotic species. Our results supported both conditions necessary for R-ERH. First, exotics were less damaged than natives, experiencing less foliar damage (insect herbivory and fungal disease) than native species, particularly in communities where soil nutrients were added. Second, fertilization increased foliar damage on native species, but not exotic species. As well as fulfilling both conditions for R-ERH, these results demonstrate the importance of considering the effects of resource availability when testing for enemy release. When both conditions are fulfilled, R-ERH predicts that increasing resource availability will increase exotic abundance only in the presence of enemies. Our results fully supported this prediction for vertebrate herbivores: fertilization increased exotic cover only in communities exposed to vertebrate herbivores. Additionally, the prediction was partially supported for insect herbivores and fungal pathogens, excluding these enemies reduced exotic cover as predicted, but inconsistent with R-ERH, this effect occurred only in unfertilized communities. These results highlight the need to consider the influence of multiple enemy guilds on community processes like exotic plant invasions. Moreover, this study experimentally demonstrates that resource availability and natural enemies can jointly influence exotic success in plant communities.
Head lice, Pediculus humanus capitis, occur in four divergent mitochondrial clades (A, B, C and D), each having particular geographical distributions. Recent studies suggest that head lice, as is the case of body lice, can act as a vector for louse-borne diseases. Therefore, understanding the genetic diversity of lice worldwide is of critical importance to our understanding of the risk of louse-borne diseases.
Here, we report the results of the first molecular screening of pygmies' head lice in the Republic of Congo for seven pathogens and an analysis of lice mitochondrial clades. We developed two duplex clade-specific real-time PCRs and identified three major mitochondrial clades: A, C, and D indicating high diversity among the head lice studied. We identified the presence of a dangerous human pathogen, Borrelia recurrentis, the causative agent of relapsing fever, in ten clade A head lice, which was not reported in the Republic of Congo, and B. theileri in one head louse. The results also show widespread infection among head lice with several species of Acinetobacter. A. junii was the most prevalent, followed by A. ursingii, A. baumannii, A. johnsonii, A. schindleri, A. lwoffii, A. nosocomialis and A. towneri.
Our study is the first to show the presence of B. recurrentis in African pygmies' head lice in the Republic of Congo. This study is also the first to report the presence of DNAs of B. theileri and several species of Acinetobacter in human head lice. Further studies are needed to determine whether the head lice can transmit these pathogenic bacteria from person to another.
In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges of drug and nutrient gradients and local populations. This ecology of a fragmented metapopulation induced the drug resistance in stage IV U87 glioblastoma cells to doxorubicin in 7 d. Exome and transcriptome sequencing of the resistant cells identified mutations and differentially expressed genes. Gene ontology and pathway analyses of the genes identified showed that they were functionally relevant to the established mechanisms of doxorubicin action. Specifically, we identified (i) a frame-shift insertion in the filamin-A gene, which regulates the influx and efflux of topoisomerase II poisons; (ii) the overexpression of aldo-keto reductase enzymes, which convert doxorubicin into doxorubicinol; and (iii) activation of NF-κB via alterations in the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway from mutations in three genes (CARD6, NSD1, and NLRP13) and the overexpression of inflammatory cytokines. Functional experiments support the in silico analyses and, together, demonstrate the effects of these genetic changes. Our findings suggest that, given the rapid evolution of resistance and the focused response, this technology could act as a rapid screening modality for genetic aberrations leading to resistance to chemotherapy as well as counter selection of drugs unlikely to be successful ultimately.
Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.
Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes.
At chemical synapses, presynaptic action potentials (APs) activate voltage-gated calcium channels, allowing calcium to enter and trigger neurotransmitter release. The duration, peak amplitude, and shape of the AP falling phase alter calcium entry, which can affect neurotransmitter release significantly. In many neurons, APs do not immediately return to the resting potential, but instead exhibit a period of depolarization or hyperpolarization referred to as an afterpotential. We hypothesized that presynaptic afterpotentials should alter neurotransmitter release by affecting the electrical driving force for calcium entry and calcium channel gating. In support of this, presynaptic calcium entry is affected by afterpotentials after standard instant voltage jumps. Here, we used the mouse calyx of Held synapse, which allows simultaneous presynaptic and postsynaptic patch-clamp recording, to show that the postsynaptic response is affected significantly by presynaptic afterpotentials after voltage jumps. We therefore tested the effects of presynaptic afterpotentials using simultaneous presynaptic and postsynaptic recordings and AP waveforms or real APs. Surprisingly, presynaptic afterpotentials after AP stimuli did not alter calcium channel responses or neurotransmitter release appreciably. We show that the AP repolarization time course causes afterpotential-induced changes in calcium driving force and changes in calcium channel gating to effectively cancel each other out. This mechanism, in which electrical driving force is balanced by channel gating, prevents changes in calcium influx from occurring at the end of the AP and therefore acts to stabilize synaptic transmission. In addition, this mechanism can act to stabilize neurotransmitter release when the presynaptic resting potential changes.
The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium current or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes.
Human immunodeficiency virus type 1 (HIV-1) infection can be effectively controlled by potent antiviral drugs, but this never results in a cure. The patient should therefore take these drugs for the rest of his/her life, which can cause drug-resistance and adverse effects. Therefore, more durable therapeutic strategies should be considered, such as a stable gene therapy to protect the target T cells against HIV-1 infection. The development of potent therapeutic regimens based on the RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) mechanisms will be described, which can be delivered by lentiviral vectors. These mechanisms attack different forms of the viral genome, the RNA and DNA, respectively, but both mechanisms act in a strictly sequence-specific manner. Early RNAi experiments demonstrated profound virus inhibition, but also indicated that viral escape is possible. Such therapy failure can be prevented by the design of a combinatorial RNAi attack on the virus and this gene therapy is currently being tested in a preclinical humanized mouse model. Recent CRISPR-Cas studies also document robust virus inhibition, but suggest a novel viral escape route that is induced by the cellular nonhomologous end joining DNA repair pathway, which is activated by CRISPR-Cas-induced DNA breaks. We will compare these two approaches for durable HIV-1 suppression and discuss the respective advantages and disadvantages. The potential for future clinical applications will be described.
Mitochondrial oxidative damage has long been known to contribute to damage in conditions such as ischaemia-reperfusion (IR) injury in heart attack. Over the past years, we have developed a series of mitochondria-targeted compounds designed to ameliorate or determine how this damage occurs. I will outline some of this work, from MitoQ to the mitochondria-targeted S-nitrosating agent, called MitoSNO, that we showed was effective in preventing reactive oxygen species (ROS) formation in IR injury with therapeutic implications. In addition, the protection by this compound suggested that ROS production in IR injury was mainly coming from complex I. This led us to investigate the mechanism of the ROS production and using a metabolomic approach, we found that the ROS production in IR injury came from the accumulation of succinate during ischaemia that then drove mitochondrial ROS production by reverse electron transport at complex I during reperfusion. This surprising mechanism led us to develop further new therapeutic approaches to have an impact on the damage that mitochondrial ROS do in pathology and also to explore how mitochondrial ROS can act as redox signals. I will discuss how these approaches have led to a better understanding of mitochondrial oxidative damage in pathology and also to the development of new therapeutic strategies.
Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.
Studies have reported that the presence of a third party observer (TPO) during neuropsychological assessments negatively affects the test performance of the examinee. The present study aimed to investigate the effects of a TPO and trait anxiety on neuropsychological performance according to Attentional Control Theory (ACT).
A sample of college students was recruited (n = 318) and then 80 participants were selected to represent the high and low trait anxiety groups. Participants of each of group were randomly assigned to either the NTPO (non-TPO) or TPO conditions. The State-Trait Anxiety Inventory - Trait measure (STAI-T), Wisconsin Card Sorting Test (WCST-64), Stroop test, and Rating Scale for Mental Effort (RSME) were administered to both groups. To analyze the data, univariate ANOVAs were conducted.
The results indicated that under the conditions without a TPO the group with high trait anxiety had poorer processing efficiency, but under the conditions with a TPO they had poorer processing efficiency and poorer performance effectiveness than the group with low trait anxiety. In addition, the group with low trait anxiety showed poorer processing efficiency in the TPO compared to non-TPO condition.
These findings provide support for the hypotheses of ACT regarding the relation between observer presence and poorer performance on neuropsychological tests, with individuals with higher trait anxiety showing greater negative effects. Implications and suggestions for further research are discussed.
Self-regulation constrains the expression of prejudice, but when self-regulation falters, the immediate environment can act as an external source of prejudice regulation. This hypothesis derives from work demonstrating that external controls and internal self-regulation can prompt goal pursuit in the absence of self-imposed controls. Across four studies we found support for this complementary model of prejudice regulation. In Study 1, self-regulatory fatigue resulted in less motivation to be non-prejudiced, compared to a non-fatigued control. In Study 2, strong (vs. weak) perceived social pressure was related to greater motivation to be non-prejudiced. In Study 3, dispositional self-regulation predicted non-prejudice motivation when perceived social pressure was weak or moderate, but not when it was strong. Finally, in Study 4 self-regulatory fatigue increased prejudice when social pressure was weak but not when it was strong.
To further enhance the intensity of deep tumor drug delivery and integrate a combined therapy, we herein report on a core-shell nanocarrier that could simultaneously overcome the double barriers of the extracellular matrix (ECM) and multiple layers of tumor cells (MLTC). A pH-triggered reversible swelling-shrinking core and an MMP2 (matrix metallopeptidase 2) degradable shell were developed to encapsulate chemotherapeutics and macroautophagy/autophagy inhibitors, respectively. MMP2 degraded the shell, which was followed by the autophagy inhibitors release. The exposed core could diffuse along the pore within the ECM to deliver chemotherapeutics into deep tumors, and it was able to swell in lysosomes and shrink back in the cytoplasm or ECM. The swelling of the core resulted in the rapid release of chemotherapeutics to kill autophagy-inhibited cells. After leaving the dead cells, the shrinking core could act on neighboring cells that were closer to the center of the tumor. The core thus could also cross MLTC layer by layer to deliver chemotherapeutics into the deep tumor.
Photosensitization of DNA by thionucleosides is a promising photo-chemotherapeutic treatment option for a variety of malignancies. DNA metabolization of thiated prodrugs can lead to cell death upon exposure to a low dose of UVA light. The exact mechanisms of thionucleoside phototoxicity are still not fully understood. In this work, we have combined femtosecond broadband transient absorption experiments with state-of-the-art molecular simulations to provide mechanistic insights for the ultrafast and efficient population of the triplet-state in the UVA-activated pyrimidine anticancer drug: 4-thiothymine. The triplet state is thought to act as a precursor to the DNA lesions and the reactive oxygen species responsible for 4-thiothymine photocytotoxicity. The electronic-structure and mechanistic results presented in this contribution reveal key molecular design criteria that can assist in developing alternative chemotherapeutic agents that may overcome some of the primary deficiencies of classical photosensitizers.
Thrombomodulin (TM) modulates the activation of protein C and coagulation. Additionally, TM regulates monocyte migration and inflammation. However, its role on monocyte differentiation is still unknown. We investigated the effects of TM on monocyte differentiation. First, we found that TM was increased when THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA). Overexpression of TM enhanced the macrophage markers, CD14 and CD68 expression in PMA-induced THP-1. TM siRNA depressed the PMA-induced increase of p21(Cip1/WAF1) via ERK1/2-NF-kB p65 signaling. TM regulated cytoskeletal reorganization via its interaction with paxillin, cofilin, LIMK1, and PYK2. In addition, PMA-induced p21(Cip1/WAF1) expression, CD14-positive cell labeling intensity and ERK1/2 phosphorylation were markedly inhibited when protein kinase C-δ (PKCδ) was knocked down. We identified that TM directly interacts with PKCδ. PKCδ was highly expressed in human atherosclerotic arteries and colocalized with TM in CD68-positive infiltrated macrophages of plaques, indicating that the coordination between TM and PKCδ in macrophages participated in atherogenesis. TM may act as a scaffold for PKCδ docking, which keeps PKCδ in the region close to the monocyte membrane to promote the activation of ERK1/2. Taken together, our findings suggest that TM-PKCδ interaction may contribute to cardiovascular disorders by affecting monocye differentiation, which may develop future therapeutic applications.
Mesenchymal stem cells (MSCs) are speculated to act at macrophage-injury interfaces to mediate efficient repair. To explore this facet in-depth this study evaluates the influence of MSCs on human macrophages existing in distinct functional states. MSCs promoted macrophage differentiation, enhanced respiratory burst and potentiated microbicidal responses in naïve macrophages (Mφ). Functional attenuation of inflammatory M1 macrophages was associated with a concomitant shift towards alternatively activated M2 state in MSC-M1 co-cultures. In contrast, alternate macrophage (M2) activation was enhanced in MSC-M2 co-cultures. Elucidation of key macrophage metabolic programs in Mo/MSC, M1/MSC and M2/MSC co-cultures indicated changes in Glucose transporter1 (GLUT1 expression/glucose uptake, IDO1 protein/activity, SIRTUIN1 and alterations in AMPK and mTOR activity, reflecting MSC-instructed metabolic shifts. Inability of Cox2 knockdown MSCs to attenuate M1 macrophages and their inefficiency in instructing metabolic shifts in polarized macrophages establishes a key role for MSC-secreted PGE2 in manipulating macrophage metabolic status and plasticity. Functional significance of MSC-mediated macrophage activation shifts was further validated on human endothelial cells prone to M1 mediated injury. In conclusion, we propose a novel role for MSC secreted factors induced at the MSC-macrophage interface in re-educating macrophages by manipulating metabolic programs in differentially polarized macrophages.
The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer's disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP's protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative 'SIP' (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer's disease.
The success of mechanism-based drug discovery depends on the definition of the drug target. This definition becomes even more important as we try to link drug response to genetic variation, understand stratified clinical efficacy and safety, rationalize the differences between drugs in the same therapeutic class and predict drug utility in patient subgroups. However, drug targets are often poorly defined in the literature, both for launched drugs and for potential therapeutic agents in discovery and development. Here, we present an updated comprehensive map of molecular targets of approved drugs. We curate a total of 893 human and pathogen-derived biomolecules through which 1,578 US FDA-approved drugs act. These biomolecules include 667 human-genome-derived proteins targeted by drugs for human disease. Analysis of these drug targets indicates the continued dominance of privileged target families across disease areas, but also the growth of novel first-in-class mechanisms, particularly in oncology. We explore the relationships between bioactivity class and clinical success, as well as the presence of orthologues between human and animal models and between pathogen and human genomes. Through the collaboration of three independent teams, we highlight some of the ongoing challenges in accurately defining the targets of molecular therapeutics and present conventions for deconvoluting the complexities of molecular pharmacology and drug efficacy.
The commercial strains of broiler chickens have genetically selected to reach market weight at younger ages. In order to sustain increased growth rate requires an efficient assimilation of nutrients from the intestinal lumen, which is permitted through nutrient assimilation through the enterocytes that is facilitated by many nutrient transporter proteins. These trans-membrane proteins have specific action, well known kinetics and assist in the partitioning of nutrients among tissues. Amino acids are transported across the plasma membrane as free amino acids or short chain peptides through several independent systems encoded by various nutrient transporter gene products, and these systems may act on multiple substrates. Any deficiency in amino acid and peptide transportation results in metabolic disorders, illustrating crucial roles being played by the body's ability to maintain amino acid homeostasis. The ability of tissues to adapt to changes in input of amino acids related to numerous external factors that negatively affect amino acids utilization appears to result from responses that alter nutrient transporter protein mRNA expression mandated for to maintain amino acid and protein homeostasis. This review summarizes the results of some recently published scientific articles on the effects of different factors that influence intestinal amino acid and peptide transporter proteins mRNA expressions in domestic fowl. Understanding the regulatory effects of diet on amino acid and peptide transport systems in tissues offers insight that ostensibly will help nutritionists to improve the efficiency of dietary protein utilization in poultry.
Immune checkpoint blockaders (ICBs) act by unbalancing the immune system, thus favoring the development of an immune-mediated antitumor effect. ICBs targeting the programmed cell death receptor-1 (PD-1) have recently been investigated in a number of advanced tumors, including non-small cell lung cancer (NSCLC). Nivolumab, a fully human IgG4 kappa directed against PD-1, has been the first ICB to be approved for second-line treatment of advanced NSCLC. Areas covered: In this review we focus on the clinical development of nivolumab for the treatment of advanced NSCLC, with an emphasis on its safety profile. In addition, we summarize the most common types of immune-related adverse events (irAEs) associated with nivolumab, mainly due to organ inflammation secondary to autoimmunity. Expert opinion: Nivolumab is the preferred treatment option for platinum-pretreated advanced NSCLC, having convincingly shown higher efficacy compared with standard docetaxel chemotherapy in phase III trials. The same trials showed far less incidence of either any grade and severe treatment-related AEs with nivolumab compared with chemotherapy. IrAEs associated with nivolumab are rarely severe in intensity, and often resolve with prompt management. Future studies will explore nivolumab in combination strategies with either platinum-based chemotherapy or other ICBs in treatment-naïve advanced NSCLC patients.
Wendelstein 7-X, a superconducting optimized stellarator built in Greifswald/Germany, started its first plasmas with the last closed flux surface (LCFS) defined by 5 uncooled graphite limiters in December 2015. At the end of the 10 weeks long experimental campaign (OP1.1) more than 20 independent diagnostic systems were in operation, allowing detailed studies of many interesting plasma phenomena. For example, fast neutral gas manometers supported by video cameras (including one fast-frame camera with frame rates of tens of kHz) as well as visible cameras with different interference filters, with field of views covering all ten half-modules of the stellarator, discovered a MARFE-like radiation zone on the inboard side of machine module 4. This structure is presumably triggered by an inadvertent plasma-wall interaction in module 4 resulting in a high impurity influx that terminates some discharges by radiation cooling. The main plasma parameters achieved in OP1.1 exceeded predicted values in discharges of a length reaching 6 s. Although OP1.1 is characterized by short pulses, many of the diagnostics are already designed for quasi-steady state operation of 30 min discharges heated at 10 MW of ECRH. An overview of diagnostic performance for OP1.1 is given, including some highlights from the physics campaigns.
Spectrometer measurements and filter upgrades to a motional Stark effect polarimeter measuring the outer half-radius of the DIII-D tokamak helped to identify asymmetries in the polarization angle of Stark-split emission. The measured polarization angle of the π components differs and is not orthogonal to the σ component. These differences persist over a range of densities and with low levels of background light. It is suggested that the difference in the polarization angle between components is from a change in the ellipticity of the emitted light across the Stark components coupled with imperfect polarization preservation from an in-vessel mirror.
Intramolecular germylene, stannylene, and plumbylene Lewis pairs were reacted with hexanal and yielded the cyclic addition products only with the germanium and tin reagents. In further reactivity studies, the hydroboration of aldehydes and ketones catalyzed by intramolecular germylene, stannylene, and plumbylene Lewis pairs was studied. In the case of the cyclic germylene Lewis pair, the product of the oxidative addition of pinacolborane at the germylene moiety was observed. According to stoichiometric as well as catalytic experiments, the intramolecular germylene Lewis pair acts as a catalyst in the hydroboration of aldehydes and ketones. The homologous stannylene Lewis pair forms a reactive tin hydride during the catalysis, which can also act as a catalyst in this transformation.
While developmental surveillance programs promote early identification of child developmental problems, evidence has indicated suboptimal uptake. This study aimed to identify predictors of developmental surveillance completion at 6 months postpartum.
Questionnaires were administered to the parents of 510 infants who were born in south western Sydney, Australia over a 22-month period. Attendance for developmental screening and completion of the Parents' Evaluation of Developmental Status (PEDS) at 6 months postpartum were modelled separately using multivariable logistic regression.
Developmental surveillance attendance was predicted by higher levels of maternal education, annual income and being informed about checks. PEDS completion at 6 months of age was predicted by higher income and being informed, as well as being married, employed, speaking English at home, full-term birth and the professional status of the practitioner completing the check.
Barriers to developmental surveillance included low socioeconomic status, linguistic diversity and possible gaps in parental knowledge and professional education. Developmental surveillance rates may be increased by the addition of targeted parental and professional support within current universal frameworks.
The discovery of the orexin system represents the single major progress in the sleep field of the last three to four decades. The two orexin peptides and their two receptors play a major role in arousal and sleep/wake cycles. Defects in the orexin system lead to narcolepsy with cataplexy in humans and dogs and can be experimentally reproduced in rodents. At least six orexin receptor antagonists have reached Phase II or Phase III clinical trials in insomnia, five of which are dual orexin receptor antagonists (DORAs) that target both OX1 and OX2 receptors (OX2Rs). All clinically tested DORAs induce and maintain sleep: suvorexant, recently registered in the USA and Japan for insomnia, represents the first hypnotic principle that acts in a completely different manner from the current standard medications. It is clear, however, that in the clinic, all DORAs promote sleep primarily by increasing rapid eye movement (REM) and are almost devoid of effects on slow-wave (SWS) sleep. At present, there is no consensus on whether the sole promotion of REM sleep has a negative impact in patients suffering from insomnia. However, sleep onset REM (SOREM), which has been documented with DORAs, is clearly an undesirable effect, especially for narcoleptic patients and also in fragile populations (e.g. elderly patients) where REM-associated loss of muscle tone may promote an elevated risk of falls. Debate thus remains as to the ideal orexin agent to achieve a balanced increase in REM and non-rapid eye movement (NREM) sleep. Here, we review the evidence that an OX2R antagonist should be at least equivalent, or perhaps superior, to a DORA for the treatment of insomnia. An OX2R antagonist may produce more balanced sleep than a DORA. Rodent sleep experiments show that the OX2R is the primary target of orexin receptor antagonists in sleep modulation. Furthermore, an OX2R antagonist should, in theory, have a lower narcoleptic/cataplexic potential. In the clinic, the situation remains equivocal, since OX2R antagonists are in early stages: MK-1064 has completed Phase I, and MIN202 is currently in clinical Phase II/III trials. However, data from insomnia patients have not yet been released. Promotional material suggests that balanced sleep is indeed induced by MIN-202, whereas in volunteers MK-1064 has been reported to act similarly to DORAs.
Soybean mosaic virus (SMV) is a devastating plant virus classified in the family Potyviridae, and known to infect cultivated soybeans (Glycine max). In this study, seven new SMVs were isolated from wild soybean samples and analyzed by whole-genome sequencing. An updated SMV phylogeny was built with the seven new and 83 known SMV genomic sequences. Results showed that three northeastern SMV isolates were distributed in clade III and IV, while four southern SMVs were grouped together in clade II and all contained a recombinant BCMV fragment (~900 bp) in the upstream part of the genome. This work revealed that wild soybeans in China also act as important SMV hosts and play a role in the transmission and diversity of SMVs.
The aim of this study was to develop an in-depth understanding of the rationale, experiences, evaluation and outcomes of using Cancer Information and Support (CIS) services in Australia, the UK and USA.
Semi-structured interviews were used to gather data between November 2015 and January 2016. Telephone interviews were recorded, de-identified, transcribed and thematically analysed. Ten users from each of three international CIS services (n = 30 in total) were recruited. Participants were eligible for inclusion if they had utilised the CIS in 2015 via telephone contact with a cancer nurse and identified as a patient or cancer survivor, or friend or family member of such a person.
Four major themes were derived and included a total of 25 sub-themes. Key themes included (i) drivers for access, (ii) experience of the service, (iii) impact and (iv) an adjunct to cancer treatment services.
Cancer Information and Support nurses internationally act as expert navigators, educators and compassionate communicators who 'listen between the lines' to enable callers to better understand and contextualise their situation and discuss it with their healthcare team and family and friends. Use of the service can result in reduced worry, extend support repertoires and enable use of new knowledge and language as a tool to getting the most from the healthcare team. The positioning of CIS alongside cancer treatment services aids fuller integration of supportive care, benefiting both patients and clinicians.
The expression of microRNA 21 (miR-21) has been reported to be upregulated in various types of cancer, including malignant gliomas. However, its functions and mechanisms in glioma remain to be fully elucidated. The present study established miRNA‑21 overexpression and knockdown cell lines using SRY‑box 2 (Sox2) small interfering RNA (siRNA) to knockdown expression and Sox2 cDNA was cloned into pcDNA 3.1 mammalian expression vector for ectopic expression. BIO and XAV‑939 were used for β‑catenin signaling activation and knockdown, respectively. Transwell chambers were used to assay the capacity of cells to migrate. The present study determined that increased expression of miR‑21 significantly promoted the migration and invasion of glioma cells, which was accompanied by an upregulated expression of the Sox2 protein. Sox2 overexpression also promoted glioma cell migration and invasion, whereas Sox2 siRNA markedly reduced the miR‑21‑enhanced migration and invasion of glioma cells, indicating Sox2 may act as a crucial mediator of miR‑21 function. Furthermore, miR‑21 also upregulated the protein expression level of β‑catenin, whereas anti‑miR‑21 and Sox2 knockdown significantly reduced β‑catenin expression. BIO, a β‑catenin specific agonist, enhanced migration and invasion of glioma cells. XAV‑939, an inhibitor of β‑catenin signaling, markedly inhibited the migration and invasion of glioma cells, suggesting that β‑catenin may be associated with miR‑21‑ and Sox2‑induced invasion of glioma cells. Notably, BIO restored the migration and invasion potential of glioma cells, which were inhibited by Sox2 siRNA and anti‑miR‑21. These findings indicated that β‑catenin may be an important downstream mediator of miR‑21 and Sox2. Therefore, the present study identified the miR‑21/Sox2/β‑catenin signaling pathway, which may regulate the migration and invasion of human glioma cells.
Silent brain lesions due to thrombogenicity of the procedure represent recognized side effects of atrial fibrillation (AF) catheter ablation. Embolic risk is higher if anticoagulation is inadequate and recent studies suggest that uninterrupted anticoagulation, ACT levels above 300 seconds and administration of a pre-transeptal bolus of heparin might significantly reduce the incidence of silent cerebral ischemia (SCI) to 2%. Asymptomatic new lesions during AF ablation should suggest worse neuropsychological outcome as a result of the association between silent cerebral infarcts and increased long-term risk of dementia in non-ablated AF patients. However, the available data are discordant. To date, no study has definitely linked post-operative asymptomatic cerebral events to a decline in neuropsychological performance. Larger volumes of cerebral lesions have been associated with cognitive decline but are uncommon findings acutely in post-ablation AF patients. Of note, the majority of acute lesions have a small or medium size and often regress at a medium-term follow-up. Successful AF ablation has the potential to reduce the risk of larger SCI that may be considered as part of the natural course of AF. Although the long-term implications of SCI remain unclear, it is conceivable that strategies to reduce the risk of SCI may be beneficial.
In contrast to the increased research interest in the benefits of mindfulness and self-compassion, relatively few studies have examined their unique and combined effects in predicting affect. This cross-sectional study examined the predictive value of mindfulness and self-compassion for depressive symptoms, negative affect, and positive affect in a large representative sample of community adults (N = 1736). The Five Facets of Mindfulness Questionnaire (FFMQ) was used as a measure of mindfulness and the Self-Compassion Scale (SCS) as a measure of self-compassion. Five FFMQ facets were explored: observe, describe, act with awareness, non-judgment, and non-reactivity. Two SCS facets were explored: its positive items (SCS Pos) and its negative items (SCS Neg). When simultaneously examining all seven facets of mindfulness and self-compassion, three of the five FFMQ facets and SCS Neg significantly predicted both depressive symptoms and negative affect, with SCS Neg and act with awareness being the strongest predictors. These findings suggest that a harsh attitude towards oneself and a lack of attention when acting have the greatest value in predicting the presence of psychological symptoms. With respect to positive affect, four of the five FFMQ facets (except non-judgment) were significant predictors, with no unique predictive value of the two SCS's facets, suggesting that mindfulness is a more important predictor of positive affect than self-compassion, as measured by the FFMQ and SCS.
Information foraging connects optimal foraging theory in ecology with how humans search for information. The theory suggests that, following an information scent, the information seeker must optimize the tradeoff between exploration by repeated steps in the search space vs. exploitation, using the resources encountered. We conjecture that this tradeoff characterizes how a user deals with uncertainty and its two aspects, risk and ambiguity in economic theory. Risk is related to the perceived quality of the actually visited patch of information, and can be reduced by exploiting and understanding the patch to a better extent. Ambiguity, on the other hand, is the opportunity cost of having higher quality patches elsewhere in the search space. The aforementioned tradeoff depends on many attributes, including traits of the user: at the two extreme ends of the spectrum, analytic and wholistic searchers employ entirely different strategies. The former type focuses on exploitation first, interspersed with bouts of exploration, whereas the latter type prefers to explore the search space first and consume later. Our findings from an eye-tracking study of experts' interactions with novel search interfaces in the biomedical domain suggest that user traits of cognitive styles and perceived search task difficulty are significantly correlated with eye gaze and search behavior. We also demonstrate that perceived risk shifts the balance between exploration and exploitation in either type of users, tilting it against vs. in favor of ambiguity minimization. Since the pattern of behavior in information foraging is quintessentially sequential, risk and ambiguity minimization cannot happen simultaneously, leading to a fundamental limit on how good such a tradeoff can be. This in turn connects information seeking with the emergent field of quantum decision theory.
The purpose of this retrospective study was to differentiate between the MRI features of normal post-operative change and those of residual or recurrent disease after intralesional treatment of an atypical cartilage tumour (ACT)/grade I chondrosarcoma.
We reviewed the case notes, radiology and histology of 75 patients, who had been treated for an ACT/grade I chondrosarcoma by curettage, phenolisation and bone allografting between 1994 and 2005. The first post-operative Gd-enhanced MRI scan was carried out within one year of surgery. Patients had a minimum of two scans and a mean follow-up of 72 months (13 to 169). Further surgery was undertaken in cases of suspected recurrence.
In 14 patients (18.6%) a second procedure was undertaken after a mean period of 59 months (8 to 114). Radio frequency ablation (RFA) was used in lesions of < 10 mm and curettage, phenolisation and bone grafting for those ≥ 10 mm. Only six of these (8% of total) had a histologically-proven recurrence. No increase in tumour grade was seen at time of recurrence.
Based on this study, we have been able to classify the post-operative MRI appearances into four groups. These groups differ in follow-up, and have a different risk of recurrence of the lesion. Follow-up and treatment vary for the patients in each group. We present a flow diagram for the appropriate and safe follow-up for this specific group of patients. Cite this article: Bone Joint J 2016;98-B:1674-81.
The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. While migraine can be treated using CGRP antagonists that act peripherally, the relevant sites of CGRP action remain unknown. To address the role of CGRP both within and outside the central nervous system, we used CGRP-induced light aversive behavior in mice as a measure of migraine-associated photophobia. Peripheral (intraperitoneal, IP) injection of CGRP resulted in light aversive behavior in wild-type CD1 mice similar to aversion previously seen following central (intracerebroventricular, ICV) injection. The phenotype was also observed in C57BL/6J mice, although to a lesser degree and with more variability. Following IP CGRP, motility was decreased in the dark only, similar to motility changes following ICV CGRP. In addition, as with ICV CGRP, there was no general increase in anxiety as measured in an open field assay following IP CGRP. Importantly, two clinically effective migraine drugs, the 5-HT1B/D agonist sumatriptan and a CGRP-blocking monoclonal antibody, attenuated the peripheral CGRP-induced light aversion and motility behaviors. To begin to address the mechanism of peripheral CGRP action, we used transgenic CGRP-sensitized mice that have elevated levels of the CGRP receptor hRAMP1 subunit in nervous tissue (nestin/hRAMP1). Surprisingly, sensitivity to low light was not seen after IP CGRP injection, but was seen after ICV CGRP injection. These results suggest that CGRP can act in both the periphery and the brain by distinct mechanisms and that CGRP actions may be transmitted to the CNS via indirect sensitization of peripheral nerves.
The neuropeptide CGRP is a central player in migraine pathogenesis, yet its site(s) of action remain unknown. Some preclinical studies have pointed to central sites in the brain and brainstem. However, a peripheral site of action is indicated by the ability of intravenous CGRP to trigger migraine in humans and the efficacy of CGRP receptor antagonists that evidently do no penetrate the CNS in effective amounts. Resolving this issue is particularly important given recent clinical trials showing that anti-CGRP monoclonal antibodies can reduce and even prevent migraine attacks. In this study, we report that CGRP can act in both the brain and the periphery of the mouse to cause migraine-like photophobia by apparently distinct mechanisms.
Proteomes of even well characterized organisms still contain a high percentage of proteins with unknown or uncertain molecular and/or biological function. A significant fraction of those proteins are predicted to have catalytic properties. Here we aimed at identifying the function of the Saccharomyces cerevisiae Ydr109c protein and of its human homolog FGGY, both of which belong to the broadly conserved FGGY family of carbohydrate kinases. Functionally identified members of this family phosphorylate 3- to 7-carbon sugars or sugar derivatives, but the endogenous substrate of S. cerevisiae Ydr109c and human FGGY has remained unknown. Untargeted metabolomics analysis of an S. cerevisiae deletion mutant of YDR109C revealed ribulose as one of the metabolites with the most significantly changed intracellular concentration as compared to a wild-type strain. In human HEK293 cells, ribulose could only be detected when ribitol was added to the cultivation medium and under this condition, FGGY silencing led to ribulose accumulation. Biochemical characterization of the recombinant purified Ydr109c and FGGY proteins showed a clear substrate preference of both kinases for D-ribulose over a range of other sugars and sugar derivatives tested, including L-ribulose. Detailed sequence and structural analyses of Ydr109c and FGGY as well as homologs thereof furthermore allowed the definition of a 5-residue D-ribulokinase signature motif (TCSLV). The physiological role of the herein identified eukaryotic D-ribulokinase remains unclear, but we speculate that S. cerevisiae Ydr109c and human FGGY could act as metabolite repair enzymes, serving to re-phosphorylate free D-ribulose generated by promiscuous phosphatases from D-ribulose-5-phosphate. In human cells, FGGY can additionally participate in ribitol metabolism.
Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity we identified and cloned forty tomato genes encoding E2 proteins. Thioester assays indicated the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the forty tomato E2 into thirteen groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv. tomato (Pst) effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking-down the expression of group III E2 genes in Nicotiana benthamiana diminished AvrPtoB-promoted degradation of the Fen kinase and AvrPtoB suppression of host immunity-associated PCD. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, PCD induced by several ETI elicitors was not affected on group-III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBC11, 28, 29, 39 and 40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart.
Transforming growth factor β (TGF-β) family signaling dictates highly complex programs of gene expression responses, which are extensively regulated at multiple levels and vary depending on the physiological context. The formation, activation, and destruction of two major functional complexes in the TGF-β signaling pathway (i.e., the TGF-β receptor complexes and the Smad complexes that act as central mediators of TGF-β signaling) are direct targets for posttranslational regulation. Dysfunction of these complexes often leads or contributes to pathogenesis in cancer and fibrosis and in cardiovascular, and autoimmune diseases. Here we discuss recent insights into the roles of posttranslational modifications in the functions of the receptor-activated Smads in the common Smad4 and inhibitory Smads, and in the control of the physiological responses to TGF-β. It is now evident that these modifications act as decisive factors in defining the intensity and versatility of TGF-β responsiveness. Thus, the characterization of posttranslational modifications of Smads not only sheds light on how TGF-β controls physiological and pathological processes but may also guide us to manipulate the TGF-β responses for therapeutic benefits.
GPR40 is generally known to signal through Gq. However, in transfected cells, certain synthetic agonists can make the receptor signal also through Gs and cAMP (Hauge et al., 2015). Here we find that, in colonic crypt cultures, the GLP-1 secretion induced by such Gq + Gs GPR40 agonists is indeed inhibited by blockers of both Gq and Gs and is eliminated by combining these. This in contrast to Gq-only GPR40 agonists which only are affected by the Gq inhibitor. Importantly, Gq-only GPR40 agonists in combination with low doses of selective synthetic agonists for Gs coupled receptors, e.g. GPR119 and TGR5 provide more than additive GLP-1 secretion both ex vivo and in vivo in mice. It is concluded that under physiological circumstances triglyceride metabolites, i.e. long chain fatty acids and 2-monoacyl glycerol plus bile acids, act synergistically through their respective receptors, GPR40, GPR119 and TGR5 to stimulate GLP-1 secretion robustly by combining Gq and Gs signaling pathways.
Colon cancer is one of the most common malignancies and its etiology closely tied to dietary habits. Recent epidemiological data shows that colon cancer incidence is shifting to a much younger population. In this regard, some dietary components from a regular human meal might have various DNA-damaging compounds. Given that not every person endure cancer, the colonic malignancy develops throughout decades, and persistent DNA damage promotes cancer when induced at the proper intensity, a critical discussion of possible novel mechanisms by which carcinogens promote these tumors is urgently needed. Robust genomic sequencing analyses showed that low and late cell cycle expressed genes are prone to undergo mutation. Moreover, detection and repair mechanisms have a particular threshold to be activated throughout the G2/M phase, and reactivation of these devices during the M phase promotes genomic instability. Conditions of combined exposure to non-genotoxic concentrations of various carcinogens seem to act effectively through these weaknesses in genomic repair mechanisms. Therefore, we suggest that the natural tolerance of body defence mechanisms eventually become overwhelmed by the chronic exposure to different combinations and intensities of dietary mutagens leading to the high incidence of colon cancer in modern society.
In the present study we have synthesized a new class of 4-aminoquinolines and evaluated against Plasmodium falciparum in vitro (3D7-sensitive strain & K1-resistant strain) and Plasmodium yoelii in vivo (N-67 strain). Among the series, eleven compounds (5, 6, 7, 8, 9, 11, 12, 13, 14, 15 and 21) showed superior antimalarial activity against K1 strain as compared to CQ. In addition, all these analogues showed 100% suppression of parasitemia on day 4 in the in vivo mouse model against N-67 strain when administered orally. Further, biophysical studies suggest that this series of compounds act on heme polymerization target.
The ethical debate surrounding transplant practices questions our societies. International recommendations set out numerous precautions which must be taken to ensure that donors act with their free will. While in most countries, including France, organ donation is a voluntary and non-commercial act, a black market exists in the world resulting in the trafficking of organs and tragic transplant tourism.
Oxidative stress caused by a sharp growth of free radicals in the organism is a major cause underlying the occurrence of all kinds of malignant formations. Selenium is an important essential trace element found in selenoproteins in the form of selenocysteine, an amino acid differing from cysteine for the presence of selenium instead of sulfur and making such proteins highly active. To date the role of selenium has been extensively investigated through studying the functions of selenoproteins in carcinogenesis. Analysis of the obtained results clearly demonstrates that selenoproteins can act as oncosuppressors, but can also, on the contrary, favor the formation of malignant tumors.
Mephedrone is a β-ketoamphetamine belonging to the family of synthetic cathinones, an emerging class of designer drugs known for their hallucinogenic and psychostimulant properties as well as for their abuse potential. The aim of this review was to examine the emerging scientific literature on the possible mephedrone-induced neurotoxicity, yet not well defined due to the limited number of experimental studies, mainly carried on animal models. Relevant scientific articles were identified from international literature databases (Medline, Scopus, etc.) using the keywords: "Mephedrone", "4-MMC," "neurotoxicity," "neuropharmacology", "patents", "monoamine transporters" and "neurochemical effects". Of the 498 sources initially found, only 36 papers were suitable for the review. Neurotoxic effect of mephedrone on 5-HT and DA systems remains controversial. Although some studies in animal models reported no damage to DA nerve endings in the striatum and no significant changes in brain monoamine levels, some others suggested a rapid reduction in 5-HT and DA transporter function. Persistent serotonergic deficits were observed after binge like treatment in a warm environment and in both serotonergic and dopaminergic nerve endings at high ambient temperature. Oxidative stress cytotoxicity and an increase in frontal cortex lipid peroxidation were also reported. In vitro cytotoxic properties were also observed, suggesting that mephedrone may act as a reductant agent and can also determine changes in mitochondrial respiration. However, due to the differences in the design of the experiments, including temperature and animal model used, the results are difficult to compare. Further studies on toxicology and pharmacology of mephedrone are therefore necessary to establish an appropriate treatment for substance abuse and eventual consequences for public health.
In recent decades, several scientists focused their process towards nanoparticles synthesis by using various sustainable approaches. Cocos nucifera (C. nucifera) was one of the versatile trees in tropical regions which also can act as a thrust quencher in all over the world. Cocos nucifera coir was one of the waste by-products in all coconut-refining industries and with the help C. nucifera coir, Palladium nanoparticles (Pd NPs) were synthesized. Green-synthesized spherical-shape Pd NPs were over layered by secondary metabolites from C. nucifera coir extract and with an average particle size of 62 ± 2 nm, which were confirmed by morphological analysis. Eco-friendly mediated Pd NPs were further subjected to several biological applications like larvicidal against Aedes aegypti (A. aegypti) and anti-feedent, ovicidal, and oviposition deterrent against agricultural pest Callasobruchus maculates (C. maculates) and compared with C. nuciferacoir methanolic extract, which results in LC50 value of 288.88 ppm and LC90 value of 483.06 ppm using LSD-Tukey's test against dengue vector (A. aegypti). Cocos nucifera coir methanolic extract shows significant output while compared with Pd NPs towards anti-feedent assays; ovicidal activity and oviposition deterrent were discussed here.
Recent reports indicate that model-based iterative reconstruction methods may improve image quality in computed tomography (CT). One difficulty with these methods is the number of options available to implement them, including the selection of the forward projection model and the penalty term. Currently, the literature is fairly scarce in terms of guidance regarding this selection step, whereas these options impact image quality. Here, the authors investigate the merits of three forward projection models that rely on linear interpolation: the distance-driven method, Joseph's method, and the bilinear method. The authors' selection is motivated by three factors: (1) in CT, linear interpolation is often seen as a suitable trade-off between discretization errors and computational cost, (2) the first two methods are popular with manufacturers, and (3) the third method enables assessing the importance of a key assumption in the other methods.
One approach to evaluate forward projection models is to inspect their effect on discretized images, as well as the effect of their transpose on data sets, but significance of such studies is unclear since the matrix and its transpose are always jointly used in iterative reconstruction. Another approach is to investigate the models in the context they are used, i.e., together with statistical weights and a penalty term. Unfortunately, this approach requires the selection of a preferred objective function and does not provide clear information on features that are intrinsic to the model. The authors adopted the following two-stage methodology. First, the authors analyze images that progressively include components of the singular value decomposition of the model in a reconstructed image without statistical weights and penalty term. Next, the authors examine the impact of weights and penalty on observed differences.
Image quality metrics were investigated for 16 different fan-beam imaging scenarios that enabled probing various aspects of all models. The metrics include a surrogate for computational cost, as well as bias, noise, and an estimation task, all at matched resolution. The analysis revealed fundamental differences in terms of both bias and noise. Task-based assessment appears to be required to appreciate the differences in noise; the estimation task the authors selected showed that these differences balance out to yield similar performance. Some scenarios highlighted merits for the distance-driven method in terms of bias but with an increase in computational cost. Three combinations of statistical weights and penalty term showed that the observed differences remain the same, but strong edge-preserving penalty can dramatically reduce the magnitude of these differences.
In many scenarios, Joseph's method seems to offer an interesting compromise between cost and computational effort. The distance-driven method offers the possibility to reduce bias but with an increase in computational cost. The bilinear method indicated that a key assumption in the other two methods is highly robust. Last, strong edge-preserving penalty can act as a compensator for insufficiencies in the forward projection model, bringing all models to similar levels in the most challenging imaging scenarios. Also, the authors find that their evaluation methodology helps appreciating how model, statistical weights, and penalty term interplay together.
False killer whales (Pseudorca crassidens) depredate pelagic longlines in offshore Hawaiian waters. On January 28, 2015 a depredation event was recorded 14 m from an integrated GoPro camera, hydrophone, and accelerometer, revealing that false killer whales depredate bait and generate clicks and whistles under good visibility conditions. The act of plucking bait off a hook generated a distinctive 15 Hz line vibration. Two similar line vibrations detected at earlier times permitted the animal's range and thus signal source levels to be estimated over a 25-min window. Peak power spectral density source levels for whistles (4-8 kHz) were estimated to be between 115 and 130 dB re 1 μPa(2)/Hz @ 1 m. Echolocation click source levels over 17-32 kHz bandwidth reached 205 dB re 1 μPa @ 1 m pk-pk, or 190 dB re 1 μPa @ 1 m (root-mean-square). Predicted detection ranges of the most intense whistles are 10 to 25 km at respective sea states of 4 and 1, with click detection ranges being 5 times smaller than whistles. These detection range analyses provide insight into how passive acoustic monitoring might be used to both quantify and avoid depredation encounters.
Synthetic progestins act as endocrine disrupters in fish but their risk to the environment is not sufficiently known. Here, we focused on an unexplored antiandrogenic progestin, chlormadinone acetate (CMA), and the antiandrogenic progestin cyproterone acetate (CPA). The aim was to evaluate whether their in vitro interaction with human and rainbowfish (Melanotaenia fluviatilis) sex hormone receptors is similar. Furthermore, we investigated their activity in zebrafish (Danio rerio) eleuthero-embryos. First, we studied agonistic and antagonistic activities of CMA, CPA, and 17α-ethinylestradiol (EE2), in recombinant yeast expressing either the human progesterone (PGR), androgen (AR), or estrogen receptor. The same compounds were also investigated in vitro in a stable transfection cell system expressing rainbowfish nuclear steroid receptors. For human receptors, both progestins exhibited progestogenic, androgenic and antiestrogenic activity with no antiandrogenic or estrogenic activity. In contrast, interactions with rainbowfish receptors showed no progestogenic, but antiandrogenic, antiglucocorticoid, and some antiestrogenic activity. Thus, interaction with and transactivation of human and rainbowfish PGR and AR were distinctly different. Second, we analyzed transcriptional alterations in zebrafish eleuthero-embryos at 96 and 144h post fertilization after exposure to CPA, CMA, EE2, and binary mixtures of CMA and CPA with EE2, mimicking the use in oral contraceptives. CMA led to slight down-regulation of the ar transcript, while CPA down-regulated ar and pgr transcripts. EE2 exposure resulted in significant transcriptional alterations of several genes, including esr1, pgr, vtg1, cyp19b, and gonadotropins (fshb, lhb). The mixture activity of CMA and EE2 followed the independent action model, while CPA and EE2 mixtures showed additive action in transcriptional alterations. Third, we analyzed the interactions of binary mixtures of CMA and CPA, and of CMA and EE2 for their joint activity in vitro and in eleuthero-embryos. Both mixtures behaved according to the concentration addition model in their in vitro interaction with human and rainbowfish receptors, often showing antagonism. In zebrafish eleuthero-embryos, binary mixtures of CMA and EE2 showed the same expression patterns as EE2 alone, indicating an independent action in vivo. Our study demonstrates that CMA and CPA interact distinctly with human and rainbowfish receptors, suggesting that activities of these and possibly additional environmental steroids determined with yeast expressing human receptors cannot simply be translated to fish. The lack of agonistic activities of both progestins to rainbowfish PGR and AR is the probable reason for the low activity found in zebrafish eleuthero-embryos.
The oxidation of s-triazines (using atrazine (ATZ) as a model compound) by a solar-enhanced Fenton-like process involving persulfate and ferrous ion was studied. A flow-through tubular photoreactor was employed for the experiments. The solar-enhanced oxidative system involving ferrous ion and persulfate (Solar/S2O8(2-)/Fe(2+)) showed the highest ATZ degradation efficiency when compared with other treatments (unactivated S2O8(2-), Solar - sunlight only, S2O8(2-)/Fe(2+), Solar/S2O8(2-)). Complete degradation of ATZ and 20% reduction in total organic carbon (TOC) content were observed after 30min of the treatment. The in situ generated (•)ОН and SO4(-•) radicals were shown to be involved in ATZ oxidation using the radical scavengers methanol and tert-butyl alcohol. Furthermore, iron compounds were shown to act not only as catalysts but also as photo-sensitizers, as the introduction of ferrous ion into the reaction mixture led to an increased absorbance of the solution and expansion of the absorption spectrum into the longer wavelength spectral region.
Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.
Evidence of the efficacy of existing psychological interventions for self-management in diabetes is limited. The current study aimed at assessing the effects of group-based ACT on self-management of patients with T2DM, considering the moderating role of coping styles.
One hundred and six patients with type 2 diabetes were randomly assigned either to the education alone (n = 53) or to a combination of education and group-based acceptance and commitment therapy (n = 53) over a period of 10 sessions. In each group, 50 participants completed a 3 month follow-up assessment.
After 3 months, compared to patients who received education alone, those in the group-based acceptance and commitment therapy condition were more likely to use effective coping strategies, reported better diabetes self-care, and optimum glycated hemoglobin (HbA1C) levels in the target range.
Consideration of the role of coping style for a more accurate evaluation of the effects of acceptance and commitment therapy may be a useful addition to services provided for patients with type 2 diabetes.
Data on sepsis prevalence on the general wards is lacking on the UK and in the developed world. We conducted a multicentre, prospective, observational study of the prevalence of patients with sepsis or severe sepsis on the general wards and Emergency Departments (ED) in Wales. During the 24-hour study period all patients with NEWS≥3 were screened for presence of 2 or more SIRS criteria. To be eligible for inclusion, patients had to have a high clinical suspicion of an infection, together with a systemic inflammatory response (sepsis) and evidence of acute organ dysfunction and/or shock (severe sepsis). There were 5317 in-patients in the 24-hour study period. Data were returned on 1198 digital data collection forms on patients with NEWS≥3 of which 87 were removed, leaving 1111 for analysis. 146 patients had sepsis and 144 patients had severe sepsis. Combined prevalence of sepsis and severe sepsis was 5.5% amongst all in-patients. Patients with sepsis had significantly higher NEWS scores (3 IQR 3-4 for non-sepsis and 4 IQR 3-6 for sepsis patients, respectively). Common organ dysfunctions in severe sepsis were hypoxia (47%), hypoperfusion (40%) and acute kidney injury (25%). Mortality at 90 days was 31% with a median (IQR) hospital free stay of 78 (36-85) days. Screening for sepsis, referral to Critical Care and completion of Sepsis 6 bundle was low: 26%, 16% and 12% in the sepsis group. Multivariable logistic regression analysis identified higher National Early Warning Score, diabetes, COPD, heart failure, malignancy and current or previous smoking habits as independent variables suggesting the diagnosis of sepsis. We observed that sepsis is more prevalent in the general ward and ED than previously suggested before and that screening and effective treatment for sepsis and severe sepsis is far from being operationalized in this environment, leading to high 90 days mortality.
Dementia is a disease characterized by cognitive impairment and physical decline that worsens over time. Exercise is one lifestyle factor that has been identified as a potential means of reducing or delaying progression of the symptoms of dementia, maximizing function and independence. The purpose of this study was to explore physical therapists' (PTs) experiences and reflections on facilitating high-intensity functional exercise with older people living with dementia, in residential care home settings. The study used a qualitative design based on interviews, individually or in small groups, with seven PTs engaged as leaders in the training of older people with dementia. The interviews were analyzed with a modified Grounded Theory method with focus on constant comparisons. To increase trustworthiness the study used triangulation within investigators and member checking. The core category "Discover and act in the moment-learn over time" reflects how the PTs continuously developed their own learning in an iterative process. They built on previous knowledge to communicate with residents and staff and to tailor the high intensity training in relation to each individual at that time point. The category "Be on your toes" highlights how the PTs searched for sufficient information about each individual, before and during training, by eliciting the person's current status from staff and by interpreting the person's body language. The category "Build a bond with a palette of strategies" describes the importance of confirmation to build up trust and the use of group members and the room to create an interplay between exercise and social interaction. These findings highlight the continuous iterative process of building on existing knowledge, sharing and reflecting, being alert to any alterations needed for individuals that day, communication skills (both with residents and staff) and building a relationship and trust with residents in the effective delivery of high intensity functional exercise to older people living with dementia in care settings.
Accumulating studies have demonstrated that long noncoding RNAs (lncRNAs) act a crucial role in the development of tumors. However, the role of lncRNAs in lung cancer remains largely unknown. In this study, we demonstrated that theexpression of RMRP was upregulated in lung adenocarcinoma tissues compared to the matched adjacent normal tissues. Moreover, of 35 lung adenocarcinoma samples, RMRP expression was upregulated in 25 cases (25/35; 71.4%) compared to the adjacent normal tissues. We also showed that RMRP expression was upregulated in lung adenocarcinoma cell lines (A549, SPC-A1, H1299 and H23) compared to the bronchial epithelial cell line (16HBE). Ectopic expression of RMRP promoted lung adenocarcinoma cell proliferation, colony formation and invasion. In addition, overexpression of RMRP inhibited the miR-206 expression in the H1299 cell and increased the KRAS, FMNL2 and SOX9 expression, which were the target genes of miR-206. Re-expression of miR-206 reversed the RMRP-induced the H1299 cell proliferation and migration. Our data proved that RMRP acted as an oncogene LncRNA to promote the expression of KRAS, FMNL2 and SOX9 by inhibiting miR-206 expression in lung cancer. These data suggested that RMRP might serve as a therapeutic target in lung adenocarcinoma.
The world is rapidly becoming urban with the global population living in cities projected to double by 2050. This increase in urbanization poses new challenges for the spread and control of communicable diseases such as malaria. In particular, urban environments create highly heterogeneous socio-economic and environmental conditions that can affect the transmission of vector-borne diseases dependent on human water storage and waste water management. Interestingly India, as opposed to Africa, harbors a mosquito vector, Anopheles stephensi, which thrives in the man-made environments of cities and acts as the vector for both Plasmodium vivax and Plasmodium falciparum, making the malaria problem a truly urban phenomenon. Here we address the role and determinants of within-city spatial heterogeneity in the incidence patterns of vivax malaria, and then draw comparisons with results for falciparum malaria.
Statistical analyses and a phenomenological transmission model are applied to an extensive spatio-temporal dataset on cases of Plasmodium vivax in the city of Ahmedabad (Gujarat, India) that spans 12 years monthly at the level of wards. A spatial pattern in malaria incidence is described that is largely stationary in time for this parasite. Malaria risk is then shown to be associated with socioeconomic indicators and environmental parameters, temperature and humidity. In a more dynamical perspective, an Inhomogeneous Markov Chain Model is used to predict vivax malaria risk. Models that account for climate factors, socioeconomic level and population size show the highest predictive skill. A comparison to the transmission dynamics of falciparum malaria reinforces the conclusion that the spatio-temporal patterns of risk are strongly driven by extrinsic factors.
Climate forcing and socio-economic heterogeneity act synergistically at local scales on the population dynamics of urban malaria in this city. The stationarity of malaria risk patterns provides a basis for more targeted intervention, such as vector control, based on transmission 'hotspots'. This is especially relevant for P. vivax, a more resilient parasite than P. falciparum, due to its ability to relapse and the operational shortcomings of delivering a "radical cure".
The Affordable Care Act (ACA), signature legislation of President Obama, was arguably the most consequential and comprehensive health care reform since Medicare was introduced as part of President Lyndon B. Johnson's great society. It has been claimed that many of the law's reforms are now so integrated in the health system that full repeal would be impractical, while others including President Elect Trump have rejected that idea and called for full repeal and replacement claiming ACA law cannot be fixed. A tsunami of increasing regulatory burden over the past 8 years, the current health care milieu has moved independent practitioners towards hospital employment in great numbers. In addition, public opinion has been slowly climbing against ObamaCare with 54% of Americans now opposing the law.President Obama has indicated that the law has accomplished many of its goals, including increasing accessibility, affordability, and quality of health care. However, others have contradicted these assertions and described the ACA as "insurance for many with coverage for few." Some believe that the ACA might be more appropriately labeled the "Medicaid Expansion Act."There are multiple plans developed over the years by republican members of the congress; however, of significant consequence and importance are President-elect Trump's proposals and the plan developed by Speaker Paul Ryan to repeal and replace the ACA. The President-elect has described the problems he perceives with the ACA; rapidly rising premiums and deductibles, narrow networks, and limits of coverage imposed by health insurance companies. The President-elect has indicated that his goal will be to create a patient-centered health care system that promotes choice, quality, and affordability with health insurance and health care, and take any needed action to alleviate the burdens imposed on American families and businesses by law.Key words: Affordable Care Act, ObamaCare, Medicaid, exchanges, Trump plan, repeal, replace.
Geographic disparities persist in access to liver transplantation. Candidates with similar urgency experience varying opportunities for transplants across the US. Policymakers are poised to act and 1 proposal entails reorganizing the current OPTN of 11 regions into 8 districts. However, redistricting has the shortcomings that OPOs are disconnected from their immediate neighbors by district borders and that it is not easily responsive to uncertainty resulting from variability in donor and listing rates.
We introduce the notion of an OPO's neighborhood - a collection of DSAs surrounding the OPO that acts as the OPO's region in the current local-regional-national framework. Districts and concentric circles are special cases. We design 58 neighborhoods for the DSAs with several attractive properties and optimize them to balance supplies and demands using 10 years of OPTN data. We conduct a simulation experiment comparing current allocation, redistricting, and neighborhoods under current sharing policies with respect to the following metrics: total mortalities, DSA-average MELD at transplant, DSA-average MELD standard deviation, and average organ transport distance. LSAM cannot accommodate neighborhoods, so we programmed a discrete-event simulator, LivSim, to approximate LSAM.
We exhibited a neighborhood solution. Compared to the current allocation, simulation results showed that neighborhoods reduce the DSA-average MELD standard deviation by 29% and save about 65 lives annually. Compared to redistricting, the neighborhoods had a smaller average transport distances that were more uniform across DSAs, saved about 20 additional lives, and reduced DSA-average MELD standard deviation by an additional 17%.
Alternatives to redistricting with desirable properties and performance are possible and should be considered.
Orexin plays an important role in pain modulation. Orexin-1 and orexin-2 receptors (Ox1r and Ox2r) are found at high density in the ventrolateral periaqueductal gray matter (vlPAG). Our previous study showed that chemical stimulation of the lateral hypothalamus with carbachol induces antinociception in the tail-flick test, a model of acute pain, and Ox1r-mediated antinociception in the vlPAG is modulated by the activity of vlPAG CB1 receptors. In the current study, TCS OX2 29, an Ox2r antagonist (5, 15, 50, 150, and 500 nmol/l), was microinjected into the vlPAG 5 min before the administration of carbachol (125 nmol/l). TCS OX2 29 dose dependently reduced carbachol-induced antinociception. In a second set of experiments, animals were treated with carbachol 5 min after intra-vlPAG administration of 15 nmol/l TCS OX2 29 and 1 nmol/l AM251 (a selective CB1 receptor antagonist), or 150 nmol/l TCS OX2 29 and 10 nmol/l AM251. The findings showed that the antinociceptive effect of orexin is partially mediated by activation of vlPAG Ox2 receptors. Furthermore, the administration of ineffective doses of Ox2 and CB1 receptor antagonists reduced the lateral hypothalamus-induced antinociception. It seems that Ox2 and CB1 receptors act through different pathways and Ox2r-mediated antinociception is not dependent on CB1 receptor activity.
Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography-mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.
Scaffolds produced by electrospinning act as supports for cell proliferation and differentiation, improved through the release of neurotrophic factors. The objective of this study was to develop aligned and random nanofiber scaffolds with and without nerve growth factor to evaluate the potential of mesenchymal stem cells (MSCs) for neural differentiation. Nanofiber morphology, diameter, degradability, cell morphology, adhesion, proliferation, viability, cytotoxicity, and neural differentiation were performed to characterize the scaffolds. The expression for nestin, β-III tubulin, and neuron-specific enolase was also evaluated. The scaffolds demonstrated a satisfactory environment for MSC growth, being nontoxic. The MSCs cultivated on the scaffolds were able to adhere and proliferate. The evaluation of neural differentiation indicated that in all groups of scaffolds the MSCs were able to upregulate neural gene expression.
This final rule implements provisions of the Affordable Care Act that expand access to health coverage through improvements in Medicaid and coordination between Medicaid, CHIP, and Exchanges. This rule finalizes most of the remaining provisions from the "Medicaid, Children's Health Insurance Programs, and Exchanges: Essential Health Benefits in Alternative Benefit Plans, Eligibility Notices, Fair Hearing and Appeal Processes for Medicaid and Exchange Eligibility Appeals and Other Provisions Related to Eligibility and Enrollment for Exchanges, Medicaid and CHIP, and Medicaid Premiums and Cost Sharing; Proposed Rule" that we published in the January 22, 2013, Federal Register. This final rule continues our efforts to assist states in implementing Medicaid and CHIP eligibility, appeals, and enrollment changes required by the Affordable Care Act.
The Administrator of the Drug Enforcement Administration is issuing this final order to temporarily schedule the synthetic opioid, N-(1-phenethylpiperidin-4-yl)-N-phenylfuran-2-carboxamide (furanyl fentanyl), and its isomers, esters, ethers, salts and salts of isomers, esters and ethers, into schedule I pursuant to the temporary scheduling provisions of the Controlled Substances Act. This action is based on a finding by the Administrator that the placement of furanyl fentanyl into schedule I of the Controlled Substances Act is necessary to avoid an imminent hazard to the public safety. As a result of this order, the regulatory controls and administrative, civil, and criminal sanctions applicable to schedule I controlled substances will be imposed on persons who handle (manufacture, distribute, reverse distribute, import, export, engage in research, conduct instructional activities or chemical analysis, or possess), or propose to handle, furanyl fentanyl.
The Food and Drug Administration (FDA or Agency) is amending its nonprescription (over-the-counter or OTC) drug regulations. This final rule supplements the time and extent application (TEA) process for OTC drugs by establishing timelines and performance metrics for FDA's review of non-sunscreen TEAs, as required by the Sunscreen Innovation Act (SIA). It also amends the existing TEA process to include filing determination and withdrawal provisions to make the TEA process more efficient.
This final rule with comment period revises the Medicare hospital outpatient prospective payment system (OPPS) and the Medicare ambulatory surgical center (ASC) payment system for CY 2017 to implement applicable statutory requirements and changes arising from our continuing experience with these systems. In this final rule with comment period, we describe the changes to the amounts and factors used to determine the payment rates for Medicare services paid under the OPPS and those paid under the ASC payment system. In addition, this final rule with comment period updates and refines the requirements for the Hospital Outpatient Quality Reporting (OQR) Program and the ASC Quality Reporting (ASCQR) Program. Further, in this final rule with comment period, we are making changes to tolerance thresholds for clinical outcomes for solid organ transplant programs; to Organ Procurement Organizations (OPOs) definitions, outcome measures, and organ transport documentation; and to the Medicare and Medicaid Electronic Health Record Incentive Programs. We also are removing the HCAHPS Pain Management dimension from the Hospital Value-Based Purchasing (VBP) Program. In addition, we are implementing section 603 of the Bipartisan Budget Act of 2015 relating to payment for certain items and services furnished by certain off-campus provider-based departments of a provider. In this document, we also are issuing an interim final rule with comment period to establish the Medicare Physician Fee Schedule payment rates for the nonexcepted items and services billed by a nonexcepted off-campus provider-based department of a hospital in accordance with the provisions of section 603.
African Americans with head and neck squamous cell carcinoma (HNSCC) have a lower survival rate than whites. This study investigated the functional importance of ancestry-informative single-nucleotide polymorphisms (SNPs) in HNSCC and also examined the effect of functionally important genetic elements on racial disparities in HNSCC survival.
Ancestry-informative SNPs, RNA sequencing, methylation, and copy number variation data for 316 oral cavity and laryngeal cancer patients were analyzed across 178 DNA repair genes. The results of expression quantitative trait locus (eQTL) analyses were also replicated with a Gene Expression Omnibus (GEO) data set. The effects of eQTLs on overall survival (OS) and disease-free survival (DFS) were evaluated.
Five ancestry-related SNPs were identified as cis-eQTLs in the DNA polymerase β (POLB) gene (false discovery rate [FDR] < 0.01). The homozygous/heterozygous genotypes containing the African allele showed higher POLB expression than the homozygous white allele genotype (P < .001). A replication study using a GEO data set validated all 5 eQTLs and also showed a statistically significant difference in POLB expression based on genetic ancestry (P = .002). An association was observed between these eQTLs and OS (P < .037; FDR < 0.0363) as well as DFS (P = .018 to .0629; FDR < 0.079) for oral cavity and laryngeal cancer patients treated with platinum-based chemotherapy and/or radiotherapy. Genotypes containing the African allele were associated with poor OS/DFS in comparison with homozygous genotypes harboring the white allele.
Analyses show that ancestry-related alleles could act as eQTLs in HNSCC and support the association of ancestry-related genetic factors with survival disparities in patients diagnosed with oral cavity and laryngeal cancer. Cancer 2016. © 2016 American Cancer Society.
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light-induced protein release occurs in less than a second, and the half-life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.
The use of femtosecond lasers to introduce controlled stress states has recently been demonstrated in silica glass. We use this technique, in combination with chemical etching, to generate and control stress-induced birefringence over a well-defined region of interest, demonstrating direct-write wave plates with precisely tailored retardance levels. This tailoring enables the fabrication of laser-written polarization optics that can be tuned to any wavelength for which silica is transparent, and with a clear aperture free of any laser modifications. Using this approach, we achieve sufficient retardance to act as a quarter-wave plate. The stress distribution within the clear aperture is analyzed and modeled, providing a generic template that can be used as a set of design rules for laser-machined polarization devices.
Single-walled carbon nanotubes (SWCNTs) are thoroughly purified and dispersed in an aqueous solution of high molecular weight poly-L-lysine (pLlys). Human intestinal epithelial Caco-2/TC7 cells are incubated with the SWCNT dispersions in pLlys, and their effects on cell viability are studied by image flow cytometry. No significant changes are observed in the cell culture wells up to pLlys concentrations of 10 μg ml(-1). However, high mortality is detected at pLlys concentrations of 100 μg ml(-1). The presence of oxygen-free SWCNTs does not modify the effects of pLlys on cell cultures at any of the tested concentrations (≤1 μg ml(-1)). In addition, SWCNTs having an 8 wt.% of surface oxygen are tested with identical results. Thus, purified SWCNTs, even bearing oxygen functional groups, act as inert particles in the cell culture medium. This result supports the applicability of SWCNTs as carriers in pharmacological formulations against digestive tract diseases.
Present study utilised textile soil isolated bacterium Pseudomonas stutzeri to synthesise extracellular silver nanoparticles (AgNPs) under optimised conditions. The synthesised AgNPs were characterised using ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Optimisation showed AgNPs synthesis within 8 h using 2mM Ag nitrate at pH9, temperature 80°C and maximum absorbance toward 400 nm. TEM analysis revealed spherical shape AgNPs and reduction in size upto 8 nm was observed under optimised conditions. FTIR spectra confirmed presence of proteins bound to AgNPs act as reducing agent. AgNPs showed strong antibacterial activity against multi-drug resistant (MDR) Escherichia coli and Klebsiella pneumoniae as demonstrated by disc diffusion and colony forming unit assays. Zone of inhibition increased with increasing concentration of AgNPs with maximum of 19 mm against E. coli and 17 mm against K. pneumoniae at concentration of 2 μg/disc. Furthermore, AgNPs did not show any cytotoxic effects on human epithelial cells as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay even at 2 μg/ml concentration of AgNPs. The results of the present study suggest that AgNPs can be synthesised rapidly under optimised conditions and show strong antimicrobial property against MDR pathogens without having toxicity effect on human epithelial cells.
Meal skipping rates may be highest during young adulthood, a period of transition and development. Although these dietary behaviours may increase future risk of chronic disease, limited research has investigated correlates of meal skipping in young adults.
A systematic literature search was conducted to identify studies that investigated correlates of meal skipping behaviours in young adults (aged 18-30 years). EBSCO host, MEDLINE Complete, Global Health, Scopus, EMBASE, Web of Science and Informit platforms were searched for eligible articles. Correlates were defined as any factor that was either associated with meal skipping or was self-reported by the participant to have an influence on meal skipping. Randomised controlled trials, prospective cohort studies, case-control studies, nested case-control studies, cross-sectional studies, and longitudinal studies were eligible for inclusion.
Three-hundred and thirty-one articles were identified, 141 full-text articles assessed for eligibility, resulting in 35 included studies. Multiple methodological and reporting weaknesses were apparent in the reviewed studies with 28 of the 35 studies scoring a negative rating in the risk of bias assessment. Meal skipping (any meal), defined as the skipping of any meal throughout the day, was reported in 12 studies with prevalence ranging between 5 and 83%. The remaining 25 studies identified specific meals and their skipping rates, with breakfast the most frequently skipped meal 14-88% compared to lunch 8-57% and dinner 4-57%. Lack of time was consistently reported as an important correlate of meal skipping, compared with correlates such as cost and weight control, while sex was the most commonly reported associated correlate. Breakfast skipping was more common among men while lunch or dinner skipping being more common among women.
This review is the first to examine potential correlates of meal skipping in young adults. Future research would benefit from stronger design and reporting strategies, using a standardised approach for measuring and defining meal skipping.
The Administrator of the Drug Enforcement Administration is issuing this final order to temporarily schedule the synthetic opioid, 3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (also known as U-47700), and its isomers, esters, ethers, salts and salts of isomers, esters and ethers, into schedule I pursuant to the temporary scheduling provisions of the Controlled Substances Act. This action is based on a finding by the Administrator that the placement of U-47700 into schedule I of the Controlled Substances Act is necessary to avoid an imminent hazard to the public safety. As a result of this order, the regulatory controls and administrative, civil, and criminal sanctions applicable to schedule I controlled substances will be imposed on persons who handle (manufacture, distribute, reverse distribute, import, export, engage in research, conduct instructional activities or chemical analysis, or possess), or propose to handle, U-47700.
Challenging conditions experienced early in life, such as a restricted diet, can detrimentally affect key life-history traits. Individuals can reduce these costs by delaying their sexual maturation, albeit at the price of the later onset of breeding, to eventually reach the same adult size as individuals that grow up in a benevolent environment. Delayed maturation can, however, still lead to other detrimental morphological and physiological changes that become apparent later in adulthood (e.g. shorter lifespan, faster senescence). In general, research focuses on the naturally selected costs of a poor early diet. In mosquitofish (Gambusia holbrooki), males with limited food intake early in life delay maturation to reach a similar adult body size to their well-fed counterparts ('catch-up growth'). Here we tested whether a poor early diet is costly due to the reduced expression of sexually selected male characters, namely genital size and ejaculate traits.
We found that a male's diet early in life significantly influenced his sperm reserves and sperm replenishment rate. Shortly after maturation males with a restricted early diet had significantly lower sperm reserves and slower replenishment rates than control diet males, but this dietary difference was no longer detectable in older males.
Although delaying maturation to reach the same body size as well fed juveniles can ameliorate some costs of a poor start in life, our findings suggest that costs might still arise because of sexual selection against these males. It should be noted, however, that the observed effects are modest (Hedges' g = 0.20-0.36), and the assumption that lower sperm production translates into a decline in fitness under sperm competition remains unconfirmed.
The Medicare Access and CHIP Reauthorization Act of 2015 (MACRA) repeals the Medicare sustainable growth rate (SGR) methodology for updates to the physician fee schedule (PFS) and replaces it with a new approach to payment called the Quality Payment Program that rewards the delivery of high-quality patient care through two avenues: Advanced Alternative Payment Models (Advanced APMs) and the Merit-based Incentive Payment System (MIPS) for eligible clinicians or groups under the PFS. This final rule with comment period establishes incentives for participation in certain alternative payment models (APMs) and includes the criteria for use by the Physician-Focused Payment Model Technical Advisory Committee (PTAC) in making comments and recommendations on physician-focused payment models (PFPMs). Alternative Payment Models are payment approaches, developed in partnership with the clinician community, that provide added incentives to deliver high-quality and cost-efficient care. APMs can apply to a specific clinical condition, a care episode, or a population. This final rule with comment period also establishes the MIPS, a new program for certain Medicare-enrolled practitioners. MIPS will consolidate components of three existing programs, the Physician Quality Reporting System (PQRS), the Physician Value-based Payment Modifier (VM), and the Medicare Electronic Health Record (EHR) Incentive Program for Eligible Professionals (EPs), and will continue the focus on quality, cost, and use of certified EHR technology (CEHRT) in a cohesive program that avoids redundancies. In this final rule with comment period we have rebranded key terminology based on feedback from stakeholders, with the goal of selecting terms that will be more easily identified and understood by our stakeholders.
This rule will better prevent and protect survivors of family violence, domestic violence, and dating violence, by clarifying that all survivors must have access to services and programs funded under the Family Violence Prevention and Services Act. More specifically, the rule enhances accessibility and non-discrimination provisions, clarifies confidentiality rules, promotes coordination among community-based organizations, State Domestic Violence Coalitions, States, and Tribes, as well as incorporates new discretionary grant programs. Furthermore, the rule updates existing regulations to reflect statutory changes made to the Family Violence Prevention and Services Act, and updates procedures for soliciting and awarding grants. The rule also increases clarity and reduces potential confusion over statutory and regulatory standards. The rule codifies standards already used by the program in the Funding Opportunity Announcements and awards, in technical assistance, in reporting requirements, and in sub-regulatory guidance.
This final rule implements section 702 (c) of the Carl Levin and Howard P. "Buck" McKeon National Defense Authorization Act for Fiscal Year 2015 which states that beginning October 1, 2015, the pharmacy benefits program shall require eligible covered beneficiaries generally to refill non-generic prescription maintenance medications through military treatment facility pharmacies or the national mail-order pharmacy program. An interim final rule is in effect. Section 702(c) of the National Defense Authorization Act for Fiscal Year 2015 also terminates the TRICARE For Life Pilot Program on September 30, 2015. The TRICARE For Life Pilot Program described in section 716(f) of the National Defense Authorization Act for Fiscal Year 2013, was a pilot program which began in March 2014 requiring TRICARE For Life beneficiaries to refill non-generic prescription maintenance medications through military treatment facility pharmacies or the national mail-order pharmacy program. TRICARE for Life beneficiaries are those enrolled in the Medicare wraparound coverage option of the TRICARE program. This rule includes procedures to assist beneficiaries in transferring covered prescriptions to the mail order pharmacy program.
The Food and Drug Administration (FDA, the Agency, or we) is amending its regulation on uses of ozone-depleting substances (ODSs), including chlorofluorocarbons (CFCs), to remove the designation for certain products as "essential uses" under the Clean Air Act. Essential-use products are exempt from the ban by FDA on the use of CFCs and other ODS propellants in FDA-regulated products and from the ban by the Environmental Protection Agency (EPA) on the use of ODSs in pressurized dispensers. The products that will no longer constitute an essential use are: Sterile aerosol talc administered intrapleurally by thoracoscopy for human use and metered-dose atropine sulfate aerosol human drugs administered by oral inhalation. FDA is taking this action because alternative products that do not use ODSs are now available and because these products are no longer being marketed in versions that contain ODSs.
The influence of π-conjugation structural changes on photoinduced electron transfer (PET) and intramolecular charge transfer (ICT) processes in π-conjugated donor (D)-acceptor (A) dyads (D-π-A) was investigated. Three types of D-π-A dyads were prepared through the modification of the structure of their π-conjugated linker, including D-π-A (1) and D-πtw-A (2) having a twisted π-conjugation, and D-π-Si-π-A (3) with a π-conjugation severed by a Si-atom. In these dyads, carbazole (Cz) and oxadiazole (Oz) moieties act as an electron donor and acceptor, respectively. The emission maxima of dyads 1 and 3 red-shifted with the increase in polarity, which could be attributed to the ICT process. The fluorescence lifetimes of dyads 1 and 3 were 2.64 and 4.29 ns in CH2Cl2, respectively. In contrast, dyad 2 showed dual emission at 350 and 470 nm in CH2Cl2. The emission of dyad 2 at 380 nm corresponded to the monomer fluorescence in the locally excited state. Moreover, the emission at 470 nm increased simultaneously with the diminishing of the fluorescence at 380 nm. This emission band can be assigned as the intramolecular exciplex emission, and showed a strong solvatochromic shift. The low emission quantum yield (<3%) of dyad 2 is due to the PET process. In dyad 2, the cationic and anionic radical species generated by the PET process were confirmed by femtosecond transient absorption (fs-TA) spectroscopy. Upon photoexcitation at 290 or 340 nm, the A or D moieties can be selectively excited. Upon excitation at 290 nm, the acceptor moiety can be excited to the (1)A* state, thus the photoinduced hole transfer (PHT) takes place from (1)A* to D through the HOMO levels within a few picoseconds. On the other hand, when the donor moiety is excited at 340 nm, the PET process occurs from (1)D* to A. Based on the fs-TA studies, it was found that the dynamics and mechanisms for the electron (or charge) transfer were strongly affected by the variation of the π-conjugation of the linker. Herein, we can conclude that the PET and ICT processes are strongly influenced by the π-conjugation properties and their mechanisms are also affected by whether selective excitation of the donor or acceptor moiety occurs. Moreover, unit electron transfers (PET or PHT) were observed dominantly in the dyads having severed/twisted linkers in π-conjugation. However, dyad 1 possessing a well-conjugated linker showed a partial charge transfer character.
In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.
Classical methods for enhancing the electromagnetic field from substrates for spectroscopic applications, such as surface-enhanced Raman spectroscopy (SERS), have involved the generation of hotspots through directed self-assembly of nanoparticles or by patterning nanoscale features using expensive nanolithography techniques. A novel large-area, cost-effective soft lithographic technique involving glancing angle deposition (GLAD) of silver on polymer gratings is reported here. This method produces hierarchical nanostructures with high enhancement factors capable of analyzing single-molecule SERS. The uniform ordered and patterned nanostructures provide extraordinary field enhancements that serve as excitatory hotspots and are herein interrogated by SERS. The high spatial homogeneity of the Raman signal and signal enhancement over a large area from a self-assembled monolayer (SAM) of 2-naphthalenethiol demonstrated the uniformity of the hotspots. The enhancement was shown to have a critical dependence on the underlying nanostructure via the surface energy landscape and GLAD angles for a fixed deposition thickness, as evidenced by atomic force microscopy and scanning electron microscopy surface analysis of the substrate. The nanostructured surface leads to an extremely concentrated electromagnetic field at sharp nanoscale peaks, here referred to as 'nano-protrusions', due to the coupling of surface plasmon resonance (SPR) with localized SPR. These nano-protrusions act as hotspots which provide Raman enhancement factors as high as 10(8) over a comparable SAM on silver. Comparison of our substrate with the commercial substrate Klarite™ shows higher signal enhancement and minimal signal variation with hotspot spatial distribution. By using the proper plasmon resonance angle corresponding to the laser source wavelength, further enhancement in signal intensity can be achieved. Single-molecule Raman spectra for rhodamine 6G are obtained from the best SERS substrate (a GLAD angle of 60°). The single-molecule spectrum is invariant over the substrate, due to the patterned ordered nanostructures (nano-protrusions).
Alcohol dependence represents a leading cause of mortality and morbidity. Understanding the variables that contribute to this diagnosis and its severity is critical. An overlap between factors that may predispose people to become obese and those that may increase the risk of alcohol dependence may exist. However, data in the literature are not conclusive. Therefore, this study aimed to identify the association between alcohol dependence and obesity-related factors, including biochemical and genetic factors.
In a case-control study with 829 participants, factors involved with metabolism and obesity were assessed, including biochemical lipid and liver markers, and the fat mass and obesity-associated (FTO) single nucleotide polymorphism (SNP) rs8050136.
Increased triglycerides, having one or two minor A alleles for rs8050136 and being a smoker were associated with increased risk of alcohol dependence, while increased low-density lipoprotein cholesterol was associated with decreased risk. In addition, having abnormal gamma-glutamyl transferase and being female were factors associated with an increased severity of alcohol dependence.
Our preliminary findings suggest a link between alcohol dependence and obesity-related biochemical and genetic factors. Future studies are needed to better understand if these factors may play a predictive role and/or may act as biomarkers for treatment response.
Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) -induced responses in human DCs and subsequent T helper type 1 (Th1) -type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti-inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte-derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS-induced secretion of interleukin-6 (IL-6) and IL-12p70, while enhancing secretion of IL-10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon-γ production was markedly suppressed, while IL-4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co-pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor-α, IL-6 and IL-12p70 while increasing IL-10 secretion. Moreover, both TsSP and PAC alone induced Th2-associated OX40L expression in DCs, and together synergized to up-regulate OX40L. These data suggest that PAC induce an anti-inflammatory phenotype in human DCs that selectively down-regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.
Coral reef ecosystems are disturbed in tandem by climatic and anthropogenic stressors. A number of factors act synergistically to reduce the live coral cover and threaten the existence of reefs. Continuous monitoring of the coral communities during 2012-2014 captured an unprecedented growth of macroalgae as a bloom at Gulf of Mannar (GoM) and Palk Bay (PB) which are protected and unprotected reefs, respectively. The two reefs varying in their protection level enabled to conduct an assessment on the response of coral communities and their recovery potential during and after the macroalgal bloom. Surveys in 2012 revealed a live coral cover of 36.8 and 14.6% in GoM and PB, respectively. Live coral cover was lost at an annual rate of 4% in PB due to the Caulerpa racemosa blooms that occurred in 2013 and 2014. In GoM, the loss of live coral cover was estimated to be 16.5% due to C. taxifolia bloom in 2013. Tissue regeneration by the foliose and branching coral morphotypes aided the recovery of live coral cover in GoM, whereas the chances for the recovery of live coral cover in PB reef were low, primarily due to frequent algal blooms, and the existing live coral cover was mainly due to the abundance of slow-growing massive corals. In combination, results of this study suggested that the recovery of a coral reef after a macroalgal bloom largely depends on coral species composition and the frequency of stress events. A further study linking macroalgal bloom to its specific cause is essential for the successful intervention and management.
This study explored the relationship between race and two key aspects of patient engagement-patient activation and working alliance-among a sample of African-American and White veterans (N = 152) seeking medication management for mental health conditions. After adjusting for demographics, race was significantly associated with patient activation, working alliance, and medication adherence scores. Patient activation was also associated with working alliance. These results provide support for the consideration of race and ethnicity in facilitating patient engagement and patient activation in mental healthcare. Minority patients may benefit from targeted efforts to improve their active engagement in mental healthcare.
Basal bodies are essential microtubule-based structures that template, anchor, and orient cilia at the cell surface. Cilia act primarily in the generation of directional fluid flow and sensory reception, both of which are utilized for a broad spectrum of cellular processes. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies of the ciliate Tetrahymena thermophila revealed important roles for two centrin family members in basal body assembly, separation of new basal bodies, and stability. Here, we characterize the basal body function of a centrin-binding protein, Sfr1, in Tetrahymena. Sfr1 is part of a large family of 13 proteins in Tetrahymena that contain Sfi1 repeats (SFRs), a motif originally identified in Saccharomyces cerevisiae Sfi1 that binds centrin. Sfr1 is the only SFR protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. In addition, Sfr1 resides predominantly at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Complete genomic knockout of SFR1 (sfr1Δ) causes a significant increase in both cortical row basal body density and the number of cortical rows, contributing to an overall overproduction of basal bodies. Reintroduction of Sfr1 into sfr1Δ mutant cells leads to a marked reduction of cortical row basal body density and the total number of cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study reveals an inhibitory role for Sfr1, and potentially centrins, in Tetrahymena basal body production. IMPORTANCE Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood. Despite the extensive study of centrins in Tetrahymena thermophila, little is known about how centrin-binding proteins contribute to centrin's roles in basal body assembly, stability, and orientation. The sole previous study of the large centrin-binding protein family in Tetrahymena revealed a role for Sfr13 in the stabilization and separation of basal bodies. In this study, we found that Sfr1 localizes to all Tetrahymena basal bodies and complete genetic deletion of SFR1 leads to overproduction of basal bodies. The uncovered inhibitory role of Sfr1 in basal body production suggests that centrin-binding proteins, as well as centrins, may influence basal body number both positively and negatively.
Pancreatic cancer is characterized by K-Ras mutations in over 90% of the cases. The mutations make the tumors aggressive and resistant to current therapies resulting in very poor prognoses. Valiant efforts to drug mutant K-Ras and related proteins for the treatment of cancers with Ras mutations have been elusive. The need thus persists for therapies to target and suppress the hyperactive K-Ras mutant proteins to normal levels of activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were designed to disrupt polyisoprenylated protein metabolism and/or functions. The potential for PCAIs to serve as targeted anticancer agents for pancreatic cancer was evaluated in pancreatic ductal adenocarcinoma (PDAC) cell lines expressing mutant (MIAPaCa-2 and Panc-1) and wild type (BxPC-3) K-Ras proteins. The PCAIs inhibited MIAPaCa-2 and BxPC-3 cell viability and induced apoptosis with EC50 values as low as 1.9 µM. The PCAIs, at 0.5 µM, inhibited MIAPaCa-2 cell migration by 50%, inhibited colony formation and disrupted F-actin filament organization. The PCAIs blocked MIAPaCa-2 cell progression at the G0/G1 phase. These results reveal that the PCAIs disrupt pertinent biological processes that lead to pancreatic cancer progression and thus have the potential to act as targeted effective treatments for pancreatic cancer.
Neointimal hyperplasia is a common pathological characteristic in diverse vascular remodeling diseases. The inflammatory response that follows vascular injury plays an important role in intimal hyperplasia. Tongxinluo (TXL), a traditional Chinese medicine, can ameliorate neointimal formation via suppressing vascular inflammatory response induced by vascular injury. However, the mechanisms underlying anti-inflammatory and anti-intimal hyperplasia of TXL are still not fully understood. The aim of present study was to examine whether the expression and post-translational modification of KLF5 were involved in the vasoprotective effects of TXL. In vivo, TXL inhibited neointimal formation induced by carotid artery injury. In vitro, TNF-α treatment of macrophages resulted in the increased proliferation and migration, but the effects of TNF-α on macrophages were blocked by TXL treatment. Next, KLF5 expression was up-regulated by carotid artery injury in vivo, as well as by exposure of macrophages to TNF-α in vitro, whereas TXL treatment abrogated the up-regulation of KLF5 by TNF-α or vascular injury. Intimal hyperplasia was strongly reduced in macrophage-specific KLF5 knockout (KLF5(ly-/-)) mice, indicating that TXL inhibits intimal hyperplasia by suppression of KLF5 expression. Furthermore, besides down-regulating KLF5 expression in macrophages, TXL also regulated KLF5 stability by ubiquitination and sumoylation of KLF5. Finally, TNF-α induced KLF5 sumoylation via PI3K/Akt signaling, whereas TXL inhibited Akt phosphorylation induced by TNF-α. We conclude that the multiple ingredients in TXL may act on different targets, which in turn generates a range of actions that manifest as a comprehensively vasoprotective effect.
This study aimed to investigate the potential molecular mechanism underlying the T3 induced vascular calcification and phenotype transformation of vascular smooth muscle cells (VSMCs).
Rat thoracic aortic smooth muscle cells (A7r5) were cultured in vitro and randomly assigned into normal control group, calcification group, T3 group and inhibitor group.
When compared with normal control group, the osteocalcin content, ALP activity, Osterix and Runx2 mRNA expression and OPN protein expression increased significantly (P<0.01), and the protein expression of SMα and SM22α reduced dramatically in A7r5 cells of calcification group (P<0.01). After T3 treatment, the osteocalcin content and ALP activity reduced markedly, mRNA expression of Osterix and Runx2 and OPN protein expression reduced significantly. However, MMI (inhibitor of T3) was able to block the above effects of T3. When compared with calcification group, Osterix and Runx2 mRNA expression and OPN protein expression increased markedly (P<0.01). In addition, the protein expression of ERK1/2, p-ERK, Akt and p-Akt increased significantly in calcification group. In the presence of integrin αvβ3/ERK blocker (PD98059) and/or PI3K/Akt antagonist (LY294002), T3 was still able to inhibit the calcification, and this effect was similar to that after treatment with inhibitors alone. Moreover, LY294002 had a better inhibitory effect as compared to PD98059.
T3 may act on PI3K/Akt signaling pathway to inhibit the phenotype transformation of VSMC, which then suppresses the calcium/phosphate induced calcification of rat VSMCs. Thus, T3 is an endogenous molecule that can protect the blood vessels against calcification.
Sphingosine kinase 1 (SphK1) is over-expressed in many cancers and therefore serves as a biomarker for cancer prognosis. SKI-5C is a new SphK1 inhibitor, and until now its molecular function in Wilms' tumor cells remained unknown. Here, using CCK-8 and nude mice experiments we assessed cell growth in Wilms' tumor cell lines (SK-NEP-1 and G401) in vitro and in vivo. We demonstrated that SphK1 is highly expressed in SK-NEP-1 and G401 cells, and through annexin V/propidium iodide staining and flow cytometry analysis, we detected cell apoptosis. Treatment with SKI-5C inhibited proliferation and induced apoptosis of SK-NEP-1 and G401 cells in a dose-dependent manner. Moreover, SKI-5C treatment inhibited the growth of SK-NEP-1 xenograft tumors in nude mice, with few side effects. Our microarray analysis revealed that SKI-5C-treated SK-NEP-1 cells mostly downregulated PRKACA and significantly inhibited phosphorylation of ERK1/2 and NF-κB p65. These results imply that SKI-5C induces apoptosis of SK-NEP-1 cells through the PRKACA/MAPK/NF-κB pathway. While, further research is required to determine the underlying details, these results provide new clues for the molecular mechanism of cell death induced by SKI-5C and suggest that SKI-5C may act as new candidate drug for Wilms' tumor.
Surrogate markers simple enough to be used by primary care workers have not been closely investigated by the community experts in rural Uttar Pradesh. We assessed the physical disabilities in activities of daily living (ADL) and unmet need in physical disabilities among rural elderly. Predictors of unmet needs in physical disabilities among the elderly were also identified.
A community based cross-sectional study was conducted among elderly residents of the rural field practice area of a tertiary care centre in rural Uttar Pradesh. Three hundred and thirty five (335) participants aged 60 years and above from 9 villages were selected using the Probability Proportional to Size (PPS) sampling technique. Study tools were the proforma regarding socio-demographic details, socio-economic status and Stanford Health Assessment Questionnaire. Multivariate logistic regression analysis was performed to identify predictors of unmet needs.
185 (55.2%) had physical disability in one or more activity limitation. Gender wise elderly females had more physical disability in one or more ADL categories than elderly males (66.8% vs. 42.0%). Almost one third (32.5%) of subjects had unmet need for one or more physical disabilities. the predictors of unmet needs that were identified in the study were female gender (P = 0.046), elderly aged 70 years and above (P = 0.032), those living alone (P = 0.035), low monthly family income (P = 0.044), financially fully dependent elderly (P = 0.0002), and those having 3 or more physical disabilities (P = 0.033).
The findings of the study highlight that large number of needs of the disabled are still unmet. Greater, targeted efforts are needed to identify at-risk elderly people living in the community. These predictors would act as surrogate markers and can be easily used by primary care workers to plan and provide services to the elderly people in rural communities.
Hybrid-natural orifice surgery combines the advantages of traditional transabdominal laparoscopic surgery, while limiting surgical trauma to the abdominal wall. Among various routes of intra-abdominal access, the transvaginal method is most appealing because of its utility and proven safety. We describe a series of 4 colonic resections performed with this approach, combined with minilaparoscopy and needlescopic approaches, and discuss the technical aspects, efficacy, and applicability of this technique.
Three patients were selected to undergo hybrid transvaginal natural-orifice right hemicolectomy. A fourth patient, who underwent a segmental resection of a splenic flexure carcinoma, was included. Transvaginal port access was obtained via posterior colpotomy, and was used for dissection, vascular ligation, bowel division, and anastomosis. We used a combination of standard laparoscopic, minilaparoscopic, and needlescopic instruments transabdominally, focusing on reduced size and number of access points.
Duration of laparoscopy, oncologic outcomes and rate of operative morbidity were comparable to the published literature. Early return of gastrointestinal function and low analgesic requirements was observed in all patients. No morbidity related to transvaginal access was observed and the procedure was performed without difficulty in all cases.
Colonic resection performed by hybrid natural-orifice technique offers several advantages over purely transabdominal laparoscopic procedures. Transvaginal access is easy to perform and offers excellent safety, efficacy, and versatility, especially for right hemicolectomy. Techniques to reduce abdominal wall surgical trauma, such as minilaparoscopy and needlescopic graspers, can be combined effectively in colonic resections, and may act synergistically to reduce postoperative pain and improve outcomes.
Research over the past decade suggested critical roles for circular RNAs in the natural growth and disease progression. However, it remains poorly defined whether the circular RNAs participate in Hirschsprung disease (HSCR). Here, we reported that the cir-ZNF609 was down-regulated in HSCR compared with normal bowel tissues. Furthermore, suppression of cir-ZNF609 inhibited the proliferation and migration of cells. We screened out several putative cir-ZNF609 ceRNAs of which the AKT3 transcript was selected. Finally, RNA immunoprecipitation and luciferase reporter assays demonstrated that cir-ZNF609 may act as a sponge for miR-150-5p to modulate the expression of AKT3. In conclusion, these findings illustrated that cir-ZNF609 took part in the onset of HSCR through the crosstalk with AKT3 by competing for shared miR-150-5p.
Long non-coding RNAs (lncRNAs) have been implicated in pathogenesis of various cancers, including lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). We used cBioPortal to analyze lncRNA alteration frequencies and their ability to predict overall survival (OS) using 504 LUSC and 522 LUAD samples from The Cancer Genome Atlas (TCGA) database. In LUSC, 624 lncRNAs had alteration rates > 1% and 64 > 10%. In LUAD 625 lncRNAs had alteration rates > 1% and 36 > 10%. Among those, 620 lncRNAs had alteration frequencies > 1% in both LUSC and LUAD, while 22 were LUSC-specific and 23 were LUAD-specific. Twenty lncRNAs had alteration frequencies > 10% in both LUSC and LUAD, while 44 were LUSC-specific and 16 were LUAD specific. Genome ontology and pathway analyses produced similar results for LUSC and LUAD. Two lncRNAs (IGF2BP2-AS1 and DGCR5) correlated with better OS in LUSC, and three (MIR31HG, CDKN2A-AS1 and LINC01600) predicted poor OS in LUAD. Chip-seq and luciferase reporter assays identified potential IGF2BP2-AS1, DGCR5 and LINC01600 promoters and enhancers. This study presented lncRNA landscapes and revealed differentially expressed, highly altered lncRNAs in LUSC and LUAD. LncRNAs that act as oncogenes and lncRNA-regulating transcription factors provide novel targets for anti-lung cancer therapeutics.
In order to quantify the amount of clinical research conducted on client-owned animals under the Veterinary Surgeons Act 1966, and the nature and extent of any ethical review of that research, a questionnaire was sent to 6 UK veterinary schools, 1 charity veterinary clinic and 12 private referral clinics. The questionnaire examined whether and how much clinical research respondents undertook, and the composition of any ethical review panels examining research proposals. The questionnaire revealed a substantial amount of clinical research was conducted in the UK, with over 200 veterinary surgeons involved in the year of the survey, with at least 170 academic papers involving clinical research published by respondents in the same year. However, it proved impossible to quantify the full extent of clinical research in the UK. All UK veterinary schools required ethical review of clinical research. The composition and working practices of their ethical review panels generally reflected skill sets in ethical review panels set-up under statute to consider the ethics of non-clinical biomedical research on animals and clinical research conducted on human patients. The process for review of clinical research in the private sector was less clear.
The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5'-CACGTG-3'. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC-Arg-Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of ∼3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.
Chronic inflammation may be a causative factor in breast cancer. One possible underlying mechanism is the generation of oxidative stress, which may favor tumorigenic processes. Antioxidant consumption may, therefore, help reduce tissue inflammation levels. However, few studies have explored this relation in breast tissue. We aimed to evaluate correlations between antioxidant (vitamin A/retinol, vitamin C, vitamin E, β-carotene, α-carotene, lycopene, lutein/zeaxanthin, β-cryptoxanthin, selenium, and zinc) intakes and protein expression levels of interleukin (IL)-6, tumor necrosis factor-α, C-reactive protein, cyclooxygenase-2, leptin, serum amyloid A1, signal transducer and activator of transcription 3, IL-8, IL-10, lactoferrin, and transforming growth factor-β measured in the normal breast tissue of 160 women diagnosed with breast cancer. Antioxidant intakes were collected using a self-administered food frequency questionnaire. Inflammation marker expression was assessed by immunohistochemistry. Correlations between antioxidant intakes and inflammatory marker expression were evaluated using Spearman's partial correlation coefficients (r) for all women and for premenopausal and postmenopausal women separately. After Bonferroni correction, negative correlations were observed between dietary β-tocopherol and IL-10 expression in all women combined (r = -0.26, P = .003) and among postmenopausal women (r = -0.39, P = .003). For all women, a negative correlation was found between total zinc intakes and IL-10 (r = -0.26, P = .002). Among postmenopausal women, dietary selenium intake was negatively correlated with the expression of lactoferrin (r = -0.39, P = .003). No associations were observed in premenopausal women. Our findings suggest that consumption of specific antioxidants, including β-tocopherol, zinc, and selenium, may act on the breast tissue through mechanisms affecting the expression of some inflammation markers, particularly among postmenopausal women.
Considerable efforts are currently devoted to understanding the regulation of primary carbon metabolism in plant leaves, which is known to change dramatically with environmental conditions, e.g. during light/dark transitions. Protein phosphorylation is believed to be a key factor in such a metabolic control. In fact, some studies have suggested modifications in the phosphorylation status of key enzymes in the dark compared with the light, or when photosynthesis varies. However, a general view of the phosphoproteome and reciprocal alterations in both the phosphoproteome and metabolome under a wide spectrum of CO2 and O2 conditions so as to vary both gross photosynthesis and photorespiration is currently lacking. Here, we used an instant sampling system and strictly controlled gaseous conditions to examine short-term metabolome and phosphoproteome changes in Arabidopsis rosettes. We show that light/dark, CO2 and O2 mole fraction have differential effects on enzyme phosphorylation. Phosphorylation events that appear to be the most important to regulate metabolite contents when photosynthesis varies are those associated with sugar and pyruvate metabolism: sucrose and starch synthesis are major phosphorylation-controlled steps but pyruvate utilization (by phosphoenolpyruvate carboxylase and pyruvate dehydrogenase) and pyruvate reformation (by pyruvate orthophosphate dikinase) are also subjected to phosphorylation control. Our results thus show that the phosphoproteome response to light/dark transition and gaseous conditions (CO2, O2) contributes to the rapid adjustment of major pathways of primary C metabolism.
PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV), however, this intrinsic defense is counteracted by the immediate-early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML, however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160 thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil mediated PML dimerization we propose that IE1 either affects PML autoSUMOylation by directly abrogating PML E3 ligase function or by preventing the access to SUMO sites. Thus, our data suggest a novel mechanism how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation.
The human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by a subnuclear structure termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism how IE1 antagonizes PML-NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility to develop small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.
Strong social bonds form between individuals in many group-living species, and these relationships can have important fitness benefits. When responding to vocalizations produced by groupmates, receivers are expected to adjust their behaviour depending on the nature of the bond they share with the signaller. Here we investigate whether the strength of the signaller-receiver social bond affects response to calls that attract others to help mob a predator. Using field-based playback experiments on a habituated population of wild dwarf mongooses (Helogale parvula), we first demonstrate that a particular vocalization given on detecting predatory snakes does act as a recruitment call; receivers were more likely to look, approach and engage in mobbing behaviour than in response to control close calls. We then show that individuals respond more strongly to these recruitment calls if they are from groupmates with whom they are more strongly bonded (those with whom they preferentially groom and forage). Our study, therefore, provides novel evidence about the anti-predator benefits of close bonds within social groups.
Ocean warming and acidification are concomitant global drivers that are currently threatening the survival of marine organisms. How species will respond to these changes depends on their capacity for plastic and adaptive responses. Little is known about the mechanisms that govern plasticity and adaptability or how global changes will influence these relationships across multiple generations. Here, we exposed the emerging model marine polychaete Ophryotrocha labronica to conditions simulating ocean warming and acidification, in isolation and in combination over five generations to identify: (i) how multiple versus single global change drivers alter both juvenile and adult life-traits; (ii) the mechanistic link between adult physiological and fitness-related life-history traits; (iii) whether observed phenotypic changes observed over multiple generations are of plastic and/or adaptive origin. Two juvenile (developmental rate; survival to sexual maturity) and two adult (average reproductive body size; fecundity) life-history traits were measured in each generation, in addition to three physiological (cellular reactive oxygen species content, mitochondrial density; mitochondrial capacity) traits. We found that multi-generational exposure to warming alone caused an increase in: juvenile developmental rate, reactive oxygen species production and mitochondrial density and decreases in: average reproductive body size, fecundity and fluctuations in mitochondrial capacity, relative to control conditions. While exposure to ocean acidification alone, had only minor effects on juvenile developmental rate. Remarkably, when both drivers of global change were present, only mitochondrial capacity was significantly affected, suggesting that ocean warming and acidification act as opposing vectors of stress across multiple generations.
Methylglyoxal (MG) is a reactive metabolite that forms adducts on lysine and arginine residues of proteins, thereby affecting their function. Methylglyoxal is detoxified by the Glyoxalase system, consisting of two enzymes, Glo1 and Glo2, that act sequentially to convert MG into D-lactate. Recently, the Parkinsonism-associated protein DJ-1 was described in vitro to have glyoxalase activity, thereby detoxifying the MG metabolite, or deglycase activity, thereby removing the adduct formed by MG on proteins. Since Drosophila is an established model system to study signaling, neurodegeneration and metabolic regulation in vivo, we asked whether DJ-1 contributes to MG detoxification in vivo. Using both DJ-1 knockdown in Drosophila cells in culture, and DJ-1b knockout flies, we could detect no contribution of DJ-1 to survival to MG challenge or to accumulation of MG protein adducts. Furthermore, we provide data suggesting that the previously reported deglycation activity of DJ-1 can be ascribed to a TRIS buffer artefact.
The smallest flying insects commonly possess wings with long bristles. Little quantitative information is available on the morphology of these bristles, and their functional importance remains a mystery. In this study, we (1) collected morphological data on the bristles of 23 species of Mymaridae by analyzing high-resolution photographs and (2) used the immersed boundary method to determine via numerical simulation whether bristled wings reduced the force required to fling the wings apart while still maintaining lift. The effects of Reynolds number, angle of attack, bristle spacing and wing-wing interactions were investigated. In the morphological study, we found that as the body length of Mymaridae decreases, the diameter and gap between bristles decreases and the percentage of the wing area covered by bristles increases. In the numerical study, we found that a bristled wing experiences less force than a solid wing. The decrease in force with increasing gap to diameter ratio is greater at higher angles of attack than at lower angles of attack, suggesting that bristled wings may act more like solid wings at lower angles of attack than they do at higher angles of attack. In wing-wing interactions, bristled wings significantly decrease the drag required to fling two wings apart compared with solid wings, especially at lower Reynolds numbers. These results support the idea that bristles may offer an aerodynamic benefit during clap and fling in tiny insects.
Baicalin and scutellarin, two flavonoid glucuronic acids isolated from Scutellaria baicalensis, exhibit beneficial effects on glucose homeostasis. Baicalin and scutellarin are similar in structure except scutellarin has an additional hydroxyl at composition C-4'. In this work, we observed that baicalin and scutellarin promoted glucose disposal in mice and in adipocytes. Baicalin selectively increased phosphorylation of AMP-activated kinase (AMPK), while scutellarin selectively enhanced Akt phosphorylation. Both of them increased AS160 phosphorylation and glucose uptake in basal condition. AMPK inhibitor or knockdown of AMPK by siRNA blocked baicalin-induced AS160 phosphorylation and glucose uptake, but showed no effects on scutellarin. In contrast, Akt inhibitor and knockdown of Akt with siRNA decreased scutellarin-stimulated glucose uptake but had no effects on baicalin. The molecular dynamic simulations analysis showed that the binding energy of baicalin to AMPK (-34.30kcal/mol) was more favorable than scutellarin (-21.27kcal/mol), while the binding energy of scutellarin (-29.81kcal/mol) to Akt was much more favorable than baicalin (4.04kcal/mol). Interestingly, a combined treatment with baicalin and scutellarin act synergistically to enhance glucose uptake in adipocytes (combination index: 0.94-0.046). In conclusion, baicalin and scutellarin, though structurally similar, promoted glucose disposal in adipocytes by differential regulation on AMPK and Akt activity. Our data provide insight that multicomponent herbal medicines may act synergistically and cooperatively on multiple targets.
Supra/interspinous ligaments connect adjacent spinous processes and act as a stabilizer of the spine. As with other spinal ligaments, it can become ossified. However, few report have discussed ossification supra/interspinous ligaments (OSIL), so its epidemiology remains unknown. We therefore aimed to investigate the prevalence and distribution of OSIL in symptomatic patients with cervical ossification of the posterior longitudinal ligament (OPLL).
The participants of our study were symptomatic patients with cervical OPLL who were diagnosed by standard radiographs of the cervical spine. The whole spine CT data as well as clinical parameters such as age and sex were obtained from 20 institutions belong to the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament (JOSL). The prevalence and distribution of OSIL and the association between OSIL and clinical parameters were reviewed. The sum of the levels involved by OPLL (OP-index) and OSIL (OSI-index) as well as the prevalence of ossification of the nuchal ligament (ONL) were also investigated.
A total of 234 patients with a mean age of 65 years was recruited. The CT-based evidence of OSIL was noted in 68 (54 males and 14 females) patients (29%). The distribution of OSIL showed a significant thoracic preponderance. In OSIL-positive patients, single-level involvement was noted in 19 cases (28%), whereas 49 cases (72%) presented multi-level involvement. We found a significant positive correlation between the OP-index grade and OSI-index. ONL was noted at a significantly higher rate in OSIL-positive patients compared to negative patients.
The prevalence of OSIL in symptomatic patients with cervical OPLL was 29%. The distribution of OSIL showed a significant thoracic preponderance.
Rapid response team (RRT) responders would benefit from training, to ensure competent and efficient management of the deteriorating patient.
We obtained delegate feedback on a pilot training course for RRTs, commissioned by the Australian and New Zealand Intensive Care Society (ANZICS), at the second ANZICS: The Deteriorating Patient Conference.
We surveyed participants on their perceptions of the course overall, and their perceptions of sessions containing presentations and videotaped and live demonstrations of simulated scenarios of patients whose conditions were deteriorating.
The survey response rate was 64% (96 of 150 potential attendees). Responses were positive, with 79.8% of responses (912/1143) agreeing that the participants had learnt something new, that the course would increase their confidence and competence during RRT calls, and that it had assisted them as an educator. The course was well received overall, with the interactive and live demonstration components of the course garnering positive feedback in the comments section of surveys.
There was unanimous agreement by participants for further development of a formalised RRT training course for responding to the deteriorating patient. Participants who were RRT educators also supported the development of an RRT train-the-trainer course.
Assertive community treatment (ACT) is one of the few evidence-based practices for adults with severe mental illness. Interest has slowly waned for ACT implementation. Yet ACT remains an appealing services platform to achieve the triple aim of health care reform (improved health outcomes, reduced cost, and improved satisfaction) through integration of primary care and behavioral health services. This review highlights the evidence for ACT to improve general medical outcomes, reduce treatment costs, and increase access to treatment.
Using a comprehensive list of relevant search terms, the authors performed a systematic literature database search for articles published through November 2015, resulting in ten articles for inclusion.
No studies reported on clinical outcomes of general medical comorbidities or on mortality of ACT clients. Half of the studies reporting utilization (three of six) found a decrease in emergency room usage, and three of four studies identified an increase in outpatient primary care visits. Most studies found no increase in overall medical care costs. Of the few studies reporting on quality of life, most found mild to moderate improvements.
To date, rigorous scientific examination of the effect of ACT on the general health of the populations it serves has not been undertaken. Given ACT's similarity to emerging chronic illness medical management models, the approach seems like a natural fit for improving general medical outcomes of persons with severe mental illnesses. More research is needed that investigates the current effect of ACT teams on general medical outcomes, treatment costs, and access to care.
Schistosomiasis is one of the most prevalent parasitic diseases impacting human health in the tropics and sub-tropics. The geographic distribution of Schistosoma mansoni, the most widespread species, includes areas in Africa, the Middle East, South America and the Caribbean. Snails of the genus Biomphalaria act as intermediate host for S. mansoni. Biomphalaria straminea is not indigenous in China but was accidentally introduced to Hong Kong from South America and has spread to other habitats in the southern parts of the country. This species is known for its great dispersal capacity that highlights the importance of the snail as a potential host for S. mansoni in China. In this connection, although no such infection has been recorded in the field so far, the continuous expansion of China's projects in endemic areas of Africa and import of the infection via returning workers or visitors deserve attention. The purpose of this study was to map and predict the spatial distribution of B. straminea in China. Snail occurrence data were assembled and investigated using MaxEnt software, along with climatic and environmental variables to produce a predictive risk map. Of the environmental variables tested, the precipitation of warmest quarter was the most contribution factor for snail's spatial distribution. Risk areas were found in southeastern China and it is expected that they will guide policies and control programmes to potential areas area of snail abundance and used for spatial targeting of control interventions for this invasive species.
Fragile X syndrome (FXS) is the most common form of familial mental retardation and one of the leading known causes of autism. The mutation responsible for FXS is a large expansion of the CGG repeats in the promoter region of the FMR1 gene, resulting in the transcriptional silencing of the gene. Leptin may be considered a cytokine-like hormone with pleiotropic actions since it may be involved in the regulation of neuroendocrine functions and the immune system response, in addition to playing a role in development. Leptin and adiponectin may act in parallel as opposing metabolic counterparts. The involvement of leptin and adiponectin in the pathophysiology of FXS was hypothesized.
Twenty-three male patients affected by FXS (full mutation in the FMR1 gene) and 24 controls were included in the study. Plasma leptin and adiponectin levels were measured by the ELISA method using commercially available kits.
Adiponectin levels in FXS patients were significantly lower than those found in controls (p < 0.04). Leptin levels in FXS patients were significantly higher than those found in controls (p = 0.03).
Adipokines may be involved in the psychiatric features observed in FXS patients. Further investigations are necessary to evaluate the role of adiponectin and leptin in FXS.
Attaching polar pharmacological modifiers to molecular imaging probes is a common strategy to modulate their pharmacokinetic profiles to improve such parameters as the clearance rate of radiotracers and/or metabolites, and to enhance signal-to-background ratios. We combined the tumor-targeting peptide sequence of bombesin (BBN) with glucuronic acid and the single-photon emission computed tomography (SPECT) radionuclide (99m) Tc by the "click-to-chelate" methodology. The (99m) Tc-tricarbonyl-labeled glucuronated BBN conjugate was compared with a reference compound lacking the carbohydrate. The radiolabeled conjugates displayed similar characteristics in vitro (cell internalization, receptor affinity), but the hydrophilicity of the glycated version was significantly increased. While the tumor uptake of the two radioconjugates in xenografted mice was similar, the glycated peptide exhibited unexpected higher uptake in organs of the hepatobiliary excretion pathway than the more lipophilic reference compound. Control experiments suggest that this may be the result of unspecific accumulation of metabolites in which the glucuronic acid moiety does not act as an innocent pharmacological modifier.
Facilitation processes constitute basic elements of vegetation dynamics in harsh systems. Recent studies in tropical alpine environments demonstrated how pioneer plant species defined as "ecosystem engineers" are capable of enhancing landscape-level richness by adding new species to the community through the modification of microhabitats, and also provided hints about the alternation of different ecosystem engineers over time. Nevertheless, most of the existing works analysed different ecosystem engineers separately, without considering the interaction of different ecosystem engineers. Focusing on the altitudinal limit of Peruvian Dry Puna vegetation, we hypothesized that positive interactions structure plant communities by facilitation cascades involving different ecosystem engineers, determining the evolution of the microhabitat patches in terms of abiotic resources and beneficiary species hosted. To analyze successional mechanisms, we used a "space-for-time" substitution to account for changes over time, and analyzed data on soil texture, composition, and temperature, facilitated species and their interaction with nurse species, and surface area of engineered patches by means of chemical analyses, indicator species analysis, and rarefaction curves. A successional process, resulting from the dynamic interaction of different ecosystem engineers, which determined a progressive amelioration of soil conditions (e.g. nitrogen and organic matter content, and temperature), was the main driver of species assemblage at the community scale, enhancing species richness. Cushion plants act as pioneers, by starting the successional processes that continue with shrubs and tussocks. Tussock grasses have sometimes been found to be capable of creating microhabitat patches independently. The dynamics of species assemblage seem to follow the nested assemblage mechanism, in which the first foundation species to colonize a habitat provides a novel substrate for colonization by other foundation species through a facilitation cascade process.
American pikas (Ochotona princeps) have been heralded as indicators of montane-mammal response to contemporary climate change. Pikas no longer occupy the driest and lowest-elevation sites in numerous parts of their geographic range. Conversely, pikas have exhibited higher rates of occupancy and persistence in Rocky Mountain and Sierra Nevada montane 'mainlands'. Research and monitoring efforts on pikas across the western USA have collectively shown the nuance and complexity with which climate will often act on species in diverse topographic and climatic contexts. However, to date no studies have investigated habitat, distribution, and abundance of pikas across hundreds of sites within a remote wilderness area. Additionally, relatively little is known about whether climate acts most strongly on pikas through direct or indirect (e.g., vegetation-mediated) mechanisms. During 2007-2009, we collectively hiked >16,000 km throughout the 410,077-ha Glacier National Park, Montana, USA, in an effort to identify topographic, microrefugial, and vegetative characteristics predictive of pika abundance. We identified 411 apparently pika-suitable habitat patches with binoculars (in situ), and surveyed 314 of them for pika signs. Ranking of alternative logistic-regression models based on AICc scores revealed that short-term pika abundances were positively associated with intermediate elevations, greater cover of mosses, and taller forbs, and decreased each year, for a total decline of 68% during the three-year study; whereas longer-term abundances were associated only with static variables (longitude, elevation, gradient) and were lower on north-facing slopes. Earlier Julian date and time of day of the survey (i.e., midday vs. not) were associated with lower observed pika abundance. We recommend that wildlife monitoring account for this seasonal and diel variation when surveying pikas. Broad-scale information on status and abundance determinants of montane mammals, especially for remote protected areas, is crucial for land and wildlife-resource managers trying to anticipate mammalian responses to climate change.
Prospective cohort study.
To determine the effects of insurance type (Medicaid vs. a specific private insurance) on patient access to spine surgeons for lumbar disc herniation as measured by (1) acceptance of insurance, (2) need for a referral, and (3) wait time for appointment.
Limited studies have been conducted to examine the issue of patient access to spine surgeons based on different insurance types (Medicaid vs. a specific private insurance), especially in relation to the Medicaid expansion that resulted from the Affordable Care Act.
Appointment success rates, the need for a referral, and waiting periods were compared between Medicaid and a specific private insurance for patients needing an evaluation for a herniated lumbar disc. The waiting period was studied in the context of comparing states that have expanded Medicaid eligibility to ones that have not, and the surgical training of the spine surgeon (orthopaedic surgeons vs. neurosurgeons).
Appointment success rate for patients seeking access to lumbar spine care was significantly higher for patients with BlueCross insurance (95.0%) vs. patients with Medicaid insurance (0.8%) (p < 0.001). The need for referrals was significantly higher for patients with Medicaid insurance (93.3%) vs. patients with BlueCross insurance (4.2%) (p < 0.001). Among BlueCross patients, wait times were longer in Medicaid-expanded states. However, the same trend was not seen among patients with Medicaid insurance.
Patients with Medicaid were less successful at scheduling an appointment and faced more barriers to care, such as the need for a referral, compared to the private insurance studied. In the states with expanded Medicaid, wait times for appointments were longer for BlueCross patients, but were not longer for patients with Medicaid insurance. Overall, this study suggests that increased coverage resulting from Medicaid expansion does not necessarily equate to increased access to care.
2.
We evaluated the effectiveness of cabotegravir (CAB; GSK1265744 or GSK744) long-acting (LA) as pre-exposure prophylaxis (PrEP) against intravenous SIV challenge in a model that mimics blood transfusions based on the per-act probability of infection.
CAB LA is an InSTI formulated as a 200 mg/mL injectable nanoparticle suspension that is an effective pre-exposure prophylaxis (PrEP) agent against rectal and vaginal SHIV transmission in macaques.
Three groups of rhesus macaques (n = 8/group) were injected intramuscularly with CAB LA and challenged intravenously with 17 AID50 SIVmac251 on week 2. Group 1 was injected with 50 mg/kg on week 0 and 4 to evaluate the protective efficacy of the CAB LA dose used in macaque studies mimicking sexual transmission. Group 2 was injected with 50 mg/kg on week 0 to evaluate the necessity of the second injection of CAB LA for protection against intravenous challenge. Group 3 was injected with 25 mg/kg on week 0 and 50 mg/kg on week 4 to correlate CAB plasma concentrations at the time of challenge with protection. Five additional macaques remained untreated as controls.
CAB LA was highly protective with 21 of the 24 CAB LA-treated macaques remaining aviremic, resulting in 88% protection. The plasma CAB concentration at the time of virus challenge appeared to be more important for protection than sustaining therapeutic plasma concentrations with the second CAB LA injection.
These results support the clinical investigation of CAB LA as PrEP in people who inject drugs.
Competitive endogenous RNAs (ceRNAs) act as molecular sponges for microRNAs (miRNAs), and are associated with tumorigenesis in various cancers, including laryngeal cancer (LC). In this work, we constructed an LC-specific inflammatory gene-related ceRNA network (IceNet). In IceNet, ceRNAs targeting inflammation-related genes tended to be network hubs. Additionally, the betweenness centralities of these hub ceRNAs were higher than those of the inflammation-related genes themselves, indicating that the hub ceRNAs in this study played critical roles in communication between IceNet molecules. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that IceNet molecules are associated with multiple cancer-related functions and signaling pathways. Using cFinder software and survival analyses, we identified a potential prognostic module within IceNet that contains 18 mRNAs and a long non-coding RNA (lncRNA), and we effectively stratified patients into high- and low-risk subgroups with different survival outcomes, independent of patient age and tumor grade. This 18-mRNA and one-lncRNA module provides a novel mechanism for potentially improving LC patient prognostic predictions. Applying the module clinically to differentiate high- and low-risk patients could inform therapeutic decision making and ultimately improve patient outcomes. In addition, these results demonstrate the potential importance of IceNet hub ceRNAs in LC development and progression.
Frequent loss of multiple regions in short arm of chromosome 3 is found in various tumors including gastric cancer (GC). RNA binding motif, single-stranded interacting protein 3 (RBMS3) is a tumor suppressor gene located in this region and mediates cancer angiogenesis. However, the role of RBMS3 in GC remains unclear.To evaluate whether RBMS3, together with HIF1A, another key regulator of angiogenesis, predicts GC prognosis, the levels of RBMS3 and HIF1A were first examined by quantitative PCR (qPCR) and western blot from 27 fresh frozen GC and paired normal gastric tissues and then tested by immunohistochemistry (IHC) from 191 GC and 46 normal controls. Moreover, uni- and multivariate analysis were employed to assess the correlations between their levels and microvessel density (MVD) and clinical prognosis. To further identify RBMS3 function in vitro, cell proliferation assay, clonogenic assay, flow cytometry analysis and endothelial cell tube formation assay were employed.We found that RBMS3 level was decreased, whereas HIF1A was elevated in GC. Furthermore, we demonstrated that RBMS3 was an independent prognostic factor and the levels of RBMS3 and HIF1A were associated with GC angiogenesis and histopathological differentiation: patients with lower RBMS3 level and higher nuclear HIF1A expression had poorer prognosis. Besides, gain- and loss-of-function study revealed RBMS3 regulation on G1/S progression, cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. These findings implicated that RBMS3 and nuclear HIF1A could act as prognostic biomarkers and therapeutic targets for GC.
Raf kinase inhibitor protein (RKIP) has been shown to be a suppressor of the mitogen-activated protein kinase pathway and is reported to be involved in human malignancy. However, the molecular mechanism of hepatitis B virus (HBV) in regulating RKIP expression is not yet clarified. In this study, we compared RKIP expression in 107 pairs of matched liver cancer and adjacent non-cancerous liver tissues. Among seven HBV-encoded proteins, we found HBV X (HBX) protein could significantly inhibit the expression level of RKIP, indicating that HBV could suppress RKIP expression through regulating HBX. To further elucidate the mechanism, analyses on transcriptional regulation and promoter methylation inhibition were conducted in Huh7 cells. Our results showed that HBX can interact with AP1 protein to inhibit the RKIP transcription. Moreover, we observed that the promoter methylation level of RKIP could be enhanced by HBV. In conclusion, our study revealed that RKIP could act as a molecular marker for HBV-infected liver cancer, but had no tumor-suppressing effect.
Autophagy is a tightly regulated catabolic process that leads to the degradation of cytoplasmatic components such as aggregated/misfolded proteins and organelles through the lysosomal machinery. Recent studies suggest that autophagy plays such a role in the context of the anti-tumor immune response, make it an attractive target for cancer immunotherapy. Defective autophagy in hematopoietic stem cells may contribute to the development of hematologic malignancies, including leukemia, myelodysplastic syndrome, and lymphoproliferative disorder. In blood cancer cells, autophagy can either result in chemoresistance or induce autophagic cell death that may act as immunogenic. Based on the successful experimental findings in vitro and in vivo, clinical trials of autophagy inhibitor such as hydroxychloroquine in combination with chemotherapy in patients with blood cancers are currently underway. However, autophagy inactivation might impair autophagy-triggered anticancer immunity, whereas induction of autophagy might become an effective immunotherapy. These aspects are discussed in this review together with a brief introduction to the autophagic molecular machinery and its roles in hematologic malignancies.
Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin (TA) gene pair. Loss of the genes depletes the cell's reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any TA gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesised that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naïve hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.
Diadenosine polyphosphates (ApnA) are thought to act as signaling molecules regulating stress responses and biofilm formation in prokaryotes. However, ApnA function in Myxococcus xanthus remains unknown. Here, we investigated the role of ApnA in M. xanthus, using the wild-type and ApnA hydrolase (apaH) mutant strains exposed to various stress conditions. In both wild-type and apaH mutant cells cultured on starvation medium (CF agar), the levels of intracellular diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) increased several fold during the first 16 h of development, and decreased gradually thereafter. The levels of Ap4A and Ap5A in the apaH mutant were about 5- and 11-fold higher than those in the wild-type strain at 16 h, respectively. ApnA hydrolase activity of the wild-type strain increased 1.5-fold during the first 8 h of development, and then gradually decreased. The apaH mutant formed spores 1-2 days after the wild-type strain did, and the yield of viable spores was 5.5% of that in the wild-type strain 5 days after inoculation onto CF agar. These results suggest the possibility that high intracellular levels of Ap4A and/or Ap5A may inhibit M. xanthus sporulation at the early stage of development, and that the bacteria reduce intracellular Ap4A and Ap5A accumulation through ApnA hydrolase activity.
Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus Magnaporthe oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome close to MoPKS19, resulted in a mutant exclusively producing dihydro pyriculol and Dihydro pyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19OXR1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion inducing secondary metabolites produced by MoWT under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.
Social media enables health professionals to network widely and is a useful tool. But there are pitfalls and hurdles that need to be navigated when using sites such as Facebook and Twitter. Guidance issued by the Nursing and Midwifery Council (NMC) clearly states that nurses who act in an unprofessional or unlawful way on social media put their registration at risk.
The announcement of nursing degree apprenticeships has been criticised as an 'act of amnesia' by a leading nurse professor.
The Mental Capacity Act 2005 was enacted with the aim of improving the protection provided by the law to the most vulnerable people in society. The principles of the act specify to whom the act can be applied, and how to ensure these individuals are placed at the centre of decision-making about their treatment and care. It covers those people who raise objections to treatments and care proposed by health and social care professionals and, crucially, it offers protection to those who raise no objection. Appropriate application of the act should ensure all treatment and care planned for vulnerable people is in those individuals' best interests. A best-interests decision involves considering the person's past and current written and spoken wishes, listening carefully to those close to the individual and being aware of biases that might influence the consultation process. Nurses have an important role in this process because of their unique position in the healthcare team.
We are extending, until December 28, 2018, the expiration date of our disability examiner authority (DEA) rule, which authorizes State agency disability examiners to make fully favorable determinations without the approval of a State agency medical or psychological consultant in claims that we consider under our quick disability determination (QDD) and compassionate allowance (CAL) processes. This is our last extension of this rule because we will phase out the use of DEA during the extension period under section 832 of the Bipartisan Budget Act of 2015 (BBA). This extension provides us the time necessary to take all of the administrative actions we need to take in order to reinstate uniform use of medical and psychological consultants. The current rule will expire on November 11, 2016. In this final rule, we are changing the November 11, 2016 expiration or "sunset" date to December 28, 2018, extending the authority for 2 years and 1 month. This is the final extension of our DEA rule. On December 28, 2018, at the conclusion of this extension, the authority for this test will terminate. We are making no other changes.
In shrimp aquaculture, reduction in the use of synthetic antibiotics is a priority due to the high incidence of resistant bacteria (Vibrio) in the white shrimp Litopenaeus vannamei. An increasing number of studies show bactericidal activity of natural treatments in aquaculture. The effectiveness of neem (Azadirachta indica) and oregano (Lippia berlandieri) aqueous extracts and colloidal silver against V. parahaemolyticus were evaluated in low salinity shrimp culture. Results show that aqueous extracts of oregano and neem each present a minimum inhibitory concentration (MIC) of 62.50 mg ml-1 and inhibitory halos of 12.0 to 19.0 mm. Colloidal silver gave a MIC of 2 mg ml-1, and the inhibitory halos were found to be between 11.8 and 18.8 mm, depending on treatment concentrations. An in vivo challenge test was conducted on white shrimp postlarvae cultured at low salinity (5 practical salinity units, PSU), and a significant increase (p < 0.05) in survival was demonstrated in the presence of the aqueous extracts (oregano 64%, neem 76% and colloidal silver 90%), when compared to the control (0%) in the challenge test. However, no significant differences were observed between treatments, suggesting that they all act as alternative bactericidal source agents against V. parahaemolyticus infections for L. vannamei postlarvae when cultured at 5 PSU.
The purpose of this study was to investigate the efficacy and safety of angiogenesis inhibitors for small-cell lung cancer (SCLC).
Totally, 16 controlled trials (1898 cases) involving angiogenesis inhibitors plus chemotherapy (ACT group) versus chemotherapy alone group (CT group) were identified from PubMed, EMBASE, Cochrane Library and Wanfang Data before March 2016.
Compared with CT group, ACT group obtained a significant benefit on objective response rate (ORR) (RR = 1.34; 95% CI = 1.19-1.51; P < 0.00001) and a trend of prolonging progression-free survival (PFS) (HR = 0.86; 95% CI = 0.73-1.01; P = 0.07) without improving overall survival (OS) (HR = 1.05; 95% CI = 0.94-1.17; P = 0.36). Remarkably, subgroup analysis showed that the antibodies targeting VEGF significantly prolonged PFS (HR = 0.76; 95% CI = 0.64-0.90; P = 0.001). With regard to toxicity, there was no significant difference in severe adverse events (AEs, Grade≥3) between two groups except that gastrointestinal symptom, hypertension, metabolic disorders, neurology and pain were higher in ACT group.
Compared with chemotherapy alone, antibodies targeting VEGF plus chemotherapy significantly improved ORR and prolonged PFS with an acceptable toxicity profile for patients with SCLC. Therefore, angiogenesis inhibitors, especially antibodies targeting VEGF, combining with chemotherapy may be a potential promising strategy in managing SCLC.
Laboratory cultures of the paralytic shellfish poisoning producing microalga Gymnodinium catenatum were subjected to a hypo-osmotic shock and changes in cell concentration were observed in two separate experiments of 8 and 24 hours duration, respectively. The increase in geomagnetic activity (GMA), radio and X-ray fluxes and solar X-ray flares were negatively correlated with cell numbers. Cell losses were observed in the short experiment, but not in the longest one. GMA action was related to the course of the experimental period, while electromagnetic radiation (EMR) was only significantly related when the previous hours before the experiments were considered. The differential action windows might be indicative of two differential disruptive mechanisms: EMR might act on DNA synthesis and mitosis phases of the cell cycle (taking place in the dark period) and GMA might be more disruptive at the end of mytosis or cytokinesis phases taking place in the light period. Formation of long chains (> 4 cells/chain) was reduced with salinity and with temperatures above 27ºC but increased with EMR and GMA, particularly when grown at the highest temperatures recorded during the study period (≥28ºC).
Diabetes mellitus and depressive disorders frequently coexist. However, this relationship has been little evaluated across stages of hyperglycemia and for a broad range of common mental disorders (CMDs). The objective here was to investigate the association between CMDs and stages of glycemia.
Cross-sectional study conducted among civil servants aged 35-74 years participating in the ELSA-Brasil cohort.
CMDs were classified using the Clinical Interview Schedule - Revised (CIS-R). Glycemia was classified in stages as normal, intermediate hyperglycemia, newly classified diabetes or previously known diabetes, based on oral glucose tolerance testing, glycated hemoglobin (HbA1c), self-reported diabetes and medication use. Blood glucose control was assessed according to HbA1c.
CMDs were most prevalent in individuals with previously known diabetes. After adjustments, associations weakened considerably and remained significant only for those with a CIS-R score ≥ 12 (prevalence ratio, PR: 1.15; 95% confidence interval, CI: 1.03-1.29). Intermediate hyperglycemia did not show any association with CMDs. For individuals with previously known diabetes and newly classified diabetes, for every 1% increase in HbA1c, the prevalence of depressive disorders became, respectively, 12% and 23% greater (PR: 1.12; 95% CI: 1.00-1.26; and PR: 1.23; 95% CI: 1.04-1.44).
Individuals with previously known diabetes had higher CIS-R scores. Among all individuals with diabetes, worse blood glucose control was correlated with depressive disorder. No relationship between intermediate hyperglycemia and CMDs was observed, thus suggesting that causal processes relating to CMDs, if present, must act more proximally to diabetes onset.
Amino ester-based benzene-1,3,5-tricarboxamides (BTAs) are widely studied experimentally for their facile self-assembly, which leads to strong three-fold hydrogen bonded supramolecular polymers. Understanding the supramolecular assembly of these BTAs is complicated by the presence of two types of dimers, based on the nature of the intermolecular hydrogen bonding pattern: amide-amide (AA) and amide-carboxylate (AC). AA dimers form three hydrogen bonds between the two molecules, are typical of BTA stacks, and act as a basic building block of assembly. In contrast, AC hydrogen bonding results in six hydrogen bonds between two molecules, and this face-to-face orientation results in a dimer that is more stable than the AA one, however, unfavorable for further assembly. We perform atomistic molecular dynamics (MD) simulations of three derivatives of BTA in order to rationalize the large body of experimental data for these systems, specifically the relative stabilities of AA and AC dimers and oligomers. We find that at zero Kelvin, the AC dimer is more stable than the AA dimer by roughly 20 kcal mol(-1). MD simulations of three BTA derivatives (BTA-Met, BTA-Nle, and BTA-Phe) under realistic conditions show that BTA-Met and BTA-Phe can aggregate to form longer assemblies via additional stabilization offered by weak CHS and CHπ hydrogen bonds, respectively. However, the aggregation of BTA-Nle, which is devoid of such functionalities, is limited to that of a dimer. We then employ umbrella sampling to show that oligomers of BTA-Met and BTA-Phe are stable over those of dimers and demonstrate that this results from such weak interactions.
The PIM family of serine/threonine kinases has three highly conserved isoforms (PIM1, PIM2 and PIM3). PIM proteins are regulated through transcription and stability by JAK/STAT pathways and are overexpressed in hematological malignancies and solid tumors. The PIM kinases possess weak oncogenic abilities, but enhance other genes or chemical carcinogens to induce tumors. We generated conditional transgenic mice that overexpress PIM1 or PIM2 in male reproductive organs and analyzed their contribution to tumorigenesis. We found an increase in alterations of sexual organs and hyperplasia in the transgenic mice correlating with inflammation. We also found that PIM1/2 are overexpressed in a subset of human male germ cells and prostate tumors correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression is a common feature of male reproductive organs tumors, which provoke tissue alterations and a large inflammatory response that may act synergistically during the process of tumorigenesis. There is also a correlation with markers of cancer stem cells, which may contribute to the therapy resistance found in tumors overexpressing PIM kinases.
Typically the disorder that alters the interference of particle waves to produce Anderson localization is potential scattering from randomly placed impurities. Here we show that disorder in the form of random gauge fields that act directly on particle phases can also drive localization. We present evidence of a superfluid bose glass to insulator transition at a critical level of this gauge field disorder in a nano-patterned array of amorphous Bi islands. This transition shows signs of metallic transport near the critical point characterized by a resistance , indicative of a quantum phase transition. The critical disorder depends on interisland coupling in agreement with recent Quantum Monte Carlo simulations. We discuss how this disorder tuned SIT differs from the common frustration tuned SIT that also occurs in magnetic fields. Its discovery enables new high fidelity comparisons between theoretical and experimental studies of disorder effects on quantum critical systems.
Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.
There is limited knowledge about human immunodeficiency virus (HIV)-positive migrants and their experiences in the Swedish health care system. It is necessary to increase our knowledge in this field to improve the quality of care and social support for this vulnerable group of patients.
The aim of this study was to describe the experiences of HIV-positive migrants and their encounters with the health care system in Sweden.
This is a Grounded Theory study based on qualitative interviews with 14 HIV-positive migrants living in Sweden, aged 29-55 years.
'A hybrid of access and adversity' was identified as the core category of the study. Three additional categories were 'appreciation of free access to treatment', 'the impact of the Swedish Disease Act on everyday life', and 'encountering discrimination in the general health care system'. The main finding indicated that participants experienced frustration and discrimination because they were required to provide sexual partners with information about their HIV status, which is compulsory under the Swedish Disease Act. The study also showed that the bias or fear regarding HIV infection among general health care professionals outside of the infectious diseases clinics limited the access to the general health care system for HIV-positive migrants.
The HIV-positive migrants appreciated the free access to antiviral therapy, but wished to have more time for patient-physician communications. The participants of this study felt discrimination in health care settings outside of the infectious diseases clinics. There is a need to reduce the discrimination in general health care services and to optimize the social support system and social network of this vulnerable group.
The needs of lesbian, gay, bisexual and trans (LGBT) people with dementia are poorly recognised. This is due partly to assumptions that all older people are heterosexual or asexual. One quarter of gay or bisexual men and half of lesbian or bisexual women have children, compared with 90% of heterosexual women and men, which means LGBT older adults are more likely to reside in care homes. Older LGBT people may be unwilling to express their sexual identities in care settings and this can affect their care. Members of older people's informal care networks must be recognised to ensure their involvement in the lives of residents in care settings continues. However, healthcare professionals may not always realise that many LGBT people rely on their families of choice or wider social networks more than on their families of origin. This article explores sociolegal issues that can arise in the care of older LGBT people with dementia, including enabling autonomy, capacity and applying legal frameworks to support their identities and relationships. It also highlights implications for practice.
Here, a click-cross-linked small intestine submucosa (SIS) drug depot is described for the treatment of rheumatoid arthritis (RA). To the best of the knowledge, there have been no studies related to the intra-articular injection of methotrexate (Met)-loaded click-cross-linkable SIS (Met-loaded Cx-SIS) for RA treatment. As the key objective of this work, injectable formulations of tetrazine-modified SIS (TE-SIS) and transcyclooctene-modified SIS (TC-SIS) are employed as drug depots. Within a few seconds, the simple mixing of equal amounts of TE-SIS and TC-SIS suspensions forms a gelatinous click-cross-linked SIS (Cx-SIS) drug depot in vitro and in vivo. The formed Cx-SIS depot is maintained in the articular joint over an extended period, while SIS alone rapidly disappears. Injectable formulations of Met-loaded Cx-SIS and Met-loaded SIS are prepared and then injected into articular joints to form drug depots. Compared to animals treated with Met-loaded SIS, RA animals treated with Met-loaded Cx-SIS show effective RA repair, as well as extensive regeneration of chondrocytes and glycosaminoglycan deposits. Collectively, these results indicate that the Met-loaded Cx-SIS depot is successfully formed after intra-articular injection of click-cross-linkable SIS, and that this formulation induces long-lasting Met release and allows Met to act effectively in the articular joint, resulting in RA repair.
Freestanding MOF films up to six-inches across and replicating various surface (micro)patterns are prepared via a templated growth method. When grown on copper supports, these films have preferred orientation of the constituent crystallites, translating into markedly different wetting properties of the film's two surfaces (water-pinning vs. water repellant). In addition, the films exhibit differential sorption of various organic solvents, can recover oil spills from seawater, and can also act as active layers of chemical sensors.
This study reports for the first time that compounds of Ge (II) can function as superbases. Two B(N=PiPr3)2 groups attached to a germanium (II) center show a gas phase proton affinity of 296.2 kcal/mol, close to the range of a hyperbase as revealed by B3LYP-D3/6-31G(2d,p) level of theory. These DFT calculations showed better agreement of geometrical parameters for the reported stable germylene compound 1 than previously reported calculations at the B3LYP/6-31G(2d,p) and PBE/DZP level of theory. A systematic study performed with different substitutions of Ge(II) revealed that such system can achieve basicity close to a hyperbase. The σ-donating substituents attached to the Ge (II) center enhance the basicity to a much higher extent compared to the combination of π and σ-donor substituents. The stabilities of these designed superbases have been examined with dimerization energy and singlet-triplet state energy difference (∆ES-T). Furthermore, the calculated gas phase proton affinity values also show good correlation with the most negative valued point (Vmin) in electron rich regions from the molecular electrostatic potential (MESP). The high PA values of compounds were also supported by ionization potential (IP), electron affinity (EA), absolute electronegativity (χ), and absolute hardness (η) calculations. The energetics for the reaction with BH3 and AlMe3 further suggest that the lone pair of Ge (II) can act as a Lewis base and display higher donor-acceptor bond strengths.
Vacuolar processing enzymes (VPEs) are cysteine proteinases that act as crucial mediators of programmed cell death (PCD) in plants. In rice, however, the role of VPEs in abiotic stress-induced PCD remains largely unknown. In this study, we generated OsVPE3 overexpression and suppression transgenic lines to elucidate the function of this gene in rice.
Survival rate and chlorophyll retention analyses showed that suppression of OsVPE3 clearly enhanced salt stress tolerance in transgenic rice compared with wild type. Furthermore, fragmentation of genomic DNA was inhibited in plants with down-regulated OsVPE3. Vital staining studies indicated that vacuole rupture occurred prior to plasma membrane collapse during salt-induced PCD. Notably, overexpression of OsVPE3 promoted vacuole rupture, whereas suppression of OsVPE3 attenuated or delayed the disintegration of vacuolar membranes. Moreover, we found that suppression of OsVPE3 caused decreased leaf width and guard cell length in rice.
Taken together, these results indicated that suppression of OsVPE3 enhances salt tolerance by attenuating vacuole rupture during PCD. Therefore, we concluded that OsVPE3 plays a crucial role in vacuole-mediated PCD and in stomatal development in rice.
Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD), psilocybin, and N,N-dimethyltryptamine (DMT), are famous for their capacity to temporally and profoundly alter an individual's visual experiences. These visual alterations show consistent attributes despite large inter- and intra-individual variances. Many reports document a common perception of colors as more saturated, with increased brightness and contrast in the environment ("Visual Intensifications"). Environmental objects might be altered in size ("Visual illusions") or take on a modified and special meaning for the subject ("Altered self-reference"). Subjects may perceive light flashes or geometrical figures containing recurrent patterns ("Elementary imagery and hallucinations") influenced by auditory stimuli ("Audiovisual synesthesia"), or they may envision images of people, animals, or landscapes ("Complex imagery and hallucinations") without any physical stimuli supporting their percepts. This wide assortment of visual phenomena suggests that one single neuropsychopharmacological mechanism is unlikely to explain such vast phenomenological diversity. Starting with mechanisms that act at the cellular level, the key role of 5-HT2A receptor activation and the subsequent increased cortical excitation will be considered. Next, it will be shown that area specific anatomical and dynamical features link increased excitation to the specific visual contents of hallucinations. The decrease of alpha oscillations by hallucinogens will then be introduced as a systemic mechanism for amplifying internal-driven excitation that overwhelms stimulus-induced excitations. Finally, the hallucinogen-induced parallel decrease of the N170 visual evoked potential and increased medial P1 potential will be discussed as key mechanisms for inducing a dysbalance between global integration and early visual gain that may explain several hallucinogen-induced visual experiences, including visual hallucinations, illusions, and intensifications.
This multi-method qualitative study examines frontline provider perspectives on consumer social relationships and barriers to social recovery in supportive housing programs for adults with serious mental illness. Thematic analyses show that guest and occupancy policies that enforce the "single" nature of single-adult supportive housing challenge consumer rights to self-determination in the realm of social recovery. Findings also highlight the ways in which providers act to reinforce and subvert these policies while mitigating risk in this service setting. Recommendations for enhancing the recovery orientation of supportive housing and implications for the design of the homeless service system are discussed.
Epac1 and Epac2 are cyclic nucleotide-binding (CNB) domain containing proteins, which were originally identified as cAMP-regulated guanine nucleotide exchange factors (GEFs) for the small G-protein Rap. Therefore, Epac proteins founded next to protein kinase A (PKA) and cyclic nucleotide-regulated ion channels the third group of cAMP-responsive proteins in higher organisms. Epac proteins are involved in the regulation of several physiological processes. In particular Epac1 mediates the regulation of molecular processes underlying cell adhesion and mobility. In the pancreas activation of Epac2 potentiates the release of glucose-induced insulin secretion and received attention as a putative target for antidiabetic treatment. While the regulation of Epac by cAMP has been analysed in structural and biochemical detail, less is known on the interaction of Epac with non-canonical cyclic nucleotides. This chapter will discuss to what extent other cyclic purines than cAMP or cyclic pyrimidine could act as Epac agonists or antagonists. The focus will be on the biophysical analysis of the interaction between Epac and these cyclic nucleotides.
Immune cells in the mammary gland play a number of important roles, including protection against infection during lactation and, after passing into milk, modulation of offspring immunity. However, little is known about the mechanism of recruitment of immune cells to the lactating gland in the absence of infection. Given the importance of prolactin to other aspects of lactation, we hypothesized it would also play a role in immune cell recruitment. Prolactin treatment of adult female mice for a period equivalent to pregnancy and the first week of lactation increased immune cell flux through the mammary gland, as reflected in the number of immune cells in mammary gland-draining, but not other lymph nodes. Conditioned medium from luminal mammary epithelial HC11 cell cultures was chemo-attractive to CD4+ and CD8+ T cells, CD4+ and CD8+ memory T cells, B cells, macrophages, monocytes, eosinophils, and neutrophils. Prolactin did not act as a direct chemo-attractant, but through effects on luminal mammary epithelial cells, increased the chemo-attractant properties of conditioned medium. Macrophages and neutrophils constitute the largest proportion of cells in milk from healthy glands. Depletion of CCL2 and CXCL1 from conditioned medium reduced chemo-attraction of monocytes and neutrophils, and prolactin increased expression of these two chemokines in mammary epithelial cells. We conclude that prolactin is an important player in the recruitment of immune cells to the mammary gland both through its activities to increase epithelial cell number as well as production of chemo-attractants on a per cell basis.
Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.
The study of enzyme function often involves a multi-disciplinary approach. Several techniques are documented in the literature towards determining secondary and tertiary structures of enzymes, and X-ray crystallography is the most explored technique for obtaining three-dimensional structures of proteins. Knowledge of three-dimensional structures is essential to understand reaction mechanisms at the atomic level. Additionally, structures can be used to modulate or improve functional activity of enzymes by the production of small molecules that act as substrates/cofactors or by engineering selected mutants with enhanced biological activity. This paper presentes a short overview on how to streamline sample preparation for crystallographic studies of treated enzymes. We additionally revise recent developments on the effects of pressurized fluid treatment on activity and stability of commercial enzymes. Future directions and perspectives on the the role of crystallography as a tool to access the molecular mechanisms underlying enzymatic activity modulation upon treatment in pressurized fluids are also addressed.
Pre-clinical and clinical studies indicated that a blockade of the NMDA receptor complex creates new opportunities for the treatment of affective disorders, including depression. The aim of the present study was to assess the influence of traxoprodil (10 mg/kg) on the activity of desipramine (10 mg/kg), paroxetine (0.5 mg/kg), milnacipran (1.25 mg/kg), and bupropion (10 mg/kg), each at sub-therapeutic doses. Moreover, brain levels of traxoprodil and tested agents were determined using HPLC. The obtained results were used to ascertain the nature of occurring interaction between traxoprodil and studied antidepressants. The experiment was carried out on naïve adult male Albino Swiss mice. Traxoprodil and other tested drugs were administered intraperitoneally. The influence of traxoprodil on the activity of selected antidepressants was evaluated in forced swim test (FST). Locomotor activity was estimated to exclude false positive/negative data. To assess the influence of traxoprodil on the concentration of used antidepressants, their levels were determined in murine brains using HPLC. Results indicated that traxoprodil potentiated activity of all antidepressants examined in FST and the observed effects were not due to the increase in locomotor activity. Only in the case of co-administration of traxoprodil and bupropion, increased bupropion concentrations in brain tissue were observed. All tested agents increased the traxoprodil levels in the brain. Administration of a sub-active dose of traxoprodil with antidepressants from different chemical groups, which act via enhancing monoaminergic transduction, caused the antidepressant-like effect in FST in mice. The interactions of traxoprodil with desipramine, paroxetine, milnacipran, and bupropion occur, at least partially, in the pharmacokinetic phase.
Diverse molecules mediate cross-kingdom communication between bacteria and their eukaryotic partners and determine pathogenic or symbiotic relationships. N-acyl-L-homoserine lactone-dependent quorum-sensing signaling represses the biosynthesis of bacterial cyclodipeptides (CDPs) that act as auxin signal mimics in the host plant Arabidopsis thaliana. In this work, we performed bioinformatics, biochemical, and plant growth analyses to identify non-ribosomal peptide synthase (NRPS) proteins of Pseudomonas aeruginosa, which are involved in CDP synthesis. A reverse genetics strategy allowed the identification of the genes encoding putative multi-modular-NRPS (MM-NRPS). Mutations in these genes affected the synthesis of the CDPs cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-Tyr), while showing wild-type-like levels of virulence factors, such as violacein, elastase, and pyocyanin. When analyzing the bioactivity of purified, naturally produced CDPs, it was found that cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were capable of antagonizing quorum-sensing-LasR (QS-LasR)-dependent signaling in a contrasting manner in the cell-free supernatants of the selected NRPS mutants, which showed QS induction. Using a bacteria-plant interaction system, we further show that the pvdJ, ambB, and pchE P. aeruginosa mutants failed to repress primary root growth, but improved root branching in A. thaliana seedlings. These results indicated that the CDP production in P. aeruginosa depended on the functional MM-NRPS, which influences quorum-sensing of bacteria and plays a role in root architecture remodeling.
Additional high-quality prospective studies are needed to better define the objective criteria used in relation to return-to-sport decisions after synthetic (ligament advanced reinforcement system [LARS]) and autograft (hamstring tendon [2ST/2GR]) anterior cruciate ligament (ACL) reconstruction in active populations.
To prospectively investigate and describe the recovery of objective clinical outcomes after autograft (2ST/2GR) and synthetic (LARS) ACL reconstructions, as well as to investigate the relationship between these clinimetric test outcomes and return-to-sport activity (Tegner activity scale [TAS] score) at 12 and 24 months postoperatively.
Case series; Level of evidence, 4.
A total of 64 patients who underwent ACL reconstruction (32 LARS, 32 2ST/2GR autograft) and 32 healthy reference participants were assessed for joint laxity (KT-1000 arthrometer), clinical outcome (2000 International Knee Documentation Committee [IKDC] knee examination), and activity (TAS score) preoperatively and at 12, 16, 20, and 24 weeks and 12 and 24 months postoperatively.
There was no significant correlation observed between clinical results using the 2000 IKDC knee examination and TAS score at 24 months (rs = 0.188, P = .137), nor were results for side-to-side difference (rs = 0.030, P = .814) or absolute KT-1000 arthrometer laxity of the surgical leg at 24 months postoperatively (rs = 0.076, P = .553) correlated with return-to-sport activity. Nonetheless, return-to-sport rates within the surgical cohort were 81% at 12 months and 83% at 24 months, respectively. No statistically significant differences were observed between physiological laxity of the uninjured knee within the surgical group compared with healthy knees within the reference group (P = .522).
The results indicate that although relatively high levels of return-to-sport outcomes were achieved at 24 months compared with those previously reported in the literature, correlations between objective clinical tests and return-to-sport outcomes may not occur. Clinical outcome measures may provide suitable baseline information; however, the results of this study suggest that clinicians may need to place greater emphasis on other outcome measures when seeking to objectively promote safe return to sport.
MicroRNA (miR)-133b has been reported to act as a tumor suppressor in multiple types of human cancers, including non small cell lung cancer (NSCLC). However, the underlying mechanism by which miR-133b inhibits NSCLC metastasis remains largely unclear. In the present study, reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect messenger RNA and protein expression. A wound healing assay and transwell assay were used to examine the cell migration and invasion. The expression level of miR-133b was found to be significantly downregulated in NSCLC cell lines compared with normal lung epithelial BEAS-2B cells. Further investigation identified fascin1 (FSCN1) as a direct target of miR-133b in NSCLC cells. The expression of FSCN1 was significantly increased in NSCLC cell lines compared with BEAS-2B cells, and its protein expression was negatively regulated by miR-133b in NSCLC A549 cells. Further investigation showed that the upregulation of miR-133b notably inhibited NSCLC cell migration and invasion, while the overexpression of FSCN1 significantly promoted NSCLC cell migration and invasion. Furthermore, the overexpression of FSCN1 reversed the suppressive effect of miR-133b overexpression on NSCLC cell migration and invasion. Accordingly, the present study suggests that miR-133b inhibits the migration and invasion of NSCLC cells via directly targeting FSCN1, and thus may be used for the treatment of NSCLC metastasis.
Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786-O and OS-RC-2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony-formation assay and flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO-EB staining and flow cytometry of the OS-RC-2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal in vitro, and this switch is associated with the inhibition of the Akt signaling pathway.
Exposure to environmental chemicals, such as dioxin, is known to have adverse effects on the homeostasis of gonadal steroids, thereby potentially altering the sexual differentiation of the brain to express autistic traits. Dioxin-like chemicals act on the aryl hydrocarbon receptor (AhR), polymorphisms, and mutations of AhR-related gene may exert pathological influences on sexual differentiation of the brain, causing autistic traits. To ascertain the relationship between AhR-related gene polymorphisms and autism susceptibility, we identified genotypes of them in patients and controls and determined whether there are different gene and genotype distributions between both groups. In addition, to clarify the relationships between the polymorphisms and the severity of autism, we compared the two genotypes of AhR-related genes (rs2066853, rs2228099) with the severity of autistic symptoms. Although no statistically significant difference was found between autism spectrum disorder (ASD) patients and control individuals for the genotypic distribution of any of the polymorphisms studied herein, a significant difference in the total score of severity was observed in rs2228099 polymorphism, suggesting that the polymorphism modifies the severity of ASD symptoms but not ASD susceptibility. Moreover, we found that a significant difference in the social communication score of severity was observed. These results suggest that the rs2228099 polymorphism is possibly associated with the severity of social communication impairment among the diverse ASD symptoms.
Arginine and Glutamate-Rich protein 1 (ARGLU1) is a protein whose function is poorly understood, but may act in both transcription and pre-mRNA splicing. We demonstrate that the ARGLU1 gene expresses at least three distinct RNA splice isoforms - a fully spliced isoform coding for the protein, an isoform containing a retained intron that is detained in the nucleus, and an isoform containing an alternative exon that targets the transcript for nonsense mediated decay. Furthermore, ARGLU1 contains a long, highly evolutionarily conserved sequence known as an Ultraconserved Element (UCE) that is within the retained intron and overlaps the alternative exon. Manipulation of the UCE, in a reporter minigene or via random mutations in the genomic context using CRISPR/Cas9, changed the splicing pattern. Further, overexpression of the ARGLU1 protein shifted the splicing of endogenous ARGLU1 mRNA, resulting in an increase in the retained intron isoform and nonsense mediated decay susceptible isoform and a decrease in the fully spliced isoform. Taken together with data showing that functional protein knockout shifts splicing toward the fully spliced isoform, our data are consistent with a model in which unproductive splicing complexes assembled at the alternative exon lead to inefficient splicing and intron retention.
Unplanned extubations can lead to iatrogenic injury and have the potential to contribute to serious safety events. We adopted lean methodology to reduce the unplanned extubation rate in a Level 3b NICU. We hypothesized that the use of a rapid-cycle PDSA (plan, do, study, act) initiative would reduce the unplanned extubation rate.
Baseline unplanned extubation data were collected from November 1, 2012 to June 6, 2014. A voice of the customer survey ascertained perceptions regarding unplanned extubation causes and impact on care. The confidential survey contained 2 open-ended and 4 closed-ended questions and was distributed to a random sample of nurses and respiratory therapists. A fishbone diagram helped to identify opportunities. Six improvements were identified and rolled out in 2 phases using didactic and kinesthetic techniques. Phase 1 standardized the process for turning intubated infants, assessing endotracheal tube (ETT) placement with growth, and communicating tube position with caregivers. Phase 2 addressed respiratory plans of care, correcting ETT migration, establishing ETT re-securement methods, and standardizing position during radiography. The Fisher exact test was used to determine differences in the number of unplanned extubations per 100 intubated days. Descriptive statistics were used to report survey results. Statistical significance was established at P < .05.
A 68% (17 of 25) survey response rate was realized. Baseline data revealed 3.8 unplanned extubations/100 intubated days, and 2.7 unplanned extubations/100 intubated days occurred in the post-improvement phase (P = .01). We noted a statistically significant decrease in the number of intubated days between the pre- and post-improvement groups (P < .001).
Staff underestimated the prevalence of unplanned extubations but recognized the need for improvement. Rapid cycle PDSA significantly reduced the unplanned extubation rate. The decrease in intubated days may have been a by-product of the post-improvement phase improvements, which encouraged practice changes.
While international research shows that receipt of welfare benefits is associated with poor mental health, less is known about the relationship between welfare receipt and mental health service use. We investigate whether within-person change in welfare recipient status is associated with change in mental health service use.
Analysis of two waves of data from an Australian national household survey. Random- and fixed-effect models considered the effect of change in welfare receipt status, and assessed whether change in mental health service use differed by type of welfare benefit or the direction of welfare transition.
Individuals were more likely to report greater mental health service use at times of welfare receipt. These associations were attenuated, but remained significant, after adjusting for mental health. Increased health service use was not tied to specific types of welfare benefits. The increase in mental health service use associated with a transition onto welfare benefits was much greater than the decline in service use associated with the transition off benefits.
Within individuals, welfare receipt is associated with greater mental health service use. While this does reflect poorer mental health at the time of welfare receipt, other factors seem to facilitate health service use.
The extracellular loops (ECLs) of G protein-coupled receptors (GPCRs) can bind directly to docked orthosteric or allosteric ligands, they can contain transient contact points for ligand entry into the transmembrane (TM) bundle and they can regulate the activation of the receptor signalling pathways. Of the three ECLs, ECL2 is the largest and most structurally diverse reflecting its functional importance. This has been shown through biochemical techniques and has been supported by the many subsequent crystal structures of GPCRs bound to both agonists and antagonists. ECL2 shares common structural features between (and sometimes across) receptor sub-families and can facilitate ligand entry to the TM core or act directly as a surface of the ligand-binding pocket. Structural similarities seem to underpin common binding mechanisms; however, where these exist, variations in primary sequence ensure ligand-binding specificity. This review will compare current understanding of the structural themes and main functional roles of ECL2 in ligand binding, activation and regulation of the major families of GPCRs.
Increasing evidences have demonstrated that the dysregulation of long non-coding RNAs (lncRNAs) may act as an important role in tumor progression. The long non-coding RNA SPRY4 intronic transcript 1 (SPRY4-IT1) has been reported in some cancer including regulating cell growth, differentiation, apoptosis, and cancer progression. However, the expression and function of SPRY4-IT1 in the progression of hepatocellular carcinoma (HCC) remain largely unknown.
The lncRNA SPRY4-IT1 was detected by quantitative real time PCR (qRT-PCR) in HCC cell lines, CCK8 cell proliferation and transwell invasion assays were performed to detect the GC cell proliferation and invasion abilities. The protein expression of E-cadherin, Vimentin and Twist1 was analyzed by Western blotting assays. Furthermore, RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays were used to analyze potential molecular mechanism of SPRY4-IT1 in HCC cells.
We found that SPRY4-IT1 was up-regulated in HCC cell lines. Further function analysis demonstrated that knockdown of SPRY4-IT1 significantly inhibited HCC cells proliferation and invasion, but over-expression of SPRY4-IT1 had the opposite effects on HCC cells in vitro. Moreover, our results also indicated that SPRY4-IT1 over-expression significantly promoted the epithelial-mesenchymal transition (EMT) by up-regulating the transcription factor Twist1 and EMT marker Vimentin and inhibited the E-cadherin expression in MHCC97L cell. Whereas, knockdown of SPRY4-IT1 suppressed the transcription factor Twist1 and EMT marker Vimentin and increased the E-cadherin expression in MHCC97H cells. Mechanisms investigations showed that SPRY4-IT1 interacted with the EZH2 and epigenetically repressed the E-cadherin expression. In vivo, we also demonstrated that the tumor growth was inhibited in SPRY4-IT1 knockdown group compared with the control group.
These results suggested that lncRNA SPRY4-IT1 might be considered as a therapeutic target in HCC.
Confabulations offer unique opportunities for establishing the neurobiological basis of delusional thinking. As regards causal factors, a review of the confabulation literature suggests that neither amnesia nor executive impairment can be the sole (or perhaps even the primary) cause of all delusional beliefs - though they may act in concert with other factors. A key perspective in the modern literature is that many delusions have an emotionally positive or 'wishful' element, that may serve to modulate or manage emotional experience. Some authors have referred to this perspective as the 'emotion dysregulation' hypothesis. In this article we review the theoretical underpinnings of this approach, and develop the idea by suggesting that the positive aspects of confabulatory states may have a role in perpetuating the imbalance between cognitive control and emotion. We draw on existing evidence from fields outside neuropsychology, to argue for three main causal factors: that positive emotions are related to more global or schematic forms of cognitive processing; that positive emotions influence the accuracy of memory recollection; and that positive emotions make people more susceptible to false memories. These findings suggest that the emotions that we want to feel (or do not want to feel) can influence the way we reconstruct past experiences and generate a sense of self - a proposition that bears on a unified theory of delusional belief states.
Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.
We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.
Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.
The aim of this report was to assess the added benefit of the drug combination dolutegravir / abacavir / lamivudine (DTG / ABC / 3TC) compared with the appropriate comparator therapy (ACT) in adults and adolescents above 12 years of age infected with human immunodeficiency virus (HIV). The Federal Joint Committee (G-BA)’s specification of the ACT for different patient groups resulted in 4 research questions. Regarding treatment-naive patients, the company followed both the differentiation between adults and adolescents, and the specification of the ACTs. In pretreated patients in contrast, the company deviated from the G-BA and summarized the population of adults and adolescents up to 12 years of age, and specified individual antiretroviral therapy as ACT. Moreover, it further specified raltegravir as ACT, together with effective antiretroviral background therapy chosen for the individual patient, for the subpopulation of pretreated adults. The assessment was based on patient-relevant outcomes. One direct comparative randomized controlled trial (RCT) was included in the assessment.
The aim of this report was to assess the added benefit of aflibercept in comparison with ranibizumab or grid laser therapy as appropriate comparator therapy (ACT) in adult patients with visual impairment due to macular oedema following branch retinal vein occlusion (BRVO). The ACT specified by the Federal Joint Committee (G-BA) as ranibizumab or grid laser therapy was followed for the present benefit assessment. This deviated from the company, which rejected grid laser therapy as ACT and specified ranibizumab as only ACT. The assessment was conducted based on patient-relevant outcomes and on the data provided by the company in the dossier. Randomized controlled trials (RCTs) with a minimum duration of 6 months were to be included in the assessment.
The aim of the present report is to assess the added benefit of apremilast in comparison with adalimumab or infliximab or ustekinumab as appropriate comparator therapy (ACT) in adult patients with moderate to severe chronic plaque psoriasis who failed to respond to or who have a contraindication to, or are intolerant to other systemic therapy including cyclosporine, methotrexate or psoralen and ultraviolet-A light (PUVA).
The aim of the present report was to assess the added benefit of dulaglutide for the treatment of adults with type 2 diabetes mellitus in comparison with the appropriate comparator therapy (ACT) specified by the Federal Joint Committee (G-BA). Four different research questions result from the different combinations with other blood-glucose lowering drugs. Deviating from the company, which only considered part of the possible combinations for research questions B to D, the assessment was conducted for the entire subindication in each case. The assessment was conducted based on patient-relevant outcomes and on the data provided by the company in the dossier. Randomized controlled trials (RCTs) with a minimum duration of 24 weeks were used for the derivation of the added benefit. This concurs with the company’s inclusion criteria.
The aim of the present report is to assess the added benefit of aclidinium bromide / formoterol (hereinafter referred to as “aclidinium / formoterol”) as a maintenance bronchodilator treatment for relief of symptoms in adult patients with chronic obstructive pulmonary disease (COPD) in comparison with the appropriate comparator therapy (ACT). From the Federal Joint Committee (G-BA)’s specification of the ACT, the following 2 research questions result for the benefit assessment (Table 2). From the options named by the G-BA, the company chose formoterol for research question 1, and formoterol and additional inhaled corticosteroids (ICS) for research question 2 as ACT. The assessment was conducted with the ACTs chosen by the company for the populations described in Table 2. The assessment was conducted based on patient-relevant outcomes and on the data provided by the company in the dossier. Randomized controlled trials (RCTs) with a minimum duration of 24 weeks were used for the derivation of the added benefit.
The aim of this report is to assess the added benefit of nintedanib in combination with docetaxel in comparison with the appropriate comparator therapy (ACT) in adult patients with locally advanced, metastatic or locally recurrent non-small cell lung cancer (NSCLC) of adenocarcinoma tumour histology after first-line chemotherapy. The Federal Joint Committee (G-BA) specified the ACT presented. The present benefit assessment was conducted in comparison with the G-BA’s ACT. The company followed the specification of the G-BA and, from the options mentioned, chose chemotherapy with docetaxel as comparator therapy. The assessment was conducted based on patient-relevant outcomes and on the evidence provided by the company in the dossier.
The aim of the present report was to assess the added benefit of enzalutamide versus the appropriate comparator therapy (ACT) for treatment of adult men with metastatic castration-resistant prostate cancer (mCRPC) who are asymptomatic or mildly symptomatic after failure of androgen deprivation therapy (ADT) in whom chemotherapy is not yet clinically indicated. The G-BA specified the following options for the ACT:watchful waiting while maintaining ongoing conventional ADT; or, if applicable,combined maximal androgen blockade with a non-steroidal anti-androgen (flutamide, bicalutamide); or: abiraterone acetate while maintaining ongoing ADT. The company concurred with the G-BA’s specification and chose watchful waiting while maintaining conventional ADT from the options mentioned. The present benefit assessment was conducted in comparison with the option chosen by the company from the options of ACT specified by the G-BA. The assessment was conducted based on patient-relevant outcomes and on the evidence provided by the company in the dossier.
The aim of the present report was the assessment of the added benefit of vildagliptin in dual oral therapy in combination with a sulfonylurea in comparison with the appropriate comparator therapy (ACT) in adult patients with type 2 diabetes mellitus with insufficient glycaemic control despite monotherapy with maximal tolerated dose of a sulfonylurea and for whom metformin is inappropriate due to contraindications or intolerance. For this therapeutic indication, the Federal Joint Committee (G-BA) specified the following ACT:human insulin in combination with a sulfonylurea (glibenclamide, glimepiride), if applicable only treatment with human insulin. The company concurred with the G-BA’s specification on the ACT and chose glimepiride as sulfonylurea. The present benefit assessment was conducted in comparison with the ACT specified by the G-BA and was based on patient-relevant outcomes and on the evidence presented by the company in the dossier. A minimum study duration of 24 weeks was applicable.
The aim of this report was to assess the added benefit of lurasidone compared with the appropriate comparator therapy (ACT) in adult patients with schizophrenia. The Federal Joint Committee (G-BA) specified amisulpride, aripiprazole, olanzapine, paliperidone, risperidone, quetiapine or ziprasidone as ACTs. The company followed this specification in principle. Instead of choosing a drug however, it presented the result versus those drugs for which direct comparative studies were available. The company planned no summarizing analysis for all drugs. In the present benefit assessment, a summarizing assessment of the added benefit is conducted versus the drugs named by the G-BA. Two research questions resulted for the assessment, which are derived from the different treatment goals in the treatment of patients with schizophrenia. On the one hand, this is the treatment of acute symptoms (e.g. after exacerbation or first diagnosis), on the other hand the prevention of relapse of a stable disease.Research question 1: acute treatment of patients with schizophrenia. Research question 2: prevention of relapse in patients with schizophrenia. The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment. Research question 1: acute treatment of patients with schizophrenia The company identified 3 studies in which lurasidone was compared with risperidone (Study D1001002), olanzapine (Study D1050231) or extended-release quetiapine (quetiapine XR; Study D1050233) respectively. In the studies in which the acute treatment of patients with schizophrenia was investigated, there were major uncertainties regarding the influence of the applied dosages of lurasidone and of the comparator therapies risperidone, olanzapine and quetiapine XR on the study results. However, the company derived an added benefit in this research question only on the basis of the reduction in adverse events (AEs). However, it could not be inferred from the available data on the studies 002, 231 and 233 that the effect of lurasidone on schizophrenia symptoms was at least similarly large as the one of the ACT. Even under the company’s assumption that fewer AEs occurred under lurasidone, overall no added benefit could be derived from this. Hence irrespective of the question whether the 3 studies were suitable for the benefit assessment at all, there is no proof of added benefit of lurasidone versus the ACT. Research question 2: prevention of relapse in patients with schizophrenia Study characteristics and risk of bias One relevant study (D1050237, hereinafter referred to as “237”) was available for the benefit assessment. Study 237 was a randomized, double-blind, active-controlled study, in which lurasidone was compared with risperidone. Adult patients with schizophrenia were enrolled. The treatment duration was 12 months. The dose of the study medication was flexible in both treatment arms. Beginning with the second treatment week, lurasidone could be administered in dose range between 40 and 120 mg / day. The dose in the risperidone arm was up-titrated to 4 mg / day within the first treatment week according to a fixed regimen. Thereafter, the patients received an individual dose, which could be adjusted to between 2 and 6 mg / day. Patients with a score of ≤ 4 on the Positive and Negative Syndrome Scale (PANSS) for the symptoms “delusions”, “conceptual disorganization”, “hallucinations” and “unusual thought content”, and concurrent Clinical Global Impression Scale of Severity (CGI-S) score of ≤ 4 could be enrolled in the study. The patients were not hospitalized. The primary objective of the study was to evaluate the long-term effects of lurasidone. It could be inferred from the sample size planning of the study that the proof of the non-inferiority of lurasidone in comparison with risperidone for the outcome “relapse rate” was a key objective. The mean PANSS total score of the patients was approximately 65, which indicates a disease severity of no more than moderate. Approximately one third of the patients had been hospitalized for schizophrenia 4 times or more before enrolment in the study. Over 10% of the patients in both study arms received other antipsychotics and / or anticholinergics as concomitant medication. The study was mainly conducted outside Europe, with the majority of the patients being from North America (66%), followed by Africa (15%) and South America (14%). Only 2% of the study participants were from Europe (Croatia); the study was not conducted in German study centres. The company’s documents contained no information on further care pathways, particularly psychotherapeutic care. The transferability of the study results to the German health care context is therefore questionable. The risk of bias at study level (and consequently also at outcome level) was rated as high. The reason for this is the high rate of patients who discontinued the study (approximately 60%) and the difference regarding the time point of discontinuation between the study arms.
The aim of the present report was to assess the added benefit of simoctocog alfa compared with the appropriate comparator therapy (ACT) in the treatment and prophylaxis of bleeding in patients with haemophilia A (congenital factor VIII deficiency). The Federal Joint Committee (G-BA) specified the ACT for the therapeutic indication as follows:recombinant or human plasma-derived coagulation factor VIII products. The company chose the recombinant coagulation factor octocog alfa as one of the options specified as ACT by the G-BA. The assessment was conducted based on patient-relevant outcomes and on the evidence provided by the company. The minimum study duration for prophylactic treatment is 6 months.
The aim of this report was to assess the added benefit of sucroferric oxyhydroxide versus sevelamer as appropriate comparator therapy (ACT) for the control of serum phosphorus levels in adult chronic kidney disease (CKD) patients on haemodialysis or peritoneal dialysis. Sucroferric oxyhydroxide should be used within the context of a multiple therapeutic approach, which could include calcium supplement, 1,25-dihydroxy vitamin D3 or one of its analogues, or calcimimetics to control the development of renal bone disease. The Federal Joint Committee (G-BA) distinguished between patients with and without contraindication to calcium-based phosphate binders in its specification of the ACT. The company followed the G-BA. The ACTs specified by the G-BA and the choice of the company are presented. The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment.
The aim of this report was to assess the added benefit of albiglutide for the treatment of adults with type 2 diabetes mellitus in the following approved subindications: Monotherapy When diet and exercise alone do not provide adequate glycaemic control in patients for whom use of metformin is considered inappropriate due to contraindications or intolerance. Combination therapy In combination with other glucose-lowering medicinal products including basal insulin, when these, together with diet and exercise, do not provide adequate glycaemic control. The assessment was conducted separately for 4 research questions versus the appropriate comparator therapy (ACT) specified by the Federal Joint Committee. Deviating from the company, the total therapeutic indication, and not only specific combinations of albiglutide within the respective indication, was considered for research questions B to D. The assessment was conducted based on patient-relevant outcomes and on direct comparative randomized controlled trials (RCTs) with a minimum duration of 24 weeks.
The aim of this report was to assess the added benefit of idelalisib compared with the appropriate comparator therapy (ACT) specified by the Federal Joint Committee (G-BA) for adult patients with chronic lymphocytic leukaemia (CLL): who have received at least one prior therapy, or as first-line treatment in the presence of 17p deletion or TP53 mutation in patients unsuitable for chemo-immunotherapy. The assessment was conducted based on patient-relevant outcomes. According to this approval of idelalisib, the G-BA distinguished between 2 subindications within the therapeutic indication CLL: pretreated patients and treatment-naive patients with 17p deletion and/or TP53 mutation. The G-BA further divided pretreated patients into 4 subpopulations. Accordingly, the assessment was conducted for a total of 5 research questions versus the ACT specified by the G-BA. The research questions and the corresponding ACTs are shown. For research questions 1a to 1d, the company deviated from the G-BA’s differentiation of the therapeutic indication. In contrast to the G-BA, the company summarized the populations of patients with relapsed and refractory CLL. This approach was not followed in the present benefit assessment. The following deviations versus the ACT specified by the G-BA result from the different division of the target population by the company for research question 1c:The company named chemotherapy in combination with rituximab specified by the physician and under consideration of the approval status as ACT for patients with refractory CLL for whom antineoplastic treatment including chemotherapy is indicated. This regimen is an option within the ACT defined by the G-BA, but it is not indicated in all patients eligible for chemotherapy in the population of research question 1c. For example, rituximab-refractory patients may be treated with a different, individually optimized treatment. For patients for whom antineoplastic treatment, but no chemotherapy is indicated, the company specified best supportive care (BSC) as comparator therapy. However, other antineoplastic treatments (except chemotherapy), which are to be used individually, are an option for these patients.
The aim of this report was to assess the added benefit of nalmefene in comparison with the appropriate comparator therapy (ACT) in patients with alcohol dependence who have a high drinking risk level (DRL), without physical withdrawal symptoms and who do not require immediate detoxification. In accordance with the limitations specified in the approval, nalmefene should only be prescribed in conjunction with continuous psychosocial support focused on treatment adherence and reducing alcohol consumption. Nalmefene should be initiated only in patients who continue to have a high DRL 2 weeks after initial assessment. For this therapeutic indication, the Federal Joint Committee (G-BA) specified the following ACT:naltrexone to support the reduction of alcoholism, according to the stipulations specified in the limitations of prescription in Appendix III Number 2 of the Pharmaceutical Directive, with psychosocial support according to the approval. According to the Pharmaceutical Directive, agents for the reduction of alcohol consumption are reimbursable in patients who are to undergo abstinence treatment, but for whom no appropriate therapy options are currently available. These agents can be prescribed for up to 3 months; in justified exceptional cases for another 3 months at the most. The company concurred with the G-BA’s specification on the ACT. The present benefit assessment was conducted in comparison with the ACT naltrexone (in each case in conjunction with psychosocial support). The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) with a minimum duration of 12 weeks. Analyses at time points between 12 and 24 weeks were primarily relevant because these reflect the stipulations specified in the Pharmaceutical Directive mentioned above and therefore also the use in patients insured in the statutory health insurance (SHI).
The aim of this report is to assess the added benefit of propranolol in comparison with the appropriate comparator therapy (ACT) in patients with proliferating infantile haemangioma requiring systemic therapy: life- or function-threatening haemangioma, ulcerated haemangioma with pain and / or lack of response to simple wound care measures, haemangioma with a risk of permanent scars or disfigurement. The Federal Joint Committee (G-BA) specified individual treatment as ACT. The specifications of the respective Summaries of Product Characteristics (SPCs) of the drugs used for treatment are to be taken into account. The company primarily concurred with the G-BA’s specification, but further specified that it considered watchful waiting to be the only treatment option for individual treatment. Watchful waiting in the sense of individual treatment may be a potential ACT for a subpopulation in the therapeutic indication of propranolol. In the present therapeutic indication, however, other individual treatment options are also conceivable (e.g. glucocorticoids for a subindication of the therapeutic indication of propranolol). The company’s specifications excluded these potential treatment options. The present benefit assessment was conducted in comparison with the ACT specified by the G-BA. The implementation of individual treatment in the studies was examined. The assessment was conducted based on patient-relevant outcomes and on direct comparative randomized controlled trials (RCTs).
The aim of the present report is to assess the added benefit of apixaban compared with the appropriate comparator therapy (ACT) for the treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE), and prevention of recurrent DVT and PE in adults. In accordance with the approval, the treatment of haemodynamically unstable PE patients is not part of the assessment. The ACT specified by the Federal Joint Committee for the initial treatment of DVT or PE consists of low molecular weight heparins (LMWHs), and for the secondary prevention (to be started in parallel) of recurrent DVT or PE of a vitamin K antagonist (VKA). It should be noted for the ACT that LMWHs are to be approved for these therapeutic indications and that the drugs are to be administered at the dosages approved for the respective therapeutic indication and optimized for the individual patient. Two research questions result for the assessment, which are derived from the subindication and the ACT. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) with a minimum duration of 3 months.
The aim of this report was to assess the added benefit of the fixed combination of canagliflozin and metformin (hereinafter referred to as canagliflozin / metformin) for the treatment of adults with type 2 diabetes mellitus in comparison with the ACT in the following approved subindications:canagliflozin/metformin: in patients with inadequate glycaemic control on their maximum tolerated dose of metformin alone; canagliflozin/metformin in combination with other blood-glucose lowering drugs including insulin: in patients with inadequate glycaemic control on their maximum tolerated dose of metformin together with other blood-glucose lowering drugs including insulin. The assessment was conducted separately for 3 research questions versus the appropriate comparator therapy (ACT). The G-BA specified the ACT. The research questions presented by the company do not cover the entire approved therapeutic indication of canagliflozin / metformin. The company noted in the dossier that further combination therapies are approved, but, referring to the lack of clinical data, did not submit any corresponding modules. An added benefit for these combination therapies is not proven.
The drug vedolizumab is approved for several therapeutic indications. The aim of the present assessment module is the assessment of the added benefit of vedolizumab in comparison with the appropriate comparator therapy (ACT) in adult patients with moderately to severely active ulcerative colitis who have had an inadequate response with, lost response to, or are intolerant to either conventional therapy or a tumour necrosis factor alpha (TNFα) antagonist. The Federal Joint Committee (G-BA) derived 2 subpopulations from the therapeutic indication (anti-TNFα-naive patients and patients with anti-TNFα failure), for which it specified a TNFα inhibitor (adalimumab or infliximab) as ACT. It is to be noted that it is possible to switch to a different TNFα inhibitor or to adjust the dose in case of treatment failure with a TNFα inhibitor. According to the approval, treatment with the respective TNFα inhibitor should not be continued or should be carefully reconsidered after a certain type of treatment failure. The company chose adalimumab as ACT for both subpopulations. For the reasons stated above, this choice does not completely cover the relevant constellations for patients with anti-TNFα failure (patients who were pretreated with adalimumab and are not to be treated anymore with adalimumab according to the approval). The present benefit assessment was conducted versus adalimumab for anti-TNFα-naive patients, and versus adalimumab or infliximab for patients with anti-TNFα failure. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) with a minimum duration of one year.
The drug insulin degludec is approved for several therapeutic indications. The aim of this assessment module was to assess the added benefit of insulin degludec in combination with short- / rapid-acting insulin compared with the appropriate comparator therapy (ACT) in adult patients with type 1 diabetes mellitus. The benefit assessment of insulin degludec in combination with bolus insulin in type 1 diabetes mellitus was conducted in comparison with the ACT human insulin specified by the Federal Joint Committee (G-BA). This deviated from the company’s approach, which specified insulin analogues as comparator therapy without providing sufficient justification for this approach. However, the company also searched for studies with human insulin. The transferability of the results of the studies with insulin analogues used by the company was viewed to be sufficient for the present research question. Hence this deviation had no consequences for the benefit assessment. The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) with a minimum study duration of 24 weeks were included in the assessment.
The aim of this report was to assess the added benefit of dolutegravir compared with the appropriate comparator therapy (ACT) in adults and adolescents above 12 years of age infected with human immunodeficiency virus type 1 (HIV-1). Four research questions arose, for which the Federal Joint Committee (G-BA) specified the ACTs. The company largely followed the G-BA’s specification of the ACT, but separated the population of pretreated patients (research question 3 and research question 4) in patients with and without integrase inhibitor (INI) resistance. For the subpopulation without INI resistance, the company specified raltegravir as component of the individual treatment as ACT. The assessment was conducted based on patient-relevant outcomes. Only direct comparative randomized controlled trials (RCTs) with a minimum duration of 48 weeks were included in the assessment.
The aim of this report was to assess the added benefit of the fixed combination of dapagliflozin and metformin (hereinafter referred to as “dapagliflozin / metformin”) for the treatment of adult patients with type 2 diabetes mellitus in comparison with the appropriate comparator therapy (ACT) in the following approved subindications:dapagliflozin / metformin: in patients with inadequate glycaemic control on their maximum tolerated dose of metformin alone; dapagliflozin / metformin in combination with other blood-glucose-lowering drugs including insulin: in patients with inadequate glycaemic control with metformin and these drugs. The assessment was conducted separately for 3 subindications versus the ACT specified by the Federal Joint Committee (G-BA). The G-BA specified sulfonylureas (glibenclamide, glimepiride) plus metformin as ACT for subindication A. According to the commission by the G-BA, direct comparative studies versus glipizide were additionally assessed. In this benefit assessment, the added benefit of dapagliflozin / metformin was therefore assessed versus the following comparator therapies:Research question A1: ACT specified by the G-BA (sulfonylureas [glibenclamide, glimepiride] plus metformin). Research question A2: glipizide plus metformin. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) with a minimum duration of 24 weeks.
The aim of the present report is to assess the added benefit of turoctocog alfa compared with the appropriate comparator therapy (ACT) in the treatment and prophylaxis of bleeding in patients with haemophilia A (congenital factor VIII deficiency). The Federal Joint Committee (G-BA) specified the ACT for the therapeutic indication as follows:recombinant or human plasma-derived coagulation factor VIII products. The company chose the recombinant coagulation factor octocog alfa as one of the options specified as ACT by the G-BA, but limited its selection to a third-generation octocog alfa (Advate). This approach was not accepted. According to the G-BA’s specification, all products containing the drug octocog alfa would have to be considered. The ACT specified by the G-BA is therefore used for the assessment. The assessment was conducted based on patient-relevant outcomes.
The aim of the present report was to assess the added benefit of the drug combination rilpivirine / emtricitabine / tenofovir disoproxil (hereinafter referred to as RPV / FTC / TDF) for the new therapeutic indication of RPV / FTC / TDF approved in November 2013 in comparison with the appropriate comparator therapy (ACT). The assessment referred to adults infected with human immunodeficiency virus type 1 (HIV-1) without known mutations associated with resistance to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, tenofovir or emtricitabine, and with a viral load ≤ 100 000 HIV-1 ribonucleic acid (RNA) copies/mL who have received previous antiretroviral treatment. The Federal Joint Committee (G-BA) specified the following ACT:individual antiretroviral therapy based on prior treatment(s) and under consideration of the reason for the switch of treatment, particularly treatment failure due to virologic failure and possible accompanying development of resistance, or due to adverse events (AEs). The respective approval of the drugs was to be considered. The company named individual antiretroviral therapy as ACT with the respective approval of the drugs to be considered. The company considered the individuality of the therapy when it specified the ACT. However, the fact that treatment switching may be advisable for certain reasons (particularly treatment failure or AEs) – as described in detail in the G-BA’s ACT – was not explicitly mentioned in the company’s ACT. The ACT specified by the G-BA was therefore used for the present dossier assessment. The assessment was to be conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs). Only studies with a minimum duration of 48 weeks (patients with previous antiretroviral treatment with several treatment options after treatment failure) or 2 years (patients with previous antiretroviral treatment who respond to their current treatment) were to be included.
The aim of this report is to assess the added benefit of radium-223 dichloride (hereinafter referred to as “radium-223”) in comparison with the appropriate comparator therapy (ACT) in patients with castration-resistant prostate cancer, symptomatic bone metastases and no known visceral metastases. The Federal Joint Committee (G-BA) specified an ACT for each different patient group:for patients with the primary treatment goal of prolongation of life: docetaxel in combination with prednisone or prednisolone; for patients with the primary treatment goal of symptom control and prevention of late complications and for patients for whom docetaxel treatment is not an option: best supportive care (BSC) (particularly adequate pain therapy, treatment with bisphosphonates and/or radionuclides). In this benefit assessment, the group of patients with the primary treatment goal of symptom control and prevention of late complications and the group of patients for whom docetaxel treatment is not an option were primarily considered jointly, also because of the identical ACT, and are hereinafter referred to as “BSC population”. The group of patients with the primary treatment goal of prolongation of life is hereinafter referred to as “docetaxel population”. Studies that investigated a comparison of radium-223 with or without BSC versus BSC could be considered for the benefit assessment of radium-223 compared with the ACT BSC. The assessment was based on patient-relevant outcomes. One direct comparative randomized controlled trial (RCT) was included in the assessment.
The aim of the present report is to assess the added benefit of trastuzumab emtansine in patients with human epidermal growth factor receptor 2 (HER2)-positive, unresectable, locally advanced or metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination. Patients should have either received prior treatment for locally advanced or metastatic disease, or developed disease recurrence during or within 6 months of completing adjuvant therapy. The assessment was conducted in comparison with the appropriate comparator therapy (ACT). The company deviated from the Federal Joint Committee (G-BA)’s specification. It used lapatinib + capecitabine as comparator therapy for the total target population. The dossier assessment was conducted with the ACTs specified by the G-BA because the company did not provide sufficient reasons for deviating from the ACTs. The assessment was based on patient-relevant outcomes. One direct comparative randomized controlled trial (RCT) was included in the assessment.
The aim of this benefit assessment is to assess the added benefit of afatinib in epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor (TKI)-naive adult patients with locally advanced and / or metastatic non-small cell lung cancer (NSCLC) with activating EGFR mutations. The assessment was conducted in comparison with the appropriate comparator therapy (ACT). In principle, the company concurred with the Federal Joint Committee (G-BA)’s specification. In research question 1 (non-pretreated patients), it chose the second option offered by the G-BA (differentiation of the ACT according to Eastern Cooperative Oncology Group Performance Status [ECOG PS]). The assessment was based on patient-relevant outcomes. One comparative randomized controlled trial (RCT) was included in the assessment.
The aim of this report is to assess the added benefit of indacaterol / glycopyrronium in comparison with the appropriate comparator therapy (ACT) in adult patients with chronic obstructive pulmonary disease (COPD) for maintenance bronchodilator treatment to relieve symptoms. The Federal Joint Committee (G-BA) specified the following ACT:The treatment of stable COPD of the severity stages “moderate” (stage II), “severe” (stage III) and “very severe” (stage IV) was conducted according to the recommendations of the most recently effective version of the German National Care Guideline COPD 4. Long-acting beta-2 agonists (formoterol, salmeterol) and / or tiotropium are recommended for long-term drug treatment from stage II. In COPD stage III/IV with more than 2 exacerbations per year, inhaled corticosteroids (ICS) should be used in addition. The company followed the specification of the G-BA and, from the options mentioned, chose formoterol in combination with tiotropium as the ACT. However, it did not consider the conditions for ICS use named above in the study inclusion. Deviating from this, the criteria for ICS treatment specified by the G-BA were used in the present benefit assessment. The assessment for patients with COPD stages II to IV was conducted based on patient-relevant outcomes. Only direct comparative randomized controlled trials (RCTs) with a minimum duration of 6 months were included in the assessment.
The aim of this report is to assess the added benefit of teriflunomide in comparison with the appropriate comparator therapy (ACT) in adult patients with relapsing remitting multiple sclerosis (RRMS). The Federal Joint Committee (G-BA) specified the ACT for the therapeutic indication of relapsing multiple sclerosis (RMS) as follows: beta interferons (1a or 1b) or glatiramer acetate, in each case under consideration of the approved therapeutic indication. Because RRMS is a subset of RMS, the G-BA’s specification also applies to the approved therapeutic indication of teriflunomide. The company chose interferon beta-1a (IFN-β1a) from the options specified by the G-BA, but limited itself to IFN-β1a 44μg subcutaneous (SC) 3 times a week (Rebif), one of the preparations from this drug group. A search targeted at the comparison with Rebif would not identify studies on the comparison with other preparations of this drug group. According to the G-BA’s specification however, all dosage forms, and thus all IFN-β1a preparations, are to be considered. Overall, the company’s limitation of the comparator therapy had no consequence for the present result of the assessment. The present benefit assessment was conducted in comparison with the ACT specified by the G-BA IFN-β1a. The assessment was based on patient-relevant outcomes.
The aim of this report was to assess the added benefit of aflibercept (Eylea) for the treatment of adults with visual impairment due to macular oedema secondary to central retinal vein occlusion (CRVO) in comparison with the appropriate comparator therapy (ACT). The company chose ranibizumab as ACT. This choice concurred with the Federal Joint Committee’s specification. The benefit assessment was conducted in comparison with ranibizumab. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs).
The aim of this report is to assess the added benefit of vemurafenib versus dacarbazine as appropriate comparator therapy (ACT) in the treatment of adult patients with BRAF V600 mutation-positive, unresectable or metastatic melanoma. The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment. In the assessment of vemurafenib from 13 June 2012 (A12-08), overall, there was an indication of considerable added benefit of vemurafenib versus the ACT dacarbazine. Based on the assessment from 13 June 2012, the Federal Joint Committee made a decision that was limited to 1 year. At the expiry of that period, the company submitted a new dossier. Accordingly, the specific research question for the present assessment was to what extent new data and findings were presented in the available dossier, and what influence these have on the assessment of the added benefit of vemurafenib from 13 June 2012.
The aim of this report is to assess the added benefit of saxagliptin in comparison with the appropriate comparator therapy (ACT) for the treatment of adult patients aged 18 years and older with type 2 diabetes mellitus for the therapeutic indication of the monotherapy that was newly approved in July 2013:as monotherapy in patients inadequately controlled by diet and exercise alone and for whom metformin is inappropriate due to contraindications or intolerance. The Federal Joint Committee (G-BA) specified the following ACT:sulfonylurea (glibenclamide or glimepiride). In principle, the company concurred with the G-BA's ACT, but additionally included the sulfonylurea glipizide in its literature search. However, because the company did not present any studies with glipizide, this deviation is without implication. The benefit assessment of saxagliptin was conducted in comparison with the ACT specified by the G-BA. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) (minimum duration ≥ 24 weeks).
The objective of this report is to assess the added benefit of vismodegib in patients with: symptomatic metastatic basal cell carcinoma (BCC); locally advanced BCC inappropriate for surgery or radiotherapy. The assessment was conducted in comparison with the appropriate comparator therapy (ACT). The Federal Joint Committee (G-BA) specified the ACT. The company deviated from the G-BA's specification in so far as it considered best supportive care (BSC) to be the only ACT for all patients with locally advanced BCC or symptomatic metastatic BCC. This was justified with the merely palliative effect of surgery and radiotherapy in the subindication "symptomatic metastatic BCC". This approach was not accepted. Since BSC is defined as palliative treatment and, moreover, can be individually optimized, there is no reason why different palliative approaches cannot be defined for different patient groups for whom different optimum treatments may be indicated. The company did not present any data for the ACT "radiotherapy" specified by the G-BA for patients with symptomatic metastatic BCC inappropriate for surgery, or for the ACT "surgery" specified for patients with symptomatic metastatic BCC inappropriate for radiotherapy. The assessment was based on patient-relevant outcomes.
The aim of this report is to assess the added benefit of EVG / COBI / FTC / TDF compared with the appropriate comparator therapy (ACT) in adults infected with human immunodeficiency virus type 1 (HIV-1). In compliance with the approval, the Federal Joint Committee (G-BA) specified separate appropriate comparator information for treatment-naive and pretreated patients. Research question A: treatment-naive patients The ACT specified by the G-BA was used for treatment-naive patients. The company claimed to concur with this ACT, but additionally referred to results on deviating comparator therapies (boosted protease inhibitors or raltegravir, each in combination with emtricitabine and tenofovir) because it considered these to be not inferior to the G-BA's ACT. This expansion was not accepted in the benefit assessment. Research question B: pretreated patients Contrary to the inclusion criteria originally formulated by the company, the company also included treatment-naive patients in the benefit assessment because it had not identified any study with pretreated patients suitable for the benefit assessment. This approach was not accepted because the company did not prove with adequate scientific research that the data from clinical studies with treatment-naive patients could be transferred to pretreated patients. Outcome-specific effect variations are conceivable in both treatment directions (larger or smaller effect differences versus the ACT). The results of studies can be regarded as "transferable" when it is proven with sufficient certainty and plausibility that the effect estimates of all patient-relevant outcomes investigated are not substantially influenced by the characteristic in question (here: prior treatment). The ACT specified by the G-BA was used for pretreated patients. Although the company claimed to follow the G-BA's ACT, it limited the ACT to few treatment regimens (EFV / FTC / TDF or ATV/r + FTC / TDF or RAL + FTC / TDF), which, from its point of view, were representative. This limitation was not accepted in the benefit assessment. The ACT was a therapy tailored to the individual patient. In this context, limitation to a small number of drugs is not advisable because individual criteria (e.g. AEs or resistance) may make it necessary to use a treatment that deviates from the company's prespecification. Summary The assessment of EVG / COBI / FTC / TDF was conducted versus the ACT specified by the G-BA for treatment-naive patients (research question A) and for pretreated patients (research question B). The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) with a minimum duration of 48 weeks. Only direct comparative studies were included in the assessment.
The aim of this report was to assess the added benefit of lisdexamfetamine as part of a comprehensive treatment programme for attention deficit / hyperactivity disorder (ADHD) in children aged 6 years of age and over when response to previous methylphenidate treatment is considered clinically inadequate, in comparison with the appropriate comparator therapy (ACT) atomoxetine. In accordance with the Federal Joint Committee, the company cited atomoxetine as ACT for lisdexamfetamine, which is indicated as part of a comprehensive treatment programme for ADHD in children aged 6 years of age and over when response to previous methylphenidate treatment is considered clinically inadequate. The benefit assessment of lisdexamfetamine was conducted in comparison with atomoxetine. The assessment was conducted based on patient-relevant outcomes and on direct comparative randomized controlled trials (RCTs).
The aim of this report is to assess the added benefit of linaclotide compared with the appropriate comparator therapy (ACT) (medically advised dietary changes and symptom-orientated treatment [constipation, bloating, cramping, pain]) in patients with moderate to severe irritable bowel syndrome with constipation (IBS-C). The assessment was conducted based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment.
The aim of this report is to assess the added benefit of ocriplasmin compared with the appropriate comparator therapy (ACT) according to the approval for the following therapeutic indication: treatment of vitreomacular traction (VMT) in adults, including when associated with a macular hole of diameter ≤ 400 microns. The Federal Joint Committee (G-BA) specified the following ACT:Subpopulation with asymptomatic VMT: watchful waiting. VMT population with mild symptoms (e.g. slight worsening of visual acuity, minor visual impairment, no progression of symptoms): watchful waiting. VMT population with severe symptoms (e.g. progressive deterioration of visual acuity, progressive retinal changes): pars plana vitrectomy. The company did not seek approval for the subpopulation with asymptomatic VMT. It did not name any ACT and excluded this subpopulation from its research question. Although, in accordance with the G-BA’s requirement, the company included the VMT population with severe symptoms in the research question, it did not present any data. The company claimed no added benefit for the two subpopulations. For the VMT populations with mild symptoms, the company followed the specification of the G-BA. The assessment was conducted based on patient-relevant outcomes. Only direct comparative randomized controlled trials (RCT) were included in the assessment.
The aim of this report is to assess the added benefit of colestilan for the treatment of hyperphosphataemia in adult patients with chronic kidney disease receiving haemodialysis or peritoneal dialysis (hereinafter referred to as CKD 5D). According to the Federal Joint Committee (G-BA)'s specification, 2 subindications are differentiated and the following appropriate comparator therapies (ACTs) are used for them:Patients without contraindications to calcium- or aluminium-based phosphate binders: calcium- or aluminium-based phosphate binders alone or in combination (subindication AI). Patients in whom calcium- and aluminium-based phosphate binders (also in combination) are contraindicated according to the Summary of Product Characteristics (SPC) (e.g. in hypercalcaemia): sevelamer or lanthanum carbonate (subindication AII). In its dossier, the company concurred with the G-BA's specification with regards to the drugs (calcium acetate for subindication AI, sevelamer hydrochloride for subindication AII). When delimiting the subindications, the company deviated in so far as it regarded aluminium-based phosphate binders as unsuitable for long-term treatment, and therefore did not use them for the delimitation. For this assessment, the ACT specified by the G-BA is followed under consideration of the options chosen by the company (calcium acetate and sevelamer hydrochloride). The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment.
The benefit assessment was conducted for the new therapeutic indication of saxagliptin / metformin (wording in the Summary of Product Characteristics [SPC]):in combination with a sulfonylurea (i.e., triple combination therapy) as an adjunct to diet and exercise to improve glycaemic control in adult patients aged 18 years and older with type 2 diabetes mellitus when the maximally tolerated dose of both metformin and the sulfonylurea does not provide adequate glycaemic control. For this new therapeutic indication, the company only requested consultation on the appropriate comparator therapy (ACT) for the individual substance saxagliptin, but not for saxagliptin / metformin. The Federal Joint Committee (G-BA) specified the following ACT for the individual substance saxagliptin:Metformin + human insulin (note: treatment only with human insulin if metformin is not sufficiently effective). The company concurred with this ACT specified by the G-BA also for saxagliptin / metformin. The benefit assessment for the therapeutic indication of saxagliptin / metformin plus sulfonylurea was conducted in comparison with the ACT specified by the G-BA. The assessment was conducted based on patient-relevant outcomes and on randomized controlled trials (RCTs) (minimum duration ≥ 24 weeks).
The benefit assessment of lixisenatide was conducted according to the approval status for the following therapeutic indication: treatment of adults with type 2 diabetes mellitus in combination with oral glucose-lowering medicinal products and/or basal insulin when these, together with diet and exercise, do not provide adequate glycaemic control. Within this therapeutic indication, different subindications for the use of lixisenatide and thus different research questions result from the type of prior treatment. According to the company's consultation request to the Federal Joint Committee (G-BA), an appropriate comparator therapy (ACT) was specified for each of the subindications. This benefit assessment concurs with the G-BA's specification.
The aim of this report is to assess the added benefit of aflibercept in combination with a chemotherapy consisting of irinotecan / 5-fluorouracil / folinic acid (FOLFIRI) in comparison with FOLFIRI as appropriate comparator therapy (ACT) in adult patients with metastatic colorectal cancer (mCRC) that has progressed during or after an oxaliplatin-containing regimen. The comparator therapy chosen by the company concurs with the ACT specified by the Federal Joint Committee. The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were to be included in the assessment.
The aim of this benefit assessment was to assess the added benefit of ingenol mebutate gel (150 μg/g) for use on the face and scalp, and of ingenol mebutate gel (500 μg/g) for use on the trunk and extremities in comparison with diclofenac / hyaluronic acid gel (3%) as appropriate comparator therapy (ACT) in adult patients with non-hyperkeratotic, non-hypertrophic (non-HK/HT) actinic keratosis. The assessment was based on patient-relevant outcomes. The company did not identify any randomized controlled trials (RCTs) with direct comparisons of ingenol mebutate gel and diclofenac / hyaluronic acid gel. It therefore aimed at determining the added benefit on the basis of indirect comparisons. For both ingenol mebutate gel and diclofenac / hyaluronic acid gel, the company identified studies comparing the intervention therapy with a vehicle gel that did not contain ingenol mebutate gel or diclofenac. The company assumed that the hyaluronic acid gel itself, which served as vehicle in the diclofenac/hyaluronic acid studies, had an effect on actinic keratosis, whereas the drug-free vehicle gel in the ingenol mebutate studies did not. It therefore rejected an adjusted indirect comparison with vehicle gel as intermediate comparator. Instead, it chose a method called "chaining of direct comparisons" for the indirect comparison (comparison of ingenol mebutate gel versus vehicle gel, vehicle gel versus hyaluronic acid gel, and hyaluronic acid gel versus diclofenac / hyaluronic acid gel on the basis of the studies mentioned above). A comparison of ingenol mebutate vehicle and hyaluronic acid gel conducted in RCTs would have been necessary, however, to draw a valid conclusion on added benefit on the basis of the method chosen by the company. The company did not identify such studies. Groups with drug-free vehicle gel from different studies were compared in one step of the method. This was an unadjusted indirect comparison, which rendered randomization ineffective. Unadjusted indirect comparisons do not constitute a valid method of analysis, however. Moreover, the company included 2 non-randomized comparative studies on ingenol mebutate versus drug-free vehicle gel in the assessment of the outcome "recurrence rate". The company did not identify any studies with diclofenac/hyaluronic acid gel that recorded this outcome. Hence there were no relevant data for the research question.
The benefit assessment of sitagliptin was conducted according to the approval for the following therapeutic indication: treatment of adult patients with type 2 diabetes mellitus as an adjunct to diet and exercise. Within this therapeutic indication, different subindications for the use of sitagliptin and thus different research questions result from the type of prior treatment. The Federal Joint Committee (G-BA) specified an appropriate comparator therapy (ACT) for each of the different subindications. This benefit assessment concurs with the G-BA's specifications. Deviations by the company The company included studies with sulfonylureas without limitation to the drugs specified by the G-BA in the following subindications: monotherapy with sitagliptin (research question A), combination of sitagliptin plus metformin (research question B) and combination of sitagliptin plus sulfonylurea (research question C). According to the commission by the G-BA, direct comparative studies versus glipizide were additionally assessed. These kinds of studies were presented for the research questions A and B and assessed separately (research question A2 and B2). In the subindication "sitagliptin plus insulin with or without metformin" (research question E), the company defined conventional insulin treatment (CT), intensified conventional insulin treatment (ICT) and insulin dose increase as comparator therapies. This constituted an appropriate specification of the ACT. Different insulin treatment regimens may be medically reasonable to optimize treatment for the individual patient. Studies in which the patients had the option to optimize their treatment on an individual basis (including switching treatment type and treatment regimen) were included in this benefit assessment.
The benefit assessment of saxagliptin was conducted according to the approval for the following therapeutic indication: treatment of adult patients with type 2 diabetes mellitus. Within this therapeutic indication, different subindications for the use of saxagliptin and thus different research questions result from the type of prior treatment. According to the company's consultation request to the Federal Joint Committee (G-BA), an appropriate comparator therapy (ACT) was specified for each of the subindications. This benefit assessment concurs with the G-BA's specifications. The company specified additionally an alternative ACT for each of the 4 subindications. The corresponding assessments were not considered any further in this dossier assessment because the company's rationales were not accepted.
The present benefit assessment concerns the following new therapeutic indication of apixaban: prevention of stroke and systemic embolism (SE) in adult patients with non-valvular atrial fibrillation (NVAF), with one or more risk factors such as prior stroke or transient ischaemic attack (TIA), age ≥ 75 years, hypertension, diabetes mellitus, symptomatic heart failure (New York Heart Association [NYHA] Class ≥ II). The Federal Joint Committee (G-BA) specified the appropriate comparator therapy (ACT) separately for the following 2 populations as:Patients who are suitable for treatment with vitamin K antagonists (VKA) (VKA population): VKA (warfarin or phenprocoumon). Patients who are not suitable for treatment with VKA (ASA population): acetylsalicylic acid (ASA). In its dossier, the company followed the specification of the G-BA. One aim of this report is therefore to assess the added benefit of apixaban in comparison with VKA (phenprocoumon or warfarin) in patients suitable for treatment with VKA, in the indication NVAF with one or more risk factors. Another aim of this report is to assess the added benefit of apixaban in comparison with ASA (at the approved dosage of 50 mg to 250 mg daily) in patients unsuitable for treatment with VKA, in the indication NVAF with one or more risk factors. The assessment was carried out on the basis of randomized controlled trials (RCT) with a minimum duration of 6 months. The following patient-relevant outcomes were used:Mortality: all-cause mortality. Morbidity: strokes (various operationalizations, including disabling strokes). SE; myocardial infarction. TIA. Health-related quality of life. Adverse events: bleeds: combination: major bleeds and clinically relevant non-major bleeds; major bleeds (intracranial and extracranial major bleeds); clinically relevant non-major bleeds; overall rate of adverse events (AEs); overall rate of serious adverse events (SAEs); overall rate of AEs that led to treatment discontinuation. Mortality, morbidity and AEs: combination: stroke, SE, major bleeds and mortality. Several operationalizations for the outcome “stroke” were used in both studies. They included strokes of ischaemic, haemorrhagic or uncertain type as well as combinations thereof. The results of all operationalizations were presented for the benefit assessment and primarily the operationalization encompassing all types was used. Both studies also used various operationalizations regarding the complex “bleeding events”. These included bleeds of differing degrees of severity (major and non-major bleeds). Since haemorrhagic strokes were also recorded under major bleeds and thus an overlap existed with the outcome “stroke”, major bleeds, especially extracranial bleeds, were used to interpret the outcome.
The aim of this report is to assess the added benefit of aflibercept compared with ranibizumab as appropriate comparator therapy (ACT) in adult patients with wet age-related macular degeneration (AMD). The assessment was based on patient-relevant outcomes. No relevant study was identified where the ACT ranibizumab was used according to its approval status. Other analyses presented by the company could not be used for the benefit assessment as they did not allow to compare the benefit of aflibercept and ranibizumab. These analyses include an unpublished mathematical simulation by the manufacturer of ranibizumab from an assessment report of the European Medicines Agency (EMA), as well as extrapolations made by the company on the risk of an ocular adverse event (AE) of each intravitreal injection, and an analysis called "descriptive indirect comparison".
The assessment of the added benefit of pixantrone was conducted according to the approval status for the following therapeutic indication: monotherapy for the treatment of adult patients with multiply relapsed or refractory aggressive B-cell non-Hodgkin's lymphoma (B-cell NHL). The approval is limited to third- and fourth-line therapy. The appropriate comparator therapy (ACT) specified by the Federal Joint Committee (G-BA) is the individual therapy assigned by the treating physician, particularly treatment containing bleomycin, cyclophosphamide, etoposide, ifosfamide, methotrexate, mitoxantrone, rituximab, trofosfamide, vinblastine, vincristine, or vindesine, provided that prior therapy allows further treatment with these drugs, and under consideration of the respective German approval status and the approved dosages. The company cited the ACT specified by the G-BA, but did not state explicitly whether it concurred with this specification. On the basis of the study called PIX301, presented by the company for a direct comparison, it became clear that the company deviated from the G-BA's specifications. This deviation particularly concerns the approval status of the comparator therapy. The randomized controlled trial (RCT) PIX301 compared pixantrone monotherapy with the individual monotherapy specified by the investigator, using one of 7 antineoplastic drugs specified in the protocol, in patients with aggressive non-Hodgkin's lymphoma (NHL) who had received at least 2 prior chemotherapeutic treatments (total population of 140 patients). The study included patients with both T-cell NHL and B-cell NHL, and patients with subsequent treatments after fourth-line therapy. Pixantrone, however, is only approved for the treatment of B-cell NHL in third- and fourth-line therapy. In its dossier, the company therefore presented an analysis of those patients for whom pixantrone is approved (subpopulation of 99 patients; pixantrone group: 50 patients; comparator treatment group: 49 patients). 4 of the 7 possible comparator therapies specified in the protocol of the study PIX301 comprise NHL in their approved therapeutic indication in Germany (etoposide, ifosfamide, mitoxantrone, and rituximab). In its dossier, the company therefore presented a further evaluation of the subpopulation for whom pixantrone is approved, considering only those patients in the comparator group who had received one of the 4 substances mentioned (subpopulation: 73 patients; pixantrone group: 50 patients; comparator treatment group: 23 patients). Only 2 of these 4 drugs are approved for monotherapy in NHL, however (mitoxantrone and rituximab). In the study PIX301, rituximab was not used in any patients, mitoxantrone was only used in of 4 patients in the total population. Hence the vast majority of patients in the comparator arm, both of the entire study PIX301 and of the subpopulation described above, were not treated according to the German approval status. According to the G-BA's specification on the ACT, however, the German approval status including the approved dosages is to be considered for the treatments chosen in the comparator group. Compliance with the approval status constitutes a necessary precondition for the ACT, pursuant to the G-BA's code of procedure. This precondition (benefit assessment strictly within the German approval status) does not inevitably mean that studies in which patients were treated outside the valid approval status are not relevant for the assessment. It has to be examined for these studies whether the study results are applicable to a treatment situation within the German approval status, i.e. whether the effects of a treatment that is not approval-compliant are sufficiently comparable to treatment within the approval status. In such a case, it would therefore be conceivable that conclusions on the added benefit of a new drug in comparison with an (approval-compliant) ACT are based on data outside the German approval status. This requires plausible, data-based considerations on why the therapy chosen in the study is an adequate option for the patients, and is at least not inferior to the ACT. For the benefit assessment of pixantrone, the company did not present any explanation for the applicability of the PIX301 study results to an approval-compliant treatment. Hence the study PIX301 presented by the company is unsuitable for answering the research question on the added benefit of pixantrone versus the ACT specified by the G-BA. For the reasons stated above, the implementation of the ACT by the company was inadequate. For the benefit assessment, the ACT specified by the G-BA was used without reservation.
The aim of this report is to assess the added benefit of axitinib compared with sorafenib as appropriate comparator therapy (ACT) in patients with advanced renal cell carcinoma (RCC) after failure of prior treatment with a cytokine (cytokine population) and everolimus as ACT after failure of prior treatment with sunitinib (sunitinib population). The assessment was based on patient-relevant outcomes. Direct comparative randomized controlled trials (RCTs) were included in the assessment.
The aim of this report is to assess the added benefit of aclidinium bromide (hereinafter abbreviated to “aclidinium”) according to the approval status for the following therapeutic indication: maintenance bronchodilator treatment to relieve symptoms in adult patients with chronic obstructive pulmonary disease (COPD). The Federal Joint Committe (G-BA) specified the following appropriate comparator therapy (ACT): The graded scheme of the current German National Care Guideline COPD is to be taken into account:From Stage II, long-acting beta-2 sympathomimetics (formoterol, salmeterol) and/or long-acting anticholinergics (tiotropium bromide), From Stage III / IV with more than 2 exacerbations per year, inhaled corticosteroids (ICS) should be used in addition. From the above-named drugs, the company chose tiotropium bromide (hereinafter abbreviated to “tiotropium”) as ACT and specified that account would be taken of the aforementioned graded scheme insofar as tiotropium plus ICS was the ACT for patients with Stage III or IV COPD with more than 2 exacerbations per year. The company’s approach with regard to the choice of ACT is appropriate. Accordingly, the dossier does not contain a comparison of aclidinium with long-acting beta-2 sympathomimetics. Only studies with a minimum duration of 6 months were considered in this benefit assessment, because only such studies are capable of contributing reliable knowledge about the benefit or added benefit of aclidinium in the approved maintenance therapy. This deviates from the company’s approach, which also included studies of shorter duration. The benefit assessment was undertaken with respect to patient-relevant outcomes.
The assessment of the added benefit of perampanel in accordance with the approval status was undertaken for the following therapeutic indication: adjunctive (add-on) treatment of partial-onset seizures with or without secondarily generalized seizures in patients with epilepsy aged 12 years and older. The Federal Joint Committee (G-BA) specified the following appropriate comparator therapy (ACT):Lamotrigine. In cases where lamotrigine is used as monotherapy, topiramate as add-on therapy is the ACT. The company adhered to the ACT specified by the G-BA, but with the restriction that topiramate is not considered as an ACT for deriving the added benefit of perampanel. The company justified this approach on the basis of the G-BA’s statement from the advisory discussion, “...a comparison versus lamotrigine as monotherapy would not be productive because of the planned therapeutic indication for perampanel as add-on therapy.” Since topiramate is supposed to be ACT only in the specific case where lamotrigine is used as monotherapy, a comparison with topiramate would also not be productive. This approach is not accepted. Topiramate is appropriate for use as comparator therapy if it is given as add-on therapy to a lamotrigine-containing basic therapy, provided perampanel is also given as add-on therapy to a lamotrigine-containing basic therapy. The assessment was therefore carried out without restriction concerning the ACT, in accordance with the G-BA’s specification. The assessment was undertaken with respect to patient-relevant outcomes.
The aim of this report is to assess the added benefit of linagliptin: in monotherapy in comparison with sulfonylureas (glimepiride, glibenclamide), in dual therapy with metformin in comparison with sulfonylureas (glimepiride, glibenclamide) plus metformin and in triple therapy with metformin and a sulfonylurea in comparison with metformin plus human insulin; in each case as appropriate comparator therapy (ACT) in patients with type 2 diabetes mellitus. The assessment was undertaken with respect to patient-relevant outcomes.
The aim of this report is to assess the added benefit of vandetanib compared to best supportive care (BSC) as appropriate comparator therapy (ACT) in patients with aggressive and symptomatic medullary thyroid carcinoma (MTC) with unresectable, locally advanced or metastatic disease. The comparator therapy chosen by the company corresponded to the ACT previously specified by the Federal Joint Committee. In the current therapeutic situation, there is a need for all patients to be treated with BSC. Studies that compared vandetanib in combination with BSC with treatment consisting of BSC alone were therefore included in the benefit assessment. If available, studies in which vandetanib as monotherapy was compared with BSC could also be included. The assessment was carried out with respect to patient-relevant outcomes.
The aim of this report is to assess the added benefit of vemurafenib versus dacarbazine as appropriate comparator therapy (ACT) in the treatment of adult patients with BRAF V600 mutation-positive, unresectable or metastatic melanoma. The assessment was carried out with respect to patient-relevant outcomes. Only randomized controlled trials (RCTs) with a direct comparator were included in the assessment.
The aim of this report was to assess the added benefit of ipilimumab compared to best supportive care (BSC) as appropriate comparator therapy (ACT) in adult patients with advanced (unresectable or metastatic) melanoma, who have received prior therapy. Given the current therapy situation, all patients need to be treated with best supportive care. Studies were therefore included in the benefit assessment that compared ipilimumab in combination with best supportive care with treatment consisting of best supportive care alone. If available, studies in which ipilimumab as monotherapy was compared with best supportive care were also included. The assessment was carried out with respect to patient-relevant outcomes. Randomized controlled trials (RCTs) with a direct comparator were considered in the assessment.
The benefit assessment of fampridine was carried out according to its approval status for the following therapeutic indication: improvement of walking in adult patients with multiple sclerosis (MS) with walking disability (EDSS (Expanded Disability Status Scale) 4-7). The Federal Joint Committee (G-BA) specified physiotherapy corresponding to the (German) Guideline on Remedies (“Heilmittelrichtlinie”) as the appropriate comparator therapy (ACT). Patients were also required to receive optimized standard therapy for MS (including symptomatic treatment with spasmolytics, if necessary). The company followed in principle the G-BA’s specification regarding the ACT and designated continuous physiotherapy with the aim of improving walking as ACT for the above-named therapeutic indication. However, the company deviated in important details from the G-BA’s specification. Firstly, it did not address the extent to which the physiotherapy used as ACT in the studies it submitted conformed to the physiotherapy described in the Guideline on Remedies as specified by the G-BA or, if this was not the case, whether it was nevertheless meaningful to consider the individual studies. Secondly, the company gave no details about the required optimized standard therapy for MS. These deviations were not justified by the company. The Institute used the ACT specified by the G-BA for the benefit assessment of fampridine.
The aim of this report is to assess the added benefit of rilpivirine compared to efavirenz as appropriate comparator therapy (ACT) for the “treatment of human immunodeficiency virus type 1 (HIV-1) infection in antiretroviral treatment-naïve adult patients with a viral load ≤ 100,000 HIV-1 RNA copies/mL”. The assessment was carried out in comparison with efavirenz as ACT with respect to patient-relevant outcomes. This deviates from the specification of the Federal Joint Committee, because the latter designates efavirenz in combination with two nucleoside reverse transcriptase inhibitors (NRTIs; tenofovir / emtricitabine or abacavir / lamivudine) as ACT. However, in the Institute’s view, it is not necessary to specify the particular backbone therapy of efavirenz for this assessment. The resulting wider consideration of backbone therapies does not contravene the approval status of rilpivirine. Only randomized controlled trials (RCTs) with a direct comparator were included in the assessment.
The aim of this report is to assess the added benefit of belatacept compared to ciclosporin A as the appropriate comparator therapy (ACT) for prophylaxis of graft rejection in adults receiving a renal transplant. The assessment was undertaken with respect to patient-relevant outcomes. Only randomized controlled trials (RCTs) with a direct comparator were included in the assessment.
In the following, “extract from Cannabis sativa” will be referred to by “THC/CBD”. The present benefit assessment of THC/CBD was carried out for the approved therapeutic indication: “...symptom improvement in patients with moderate to severe spasticity due to multiple sclerosis (MS) who have not responded adequately to other anti-spasticity medication and who demonstrate clinically significant improvement in spasticity related symptoms during an initial trial of therapy” (Summary of Product Characteristics). According to the SPC, THC/CBD is intended to be used as add-on treatment to the patient's previous anti-spasticity medication. The benefit assessment was carried out in comparison with the appropriate comparator therapy (ACT) specified by the Federal Joint Committee (G-BA). This is the optimized standard therapy with baclofen or tizanidine, or drugs approved for the treatment of spasticity in underlying neurological disease, taking into account the approved dosages. At least 2 previous trials of therapy were to have taken place, in each of which different oral spasmolytics - at least one of which had to be a product containing baclofen or tizanidine - were used in an optimum way. The aim of this report is therefore to assess the added benefit of THC/CBD in comparison with an optimized standard therapy (as specified by the G-BA) in patients with spasticity due to MS (as described in the approval status). The company deviated from the ACT specified by the G-BA. At first it designated the continuation of the individual previous medication as the ACT. In justifying its choice of the ACT, the company widened its definition of the ACT on the basis of the assumption that an optimized therapy could be presumed in chronically pre-treated patients. It designated the continuation of the previous optimized anti-spasticity medication as the ACT. According to the company, the previous therapy results from the sum of the percentage proportions of all the drugs approved for the indication of THC/CBD that were administered to patients. With both definitions, the company deviated from the G-BA’s specification of the ACT. However, these deviations are not adequately justified.
The benefit assessment of apixaban was undertaken in accordance with the approval status in the following therapeutic indication: “Prevention of venous thromboembolic events (VTE) in adult patients who have undergone elective hip or knee replacement surgery”. The assessment was conducted in comparison with enoxaparin (customized to the individual patient) as appropriate comparator therapy (ACT) with respect to patient-relevant outcomes. Only randomized controlled trials (RCTs) with a direct comparator were included in the assessment.
The benefit assessment of the active substance collagenase was carried out for the approved therapeutic indication “Dupuytren’s contracture in adult patients with a palpable cord”. The Federal Joint Committee (G-BA) specified appropriate comparator therapies (ACTs), depending on the severity of the disease (Tubiana’s classification). The pharmaceutical company did not follow the G-BA specifications with regard to the comparator therapies, but instead chose partial fasciectomny as the sole ACT for all treatment-requiring stages of the disease. The Institute considers this deviation from the G-BA requirements as inadequately justified. The benefit assessment of collagenase was therefore carried out using the ACTs specified by the G-BA, thus producing a total of 4 research questions (divided according to severity).
The present benefit assessment relates to the treatment of metastatic hormone-refractory prostate cancer (mHRPC) in patients previously treated with a docetaxel-containing treatment regimen and was carried out separately for 2 patient populations. Best supportive care population The best supportive care population consists of patients for whom further treatment with docetaxel is no longer an option. The appropriate comparator therapy (ACT) for this patient population is palliative treatment with dexamethasone, prednisone, prednisolone or methylprednisolone, as well as best supportive care (BSC) (e.g. adequate pain therapy). BSC means the therapy that provides the patient with the best possible individually optimized supportive treatment to alleviate symptoms and improve the quality of life. The first objective of the present report is therefore to assess the added benefit of cabazitaxel in combination with prednisone or prednisolone compared to dexamethasone, prednisone, prednisolone or methylprednisolone as well as BSC in patients with metastatic, hormone- refractory prostate cancer, for whom further treatment with docetaxel is no longer an option. This benefit assessment was able to take account of studies that compared cabazitaxel together with prednisone or prednisolone in combination with BSC or without BSC versus treatment with the ACT. One study could be included in the assessment. This study compared cabazitaxel in combination with prednisone and BSC (cabazitaxel / prednisone / BSC) with a treatment consisting of mitoxantrone in combination with prednisone and BSC (mitoxantrone / prednisone / BSC). Here, mitoxantrone was classed as a component of BSC. The assessment was undertaken in respect of patient-relevant outcomes. Docetaxel retreatment population The docetaxel retreatment population comprises patients for whom further treatment with docetaxel is still an option. The ACT for this patient population is docetaxel in combination with prednisone or prednisolone. A further objective of the present report is therefore to assess the added benefit of cabazitaxel in combination with prednisone or prednisolone compared to docetaxel in combination with prednisone or prednisolone in patients with metastatic, hormone-refractory prostate cancer, for whom further treatment with docetaxel is still an option.
The present benefit assessment relates to the treatment of metastatic castration-resistant prostate cancer (mCRPC) of adult men and was carried out separately for 2 patient populations. Best supportive care population The “best supportive care” population comprises patients who are not eligible for further treatment with docetaxel. The appropriate comparator therapy (ACT) for this patient population is palliative treatment with dexamethasone, prednisone, prednisolone or methylprednisolone as well as best supportive care (BSC) (e.g. adequate pain therapy). BSC refers to the therapy that provides the patient with the best possible individually optimized supportive treatment to alleviate symptoms and improve the quality of life. The first objective of the present report is therefore to assess the added benefit of abiraterone acetate in combination with prednisone or prednisolone compared with dexamethasone, prednisone, prednisolone or methylprednisolone as well as BSC in patients with mCRPC who are not eligible for further treatment with docetaxel. This benefit assessment considered studies that investigated the comparison of abiraterone acetate together with prednisone / prednisolone in combination with BSC or without BSC versus treatment with the ACT. One study was included in the assessment. The assessment was carried out by means of the comparison performed in the included study, i.e. abiraterone acetate in combination with prednisone and BSC (abiraterone / prednisone / BSC) versus prednisone and BSC (placebo / prednisone / BSC). The assessment was undertaken in respect of patient-relevant outcomes and the study included was a direct comparative randomized controlled trial. Docetaxel retreatment population The “docetaxel retreatment population” comprises patients for whom further treatment with docetaxel is still an option. The ACT for this patient population is docetaxel in combination with prednisone or prednisolone. A further objective of the present report is therefore to assess the added benefit of abiraterone acetate in combination with prednisone or prednisolone compared with docetaxel in combination with prednisone or prednisolone in patients with mCRPC who are not eligible for further treatment with docetaxel.
During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. We have characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-à-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, we demonstrate that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, we find that Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis.
Radical environmental groups and their members have a wide and varied agenda which often encompasses both local and global issues. In their efforts to call attention to environmental problems, communicate with like-minded groups, and mobilize support for their activities, radical environmental organizations also produce an enormous amount of text, which can be used to estimate the complex communications and task-based networks that underlie these organizations. Moreover, the tactics employed to garnish attention for these groups' agenda can range from peaceful activities such as information dissemination to violent activities such as fire-bombing buildings. To obtain these varied objectives, radical environmental organizations must harness their networks, which have an important spatial component that structures their ability to communicate, coordinate and act on any given agenda item. Here, we analyze a network built from communications and information provided by the semi-annual "Do or Die" (DoD) magazine published in the UK over a 10 year period in the late 1990s and early 2000s. We first employ structural topic model methods to discover violent and nonviolent actors within the larger environmental community. Using this designation, we then compare the spatial structure of these groups, finding that violent groups are especially likely to engage in coordination and/or communication if they are sufficiently close, but exhibit a quickly decreasing probability of interaction over even a few kilometers. Further, violent and nonviolent groups each have a higher probability of coordination with their own group than across groups over even short distances. In these respects, we see that violent groups are especially local in their organization and that their geographic reach is likely very limited. This suggests that nonviolent environmental groups seek each other out over both large and short distances for communication and coordination, but violent groups tend to be highly localized.
BACKGROUND Muscle atrophy due to disuse occurs along with adverse physiological and functional changes, but bone marrow stromal cells (MSCs) may be able to act as muscle satellite cells to restore myofibers. Thus, we investigated whether MSCs could enhance the proliferation of satellite cells and suppress myonuclear apoptosis during immobilization. MATERIAL AND METHODS We isolated, purified, amplified, and identified MSCs. Rats (n=48) were randomized into 3 groups: WB group (n=16), IM-PBS group (n=16), and IM-MSC (n=16). Rat hind limbs were immobilized for 14 d, treated with MSCs or phosphate-buffered saline (PBS), and then studied using immunohistochemistry and Western blot analysis to characterize the proteins involved. Apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) method. RESULTS We compared muscle mass, cross-sectional areas, and peak tetanic forces and noted insignificant differences between PBS- and MSC-treated animals, but satellite cell proliferation was significantly greater after MSC treatment (p<0.05). Apoptotic myonuclei were reduced (p<0.05) after MSC treatment as well. Pro-apoptotic Bax was down-regulated and anti-apoptotic Bcl-2 and p-Akt protein were upregulated (p<0.05). CONCLUSIONS MSCs injected during hind limb immobilization can maintain satellite cell activity by suppressing myonuclear apoptosis.
Standardising a dual x-ray absorptiometry (DXA) protocol is thought to provide a reliable measurement of body composition.
We investigated the effects of manipulating muscle glycogen and creatine content independently and additively on DXA estimates of lean mass.
Eighteen well-trained male cyclists undertook a parallel group application of creatine loading (n=9) (20 g/d for 5 d loading; 3 g/d maintenance) or placebo (n=9) with crossover application of glycogen loading (12 v 6 g/kg BM/d for 48 h) as part of a larger study involving a glycogen-depleting exercise protocol. Body composition, total body water, muscle glycogen and creatine content were assessed via DXA, bioelectrical impedance spectroscopy and standard biopsy techniques. Changes in the mean were assessed using the following effect-size scale: >0.2 small, >0.6, moderate, >1.2 large and compared with the threshold for the smallest worthwhile effect of the treatment.
Glycogen loading, both with and without creatine loading, resulted in substantial increases in estimates of lean body mass (mean ± SD; 3.0 ± 0.7 % and 2.0 ± 0.9 %) and leg lean mass (3.1 ± 1.8 %and 2.6 ± 1.0 %) respectively. A substantial decrease in leg lean mass was observed following the glycogen depleting condition (-1.4 ± 1.6 %). Total body water showed substantial increases following glycogen loading (2.3 ± 2.3 %), creatine loading (1.4 ± 1.9 %) and the combined treatment (2.3 ± 1.1 %).
Changes in muscle metabolites and water content alter DXA estimates of lean mass during periods in which minimal change in muscle protein mass is likely. This information needs to be considered in interpreting the results of DXA-derived estimates of body composition in athletes.
Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), an important hormone in regulating salt-water balance and blood pressure. This review focuses on the regulation of corin function and potential roles of corin defects in hypertensive, heart, and renal diseases.
Proprotein convertase subtilisin/kexin-6 has been identified as a primary enzyme that converts zymogen corin to an active protease. Genetic variants that impair corin intracellular trafficking, cell surface expression, and zymogen activation have been found in patients with hypertension, cardiac hypertrophy, and pre-eclampsia. Reduced corin expression has been detected in animal models of cardiomyopathies and in human failing hearts. Low levels of circulating soluble corin have been reported in patients with heart disease and stroke. Corin, ANP and natriuretic peptide receptor-A mRNAs, and proteins have been colocalized in human renal segments, suggesting a corin-ANP autocrine function in the kidney.
Corin is a key enzyme in the natriuretic peptide system. The latest findings indicate that corin-mediated ANP production may act in a tissue-specific manner to regulate cardiovascular and renal function. Corin defects may contribute to major diseases such as hypertension, heart failure, pre-eclampsia, and kidney disease.
Long non-coding RNAs (lncRNAs) have been reported to play pivotal roles in multiple tumors and can act as tumor biomarkers. In this study, we explored the association of the expression of an lncRNA, DGCR5 with clinicopathological features and prognosis in HCC.
Expression levels of DGCR5 were detected by quantitative real-time PCR (qRT-PCR) and the clinical data was obtained, including basic information, data of clinicopathology and cancer specific survival rate. Receiver operating characteristic (ROC) curve, Kaplan-Meier methods and multivariable Cox regression models were used to analyze predictive efficiency, long-term survival outcomes and risk factors.
DGCR5 was found down-regulated in HCC tissues (P<0.001) and serum (P = 0.0035) and low expression of DGCR5 was correlated with a poor cancer specific survival (CSS) (P = 0.0019), as the overall 5-year CSS rates were 10.3% (low expression group) and 36.6% (high expression group), respectively. A stratified analysis demonstrated that low DGCR5 expression was an independent negative prognostic factor for HCC. In addition, the area under the ROC curve was 0.782 with a sensitivity of 0.633 and a specificity of 0.833.
Our results suggest that DGCR5 may be a participator in HCC and can serve as potential biomarker for the diagnosis and prognosis in HCC.
Improved micropollutant (MP) biotransformation during biological wastewater treatment has been associated with high ammonia oxidation activities, suggesting co-metabolic biotransformation by ammonia oxidizing bacteria as an underlying mechanism. The goal of this study was to clarify the contribution of ammonia oxidizing bacteria to increased MP degradation in nitrifying activated sludge (NAS) communities using a series of inhibition experiments. To this end, we treated a NAS community with two different ammonia oxidation inhibitors, namely octyne (OCT), a mechanistic inhibitor that covalently binds to ammonia monooxygenases, and allylthiourea (ATU), a copper chelator that depletes copper ions from the active center of ammonia monooxygenases. We investigated the biotransformation of 79 structurally different MPs by the inhibitor-treated and untreated sludge communities. Fifty-five compounds exhibited over 20% removal in the untreated control after a 46 h-incubation. Of these, 31 compounds were significantly inhibited by either ATU and/or OCT. For 17 of the 31 MPs, the inhibition by ATU at 46 h was substantially higher than by OCT despite the full inhibition of ammonia oxidation by both inhibitors. This was particularly the case for almost all thioether and phenylurea compounds tested, suggesting that in nitrifying activated sludge communities, ATU does not exclusively act as an inhibitor of bacterial ammonia oxidation. Rather, ATU also inhibited enzymes contributing to MP biotransformation but not to bulk ammonia oxidation. Thus, inhibition studies with ATU tend to overestimate the contribution of ammonia-oxidizing bacteria to MP biotransformation in nitrifying activated sludge communities. Biolog tests revealed only minor effects of ATU on the heterotrophic respiration of common organic substrates by the sludge community, suggesting that ATU did not affect enzymes that were essential in energy conservation and central metabolism of heterotrophs. By comparing ATU- and OCT-treated samples, as well as before and after ammonia oxidation was recovered in OCT-treated samples, we were able to demonstrate that ammonia-oxidizing bacteria were highly involved in the biotransformation of four compounds: asulam, clomazone, monuron and trimethoprim.
The capability of clostridia spores to act as pathogen indicators in sewage sludge treatment was investigated. Sulfite-reducing clostridia and E. coli levels were monitored during waste activated sludge pre-treatments (alkali and ultrasound) and its subsequent mesophilic anaerobic digestion. E. coli was maintained or reduced depending on treatment type and intensity. However, alkali pre-treatment (35.3gNaOH/kgTS) by itself and alkali (157gNaOH/kgTS) and ultrasound (27,000kJ/kgTS) pre-treatments followed by anaerobic digestion provoked reproducible clostridia increases. Specifically, up to 2.7log10 after 35.3gNaOH/kgTS pre-treatment and up to 1.9 and 1.1log10 after digesting the 157gNaOH/kg TS and 27,000kJ/kgTS pre-treated sludge, respectively. Having rejected the hypotheses of sporulation and floc dissipation, the most plausible explanation for these clostridia increases is re-growth. These results question the suitability of clostridia spores as indicators of sludge treatment and other biological treatments where clostridia may have a role.
Worldwide, the sustainability of public health systems is challenged by the increasing number and cost of personalized therapies. Quality biological samples stored in biobanks are essential for the provision of appropriate health services and also act as a reservoir for the development of precision medicine and biotechnological innovation. Economic sustainability is a crucial factor in the maintenance of biobanking activities. Traditionally, management of biobanking is performed by health researchers and/or clinicians whose knowledge of economic issues is inadequate. On the other hand, familiarity with financial instruments used by economists is not often accompanied by a consolidated understanding of biobanking features. This article aims to be a guide for the implementation of business plans in biobanking and proposes models for the facilitation of their preparation, thus contributing to recognition of the importance of efficient management of resources of public health services.
Abnormal redox homeostasis and oxidative stress have been proposed to play a role in the etiology of several neuropsychiatric spectrum disorders. Emerging interest has recently focused on markers of oxidative stress and neuroinflammation in schizophrenic spectrum disorders, at least in particular subgroups of patients. Altered expression of genes related to oxidative stress, oxidative damage to DNA, protein and lipids, as well as reduced glutathione levels in central and peripheral tissues could act synergistically, and contribute to the course of the disease.  Herein, we discuss cellular mechanisms that may be operative in neuroinflammation and contributory to schizophrenia. We address modulation of endogenous cellular defense mechanisms as a potentially innovative approach to therapeutics for schizophrenia, and other neuropsychiatric conditions that are associated with neuroinflammation. Specifically, we discuss the emerging role of heme oxygenase as prominent member of neuroprotective network in redox stress responsive mechanisms, as well as the importance of glutathione relevant in schizophrenia pathophysiology. Finally we introduce the hormetic dose response concept as relevant and important to neuroprotection, and review hormetic mechanisms as possible approaches to manipulation of neuroinflammatory targets that may be viable for treating schizophrenia spectrum disorders. © 2016 Wiley Periodicals, Inc.
The importance of Glucagon like peptide 1 (GLP-1) for metabolic control and insulin release sparked the evolution of genes mimicking GLP-1 action in venomous species (e.g. Exendin-4 in Heloderma suspectum (gila monster)). We discovered that platypus and echidna express a single GLP-1 peptide in both intestine and venom. Specific changes in GLP-1 of monotreme mammals result in resistance to DPP-4 cleavage which is also observed in the GLP-1 like Exendin-4 expressed in Heloderma venom. Remarkably we discovered that monotremes evolved an alternative mechanism to degrade GLP-1. We also show that monotreme GLP-1 stimulates insulin release in cultured rodent islets, but surprisingly shows low receptor affinity and bias toward Erk signaling. We propose that these changes in monotreme GLP-1 are the result of conflicting function of this peptide in metabolic control and venom. This evolutionary path is fundamentally different from the generally accepted idea that conflicting functions in a single gene favour duplication and diversification, as is the case for Exendin-4 in gila monster. This provides novel insight into the remarkably different metabolic control mechanism and venom function in monotremes and an unique example of how different selective pressures act upon a single gene in the absence of gene duplication.
Two novel chlorinated alkane-respiring Dehalobacter restrictus strains CF and DCA were isolated from the same enrichment culture, ACT-3, and characterized. The closed genomes of these highly similar sister strains were previously assembled from metagenomic sequence data and annotated. The isolation of the strains enabled experimental verification of predicted annotations, particularly focusing on irregularities or predicted gaps in central metabolic pathways and cofactor biosynthesis. Similar to D. restrictus strain PER-K23, strains CF and DCA require arginine, histidine and threonine for growth, although the corresponding biosynthesis pathways are predicted to be functional. Using strain CF to experimentally verify annotations, we determined that the predicted defective serine biosynthesis pathway can be rescued with a promiscuous serine hydroxymethyltransferase. Strain CF grew without added thiamine although the thiamine biosynthesis pathway is predicted to be absent; intracellular thiamine diphosphate, the cofactor of carboxylases in central metabolism, was not detected in cell extracts. Thus, strain CF may use amino acids to replenish central metabolites, portending entangled metabolite exchanges in ACT-3. Consistent with annotation, strain CF possesses a functional corrinoid biosynthesis pathway, demonstrated by increasing corrinoid content during growth and guided cobalamin biosynthesis in corrinoid-free medium. Chloroform toxicity to corrinoid-producing methanogens and acetogens may drive the conservation of corrinoid autotrophy in Dehalobacter strains. Heme detection in strain CF cell extracts suggests the 'archaeal' heme biosynthesis pathway also functions in anaerobic Firmicutes. This study reinforces the importance of incorporating enzyme promiscuity and cofactor availability in genome-scale functional predictions and identifies essential nutrient interdependencies in anaerobic dechlorinating microbial communities.The ISME Journal advance online publication, 29 November 2016; doi:10.1038/ismej.2016.158.
"Sleep-low" consists of a sequential periodization of carbohydrate (CHO) availability-low glycogen recovery after "train high" glycogen-depleting interval training, followed by an overnight-fast and light intensity training ("train low") the following day. This strategy leads to an upregulation of several exercise-responsive signaling proteins, but the chronic effect on performance has received less attention. We investigated the effects of short-term exposure to this strategy on endurance performance.
Following training familiarization, 11 trained cyclists were divided into two groups for a one-week intervention-one group implemented three cycles of periodized CHO intake to achieve the sleep-low strategy over six training sessions (SL, CHO intake: 6 g·kg(-1)·day(-1)), whereas the control group consumed an even distribution of CHO over the day (CON). Tests were a 2 h submaximal ride and a 20 km time trial.
SL improved their performance (mean: +3.2%; p < 0.05) compared to CON. The improvement was associated with a change in pacing strategy with higher power output during the second part of the test. No change in substrate utilization was observed after the training period for either group.
Implementing the "sleep-low" strategy for one week improved performance by the same magnitude previously seen in a three-week intervention, without any significant changes in selected markers of metabolism.
Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal β-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.
A growing body of evidence has demonstrated that multiple sources of salience tune attentional sets toward aspects of the environment, including affectively and motivationally significant categories of stimuli such as angry faces and reward-associated target locations. Recent evidence further indicates that objects that have gained personal significance through ownership can elicit similar attentional prioritization. Here we discuss current research on sources of attentional prioritization that shape our awareness of the visual world from moment to moment and the underlying neural systems and contextualize what is known about attentional prioritization of our possessions within that research. We review behavioral and neuroimaging research on the influence of self-relevance and ownership on cognition and discuss challenges to this literature stemming from different modes of conceptualizing and operationalizing the self. We argue that ownership taps into both "self-as-object," which characterizes the self as an object with a constellation of traits and attributes, and "self-as-subject," which characterizes the self as an agentic perceiver and knower. Despite an abundance of research probing neural and behavioral indices of self-as-object and its effects on attention, there exists a paucity of research on the influence of self-relevance of attention when self is operationalized from the perspective of a first-person subject. To begin to address this gap, we propose the Self as Ownership in Attentional Prioritization (SOAP) framework to explain how ownership increases salience through attention to external representations of self-identity (i.e., self as object) and attention to contextually mediated permission to act (i.e., self as subject).
Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia.
Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1.
We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness.
We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.
The exact impact of ageing on skeletal muscle phenotype and mitochondrial and lipid content remains controversial, probably because physical activity, which greatly influences muscle physiology, is rarely accounted for. The present study was therefore designed to investigate the effects of ageing, physical activity, and pre-frailty on skeletal muscle phenotype, and mitochondrial and intramyocellular lipid content in men.
Recreationally active young adult (20-30 yo; YA); active (ACT) and sedentary (SED) middle-age (50-65 yo; MA-ACT and MA-SED); and older (65 + yo; 65 + ACT and 65 + SED) and pre-frail older (65 + PF) men were recruited. Muscle biopsies from the vastus lateralis were collected to assess, on muscle cross sections, muscle phenotype (using myosin heavy chain isoforms immunolabelling), the fibre type-specific content of mitochondria (by quantifying the succinate dehydrogenase stain intensity), and the fibre type-specific lipid content (by quantifying the Oil Red O stain intensity).
Only 65 + SED and 65 + PF displayed significantly lower overall and type IIa fibre sizes vs. YA. 65 + SED displayed a lower type IIa fibre proportion vs. YA. MA-SED and 65 + SED displayed a higher hybrid type IIa/IIx fibre proportion vs. YA. Sedentary and pre-frail, but not active, men displayed lower mitochondrial content irrespective of fibre type vs. YA. 65 + SED, but not 65 + ACT, displayed a higher lipid content in type I fibres vs. YA. Finally, mitochondrial content, but not lipid content, was positively correlated with indices of muscle function, functional capacity, and insulin sensitivity across all subjects.
Taken altogether, our results indicate that ageing in sedentary men is associated with (i) complex changes in muscle phenotype preferentially affecting type IIa fibres; (ii) a decline in mitochondrial content affecting all fibre types; and (iii) an increase in lipid content in type I fibres. They also indicate that physical activity partially protects from the effects of ageing on muscle phenotype, mitochondrial content, and lipid accumulation. No skeletal specific muscle phenotype of pre-frailty was observed.
Police have historically been responsible for transporting people during a mental health crisis in Australia. A major change to the New South Wales (NSW) Mental Health Act (MHA) in 2007 expanded the range of coercive transportation agencies to include NSW Ambulance (paramedics) and NSW Health (mental health nurses). Anecdotal reports, however, describe a lack of clarity around how these changes should be implemented in practice. This research aims to explore this lack of clarity through qualitative analysis of interviews with people with the lived experience of involuntary transport under the MHA. Sixteen interviews were conducted; most (n = 14) interviews in northern NSW regions: six with people who had been transported (consumers), four with carers, and six with service providers (two police, one paramedic, and three mental health nurses). For consumers and carers, the police response was often perceived as too intense, particularly if the person was not violent. Carers were often conflicted by having to call for emergency intervention. Service providers were frustrated by a lack of a coordinated interagency response, resourcing issues, delays at emergency departments, and lack of adequate training. A central theme across all groups was the importance of communication styles. As one participant (consumer) said: 'Everybody needs a lesson in kindness'. All groups agreed that high-risk situations necessitate police involvement. However, invocation of the MHA during a high-risk situation is fraught with stress and difficulties, leaving little room for empathetic communications. Effective and diverse, evidence-based, early intervention strategies - both consensual and non-consensual - are necessary to reduce the requirement for police involvement in mental health transports.
Self-standing TiO2 nanotube layers in the form of membranes are fabricated by self-organizing anodization of Ti metal and a potential shock technique. The membranes were then decorated by sputtering different Pt amounts i) only at the top, ii) only at the bottom or iii) at both top and bottom of the tube layers. The Pt-decorated membranes are transferred either in tube top up or in tube top down configuration onto FTO slides and investigated after crystallization as photocatalysts for H2 generation using either front or back-side light irradiation. Double-side Pt-decoration of the tube membranes leads to higher H2 generation rates (independent of tube and light irradiation configuration) compared to membranes decorated at only one side with similar overall Pt amounts. The results suggest that this effect is not ascribed to the overall amount of Pt cocatalyst as such but rather to its distribution at both tube extremities. This leads to optimized light absorption and electron diffusion/transfer dynamics: the central part of the membranes act as light harvesting zone and electrons therein generated can diffuse towards the Pt/TiO2 active zones (tube extremities) where they can react with the environment and generate H2 gas.
Type II polyketide synthase (PKS) iteratively generates a nascent polyketide thioester of acyl carrier protein (ACP), which is structurally modified to produce an ACP-free intermediate towards its final metabolite. However, the timing of ACP off-loading is not well defined due to the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess a TE activity in vitro with a model substrate, anthraquinone-2-carboxylic acid-N-acetylcysteamine. To further investigate its function, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was stepwisely reconstituted in vitro up to (S)-DNPA, and the product of ActIV reaction was characterized as an ACP-free bicyclic intermediate.These findings indicate that ActIV is a bifunctional CYC-TE and provide clear-cut evidence for the release timing of the intermediate from the ACP anchor.
The polymorphism of two dimensional MoS2 promises new possibilities for nanoelectronics. The realization of those possibilities necessitates techniques to enable flexible and controllable phase engineering of MoS2. In the present study, based on first-principles calculations, a new and flexible route to engineer the phase stability of MoS2 by interfacing it with a GaN or AlN substrate is reported. Depending on the surface termination of the underlying substrate, MoS2 may exhibit either the 2H or 1T' (1T'') phase. The interface coupling between MoS2 and the substrate also affects the phase transition kinetics. In addition, electron doping can act as another means to influence MoS2-substrate interactions and enable further phase engineering of MoS2. The present findings contribute to new knowledge towards phase engineering of MoS2 and the design of hybrid nanodevices comprising both 2D and 3D optoelectronic materials.
Carbon exhibits a large number of allotropes and its phase behaviour is still subject to significant uncertainty and intensive research. The hexagonal form of diamond, also known as lonsdaleite, was discovered in the Canyon Diablo meteorite where its formation was attributed to the extreme conditions experienced during the impact. However, it has recently been claimed that lonsdaleite does not exist as a well-defined material but is instead defective cubic diamond formed under high pressure and high temperature conditions. Here we report the synthesis of almost pure lonsdaleite in a diamond anvil cell at 100 GPa and 400 °C. The nanocrystalline material was recovered at ambient and analysed using diffraction and high resolution electron microscopy. We propose that the transformation is the result of intense radial plastic flow under compression in the diamond anvil cell, which lowers the energy barrier by "locking in" favourable stackings of graphene sheets. This strain induced transformation of the graphitic planes of the precursor to hexagonal diamond is supported by first principles calculations of transformation pathways and explains why the new phase is found in an annular region. Our findings establish that high purity lonsdaleite is readily formed under strain and hence does not require meteoritic impacts.
The goal of this project was to characterize the molecular and cellular roles of various gene targets regulated by 8 miRNAs in differentiating macrophages. Among a number of miRNAs that are found to be expressed in avian macrophages, we focused on eight specific miRNAs (miR-1618, miR-1586, miR-1633, miR-1627, miR-1646, miR-1649, miR-1610, miR-1647) associated with macrophage activation through Wnt signaling, ubiquitination, PPAR mediated macrophage function, vesicle mediated cytokine trafficking, and WD40 domain proteins in macrophage differentiation. The results of our analysis identified a global theme for macrophage function: Differentiation and activation of macrophages requires a comprehensive redistribution of the cell's protein repertoire. This redistribution involves two processes: 1) the degradation and recycling of unneeded cytoplasmic and membrane components and 2) the mobilization of newly synthesized cellular components via vesicular trafficking. Ultimately, this leads to a change in the membrane surface expression profile of the cell as well as to a change in proteins regulating phagosomal and lysosomal compartments facilitating increased efficiency of phagocytic activity. In this manner, a monocyte tooled with chemokine surface receptors and an internal cytoskeletal structure geared towards mobility may efficiently sense, react, and migrate toward a site of infection. Once a monocyte arrives to a site of infection, local signals induce a redistribution of resources into a pro-phagocytic phenotype. This may involve upregulating pathogen pattern recognizing receptors and increasing the efficiency of lysosomal biogenesis, while simultaneously recycling components involved in circulatory migration and leukocyte extravasation. In parallel, Wnt and NF-κB signal transduction induces expression of cytokine signals that act in an autocrine and paracrine manner, driving this process of self-differentiation as well as inducing differentiation of nearby monocytes into macrophages. In this study we focused on two main phases: miRNA associated with genes modulating monocyte to macrophage differentiation and miRNA mediating macrophage function/response to infection or inflammation. Among the hundreds of genes that these miRNA target, our analysis identified a set of them that provide insight into avian macrophage biology.
Glioblastoma multiforme (GBM) is the most common and fatal intracranial cancer in humans and exhibits intense and aberrant angiogenesis that sustains its malignancy and involves several angiogenic signals. Among them, vascular endothelial growth factor (VEGF) plays a key role and is overexpressed in GBM. Different cells appear to act as triggers of the aberrant angiogenesis, and, among them, platelets act as key participants. In order to provide further insights into the platelet features and angiogenic role in GBM, this study investigated the effects of platelet releasate on GBM-derived endothelial cells (GECs) and the levels of VEGF and endostatin, as pro- and anti-angiogenic components of platelet releasate from GBM patients. We demonstrate for the first time that: 1) platelet releasate exerts powerful pro-angiogenic effect on GECs, suggesting it might exert a role in the aberrant angiogenesis of GBM; 2) ADP and thrombin stimulation leads to significantly higher level of VEGF, but not of endostatin, in the releasate of platelets from GBM patients than those from healthy subjects; and 3) the intraplatelet concentrations of VEGF were significantly elevated in GBM patients as compared to controls. Moreover, we found a direct correlation between platelet-released VEGF and overall survival in our patient cohort. Although preliminary, these findings prompt further investigations to clarify the biologic relevance of platelet VEGF in GBM and prospective studies for screening GBM patients for anti-VEGF therapy and/or to optimize this treatment.
The treatment of B cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials<sup>1-6</sup>. The anti-CD19 CAR T cell production method used to support initial trials relied on numerous manual, open process steps, cell culture media supplemented with human serum and 10 days of cell culture to achieve a clinical dose <sup>7</sup>. This approach limited the ability to support large multicenter clinical trials, as well as scale-up for commercial cell production. Therefore, studies were completed to streamline and optimize the original NCI production process by removing human serum from the process to minimize risk of viral contamination, moving process steps from an open system to closed system operations to minimize the risk of microbial contamination, and standardizing additional process steps to maximize process consistency. In this study, we report a procedure for generating CD19 CAR-T cells in 6 days, using a predominantly closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.
Electronic health records (EHRs) have become a vital source of patient outcome data but the widespread prevalence of missing data presents a major challenge. Different causes of missing data in the EHR data may introduce unintentional bias. Here, we compare the effectiveness of popular multiple imputation strategies with a deeply learned autoencoder using the Pooled Resource Open-Access ALS Clinical Trials Database (PRO-ACT). To evaluate performance, we examined imputation accuracy for known values simulated to be either missing completely at random or missing not at random. We also compared ALS disease progression prediction across different imputation models. Autoencoders showed strong performance for imputation accuracy and contributed to the strongest disease progression predictor. Finally, we show that despite clinical heterogeneity, ALS disease progression appears homogenous with time from onset being the most important predictor.
With continued rapid growth in the number and quality of fully sequenced and accurately annotated bacterial genomes, we have unprecedented opportunities to understand metabolic diversity. We selected 101 diverse and representative completely sequenced bacteria and implemented a manual curation effort to identify 846 unique metabolic variants present in these bacteria. The presence or absence of these variants act as a metabolic signature for each of the bacteria, which can then be used to understand similarities and differences between and across bacterial groups. We propose a novel and robust method of summarizing metabolic diversity using metabolic signatures and use this method to generate a metabolic tree, clustering metabolically similar organisms. Resulting analysis of the metabolic tree confirms strong associations with well-established biological results along with direct insight into particular metabolic variants which are most predictive of metabolic diversity. The positive results of this manual curation effort and novel method development suggest that future work is needed to further expand the set of bacteria to which this approach is applied and use the resulting tree to test broad questions about metabolic diversity and complexity across the bacterial tree of life.
Most phylogenetic methods are model-based and depend on models of evolution designed to approximate the evolutionary processes. Several methods have been developed to identify suitable models of evolution for phylogenetic analysis of alignments of nucleotide or amino acid sequences and some of these methods are now firmly embedded in the phylogenetic protocol. However, in a disturbingly large number of cases, it appears that these models were used without acknowledgement of their inherent shortcomings. In this chapter, we discuss the problem of model selection and show how some of the inherent shortcomings may be identified and overcome.
Sometimes ignorance is an excuse. If an agent did not know and could not have known that her action would realize some bad outcome, then it is plausible to maintain that she is not to blame for realizing that outcome, even when the act that leads to this outcome is wrong. This general thought can be brought to bear in the context of climate change insofar as we think (a) that the actions of individual agents play some role in realizing climate harms and (b) that these actions are apt targets for being considered right or wrong. Are agents who are ignorant about climate change and the way their actions contribute to it excused because of their ignorance, or is their ignorance culpable? In this paper I examine these questions from the perspective of recent developments in the theories of responsibility for ignorant action and characterize their verdicts. After developing some objections to existing attempts to explore these questions, I characterize two influential theories of moral responsibility and discuss their implications for three different types of ignorance about climate change. I conclude with some recommendations for how we should react to the face of the theories' conflicting verdicts. The answer to the question posed in the title, then, is: "Well, it's complicated."
As of October 2016, use of federal Older Americans Act funds for health promotion and disease prevention will be restricted to the Administration on Aging's criteria for high-level evidence-based health promotion programs. Dissemination of these programs to rural communities remains limited. Therefore a strong need exists to identify strategies that facilitate program implementation and sustainability. The objective of this study was to compare organizational readiness and implementation strategies used by rural communities that achieved varying levels of success in sustaining evidence-based health promotion programs for older adults. We utilized a qualitative multi-site case study design to analyze the longitudinal experiences of eight rural sites working to implement evidence-based health promotion program over 3 years (8/2012-7/2015). Multiple sources of data (interviews, documents, reports, surveys) from each site informed the analysis. We used conventional content analysis to conduct a cross-case comparison to identify common features of rural counties that successfully implemented and sustained their target evidence-based health promotion program. Readiness to implement evidence-based programs as low at baseline as all site leaders described needing to secure additional resources for program implementation. Sites that successfully utilized six essential resources implemented and sustained greater numbers of workshops: (1) External Partnerships, (2) Agency Leadership Commitment, (3) Ongoing Source of Workshop Leaders, (4) Health Promotion Coordination Tasks Assigned to Specific Staff, (5) Organizational Stability, and (6) Change Team Engagement. The six essential resources described in this study can help rural communities assess their readiness to implement health promotion programs and work secure the resources necessary for successful implementation.
Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of aging, was long believed to be a non-genetic sporadic origin syndrome. The proof that several genetic loci are responsible for rare Mendelian forms has represented a revolutionary breakthrough, enabling to reveal molecular mechanisms underlying this debilitating still incurable condition. While single nucleotide polymorphisms (SNPs) and small indels constitute the most commonly investigated DNA variations accounting for only a limited number of PD cases, larger genomic molecular rearrangements have emerged as significant PD-causing mutations, including submicroscopic Copy Number Variations (CNVs). CNVs constitute a prevalent source of genomic variations and substantially participate in each individual's genomic makeup and phenotypic outcome. However, the majority of genetic studies have focused their attention on single candidate-gene mutations or on common variants reaching a significant statistical level of acceptance. This gene-centric approach is insufficient to uncover the genetic background of polygenic multifactorial disorders like PD, and potentially masks rare individual CNVs that all together might contribute to disease development or progression. In this review, we will discuss literature and bioinformatic data describing the involvement of CNVs on PD pathobiology. We will analyze the most frequent copy number changes in familiar PD genes and provide a "systems biology" overview of rare individual rearrangements that could functionally act on commonly deregulated molecular pathways. Assessing the global genome-wide burden of CNVs in PD patients may reveal new disease-related molecular mechanisms, and open the window to a new possible genetic scenario in the unsolved PD puzzle.
The recent influx of refugees and asylum seekers into Germany poses a challenge for the national healthcare system. In compliance with the present Asylum Seekers Benefits Act, the national healthcare system can be expected to have 1.5 million new members by early 2017. Providing adequate care particularly for people with mental illnesses or disorders will represent an immense challenge for all actors in the system. The circumstances of the flight combined with the foreign linguistic and socio-cultural background increase the severity of the cases and the difficulties of treatment. No procedures or guidelines for treatment have yet been established to ensure a standardized, cost-efficient and therapeutically effective treatment of patients with this background. This article describes the components of a stepped treatment procedure and proposes a stepped and collaborative care model (SCCM) that could be evaluated in nationwide studies. This approach is based on national and international treatment guidelines and aims to provide target-group specific, culturally sensitive methods of diagnosis and treatment. The various steps of the model build on each other, with the first steps relying on technological aids (e.g. online or smartphone options) and support from lay helpers and the more expensive specialist psychiatric and psychotherapeutic therapy only being initiated in cases of more severe mental disorders.
The most trusted hypothesis to explain how α2-adrenergic agonists may preserve pulmonary functions in critically ill patients is that they directly act on macrophages by interfering with an autocrine/paracrine adrenergic system that controls cytokine release through locally synthetized noradrenaline and α1- and α2-adrenoreceptors. We tested this hypothesis in primary cultures of resident macrophages from human lung (HLMs). HLMs were isolated by centrifugation on percoll gradients from macroscopically healthy human lung tissue obtained from four different patients at the time of lung resection for cancer. HLMs from these patients showed a significant expression of α2A, α2B and α2C adrenoreceptors both at the mRNA and at the protein level. To evaluate whether α2 adrenoreceptors controlled cytokine release from HMLs, we measured IL-6, IL-8 and TNF-α concentrations in the culture medium in basal conditions and after preincubation with several α2-adrenergic agonists or antagonists. Neither the pretreatment with the α2-adrenergic agonists clonidine, medetomidine or dexdemetomidine or with the α2-adrenergic antagonist yohimbine caused significant changes in the response of any of these cytokines to LPS. These results show that, different from what reported in rodents, clonidine and dexdemetomidine do not directly suppress cytokine release from human pulmonary macrophages. This suggests that alternative mechanisms such as effects on immune cells activation or the modulation of autonomic neurotransmission could be responsible for the beneficial effects of these drugs on lung function in critical patients.
Melanoma has emerged as a paradigm of a highly aggressive and plastic cancer, capable to co-opt the tumor stroma in order to adapt to the hostile microenvironment, suppress immunosurveillance mechanisms, and disseminate. In particular, oncogene- and aneuploidy-driven dysregulations of proteostasis in melanoma cells impose a rewiring of central proteostatic processes, such as the heat shock and unfolded protein responses, autophagy, and the endo-lysosomal system, to avoid proteotoxicity. Research over the past decade has indicated that alterations in key nodes of these proteostasis pathways act in conjunction with crucial oncogenic drivers to increase intrinsic adaptations of melanoma cells against proteotoxic stress, modulate the high metabolic demand of these cancer cells and the interface with other stromal cells, through the heightened release of soluble factors or exosomes. Here, we overview and discuss how key proteostasis pathways and vesicular trafficking mechanisms are turned into vital conduits of melanoma progression, by supporting cancer cell's adaptation to the microenvironment, limiting or modulating the ability to respond to therapy and fueling melanoma dissemination.
A pressure sore wound is often extensive or complicated by local infection involving adjacent soft tissue and bone. In this case, a regional flap after simple debridement is not adequate. Here, we present a case of an extensive pressure sore in the sacral area with deep tissue infection. A 43-year-old female patient with a complicated sore with deep tissue infection had a presacral abscess, an iliopsoas abscess, and an epidural abscess in the lumbar spine. After a multidisciplinary approach performed in stages, the infection had subsided and removal of the devitalized tissue was possible. The large soft tissue defect with significant depth was reconstructed with a free latissimus dorsi musculocutaneous flap, which was expected to act as a local barrier from vertical infection and provide tensionless skin coverage upon hip flexion. The extensive sacral sore was treated effectively without complication, and the deep tissue infection completely resolved. There was no evidence of donor site morbidity, and wheelchair ambulation was possible by a month after surgery.
Fragranced consumer products-such as air fresheners, cleaning supplies, and personal care products- pervade society. This study investigated the occurrence and types of adverse effects associated with exposure to fragranced products in Australia, and opportunities for prevention. Data were collected in June 2016 using an on-line survey with a representative national sample (n = 1098). Overall, 33% of Australians report health problems, such as migraine headaches and asthma attacks, when exposed to fragranced products. Of these health effects, more than half (17.1%) could be considered disabling under the Australian Disability Discrimination Act. Additionally, 7.7% of Australians have lost workdays or a job due to illness from fragranced product exposure in the workplace, 16.4% reported health problems when exposed to air fresheners or deodorizers, 15.3% from being in a room after it was cleaned with scented products, and 16.7% would enter but then leave a business as quickly as possible due to fragranced products. About twice as many respondents would prefer that workplaces, health care facilities and professionals, hotels, and airplanes were fragrance-free rather than fragranced. While 73.7% were not aware that fragranced products, even ones called green and organic, emitted hazardous air pollutants, 56.3% would not continue to use a product if they knew it did. This is the first study in Australia to assess the extent of adverse effects associated with exposure to common fragranced products. It provides compelling evidence for the importance and value of reducing fragranced product exposure in order to reduce and prevent adverse health effects and costs.
The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.
Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.
Graves' hyperthyroidism is caused by autoantibodies directed against the thyroid-stimulating hormone receptor (TSHR) that mimic the action of TSH. The establishment of Graves' hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs). "Shimojo's model" was the first successful Graves' animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC) class II molecule, but not either alone, induced TSAb production in AKR/N (H-2(k)) mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves' hyperthyroidism. These data are also in agreement with the observation that Graves' thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves' animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves' animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves' disease triggers.
Genetic improvement of photosynthetic performance of cereal crops and increasing the efficiency with which solar radiation is converted into biomass has recently become a major focus for crop physiologists and breeders. The pulse amplitude modulated chlorophyll fluorescence technique (PAM) allows quantitative leaf level monitoring of the utilization of energy for photochemical light conversion and photo-protection in natural environments, potentially over the entire crop lifecycle. Here the diurnal relationship between electron transport rate (ETR) and irradiance was measured in five cultivars of Oryza sativa in canopy conditions with PAM fluorescence under natural solar radiation. This relationship differed substantially from that observed for conventional short term light response curves measured under controlled actinic light with the same leaves. This difference was characterized by a reduced curvature factor when curve fitting was used to model this diurnal response. The engagement of photo-protective processes in chloroplast electron transport in leaves under canopy solar radiation was shown to be a major contributor to this difference. Genotypic variation in the irradiance at which energy flux into photo-protective dissipation became greater than ETR was observed. Cultivars capable of higher ETR at mid range light intensities were shown to produce greater leaf area over time, estimated by non-invasive imaging.
Scotland's parochial asylums are unfamiliar institutional spaces. Representing the concrete manifestation of the collision between two spheres of legislation, the Poor Law and the Lunacy Law, six such asylums were constructed in the latter half of the nineteenth century. These sites expressed the enduring mandate of the Scottish Poor Law 1845 over the domain of 'madness'. They were institutions whose very existence was fashioned at the directive of the local arm of the Poor Law, the parochial board, and they constituted a continuing 'Scottish Poor Law of Lunacy'. Their origins and operation significantly subverted the intentions and objectives of the Lunacy Act 1857, the aim of which had been to institute a public district asylum network with nationwide coverage.
Cognate interactions between T follicular helper (Tfh) cells and B cells are essential for promoting protective Ab responses. Whereas costimulatory receptors such as ICOS are accepted as being important for the induction of Tfh cell fate decision, other molecules may play key roles in amplifying or maintaining the Tfh phenotype. In this study, with vaccinia virus infection in mice, we show that OX40 was expressed on Tfh cells that accumulated at the T/B borders in the white pulp of the spleen and that OX40-dependent signals directly shaped the magnitude and quality of the their response to viral Ags. OX40 deficiency in Tfh cells profoundly impaired the acquisition of germinal center (GC) B cell phenotype, plasma cell generation, and virus-specific Ab responses. Most significantly, we found that sustained interactions between OX40 and its ligand, OX40L, beyond the time of initial encounter with dendritic cells were required for the persistence of high numbers of Tfh and GC B cells. Interestingly, OX40 was coexpressed with ICOS on Tfh cells in and around the GC, and ICOS-ICOSL interactions were similarly crucial at late times for maintenance of the Tfh and GC B cells. Thus, OX40 and ICOS act in a cooperative, nonredundant manner to maximize and prolong the Tfh response that is generated after acute virus infection.
Malignant features such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility and metastasis can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions such as cell proliferation, survival or motility, and is involved in metabolic homeostasis, inflammation or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an onco-modifier, able to modulate carcinogenesis in a cell-type specific way.
For several decades, glucocorticoids have been used empirically to treat rapid progressive GN. It is commonly assumed that glucocorticoids act primarily by dampening the immune response, but the mechanisms remain incompletely understood. In this study, we inactivated the glucocorticoid receptor (GR) specifically in kidney epithelial cells using Pax8-Cre/GR(fl/fl) mice. Pax8-Cre/GR(fl/fl) mice did not exhibit an overt spontaneous phenotype. In mice treated with nephrotoxic serum to induce crescentic nephritis (rapidly progressive GN), this genetic inactivation of the GR in kidney epithelial cells exerted renal benefits, including inhibition of albuminuria and cellular crescent formation, similar to the renal benefits observed with high-dose prednisolone in control mice. However, genetic inactivation of the GR in kidney epithelial cells did not induce the immunosuppressive effects observed with prednisolone. In vitro, prednisolone and the pharmacologic GR antagonist mifepristone each acted directly on primary cultures of parietal epithelial cells, inhibiting cellular outgrowth and proliferation. In wild-type mice, pharmacologic treatment with the GR antagonist mifepristone also attenuated disease as effectively as high-dose prednisolone without the systemic immunosuppressive effects. Collectively, these data show that glucocorticoids act directly on activated glomerular parietal epithelial cells in crescentic nephritis. Furthermore, we identified a novel therapeutic approach in crescentic nephritis, that of glucocorticoid antagonism, which was at least as effective as high-dose prednisolone with potentially fewer adverse effects.
Biased agonism at G protein coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study, we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high-affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12. Binding of J113863 to CCR2 or CCR5 also induced the recruitment of β-arrestin 2 whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and β-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist or full agonist according to the receptor, the enantiomer considered and the signaling pathway investigated.
Transcriptional enhancers are critical for maintaining cell type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a sub-cluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by co-binding of 5 or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified which regulated clusters of paralogous genes. We conclude that whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.
To study the changes in prevalence, characteristics and outcomes of pregnant smokers over time and legislative changes.
Retrospective nationwide cohort.
Our study consisted of 9627 randomly selected pregnancies from the Finnish Maternity Cohort (1987-2011), with demographic characteristics and pregnancy and perinatal data obtained from the Medical Birth Registry and early pregnancy serum samples analysed for cotinine levels. Women were categorised based on their self-reported smoking status and measured cotinine levels (with ≥4.73 ng/mL deemed high). Data were stratified to three time periods based on legislative changes in the Tobacco Act.
Prevalence of pregnant smokers and demographics, and perinatal and pregnancy outcomes of pregnant smokers over time.
Overall, 71.6% of women were non-smokers, 16.2% were active cigarette smokers, 7.7% undisclosed smoking but had high cotinine levels and 4.5% were inactive cigarette smokers. The prevalence of active cigarette smokers decreased from mid-1990s onwards among women aged ≥30 years, probably due to the ban of cigarette smoking in most workplaces. We observed no changes in the prevalence of inactive smokers or women who undisclosed smoking by time or legislative changes.Women who undisclosed smoking had similar characteristics and perinatal outcomes as inactive and active smokers. Compared with non-smokers, women who undisclosed smoking were more likely to be young, unmarried, have a socioeconomic status lower than white-collar worker and have a preterm birth.
Women who undisclosed smoking were very similar to pregnant cigarette smokers. We observed a reduction in the prevalence of active pregnant cigarette smokers after the ban of indoor smoking in workplaces and restaurants, mostly among women aged ≥30 years.
Herbal medicine is a concoction of numerous chemical ingredients, and it exhibits polypharmacological effects to act on multiple pharmacological targets, regulating different biological mechanisms and treating a variety of diseases. Thus, this complexity is impossible to deconvolute by the reductionist method of extracting one active ingredient acting on one biological target.
To dissect the polypharmacological effects of herbal medicines and their underling pharmacological targets as well as their corresponding active ingredients.
We propose a system-biology strategy that combines omics and bioinformatical methodologies for exploring the polypharmacology of herbal mixtures. The myocardial ischemia model was induced by Ameroid constriction of the left anterior descending coronary in Ba-Ma miniature pigs. RNA-seq analysis was utilized to find the differential genes induced by myocardial ischemia in pigs treated with formula QSKL. A transcriptome-based inference method was used to find the landmark drugs with similar mechanisms to QSKL.
Gene-level analysis of RNA-seq data in QSKL-treated cases versus control animals yields 279 differential genes. Transcriptome-based inference methods identified 80 landmark drugs that covered nearly all drug classes. Then, based on the landmark drugs, 155 potential pharmacological targets and 57 indications were identified for QSKL.
Our results demonstrate the power of a combined approach for exploring the pharmacological target and chemical space of herbal medicines. We hope that our method could enhance our understanding of the molecular mechanisms of herbal systems and further accelerate the exploration of the value of traditional herbal medicine systems.
Depression is one of the major contributors to poorer quality of life amongst individuals with psychosis and schizophrenia. The study was designed as a Pilot Trial to determine the parameters of a larger, definitive pragmatic multi-centre randomised controlled trial of Acceptance and Commitment Therapy for depression after psychosis (ACTdp) for individuals with a diagnosis of schizophrenia who also meet diagnostic criteria for major depression.
Participants were required to meet criteria for schizophrenia and major depression. Blinded follow-ups were undertaken at 5-months (end of treatment) and at 10-months (5-months posttreatment). Primary outcomes were depression as measured by the Calgary Depression Scale for Schizophrenia (CDSS) and the Beck Depression Inventory (BDI).
A total of 29 participants were randomised to ACTdp + Standard Care (SC) (n=15) or SC alone (n=14). We did not observe significant differences between groups on the CDSS total score at 5-months (Coeff=-1.43, 95%CI -5.17, 2.32, p=0.45) or at 10-months (Coeff=1.8, 95%CI -2.10, 5.69, p=0.36). In terms of BDI, we noted a statistically significant effect in favour of ACTdp+SC at 5-months (Coeff=-8.38, 95%CI -15.49, -1.27, p=0.02) but not at 10-months (Coeff=-4.85, 95%CI -12.10, 2.39, p=0.18). We also observed significant effects on psychological flexibility at 5-months (Coeff=-8.83, 95%CI -14.94, -2.71, p<0.01) but not 10-months (Coeff=-4.92, 95%CI -11.09, 1.25, p=0.11).
In this first RCT of a psychological therapy with depression as the primary outcome, ACT is a promising intervention for depression in the context of psychosis. A further large-scale definitive randomised controlled trial is required to determine effectiveness.
ISRCTN: 33306437.
Millions of humans are exposed occupationally and environmentally to lead, mercury and cadmium compounds. Mercury compounds are less abundant but some of them belong to the most toxic chemicals which are known. We evaluated the literature to find out if these metals act in humans as genotoxic carcinogens and if their health effects can be predicted by use of micronucleus (MN) assays with lymphocytes and/or with other genotoxicity tests. Numerous studies showed that lead and mercury induce cancer in humans and also in animals, in vitro experiments with cultured cells indicate that they cause DNA damage via different molecular mechanisms including release of reactive oxygen species and interactions with DNA repair processes. Also in most human studies, positive results were obtained in MN tests with lymphocytes (all 15 occupational studies with lead yielded positive results, with mercury 6 out of 7 investigations were positive). For cadmium, there is clear evidence that it causes cancer in humans; however, induction of chromosomal damage was only seen in high dose experiments with mammalian cells while results of animal and human studies yielded conflicting results (only in 2 of 5MN trials with humans positive findings were reported). Possibly, non-genotoxic mechanisms such as inhibition of apoptosis and interaction with signaling pathways account for the carcinogenic properties of cadmium species. The findings of MN studies with lead and mercury are in excellent agreement with results which were obtained with other endpoints (e.g. chromosomal aberrations and comet formations) and it is evident that this approach can be used for occupational and environmental monitoring of exposed individuals. Important future tasks will be the realization of larger studies with a uniform standardized protocol, the additional evaluation of anomalies other than MN (nuclear buds and bridges) and the combination of such trials with investigations which allow to define the molecular mechanisms relevant for exposed humans.
It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.
To identify barriers to fruit and vegetable intake for Indigenous Australian children and quantify factors related to these barriers, to help understand why children do not meet recommendations for fruit and vegetable intake.
We examined factors related to carer-reported barriers using multilevel Poisson models (robust variance); a key informant focus group guided our interpretation of findings.
Eleven diverse sites across Australia.
Australian Indigenous children and their carers (N 1230) participating in the Longitudinal Study of Indigenous Children.
Almost half (45 %; n 555/1230) of carers reported barriers to their children's fruit and vegetable intake. Dislike of fruit and vegetables was the most common barrier, reported by 32·9 % of carers; however, we identified few factors associated with dislike. Carers were more than ten times less likely to report barriers to accessing fruit and vegetables if they lived large cities v. very remote areas. Within urban and inner regional areas, child and carer well-being, financial security, suitable housing and community cohesion promoted access to fruit and vegetables.
In this national Indigenous Australian sample, almost half of carers faced barriers to providing their children with a healthy diet. Both remote/outer regional carers and disadvantaged urban/inner regional carers faced problems accessing fruit and vegetables for their children. Where vegetables were accessible, children's dislike was a substantial barrier. Nutrition promotion must address the broader family, community, environmental and cultural contexts that impact nutrition, and should draw on the strengths of Indigenous families and communities.
Understanding dietary patterns in obese pregnant women will inform future intervention strategies to improve pregnancy outcomes and the health of the child. The aim of this study was to investigate the effect of a behavioral intervention of diet and physical activity advice on dietary patterns in obese pregnant woman participating in the UPBEAT study, and to explore associations of dietary patterns with pregnancy outcomes.
In the UPBEAT randomized controlled trial, pregnant obese women from eight UK multi-ethnic, inner-city populations were randomly assigned to receive a diet/physical activity intervention or standard antenatal care. The dietary intervention aimed to reduce glycemic load and saturated fat intake. Diet was assessed using a food frequency questionnaire (FFQ) at baseline (15(+0)-18(+6) weeks' gestation), post intervention (27(+0)-28(+6) weeks) and in late pregnancy (34(+0)-36(+0) weeks). Dietary patterns were characterized using factor analysis of the baseline FFQ data, and changes compared in the control and intervention arms. Patterns were related to pregnancy outcomes in the combined control/intervention cohort (n = 1023).
Four distinct baseline dietary patterns were defined; Fruit and vegetables, African/Caribbean, Processed, and Snacks, which were differently associated with social and demographic factors. The UPBEAT intervention significantly reduced the Processed (-0.14; 95% CI -0.19, -0.08, P <0.0001) and Snacks (-0.24; 95% CI -0.31, -0.17, P <0.0001) pattern scores. In the adjusted model, baseline scores for the African/Caribbean (quartile 4 compared with quartile 1: OR = 2.46; 95% CI 1.41, 4.30) and Processed (quartile 4 compared with quartile 1: OR = 2.05; 95% CI 1.23, 3.41) patterns in the entire cohort were associated with increased risk of gestational diabetes.
In a diverse cohort of obese pregnant women an intensive dietary intervention improved Processed and Snack dietary pattern scores. African/Caribbean and Processed patterns were associated with an increased risk of gestational diabetes, and provide potential targets for future interventions.
Current controlled trials; ISRCTN89971375.
Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P < 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03-2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02-1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators.
Fatigue in hospital nurses is associated with decreased nurse satisfaction, increased turnover and negative patient outcomes. Addressing fatigue in nurses has been identified as a priority by many organizations worldwide in an effort to promote both a culture of patient safety and a healthy nursing workforce.
The overall aim of this study was to explore barriers and facilitators within the hospital nurse work system to nurse coping and fatigue. The purpose of this paper is to describe emergent themes that offer new insight describing the relationships among nurse perceptions of fatigue, nursing professional culture, and implications for the nursing workforce.
A qualitative exploratory study was used to explore nurse identified sources, barriers to addressing, and consequences of fatigue.
Twenty-two nurses working in intensive care and medical-surgical units within a large academic medical center in the United States participated in the interviews.
Interviews with the participants followed a semi-structured interview guide that included questions eliciting participants' views on nurse fatigue levels, consequences of fatigue, and barriers to addressing fatigue. The interview transcripts were analyzed using directed content analysis guided by the Systems Engineering Initiative for Patient Safety (SEIPS) model. Additional themes that did not directly align with the SEIPS model were also identified.
All nurses in the current study experienced fatigue; yet they had varying perspectives on the importance of addressing fatigue in relation to other health systems challenges. A new construct related to nursing professional culture was identified and defined as "Supernurse". Identified subthemes of Supernurse include: extraordinary powers used for good; cloak of invulnerability; no sidekick; Kryptonite, and an alterego. These values, beliefs, and behaviors define the specific aspects of nursing professional culture that can act as barriers to fatigue risk management programs and achieving safety culture in hospital organizations. Nurse fatigue and attributes of nurse professional culture also have implications for nurse satisfaction and retention.
Findings from this study further support the role of nursing professional culture as an important barrier to effectively addressing fatigue in nursing work systems. Future work is needed to identify and evaluate innovative culture change models and strategies to target these barriers.
Both obesity (body mass index ≥ 30) and educational attainment have increased dramatically in the United States since the 1970s. This study analyzed the influences of educational inequalities in obesity and population improvements in education on national obesity trends between 1970 and 2010. For non-Hispanic white and black males and females aged 25-74 years, educational differences in the probability of being obese were estimated from the 1971-2012 National Health and Nutrition Examination Surveys, and population distributions of age and educational groups, from the 1970 Census and 2010 American Community Survey. In the total population, obesity increased from 15.7% to 38.8%, and there were increases in the greater obese probabilities of non-college graduates relative to four-year college graduates. The increase in obesity would have been lower by 10% (2.2 percentage points) if educational inequalities in obesity had stayed at their 1970 values and lower by one third (7.9 points) if obesity inequalities had been eliminated. Obesity inequalities were larger for females than males and for whites than blacks, and obesity did not differ by education among black males. As a result, the impact of obesity inequalities on the obesity trend was largest among white females (a 47% reduction in the obesity increase if obesity inequalities had been eliminated), and virtually zero among black males. On the other hand, without educational improvements, the obesity increase would have been 9% more in the total population, 23% more among white females and not different in the other three subpopulations. Results indicate that obesity inequalities made sizable contributions to the obesity trends, and the obesity reductions associated with educational improvements were more limited.
Microsatellites or simple sequence repeats (SSR) are abundant, highly diverse stretches of short DNA repeats present in all genomes. Tandem mono/tri/hexanucleotide repeats in the coding regions contribute to single amino acids repeats (SAARs) in the proteome. While SSRs in the coding region always result in amino acid repeats, a majority of SAARs arise due to a combination of various codons representing the same amino acid and not as a consequence of SSR events. Certain amino acids are abundant in repeat regions indicating a positive selection pressure behind the accumulation of SAARs. By analysing 22 proteomes including the human proteome, we explored the functional and structural relationship of amino acid repeats in an evolutionary context. Only ~15% of repeats are present in any known functional domain, while ~74% of repeats are present in the disordered regions, suggesting that SAARs add to the functionality of proteins by providing flexibility, stability and act as linker elements between domains. Comparison of SAAR containing proteins across species reveals that while shorter repeats are conserved among orthologs, proteins with longer repeats, >15 amino acids, are unique to the respective organism. Lysine repeats are well conserved among orthologs with respect to their length and number of occurrences in a protein. Other amino acids such as glutamic acid, proline, serine and alanine repeats are generally conserved among the orthologs with varying repeat lengths. These findings suggest that SAARs have accumulated in the proteome under positive selection pressure and that they provide flexibility for optimal folding of functional/structural domains of proteins. The insights gained from our observations can help in effective designing and engineering of proteins with novel features.
Trust has been considered the "cement" of a society and is much studied in sociology and other social sciences. Most studies, however, have neglected one important aspect of trust: it involves an act of forgiving and showing tolerance toward another's failure. In this study, we refer to this concept as "generous trust" and examine the conditions under which generous trust becomes a more viable option when compared to other types of trust. We investigate two settings. First, we introduce two types of uncertainties: uncertainty as to whether trustees have the intention to cooperate, and uncertainty as to whether trustees have enough competence to accomplish the entrusted tasks. Second, we examine the manner in which trust functions in a broader social context, one that involves matching and commitment processes. Since we expect generosity or forgiveness to work differently in the matching and commitment processes, we must differentiate trust strategies into generous trust in the matching process and that in the commitment process. Our analytical strategy is two-fold. First, we analyze the "modified" trust game that incorporates the two types of uncertainties without the matching process. This simplified setting enables us to derive mathematical results using game theory, thereby giving basic insight into the trust mechanism. Second, we investigate socially embedded trust relationships in contexts involving the matching and commitment processes, using agent-based simulation. Results show that uncertainty about partner's intention and competence makes generous trust a viable option. In contrast, too much uncertainty undermines the possibility of generous trust. Furthermore, a strategy that is too generous cannot stand alone. Generosity should be accompanied with moderate punishment. As for socially embedded trust relationships, generosity functions differently in the matching process versus the commitment process. Indeed, these two types of generous trust coexist, and their coexistence enables a society to function well.
γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.
This report demonstrates a unique case of conjunctival melanoma harboring a BRAF V600E mutation responsive to systemic therapy with BRAF and MEK inhibitors. While systemic therapy would not be appropriate in patients with local disease alone, it may act therapeutically in cases of higher stage ocular surface and eyelid melanoma.
Improving patient care through enhanced electronic communication among health care providers is aimed at reducing the number of medication and medical errors. The American Reinvestment and Recovery Act (ARRA) was signed into law in 2009, supporting the federal government's commitment to the improvement of health care quality, safety, and efficiency through requirements to implement an electronic health record by October 2015 or hospitals and eligible providers potentially realizing penalties or reduced reimbursement rates. In addition to ARRA, Congress presented another initiative to further advance the delivery of high-quality health care, the Health Information Technology for Economic and Clinical Health Act (HITECH), leading to the authorization of $27 billion to encourage health care providers to achieve meaningful use of the electronic health record. However, the conversion of the paper medical records to an electronic version has been challenging, particularly in specialty departments. The burn unit of a tertiary hospital located in the Pittsburgh area experienced such challenges. A project plan, developed in 2009 prior to the electronic medical record going live, involved a multidisciplinary team, consisting of providers, nurses, and information system builders who came together to determine how to capture the totality of the burn unit documentation. The goal of the project was to develop an electronic documentation tool and provide a system to accurately and efficiently evaluate documentation compliance with the staff. The Lund Browder documentation tool, used with the paper medical record, was the selected tool for the electronic conversion. This tool has been regarded by most health care organizations as being the most accurate in measuring the extent and degree of the burn. With the paper documentation tool, the staff was, on average, 74% compliant with the Lund Browder tool. The electronic version and availability of the tool increased compliance to 100% in the fourth quarter of 2015.
Glioblastoma multiforme (GBM) continues to be associated with a dismal prognosis despite aggressive treatment. Significant efforts are being made to develop new nanotechnology-based therapeutic and diagnostic agents. Nanoparticles can act directly on cancer cells or as drug carriers to enhance the cancer therapeutic effect. In this study, we investigated the effect of silver nanoparticles (AgNPs) on human glioma U251 cells and its role in the combinational use with Temozolomide (TMZ), an imidazotetrazine derivative of the alkylating agent dacarbazine, against glioma cells. AgNPs were synthesized in the sodium citrate system and the mean size were 26 nm in diameter. The AgNP particles showed dose-dependent cytotoxicity on U251 cells. They also showed the ability to enhance the drug-sensitivity of TMZ on U251 cells. Our results revealed that AgNPs could have a potential application in enhancing chemotherapy for glioma.
Taking their inspiration from a case history, the authors explore the effects of a writing workshop led by a professional writer for patients in a psychiatric hospital. This workshop allowed different modes of transference to unfold: transference to the analyst-therapist, transference to the writer who led the workshop, and transference to the other members of the group. The writing activity created conditions in which there could be a movement from hallucination to delusion-a delusion expressed in fiction through the act of writing. Psychotic patients "invent" a writing that remains unfinished and that relates to the experiences of persecution. Writing thus makes it possible for them to tolerate language, through its transformation into writing.
The packaging of genetic information in form of chromatin within the nucleus provides cells with the ability to store and protect massive amounts of information within a compact space. Storing information within chromatin allows selective access to specific DNA sequences by regulating the various levels of chromatin structure from nucleosomes, to chromatin fibers, loops and topological associating domains (TADs) using mechanisms that are being progressively unravelled. However, a relatively unexplored aspect is the energetic cost of changing the chromatin configuration to gain access to DNA information. Among the enzymes responsible for regulating chromatin access are the ATP-dependent chromatin remodellers that act on nucleosomes and use the energy of ATP hydrolysis to make chromatin DNA more accessible. It is assumed that the ATP used by these enzymes is provided by the mitochondria or by cytoplasmic glycolysis. We hypothesize that though this may be the case for cells in steady state, when gene expression has to be globally reprogramed in response to externals signals or stress conditions, the cell directs energy production to the cell nucleus, where rapid chromatin reorganization is needed for cell survival. We discovered that in response to hormones a nuclear ATP synthesis mechanism is activated that utilizing ADP-ribose and pyrophosphate as substrates. (1) This extra view aims to put this process within its historical context, to describe the enzymatic steps in detail, to propose a possible structure of the ATP synthesising enzyme, and to shed light on how this may link to other reactions within the cell providing a perspective for future lines of investigation.
Sleep spindles act as a powerful marker of individual differences in cognitive ability. Sleep spindle parameters correlate with both age-related changes in cognitive abilities and with the age-independent concept of IQ. While some studies have specifically demonstrated the relationship between sleep spindles and intelligence in young children, our previous work in older subjects revealed sex differences in the sleep spindle correlates of IQ, which was never investigated in small children before. We investigated the relationship between age, Raven Colored Progressive Matrices (CPM) scores and sleep spindles in 28 young children (age 4-8 years, 15 girls). We specifically investigated sex differences in the psychometric correlates of sleep spindles. We also aimed to separate the correlates of sleep spindles that are because of age-related maturation from other effects that reflect an age-independent relationship between sleep spindles and general intelligence. Our results revealed a modest positive correlation between fast spindle amplitude and age. Raven CPM scores positively correlated with both slow and fast spindle amplitude, but this effect remained a tendency in males and vanished after correcting for the effects of age. Age-corrected correlations between Raven CPM scores and both slow and fast spindle amplitude were only significant in females. Overall, our results show that in male children sleep spindles are a maturational marker, but in female children they indicate trait-like intelligence, in line with previous studies in adolescent and adult subjects. Thalamocortical white matter connectivity may be the underlying mechanism behind both higher spindle amplitude and higher intelligence in female, but not male subjects. (PsycINFO Database Record
We aimed to establish the prevalence of parental consanguinity among patients with primary immunodeficiency diseases (PID), and compare the prevalence with the general population.
We searched PubMed, EMBASE, and Scopus for studies mentioning parental consanguinity prevalence in PID patients and calculated the prevalence odds ratio (POR) of parental consanguinity in each study, compared to a matched healthy population.
We identified 21 eligible studies with a total population of 18091 accounting for sample overlap. The POR among studies on a sample of mixed PID patients ranged from 0.6 to 21.9 with the pooled POR of 3.0 (P<0.001; I(2) =89%, 95% CI 2.5-3.7).
PIDs with an autosomal recessive pattern of inheritance had significant odds of parental consanguinity compared to the healthy population; a phenomenon not observed in other inheritance patterns. Determining the extent of the impact that consanguinity impose upon the progeny paves the way for convincing healthcare policy makers in highly consanguineous communities to act more diligently in informing the masses about the consequences of practicing inbreeding. This article is protected by copyright. All rights reserved.
Atmospheric CO2 enrichment is expected to often benefit plant growth, despite causing global warming and nitrogen (N) dilution in plants. Most plants primarily procure N as inorganic nitrate (NO3(-) ) or ammonium (NH4(+) ), using membrane-localized transport proteins in roots, which are key targets for improving N use. Though interactive effects of elevated CO2 , chronic warming, and N form on N relations are expected, these have not been studied. In this study, tomato (Solanum lycopersicum) plants were grown at two levels of CO2 (400 or 700 ppm) and two temperature regimes (30 or 37 °C), with NO3(-) or NH4(+) as the N source. Elevated CO2 plus chronic warming severely inhibited plant growth, regardless of N form, while individually they had smaller effects on growth. Although %N in roots was similar among all treatments, elevated CO2 plus warming decreased (1) N uptake rate by roots, (2) total protein concentration in roots, indicating an inhibition of N assimilation, and (3) shoot %N, indicating a potential inhibition of N translocation from roots to shoots. Under elevated CO2 plus warming, reduced NO3(-) uptake rate per g root was correlated with a decrease in the concentration of NO3(-) -uptake proteins per g root, reduced NH4(+) uptake was correlated with decreased activity of NH4(+) -uptake proteins, and reduced N assimilation was correlated with decreased concentration of N-assimilatory proteins. These results indicate that elevated CO2 and chronic warming can act synergistically to decrease plant N uptake and assimilation; hence, future global warming may decrease both plant growth and food quality (%N).
The Patient Protection and Affordable Care Act of 2010 brought attention to adverse drug events in national patient safety efforts. Updated, detailed, nationally representative data describing adverse drug events can help focus these efforts.
To describe the characteristics of emergency department (ED) visits for adverse drug events in the United States in 2013-2014 and describe changes in ED visits for adverse drug events since 2005-2006.
Active, nationally representative, public health surveillance in 58 EDs located in the United States and participating in the National Electronic Injury Surveillance System-Cooperative Adverse Drug Event Surveillance project.
Drugs implicated in ED visits.
National weighted estimates of ED visits and subsequent hospitalizations for adverse drug events.
Based on data from 42 585 cases, an estimated 4.0 (95% CI, 3.1-5.0) ED visits for adverse drug events occurred per 1000 individuals annually in 2013 and 2014 and 27.3% (95% CI, 22.2%-32.4%) of ED visits for adverse drug events resulted in hospitalization. An estimated 34.5% (95% CI, 30.3%-38.8%) of ED visits for adverse drug events occurred among adults aged 65 years or older in 2013-2014 compared with an estimated 25.6% (95% CI, 21.1%-30.0%) in 2005-2006; older adults experienced the highest hospitalization rates (43.6%; 95% CI, 36.6%-50.5%). Anticoagulants, antibiotics, and diabetes agents were implicated in an estimated 46.9% (95% CI, 44.2%-49.7%) of ED visits for adverse drug events, which included clinically significant adverse events, such as hemorrhage (anticoagulants), moderate to severe allergic reactions (antibiotics), and hypoglycemia with moderate to severe neurological effects (diabetes agents). Since 2005-2006, the proportions of ED visits for adverse drug events from anticoagulants and diabetes agents have increased, whereas the proportion from antibiotics has decreased. Among children aged 5 years or younger, antibiotics were the most common drug class implicated (56.4%; 95% CI, 51.8%-61.0%). Among children and adolescents aged 6 to 19 years, antibiotics also were the most common drug class implicated (31.8%; 95% CI, 28.7%-34.9%) in ED visits for adverse drug events, followed by antipsychotics (4.5%; 95% CI, 3.3%-5.6%). Among older adults (aged ≥65 years), 3 drug classes (anticoagulants, diabetes agents, and opioid analgesics) were implicated in an estimated 59.9% (95% CI, 56.8%-62.9%) of ED visits for adverse drug events; 4 anticoagulants (warfarin, rivaroxaban, dabigatran, and enoxaparin) and 5 diabetes agents (insulin and 4 oral agents) were among the 15 most common drugs implicated. Medications to always avoid in older adults according to Beers criteria were implicated in 1.8% (95% CI, 1.5%-2.1%) of ED visits for adverse drug events.
The prevalence of emergency department visits for adverse drug events in the United States was estimated to be 4 per 1000 individuals in 2013 and 2014. The most common drug classes implicated were anticoagulants, antibiotics, diabetes agents, and opioid analgesics.
One of the most difficult undertakings for any employer is carrying out a decision to terminate an employee. Of all the employment-related actions taken by employers, the act of termination creates the greatest risk of legal liability. Many claims of employment discrimination filed with the Equal Employment Opportunity Commission arise from the act of termination. In many federal courts, employment-related lawsuits account for more than 50% of all court filings; these lawsuits cover a wide range of subjects, such as failure to hire, defamation, breach of contract, and harassment, to name a few. However, most employees sue because they have lost their job or fear they will lose their job. Because these individuals have virtually nothing to lose, they often see filing a claim with the Equal Employment Opportunity Commission or filing a lawsuit as the only viable option-often suing for wrongful discharge. With a thoughtful review of the issues and the legislation addressed in this article, health care managers can reduce the unnecessary risk of expensive, time-consuming litigation.
The infection caused by the zoonotic opportunistic pathogen Mycobacterium avium subsp. hominissuis (Mah) was reported for the first time in a pet ferret. Both owners were HIV-positive. Euthanasia of the pet was recommended due to medical reasons and as a preventive action. Disseminated and open tuberculosis lesions were observed in the gastrointestinal and respiratory systems of the ferret. Ecographic and radiographic surveys showed a severe generalized lymphadenopathy, strong thickening of the gastric wall and peritoneum layer. The histopathological findings revealed a disseminated, granulomatous, chronic inflammation affecting the gastrointestinal tract, lungs, lymphoid tissues (spleen, tonsils and lymph nodes) and liver. Ziehl-Neelsen staining displayed the presence of positive acid-fast bacilli within these granulomas. Bacteriology and sequencing of the isolates yielded Mah sequevar code 3. Ferrets can act as reservoirs of mycobacteria exposing their owners to the infection, which is of major concern in immunodeficient individuals, as those HIV-infected.
Mosquitoes act as vectors of devastating pathogens and parasites, representing a key threat for millions of humans and animals worldwide. Eco-friendly control tools are urgently required. We proposed a novel method of fern-mediated biosynthesis of silver nanoparticles (AgNP) using Dicranopteris linearis, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), zeta potential and particle size analysis. In mosquitocidal assays, the LC50 of D. linearis extract against Aedes aegypti ranged from 165.213 (larva I) to 255.055ppm (pupa). LC50 of D. linearis-synthesized AgNP ranged from 18.905 (larva I) to 29.328ppm (pupa). In the field, the application of D. linearis extract and AgNP (10×LC50) led to 100% larval reduction after 72h. Smoke toxicity experiments conducted against A. aegypti adults showed that D. linearis leaf-, stem- and root-based coils evoked mortality rates comparable to the permethrin-based positive control (58%, 47%, 34%, and 48% respectively). In ovicidal experiments, egg hatchability was reduced by 100% after treatment with 25ppm of AgNP and 300ppm of D. linearis extract. Interestingly, oviposition deterrent assays highlighted that 100ppm of fern extract reduced oviposition rates of more than 65%, while 10ppm of fern-fabricated AgNP reduced oviposition rates of more than 70% in A. aegypti (OAI were -0.52 and -0.55, respectively). Overall, our results highlighted that D. linearis-synthesized AgNP could be useful candidates to develop nano-formulated oviposition deterrents effective against dengue vectors.
Chrysobalanus icaco L. (Chrysobalanaceae) has been used for the treatment of abdominal pain and cramps.
Assess the chemical and pharmacological profile of the lyophilized aqueous extract from C. icaco leaves (AEC).
Chromatographic methods were used to assess compounds from AEC. Mice were treated with vehicle (control group) or AEC (100, 200 or 400 mg/kg, p.o.) (group with 7-8 mice) and the analgesic profile was assessed employing the acetic acid-induced writhing, formalin, hot plate tests and hyperalgesia induced by carrageenan (CG) or tumour necrosis factor-alpha. The animal motor performance was assessed using rota-rod and grip strength tests.
The chromatographic profile of AEC demonstrated the presence of terpenoid compounds. The acute pretreatment with AEC, at all doses, produced a significant (p < 0.01) inhibition of painful bahaviour (11.4 ± 3.6; 10.3 ± 2.8; 11.3 ± 2.2) when compared to the control group (24.7 ± 4.7) in acetic acid-induced writhing test. In the formalin test, AEC were effective in the second phase (p < 0.01) (57.2 ± 10.3; 56.3 ± 9.2; 54.7 ± 8.9) when compared to control group (121.9 ± 18.5). No response was observed in the hot plate test. The higher dose of AEC produced a significant (p < 0.01 or p < 0.05) inhibitory effect on the mechanical hyperalgesia test. AEC did not affect the motor performance of the mice.
The terpenoids from AEC are known for its analgesic and anti-inflammatory properties. So, these results corroborate the experiments using the AEC in inflammatory pain protocols.
Our results suggest that AEC act against inflammatory pain.
Building upon the Foucauldian insight that sexuality is a discourse and thereby refusing to be chained to the Freudian repressive hypothesis, this paper aims to ascertain how the closet is made and how the homosexual comes to be seen in the act of reading the literary text, "The Beast in the Jungle," written by Henry James. It will examine the power relationships between the characters and between the narrator and the reader, surrounding the protagonist's sexual secret, which is linked to fear.
D-Fraction is protein-bound β-1,6 and β-1,3 glucans (proteoglucan) extracted from the edible and medicinal mushroom Grifola frondosa (Maitake). The antitumoral effect of D-Fraction has long been exclusively attributed to their immunostimulatory capacity. However, in recent years increasing evidence showed that D-Fraction directly affects the viability of canine and human tumor cells, independent of the immune system. Previously, we have reported that D-Fraction modulates the expression of genes associated with cell proliferation, cell death, migration, invasion, and metastasis in MCF7 human breast cancer cells. Therefore, the purpose of the current study is to investigate if this modulation of gene expression by Maitake D-Fraction really modulates tumor progression. In the present work, we demonstrate for the first time that Maitake D-Fraction is able to act directly on mammary tumor cells, modulating different cellular processes involved in the development and progression of cancer. We demonstrate that D-Fraction decreases cell viability, increases cell adhesion, and reduces the migration and invasion of mammary tumor cells, generating a less aggressive cell behavior. In concordance with these results, we also demonstrate that D-Fraction decreases tumor burden and the number of lung metastases in a murine model of breast cancer.
Chronic pruritus is difficult to treat. Current treatment options are frequently ineffective and new therapeutic approaches are urgently needed. Avenanthramides are active substances in oats that exhibit anti-inflammatory effects. Their potential to interrupt pruritus mechanisms was investigated in this study. It was found that the synthetic analog dihydroavenanthramide D (DHAvD) can interact with the neurokinin-1 receptor (NK1R) and inhibit mast cell degranulation. DHAvD also affects inflammatory processes and reduces secretion of the cytokine interleukin-6. Our findings indicate that DHAvD may act as a NK1R inhibitor and could be a promising candidate for topical treatments of chronic pruritus. This article is protected by copyright. All rights reserved.
The hydrogen spill-over mechanism was studied by applying Density Functional Theory. We used small palladium clusters to act as the catalyst supported on the substrate (comprised of pyridinic and pyrrolic nitrogen doped graphene), in order to study hydrogen dissociation, migration and diffusion. Charge transfer and strong binding between the catalyst and the substrate lead to dissociated states of H2 and prevent clusters from detaching and coalescing. In dissociated cases of H2 on Pd clusters, energy barriers below 0.6 eV were found. Likewise, concerning hydrogen migration from the catalyst to the substrate, energy barrier values of 0.8 eV (pyridinic defect) and 0.5 eV (pyrrolic defect) were apparent in the case of the Pd4 cluster at full hydrogen saturation. This indicates that hydrogen dissociation and migration may occur spontaneously at room temperature. This result shows that the interaction between the defects and the small metal clusters may explain the role that defects play in hydrogen migration from the catalyst to the substrate. Subsequently, it was found that thermal desorption does not limit chemisorbed hydrogen diffusion on the substrate. This work may thus help to determine experimental strategies with the capacity to enhance hydrogen storage.
In addition to their antioxidant function, the eukaryotic peroxiredoxins (Prxs) facilitate peroxide-mediated signaling by undergoing controlled inactivation by peroxide-driven over-oxidation. In general, the bacterial enzyme lacks this controlled inactivation mechanism, making it more resistant to high H2O2 concentrations. During peroxide reduction, the active site alternates between reduced, fully folded (FF), and oxidized, locally unfolded (LU) conformations. Here we present novel insights into the divergence of bacterial and human Prxs in robustness and sensitivity to inactivation, respectively. Structural details provide new insights into sub-steps during the catalysis of peroxide reduction, enabling the transition from an FF to a LU conformation. Complementary to mutational and enzymatic results, these data unravel the essential role of the C-terminal tail of bacterial Prxs to act as a molecular switch, mediating the transition from an FF to a LU state. In addition, we propose that the C-terminal tail has influence on the propensity of the disulphide bond formation, indicating that as a consequence on the robustness and sensitivity to over-oxidation. Finally, a physical linkage between the catalytic site, the C-terminal tail and the oligomer interface is described.
Epigenetic inactivation of GPX3 has been identified in various cancers including leukemia. Moreover, aberrant DNA methylation was also found as a dominant mechanism of disease progression in myelodysplastic syndrome (MDS). This study intended to explore GPX3 promoter methylation and its clinical relevance in 110 patients with MDS. GPX3 methylation was examined by real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP). GPX3 methylation was identified in 15% (17/110) MDS patients, and significantly higher than controls, and lower than acute myeloid leukemia (AML) patients (P = 0.024 and 0.041). GPX3 methylated patients had older age and higher frequency of DNMT3A mutation (P = 0.015 and 0.066). Cases with GPX3 methylation showed significantly shorter overall survival (OS) time than those with GPX3 unmethylation analyzed with Kaplan-Meier analysis (P = 0.012). Moreover, Cox regression analysis revealed that GPX3 methylation might act as an independent prognostic indicator in MDS (HR = 1.847, P = 0.072). GPX3 methylation density was significantly increased during the progression from MDS to secondary acute myeloid leukemia (sAML) in three follow-up paired patients. Our study concludes that GPX3 methylation in bone marrow is associated with adverse prognosis and leukemia transformation in MDS.
Accurate representation of photosynthesis in terrestrial biosphere models (TBMs) is essential for robust projections of global change. However, current representations vary markedly between TBMs, contributing uncertainty to projections of global carbon fluxes. Here we compared the representation of photosynthesis in seven TBMs by examining leaf and canopy level responses of photosynthetic CO2 assimilation (A) to key environmental variables: light, temperature, CO2 concentration, vapor pressure deficit and soil water content. We identified research areas where limited process knowledge prevents inclusion of physiological phenomena in current TBMs and research areas where data are urgently needed for model parameterization or evaluation. We provide a roadmap for new science needed to improve the representation of photosynthesis in the next generation of terrestrial biosphere and Earth system models.
Cryptochromes are evolutionarily related to the light-dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types of cryptochromes in the animal kingdom: Drosophila-like CRYs that act as non-visual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate-like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C-terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C-termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression, and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes, and explore some of the divergent roles for this class of proteins. This article is protected by copyright. All rights reserved.
Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently we have shown that celastrol prevents Cd-induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here we show that celastrol attenuated Cd-elevated intracellular free calcium ([Ca(2+) ]i ) level and apoptosis in neuronal cells. Celastrol prevented Cd-induced neuronal apoptosis by inhibiting Akt-mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd-induced phosphorylation of S6K1/4E-BP1 and cell apoptosis. Furthermore, chelating intracellular Ca(2+) with BAPTA/AM or preventing [Ca(2+) ]i elevation using EGTA potentiated celastrol's repression of Cd-induced [Ca(2+) ]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd-elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd-increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca(2+) ]i -mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd-induced neuronal apoptosis via inhibiting Ca(2+) -CaMKII-dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd-induced neurodegenerative disorders. This article is protected by copyright. All rights reserved.
Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.
Sodium hypochlorite (NaOCl), the most commonly used irrigant, has many potential properties like its unique ability to dissolve pulp tissue, excellent antimicrobial activity, but has a cytotoxic effect when injected into periapical tissues. It is also known to produce allergic reactions, foul smell and taste, and potential for corrosion. Facultative organisms such as Enterococcus faecalis and aerobes like Staphylococcus aureus are considered to be the most resistant species and one of the possible causes of root canal treatment failure. So there is a need to find an alternative to sodium hypochlorite to act against these resistant microorganisms.
To evaluate and compare the antibacterial efficacy of morinda citrifolia and turmeric extract with 3% NaOCl as a root canal irrigant, against E. faecalis and S.aureus.
The antimicrobial efficacy was assessed in vitro using agar well diffusion method. Agar plates were prepared using Brain-Heart Infusion (BHI) agar. Cultures of E.faecalis and S.aureus were grown in nutrient broth at 37°C. Plates were incubated for 24 hours at 37°C and microbial zones of inhibition were recorded. Statistical analysis was performed using ANOVA.
NaOCl (3%) showed larger zones of inhibition than herbal irrigants against both the microorganisms. Among the herbal irrigants, morinda citrifolia showed larger zones of inhibition than turmeric hydro-alcoholic extract and turmeric water extract which was statistically significant (p<0.05).
NaOCl (3%) showed maximum antibacterial activity against E. faecalis, followed by morinda citrifolia and turmeric extracts. Considering the potential for undesirable properties of NaOCl, use of herbal alternatives in endodontics might prove to be advantageous.
Scrape cytology is an important diagnostic tool. It has been used in many tissue types as an adjunct or replacement for frozen section/ intra operative consultation.
This study was done to evaluate the role of scrape cytology in the diagnosis of thyroid lesions, its role as an adjunct to Fine Needle Aspiration Cytology (FNAC) and application of this technique for intra-operative consultation.
A prospective study on 50 thyroid neoplasms received over a period of 18 months (Nov 2014- March 2016) was conducted. Scrapings obtained from the fresh cut surface of thyroid specimens before formalin fixation, were smeared uniformly on to glass slides, and immediately fixed in 95% ethyl alcohol for rapid Haematoxylin and Eosin (H&E). Cytological findings were compared with pre-operative FNAC findings and histopathological diagnosis. Variables taken in to consideration while assessing the smears included cellularity, nuclear, cytological details and amount of colloid.
There were total of 50 patients. Out of the 50 patients, 15 were diagnosed as benign on scrape; of which 100% of cases were true negative for malignancy and five malignant cases were diagnosed as benign-false negative rate of 16.1%; four (8%) were deferred (non-diagnostic) with a true positive rate of 83.3%. Histopathological correlation was available in all cases. The overall diagnostic accuracy of scrape cytology was 89.1% with sensitivity and specificity of 83.87% (C.I.; 66.27% to 94.55%) and 100% (C.I; 76.84% to 100.00%) respectively. Fine Needle Aspiration results were available in 41 cases of which 2 were non-diagnostic. Of the remaining 39 cases 19 were benign and 20 were malignant with false negative rate of 40% and true positive rate of 60%. The overall diagnostic accuracy of FNAC was 64.1% with sensitivity and specificity of 60% (C.I.; 40.6% to77.3 %) and 77.78% (C.I; 39.9% to 97.1%) respectively.
We conclude that scrape cytology can act as a useful adjunct to FNAC and is a good procedure for intra-operative consultation. It is especially helpful in neoplasms diagnosed as suspicious for papillary carcinoma on aspiration and may obviate the need for a second procedure for completion thyroidectomy.
This case report summarizes the first use of intravenous vitamin C employed as an adjunctive interventional agent in the therapy of recurrent acute respiratory distress syndrome (ARDS). The two episodes of ARDS occurred in a young female patient with Cronkhite-Canada syndrome, a rare, sporadically occurring, noninherited disorder that is characterized by extensive gastrointestinal polyposis and malabsorption. Prior to the episodes of sepsis, the patient was receiving nutrition via chronic hyperalimentation administered through a long-standing central venous catheter. The patient became recurrently septic with Gram positive cocci which led to two instances of ARDS. This report describes the broad-based general critical care of a septic patient with acute respiratory failure that includes fluid resuscitation, broad-spectrum antibiotics, and vasopressor support. Intravenous vitamin C infused at 50 mg per kilogram body weight every 6 hours for 96 hours was incorporated as an adjunctive agent in the care of this patient. Vitamin C when used as a parenteral agent in high doses acts "pleiotropically" to attenuate proinflammatory mediator expression, to improve alveolar fluid clearance, and to act as an antioxidant.
Despite decades of studying, the mechanisms maintaining high diversity in the genes of the Major Histocompatibility Complex (MHC) are still puzzling scientists. In addition to pathogen recognition and other functions, MHC molecules may act prenatally in mate choice and in maternal-foetal interactions. These interactions are potential selective mechanisms that increase genetic diversity in the MHC. During pregnancy, immune response has a dual role: the foetus represents foreign tissue compared to mother, but histo-incompatibility is required for successful pregnancy. We have studied the prenatal selection in MHC class II loci (DLA-DQA1, DLA-DQB1 and DLA-DRB1) in domestic dogs by comparing the observed and expected offspring genotype proportions in 110 dog families. Several potential selection targets were addressed, including the peptide-binding site, the MHC locus, three-locus haplotype and supertype levels. For the supertype analysis, the first canine supertype classification was created based on in silico analysis of peptide-binding amino-acid polymorphism.
In most loci and levels, no deviation from the expected genotype frequencies was observed. However, one peptide-binding site in DLA-DRB1 had an excess of heterozygotes among the offspring. In addition, if the father shared a DLA-DRB1 allele with the mother, that allele was inherited by the offspring more frequently than expected, suggesting the selective advantage of a histo-compatible foetus, in contrast to our expectations.
We conclude that there is some evidence of post-copulatory selection at nucleotide site level in the MHC loci of pet dogs. But due to no indication of selection at locus, three-locus, or supertype levels, we estimated that the prenatal selection coefficient is less than 0.3 in domestic dogs and very likely other factors are more important in maintaining the genetic diversity in MHC loci.
Autism spectrum disorder (ASD) is a developmental condition that affects approximately four times as many males as females, a strong sex bias that has not yet been fully explained. Understanding the causes of this biased prevalence may highlight novel avenues for treatment development that could benefit patients with diverse genetic backgrounds, and the expertise of sex differences researchers will be invaluable in this endeavor. In this review, I aim to assess current evidence pertaining to the sex difference in ASD prevalence and to identify outstanding questions and remaining gaps in our understanding of how males come to be more frequently affected and/or diagnosed with ASD. Though males consistently outnumber females in ASD prevalence studies, prevalence estimates generated using different approaches report male/female ratios of variable magnitude that suggest that ascertainment or diagnostic biases may contribute to the male skew in ASD. Here, I present the different methods applied and implications of their findings. Additionally, even as prevalence estimations challenge the degree of male bias in ASD, support is growing for the long-proposed female protective effect model of ASD risk, and I review the relevant results from recurrence rate, quantitative trait, and genetic analyses. Lastly, I describe work investigating several sex-differential biological factors and pathways that may be responsible for females' protection and/or males' increased risk predicted by the female protective effect model, including sex steroid hormone exposure and regulation and sex-differential activity of certain neural cell types. However, much future work from both the ASD and sex differences research communities will be required to flesh out our understanding of how these factors act to influence the developing brain and modulate ASD risk.
Rice is more sensitive to salinity, particularly at its early vegetative and later productive stages. Wild plants growing in harsh environments such as wild barley from Qinghai-Tibet Plateau adapt to the adverse environment with allelic variations at the loci responsible for stressful environment, which could be used for rice genetic improvement. In this study, we overexpressed HsCBL8 encoding a calcium-sensor calcineurin B-like (CBL) protein in rice. The gene was isolated from XZ166, a wild-barley (Hordeum spontanum) line originated from Qinghai-Tibet Plateau. We found that XZ166 responded to high NaCl concentration (200 mM) with more HsCBL8 transcripts than CM72, a cultivated barley line known for salinity tolerance. XZ166 is significantly different from CM72 with nucleotide sequences at HsCBL8. The overexpression of HsCBL8 in rice resulted in significant improvement of water protection in vivo and plasma membrane, more proline accumulation, and a reduction of overall Na(+) uptake but little change in K(+) concentration in the plant tissues. Notably, HsCBL8 did not act on some genes downstream of the rice CBL family genes, suggesting an interesting interaction between HsCBL8 and unknown factors to be further investigated.
Natural killer (NK) cells play a pivotal role in the first line of defense against cancer. NK cells that are deficient in CD3 and a clonal T cell receptor (TCR) can be subdivided into two major subtypes, CD56(dim)CD16(+) cytotoxic and CD56(bright)CD16(-) immunoregulatory NK cells. Cytotoxic NK cells not only directly kill tumor cells without previous stimulation by cytotoxic effector molecules, such as perforin and granzymes or via death receptor interactions, but also act as regulatory cells for the immune system by secreting cytokines and chemokines. The aim of this review is to highlight therapeutic strategies utilizing autologous and allogenic NK cells, combinations of NK cells with monoclonal antibodies to induce antibody-dependent cellular cytotoxicity, or immune checkpoint inhibitors. Additionally, we discuss the use of chimeric antigen receptor-engineered NK cells in cancer immunotherapy.
Craniovertebral junction (CVJ) can be approached from various corridors depending on the location and extent of disease. A three-dimensional understanding of anatomy of CVJ is paramount for safe surgery in this region. Aim of this cadaveric study is to elucidate combined microscopic and endoscopic anatomy of critical neurovascular structures in this area in relation to bony and muscular landmarks.
Eight fresh-frozen cadaveric heads injected with color silicon were used for this study. A stepwise dissection was done from anterior, posterior, and lateral sides with reference to bony and muscular landmarks. Anterior approach was done endonasal endoscopically. Posterior and lateral approaches were done with a microscope. In two specimens, both anterior and posterior approaches were done to delineate the course of vertebral artery and lower cranial nerves from ventral and dorsal aspects.
CVJ can be accessed through three corridors, namely, anterior, posterior, and lateral. Access to clivus, foreman magnum, occipital cervical joint, odontoid, and atlantoaxial joint was studied anteriorly with an endoscope. Superior and inferior clival lines, supracondylar groove, hypoglossal canal, arch of atlas and body of axis, and occipitocervical joint act as useful bony landmarks whereas longus capitis and rectus capitis anterior are related muscles to this approach. In posterior approach, spinous process of axis, arch of atlas, C2 ganglion, and transverse process of atlas and axis are bony landmarks. Rectus capitis posterior major, superior oblique, inferior oblique, and rectus capitis lateralis (RCLa) are muscles related to this approach. Occipital condyles, transverse process of atlas, and jugular tubercle are main bony landmarks in lateral corridor whereas RCLa and posterior belly of digastric muscle are the main muscular landmarks.
With advances in endoscopic and microscopic techniques, access to lesions and bony anomalies around CVJ is becoming easier and straightforward. A combination of microscopic and endoscopic techniques is more useful to understand this anatomy and may aid in the development of future combined approaches.
Clinical neuroscience has made tremendous advances over the last century. Neurology as a discipline is still considered challenging and at times risky due to the natural history and progressive course of few of the neurological diseases. Encouragingly, the patient and their caregivers are now increasingly willing to be actively involved in making decisions. The patients' relationship with the doctor is a reflection of the society. A society that is orienting itself toward "rating" and "feedback" has made this doctor-patient relationship, a consumer-service provider relationship. This perhaps is due to commercialization of health that usually accompanies globalization. Moreover, a rapid influx of information from potential erroneous sources such as the Internet has also made patient and caregivers not being hesitant to taking legal course in the case of adverse events during treatment or simply because of dissatisfaction. The purpose of the legal process initiated by patients with neurological ailments is more often to compensate for the income lost, physical and psychological anguish that accompanies disease and its treatment, and to fund treatment or rehabilitation requirements. However, it is not clearly established if monetary benefits acquired lead to better opportunities for recovery of the patient. The consumer protection act and commercialization of medical services may well have an adverse effect on the doctor and patient relationship. Hence, there is a great need for all medical professionals to mutually complement and update each other. This review examines legal (litigation) processes with special interest on medicolegal system in patients with neurological ailments and the challenges faced by the neurologist during day-to-day clinical practice.
The Tamil Nadu model of public health is renowned for its success in providing quality health services at an affordable cost especially to the rural people. Tamil Nadu is the only state with a distinctive public health cadre in the district level and also the first state to enact a Public Health Act in 1939. Tamil Nadu has gained significant ground in the various aspects of health in the last few decades largely because of the significant reforms in its health sector which dates back to 1980s which saw rigorous expansion of rural health infrastructure in the state besides deployment of thousands of multipurpose health workers as village health nurses in rural areas. Effective implementation of Universal Immunization Programme, formation of Tamil Nadu Medical Services Corporation for regulating the drug procurement and promoting generic drugs, early incorporation of indigenous system of medicine into health care service, formulation of a health policy in 2003 by the state with special emphasis on low-income, disadvantaged communities alongside efficient implementation of The Tamil Nadu Health Systems Project (TNHSP) are the major factors which contributed for the success of the state. The importance of good political commitment and leadership in the health gains of the state warrants special mention. Moreover, the economic growth of the state, improved literacy rate, gender equality, and lowered fertility rate in the last few decades and contributions from the private sector have their share in the public health success of the state. In spite of some flaws and challenges, the Tamil Nadu Model remains the prototype health care delivery system in resource-limited settings which can be emulated by other states also toward a better health care delivery system.
Carcinogenesis caused by human papillomavirus (HPV) leads to over-expression of p16 protein. p16 may act as a marker of HPV integration with host genome and serve as a surrogate marker of HPV oncogenesis.
A single center study of 75 women (35 HIV-positive and 40 HIV-negative women) was conducted. Anal and cervical specimens were obtained for cytology and p16 immunostaining.
The sensitivity of p16 to diagnose anal and cervical dysplasia was 50% and 58.8%, respectively, whereas specificity was 98.6% and 100%, respectively. Positive predictive value for anal and cervical was 75% and 100%, whereas negative predictive value was 95.8% and 89.2%, respectively. A strong relationship between the grade of dysplasia and intensity of p16 immunoscore was observed (Pearson correlation r = 0.666, P < 0.0001 and r = 0.496, P < 0.0001 for anal and cervical, respectively).
p16 immunostaining with greater specificity for high-grade lesions may improve the diagnostic accuracy, especially for high-grade lesions which have a high risk of progression to malignancy and thereby necessitate treatment.
In this review, we demonstrate that the practical applications of a novel long-chain amidoamine derivative with a simple molecular structure (C18AA) are equivalent to or higher in number than those of supramolecules with complex molecular structures. Molecular assemblies of C18AA exhibit distinctive thermal responsiveness; namely, C18AA can form a gel with apolar organic solvents, while O/W emulsions of C18AA act as a heat-induced gelator that undergoes a phase transition from solution to gel upon heating. We also show that C18AA emulsions containing a quaternary ammonium salt develop an iridescent color over a specific temperature range. Further, molecular assemblies of C18AA serve as high-performance soft templates for the preparation of shape-controlled metal nanocrystals such as ultrathin Au nanowires and Pd nanowires.
Given the significant time and financial costs of developing a commercial drug, it remains important to constantly reform the drug discovery pipeline with novel technologies that can narrow the candidates down to the most promising lead compounds for clinical testing. The past decade has witnessed tremendous growth in computational capabilities that enable in silico approaches to expedite drug discovery processes. Molecular dynamics (MD) has become a particularly important tool in drug design and discovery. From classical MD methods to more sophisticated hybrid classical/quantum mechanical (QM) approaches, MD simulations are now able to offer extraordinary insights into ligand-receptor interactions. In this review, we discuss how the applications of MD approaches are significantly transforming current drug discovery and development efforts.
Relatively low bioavailability of plant-derived nutraceuticals with anticancer properties may limit their usefulness for prevention and therapy of cancer. In the present study, we have screened for nutraceuticals (n=30) that would act at low micromolar range against phenotypically distinct breast cancer cell lines, namely MCF-7 (ER(+), PR(+/-), HER2(-)), MDA-MB-231 (ER(-), PR(-), HER2(-)) and SK-BR-3 (ER(-), PR(-), HER2(+)), and diosmin, a citrus fruit flavonoid belonging to a flavone subclass, was selected. MCF-7 cell line was found to be the most sensitive to diosmin treatment. Diosmin caused G2/M cell cycle arrest, elevation in p53, p21 and p27 levels and stress-induced premature senescence when used at lower concentrations (5 and 10μM). Diosmin (20μM) also promoted apoptosis that was not observed in normal human mammary epithelial cells (HMEC). Diosmin stimulated oxidative and nitrosative stress, DNA damage and changes in global DNA methylation patterns. The status of p53 (wild type versus mutant) and the levels of phosphorylated ERK1/2 in a steady state, and diosmin-induced autophagy may reflect diverse response to diosmin treatment in MCF-7, MDA-MB-231 and SK-BR-3 cells, which in turn results in different cell fates. Taken together, diosmin that acts at low micromolar range against breast cancer cells may be considered as a promising candidate for anticancer therapy.
In cellular organisms composition of DNA is constrained to only four nucleobases A, G, T and C, except for minor DNA base modifications such as methylation which serves for defence against foreign DNA or gene expression regulation. Interestingly, this severe evolutionary constraint among other things demands DNA repair systems to discriminate between regular and modified bases. DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, the mismatched base pairs in DNA can occur from a spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated BER and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Importantly, in DNA repair deficient cells bacterial MutY, human TDG and mammalian MMR can act in the aberrant manner: MutY and TDG removes adenine and thymine opposite misincorporated 8-oxoguanine and damaged adenine, respectively, whereas MMR removes thymine opposite to O(6)-methylguanine. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences accumulated showing that in addition to the accumulation of oxidatively damaged DNA in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. For example, the aberrant BER and MMR pathways for oxidized guanine residues can lead to trinucleotide expansion that underlies Huntington's disease, a severe hereditary neurodegenerative syndrome. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidized base modifications and their possible role in age-related diseases.
Upon exposure to light, developing seedlings undergo photomorphogenesis, as illustrated by inhibition of hypocotyl elongation, cotyledon opening, and leaf greening. Hypocotyl growth is a well-established model for photomorphogenesis studies. During hypocotyl photomorphogenesis, light signals are sensed by multiple photoreceptors, among which the red/far-red light-sensing phytochromes have been extensively studied. However, it is not fully understood how the phytochromes modulate hypocotyl growth. Here, we demonstrated that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), which is known to either act as E3 ubiquitin ligase or affect chromatin organization, inhibits the transcriptional activation activity of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) that promotes hypocotyl growth. Consistent with the negative regulatory role of HOS1 in hypocotyl growth, HOS1-defective mutants exhibited elongated hypocotyls in the light. Notably, phyB induces HOS1 activity in inhibiting PIF4 function. These observations provide a molecular basis for the phyB-mediated suppression of hypocotyl growth in Arabidopsis.
The consensus of epidemiologic evidence indicates that an abundant intake of foodstuffs rich in folate conveys protection against the development of colorectal cancer, and perhaps some other common cancers as well. Pre-clinical models substantiate that the relationship is a genuinely causal one. Pre-clinical models have also lent mechanistic insights into the biochemical and molecular pathways by which adequate folate exposure conveys these protective effects, although human studies are beginning to establish the relevance of our mechanistic understanding to human cancer biology. Enhancement of genetic stability appears to be a major mechanism by which folate sufficiency protects against carcinogenesis. To date, the Wnt signaling cascade has been the pathway most examined in this regard. The relationship between folate exposure and colorectal cancer risk is a complex one, in part because a number of extrinsic and intrinsic factors act as effect modifiers. This review discusses how the intake of the other three B-vitamins integral to the 1-carbon pathway acts as one such effect modifier. In addition, two concepts that remain matters of considerable debate are whether parental intake of folate impacts on subsequent cancer risk in the offspring, and whether excessive intakes of folate may have a paradoxical cancer-promoting effect: observations underlying these two concepts are presented as well.
A significant portion of the key biological functions of αKlotho (αKL) and its cognate ligand Fibroblast growth factor-23 (FGF23) have been revealed through the study of rare diseases of mineral metabolism. These findings have far reaching implications for common disorders such as chronic kidney disease-mineral bone disorder (CKD-MBD). αKL's predominant effect on mineral homeostasis is through its actions in the kidney as a co-receptor for FGF23, however emerging data has shed light on its capacity to act as a circulating factor through the cleavage of the transmembrane form of αKL ('mKL') to produce 'cleaved KL' or 'cKL'. This review summarizes new findings from studies using extended delivery of cKL to mouse models with phenotypes reflecting those arising in CKD-MBD.
In this study, we investigate changes in the glaciated surface and the formation of lakes in the headwater of the Querococha watershed in Cordillera Blanca (Peru) using 24 Landsat images from 1975 to 2014. Information of glacier retreat was integrated with available climate data, the first survey of recent depositional dynamics in proglacial Yanamarey Lake (4600m a.s.l.), and a relatively short hydrological record (2002-2014) at the outlet of Yanamarey Lake. A statistically significant temperature warming (0.21°C decade(-1) for mean annual temperature) has been detected in the region, and it caused a reduction of the glacierized area since 1975 from 3.5 to 1.4km(-2). New small lakes formed in the deglaciated areas, increasing the flooded area from1.8ha in 1976 to 2.8ha in 2014. A positive correlation between annual rates of glacier recession and runoff was found. Sediment cores revealed a high sedimentation rate (>1cmyr(-1)) and two contrasted facies, suggesting a shift toward a reduction of meltwater inputs and higher hydrological variability likely due to an increasing role of precipitation on runoff during the last decades. Despite the age control uncertainties, the main transition likely occurred around 1998-2000, correlating with the end of the phase with maximum warming rates and glacier retreat during the 1980s and 1990s, and the slowing down of expansion of surface lake-covered surface. With this hydrological - paleolimnological approach we have documented the association between recent climate variability and glacier recession and the rapid transfer of hydroclimate signal to depositional and geochemical processes in high elevation Andean environments. This, study also alerts about water quality risks as proglacial lakes act as secondary reservoirs that trap trace and minor elements in high altitude basins.
This study investigated the geochemistry of arsenic (As) in low sulfide-high carbonate coal waste rock of the Elk Valley, British Columbia, Canada. Its abundance and mineralogical associations in waste rock of different placement periods were determined in addition to its mobilization into porewater and rock-drain effluent. The mean (5.34mg/kg; 95% confidence interval: 4.95-5.73mg/kg) As concentration in the waste rock was typical of sedimentary rock. Electron microprobe and As K-edge X-ray absorption near-edge spectroscopic analyses showed the As is predominantly associated with primary pyrites in both source and freshly blasted waste rock. However, in aged waste rock the As is associated with both primary pyrites and secondary Fe oxyhydroxides. Oxidation of pyrite in waste rock dumps was reflected by the presence of high concentrations of SO4(2-) in porewater and oxidation rims of Fe oxyhydroxides around pyrite grains. Acid released from pyrite oxidation to Fe oxyhydroxides is neutralized by carbonate mineral dissolution that buffers the pH in the waste rock to circumneutral values. Adsorption of As onto secondary Fe oxyhydroxides provides an internal geochemical control on As release during pyrite oxidation and porewater flushing from the dump, resulting in the low As concentrations observed in porewater (median: 9.91μg/L) and rock-drain effluent (median: 0.31μg/L). Secondary Fe oxyhydroxides act as a long-term sink for As under present day hydrologic settings in waste rock dumps in the Elk Valley.
Antimicrobial peptides are central effector molecules in skin immunology. The functions of antimicrobial peptides in skin diseases include the ability to act as cytokines or growth factors, driving disorders such as psoriasis and rosacea, as well as their action as natural antibiotics to control bacteria that influence diseases such as atopic dermatitis and acne.
In 1969, Butler (1969) first coined the term "ageism" to confront "prejudice by one age group toward other age groups" (pp. 243). As with other "isms," such as racism and sexism, ageism leads to bigotry and discrimination, though it is a very distinct beast in that, for the most part, other "isms" refer to those different from ourselves: distinct, mutually exclusive, and impervious groups. Conversely, age is a fluid social construct in which we are all intimately bound as we move through the lifespan, transitioning in and out of different age-groups. Unlike other "isms," individuals negotiate shifts from the "in-group" of youth to the "out-group" of old age. Yet we are all immersed, largely unconsciously, in this ubiquitous but too often unrecognized "ism" that needs to be named and challenged.
Periprosthetic osteolysis induced by wear particles can lead to aseptic loosening, one main reason of arthroplasty failure. However, the role of microRNA-130b (miR-130b) in particle-induced osteolysis (PIO) has not been explored yet. In this study, PIO models were established in C57BL/J6 mice via the implantation of Co-Cr-Mo alloy particles, and evaluated by detecting tartrate-resistant acid phosphatase (TRAP) activity and bone resorption in the calvaria. Mouse preosteoblast MC3T3-E1 cells were cultured to receive particle stimulation in vitro. Real time PCR and western blotting were performed to determine the expression levels of miR-130b and frizzled-related protein (FRZB), one potential target of miR-130b. Results showed upregulated miR-130b and downregulated FRZB in both PIO mice with remarkable osteolysis and particle-treated MC3T3-E1 cells showing inhibited proliferation and differentiation assayed by bromodeoxy urodine (BrdU) incorporation and alkaline phosphatase (ALP) activity respectively. Functional studies were conducted by transfection of miR-130b inhibitor in vitro or the injections of miR-130b inhibitor or small interfering RNA (siRNA) targeting FRZB in vivo. Interestingly, particle-induced inhibition on cell proliferation, differentiation and FRZB expression were all reversed by miR-130b silence. Luciferase report assays demonstrated that miR-130b indeed negatively regulated FRZB expression by targeting, while FRZB could reverse the opposed effect of miR-130b silence on PIO development. Therefore, the upregulated miR-130b in PIO models could act as one key regulator of PIO development, partly due to its negative regulation on FRZB.
Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.
The central nervous system (CNS) is a large network of intercommunicating cells that function to maintain tissue health and homeostasis. Considerable evidence suggests that glucocorticoids exert both neuroprotective and neurodegenerative effects on the CNS. Glucocorticoids act by binding two related receptors in the cytoplasm, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The glucocorticoid receptor complex mediates cellular responses by transactivating target genes and by protein: protein interactions. The paradoxical effects of glucocorticoids on neuronal survival and death have been attributed to the concentration and the ratio of mineralocorticoid to glucocorticoid receptor activation. Glucocorticoid-induced leucine zipper (GILZ) is a recently identified protein transcriptionally upregulated by glucocorticoids. Constitutively, expressed in many tissues including brain, GILZ mediates many of the actions of glucocorticoids. It mimics the anti-inflammatory and anti-proliferative effects of glucocorticoids but exerts differential effects on stem cell differentiation and lineage development. Recent experimental data on the effects of GILZ following induced stress or trauma suggest potential roles in CNS diseases. Here, we provide a short overview of the role of GILZ in CNS health and discuss three potential rationales for the role of GILZ in Alzheimer's disease pathogenesis.
Many donors and recipients report an improved relationship after transplantation; however, tension, neglect, guilt, and proprietorial concern over the recipient can impede donor and recipient well-being and outcomes. We aimed to describe donor and recipient expectations and experiences of their relationship in the context of living kidney donation.
Thematic synthesis of qualitative studies.
Living kidney donors and recipients.
Electronic databases were searched to October 2015.
Thematic synthesis.
From 40 studies involving 1,440 participants (889 donors and 551 recipients) from 13 countries, we identified 6 themes. "Burden of obligation" described the recipient's perpetual sense of duty to demonstrate gratitude to the donor. "Earning acceptance" was the expectation that donation would restore relationships. "Developing a unique connection" reflected the inexplicable bond that donor-recipient dyads developed postdonation. "Desiring attention" was expressed by donors who wanted recognition for the act of donation and were envious and resentful of the attention the recipient received. "Retaining kidney ownership" reflected the donor's inclination to ensure that the recipient protected "their" kidney. "Enhancing social participation" encompassed relieving both the caregiver from the constraints of dialysis and the recipient from increased involvement and contribution in family life.
Non-English articles were excluded.
Living kidney donation can strengthen donor-recipient relationships but may trigger or exacerbate unresolved angst, tension, jealousy, and resentment. Facilitating access to pre- and posttransplantation psychological support that addresses potential relationship changes may help donors and recipients better adjust to changes in the relationship dynamics, which in turn may contribute to improved psychosocial and transplantation outcomes following living kidney donation.
The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.
Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.
This article explores the experience of workers on Assertive Community Treatment (ACT) teams surrounding their efforts to facilitate social integration for their clients.
Sixteen workers were individually interviewed and eight additional workers participated in two focus groups.
The formation of caring relationships between worker and client was an important first step towards social integration for ACT clients. Community activities offer opportunities for social interaction.
The frequency of community based activities should be increased. Social integration should be a targeted focus of service by structurally embedding a social integration specialist onto the ACT model.
Differences in caregiver input across socioeconomic status (SES) predict syntactic development, but the mechanisms are not well understood. Input effects may reflect the exposure needed to acquire syntactic representations during learning (e.g., does the child have the relevant structures for passive sentences?) or access this knowledge during communication (e.g., can she use the past participle to infer the meaning of passives?). Using an eye-tracking and act-out paradigm, the current study distinguishes these mechanisms by comparing the interpretation of actives and passives in 3- to 7-year-olds (n=129) from varying SES backgrounds. During the presentation of spoken sentences, fixations revealed robust disambiguation of constructions by children from higher-SES backgrounds, but less sensitivity by lower-SES counterparts. After sentence presentation, decreased sensitivity generated interpretive challenges and average SES-related differences for passives requiring syntactic revision ("The seal is quickly eaten by it"). Critically, no differences were found when revision was not needed ("It is quickly eaten by the seal"). These results suggest that all children shared an ability to acquire passives, but SES-related differences in real-time processing can impact the accuracy of utterance interpretation.
American serpentine leafminer (ASL), Liriomyza trifolii (Burgess), are a significant pest of greenhouse ornamental crops and females damage leaf tissue with their ovipositor during feeding and oviposition. The sterile insect technique has been advocated as a non-chemical alternative to currently available control methods. In area-wide sterile insect release programs, males act as true vectors of sterility. Females should be eliminated from a cohort of pupae prior to irradiation to maximize production economics and sterility spread. The aim of this research was to develop a mechanical sexing system based on pupal size to decrease the proportion of ASL females.
Cumulative frequency distributions were used to examine significant differences in male and female pupal length, dorsal and lateral width distributions. Optimum size cut-off points based on the largest differences in distribution curves were used to determine dimensions of three different sieve designs. Sieve pores measuring 1.543 mm by 0.765 mm excluded 76% of female pupae and doubled the proportion of males in the throughput sample.
Pupal sexual dimorphisms identified in this research can be used to design a sieve to aid in reducing the proportion of females prior to irradiation thus improving the efficacy of an area-wide sterile insect release program.
Organ donation should neither enrich nor impose financial burdens on donors. We describe the scope of health care required for all living kidney donors that reflects contemporary understanding of long-term donor health outcomes, propose an approach to identify donor health conditions which should be covered within the framework of financial neutrality, and propose strategies to pay for this care. Despite the Affordable Care Act in the United States, donors continue to have inadequate coverage for important health conditions that are either donation related or may compromise post-donation kidney function. Amendment of Medicare regulations is needed to clarify that surveillance and treatment of conditions that may compromise post-donation kidney function following donor nephrectomy will be covered without expense to the donor. In other countries lacking health insurance for all residents, sufficient data exist to allow creation of a compensation fund or donor insurance policies to ensure appropriate care. Providing coverage for donation-related sequelae as well as care to preserve post-donation kidney function ensures protection against the financial burdens of health care encountered by donors throughout their lifetime. Providing coverage for this care should thus be cost-effective even without considering the health care cost savings that occur in living donor transplant recipients. This article is protected by copyright. All rights reserved.
Despite abundant information on the negative impacts of smoking, more than 40 million adult Americans continue to smoke. The Affordable Care Act (ACA) requires tobacco cessation as a preventive service with no patient cost share for all FDA-approved cessation medications. Health plans have a vital role in supporting smoking cessation by managing medication access, but uncertainty remains on the gaps between smoking cessation requirements and what is actually occurring in practice. This study presents current cessation patterns, real-world drug costs and plan benefit design data, and estimates the 1- to 5-year pharmacy budget impact of providing ACA-required coverage for smoking cessation products to understand the fiscal impact to a US healthcare plan.
A closed cohort budget impact model was developed in Microsoft Excel(®) to estimate current and projected costs for US payers (commercial, Medicare, Medicaid) covering smoking cessation medicines, with assumptions for coverage and smoking cessation product utilization based on current, real-world national and state-level trends for hypothetical commercial, Medicare, and Medicaid plans with 1 million covered lives. A Markov methodology with five health states captures quit attempt and relapse patterns. Results include the number of smokers attempting to quit, number of successful quitters, annual costs, and cost per-member per-month (PMPM).
The projected PMPM cost of providing coverage for smoking cessation medications is $0.10 for commercial, $0.06 for Medicare, and $0.07 for Medicaid plans, reflecting a low incremental PMPM impact of covering two attempts ranging from $0.01 for Medicaid to $0.02 for commercial and Medicare payers.
The projected PMPM impact of covering two quit attempts with access to all seven cessation medications at no patient cost share remains low. Results of this study reinforce that the impact of adopting the ACA requirements for smoking cessation coverage will have a limited near-term impact on health plan's budgets.
Pfizer Inc.
Gallstone is a high-risk factor for gallbladder pre-malignancy or malignancy (GB PM-M) but which substances of gallstones definitely assist to turn out in to GB PM-M, remains unclear. This study aimed to find out the presence of carcinogenic heavy metals in gallstones and to explore the aetiopathogenesis of gallbladder pre-malignancy and malignancy.
Presence of elements in gallstones was detected by energy dispersive X-ray spectroscopy (EDS) with scanning electron microscopy (SEM) and then level of carcinogenic heavy metals was estimated in gallstones using atomic absorption spectroscopy (AAS). The experiment was carried out in gallstone samples of 46 patients with gallbladder pre-malignant and malignant condition (PM-M group) and 65 sex and age-matched patients with chronic cholecystitis (C-C group). Gallstones were also classified in to three types such as cholesterol stone, mixed stone, and black pigment stone.
EDS analysis detected presence of mercury, lead, and cobalt elements in all types of gallstones of both PM-M and C-C groups. AAS analysis revealed significantly higher amount of mercury (p < 0.001), lead (p < 0.0001), cobalt (p < 0.01), and cadmium (p < 0.01) in the gallstones of PM-M than C-C groups. The presence of these heavy metals also varied among stone types of both groups. EDS phase analysis showed 'dense deposits' of these metals in gallstones.
Presence of significantly higher amount of mercury, lead, cobalt, and cadmium in gallstones may play a pivotal role as risk factors in the development of gallbladder malignancy or pre-malignancy. 'Dense deposits' of these metals in the gallstones which is the first observation, may act as crucial doses of carcinogens.
Tight junction (TJ) strands between epithelial or endothelial cells are formed by claudins, a protein family comprising up to 27 members in mammals. Although many more proteins are involved in the formation of TJ complexes, claudins are the only TJ proteins that are able to form TJ-like strands when overexpressed in cells that are normally devoid of TJs (e.g., fibroblasts). Within the paracellular cleft, the extracellular domains of claudins provide the matrix that seals the paracellular pathway. However, within this matrix, some claudins act as channels that specifically allow certain ions to cross this barrier. Barrier-forming claudins predominate in epithelia that enclose compartments containing harmful ion concentrations (e.g., H(+) in the stomach, K(+) in the inner ear endolymph) or high pressures (e.g., in blastocoel or brain ventricle formation during development). Here, even seemingly minor alterations in TJ composition may be detrimental to the organism. In contrast, in many transporting epithelia, channel-forming claudins are essential for transcellular and paracellular transport coupling. Mutation or knockout of channel-forming claudins in these tissues brings both transcellular and paracellular transports to a standstill. The present review will present examples to illustrate the importance of single members of the claudin family in general epithelial transport physiology.
Recently, long non-coding RNAs (lncRNAs) have been shown to have critical regulatory roles in tumourigenesis. Increasing evidence has suggested that lncRNA NEAT1 has been implicated in various types of human cancer. However, the potential biological roles and regulatory mechanisms of NEAT1 in pancreatic cancer (PC) remains unclear. Here, we found that the expression level of NEAT1 was higher in PC tissues compared to the corresponding non-tumor tissues. Besides, our findings indicate that high NEAT1 expression level is closely correlated with tumor progression and poor survival in PC patients. Furthermore, we also found that knockdown of NEAT1 remarkably suppressed cell proliferation by inducing cell cycle arrest and apoptosis promotion in PC cells. Moreover, bioinformatics analysis and luciferase reporter assay revealed that NEAT1 directly bound to the miR-506-3p, which has been reported to act as a tumor suppressor in diverse cancers. Additionally, our results confirmed that the tumor-promoting effects of NEAT1 in PC cells is at least partly through negative modulation of miR-506-3p. Overall, our results suggested that NEAT1 functions as an oncogenic lncRNA in PC, which could be a novel diagnostic and therapeutic target for PC.
Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses.
Primary nocturnal enuresis is one of the sleep related phenomena characterized by disruption in the relationship between arousal and urination. Corticotropin-releasing factor (CRF) is a neurohormone released from the paraventricular nucleus of the hypothalamus into the median eminence to elicit release of adrenocorticotrophin from the anterior pituitary. It may act to modulate autonomic function and behavior in concert with the endocrine effects. Conflicting animal studies about the role of CRF in micturition, either facilitating or inhibiting, have been raised. It was suggested to be a novel target for treatment of urinary disorders based on the finding that manipulation of CRF in the pontine micturition circuit could affect urodynamic function.
The aim was to throw light on the possible role of CRF in primary monosymptomatic nocturnal enuresis by assessing its serum level.
Twenty-nine children aged 8-14 years complaining of primary monosymptomatic nocturnal enuresis and 16 age- and sex-matched healthy children with good toilet control day and night were recruited to the study. History taking, clinical examination, and assessment of serum CRF levels in the morning and evening (9 a.m. and 9 p.m.) were carried out for all patients and controls.
A positive family history of enuresis was detected in 82.8% of enuretic patients. Serum levels of CRF (both morning and evening) were significantly lower in patients than in controls. Several animal studies suggested that CRF in descending projections from Barrington's nucleus to the lumbosacral parasympathetic neurons is inhibitory to micturition, which supports our results and the assumption that reduction of the evening serum CRF level could have a role in the occurrence of primary monosymptomatic nocturnal enuresis. No significant difference was found between morning and evening CRF serum levels in either cases or controls, which negates our assumption of having a rhythmic pattern of release (figure). No correlations with age were found. According to their history, all our enuretic patients were deep sleepers. Deep sleep and difficult arousal were found to have a major role in primary monosymptomatic nocturnal enuresis. It was proposed that CRF function may allow arousal to occur before micturition to facilitate preparative behaviors. A lower CRF level may explain deep-sleep pattern in children with enuresis.
CRF was deficient in our enuretic children, which may draw attention to the possible pathophysiological implications in primary nocturnal enuresis (either at the level of loss of inhibitory effect on micturition or lack of arousal in response to bladder distension). Further proof studies are recommended.
The Canadian 24-h movement guidelines were developed with the hope of improving health and future health outcomes in children and youth. The purpose of this study was to evaluate adherence to the 3 recommendations most strongly associated with health outcomes in new 24-h movement guidelines and their relationship with adiposity (obesity and body mass index z-score) across countries participating in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE).
Cross-sectional results were based on 6128 children aged 9-11 years from the 12 countries of ISCOLE. Sleep duration and moderate-to-vigorous physical activity (MVPA) were assessed using accelerometry. Screen time was measured through self-report. Body weight and height were measured. Body mass index (BMI, kg · m(-2)) was calculated, and BMI z-scores were computed using age- and sex-specific reference data from the World Health Organization. Obesity was defined as a BMI z-score > +2 SD. Meeting the overall 24-h movement guidelines was defined as: 9 to 11 h/night of sleep, ≤2 h/day of screen time, and at least 60 min/day of MVPA. Age, sex, highest parental education and unhealthy diet pattern score were included as covariates in statistical models. Associations between meeting vs. not meeting each single recommendation (and combinations) with obesity were assessed with odds ratios calculated using generalized linear mixed models. A linear mixed model was used to examine the differences in BMI z-scores between children meeting vs. not meeting the different combinations of recommendations.
The global prevalence of children meeting the overall recommendations (all three behaviors) was 7%, with children from Australia and Canada showing the highest adherence (15%). Children meeting the three recommendations had lower odds ratios for obesity compared to those meeting none of the recommendations (OR = 0.28, 95% CI 0.18-0.45). Compared to not meeting the 24-h movement recommendations either independently or combined, meeting them was significantly associated with a lower BMI z-score. Whenever the MVPA recommendation was included in the analysis the odds ratios for obesity were lower.
For ISCOLE participants meeting these 3 healthy movement recommendations the odds ratios of being obese or having high BMI z-scores were lower. However, only a small percentage of children met all recommendations. Future efforts should aim to find promising ways to increase daily physical activity, reduce screen time, and ensure an adequate night's sleep in children.
The International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) was registered at ClinicalTrials.gov (Identifier NCT01722500) (October 29, 2012).
The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT).
Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2.
None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0-1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases.
This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.
Jerimalai is a rock shelter in East Timor with cultural remains dated to 42,000 years ago, making it one of the oldest known sites of modern human activity in island Southeast Asia. It has special global significance for its record of early pelagic fishing and ancient shell fish hooks. It is also of regional significance for its early occupation and comparatively large assemblage of Pleistocene stone artefacts. Three major findings arise from our study of the stone artefacts. First, there is little change in lithic technology over the 42,000 year sequence, with the most noticeable change being the addition of new artefact types and raw materials in the mid-Holocene. Second, the assemblage is dominated by small chert cores and implements rather than pebble tools and choppers, a pattern we argue pattern, we argue, that is common in island SE Asian sites as opposed to mainland SE Asian sites. Third, the Jerimalai assemblage bears a striking resemblance to the assemblage from Liang Bua, argued by the Liang Bua excavation team to be associated with Homo floresiensis. We argue that the near proximity of these two islands along the Indonesian island chain (c.100 km apart), the long antiquity of modern human occupation in the region (as documented at Jerimalai), and the strong resemblance of distinctive flake stone technologies seen at both sites, raises the intriguing possibility that both the Liang Bua and Jerimalai assemblages were created by modern humans.
Degradation pathway and surface biosorption of triphenyltin (TPT) by effective microbes have been investigated in the past. However, unclear interactions among membrane components and TPT binding and transport are still obstacles to understanding TPT biotransformation. To reveal the mechanism involved, the phospholipid expression, membrane potential, cellular mechanism and molecular dynamics between TPT and fatty acids (FAs) during the TPT degradation process in the presence of d-malic acid (DMA) were studied. The results show that the degradation efficiency of 1 mg L(-1) TPT by Bacillus thuringiensis (1 g L(-1)) with 0.5 or 1 mg L(-1) DMA reached values up to approximately 90% due to the promotion of element metabolism and cellular activity, and the depression of FA synthesis induced by DMA. The addition of DMA caused conversion of more linoleic acid into 10-oxo-12(Z)-octadecenoic acid, increased the membrane permeability, and alleviated the decrease in membrane potential, resulting in TPT transport and degradation. Fluorescence analysis reveals that the endospore of B. thuringiensis could act as an indicator for membrane potential and cellular activities. The current findings are advantageous for acceleration of biosorption, transport and removal of pollutants from natural environments.
Metal deposition on oxide surfaces usually results in adatoms, clusters, or islands of the deposited material, where defects in the surface often act as nucleation centers. Here an alternate configuration is reported. After the vapor deposition of Fe on the In_{2}O_{3}(111) surface at room temperature, ordered adatoms are observed with scanning tunneling microscopy. These are identical to the In adatoms that form when the sample is reduced by heating in ultrahigh vacuum. Density functional theory calculations confirm that Fe interchanges with In in the topmost layer, pushing the excess In atoms to the surface where they arrange as a well-ordered adatom array.
Increasing use of engineered nanoparticles has led to extensive research into their potential hazards to the environment and human health. Cellular uptake from the gut is sparsely investigated and microscopy techniques applied for uptake studies can result in misinterpretations. Various microscopy techniques are used to investigate internalization of 10 nm gold nanoparticles in Daphnia magna gut lumen and gut epithelial cells upon 24h exposure and outline potential artefacts, i.e. high contract precipitates from sample preparation related to these techniques. Light sheet microscopy confirmed accumulation of gold nanoparticles in the gut lumen. Scanning transmission electron microscopy and elemental analysis revealed gold nanoparticles attached to the microvilli of gut cells. Interestingly, the peritrophic membrane appeared to act as a semipermeable barrier between the lumen and the gut epithelium, permitting only single particles through. Structures resembling nanoparticles were also observed inside gut cells. As elemental analysis could not verify these to be gold they were likely artifacts from the preparation, such as osmium and iron. Importantly, gold nanoparticles were in fact found inside holocrine cells with disrupted membranes. Thus, false positive observations of nanoparticle internalization may result from either preparation artefacts or by mistaking disrupted cells for intact. These findings emphasize the importance of cell integrity and combining elemental analysis with the localization of internalized nanoparticles using transmission electron microscopy. This article is protected by copyright. All rights reserved.
Several foods on the market, such as yogurt and fermented milk, include mixtures of prebiotics and probiotic microorganisms effective in promoting the proliferation and equilibrium of intestinal bacteria, thus improving gut health. Particularly, researchers and the public have shown increasing interest in the combination of probiotics with natural substances that promote health or that can act as substrates to promote bacterial growth. The aim of this study is to evaluate the effects of different extracts of Aloe barbadensis and Aloe arborescens in fermented milk, taking into account both the prebiotic effect of aloe polysaccharides and the antimicrobial activity of several secondary metabolites. The results demonstrate a beneficial effect of 5% aloe inner gel on Lactobacillus growth and confirm the antimicrobial activity of the phenolic compounds peculiar of green rind extracts.
In addition to dopamine neuron firing, cholinergic interneurons (ChIs) regulate dopamine release in the striatum via presynaptic nicotinic receptors (nAChRs) on dopamine axon terminals. Synchronous activity of ChIs is necessary to evoke dopamine release through this pathway. The frequency-dependence of disynaptic nicotinic modulation has led to the hypothesis that nAChRs act as a high-pass filter in the dopaminergic microcircuit. Here, we used optogenetics to selectively stimulate either ChIs or dopamine terminals directly in the striatum. To measure the functional consequence of dopamine release, D2-receptor synaptic activity was assessed via virally overexpressed potassium channels (GIRK2) in medium spiny neurons (MSNs). We found that nicotinic-mediated dopamine release was blunted at higher frequencies because nAChRs exhibit prolonged desensitization after a single pulse of synchronous ChI activity. However, when dopamine neurons alone were stimulated, nAChRs had no effect at any frequency. We further assessed how opioid receptors modulate these two mechanisms of release. Bath application of the κ opioid receptor agonist U69593 decreased D2-receptor activation through both pathways, whereas the μ opioid receptor agonist DAMGO decreased D2-receptor activity only as a result of cholinergic-mediated dopamine release. Thus the release of dopamine can be independently modulated when driven by either dopamine neurons or cholinergic interneurons.
Diabolical points (spectral degeneracies) can naturally occur in spectra of two-dimensional quantum systems and classical wave resonators due to simple symmetries. Geometric Berry phase is associated with these spectral degeneracies. Here, we demonstrate a diabolical point and the corresponding Berry phase in the spectrum of hybrid light-matter quasiparticles-exciton-polaritons in semiconductor microcavities. It is well known that sufficiently strong optical pumping can drive exciton-polaritons to quantum degeneracy, whereby they form a macroscopically populated quantum coherent state similar to a Bose-Einstein condensate. By pumping a microcavity with a spatially structured light beam, we create a two-dimensional quantum billiard for the exciton-polariton condensate and demonstrate a diabolical point in the spectrum of the billiard eigenstates. The fully reconfigurable geometry of the potential walls controlled by the optical pump enables a striking experimental visualization of the Berry phase associated with the diabolical point. The Berry phase is observed and measured by direct imaging of the macroscopic exciton-polariton probability densities.
In marine organisms primarily intended for human consumption, the quality of the muscle and the extracted oils may be affected by lipid oxidation during storage, even at low temperatures. This has led to a search for alternatives to maintain quality. In this sense, antioxidant compounds have been used to prevent such lipid deterioration. Among the most used compounds are tocopherols, which, due to their natural origin, have become an excellent alternative to prevent or retard lipid oxidation and maintain the quality of marine products. Tocopherols as antioxidants have been studied both exogenously and endogenously. Exogenous tocopherols are often used by incorporating them into plastic packaging films or adding them directly to fish oil. It has been observed that exogenous tocopherols incorporated in low concentrations maintain the quality of both muscle and the extracted oils during food storage. However, it has been reported that tocopherols applied at higher concentrations act as a prooxidant molecule, probably because their reactions with singlet oxygen may generate free radicals and cause the oxidation of polyunsaturated fatty acids in fish oils. However, when tocopherols are included in a fish diet (endogenous tocopherols), the antioxidant effect on the muscle lipids is more effective due to their incorporation into the membrane lipids, which can help extend the shelf life of seafood by reducing the lipid deterioration that occurs due to antioxidant synergy with other phenolic compounds used supplements in fish muscle. This review focuses on the most important studies in this field and highlights the potential of using tocopherols as antioxidants in marine oils.
Increasing evidence has demonstrated a significant role for long non-coding RNAs (lncRNAs) in tumorigenesis. However, their functions in nasopharyngeal carcinoma (NPC) metastasis remain largely unknown. In this study, a model comparing high and low metastatic NPC cell lines (5-8F vs. 6-10B and S18 vs. S26) was constructed to determine the expression profile of lncRNAs using the microarray analysis, and we found 167 lncRNAs and 209 mRNAs were differentially expressed. Bioinformatic analysis indicated that the dysregulated mRNAs participated in important biological regulatory functions in NPC. Validation of 26 significantly dysregulated lncRNAs by qRT-PCR showed the expression patterns of 22 lncRNAs were in accordance with the microarray data. Furthermore, the expression level of ENST00000470135, which was the most upregulated lncRNA in high metastatic cell lines, was significantly higher in NPC cell lines and tissues with lymph node metastasis (LNM) and knocking down ENST00000470135 suppressed the migration, invasion and proliferation of NPC cells in vitro. In conclusion, our study revealed expression patterns of lncRNAs in NPC metastasis. The dysregulated lncRNAs may act as novel biomarkers and therapeutic targets for NPC.
Internet of Things (IoT) is the logical further development of today's Internet, enabling a huge amount of devices to communicate, compute, sense and act. IoT sensors placed in Ambient Assisted Living (AAL) environments, enable the context awareness and allow the support of the elderly in their daily routines, ultimately allowing an independent and safe lifestyle. The vast amount of data that are generated and exchanged between the IoT nodes require innovative context modeling approaches that go beyond currently used models. Current paper presents and evaluates an open interoperable platform architecture in order to utilize the technical characteristics of IoT and handle the large amount of generated data, as a solution to the technical requirements of AAL applications.
Salivary antibodies may act as non-invasive marker of systemic immunity enabling assessment of vaccination and protection against bacterial infections.
To assess if levels of anti-pneumococcal antibodies in saliva reflect concentrations in serum and determine whether saliva can accurately identify protective concentrations in serum.
IgG, IgA and IgM antibody levels in paired saliva and serum samples were measured against 12 pneumococcal polysaccharide antigens in 72 healthy adults.
Antibody levels in saliva correlated positively with serum across immunoglobulin classes, most strongly for IgA. Individuals who had protective antibody levels in serum demonstrated significantly higher IgG and IgA salivary antibody concentrations/secretion rates. Salivary IgG and IgA pneumococcal antibodies were able to distinguish between those with/without protective levels in serum for the majority of serotypes. Salivary IgM antibodies were not able to differentiate protective status. Median IgG and IgA pneumococcal salivary parameters were able to identify individuals who had protective levels in serum on ≥ 8/12 serotypes with moderate accuracy: median IgA secretion rates provided the best sensitivity (73%) and specificity (71%).
These findings suggest that IgG and IgA pneumococcal specific antibodies in saliva may be useful surrogate markers of antibody status in serum.
Debate continues regarding the benefits versus risks of initial resection of the primary tumor in metastatic colorectal cancer (mCRC) patients with an asymptomatic primary tumor. Although the benefit of the anti-vascular endothelial growth factor agent bevacizumab alongside first-line chemotherapy in mCRC is established, the impact of bevacizumab on the intact primary tumor (IPT) is less well understood.
Data from an Australian mCRC registry were used to assess the impact of bevacizumab-based regimens in the presence of an IPT, to see if this differs from effects in resected primary tumor (RPT) patients and to understand the safety profile of bevacizumab in patients with IPT. Progression-free survival (PFS), overall survival (OS) and safety endpoints were analyzed.
Of 1204 mCRC patients, 826 (69%) were eligible for inclusion. Bevacizumab use was similar in both arms (IPT (64%) versus RPT (70%)); compared with chemotherapy alone, bevacizumab use was associated with significantly longer PFS (IPT: 8.5 months vs 4.7 months, P = 0.017; RPT: 10.8 months vs 5.8 months, P < 0.001) and OS (IPT: 20 months vs 14.8 months, P = 0.005; RPT: 24.4 months vs 17.3 months, P = 0.004)).(1) Bevacizumab use in an IPT was associated with more GI perforations (4.5% vs 1.8%, P = 0.210) but less frequent bleeding (1.5% vs 5.3%, P = 0.050) and thrombosis (1.5% vs 2.7%, P = 0.470), versus chemotherapy alone. Median survival was equivalent between patients that did or did not experience bevacizumab-related adverse events - 20.0 months versus 19.9 months, hazard ratio = 0.98, P = 0.623.(1) CONCLUSIONS: The addition of bevacizumab significantly improved survival outcomes in mCRC with an IPT. The occurrence of bevacizumab-related adverse events did not significantly impact survival outcomes.
p53-Transcriptional-regulated proteins interact with a large number of other signal transduction pathways in the cell, and a number of positive and negative autoregulatory feedback loops act upon the p53 response. P53 directly controls the POMC/α-MSH productions induced by ultraviolet (UV) and is associated with UV-independent pathological pigmentation. When identifying the causative gene of dyschromatosis universalis hereditaria (DUH), we found three mutations encoding amino acid substitutions in the gene SAM and SH3 domain containing 1 (SASH1), and SASH1 was associated with guanine nucleotide-binding protein subunit-alpha isoforms short (Gαs). However, the pathological gene and pathological mechanism of DUH remain unknown for about 90 years. We demonstrate that SASH1 is physiologically induced by p53 upon UV stimulation and SASH and p53 is reciprocally induced at physiological and pathophysiological conditions. SASH1 is regulated by a novel p53/POMC/α-MSH/Gαs/SASH1 cascade to mediate melanogenesis. A novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype. Our study demonstrates that a novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype.
Even species within the same assemblage have varied responses to climate change, and there is a poor understanding for why some taxa are more sensitive to climate than others. In addition, multiple mechanisms can drive species' responses, and responses may be specific to certain life stages or times of year. To test how marine species respond to climate variability, we analyzed 73 diverse taxa off the southeast U.S. coast in 26 years of scientific trawl survey data and determined how changes in distribution and biomass relate to temperature. We found that winter temperatures were particularly useful for explaining interannual variation in species' distribution and biomass, though the direction and magnitude of the response varied among species from strongly negative, to little response, to strongly positive. Across species, the response to winter temperature varied greatly, with much of this variation being explained by thermal preference. A separate analysis of annual commercial fisheries landings revealed that winter temperatures may also impact several important fisheries in the southeast U.S. Based on the life stages of the species surveyed, winter temperature appears to act through overwinter mortality of juveniles or as a cue for migration timing. We predict that this assemblage will be responsive to projected increases in temperature, and that winter temperature may be broadly important for species relationships with climate on a global scale. This article is protected by copyright. All rights reserved.
2,4-dichlorophanoxyacetic acid (2,4-D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, Indole-3-acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4-D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4-D specific mutants suggested that 2,4-D and IAA might also use distinct pathways to modulate Arabidopsis root growth. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4-D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4-D but not IAA altered the actin structure in long term and short term assays. Analysis of the 2,4-D specific mutant aar1-1 revealed that Small Acidic Protein 1 (SMAP1) functions positively to facilitate the 2,4-D-induced depolymerization of actin. The ubiquitin proteasome mutants, tir1-1 and axr1-12, which show enhanced resistance to 2,4-D compared with IAA for root growth inhibition, were also found to have less disrupted actin filament networks after 2,4-D exposure. Consistently, chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4-D on the organization of actin filaments. Roots of the double mutant aar1-1 tir1-1 also showed enhanced resistance to 2,4-D-induced root growth inhibition and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4-D on actin filament organization and root growth is mediated through synergistic interactions between SMAP1 and SCF(TIR)(1) ubiquitin proteasome components. This article is protected by copyright. All rights reserved.
To investigate the role of methylation levels at promoter regions of placental vascularization genes (VEGF, EGFR and c-jun) in pathogenesis and diagnosis of placental disorders.
We analyzed DNA and histone methylation at promoters of VEGF, EGFR and c-jun via methylation-sensitive high resolution melting and chromatin immunoprecipitation assay in pregnant women with normal pregnancy in first, second and third trimester (n = 30 in each group) and pregnant women with pregnancy complicated with preeclampsia (n = 30) and hydatidiform mole (n = 15).
The higher expression of VEGF, EGFR and c-jun in early pregnancy was observed to be independent of DNA methylation while it was associated with H3 K9/K27 trimethylations. Also, abnormally higher expression of c-jun in GTDs was associated with lower H3K9me3 level at its promoter. Under preeclampsia conditions, we observed dysregulation of both DNA methylation and H3 trimethylation and subsequent low expression of VEGF, EGFR and c-jun. Importantly, our promoter methylation data indicated that VEGF may act as novel fetal DNA diagnostic marker for preeclampsia and molar pregnancies in maternal plasma.
These findings emphasize the importance of dysregulated epigenetic phenomenon behind the pathologies of placental disorders and use of promoter region DNA methylation as an epigenetic marker for these pathological pregnancies. This article is protected by copyright. All rights reserved.
Mammalian glutaminases catalyze the stoichiometric conversion of L-glutamine to L-glutamate and ammonium ions. In brain, glutaminase is considered the prevailing pathway for synthesis of the neurotransmitter pool of glutamate. Besides neurotransmission, the products of glutaminase reaction also fulfill crucial roles in energy and metabolic homeostasis in mammalian brain. In the last years, new functional roles for brain glutaminases are being uncovered by using functional genomic and proteomic approaches. Glutaminases may act as multifunctional proteins able to perform different tasks: the discovery of multiple transcript variants in neurons and glial cells, novel extramitochondrial localizations, and isoform-specific proteininteracting partners strongly support possible moonlighting functions for these proteins. In this chapter, we present a critical account of essential works on brain glutaminase 80 years after its discovery. We will highlight the impact of recent findings and thoughts in the context of the glutamate/glutamine brain homeostasis.
The Affordable Care Act (ACA) has expanded health coverage for thousands of Illinois residents. Expanded coverage, however, does not guarantee appropriate health care. Diabetes and its ocular complications serve as an example of how providers in underserved urban areas may not be able to keep up with new demand for labor- and technology-intensive health care unless changes in reimbursement policies are instituted.
A retrospective cohort study was conducted using medical encounter information from the Chicago HealthLNK Data Repository (HDR), an assembly of non-duplicated and de-identified patient medical records. We used a method of estimating the geographic distribution of undiagnosed diabetic retinopathy in the city of Chicago to illustrate the magnitude of potentially preventable eye disease. All rates were calculated for all ZIP Codes within Chicago (Cook County), and statistical differences between observed and geographically adjusted expected rates (p < 0.10, p < 0.05, p < 0.01) were highlighted as underserved areas.
This analysis included 150,661 patients with diabetes identified from a total of nearly two million patients in Chicago. High rates of undetected diabetic retinopathy were found in low-income and minority areas. Within these areas, 37% of the identified diabetics were uninsured, with rates ranging widely from 20% to 68.6%. Among those with insurance, 32.8% were covered by Medicare and only 10% by Medicaid. Most patients with untreated diabetic retinopathy were found to reside in areas where primary health care is provided through Federally Qualified Health Centers.
With 150,661 diabetics identified in the city of Chicago, and this number continuing to rise each year, a manpower approach with ophthalmologist screening for diabetic retinopathy is not realistic. The ability to identify the growing number of diabetic patients with retinopathy in low-income areas will likely require the adoption of cost-effective screening technologies that are currently not funded by Medicare and Medicaid.
Tight junctions are critically important for many physiological functions, including the maintenance of cell polarity, regulation of paracellular permeability, and involvement in signal transduction pathways to regulate integral cellular processes. Furthermore, tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection. Viruses, in turn, have evolved mechanisms to exploit tight junction proteins to gain access to cells or spread through tissues in an infected host. Claudin family proteins are integral components of tight junctions and are thought to play crucial roles in regulating their permeability. Claudins have been implicated in the infection process of several medically important human pathogens, including hepatitis C virus, dengue virus, West Nile virus, and human immunodeficiency virus, among others. In this review, we summarize the role of claudins in viral infections and discuss their potential as novel antiviral targets. A better understanding of claudins during viral infection may provide insight into physiological roles of claudins and uncover novel therapeutic antiviral strategies.
In Finland, forensic age assessment is strictly regulated by legislation. According to the Aliens Act (301/2004) and the amendment of the Act (549/2010), the police authorities, the frontier guard authorities, and the immigration authorities have the right to refer asylum seekers to the University of Helsinki, Department of Forensic Medicine, for age assessment. These assessments are especially performed to solve if the person is of major age, the cutoff being 18 completed years. The forensic age assessment is largely based on dental development, since the special permit of the Radiation and Nuclear Safety Authority (STUK) to the Department of Forensic Medicine of the University of Helsinki, allowing the use of ionizing radiation for non-medical purposes, includes dental and hand X-rays. Forensic age assessment is always performed by two forensic odontologists. In 2015, the total number of forensic age assessment examinations was 149, and the countries of origin of the asylum seekers were most commonly Iraq, Afghanistan, and Somalia. The current legislation on forensic age assessment has been well received and approved. Radiological and other examinations can be performed in different parts of Finland, but the forensic odontologist at the University of Helsinki is always involved in the process and ensures joint quality standards for the forensic age assessment.
In 2000, Congress passed the Breast and Cervical Cancer Prevention and Treatment Act (BCCPTA) to provide coverage through Medicaid to women who screened positive for breast and cervical cancer. We aimed to determine if late-stage breast cancer prevalence decreased among Oklahoma women after passage of BCCPTA. Data were obtained from the Oklahoma Central Cancer Registry during 2000-2011. We estimated prevalence proportion ratios (PPR) using modified Poisson regression between the proportion of women with late-stage breast cancer and timing of diagnosis related to BCCPTA. Among uninsured women, the probability of being diagnosed with late-stage cancer after enactment of the BCCPTA was 0.80 (95% CI: 0.67, 0.96) times the probability before enactment. This was significant among uninsured women living in metro counties (PPR: 0.74, 95% CI: 0.61, 0.90) but not in non-metro counties (PPR: 1.05, 95% CI: 0.71, 1.56). These findings may be similar to other rural states with large uninsured populations.
To date adult-onset asthmatic patients who lack a clear stridor and show prolonged coughs and chest discomfort caused by small-airways dysfunction have increased. We examined the small-airways function of these cases and the effectiveness of slow and deep inhalation of FP/FM-pMDI.
62 adult-onset asthmatic patients who had prolonged coughs and chest discomfort with the middle or high dose of ICS/LABA combination agents under well technique (32 of BUD/FM-DPI group and 30 of FP/SM-pMDI group) were included into this study. ICS/LABAs were switched to FP/FM-pMDI and slow and deep inhalation for 2-3 seconds was carried out thoroughly. The dose of FP/FM-pMDI was reduced depending on the improvement of symptoms. ACT score, respiratory function tests and respiratory resistance were measured after approximately six months from switching (stable condition after switching) and were compared with the values of the same period of the last year (stable condition under the previous ICS/LABA).
After switching to FP/FM-pMDI, asthmatic symptoms and plural values of small-airways function were improved in 93.7% (30/32 cases) of BUD/FM-DPI group and in 86.6% (26/30 cases) of FP/SM-pMDI group. Moreover, mean daily inhalation doses were decreased from 5.0 to 4.3 in BUD/FM-DPI group and decreased from 5.7 to 3.7 in FP/SM-pMDI group.
Slow and deep inhalation of FP/FM-pMDI is effective in many asthmatic patients who have prolonged small-airways dysfunction. A prospective, multi-centered contrastive study is warranted to confirm the effectiveness of this inhalational method.
Stanford type A acute aortic dissection (A-AAD) extends to the brachiocephalic branches in some patients. After ascending aortic replacement, a remaining re-entry tear in the distal brachiocephalic branches may act as an entry and result in a patent false lumen in the aortic arch. However, the effect of brachiocephalic branch re-entry concomitant with A-AAD remains unknown.Methods and Results:Eighty-five patients with A-AAD who underwent ascending aortic replacement in which both preoperative and postoperative multiple-detector computed tomography (MDCT) scans could be evaluated were retrospectively studied. The presence of a patent false lumen in at least one of the brachiocephalic branches on preoperative MDCT was defined as brachiocephalic branch re-entry, and 41 patients (48%) had this. Postoperatively, 47 of 85 (55%) patients had a patent false lumen in the aortic arch. False lumen remained patent after operation in 34 out of the 41 (83%) patients with brachiocephalic branch re-entry, as compared to that in 13 of the 44 (30%) patients without such re-entry (P<0.001). Brachiocephalic branch re-entry was a significant risk factor for a late increase in the aortic arch diameter greater than 10 mm (P=0.047).
Brachiocephalic branch re-entry in patients with A-AAD is related to a patent false lumen in the aortic arch early after ascending aortic replacement and is a risk factor for late aortic arch enlargement.
More than a decade of research has supported a robust consensus: Acute stress impairs memory retrieval. We aimed to determine whether a highly effective learning technique could strengthen memory against the negative effects of stress. To bolster memory, we used retrieval practice, or the act of taking practice tests. Participants first learned stimuli by either restudying or engaging in retrieval practice. Twenty-four hours later, we induced stress in half of the participants and assessed subsequent memory performance. Participants who learned by restudying demonstrated the typical stress-related memory impairment, whereas those who learned by retrieval practice were immune to the deleterious effects of stress. These results suggest that the effects of stress on memory retrieval may be contingent on the strength of the memory representations themselves.
The annual cost of healthcare delivery in the USA now exceeds US$3 trillion. Fee for service methodology is often implicated as a cause of this exceedingly high figure. The Affordable Care Act created the Center for Medicare and Medicaid Innovation (CMMI) to pilot test value based alternative payments for reimbursing physician services. In 2015, the Medicare Access and CHIP Reauthorization Act (MACRA) was passed into law. MACRA has dramatic implications for all US based healthcare providers. MACRA permanently repealed the Medicare Sustainable Growth Rate so as to stabilize physician part B Medicare payments, consolidated pre-existing federal performance programs into the Merit based Incentive Payments System (MIPS), and legislatively mandated new approaches to paying clinicians. Neurointerventionalists will predominantly participate in MIPS. MIPS unifies, updates, and streamlines previously existing federal performance programs, thereby reducing onerous redundancies and overall administrative burden, while consolidating performance based payment adjustments. While MIPS may be perceived as a straightforward continuation of fee for service methodology with performance modifiers, MIPS is better viewed as a stepping stone toward eventually adopting alternative payment models in later years. In October 2016, the Centers for Medicare and Medicaid Services (CMS) released a final rule for MACRA implementation, providing greater clarity regarding 2017 requirements. The final rule provides a range of options for easing MIPS reporting requirements in the first performance year. Nonetheless, taking the newly offered 'minimum possible' approach toward meeting the requirements will still have negative consequences for providers.
The Word Cloud is a frequent wish in the 3 Wishes Project developed to nurture peace and ease the grieving process for dying critically ill patients. The objective was to examine whether Word Clouds can act as a heuristic approach to encourage a narrative orientation to medicine. Narrative medicine is an approach which can strengthen relationships, compassion and resilience.
Word Clouds were created for 42 dying patients, and we interviewed 37 family members and 73 clinicians about their impact. We conducted a directed qualitative content analysis, using the 3 stages of narrative medicine (attention, representation, affiliation) to examine the narrative medicine potential of Word Clouds.
The elicitation of stories for the Word Cloud promotes narrative attention to the patient as a whole person. The distillation of these stories into a list of words and the prioritisation of those words for arrangement in the collage encourages a representation that did not enforce a beginning, middle or end to the story of the patient's life. Strong affiliative connections were achieved through the honouring of patients, caring for families and sharing of memories encouraged through the creation, sharing and discussion of Word Clouds.
In the 3 Wishes Project, Word Clouds are 1 way that families and clinicians honour a dying patient. Engaging in the process of making a Word Cloud can promote a narrative orientation to medicine, forging connections, making meaning through reminiscence and leaving a legacy of a loved one. Documenting and displaying words to remember someone in death reaffirms their life.
Cell adhesion molecules (CAM) play essential roles in the central nervous system, where some families are involved in synaptic development and function. These synaptic adhesion molecules (SAMs) are involved in the regulation of synaptic plasticity, and the formation of neuronal networks. Recent findings from studies examining the consequences of sleep loss suggest that these molecules are candidates to act in sleep regulation. This review highlights the experimental data that lead to the identification of SAMs as potential sleep regulators, and discusses results supporting that specific SAMs are involved in different aspects of sleep regulation. Further, some potential mechanisms by which SAMs may act to regulate sleep are outlined, and the proposition that these molecules may serve as molecular machinery in the two sleep regulatory processes, the circadian and homeostatic components, is presented. Together, the data argue that SAMs regulate the neuronal plasticity that underlies sleep and wakefulness.
Scavenger receptor A (SRA) has been known as an immunosuppressive factor and therefore therapeutic inhibition of SRA may be potentially exploited for cancer immunotherapy. Our previously work suggested that rhein may act as an inhibitor of SRA in reversing immunosuppression of SRA during T cells activation. Herein, three deconstruction analogs of rhein, compound 1, 2, and 3, were further studied as inhibitors of SRA. These three compounds, particularly compound 1, also known as a natural product danthron, enhanced T cells activation, indicated by increased transcriptional activation of interleukin 2 (Il2) gene, production of IL-2 protein, and proliferation of T cells. Additionally, the interaction between these compounds and SRA was studied by molecular modeling. Compound 1 showed a favorable binding mode with the cysteine rich domain of SRA protein compared to compound 2 and 3. Collectively, those results would provide insight for future design and development of next generation rhein derivatives as SRA inhibitors.
Although conventional antimicrobial drugs have been viewed as miraculous cure-alls for the past 80 years, increasing antimicrobial drug resistance requires a major and rapid intervention. However, the development of novel but still conventional systemic antimicrobial agents, having only a single mode or site of action, will not alleviate the situation because it is probably only a matter of time until any such agents will also become ineffective. To continue to produce new agents based on this notion is unacceptable, and there is an increasing need for alternative approaches to the problem. By contrast, light-activated molecules called photoantimicrobials act locally via the in-situ production of highly reactive oxygen species, which simultaneously attack various biomolecular sites in the pathogenic target and therefore offer both multiple and variable sites of action. This non-specificity at the target circumvents conventional mechanisms of resistance and inhibits the development of resistance to the agents themselves. Photoantimicrobial therapy is safe and easy to implement and, unlike conventional agents, the activity spectrum of photoantimicrobials covers bacteria, fungi, viruses, and protozoa. However, clinical trials of these new, truly broad-spectrum, and minimally toxic agents have been few, and the funding for research and development is almost non-existent. Photoantimicrobials constitute one of the few ways forward through the morass of drug-resistant infectious disease and should be fully explored. In this Personal View, we raise awareness of the novel photoantimicrobial technologies that offer a viable alternative to conventional drugs in many relevant application fields, and could thus slow the pace of resistance development.
The aim of this study was to assess the influence of different factors on the final reimbursement recommendations for drugs in Poland and to identify the correlation between these factors and the probability of a positive reimbursement recommendation for an applicant drug issued by the President of the Agency for Health Technology Assessment and Tariff System (AOTMiT). We analysed all recommendations for the period of 2012-2014 in Poland, three years following the launch of the new Reimbursement Act of Medicines, Foodstuffs Intended for Particular Nutritional Uses and Medical Devices. For each recommendation we collected data on efficacy, safety, cost of therapy, cost-effectiveness, quality of evidence, orphan drug status and others. Logistic regression was used to identify factors that increase the odds of a positive reimbursement recommendation. We analysed 221 recommendations for drugs, of which 78% were positive. We observed significant associations of all selected factors with positive recommendations. Proven efficacy and safety were associated with much greater odds for a positive reimbursement recommendation (123.5 and 42.6, respectively) than cost factors, which may suggest that patient outcome is much more important than the results of the cost-effectiveness analysis (odds ratio of 3.5) and the general cost of therapy (odds ratio of 3) in the analysed period.
The pathological features of Parkinson's disease (PD) include an abnormal accumulation of α-synuclein in the surviving dopaminergic neurons. Though PD is multifactorial, several epidemiological reports show an increased incidence of PD with co-exposure to pesticides such as Maneb and paraquat (MP). In pesticide-related PD, mitochondrial dysfunction and α-synuclein oligomers have been strongly implicated, but the link between the two has not yet been understood. Similarly, the biological effects of α-synuclein or its radical chemistry in PD is largely unknown. Mitochondrial dysfunction during PD pathogenesis leads to release of cytochrome c in the cytosol. Once in the cytosol, cytochrome c has one of two fates: It either binds to apaf1 and initiates apoptosis or can act as a peroxidase. We hypothesized that as a peroxidase, cytochrome c leaked out from mitochondria can form radicals on α-synuclein and initiate its oligomerization.
Samples from controls, and MP co-exposed wild-type and α-synuclein knockout mice were studied using immuno-spin trapping, confocal microscopy, immunohistochemistry, and microarray experiments.
Experiments with MP co-exposed mice showed cytochrome c release in cytosol and its co-localization with α-synuclein. Subsequently, we used immuno-spin trapping method to detect the formation of α-synuclein radical in samples from an in vitro reaction mixture consisting of cytochrome c, α-synuclein, and hydrogen peroxide. These experiments indicated that cytochrome c plays a role in α-synuclein radical formation and oligomerization. Experiments with MP co-exposed α-synuclein knockout mice, in which cytochrome c-α synuclein co-localization and interaction cannot occur, mice showed diminished protein radical formation and neuronal death, compared to wild-type MP co-exposed mice. Microarray data from MP co-exposed wild-type and α-synuclein knockout mice further showed that the absence of α-synuclein per se or its co-localization with cytochrome c confers protection from MP co-exposure, as several important pathways were unaffected in α-synuclein knockout mice.
Altogether, these results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein, through its co-localization with cytochrome c or on its own, affects several biological pathways which contribute to increased neuronal death in an MP-induced model of PD.
Youth trauma exposure is associated with syndemic HIV risk. We measured lifetime prevalence, type, and correlates of trauma experience by gender among adolescents living in the HIV hyper-endemic setting of Soweto, South Africa.
Using data from the Botsha Bophelo Adolescent Health Survey (BBAHS), prevalence of "ever" experiencing a traumatic event among adolescents (aged 14-19) was assessed using a modified Traumatic Event Screening Inventory-Child (TESI-C) scale (19 items, study alpha = 0.63). We assessed self-reported number of potentially traumatic events (PTEs) experienced overall and by gender. Gender-stratified multivariable logistic regression models assessed independent correlates of 'high PTE score' (≥7 PTEs).
Overall, 767/830 (92%) participants were included (58% adolescent women). Nearly all (99.7%) reported experiencing at least one PTE. Median PTE was 7 [Q1,Q3: 5-9], with no gender differences (p = 0.19). Adolescent men reported more violent PTEs (e.g., "seen an act of violence in the community") whereas women reported more non-violent HIV/AIDS-related PTEs (e.g., "family member or someone close died of HIV/AIDS"). High PTE score was independently associated with high food insecurity among adolescent men and women (aOR = 2.63, 95%CI = 1.36-5.09; aOR = 2.57, 95%CI = 1.55-4.26, respectively). For men, high PTE score was also associated with older age (aOR = 1.40/year, 95%CI = 1.21-1.63); and recently moving to Soweto (aOR = 2.78, 95%CI = 1.14-6.76). Among women, high PTE score was associated with depression using the CES-D scale (aOR = 2.00, 95%CI = 1.31-3.03,) and inconsistent condom use vs. no sexual experience (aOR = 2.69, 95%CI = 1.66-4.37).
Nearly all adolescents in this study experienced trauma, with gendered differences in PTE types and correlates, but not prevalence. Exposure to PTEs were distributed along social and gendered axes. Among adolescent women, associations with depression and inconsistent condom use suggest pathways for HIV risk. HIV prevention interventions targeting adolescents must address the syndemics of trauma and HIV through the scale-up of gender-transformative, youth-centred, trauma-informed integrated HIV and mental health services.
Cancer has become a global burden due to its high incidence and mortality rates, with an estimated 14.1 million cancer cases reported worldwide in 2012 particularly as a result of metastasis. Metastasis involves two crucial steps: adhesion and invasion, and the non-integrin receptor; the 37-kDa/67-kDa laminin receptor precursor/ high affinity laminin receptor (LRP/LR) has been shown to be overexpressed on the surface of tumorigenic cells, thus being implicated in the enhancement of these two crucial steps. The current study investigated the role of LRP/LR on the aggressiveness of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells with respect to their adhesive and invasive potential.
AsPC-1 and IMR-32 cells were utilized as the experimental cell lines for the study. Cell surface LRP/LR levels were visualised and quantified on the experimental and control (MCF-7) cell lines via confocal microscopy and flow cytometry, respectively. Total LRP/LR levels in the cell lines were assessed by Western blotting and the adhesive and invasive potential of the above-mentioned cell lines was determined before and after supplementation with the anti-LRP/LR specific antibody IgG1-iS18. Statistical significance of the data was confirmed via the use of the two-tailed student's t-test and Pearson's correlation coefficient.
Flow cytometry revealed that AsPC-1 and IMR-32 cells displayed significantly higher cell surface LRP/LR levels in comparison to the MCF-7 control cell line. However, Western blotting and subsequent densitometric analysis revealed that all three tumorigenic cell lines displayed no significant difference in total LRP/LR levels. The treatment of AsPC-1 and IMR-32 cells with IgG1-iS18 caused a significant reduction in the adhesive and invasive potential of the cells to laminin-1 and through the ECM-like Matrigel™, respectively. Pearson's correlation coefficients indicated a high correlation, thus suggesting a directly proportional relationship between cell surface LRP/LR levels and the adhesive and invasive potential of AsPC-1 and IMR-32 cells.
These findings suggest that through the interference of the LRP/LR-laminin-1 interaction, the anti-LRP/LR specific antibody IgG1-iS18 may act as an alternative therapeutic tool for the treatment of metastatic pancreatic cancer and neuroblastoma.
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a widespread highly toxic environmental contaminant, suppresses immune response and leads to an increased susceptibility to infectious agents. In particular, several studies have provided evidence that TCDD decreases resistance to numerous viruses. Indeed, in vivo and in vitro investigations showed that the presence of TCDD is able to interfere with the replication of both human and animal viruses, such as influenza A viruses, coxsackie virus B3, immunodeficiency virus type-1 (HIV-1), cytomegalovirus (CMV), herpes simplex II, and bovine herpesvirus 1. Moreover, TCDD could induce an exacerbation of latent infection produced by HIV-1, CMV or Epstein-Barr virus. In this review, we first describe the general effects of TCDD exposure on mammalian cells, then we focus on its influence on the viral infections. Overall, the available data support the concept that TCDD exposure may act as an additional risk factor in promoting of viral diseases.
The clinical placement learning environment is a critical component of nursing education where Australian nursing students spend a minimum of 800h. Identifying components of successful clinical placements for undergraduate nursing students is therefore paramount.
To assess nursing students' views of the learning environment during clinical placement with an emphasis on the pedagogical atmosphere, leadership style of the ward manager, and premises of nursing on the unit or ward.
The study used Clinical Learning Environment, Supervision and nurse teacher (CLES+T) questionnaire to examine 150 final year undergraduate students' perceptions of the clinical placement learning environment. The questionnaire was anonymous and completed by the students at the end of their clinical placement. The statistical program SPSS v22 was used. Principal components analysis (PCA) for data reduction was run on the 42-question section of the first dimension ('pedagogical atmosphere on the ward') of the questionnaire that measured the perceptions of the learning environment of the clinical placement of the 150 final-year undergraduate nursing students. The comments sections of the factors were subjected to interpretive content analysis to create the themes for the two components.
Principle Component Analysis revealed two components that had eigenvalues greater than one: 'Happy to Help' Component 1 and 'Happy to be Here' Component 2. These components were statistically significant (p<0.0005), using Bartlett's Test of Sphericity indicating that the data was likely factorizable. These components scored higher than any other related factors.
Student nurses value a welcoming workplace where staff and educators are happy to help and have a positive attitude to student presence on the wards. More than any other factors these ward-based factors appear to have the strongest influence on student satisfaction.
Reticulon 3 (RTN3), which is a member of the reticulon family of proteins, has a biochemical function of shaping tubular endoplasmic reticulum. RTN3 has also been found to interact with β-site amyloid precursor protein cleaving enzyme 1 (BACE1), which initiates the generation of β-amyloid peptides (Aβ) from amyloid precursor protein. Aβ is the major proteinaceous component in neuritic plaques, which constitute one of the major pathological features in brains of Alzheimer's disease (AD) patients. Mice deficient in or overexpressing RTN3 have altered amyloid deposition through effects on BACE1 expression and activity. In this review, we will summarize the current findings concerning the role of RTN3 in AD pathogenesis and demonstrate that RTN3 protein levels act as age-dependent modulators of BACE1 activity and Aβ deposition during the pathogenic progression of AD.
Clinical manifestations of influenza range from relatively mild and self-limiting respiratory infections to severe clinical manifestations with significant morbidity and mortality. The awareness of predictive indicators for the lethal outcome of influenza is of particular significance in making timely and exact decision for adequate treatment. The aim of this study was to identify the factors in patients with a severe form of influenza, resulting in lethal outcome.
The investigation was a prospective group comparison conducted at the University Clinic for Infectious Diseases in Skopje, R. Macedonia in the period from January 01, 2012 to January 01, 2015. The study included adult patients with a severe form of influenza who were further categorized into a group of either survived patients or a group of deceased patients. Demographic, clinical and biochemical data were noted in all patients included in the study on admission. The variables of the univariate analysis that showed a significant difference in terms of the outcome were used for creating multivariate logistic and regression analysis of the outcome as dependent factors. The independent predictors for lethal outcome in severe cases of influenza were identified by using logistic regression.
The study included 87 patients with a severe form of clinical and laboratory confirmed influenza. The patients were divided in two groups: survived (n = 75) and deceased (n = 75). The overall mortality was 13.79%. Multivariate analysis conducted on admission to hospital identified cardiovascular comorbid diseases (p = 0.014), urea values higher than 8.3 U/L (p = 0.045) and SAPS score (p = 0.048) as independent predictors of the outcome in patients with severe form of influenza. Influenza patients with cardiovascular diseases had 2.024 times greater risk of death from influenza in comparison to the patients having influenza without history of such a disease (OR = 2.024 95% CI 1.842-17.337). Patients with serum urea values higher than 8.3 U/L had 1.89 times higher chance of death compared to patients with normal values (OR = 1.89 95% CI 1.091-11.432). The increase of the SAPS score in one point increased the chance of death in patients with influenza by 1.2% (OR = 1.12 95% CI 1.01-2.976). The ROC analysis indicated that cardiovascular diseases, increased urea values and SAPS score in combination act as a good prognostic model for the fatal outcome. The global authenticity of this predictive model to foresee lethal outcome amounts to 80%, sensitivity being 82%, and specificity 70%.
Cardiovascular diseases, increased values of urea over 8.3 mmol/l and SAPS score are independent predictive indicators for lethal outcome in severe influenza. Early identification of the outcome predictors in patients with severe influenza will allow implementation of adequate medical treatment and will contribute to decreasing of mortality in patients with severe form of influenza.
Sorting nexins are PX domain-containing proteins that bind phospholipids and often act in membrane trafficking where they help to select cargo. However, the functions and cargo specificities of many sorting nexins are unknown. Here, a high-throughput imaging screen was used to identify new sorting nexin cargo in the yeast Saccharomyces cerevisiae. Deletions of nine different sorting nexins were screened for mislocalization of a set of GFP-tagged membrane proteins found at the plasma membrane, Golgi or endosomes. This identified 27 proteins that require one or more sorting nexins for their correct localization, 23 of which represent novel sorting nexin cargo. Nine hits whose sorting was dependent on Snx4, the sorting nexin-containing retromer complex, or both retromer and Snx3, were examined in detail to search for potential sorting motifs. We identified cytosolic domains of Ear1, Ymd8 and Ymr010w that conferred retromer-dependent sorting on a chimeric reporter and identified conserved residues required for this sorting in a functional assay. This work defined a consensus sequence for retromer and Snx3-dependent sorting.
The 50th anniversary of the Swampscott Conference offers an opportunity to reflect on a community psychology setting, The Consultation Center at Yale, that was formed in response to the 1963 Community Mental Health Act and the 1965 Swampscott Conference. The Center has flourished as a community psychology setting for practice, research, and training for 39 of the 50 years since Swampscott. Its creation and existence over this period offers an opportunity for reflection on the types of settings needed to sustain the field into the future.
Blocking the TRPV1 receptor is an interesting approach for the treatment of sensitive skin. Here we investigated the potential of grifolin derivatives from Albatrellus ovinus to act as TRPV1 receptor blockers and their potential to serve as cosmetic active ingredients.
Binding characteristics of grifolin derivatives from Albatrellus ovinus were determined in competitive and functional in vitro assays to achieve IC50 values. The TRPV1 receptor was activated in vivo with capsaicin and noxious heat to investigate skin reddening, microcirculation, skin sensations and heat pain thresholds.
Grifolin derivatives extracted from Albatrellus ovinus proved to inhibit the TRPV1 receptor in vitro and in vivo. Besides suppression of the TRPV1 receptor activity upon chemical stimulation with capsaicin, thermal activation was shown to be inhibited as well by application of cosmetic formulations containing 3% Albatrellus ovinus extract. The reduction of stinging and burning sensations as well as reduction of reddening and microcirculation upon irritation with capsaicin or thermal stress proved efficacy in vivo.
Grifolin derivatives from Albatrellus ovinus are able to serve as fungal derived TRPV1 receptor blockers with capability to serve as a cosmetic active ingredient on sensitive skin. This article is protected by copyright. All rights reserved.
This study examined social influences on 3-year-old children's decisions to help an experimenter gain another person's attention (N = 32). Children were slower to help the experimenter when the target had previously expressed disinterest in attending to her. Shy children were less likely to support the experimenter's attempts to communicate with the target; however, this association was not influenced by children's knowledge of the target's disinterest, and there was no relation between shyness and children's support for a separate physical goal. Therefore, young children's decisions to act helpfully incorporate consideration for others beyond a focal person with an unmet need, and they are further constrained by children's own comfort with the actions required to help.
The Mediterranean Diet (MD) has been proved to exert benefits with respect to the maintenance of the redox balance, and wine is a representative component. Bioactive compounds such as polyphenols, melatonin and hydroxytyrosol act as radical scavengers and regulate the oxidation status of organisms. Oxidative damage to DNA yields a large range of end products. The repair of oxidized DNA entails the removal of the useless bases and/or nucleotides as well as the release of circulating nucleotides and nucleosides. The current research aims to elucidate, for the first time, the DNA protection against oxidative stress provided by three types of red wine - relating it to the intake of bioactive compounds - after the intake of a serving of red wine/must by 18 healthy female volunteers during a short term double-blind, crossover and placebo-controlled study. The novelty of our work is to describe the importance of melatonin and hydroxytyrosol and its metabolites (from gut microflora) in comparison with polyphenols in a red wine matrix (excluding colon derivatives). The results show that the intake of red wine and must secondarily reduces oxidative stress and carcinogenesis due to their content of homovanillic acid, as measured by decreases in the plasmatic concentration of 8-hydroxy-2'deoxyguanosine, 8-hydroxyguanine, and 8-nitroguanosine. Moreover, the intake of wine appears to exert vasodilatory effects, mediated by the action of nitric oxide and increased plasma guanosine-3'-5'-cyclic monophosphate plasmatic levels, owing to the intake of wines higher in melatonin and homovanillic acid. Therefore, the results obtained in the present study revealed that polyphenols, despite being the major compounds in the red wine matrix, are not the most effective compounds protecting DNA from oxidative attack.
The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8(+) T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4(+) subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat's immunity, opening up new perspectives of therapeutic interventions for humans.
Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6C(low) monocytes and CD4(+) CD25(+) FoxP3(+) T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.
The aim was to clarify the associations of five adipocytokines: Sfrp5, Wnt5a, adiponectin, chemerin and high-sensitivity C-reactive protein (hsCRP) with blood pressure (BP), and to examine whether BP can be influenced by changes in these adipocytokines in obese children after a 6-month lifestyle intervention. We conducted a cross-sectional study in 263 obese children and performed a 6-month lifestyle intervention in a subgroup of 89 obese children with hypertension. Anthropometric data, adiponectin, chemerin, Sfrp5 and Wnt5a were assessed at baseline and after 6-month lifestyle intervention. Sfrp5 and adiponectin serum levels were significantly lower in obese children with hypertension, but Wnt5a, hsCRP and chemerin serum levels were elevated in obese children with hypertension. In multivariable linear regression analysis, Sfrp5, Wnt5a, adiponectin, chemerin and hsCRP were associated with both standard deviation score-systolic blood pressure (SDS-SBP) and -diastolic blood pressure (SDS-DBP). Lifestyle intervention resulted in a significant improvement in BP and weight loss. These were accompanied by significant decreases in hsCRP and chemerin, and significant increases in Sfrp5 and adiponectin, whereas Wnt5a was not changed. Furthermore, the changes in Sfrp5 and adiponectin act as partial mediators of the relationship between weight loss and BP reduction after controlling for covariates. Although Sfrp5, Wnt5a, adiponectin, chemerin and hsCRP levels are correlated with BP at baseline, after lifestyle intervention, the relationship between weight loss and BP reduction were partially mediated by changes in Sfrp5 and adiponectin after controlling for covariates. So we speculate that Sfrp5 and adiponectin may have some influence on BP.Journal of Human Hypertension advance online publication, 24 November 2016; doi:10.1038/jhh.2016.76.
The efficient and precise delivery of siRNA to target cells is critical to successful gene therapy. While novel nanomaterials enhance delivery efficiency, it still remains challenging for precise gene delivery to overcome nonspecific adsorption and off-target effect. Here we design a dual lock-and-key system to perform cell-subtype-specific recognition and siRNA delivery. The siRNA is self-assembled in an oligonucleotide nano vehicle that is modified with a hairpin structure to act as both the 'smart key' and the delivery carrier. The auto-cleavable hairpin structure can be activated on site at target cell membrane by reacting with two aptamers as 'dual locks' sequentially, which leads to cell-subtype discrimination and precise siRNA delivery for high efficient gene silencing. The success of this strategy demonstrates the precise delivery of siRNA to specific target cells by controlling multiple parameters, thus paving the way for application of RNAi in accurate diagnosis and intervention.
Porphyromonas gingivalis, a keystone pathogen in chronic periodontitis, has been found to associate with remote body organ inflammatory pathologies, including atherosclerosis and Alzheimer's disease (AD). Although P. gingivalis has a plethora of virulence factors, much of its pathogenicity is surprisingly related to the overall immunosuppression of the host. This review focuses on P. gingivalis aiding suppression of the host's adaptive immune system involving manipulation of cellular immunological responses, specifically T cells and B cells in periodontitis and related conditions. In periodontitis, this bacterium inhibits the synthesis of IL-2 and increases humoral responses. This reduces the inflammatory responses related to T- and B-cell activation, and subsequent IFN-γ secretion by a subset of T cells. The T cells further suppress upregulation of programmed cell death-1 (PD-1)-receptor on CD(+)cells and its ligand PD-L1 on CD11b(+)-subset of T cells. IL-2 downregulates genes regulated by immune response and induces a cytokine pattern in which the Th17 lineage is favored, thereby modulating the Th17/T-regulatory cell (Treg) imbalance. The suppression of IFN-γ-stimulated release of interferon-inducible protein-10 (IP-10) chemokine ligands [ITAC (CXCL11) and Mig (CXCL9)] by P. gingivalis capsular serotypes triggers distinct T cell responses and contributes to local immune evasion by release of its outer membrane vesicles. In atherosclerosis, P. gingivalis reduces Tregs, transforms growth factor beta-1 (TGFβ-1), and causes imbalance in the Th17 lineage of the Treg population. In AD, P. gingivalis may affect the blood-brain barrier permeability and inhibit local IFN-γ response by preventing entry of immune cells into the brain. The scarcity of adaptive immune cells in AD neuropathology implies P. gingivalis infection of the brain likely causing impaired clearance of insoluble amyloid and inducing immunosuppression. By the effective manipulation of the armory of adaptive immune suppression through a plethora of virulence factors, P. gingivalis may act as a keystone organism in periodontitis and in related systemic diseases and other remote body inflammatory pathologies.
In this article, we sought reconciliation between the "body-as-representation" and the "body-as-experience," that is, how the body is represented in discourse and how the body of older people with cognitive impairment is experienced. We identified four contemporary "technologies" and gave examples of these to show how they influence how older people with cognitive impairment are often represented in acute care settings. We argued that these technologies may be mediated further by discourses of ageism and ableism which can potentiate either the repressive or productive tendencies of these technologies resulting in either positive or negative care experiences for the older person and/or their carer, including nurses. We then provided examples from research of embodied experiences of older people with dementia and of how nurses and other professionals utilized their inter-bodily experiences to inform acts of caring. The specificity and individuality of these experiences were more conducive to positive care experiences. We conclude the article by proposing that the act of caring is one way nurses seek to reconcile the "body-as-representation" with the "body-as-experience" to mitigate the repressive effects of negative ageism and ableism. The act of caring, we argue, is the essence of caring enacted through the provision of person-centred care which evokes nurses to respond appropriately to the older person's "otherness," their "variation of being" while enabling them to enact a continuation of themselves and their own version of normality.
Proliferating Ki-67(+) cardiomyocytes were detected in the interventricular septum myocardium of adult patients with hypertrophic cardiomyopathy. In the same patients, the severity of hypertrophy and the degree of cardiomyocyte differentiation were assessed by the content of myofibrils, ultrastructural morphology, and the pattern of connexin 43-containing gap junction distribution. Adult Ki-67(+) cardiomyocytes containing sarcomeric α-actin (sarc α-act(+)) in the sarcoplasm (diameter 23.9±6.9 μ) were detected in the myocardium of patients with hypertrophic cardiomyopathy; their relative content varied from 2 to 3084 cells per 1 million cardiomyocytes. Small early differentiating Ki-67(+)/sarc α-act(+) cardiomyocytes with a thin cytoplasm layer (diameter 5.9±1.7 μ) constituted from 3 to 2262 cells per 1 million cardiomyocytes. These cells were found in the myocardium with the most pronounced structural changes: hypertrophy of cardiomyocytes with signs of their partial dedifferentiation.
Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.
The adult central nervous system (CNS) was considered a comparatively static tissue with little cell turnover. It is now well established that there is more plasticity than previously thought and that astrocytes act as neural stem/precursor cells (NSPCs) in the subventricular zone (SVZ). The discovery that these NSPCs can give rise to a limited number of new neurons, reactive astrocytes and oligodendrocytes contributing to brain repair in CNS disease, has raised hopes toward harnessing these cells for therapeutic interventions. Here, we will discuss the transcriptional control of adult NSPC differentiation into astrocytes in CNS disease focusing on the helix-loop-helix transcription factor protein family. In our recent study, we reported that elevated BMP-2 levels are translated into an increase in Id3 expression in adult NSPC subpopulations after cortical injury. Id3 then heterodimerizes with the basic helix-loop-helix transcription factor E47 and releases the E47-mediated repression of astrocyte-specific gene expression. Consequently, adult NSPCs preferentially differentiate into astrocytes. We believe that understanding the in vivo differentiation potential and the molecular underpinnings of NSPCs in the adult mammalian brain will help us to evaluate their contributions to brain repair and may lead to new concepts in treating human CNS diseases.
Coronary artery disease (CAD) and its complication remain the leading cause of mortality in industrialized countries despite great advances in terms of diagnosis, prognosis, and treatment options. MicroRNAs (miRNAs), small noncoding RNAs, act as posttranscriptional gene expression modulators and have been implicated as key regulators in several physiological and pathological processes linked to CAD. Circulating miRNAs have been evaluated as promising novel biomarkers of CAD, acute coronary syndromes, and acute myocardial infarction, with prognostic implications. Several challenges related to technical aspects, miRNAs normalization, drugs interaction, and quality reporting of statistical multivariable analysis of the miRNAs observational studies remain unresolved. MicroRNA-based therapies in cardiovascular diseases are not ready yet for human trials but definitely appealing. Through this review we will provide clinicians with a concise overview of the pros and cons of microRNAs.
The aim of this study was to test the action of Porphyrin compounds, Tetraphenylporphine sulfonate (TPPS), 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) Chloride (FeTPPS) and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) nitrosyl Chloride (FeNOTPPS), on Na+, K+ -ATPase of cell membrane of erythrocytes.
Enzymatic assays, measuring the amount of inorganic phosphate produced, were used to estimate the activity of Na(+), K(+)-ATPase.
The results show that Porphyrin compounds exert an insulin-like effect on Na(+), K(+)-ATPase. They act by increasing the activity of the membrane-bound enzyme.
All the three Porphyrin compounds increased the activity of erythrocyte Na(+), K(+)-ATPase. The exact mechanism of action of these compounds is not clear.
Previous studies have found that predators utilise habitat corridors to ambush prey moving through them. In the marine environment, coastal channels effectively act as habitat corridors for prey movements, and sightings of predators in such areas suggest that they may target these for foraging. Unlike terrestrial systems where the underlying habitat structure is generally static, corridors in marine systems are in episodic flux due to water movements created by tidal processes. Although these hydrographic features can be highly complex, there is generally a predictable underlying cyclic tidal pattern to their structure. For marine predators that must find prey that is often patchy and widely distributed, the underlying temporal predictability in potential foraging opportunities in marine corridors may be important drivers in their use. Here, we used data from land-based sightings and 19 harbour seals (Phoca vitulina) tagged with high-resolution GPS telemetry to investigate the spatial and temporal distribution patterns of seals in a narrow tidal channel. These seals showed a striking pattern in their distribution; all seals spent a high proportion of their time around the narrowest point of the channel. There was also a distinctive tidal pattern in the use of the channel; sightings of seals in the water peaked during the flood tide and were at a minimum during the ebb tide. This pattern is likely to be related to prey availability and/or foraging efficiency driven by the underlying tidal pattern in the water movements through the channel.
To maximise foraging efficiency, predators often make use of narrow constrictions in habitat to intercept prey using these corridors for movement. In the marine environment, narrow channels may act as corridors, and sightings of predators suggest that they may target these for foraging. Despite this, there is little information on how individual predators use such areas. Here, we investigate how individual harbour seals use a narrow coastal channel subject to strong tidal currents; results showed that seals spent the majority of their time at the narrowest point of the channel foraging during peak tidal currents. This highlights the importance of narrow channels for marine predators and suggests that this usually wide-ranging predator may restrict its geographic range to forage in the channel as a result of increased prey availability and/or foraging efficiency driven by water movements through the narrow corridor.
The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4+ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine to adenine hypermutation through deamination of cytosine to form uracil in (-)DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity than A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 alone, A3G and A3F when combined had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer, in the absence of RNA, which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded (-)DNA exposed for longer periods of time enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties when compared to each individual counterpart.
The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 viral infectivity factor (Vif) protein. After APOBEC3 enzymes are encaspidated into virions they deaminate cytosines in (-) DNA which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found demonstrating that APOBEC3s still have discernable activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate than an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we may still not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.
Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that activation/inactivation represents an antiviral mechanism common to all tested CD4mc. Biphasic dose-response curves indicate that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on-rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility.
The trimeric envelope glycoproteins of the human immunodeficiency virus (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, trigger changes in the conformation of the HIV-1 envelope glycoprotein trimer important for virus entry. Small-molecule CD4-mimetic compounds inhibit HIV-1 infection by multiple mechanisms: 1) Direct blockade of the interaction between the gp120 exterior envelope glycoprotein and CD4; 2) Premature triggering of conformational changes in the envelope glycoproteins, leading to irreversible inactivation; and 3) Exposure of cryptic epitopes to antibodies, allowing virus neutralization. The consequences of the binding of the CD4-mimetic compound to the HIV-1 envelope glycoproteins depends upon how many of the three subunits of the trimer are bound and upon the propensity of the envelope glycoproteins to undergo conformational changes. Understanding the mechanistic factors that influence the activity of CD4-mimetic compounds can help to improve their potency and coverage of diverse HIV-1 strains.
IL-37 is a cytokine belonging to the IL-1 family. Although discovered in silico in 2000, significant advances in the understanding of its biology were made only in recent years. It is a member of the family with potent anti-inflammatory and immunosuppressive properties. It is produced as a precursor without a classic signal peptide. The precursor is cleaved into mature form in the cytoplasm by caspase-1. A small fraction of the cleaved IL-37 binds SMAD-3, translocates to the nucleus, and suppresses transcription of several proinflammatory genes. Both precursor and cleaved forms of IL-37 are secreted. They bind IL-18Rα chain (also used by IL-18 as a receptor subunit) and recruit Toll/IL-1R (TIR)-8 for transducing intracellular signaling. TIR-8 is a member of the IL-1 receptor family (IL-1RF) and was previously known as an orphan receptor. IL-37 suppresses activation of NF-κB and MAPK and activates Mer-PTEN-DOK pathway. It negatively regulates signaling mediated by TLR agonists, proinflammatory cytokines, and IL-1RF ligands. It also affects cell metabolism by inhibiting mTOR, GSK-3α/β, and activating AMPK. Despite having the ability to dampen its host's immune responses, the cytokine has been shown to exert antitumor effects, and it has been suggested that it may act as a prognostic marker in a variety of human cancers. Recent studies have suggested that IL-37 may represent a novel therapeutic tool in patients with cancer. In this review, we provide an overview of the cytokine biology, discuss recent advances made in unraveling its anti-cancer effects, and suggest guidelines for future research.
RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.
Neighborhood walkability has been shown to be associated with walking behavior. However, the availability of geographical data necessary to construct it remains a limitation. Building on the concept of space syntax, we propose an alternative walkability index, space syntax walkability (SSW). This study examined associations of the full walkability index and SSW with walking for transport (WT).
Data were collected in 2003-2004 from 2544 adults living in 154 Census Collection Districts (CCD) in Adelaide, Australia. Participants reported past week WT frequency. Full walkability (consisting of net residential density, intersection density, land use mix, and net retail area ratio) and SSW (consisting of gross population density and a space syntax measure of street integration) were calculated for each CCD using geographic information systems and space syntax software. Generalized linear models with negative binomial variance and logarithmic link functions were employed to examine the associations of each walkability index with WT frequency, adjusting for socio-demographic variables.
Two walkability indices were closely correlated (ρ = 0.76, p < 0.01). The associations of full walkability and SSW with WT frequency were positive, with regression coefficients of 1.12 (95% CI: 1.08, 1.17) and 1.14 (95% CI: 1.10, 1.19), respectively.
SSW employs readily-available geographic data, yet is comparable to full walkability in its association with WT. The concept and methods of space syntax provide a novel approach to further understanding how urban design influences walking behaviors.
Many malaria-endemic countries have implemented national community health worker (CHW) programmes to serve remote populations that have poor access to malaria diagnosis and treatment. Despite mounting evidence of CHWs' ability to adhere to malaria rapid diagnostic tests (RDTs) and treatment guidelines, there is limited evidence whether CHWs adhere to the referral guidelines and refer severely ill children for further management. In southwest Uganda, this study examined whether CHWs referred children according to training guidelines and described factors associated with adherence to the referral guideline.
A secondary analysis was undertaken of data collected during two cluster-randomized trials conducted between January 2010 and July 2011, one in a moderate-to-high malaria transmission setting and the other in a low malaria transmission setting. All CHWs were trained to prescribe artemisinin-based combination therapy (ACT) and recognize symptoms in children that required immediate referral to the nearest health centre. Intervention arm CHWs had additional training on how to conduct an RDT; CHWs in the control arm used a presumptive diagnosis for malaria using clinical signs and symptoms. CHW treatment registers were reviewed to identify children eligible for referral according to training guidelines (temperature of ≥38.5 °C), to assess whether CHWs adhered to the guidelines and referred them. Factors associated with adherence were examined with logistic regression models.
CHWs failed to refer 58.8% of children eligible in the moderate-to-high transmission and 31.2% of children in the low transmission setting. CHWs using RDTs adhered to the referral guidelines more frequently than CHWs not using RDTs (moderate-to-high transmission: 50.1 vs 18.0%, p = 0.003; low transmission: 88.5 vs 44.1%, p < 0.001). In both settings, fewer than 20% of eligible children received pre-referral treatment with rectal artesunate. Children who were prescribed ACT were very unlikely to be referred in both settings (97.7 and 73.3% were not referred in the moderate-to-high and low transmission settings, respectively). In the moderate-to-high transmission setting, day and season of visit were also associated with the likelihood of adherence to the referral guidelines, but not in the low transmission setting.
CHW adherence to referral guidelines was poor in both transmission settings. However, training CHWs to use RDT improved correct referral of children with a high fever compared to a presumptive diagnosis using sign and symptoms. As many countries scale up CHW programmes, routine monitoring of reported data should be examined carefully to assess whether CHWs adhere to referral guidelines and take remedial actions where required.
Repeated self-harm represents the single strongest risk factor for suicide. To date no study with full national coverage has examined the pattern of hospital repeated presentations due to self-harm among young people.
Data on consecutive self-harm presentations were obtained from the National Self-Harm Registry Ireland. Socio-demographic and behavioural characteristics of individuals aged 10-29 years who presented with self-harm to emergency departments in Ireland (2007-2014) were analysed. Risk of long-term repetition was assessed using survival analysis and time differences between the order of presentations using generalised estimating equation analysis.
The total sample comprised 28,700 individuals involving 42,642 presentations. Intentional drug overdose was the most prevalent method (57.9%). Repetition of self-harm occurred in 19.2% of individuals during the first year following a first presentation, of whom the majority (62.7%) engaged in one repeated act. Overall, the risk of repeated self-harm was similar between males and females. However, in the 20-24-year-old age group males were at higher risk than females. Those who used self-cutting were at higher risk for repetition than those who used intentional drug overdose, particularly among females. Age was associated with repetition only among females, in particular adolescents (15-19 years old) were at higher risk than young emerging adults (20-24 years old). Repeated self-harm risk increased significantly with the number of previous self-harm episodes. Time differences between first self-harm presentations were detected. Time between second and third presentation increased compared to time between first and second presentation among low frequency repeaters (patients with 3 presentations only within 1 year following a first presentation). The same time period decreased among high frequency repeaters (patients with at least 4 to more than 30 presentations).
Young people with the highest risk for repeated self-harm were 15-19-year-old females and 20-24-year-old males. Self-cutting was the method associated with the highest risk of self-harm repetition. Time between first self-harm presentations represents an indicator of subsequent repetition. To prevent risk of repeated self-harm in young people, all individuals presenting at emergency departments due to self-harm should be provided with a risk assessment including psychosocial characteristics, history of self-harm and time between first presentations.
An increase in the activity of the pituitary-gonad axis (PG-axis) and gonad development are essential for the onset of spawning migration in teleosts. In the fish Coilia nasus, gonad development and spawning migration up the Yangtze River occurs by the end of each summer. We hypothesized that gonadotropin releasing hormones receptor 2 (GnRH-R2), which together produce a signal that interacts with the PG-axis, may help to regulate spawning migration processes.
In this regard, we (1) characterized the gonadosomatic index (GSI) in the anadromous fish C. nasus; (2) analyzed the GnRH-R2 mRNA expression levels in ovary and brain, and concentrations in the serum; and (3) identified the GnRH-R2 protein distribution in the brain and ovaries. We found strong relationships between all of these indices.
The results indicate that GnRH-R2 could act together to promote spawning during the anadromous migration. There is some evidence that the GnRH-R2 gene expression levels and protein distributions change in association with the migratory behavior.
The proteasome is the major proteolytic site on the eukaryotic cell, degrading most of its short-lived or misfolded polypeptides. The ubiquitin-proteasome pathway has been found to play a fundamental role in the development of several pathologies, from cancer to neurodegenerative diseases, or even retroviral infections. Nature remains a powerful source for the discovery of bioactive compounds. Recently, a number of molecules of natural origin, as well as natural product derivatives, have been described as proteasome inhibitors. Most of these molecules directly block one or more catalytic sites of the 20S proteasome, but some of them act upstream of proteolytic degradation, for instance, inhibiting the ubiquitin tagging process.
The present review focuses on recent patents on proteasome inhibitors of natural origin, their derivatives and synthetic routes to obtain such molecules, as well as their application as a tool in chemotherapy.
With several of these modulators of the ubiquitin-proteasome system under clinical trials, we hope that the next few years lead to the development of new pharmaceutical drugs and characterization of new proteasome inhibitors of natural origin or inspiration.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer associated mortality. Accumulating evidence has shown that microRNAs (miRNAs) act as critical factors for tumor recurrence and metastasis. MiR-508-5p has been reported as a down-regulated miRNA in the primary gastric cancer tissues. However, the role of miR-508-5p on HCC has not been well elucidated. In this study, we observed that miR-508-5p was downregulated in HCC tissues when compared to the non-tumorous tissues. We then demonstrated that overexpression of miR-508-5p attenuated HepG2 cells proliferation and invasion and induced cell apoptosis in vitro. Furthermore, our further investigations revealed that mesoderm development candidate 1 (MESDC1) is a potential target of miR-508-5p, as well as miR-508-5p overexpression downregulated MESDC1 expression. Overexpression of MESDC1 promoted HepG2 cells migration, invasion and proliferation in vitro. In addition, miR-508-5p markedly suppressed the tumor growth in xenograft model, while MESDC1 promoted the tumor growth in xenograft model. This study provides new insight into molecular mechanisms that miR-508-5p acts as a tumor suppressor by targeting MESDC1 in HCC progression.
BST2 (bone marrow stromal antigen 2)/tetherin is a restriction factor of enveloped viruses, which blocks the release of viral particles. HIV-1 encodes proteins that antagonize this innate barrier, including the accessory protein Vpu. Here, we investigate whether the autophagy pathway and/or ATG proteins are hijacked by HIV-1 Vpu to circumvent BST2 restriction of viral release. We report that BST2 and Vpu are present in LC3-positive compartments. We found that Vpu selectively interacts with the ATG8 ortholog LC3C through the Vpu L63VEM66 sequence. This sequence is required for Vpu to antagonize BST2 restriction. LC3C expression favors the removal of BST2 from the HIV-1 budding site, and thus HIV-1 release in BST2-expressing cells. Additionally, ATG5 and beclin 1/ATG6, but not all the components of the autophagy pathway, act with LC3C to facilitate Vpu antagonism of BST2 restriction. Altogether, our data support the view that a non-canonical autophagy pathway reminiscent of LC3-associated phagocytosis contributes to Vpu counteraction of BST2 restriction.
Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3'-end of E1 highly conserved and the 5'-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone.
Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel's cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.
Deletion of chromosome 8p is the second most frequent genomic alteration in prostate cancer. To better understand its clinical significance, 8p deletion was analyzed by fluorescence in-situ hybridization on a prostate cancer tissue microarray. 8p deletion was found in 2,581 of 7,017 cancers (36.8%), and was linked to unfavorable tumor phenotype. 8p deletion increased from 29.5% in 4,456 pT2 and 47.8% in 1,598 pT3a to 53.0% in 931 pT3b-pT4 cancers (P < 0,0001). Deletions of 8p were detected in 25.5% of 1,653 Gleason ≤ 3 + 3, 36.6% of 3,880 Gleason 3 + 4, 50.2% of 1,090 Gleason 4 + 3, and 51.1% of 354 Gleason ≥ 4 + 4 tumors (P < 0,0001). 8p deletions were strongly linked to biochemical recurrence (P < 0.0001) independently from established pre- and postoperative prognostic factors (P = 0.0100). However, analysis of morphologically defined subgroups revealed, that 8p deletion lacked prognostic significance in subgroups with very good (Gleason ≤ 3 + 3, 3 + 4 with ≤ 5% Gleason 4) or very poor prognosis (pT3b, Gleason ≥ 8, pN1). 8p deletions were markedly more frequent in cancers with (53.5%) than without PTEN deletions (36.4%; P < 0,0001) and were slightly more frequent in ERG-positive (40.9%) than in ERG-negative cancers (34.7%, P < 0.0001) due to the association with the ERG-associated PTEN deletion. Cancers with 8p/PTEN co-deletions had a strikingly worse prognosis than cancers with deletion of PTEN or 8p alone (P ≤ 0.0003). In summary, 8p deletion is an independent prognostic parameter in prostate cancer that may act synergistically with PTEN deletions. Even statistically independent prognostic biomarkers like 8p may have limited clinical impact in morphologically well defined high or low risk cancers.
RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.
Risk of obstetric complications increases linearly with rising maternal glycaemia. Testing HbA1c is an effective option to detect hyperglycemia but its association with adverse pregnancy outcomes remains unclear. Emerging data sustains that an early HbA1c≥5.9% could act as a pregnancy risk marker.
To determine, in a multi-ethnic cohort, whether an early ≥5.9% HbA1c could be useful to identify women without diabetes mellitus at increased pregnancy risk.
A prospective study was conducted at Hospital del Mar, Barcelona, between April 2013-September 2015.
1,631 pregnant women had an HbA1c measurement added to their first antenatal bloods and were screened for gestational diabetes mellitus at 24-28 weeks' gestation.
Primary outcome was macrosomia. Secondary outcomes were pre-eclampsia, preterm birth and Caesarean section rate.
1,228 pregnancies were included for outcome analysis. Women with HbA1c≥5.9% (n= 48) showed a higher rate of macrosomia (16.7% vs. 5.9%,p= 0.008) and a tendency towards higher rate of preeclampsia (9.32% vs. 3.9% ,p= 0.092). There were no significant differences in other pregnancy outcomes. After adjusting for potential confounders an HbA1c≥5.9% was independently associated with a three-fold increased risk of macrosomia (95% CI: 1.127-8.603,p= 0.028) and preeclampsia (95% CI: 1.086-11.532,p= 0.036).
In a multiethnic population, an early HbA1c≥5.9% measurement identifies women at high risk for poorer pregnancy outcomes independently of GDM diagnosis later in pregnancy. Further studies are required to establish cutoff points adapted to each ethnic group and to assess whether early detection and treatment are of benefit.
Hybrids between the Arabidopsis ecotypes C24 and Ler have high levels of hybrid vigour, heterosis, in both biomass and seed yield. Heterosis can be detected throughout the development of the plant and in different tissues. We examined developing embryos and seeds of C24/Ler reciprocal hybrids with the aim of detecting the earliest time at which heterotic gene activity occurs. In the transcriptomes of 4 dap (dermatogen to globular) and 6 dap (heart) embryos from both parents and hybrids, 95% of expressed genes were at the Mid Parent Value (MPV) and 95% of the genes with SNPs between C24 and Ler retained the same relative allelic expression levels in the hybrids as existed in the parents. This included loci that had equivalent levels of transcription in the two parents, together with loci which had different levels of expression in the parents. Amongst the genes which did not have MPV expression levels in the hybrids (non-additively expressed genes), approximately 40 in the globular embryo stage and 89 in the heart embryos had altered levels of transcription in both reciprocal hybrids; these genes could contribute to the heterotic phenotype of the hybrid embryo. Many of the non-additively expressed genes had expression levels shifted towards maternal levels of transcription and these differed in the reciprocal hybrids. Allelic expression analysis indicated that most genes with altered allelic contributions in the hybrids had an increase in the expression level of the hybrid's maternal allele. Consistent with the maternal pattern of gene expression, embryo and seed also show maternally influenced phenotypes. This article is protected by copyright. All rights reserved.
MicroRNAs (miRNAs) play key roles in tumor development and progression. The aim of this study was to explore the expression levels of miR-34a and miR-217 in hepatocellular carcinoma (HCC) and to further investigate the clinicopathological and prognostic value of miR-34a and miR-217.
The expression levels of miR-34a and miR-217 were evaluated using quantitative real-time PCR (qRT-PCR). Associations between these miRNAs expression and clinicopathological features were analyzed. Survival rate was determined with Kaplan-Meier and statistically analyzed with the log-rank method between groups.
We found that miR-34a expression was significantly downregulated in HCC tissues (P<0.05). Reduced expression of miR-34a was associated with vascular invasion, and advanced TNM stage (P<0.05). Kaplan-Meier revealed that reduced expression of miR-34a was associated with poor overall survival (log-rank test, P<0.05). We found that miR-217 was downregulated in HCC tissues. Decreased expression of miR-217 was remarkably correlated vascular invasion, and advanced TNM stage (P<0.05). Kaplan-Meier analysis and log-rank test showed that HCC patients with low expression of miR-217 was associated with shorter overall survival than patients with high expression (log-rank test, P<0.05).
Our data showed that downregulation of miR-34a and miR-217 was associated with HCC progression and both of them may act as tumor suppressor in HCC.
Carbon-based nanomaterials serve as a type of smart material for photo-triggered disease theranostics. The inherent physicochemical properties of these nanomaterials facilitate their use for less invasive treatments. This review summarizes the properties and applications of materials including fullerene, nanotubes, nanohorns, nanodots and nanographenes for photodynamic nanomedicine in cancer and antimicrobial therapies. Carbon nanomaterials themselves do not usually act as photodynamic therapy (PDT) agents owing to the high hydrophobicity, however, when the surface is passivated or functionalized, these materials become great vehicles for PDT. Moreover, conjugation of carbonaceous nanomaterials with the photosensitizer (PS) and relevant targeting ligands enhances properties such as selectivity, stability, and high quantum yield, making them readily available for versatile biomedical applications.
Surgical repair of orbital fractures with implants is a widely used treatment modality. While a variety of established complications are associated with this technique, most are directly understood and treated. Bloody epiphora is a finding with potentially ominous causes. The authors present a unique case of bloody epiphora, accompanied by orbital hemorrhage arising several years after orbital floor fracture repair, due to erosion of the nasolacrimal drainage system by a displaced implant combined with anticoagulation. The collection and evaluation of all protected patient health information was compliant with the regulations and conditions set forth in the Health Insurance Portability and Availability Act of 1996.
Novel carbazole aminoalcohols were designed and synthesized as anticancer agents. Among them, alkylamine-chain-substituted compounds showed the most promising antiproliferative activity, with IC50 values in the single-digit micromolar range against two human tumor cell lines. Topoisomerase I (topo I) is likely to be one of the targets of these compounds. Results of comet assays and molecular docking indicate that the representative compounds may act as topo I poisons, causing single-strand DNA damage by stabilizing the topo I-DNA cleavage complex. In particular, the most potent compound, 1-(butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (6), was shown to be able to induce G2 -phase cell-cycle arrest and apoptosis in HeLa cells.
Drotaverine (also known as dihydroperparine or No-Spa(®) ) is an antispasmodic drug closely related to papaverin. Drotaverin also acts as a cytostatic compound for several human tumor cell lines and nonmalignant mouse fibroblasts, and EC50 values as low as 3.0 μM were observed in SRB assays for HT-29 human colorectal carcinoma cells. Small structural changes (e.g., aromatization, benzylic oxidation) led to a reduced activity or a complete loss of cytotoxicity. Staining of the cells with acridine orange showed the cell membrane of the dead cells to be still intact, and a slight G1/G0 arrest in the treated cells was observed after 24 h. Extra annexin V-FITC/PI assays and flow cytometry revealed drotaverine mainly to act as a cytostatic and only to a minor extent as cytotoxic agent.
The recalcitrance of lignocellulose forms a strong barrier for the bioconversion of lignocellulosic biomass in chemical or biofuel industries. Filamentous fungi are major plant biomass decomposer, and capable of forming all the required enzymes. Here, we characterized the GH10 and GH11 endo-xylanases and a CE1 acetyl-xylan esterase (Axe1) from a superior biomass-degrading strain, Aspergillus fumigatus Z5, and examined how they interact in xylan degradation. Cellulose-binding (CBM1) domain inhibited GH10 xylanase activities for pure xylan, but afforded them an ability to hydrolyze washed corncob particles (WCCP). CBM1-containing GH10 xylanases also showed synergism with CBM1-containing Axe1 in WCCP hydrolysis, and this synergy was strictly dependent on the presence of their CBM1 domains. In contrast, GH11 xylanases had no CBM1, but still could bind xylan and hydrolyzed WCCP, however, no synergism displayed with Axe1. GH10 xylanases and GH11 xylanases showed a pronounced synergism in WCCP hydrolysis, which was dependent on the presence of the CBM1 in GH10 xylanases and absence from GH11 xylanases. They exhibit different mechanisms to bind to cellulose and xylan, and act in synergy when these two structures are intact. These findings will be helpful for the further development of highly efficient enzyme mixtures for lignocellulosic biomass conversion. This article is protected by copyright. All rights reserved.
Extracted human teeth have been used to practice operative techniques for a very long time. As a natural surrogate for a live tooth in vivo, their use has traditionally been very important for the development of skills in trainee dentists, as well as their qualified colleagues who wish to practice existing or new skills. As synthetic alternatives develop greater authenticity, alongside a society in which many retain their natural dentition well into old age, the current paradigm relating to how extracted teeth in dental education are used needs to be re-visited. An ethical and legal dilemma that must be addressed within dental education relates to where and how teeth might be sourced. This article will seek to question whether there is a legal or ethical requirement to gain consent for the use of extracted teeth from patients, as well as exploring the status of whether extracted dental tissue can be considered to be the property of either patient or surgeon. Whilst synthetic alternatives are being utilised more frequently in education, it is unlikely that they will completely replace extracted natural teeth in the immediate future. It is therefore imperative that their use complies with legal doctrine and contemporary ethical thought. This article is protected by copyright. All rights reserved.
Recent findings on holdfast development in the giant kelp highlighted its key importance for Macrocystis vegetative propagation. We report here for the first time the development of adventitious holdfasts from Macrocystis stipes. Swellings emerge spontaneously from different areas of the stipes, especially in senescent or creeping individuals. After being manually fastened to solid substrata, these swellings elongated into haptera, which became strongly attached after one month. Within 4 months new thalli increased in size and vitality, and developed reproductive fronds. Our results suggest the usage of these structures for auxiliary attachment techniques. These could act as a backup, when primary holdfasts are weak, and thus improve the survival rate of the giant kelp in natural beds. This article is protected by copyright. All rights reserved.
The U.S. hospital industry has recently witnessed a number of policy changes aimed at aligning hospital payments to costs and these can be traced to significant concerns regarding selection of profitable patients and procedures by physician-owned specialty hospitals. The policy responses to specialty hospitals have alternated between payment system reforms and outright moratoriums on hospital operations including one in the recently enacted Affordable Care Act. A key issue is whether physician-owned specialty hospitals pose financial strain on the larger group of general hospitals through cream-skimming of profitable patients, yet there is no study that conducts a systematic analysis relating such selection behavior by physician-owners to financial impacts within hospital markets. The current paper takes into account heterogeneity in specialty hospital behavior and finds some evidence of their adverse impact on profit margins of competitor hospitals, especially for-profit hospitals. There is also some evidence of hospital consolidation in response to competitive pressures by specialty hospitals. Overall, these findings underline the importance of the payment reforms aimed at correcting distortions in the reimbursement system that generate incentives for risk-selection among providers groups. The identification techniques will also inform empirical analysis on future data testing the efficacy of these payment reforms.
This paper examines the impact of local (county-level) house prices on individual self-reported mental health using individual level data from the United States Behavioral Risk Factor Surveillance System between 2005 and 2011. Exploiting a fixed-effects model that relies on within-county variations, relative to the corresponding changes in other counties, I find that while individuals are likely to experience worse self-reported mental health when local house prices decline, this association is most pronounced for individuals who are least likely to be homeowners. This finding is not consistent with a prediction from a pure wealth mechanism but rather with the hypothesis that house prices act as an economic barometer. I also demonstrate that the association between self-reported mental health and local house prices is not driven by unemployment or foreclosure. The primary result-that lower local house prices have adverse impact on self-reported mental health of homeowners and renters-is consistent with studies using data from the United Kingdom.
To investigate the ability of the proteases, subtilisin and α-chymotrypsin (aCT), to inhibit the adhesion of Candida albicans biofilm to a polypropylene surface.
The proteases were immobilized on plasma-treated polypropylene by covalently linking them with either glutaraldehyde (GA) or N'-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS). The immobilization did not negatively affect the enzyme activity and in the case of subtilisin, the activity was up to 640% higher than that of the free enzyme when using N-acetyl phenylalanine ethyl ester as the substrate. The efficacies against biofilm dispersal for the GA-linked SubC and aCT coatings were 41 and 55% higher than the control (polypropylene coated with only GA), respectively, whereas no effect was observed with enzymes immobilized with DIC and NHS. The higher dispersion efficacy observed for the proteases immobilized with GA could be both steric (proper orientation of the active site) and dynamic (higher protein mobility/flexibility).
Proteases immobilized on a polypropylene surface reduced the adhesion of C. albicans biofilms and therefore may be useful in developing anti-biofilm surfaces based on non-toxic molecules and sustainable strategies.
Small RNA-mediated gene silencing encompasses diverse developmental events, stress responses, defense against pathogens, and maintenance of genome integrity. Extensive studies in model organisms have unveiled the molecular mechanisms underpinning the RNA silencing phenomena, and the accumulating knowledge have characterized the intricate pathways and the repertoire of proteins responsible for the actions of small RNAs characterized as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Although the single-stranded, matured guide small RNAs direct the effector ribonucleoprotein complexes to induce gene silencing in sequence-specific manner, the double-stranded intermediate, the small RNA duplexes, which are processed as nascent products of the RNase III enzyme activities, act as key to determine the downstream molecular pathways and the fate of small RNAs. Based at the small RNA duplex-centered view, this review describes the recent advances in understanding the small RNA pathways in plants.
Since 2004 a single varicella vaccination for all infants aged 11-14 months has been recommended in Germany and since 2009 a second dose at the age of 15-23 months is recommended. Vaccination coverage after 24 months rose from 43% in 2006 to 87.5% in 2012. A mandatory notification system was introduced in the New Federal States (NFS) between 2002 and 2009 and nationwide in 2013. A national sentinel system has been in place since 2005.
We analyzed both data sources to describe the varicella epidemiology related to vaccination coverage after initiation of routine childhood varicella vaccination and to evaluate both notification systems regarding informative value and data quality. We looked at trends, age distribution and incidences using Microsoft Excel and Stata12. Vaccination coverage data were available from health insurance claims data.
By 2013 a decrease of cases/medical practice/month from 3.47 to 0.43 was observed. The incidence in the NFS declined from 32 to 12 out of 100,000. Sentinel and mandatory notification data showed the largest decrease among the 1-4 year-olds (-94 and -90% resp.). In 2014, varicella incidences increased in all age groups, but not the cases/medical-practice/month by age in the sentinel.
Increasing vaccination coverage and decreasing varicella cases demonstrate the success of routine childhood varicella vaccination. Mandatory notification data allow incidence calculation; The sentinel system has been providing more detailed information about vaccination status, better data quality and continuous national data since 2005, irrespective of the Infectious disease protection act. Trends and age distribution can be continuously calculated, whereas the nationwide mandatory data collected in the short period since April 2013 can only be evaluated to a limited extent.
To report the impact of an inpatient anticoagulation stewardship program at a community hospital to promote optimal anticoagulant use. The anticoagulation team (ACT) stewardship program consists of two clinical pharmacists and hematologists to provide oversight of anticoagulants, high cost reversal agents including prothrombin complex concentrate (PCC, Kcentra™), and heparin-induced thrombocytopenia (HIT) management. Intervention data and number of charts reviewed were collected. Average cost avoidance data was applied to ACT interventions to estimate cost savings. The PCC analysis was conducted via retrospective chart review during the pre-intervention period. Prospective monitoring continued in the post-intervention period to determine the percentage of PCC use within the institution's guidelines or approved by ACT or hematology. A total of 19,445 patient charts were reviewed, and 1930 (10%) contained stewardship opportunity. Of the interventions, 71% were provided to the medical service and 22% to surgical services with acceptance rates of 91 and 83%, respectively. Intervention cost-avoidance calculated to be $694,217. Regarding HIT interventions, 52% of interventions involved pharmacokinetic/pharmacodynamics optimization in 18 patients with suspected or confirmed HIT. Regarding PCC use, 55.8% of PCC orders were considered inappropriate in the pre-invention period versus 2.6% post-intervention. Appropriate PCC doses per month post-intervention were consistent with pre-intervention doses (7.67 vs. 6.73, respectively). The projected annual PCC cost savings is $385,473. The overall estimated financial impact of ACT is $799,690 saved. Implementation of an anticoagulation stewardship program reduced costs and improved clinical outcomes. It is also expected that anticoagulant optimization and provider education improved overall safety.
Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is modified by cyclooxygenases into active compounds called eicosanoids that act as signaling molecules in a number of physiological processes. Excessive production of eicosanoids leads to several pathological conditions such as inflammation. In order to block the inflammatory effect of these compounds, upstream enzymes such as PLA2 are valid targets. In the present contribution, molecular dynamic analysis was performed to evaluate the binding of diclofenac, 9-hydroxy aristolochic acid (9-HAA) and indomethacin to PLA2. Obtained results revealed that 9-HAA could form a more stable complex with PLA2 when compared to diclofenac and indomethacin. Furthermore, analysis of intermolecular binding energy components indicated that hydrophobic interactions were dominant in binding process. On the basis of obtained data, inhibitors bearing fused rings with hydrogen acceptor/donor substituent(s) interacted with His48 and Asp49 residues of the active site. More affinity toward PLA2 might be envisaged through negatively charged moieties via interaction with Trp31, Lys34 and Lys69.
Long non-coding RNAs (lncRNAs) have been demonstrated to act as a critical regulator in the processes of tumor biology. In this study, whether lncRNA-ATB is a potential indicator for non-small cell lung cancer (NSCLC) was investigated and its biological function in NSCLC was also determined.
The expression levels of lncRNA-ATB in NSCLC tissues and cell lines were measured. A549 cell line was explored to investigate the functions of lncRNA-ATB in NSCLC.
Real-time PCR results showed that lncRNA-ATB expression was up-regulated in both in NSCLC tissues and cell lines. High lncRNA-ATB expression in tumor tissue was associated with larger tumor size, lymph node metastasis, and distant metastasis in patients with NSCLC, respectively. In addition, the patients with high expression of lncRNA-ATB presented a lower survival probability. In vitro experiments showed that down-regulation of lncRNA-ATB promoted the cell apoptosis, whereas inhibited the cell viability, cell migration, and cell invasion.
High expression of lncRNA-ATB indicated a poor prognosis and led to the cell proliferation and metastasis in NSCLC.
Dinitrosyl iron complexes (DNICs) are physiological NO derivatives and account for many NO functions in biology. Polyfunctional dipeptide carnosine (beta-alanyl-L-histidine) is considered to be a very promising pharmacological agent. It was shown that in the system containing carnosine, iron ions and Angeli's salt, a new type of DNICs bound with carnosine as ligand {(carnosine)2-Fe-(NO)2}, was formed. We studied how the carbonyl compound methylglyoxal influenced this process. Carnosine-bound DNICs appear to be one of the cell's adaptation mechanisms when the amount of reactive carbonyl compounds increases at hyperglycemia. These complexes can also participate in signal and regulatory ways of NO and can act as protectors at oxidative and carbonyl stress conditions.
Human immunodeficiency virus (HIV) infection among men who have sex with men (MSM) has increased to a drastic proportion throughout India in the last couple of years due to a lack of productive identification and management framework. In apprehension of social disgrace these men attempt to live a normal hetero conjugal life and, in the process, act as a bridge in spreading the virus to their women partners. In this case report we have highlighted two cases which clearly distinguished the adequacy of HIV treatment among MSM when they are diagnosed during early or late phases of infection. An intensive and ample counseling to comprehend the psychology and sexual behavior of these men was found to be critically important in both the cases. Our study, which is actually the first of its kind, recorded and documented evidence of HIV infected MSM from Eastern India and renders a ray of hope among this marginally isolated group to comprehend the challenges and health risks faced by the MSM population. It also provides a format for the medical practitioners here in managing and treating related cases.
We investigated the interacting impacts of urban landscape and gardening practices on the species richness and total abundance of communities of common butterfly communities across France, using data from a nationwide monitoring scheme. We show that urbanization has a strong negative impact on butterfly richness and abundance but that at a local scale, such impact could be mitigated by gardening practices favoring nectar offer. We found few interactions among these landscape and local scale effects, indicating that butterfly-friendly gardening practices are efficient whatever the level of surrounding urbanization. We further highlight that species being the most negatively affected by urbanization are the most sensitive to gardening practices: Garden management can thus partly counterbalance the deleterious effect of urbanization for butterfly communities. This holds a strong message for park managers and private gardeners, as gardens may act as potential refuge for butterflies when the overall landscape is largely unsuitable.
CuAlO2 has been examined as a potential luminescent material by substituting Eu for Al cations in the delafossite structure. CuAlO2:Eu(3+) nanofibers have been prepared via electrospinning for the ease of mitigating synthesis requirements and for future optoelectronics and emerging applications. Single-phase CuAlO2 fibers could be obtained at a temperature of 1100 °C in air. The Eu was successfully doped in the delafossite structure and two strong emission bands at ~405 and 610 nm were observed in the photoluminescence spectra. These bands are due to the intrinsic near-band-edge transition of CuAlO2 and the f-f transition of the Eu(3+) activator, respectively. Further electrical characterization indicated that these fibers exhibit semiconducting behavior and the introduction of Eu could act as band-edge modifiers, thus changing the thermal activation energies. In light of this study, CuAlO2:Eu(3+) fibers with both strong photoluminescence and p-type conductivity could be produced by tailoring the rare earth doping concentrations.
Stem-chicory of the "Catalogna" group is a vegetable consumed for bitter-flavored stems. Type and levels of bitter sesquiterpene lactones (STLs) participate in conferring bitterness in vegetables. The content of lactucin-and lactucopocrin-like STLs was higher in "Molfettese" than "Galatina" landrace stalks, regardless of the cultivation sites, consistently with bitterness scores and gustative differences. The "Galatina" transcriptome assembly resulted in 58,872 unigenes, 77% of which were annotated, paving the way to molecular investigation of the STL pathway. Comparative transcriptome analysis allowed the identification of 69,352 SNPs and of 1640 differentially expressed genes that maintained the pattern independently of the site. Enrichment analyses revealed that 4 out of 29 unigenes were up-regulated in "Molfettese" vs "Galatina" within the sesquiterpenoid pathway. The expression of two germacrene A -synthase (GAS) and one -oxidase (GAO) genes of the costunolide branch correlated positively with the contents of lactucin-like molecules, supporting that STL biosynthesis regulation occurs at the transcriptional level. Finally, 46 genes encoding transcription factors (TFs) maintained a differential expression pattern between the two varieties regardless of the growth site; correlation analyses among TFs, GAS, GAO gene expressions and STLs contents suggest that one MYB and one bHLH may act in the pathway.
Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are endowed with several different functions besides being carriers for pheromones and odorants. Based on a previous report of a CSP acting as surfactant in the proboscis of the moth Helicoverpa armigera, we revealed the presence of orthologue proteins in two other moths Plutella xylostella and Chilo suppressalis, as well as two butterflies Papilio machaon and Pieris rapae, using immunodetection and proteomic analysis. The unusual conservation of these proteins across large phylogenetic distances indicated a common specific function for these CSPs. This fact prompted us to search for other functions of these proteins and discovered that CSPs are abundantly expressed in the eyes of H. armigera and possibly involved as carriers for carotenoids and visual pigments. This hypothesis is supported by ligand-binding experiments and docking simulations with retinol and β-carotene. This last orange pigment, occurring in many fruits and vegetables, is an antioxidant and the precursor of visual pigments. We propose that structurally related CSPs solubilise nutritionally important carotenoids in the proboscis, while they act as carriers of both β-carotene and its derived products 3-hydroxyretinol and 3-hydroxyretinal in the eye. The use of soluble olfactory proteins, such as CSPs, as carriers for visual pigments in insects, here reported for the first time, parallels the function of retinol-binding protein in vertebrates, a lipocalin structurally related to vertebrate odorant-binding proteins.
Keloids and hypertrophic scars are the most common types of pathological scarring. Traditionally, keloids have been considered as a result of aberrant wound healing, involving excessive fibroblast participation that is characterized by hyalinized collagen bundles. However, the usefulness of this characterization has been questioned. In recent years, studies have reported the appropriate use of verapamil for keloids and hypertrophic scars.
Searches were conducted on the databases Medline, Embase, Cochrane, PubMed, and China National Knowledge Infrastructure from 2006 to July 2016. State12.0 was used for literature review, data extraction, and meta-analysis. Treatment groups were divided into verapamil and nonverapamil group. Nonverapamil group includes steroids and intense pulsed light (IPL) therapy. Total effective rates include cure rate and effective rate. Cure: skin lesions were completely flattened, became soft and symptoms disappeared. Efficacy: skin lesions subsided, patient significantly reduced symptoms. Inefficient definition of skin was progression free or became worse. Random-effects model was used for the meta-analysis.
Six studies that included 331 patients with keloids and hypertrophic scars were analyzed. Analysis of the total effective rate of skin healing was performed. The total effective rates in the two groups were 54.07% (verapamil) and 53.18% (nonverapamil), respectively. The meta-analysis showed that there was no difference between the two groups. We also compared the adverse reactions between the verapamil treatment group and the steroids treatment group in two studies, and the result indicated that the verapamil group showed less adverse reactions.
There were no differences between the application of verapamil and nonverapamil group in keloids and hypertrophic scars treatment. Verapamil could act as an effective alternative modality in the prevention and treatment of keloid and hypertrophic scars. A larger number of studies are required to confirm our conclusion.
Current treatment of inflammatory bowel disease is based on the use of immunosuppressants or anti-inflammatory drugs, which are characterized by important side effects that can limit their use. Previous research has been performed by administering these drugs as nanoparticles that target the ulcerated intestinal regions and increase their bioavailability. It has been reported that silk fibroin can act as a drug carrier and shows anti-inflammatory properties.
This study was designed to enhance the interaction of the silk fibroin nanoparticles (SFNs) with the injured intestinal tissue by functionalizing them with the peptide motif RGD (arginine-glycine-aspartic acid) and to evaluate the intestinal anti-inflammatory properties of these RGD-functionalized silk fibroin nanoparticles (RGD-SFNs) in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis.
SFNs were prepared by nanoprecipitation in methanol, and the linear RGD peptide was linked to SFNs using glutaraldehyde as the crosslinker. The SFNs (1 mg/rat) and RGD-SFNs (1 mg/rat) were administered intrarectally to TNBS-induced colitic rats for 7 days.
The SFN treatments ameliorated the colonic damage, reduced neutrophil infiltration, and improved the compromised oxidative status of the colon. However, only the rats treated with RGD-SFNs showed a significant reduction in the expression of different pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and IL-12) and inducible nitric oxide synthase in comparison with the TNBS control group. Moreover, the expression of both cytokine-induced neutrophil chemoattractant-1 and monocyte chemotactic protein-1 was significantly diminished by the RGD-SFN treatment. However, both treatments improved the intestinal wall integrity by increasing the gene expression of some of its markers (trefoil factor-3 and mucins).
SFNs displayed intestinal anti-inflammatory properties in the TNBS model of colitis in rats, which were improved by functionalization with the RGD peptide.
Screening of 83 mature wild rodents of Bandicota bengalensis (52 male and 31 females), live trapped from premises near railway station, fish market and agricultural fields in Ludhiana district of Punjab province, India was carried out to assess the natural occurrence of helminth parasites and their potential as reservoirs of zoonoses. On necropsy, intestine of rats was found infected with adult cestode parasites of Hymenolepis diminuta and H. nana. Liver was found infected with Capillaria hepatica and Cysticercus fasciolaris. These parasites were present either alone or as concurrent infection. In overall, 68.67 % rats were found infected of which 59.65 % were infected with nematode parasite, C. hepatica and 75.44 % with cestode parasites. Grossly, pathognomonic lesions of C. hepatica and C. fasciolaris were seen clearly and microscopic changes were observed only in severely infected cases. Overall investigation on the effect of these parasitic infections on pathophysiology of the host revealed that rodents serve as reservoirs of these parasitic infections without having much pathogenic effect on their vital organs, fecundity and enzyme activities. Because of its close association with human habitations, B. bengalensis may act as an important source of zoonotic infections and thus direct or indirect contact with excrements and carcasses of this rodent species, should be avoided.
Gastrointestinal parasites are among the most common parasitic infections found in stray cats, which might act potential helminthic parasites to domestic cats. The objective of this study was to determine the prevalence of gastrointestinal parasites in stray cats in the city of Azarshahr, which is located in East Azerbaijan province, Iran. A cross-sectional study was conducted on 50 necropsied stray cats, trapped and collected from different geographic regions of Azarshahr. From a total 50 stray cats examined, 15 (30 %) were female and 35 (70 %) were male. Overall 47 cats (94 %) were identified as infected with at least one of the endoparasites. The prevalence of parasites found were: Taenia taeniaeformis (60 %), Dipylidium caninum (58 %), Taenia hydatigera (24 %), Mesocestoides lineatus (78 %), Ancylostoma tubaeforme (14 %), Toxascaris leonina (30 %), Toxocara cati (78 %), Physaloptera praeputialis (10 %), and Syphacia obvelata (10 %). Contamination rate for zoonotic parasites of cat was greater than expected in AzarShahr region. Therefore, appropriate control measures should be taken and preventive methods should be applied.
Sodium deficit poses a life-threatening challenge to body fluid homeostasis and generates a sodium appetite - the behavioral drive to ingest sodium. Dr. Randall R. Sakai greatly contributed to our understanding of the hormonal responses to negative sodium balance and to the central processing of these signals. Reactivity to the taste of sodium solutions and the motivation to seek and consume sodium changes dramatically with body fluid balance. Here, we review studies that collectively suggest that sodium deficit recruits the mesolimbic system to play a role in the behavioral expression of sodium appetite. The recruitment of the mesolimbic system likely contributes to intense sodium seeking and reinforces sodium consumption observed in deficient animals. Some of the hormones that are released in response to sodium deficit act directly on both dopamine and nucleus accumbens elements. Moreover, the taste of sodium in sodium deficient rats evokes a pattern of dopamine and nucleus accumbens activity that is similar to responses to rewarding stimuli. A very different pattern of activity is observed in non-deficient rats. Given the well-characterized endocrine response to sodium deficit and its central action, sodium appetite becomes an ideal model for understanding the role of mesolimbic signaling in reward, reinforcement and the generation of motivated behavior.
Strenuous exercise is followed by an elevation of many cytokines with inflammation regulating properties. Since most cytokines act at pico- or nanomolar concentrations many investigations failed to detect their concentrations in vivo. Hence, the aim of this study was to evaluate the significance of cytokine measurements (IL-1β, TNF-α, IL-1ra, IL-6, CCL2 and CXCL8) in a stimulated whole-blood culture (sWBC) compared to serum with respect to their exercise-induced kinetics and detection rates. 40 male volunteers (age: 25,5±4,3years, BMI: 24,00±2,24, VO2peak: 46,9±4,1mL/kg×min) performed 60min of intensive bicycle exercise (80% VO2peak). Blood samples were taken before and for up to 24h after exercise. All cytokines were determined by a multiplex ELISA. There were weak to moderate correlations between cytokines in sWBC and serum. While exercise did not affect pro-inflammatory cytokines in serum, in sWBC only IL-1β was increased 1.2-fold at 3h (p<0,05). All other cytokines increased both in sWBC and serum. The detection rate was superior in sWBC vs serum for most cytokines. Exercise-induced cytokine kinetics in sWBC do not reflect systemic changes. Both approaches provide a synergistic insight into inflammatory processes on the cytokine level.
Water Quality (WQ) condition is based on ecosystem stressor indicators (e.g. water clarity) which are biogeochemically important and critical when considering the Deepwater Horizon oil spill restoration efforts under the 2012 RESTORE Act. Nearly all of the proposed RESTORE projects list restoring WC as a goal, but 90% neglect water clarity. Here, dynamics of optical constituents impacting clarity are presented from a 2009-2011 study within Pensacola, Choctawhatchee, St. Andrew and St. Joseph estuaries (targeted RESTORE sites) in Northwest Florida. Phytoplankton were the smallest contribution to total absorption (at-wPAR) at 412nm (5-11%), whereas colored dissolved organic matter was the largest (61-79%). Estuarine at-wPAR was significantly related to light attenuation (KdPAR), where individual contributors to clarity and the influence of climatic events were discerned. Provided are conversion equations demonstrating interoperability of clarity indicators between traditional State-measured WQ measures (e.g. secchi disc), optical constituents, and even satellite remote sensing for obtaining baseline assessments.
Before the 2009 Biologics Price Competition and Innovation Act that enabled the U.S. Federal Drug Administration (FDA) to create the 351(k) Biologic License Application-an abbreviated biosimilar approval process, FDA approved follow-on biomolecule products such as beta-interferon, glucagon, hyaluronidase, and somatropin (human growth hormone) under varying and evolving rules. With the 351(k) Biologic License Application biosimilar approval process in place, currently, there are 4 (licensed in 2015-2016) biosimilars available, namely Neupogen (filgrastim; $1 B/y), Humira (adalumumab; $14.2 B/y), Enbrel (etanercept; $8.7 B/y), and Remicade (infliximab; $6.5 B/yr). With well-established product market capitalization of these and other top income producers-such as Rituxan (rituximab; $6.8 B/y), Herceptin (trastuzumab; $6.5 B/y), and Avastin (bevacizumab; $5.8 B/y), and a price differential of 15%-30% compared to branded products, there is an intense interest in development of biosimilars by established pharmaceutical companies. Currently, there are 160 biosimilar candidates in clinical studies, many of which are sponsored by large pharmaceutical companies known for product innovation. This trend will likely continue. Additional information on a biomolecule platform is presented in the Journal of Pharmaceutical Sciences Drug Delivery Clinical Trials Database (jpharmscidatabase.org). There are 44,789, 18,456, and 12,897 clinical trials registered to evaluate (1) drug delivery technology, (2) biomolecule platform, and (3) drug metabolism and pharmacokinetic-pharmacodynamic interactions; representing 19%-60% increase over the last 3 years.
In November 2005, hepatitis A vaccine was funded under the Australian National Immunisation Program for Aboriginal and Torres Strait Islander (Indigenous) children aged 12-24months in the targeted jurisdictions of Queensland, South Australia, Western Australia and the Northern Territory. We reviewed the epidemiology of hepatitis A from 2000 to 2014 using data from the Australian National Notifiable Diseases Surveillance System, the National Hospital Morbidity Database, and Australian Bureau of Statistics causes-of-death data. The impact of the national hepatitis A immunisation program was assessed by comparison of pre-vaccine (2000-2005) and post-vaccine time periods (2006-2014), by age group, Indigenous status and jurisdiction using incidence rate ratios (IRR) per 100,000 population and 95% confidence intervals (CI). The national pre-vaccine notification rate in Indigenous people was four times higher than the non-Indigenous rate, and declined from 8.41 per 100,000 (95% CI 5.03-11.79) pre-vaccine to 0.85 per 100,000 (95% CI 0.00-1.99) post-vaccine, becoming similar to the non-Indigenous rate. Notification and hospitalisation rates in Indigenous children aged <5years from targeted jurisdictions declined in the post-vaccine period when compared to the pre-vaccine period (notifications: IRR=0.07; 95% CI 0.04-0.13; hospitalisations: IRR=0.04; 95% CI 0.01-0.16). As did notification rates in Indigenous people aged 5-19 (IRR=0.08; 95% CI 0.05-0.13) and 20-49years (IRR=0.06; 95% CI 0.02-0.15) in targeted jurisdictions. For non-Indigenous people from targeted jurisdictions, notification rates decreased significantly in children aged <5years (IRR 0.47; 95% CI 0.31-0.71), and significantly more overall (IRR=0.43; 95% CI 0.39-0.47) compared to non-Indigenous people from non-targeted jurisdictions (IRR=0.60; 95% CI 0.56-0.64). The national hepatitis A immunisation program has had a significant impact in the targeted population with relatively modest vaccine coverage, with evidence suggestive of substantial herd protection effects.
To investigate whether clinical or laboratory variables on admission of patients with odontogenic infections are associated with a severe clinical course and a prolonged hospital stay, we hypothesised that specific factors such as the serum concentration of C-reactive protein (CRP) may act as predictors of the duration of stay. We designed a prospective patient-oriented study that included all those treated for maxillofacial infections of odontogenic origin in the Oral and Maxillofacial Surgery Department of Northampton General Hospital between November 2013 and December 2014. A total of 71 were enrolled. We found that the concentration of CRP was a significant predictor of hospital stay (p=0.01). Its measurement on admission can predict the likely duration of stay of these patients and enable beds to be managed more efficiently.
Recent studies based on biography analysis provide support for the notion that the prevalence of mental illness in the creative geniuses of art, literature and science is higher than it is in more ordinary folk. However, this relationship between madness and genius, which was also addressed by the classical philosophers, has been generalized to all branches of professional endeavour. Whilst it may hold true for illustrious personalities of the fine arts, we found that the relationship proves inappropriate to the biographies of ten individuals renowned in history for their innovative contributions to medical science. Furthermore, examination of these ten biographies invites the hypothesis that certain personality traits - especially, agreeableness, conscientiousness and openness to new experience - can act to enhance creativity and protect against mental illness.
Excessive dermal scarring (EDS) is a wound healing complication, characterized by protruded erythematous and inelastic 'proliferative scar tissue' which is associated with increased and prolonged inflammation process within the wound microenvironment. As inflammation plays a key role in this process, methods to contain or attenuate excessive inflammation hold promise in treatment and prophylaxis of EDS conditions. While cold exposure is notorious as the causative agent a wide array of morbidities and fatalities, its tempered use is exploited in medicine for ablative and therapeutic applications. "Subphysiological cold" has been administered for its antiinflammatory effects which act via decreasing vascular permeability and downregulating proliferation of cells in the wound environment; this knowledge supports our hypothesis that "subphysiological cold application" can also be utilized in human EDS prophylaxis and treatment. In this study, we are reviewing the mechanisms of its both deleterious and therapeutic actions and suggesting another possible application for prevention and/or treatment of human EDS conditions.
The treatment and prevention of migraine within the last decade has become largely pharmacological. While there is little doubt that the advent of drugs (e.g. triptans) has helped many migraine sufferers to lead a normal life, there is still little knowledge with respect to the factors responsible for precipitating a migraine attack. Evidence from biochemical and behavioural studies from a number of disciplines is integrated to put forward the proposal that migraine is part of a cascade of events, which together act to protect the organism when confronted by a metabolic challenge.
The cotton bollworm, Helicoverpa armigera, is a major pest of many agricultural crops in several countries, including Australia. Transgenic cotton, expressing a single Bt toxin, was first used in the 1990s to control H. armigera and other lepidopteran pests. Landscape scale or greater pest suppression has been reported in some countries using this technology. However, a long-term, broad-scale pheromone trapping program for H. armigera in a mixed cropping region in eastern Australia caught more moths during the deployment of single Bt toxin cotton (Ingard®) (1996-2004) than in previous years. This response can be attributed, at least in part, to (1) a precautionary cap (30% of total cotton grown, by area) being applied to Ingard® to restrict the development of Bt resistance in the pest, and (2) during the Ingard® era, cotton production greatly increased (as did that of another host plant, sorghum) and H. armigera (in particular the 3rd and older generations) responded in concert with this increase in host plant availability. However, with the replacement of Ingard® with Bollgard II® cotton (containing two different Bt toxins) in 2005, and recovery of the cotton industry from prevailing drought, H. armigera failed to track increased host-plant supply and moth numbers decreased. Greater toxicity of the two gene product, introduction of no cap on Bt cotton proportion, and an increase in natural enemy abundance are suggested as the most likely mechanisms responsible for the suppression observed.
Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution.
Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed.
One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes.
Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.
Prenatal diagnosis involves methods used in early pregnancy as either screening tests or diagnostic methods. The aims of the study were to i) investigate guidelines on prenatal diagnosis in the counties of Sweden, ii) investigate uptake of prenatal diagnosis, and iii) background characteristics and pregnancy outcomes in relation to different prenatal diagnostic methods.
A retrospective cross-sectional study using data from the Swedish Pregnancy Register 2011 to 2013 (284,789 pregnancies) was performed. Additionally, guidelines on prenatal diagnosis were collected. Biostatistical and epidemiological analyses were performed including calculation of odds ratios (OR) and their 95% confidence intervals in univariate and multivariate logistic regression analyses.
The national uptake of routine ultrasound examination, Combined Ultrasound and Biochemical test (CUB), Amniocentesis (AC) and Chorionic Villus Sampling (CVS) were 97.6, 33.0, 2.6 and 1.1%, respectively. From 2012, 6/21 counties offered CUB test to all pregnant women, nine counties at specific indications, and five counties did not offer CUB at all. Advanced maternal age demonstrated the highest impact on uptake of prenatal diagnosis. Further, university educational level in relation to lower educational level was associated with an increased likelihood of undergoing CUB (OR 2.30, 95% CI 2.26-2.35), AC (OR 1.54, 95% CI 1.46-1.63) and CVS (OR 2.68, 95% CI 2.44-2.93).
Offers of prenatal diagnosis varied considerably between counties resulting in unequal access to prenatal diagnosis for pregnant women. The intentions of the Swedish Health and Medical Services Act stating equal care for all, was thus not fulfilled.
Plant allelochemicals act as toxins, inhibitors of digestion, and deterrents that affect the fecundity of insects. These compounds have attracted significant research attention in recent decades, and much is known about the effects of these xenobiotic plant secondary metabolites on insect development. To date, although ecological interactions between xenobiotic plant secondary chemicals that retard insect growth have been observed in many species, it remains unclear how particular allelochemicals influence insect development in a life stage-dependent manner.
We found that 2-tridecanone can affect insect development; this effect appears similar to the symptoms induced by the physiological imbalance between juvenile and molting hormones in cotton bollworm. We later detected that a decrease in the concentration of 20-hydroxyecdysone occurred alongside the observed symptoms. We next identified the transcriptome of Helicoverpa armigera and eightdigital gene expression libraries for shading light on how 2-tridecanone retarded the development of cotton bollworm. The expression of CYP314A1, CYP315A1, CYP18A1, CYP307A1, and CYP306A1 (unigenes 16487, 15409, 40026, 41217, 35643, 16953, 8199, 13311, and 13036) were found to be induced by 2-tridecanone; these are known to be related to the biosynthesis or metabolism of 20-hydroxyecdysone. Expression analysis and RNA interference studies established that the retardant effect of 2-tridecanone on the development of cotton bollworm is mediated by P450 genes.
The candidate P450 gene approach described and exploited here is useful for identifying potential causal genes for the influence of plant allelochemicals on insect development.
Spirochetes are suspected to be linked to the genesis of neurological diseases, including neurosyphillis or neurodegeneration (ND). Impaired iron homeostasis has been implicated in loss of function in several enzymes requiring iron as a cofactor, formation of toxic oxidative species, inflammation and elevated production of beta-amyloid proteins. This review proposes to discuss the link that may exist between the involvement of Treponema spp. in the genesis or worsening of ND, and iron dyshomeostasis. Proteins secreted by Treponema can act directly on iron metabolism, with hemin binding ability (HbpA and HbpB) and iron reductase able to reduce the central ferric iron of hemin, iron-containing proteins (rubredoxin, neelaredoxin, desulfoferrodoxin metalloproteins, bacterioferritins etc). Treponema can also interact with cellular compounds, especially plasma proteins involved in iron metabolism, contributing to the virulence of the syphilis spirochetes (e.g. treponemal motility and survival). Fibronectin, transferrin and lactoferrin were also shown to be receptors for treponemal adherence to host cells and extracellular matrix. Association between Treponema and iron binding proteins results in iron accumulation and sequestration by Treponema from host macromolecules during systemic and mucosal infections.
In this paper, a novel foaming methodology consisting of turbulent mixing and thermally induced phase separation (TIPS) was used to generate scaffolds for tissue engineering. Air bubbles were mechanically introduced into a chitosan solution which forms the continuous polymer/liquid phase in the foam created. The air bubbles entrained in the foam act as a template for the macroporous architecture of the final scaffolds. Wet foams were crosslinked via glutaraldehyde and frozen at -20 °C to induce TIPS in order to limit film drainage, bubble coalescence and Ostwald ripening. The effects of production parameters, including mixing speed, surfactant concentration and chitosan concentration, on foaming are explored. Using this method, hydrogel scaffolds were successfully produced with up to 80% porosity, average pore sizes of 120 μm and readily tuneable compressive modulus in the range of 2.6 to 25 kPa relevant to soft tissue engineering applications. These scaffolds supported 3T3 fibroblast cell proliferation and penetration and therefore show significant potential for application in soft tissue engineering.
Young adults with higher trait urgency (i.e., a tendency to act rashly in response to heightened affect) may be especially vulnerable to heavy drinking. The current study examined 1) the influence of urgency on daily relations between affect and drinking to intoxication, and 2) whether urgency influenced the effectiveness of naltrexone (vs. placebo) for reducing alcohol use.
This study is a secondary analysis of data from 126 (n=40 female) heavy drinking young adults, ages 18-25, enrolled in a double-blind, 8-week clinical trial comparing brief motivational intervention and either naltrexone or placebo. Multilevel models examined whether trait urgency moderated daily relations between positive and negative affect and drinking to intoxication, measured by an estimated blood-alcohol concentration (eBAC) at or above the legal limit (≥0.08g%). Person-level interactions examined whether naltrexone was more effective than placebo at reducing the odds of eBAC≥0.08g% for individuals with higher vs. lower trait urgency.
On days of greater within-person positive or negative affect, young adults with higher urgency were more likely to drink to intoxication than those with lower urgency. Naltrexone reduced the odds of drinking to intoxication significantly more than placebo, independent of positive or negative urgency.
Although naltrexone treatment reduced drinking overall, young adults with higher trait urgency were still at increased risk for hazardous drinking following times of strong positive or negative mood. Targeted interventions are needed to reduce the risk of heavy drinking among young adults with high trait urgency.
Though only occurring rarely, synergistic interactions between chemicals in mixtures have long been a point of focus. Most studies analyzing synergistic interactions used unrealistically high chemical concentrations. The aim of the present study is to determine the threshold concentration below which proven synergists cease to act as synergists towards the aquatic crustacean Daphnia magna. To do this, we compared several approaches and test-setups to evaluate which approach gives the most conservative estimate for the lower threshold for synergy for three known azole synergists. We focus on synergistic interactions between the pyrethroid insecticide, alpha-cypermethrin, and one of the three azole fungicides prochloraz, propiconazole or epoxiconazole measured on Daphnia magna immobilization. Three different experimental setups were applied: A standard 48h acute toxicity test, an adapted 48h test using passive dosing for constant chemical exposure concentrations, and a 14-day test. Synergy was defined as occuring in mixtures where either EC50 values decreased more than two-fold below what was predicted by concentration addition (horizontal assessment) or as mixtures where the fraction of immobile organisms increased more than two-fold above what was predicted by independent action (vertical assessment). All three tests confirmed the hypothesis of the existence of a lower azole threshold concentration below which no synergistic interaction was observed. The lower threshold concentration, however, decreased with increasing test duration from 0.026±0.013μM (9.794±4.897μgL(-1)), 0.425±0.089μM (145.435±30.46μgL(-1)) and 0.757±0.253μM (249.659±83.44μgL(-1)) for prochloraz, propiconazole and epoxiconazole in standard 48h toxicity tests to 0.015±0.004μM (5.651±1.507μgL(-1)), 0.145±0.025μM (49.619±8.555μgL(-1)) and 0.122±0.0417μM (40.236±13.75μgL(-1)), respectively, in the 14-days tests. Testing synergy in relation to concentration addition provided the most conservative values. The threshold values for the vertical assessments in tests where the two could be compared were in general 1.2 to 4.7 fold higher than the horizontal assessments. Using passive dosing rather than dilution series or spiking did not lower the threshold significantly. Below the threshold for synergy, slight antagony could often be observed. This is most likely due to induction of enzymes active in metabolization of alpha-cypermethrin. The results emphasize the importance of test duration when assessing synergy, but also show that azole concentrations within the typically monitored range of up to 0.5μgL(-1) are not likely to cause severe synergy concerning Daphnia magna immobilization.
On July 1, 2012, the Illinois legislature passed the Save Medicaid Access and Resources Together (SMART) Act, which restricts adult public dental insurance coverage to emergency-only treatment. The purpose of this study was to measure the effect of this restriction on the volume, severity, and treatment costs of odontogenic infections in an urban hospital.
A retrospective cohort study of patients presenting for odontogenic pain or infection at the University of Illinois Hospital was performed. Data were collected using related International Classification of Diseases, Ninth Revision codes from January 1, 2011 through December 31, 2013 and divided into 2 cohorts over consecutive 18-month periods. Outcome variables included age, gender, insurance status, oral and maxillofacial surgery (OMS) consultation, imaging, treatment, treatment location, number of hospital admission days, and inpatient care level. Severity was determined by the presence of OMS consultation, incision and drainage, hospital admission, and cost per encounter. Hospital charges were used to compare the cost of care between cohorts. Between-patients statistics were used to compare risk factors and outcomes between cohorts.
Of 5,192 encounters identified, 1,405 met the inclusion criteria. There were no significant differences between cohorts for age (P = .28) or gender (P = .43). After passage of the SMART Act, emergency department visits increased 48%, surgical intervention increased 100%, and hospital admission days increased 128%. Patients were more likely to have an OMS consult (odds ratio [OR] = 1.42; 95% confidence interval [CI], 1.11-1.81), an incision and drainage (OR = 1.48; 95% CI, 1.13-1.94), and a longer hospital admission (P = .04). The average cost per encounter increased by 20% and the total cost of care increased by $1.6 million.
After limitation of dental benefits, there was an increase in the volume and severity of odontogenic infections. In addition, there was an escalated health care cost. The negative public health effects and increased economic impact of eliminating basic dental care show the importance of affordable and accessible preventative oral health care.
Recent media reports have drawn widespread attention to the experiences of patients who are denied reproductive services at Catholic hospitals. For some patients, such as those experiencing miscarriage, denial of appropriate treatment can lead to serious health consequences. However, many patients are unaware of the limitations on services available at religiously affiliated health care institutions. As a result, patients' ability to make informed and autonomous decisions about where to seek treatment is hindered. There are currently no federal or state laws requiring health care institutions to inform patients directly of the full scope of medical services they are unable to provide on the basis of religion or conscience. Given religious hospitals' significant role in the provision of health care in the United States, it is essential that patients be clearly informed about institutional limitations on care. This goal would be best served by adopting a federal disclosure law similar to the Patient Self-Determination Act.
Inducing apoptosis is an interesting therapeutic approach to develop drugs that act against helminthic parasites. Researchers have investigated how curcumin (CUR), a biologically active compound extracted from rhizomes of Curcuma longa, affects Schistosoma mansoni and several cancer cell lines. This study evaluates how CUR influences the induction of apoptosis and oxidative stress in couples of adult S. mansoni worms. CUR decreased the viability of adult worms and killed them. The tegument of the parasite suffered morphological changes, the mitochondria underwent alterations, and chromatin condensed. Different apoptotic parameters were determined in an attempt to understand how CUR affected adult S. mansoni worms. CUR induced DNA damage and fragmentation and increased the expression of SmCASP3/7 transcripts and the activity of Caspase 3 in female and male worms. However, CUR did not intensify the activity of Caspase 8 in female or male worms. Evaluation of the superoxide anion and different antioxidant enzymes helped to explore the mechanism of parasite death further. The level of superoxide anion and the activity of Superoxide Dismutase (SOD) increased, whereas the activity of Glutathione-S-Transferase (GST), Glutathione reductase (GR), and Glutathione peroxidase (GPX) decreased, which culminated in the oxidation of proteins in adult female and male worms incubated with CUR. In conclusion, CUR generated oxidative stress followed by apoptotic-like-events in both adult female and male S. mansoni worms, ultimately killing them.
: Nurses across all care settings and experience levels are being called upon to lead. In a 2011 report, The Future of Nursing: Leading Change, Advancing Health, the Institute of Medicine examined the ways nurses could more fully apply their knowledge of direct patient care to address the increasing demands placed on the health care system since the passage of the Affordable Care Act. The report asked: "What roles can nursing assume to address the increasing demand for safe, high-quality, and effective health care services?"Multiple variables influence a nurse's ability to assume a leadership role, and multiple barriers to these roles continue to exist. This article uses the first-person voice to share the experience of a new graduate nurse in a formal nurse residency program who found himself in a position to identify the need for, advocate for, and ultimately influence a policy change in the staffing practice of floating as it was applied to new RNs on his unit. In a retrospective analysis of the process, the new graduate RN and his former professor acting as a writing mentor developed a leadership framework for nurses called RN LEADER, which they hope will empower and guide other nurses to lead evidence-based change in their workplaces.
To summarize why there are polarized opinions regarding the management of patients with asymptomatic carotid disease and whether it is possible to identify patients who might benefit from carotid interventions.
Carotid Revascularization Endarterectomy Versus Stenting Trial and Asymptomatic Carotid Trial 1 (ACT-1) recently concluded that outcomes after carotid endarterectomy and carotid stenting were not significantly different in asymptomatic patients and that procedural risks were below the accepted 3% threshold. However, systematic reviews suggest that Carotid Revascularization Endarterectomy Versus Stenting Trial/ACT-1 results may not be generalizable into routine practice. In parallel, meta-analyses suggest that stroke rates on medical therapy may be declining, suggesting that Asymptomatic Carotid Atherosclerosis Study/Asymptomatic Carotid Surgery Trial data, which have underpinned every practice guideline since 1995, are too historical for use in 2017. A recent review has, however, identified a number of clinical/imaging features that may be associated with higher rates of stroke on medical therapy.
The majority of surgeons/interventionists are unlikely to accept radical changes in practice until new randomized trials confirm that the risk of stroke on modern medical therapy is significantly lower than that previously accepted. In the interim, it would be preferable to target interventions into a smaller cohort who present with clinical/imaging features that might render them 'higher risk for stroke' on medical therapy.
We report methyl ammonium lead iodide (MAPbI3) solar cells with an ultra-porous TiO2 electron transport layer fabricated using sequential flame aerosol and atomic layer depositions of porous and compact TiO2 layers. Flame aerosol pyrolysis allows rapid deposition of nanostructured and ultra-porous TiO2 layers that could be easily scaled-up for high-throughput low-cost industrial solar cell production. An efficiency of 13.7% was achieved with a flame-made nanostructured and ultra-porous TiO2 electrode that was coated with a compact 2 nm TiO2 layer. This demonstrates that MAPbI3 solar cells with a flame-made porous TiO2 layer can have a comparable efficiency to that of the control MAPbI3 solar cell with the well-established spin-coated porous TiO2 layer. The combination of flame aerosol and atomic layer deposition provides precise control of the TiO2 porosity. Notably, the porosity of the as-deposited flame-made TiO2 layers was 97% which was then fine-tuned down to 87%, 56% and 35% by varying the thickness of the subsequent compact TiO2 coating step. The effects of the decrease in porosity on the device performance are discussed. It is also shown that MAPbI3 easily infiltrates into the flame-made porous TiO2 nanostructure thanks to their high porosity and large pore size.
GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces. In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA-binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII. To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces. Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.
(-)-Englerin A ((-)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing Transient Receptor Potential Canonical 4 protein (TRPC4). Since these channels are Ca2+ permeable it was initially thought that the cytotoxicity arose as a consequence of Ca2+ overload. Here we show that this is not the case and that the effect of (-)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (-)-EA cytotoxicity, however, although TRPC4 was necessary for the (-)-EA-evoked Ca2+-elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca2+ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (-)-EA and (-)-EA-evoked cell death was dependent upon entry of the monovalent cation Na+. We therefore hypothesized that Na+/K+-ATPase might act protectively by counteracting the Na+ load resulting from sustained Na+ entry. Indeed, inhibition of Na+/K+-ATPase by ouabain potently and strongly increased (-)-EA-evoked cytotoxicity. The data suggest that (-)-EA achieves cancer cell cytotoxicity by inducing sustained Na+ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (-)-EA can be potentiated by Na+/K+-ATPase inhibition.
Ab initio MP2/aug'-cc-pVTZ calculations have been carried out to investigate hydrogen bonding, halogen bonding, and pnicogen bonding involving tetrahedral P4 and the FH, ClH, and FCl molecules. P4 has three unique interaction sites: at a vertex (designated the P1 atom); at an edge (the P2-P3 bond); and at the P2-P3-P4 face. The uniqueness of molecular P4 is its ability to act as an electron donor and an electron acceptor at the same site, except for the P2-P3 bond, which is only an electron donor. FCl and FH form five different complexes with P4, but ClH forms only three. The type of complex formed and its binding energy depend on both the interaction site of molecular P4 and the interacting molecule. For all complexes with FH, ClH, and FCl, the binding energies at a given site with the P4 molecule acting as the base are greater than the binding energies when P4 is the acid. Thus, P4 is a better electron donor than an electron acceptor. Charge-transfer interactions and EOM-CCSD spin-spin coupling constants across hydrogen, halogen, and pnicogen bonds are reported for all of the P4 complexes. Relative to (1)J(Pi-Pj) in molecular P4, (1)J(P1-P2) coupling constants decrease in absolute value and (1)J(P2-P3) coupling constants increase in pnicogen-bonded complexes and the complex with FCl that has a PF halogen bond. Absolute values of (1)J(P1-P2) increase and those of (1)J(P2-P3) decrease in hydrogen-bonded complexes and complexes with PCl halogen bonds. (1)J(P1-P2) and (1)J(P2-P3) exhibit a single linear correlation with the corresponding Pi-Pj distances.
The network structure of biological systems suggests that effective therapeutic intervention may require combinations of agents that act synergistically. However, a dearth of systematic chemical combination datasets have limited the development of predictive algorithms for chemical synergism. Here, we report two large datasets of linked chemical-genetic and chemical-chemical interactions in the budding yeast Saccharomyces cerevisiae. We screened 5,518 unique compounds against 242 diverse yeast gene deletion strains to generate an extended chemical-genetic matrix (CGM) of 492,126 chemical-gene interaction measurements. This CGM dataset contained 1,434 genotype-specific inhibitors, termed cryptagens. We selected 128 structurally diverse cryptagens and tested all pairwise combinations to generate a benchmark dataset of 8,128 pairwise chemical-chemical interaction tests for synergy prediction, termed the cryptagen matrix (CM). An accompanying database resource called ChemGRID was developed to enable analysis, visualisation and downloads of all data. The CGM and CM datasets will facilitate the benchmarking of computational approaches for synergy prediction, as well as chemical structure-activity relationship models for anti-fungal drug discovery.
Natural Killer (NK) cells are essential for control of viral infection and cancer. NK cells express NKG2D, an activating receptor that directly recognizes NKG2D ligands. These are expressed at low level on healthy cells, but are induced by stresses like infection and transformation. The physiological events that drive NKG2D ligand expression during infection are still poorly understood. We observed that the mouse cytomegalovirus encoded protein m18 is necessary and sufficient to drive expression of the RAE-1 family of NKG2D ligands. We demonstrate that RAE-1 is transcriptionally repressed by histone deacetylase inhibitor 3 (HDAC3) in healthy cells, and m18 relieves this repression by directly interacting with Casein Kinase II and preventing it from activating HDAC3. Accordingly, we found that HDAC inhibiting proteins from human herpesviruses induce human NKG2D ligand ULBP-1. Thus our findings indicate that virally mediated HDAC inhibition can act as a signal for the host to activate NK-cell recognition.
Nucleolin, a multifunctional protein distributed in the nucleolus, participates in many modulations including rDNA transcription, RNA metabolism, and ribosome assembly. Nucleolin is also found in the cytoplasm and on the cell membrane, and surface nucleolin can bind to various ligands to affect many physiological functions. The expression and localization of nucleolin is often abnormal in cancers, as the differential distribution of nucleolin in cancer can influence the carcinogenesis, proliferation, survival, and metastasis of cancer cells, leading to the cancer progression. Thus, nucleolin may be a novel and promising target for anti-cancer treatment. Here, we describe how nucleolin act functions in cancer development and describe nucleolin-dependent anti-cancer therapies.
The aim of this study is to clarify whether improvement of cognitive functioning by cognitive remediation therapy can improve work outcome in schizophrenia and other severe mental illness when combined with supported employment.
The Subjects of this study were persons with severe mental illness diagnosed with schizophrenia, major depression, or bipolar disorder (ICD-10) and cognitive dysfunction who participated in both cognitive remediation using the Thinking Skills for Work program and supported employment program in a multisite, randomized controlled study. Logistic and multiple linear regression analyses were performed to clarify the influence of cognitive functioning on vocational outcomes, adjusting for demographic and clinical variables.
Improvement of cognitive functioning with cognitive remediation significantly contributed to the total days employed and total earnings of competitive employment in supported employment service during the study period. Any baseline demographic and clinical variables did not significantly contribute to the work-related outcomes.
A cognitive remediation program transferring learning skills into the real world is useful to increase the quality of working life in supported employment services for persons with severe mental illness and cognitive dysfunction who want to work competitively.
The advancement of knowledge and development of policy in the field of medical education require critical academic discourse among the most intelligent medical educators; and critical academic discourse requires coffee. In this essay, we reflect on the state of professional development conferences in the field of medical education and the rituals that surround their success. Having begun in ancient Greece, symposia were ripe with debauchery. Today, sedated by the light brown walls of hotel conference centres, symposia are more serious endeavours, engaging men and women in the sometimes turbulent waters of epistemological debate. The abstract submission process (summed up by: 'Yay! It was accepted for presentation' [Deep breath] 'Oh no…it was accepted for presentation'), the 'juggling act' of parent attendees, the acting prowess of abstract presenters and the unapologetic approach to buffet eating are all by-products of the collision of true intellects among medical education scholars. We hold these rituals in high regard and argue that they are required to advance the field of medical education. These rituals bind the walls supporting true progressive thought and innovative research, all fuelled by the glass of wine purchased with that one coveted drink ticket.
To highlight the organisation-level management's role in building leadership capacity in advanced nurse practitioners and the need for appropriate supports to increase their becoming leaders.
Little is published about the role of organisation-level management in building leadership capacity and in developing the next generation of nurse leaders. In times of economic constraint, organisations need to focus their efforts on targeted leadership initiatives. Advanced nurse practitioners are ideally positioned to act as leaders both within and beyond the health care organisation.
From the available research evidence, several support structures and mechanisms are identified as enablers for advanced nurse practitioners to enact their leadership role.
Health care organisations need to include building leadership capacity as a priority in their strategic plan and take action to build-up the level of advanced nurse practitioner leadership.
Nurse executives have a vital role in influencing the organisation's strategic plan and making a business case for prioritising leadership capacity building within advanced nurse practitioners. A challenge for nurse executives faced with competing service and leadership development demands, involves strategic decision-making regarding whether the advanced nurse practitioner's role is limited to service delivery or its potential in leading health care reforms is realised.
Preeclampsia is a life-threatening vascular disorder of pregnancy due to a failing stressed placenta. Millions of women risk death to give birth each year and globally each year, almost 300,000 lose their life in this process and over 500,000 babies die as a consequence of preeclampsia. Despite decades of research, we lack pharmacological agents to treat it. Maternal endothelial oxidative stress is a central phenomenon responsible for the preeclampsia phenotype of high maternal blood pressure and proteinuria. In 1997, it was proposed that preeclampsia arises due to the loss of VEGF activity, possibly due to elevation in anti-angiogenic factor, soluble Flt-1 (sFlt-1). Researchers showed that high sFlt-1 and soluble endoglin (sEng) elicit the severe preeclampsia phenotype in pregnant rodents. We demonstrated that heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway prevents placental stress and suppresses sFlt-1 and sEng release. Likewise, hydrogen sulphide (H2S)/cystathionine-γ-lyase (Cth) systems limit sFlt-1 and sEng and protect against the preeclampsia phenotype in mice. Importantly, H2S restores placental vasculature, and in doing so improves lagging fetal growth. These molecules act as the inhibitor systems in pregnancy and when they fail, preeclampsia is triggered. In this review, we discuss what are the hypotheses and models for the pathophysiology of preeclampsia on the basis of Bradford Hill causation criteria for disease causation and how further in vivo experimentation is needed to establish 'proof of principle'. Hypotheses that fail to meet the Bradford Hill causation criteria include abnormal spiral artery remodelling and inflammation and should be considered associated or consequential to the disorder. In contrast, the protection against cellular stress hypothesis that states that the protective pathways mitigate cellular stress by limiting elevation of anti-angiogenic factors or oxidative stress and the subsequent clinical signs of preeclampsia appear to fulfil most of Bradford Hill causation criteria. Identifying the candidates on the roadmap to this pathway is essential in developing diagnostics and therapeutics to target the pathogenesis of preeclampsia.
Cellular organization and response to internal and external stimuli are mediated by an intricate web of protein interactions. Some of these interactions are regulated by covalent posttranslational modifications such as phosphorylation and acetylation. These modifications can change the chemical nature of the interaction interfaces and modulate the binding affinity of the interacting partners. In signal transduction, the most frequent modification is reversible phosphorylation of tyrosine, serine or threonine residues. Protein phosphorylation may modulate the activity of enzymes by modifying their conformation, or regulate the formation of complexes by creating docking sites to recruit downstream effectors. Families of modular domains, such as SH2, PTB, and 14-3-3, act as "readers" of the modification event. Specificity between closely related domains of the same family is mediated by the chemical properties of the domain binding surface that, aside from offering a hydrophilic pocket for the phosphorylated residue, shows preference for specific sequences. Although the protein structure and the cell context are also important to ensure specificity, the amino acid sequence flanking the phosphorylation site defines the accuracy of the recognition process, and it is therefore essential to define the binding specificity of phosphopeptide binding domains in order to understand and to infer the interaction web mediated by phosphopeptides. Methods commonly used to discover new interactions (such as yeast two hybrid and phage display) are not suited to study interactions with phosphorylated proteins. On the other hand, peptide arrays are a powerful approach to precisely identify the binding preference of phosphopeptide recognition domains. Here we describe a detailed protocol to assemble arrays of hundreds to thousands phospho-peptides and to screen them with any modular domain of interest.
Unlike physical training, skill acquisition does not currently utilise periodisation to plan, monitor and evaluate programs. Development of a skill acquisition periodisation framework would allow for systematic investigation into the acute and longitudinal effectiveness of such interventions. Using the physical training literature as a reference point, a skill-training periodisation framework was developed for use in high-performance sport. Previous research undertaken in skill acquisition was used to provide support for the framework. The specificity, progression, overload, reversibility and tedium (SPORT) acronym was adopted. Each principle was then re-conceptualised so that it related to relevant skill acquisition principles. Methods for the measurement and analysis of each principle are provided and future directions for the longitudinal assessment of skill acquisition are discussed. The skill acquisition periodisation framework proposed in this study represents an opportunity for the principles relating to skill acquisition training to be measured in a systematic and holistic manner. This can also allow for a more sophisticated evaluation of the efficacy of longitudinal training programmes and interventions designed for sustained skill enhancement.
There is compelling evidence that initiation and maintenance of epileptic seizures in temporal lobe epilepsy (TLE) is facilitated by excessive accumulation in the extracellular (perisynaptic) space of the excitatory neurotransmitter glutamate (Glu). This review discusses the mechanisms underlying this phenomenon. Glu released from neurons is taken up by astrocytes and activated there by glutamine synthetase (GS) to form glutamine (Gln) which upon entry to neurons is degraded back to Glu by phosphate-activated glutaminase (PAG): this chain of reactions has been defined as the glutamine/glutamate/cycle (GGC). In the initial phase of epileptogenesis, increased Glu supply is a consequence of activation of its turnover in GGC by Glu released by a primary chemical or physical stimulus. In chronic TLE, profound astrogliosis and demise of neurons which culminate in hippocampal sclerosis, are associated with changes in GGC which act in concert towards increasing the extracellular Glu concentration. Deficiency of GS and of the astrocytic Glu transporter, GLT-1, impede Glu inactivation, whereas Glu release from neurons appears facilitated by activation of PAG and increased activity of the neuronal Glu transporter EAAC1. Conclusions derived from measurements of activities/expression patterns of the GGC enzymes and transporter moieties find support in metabolic studies employing (13)C labeled Glu precursors. Glu reuptake by astrocytes is additionally impeded by unfavorable ion gradients resulting from ion and water dyshomeostasis, and extracellular Glu concentration is further increased by reduction of extracellular space due to edema and altered cytoarchitecture of the hippocampus. Missing links in the scenario are discussed in concluding comments.
Living in high-density groups of animals has advantages and disadvantages for mating. The advantage of facilitated mate finding is compromised by difficulties in protecting a suitable partner from competitors. Thus, males regularly are faced with increased competition for sperm, and females with harassment by males at high population densities. To cope with these problems, mating tactics and mate choice mechanisms have to be adjusted. An adaptation to gregarious condition observed in locusts includes the use of male-emitted pheromones. Males of the Central American locust, Schistocerca piceifrons, release sex-specific volatiles, which were identified as phenethyl alcohol (synonym: phenyl-ethyl-alcohol, 2-phenyl-1-ethanol, 2-phenylethanol, PEA), (Z)-3-nonen-1-ol (3-Nol), and (Z)-2-octen-1-ol (2-Ool). The emission of the two major compounds, PEA and 3-Nol, was restricted to crowded conditions. Furthermore, the release of both volatiles was coupled to males reaching sexual maturity, indicating a function in reproductive behavior. However, neither the single substances nor their mixtures were attractive or repellent to the locusts. Instead, females prefer the sperm of high pheromone-emitting males to fertilize their ova. In this way, the male-specific volatiles act as mate assessment pheromones utilized in a context of cryptic female choice. This function is well supported by the highly variable but individual-specific emission rates of the three compounds. Schistocerca piceifrons males release a virtually unique personal pheromone signature, a prerequisite for mate assessment pheromones.
Bisexual men experience significant health disparities likely related to biphobia. Biphobia presents via several preconceptions, including that bisexuality is transitory, and that bisexual men act as viral bridges between men who have sex with men and heterosexual populations. We analyzed data from a prospective cohort of gay and bisexual men, the Multicenter AIDS Cohort Study, to test these preconceptions. Men reporting both male and female sexual partners (MSMW) between 2002 and 2009 (n = 111) were classified as behaviorally bisexual. We assessed five hypotheses over two domains (transience of bisexual behavior and viral bridging). No evidence was found supporting the transitory nature of bisexuality. Trajectories of bisexual behavior were not transient over time. We found little evidence to support substantial viral bridging behavior. Notably, HIV-positive MSMW reported lower proportions of female partners than HIV-negative MSMW. Our results provide no empirical support for bisexual transience and scant support for viral bridging hypotheses. Our results provide key data showing that male bisexual behavior may be stable over long time periods and that behaviorally bisexual men's risk to female sexual partners may be lower than expected.
Marine microorganisms such as phytoplanktons are a rich resource of bioactive components with antioxidant and anti-proliferative activities that can act as novel functional food ingredients. In this study, the pigment profiles, total mycosporine-like amino acids (MAAs) and total phenolic contents (TPCs) in solvent extracts including 90% acetone and methanol from five marine phytoplanktons including Nitzschia closterium (Bacillariophyta), Isochrysis zhangjiangensis (Haptophyta), Platymonas subcordiformis (Chlorophyta), Porphyridium cruentum (Rhodophyta) and Synechocystis pevalekii (Cyanobacteria) were analyzed. Each phytoplankton from different phyla had its unique compositions of carotenoids and chlorophylls. The 90% acetone extract from I. zhangjiangensis had the highest MAA content (508.30 μg per g DW) while the methanol extract from N. closterium had the highest level of TPCs (6.15 mg GAE per g DW) among all the phytoplanktons investigated. The amounts of total carotenoids in all the 90% acetone extracts from the five phytoplanktons as well as total MAAs in those from within the four microalgae except S. pevalekii were found to be strongly correlated with their antioxidant activities evaluated by the DPPH, TEAC and FRAP assays. Only the level of total carotenoids in the phytoplanktons was correlated with their anti-proliferative activities assessed by the MTT assays using MCF-7 cells. Therefore, individual carotenoid pigments seemed to be mainly responsible for the antioxidant and anti-proliferative (or anticancer) activities found in the solvent extracts of the five phytoplanktons. Hence these phytoplanktons have the potential as novel sources of natural food antioxidants and anticancer agents to be used as active ingredients in functional food products.
North-east region of India has consistent role in the spread of multi drug resistant Plasmodium (P.) falciparum to other parts of Southeast Asia. After rapid clinical treatment failure of Artemisinin based combination therapy-Sulphadoxine/Pyrimethamine (ACT-SP) chemoprophylaxis, Artemether-Lumefantrine (ACT-AL) combination therapy was introduced in the year 2012 in this region for the treatment of uncomplicated P. falciparum malaria. In a DNA sequencing based polymorphism analysis, seven codons of P. falciparum dihydropteroate synthetase (Pfdhps) gene were screened in a total of 127 P. falciparum isolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes.
Sequences were analyzed to rearrange both nucleotide and protein haplotypes. Molecular diversity indices were analyzed in DNA Sequence Polymorphism software (DnaSP) on the basis of Pfdhps gene sequences. Disappearance from selective neutrality was assessed based on the ratio of non-synonomous to synonomous nucleotide substitutions [dN/dS ratio]. Moreover, two-tailed Z test was performed in search of the significance for probability of rejecting null hypothesis of strict neutrality [dN = dS]. Presence of mutant P. falciparum multidrug resistance protein1 (Pfmdr1) was also checked in those isolates that were present with new Pfdhps haplotypes. Phylogenetic relationship based on Pfdhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA).
Among eight different sulfadoxine resistant haplotypes found, IS GNG A haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538 R allele. Sequence analysis of Pfmdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86 Y ; Y YSND) in the multidrug resistance protein. Molecular diversity based on Pfdhps haplotypes revealed that P. falciparum populations in Assam and Tripura were under balancing selection for sulfadoxine resistant haplotypes but population from Arunachal Pradesh was under positive selection with comparatively high haplotype diversity (h = 0.870). In reconstructed phylogenetic analysis, isolates having IS GNG A haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities.
This study suggests that sulfadoxine resistant isolates are still migrating from its epicenter to the other parts of Southeast Asia and hence control and elimination of the drug resistant isolates have become impedimental. Moreover, P. falciparum populations in different areas may undergo selection of particular sulfadoxine resistant haplotypes either in the presence of drug or after its removal to maintain their plasticity.
Exosomes, as a mediator of cell-to-cell transfer of genetic information, act an important role in intercommunication between tumor cells and their niche including fibroblasts, endothelial cells, adipocytes and monocytes. Several studies have shown that tumor cells can influence their neighboring cells by releasing exosomes. These exosomes provide signaling cues for stimulation, activation, proliferation and differentiation of cells. Exosomes contain mRNAs, microRNAs (miRNA), and proteins that could be transferred to target cells inducing genetic and epigenetic changes. By facilitating the horizontal transfer of bioactive molecules such as proteins, RNAs and microRNAs, they are now thought to have vital roles in tumor invasion and metastases, inflammation, coagulation, and stem cell renewal and expansion. The aim of this review article is to discuss the significance of exosome-mediated intercellular communication within the tumor biology.
Extracellular microRNAs are released from cells both passively and actively. The presence of these microRNAs in the tumour microenvironment (TME) can significantly impact on the plasticity of cancer cells leading to the promotion of metastatic and angiogenic processes. These extracellular microRNAs can act not only on other cancer cells, but also cells present in the TME, such as immune cells, endothelial cells, fibroblasts, and others acting to subvert the host immune system and drive tumour progression. In this review we highlight the current understanding of both the mechanisms by which microRNAs are released from tumour cells and the downstream functional effects that extracellular microRNAs have on recipient cells.
As an important component of ascending activating systems, brainstem cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg) are involved in the regulation of motor control (locomotion, posture and gaze) and cognitive processes (attention, learning and memory). The PPTg is highly interconnected with several regions of the basal ganglia, and one of its key functions is to regulate and relay activity from the basal ganglia. Together, they have been implicated in the motor control system (such as voluntary movement initiation or inhibition), and modulate aspects of executive function (such as motivation). In addition to its intimate connection with the basal ganglia, projections from the PPTg to the cerebellum have been recently reported to synaptically activate the deep cerebellar nuclei. Classically, the cerebellum and basal ganglia were regarded as forming separated anatomical loops that play a distinct functional role in motor and cognitive behavioral control. Here, we suggest that the PPTg may also act as an interface device between the basal ganglia and cerebellum. As such, part of the therapeutic effect of PPTg deep brain stimulation (DBS) to relieve gait freezing and postural instability in advanced Parkinson's disease (PD) patients might also involve modulation of the cerebellum. We review the anatomical position and role of the PPTg in the pathway of basal ganglia and cerebellum in relation to motor control, cognitive function and PD.
Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L) on HPFs was lower than on HCFs (100 μmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.
In clinical chemistry, harmonisation of the testing process is a global initiative with the purpose of improving patient safety, allowing better integration of research data and enabling the use of national electronic heath records. In Australia, as in other countries, the initial focus has been on the harmonisation of the more commonly measured analytes. There are also a number of calculated parameters, derived from these measured analytes, which could also be considered for harmonisation. Calculated parameters that are reported by laboratories and used for clinical decision-making should undergo the same robust process of harmonisation as is the case for the measured analytes. Aspects that should be considered for harmonisation are: terminology, the formulae used and where possible the use of common reference intervals. To investigate pathways towards the harmonisation of calculated parameters, three commonly reported parameters are considered. Calculated osmolality, the anion gap and albumin-adjusted calcium are all derived from common analytes which have individually been considered for harmonisation. They present different methodological, measurement uncertainty and terminological hurdles to harmonisation and are likely to require different pathways and solutions.
Reference intervals (RIs) are used to help clinicians determine if a patient can be classified as being in a diseased or healthy state and there are often sound scientific and clinical reasons for differences in RIs. One of the current strategic priorities for the Australasian Association of Clinical Biochemists is to encourage and assist laboratories to achieve harmonisation of RIs for common clinical chemistry analytes where sound calibration and traceability are in place. This need is based on good laboratory practice, providing the clinician with results that allow appropriate and reliable clinical interpretation and progression further toward the national e-health framework and a single electronic health record. After reviewing and considering studies related to bias as well as both a priori and a posteriori RI studies nationally and internationally and the consideration of flagging rates and clinical relevance, an initial group of 12 harmonised RIs were endorsed by the Royal College of Pathologists of Australasia in 2014. In 2015, after further stakeholder consultation, a second group of six harmonised RIs for common chemistry analytes has been proposed for adults which includes ALT and AST where methods do not use pyridoxal-5'-phosphate as an activator and lipase excluding the Ortho Clinical Diagnostics and Siemens Dimension assays.
For more than a decade there has been a global effort to harmonise all phases of the testing process, with particular emphasis on the most frequently utilised measurands. In addition, it is recognised that calculated parameters derived from these measurands should also be a target for harmonisation. Using data from the Aussie Normals study we report reference intervals for three calculated parameters: serum osmolality, serum anion gap and albumin-adjusted serum calcium. The Aussie Normals study was an a priori study that analysed samples from 1856 healthy volunteers. The nine analytes used for the calculations in this study were measured on Abbott Architect analysers. The data demonstrated normal (Gaussian) distributions for the albumin-adjusted serum calcium, the anion gap (using potassium in the calculation) and the calculated serum osmolality (using both the Bhagat et al. and Smithline and Gardner formulae). To assess the suitability of these reference intervals for use as harmonised reference intervals, we reviewed data from the Royal College of Pathologists of Australasia/Australasian Association of Clinical Biochemists (RCPA/AACB) bias survey. We conclude that the reference intervals for the calculated serum osmolality (using the Smithline and Gardner formulae) may be suitable for use as a common reference interval. Although a common reference interval for albumin-adjusted serum calcium may be possible, further investigations (including a greater range of albumin concentrations) are needed. This is due to the bias between the Bromocresol Green (BCG) and Bromocresol Purple (BCP) methods at lower serum albumin concentrations. Problems with the measurement of Total CO2 in the bias survey meant that we could not use the data for assessing the suitability of a common reference interval for the anion gap. Further study is required.
The transfer-free fabrication of the high quality graphene on the metallic nanostructures, which is highly desirable for device applications, remains a challenge. Here, we develop the transfer-free method by direct chemical vapor deposition of the graphene layers on copper (Cu) nanoparticles (NPs) to realize the hybrid nanostructures. The graphene as-grown on the Cu NPs permits full electric contact and strong interactions, which results in a strong localization of the field at the graphene/copper interface. An enhanced intensity of the localized surface plasmon resonances (LSPRs) supported by the hybrid nanostructures can be obtained, which induces a much enhanced fluorescent intensity from the dye coated hybrid nanostructures. Moreover, the graphene sheets covering completely and uniformly on the Cu NPs act as a passivation layer to protect the underlying metal surface from air oxidation. As a result, the stability of the LSPRs for the hybrid nanostructures is much enhanced compared to that of the bare Cu NPs. The transfer-free hybrid nanostructures with enhanced intensity and stability of the LSPRs will enable their much broader applications in photonics and optoelectronics.
Dexmedetomidine (DEX) may act as an antioxidant through regulation of TRPM2 and TRPV1 channel activations in the neurons by reducing cerebral ischemia-induced oxidative stress and apoptosis. The neuroprotective roles of DEX were tested on cerebral ischemia (ISC) in the cultures of rat primary hippocampal and DRG neurons. Fifty-six rats were divided into five groups. A placebo was given to control, sham control, and ISC groups, respectively. In the third group, ISC was induced. The DEX and ISC+DEX groups received intraperitoneal DEX (40 μg/kg) 3, 24, and 48 hours after ISC induction. DEX effectively reversed capsaicin and cumene hydroperoxide/ADP-ribose-induced TRPV1 and TRPM2 densities and cytosolic calcium ion accumulation in the neurons, respectively. In addition, DEX completely reduced ISC-induced oxidative toxicity and apoptosis through intracellular reactive oxygen species production and depolarization of mitochondrial membrane. The DEX and ISC+DEX treatments also decreased the expression levels of caspase 3, caspase 9, and poly (ADP-ribose) polymerase in the hippocampus and DRG. In conclusion, the current results are the first to demonstrate the molecular level effects of DEX on TRPM2 and TRPV1 activation. Therefore, DEX can have remarkable neuroprotective impairment effects in the hippocampus and DRG of ISC-induced rats.
More than 40 antimicrobial peptides and proteins (AMPs) are expressed in the oral cavity. These AMPs have been organized into 6 functional groups, 1 of which, cationic AMPs, has received extensive attention in recent years for their promise as potential antibiotics. The goal of this review is to describe recent advances in our understanding of the diverse mechanisms of action of cationic AMPs and the bacterial resistance against these peptides. The recently developed peptide GL13K is used as an example to illustrate many of the discussed concepts. Cationic AMPs typically exhibit an amphipathic conformation, which allows increased interaction with negatively charged bacterial membranes. Peptides undergo changes in conformation and aggregation state in the presence of membranes; conversely, lipid conformation and packing can adapt to the presence of peptides. As a consequence, a single peptide can act through several mechanisms depending on the peptide's structure, the peptide:lipid ratio, and the properties of the lipid membrane. Accumulating evidence shows that in addition to acting at the cell membrane, AMPs may act on the cell wall, inhibit protein folding or enzyme activity, or act intracellularly. Therefore, once a peptide has reached the cell wall, cell membrane, or its internal target, the difference in mechanism of action on gram-negative and gram-positive bacteria may be less pronounced than formerly assumed. While AMPs should not cause widespread resistance due to their preferential attack on the cell membrane, in cases where specific protein targets are involved, the possibility exists for genetic mutations and bacterial resistance. Indeed, the potential clinical use of AMPs has raised the concern that resistance to therapeutic AMPs could be associated with resistance to endogenous host-defense peptides. Current evidence suggests that this is a rare event that can be overcome by subtle structural modifications of an AMP.
Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering.
To describe the impact of Street Triage (ST) on the number and rate of Section 136 Mental Health Act (S136) detentions in one NHS Mental Health and Disability Trust (Northumberland, Tyne and Wear (NTW)).
Comparative descriptive study of numbers and rates of S136 detentions prior to and following the introduction of ST in NTW. More detailed data were obtained from one local authority in the NTW area.
NTW, a secondary care NHS Foundation Trust providing mental health and disability services in the north-east of England, in conjunction with Northumbria Police Service.
People being detained under S136 Mental Health Act (MHA). Routine data on S136 detentions and ST interventions were obtained from NTW, Northumbria Police, Sunderland Hospitals NHS Foundation Trust and Sunderland Local Authority.
Introduction of a ST service in NTW. The main outcome measures were routinely collected data on the number and rate of ST interventions as well as patterns of the numbers and rates of S136 detentions. These were collected retrospectively.
The annual rate of S136 detentions reduced by 56% in the first year of ST (from 59.8 per 100 000 population to 26.4 per 100 000). There was a linear relationship between the rate of ST in each locality and the reduction in rate of S136 detentions. There were 1623 ST contacts in the first 3 localities to have a ST service during its first year; there were also 403 fewer S136 detentions. Data from Sunderland indicate a 78% reduction in S136 use and a significant reduction in the number and proportion of adult admissions that originated from S136 detentions.
There is evidence to support the hypothesis that ST decreases the rate of s136 detention. When operating across the whole of NTW, ST resulted in 50 fewer S136 detentions a month, which represents a substantial reduction.
Earlier cancer diagnosis is crucial in improving cancer survival. The International Cancer Benchmarking Partnership Module 4 (ICBP4) is a quantitative survey study that explores the reasons for delays in diagnosis and treatment of breast, colorectal, lung, and ovarian cancer. To further understand the associated diagnostic processes, it is also important to explore the patient perspectives expressed in the free-text comments.
To use the free-text data provided by patients completing the ICBP4 survey to augment the understanding of patients' perspectives of their diagnostic journey.
Qualitative analysis of the free-text data collected in Wales between October 2013 and December 2014 as part of the ICBP4 survey. Newly-diagnosed patients with either breast, ovarian, colorectal, or lung cancer were identified from registry data and then invited by their GPs to participate in the survey.
A thematic framework was used to analyse the free-text comments provided at the end of the ICBP4 survey. Of the 905 patients who returned a questionnaire, 530 included comments.
The free-text data provided information about patients' perspectives of the diagnostic journey. Analysis identified factors that acted as either barriers or facilitators at different stages of the diagnostic process. Some factors, such as screening, doctor-patient familiarity, and private treatment, acted as both barriers and facilitators depending on the context.
Factors identified in this study help to explain how existing models of cancer diagnosis (for example, the Pathways to Treatment Model) work in practice. It is important that clinicians are aware of how these factors may interact with individual clinical cases and either facilitate, or act as a barrier to, subsequent cancer diagnosis. Understanding and implementing this knowledge into clinical practice may result in quicker cancer diagnoses.
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with survival averaging only 3months if untreated following diagnosis. A major limitation in effectively treating PDAC using conventional and targeted chemotherapeutic agents, is inadequate drug delivery to the target location, predominantly due to a poorly vascularised, desmoplastic tumour microenvironment. Ultrasound in combination with ultrasound contrast agents, i.e., microbubbles, that flow through the vasculature and capillaries can be used to disrupt such mechanical barriers, potentially allowing for a greater therapeutic efficacy. This phenomenon is commonly referred to as sonoporation. In an attempt to improve the efficacy of sonoporation, novel microbubble formulations are being developed to address the limitation of commercially produced clinical diagnostic ultrasound contrast agents. In our work here we evaluate the ability of a novel formulation; namely Acoustic Cluster Therapy (ACT®) to improve the therapeutic efficacy of the chemotherapeutic agent paclitaxel, longitudinally in a xenograft model of PDAC. Results indicated that ACT® bubbles alone demonstrated no observable toxic effects, whilst ACT® in combination with paclitaxel can transiently reduce tumour volumes significantly, three days posttreatment (p=0.0347-0.0458). Quantitative 3D ultrasound validated the calliper measurements. Power Doppler ultrasound imaging indicated that ACT® in combination with paclitaxel was able to transiently sustain peak vasculature percentages as observed in the initial stages of tumour development. Nevertheless, there was no significant difference in tumour vasculature percentage at the end of treatment. The high vascular percentage correlated to the transient decrease and overall inhibition of the tumour volumes. In conclusion, ACT® improves the therapeutic efficacy of paclitaxel in a PDAC xenograft model allowing for transient tumour volume reduction and sustained tumour vasculature percentage.
Nonsynaptic mechanism changes, particularly the enhancement of NKCC1 expression in the dentate gyrus (DG) after 4weeks of ethanol consumption, motivate the present work, in which rats were submitted to a period of chronic consumption (12weeks). Four groups of six animals (6-week-old male Wistar rats) were formed, including the control (C), ethanol 1 (E1), ethanol 2 (E2) and ethanol 3 (E3) groups. The rats in the E1, E2 and E3 groups were treated daily with a 30% v/v solution of ethanol, administered via oral gavage (1.0, 2.0 and 3.0g/kg, respectively). Nonsynaptic epileptiform activities (NEA) were induced by means of the zero-Ca(2+) and high-K(+) model using hippocampal slices and were recorded in the DG. The presence of NKCC1, KCC2, α1-Na(+)/K(+)-ATPase and GFAP immunoreactivity was analyzed. The results demonstrate that alcohol consumption changes NEA, and these changes are more prominent at the lower dosage. An increase in the DC shifts associated with epileptiform discharges was present with the low dose. This increase was correlated with the increment of NKCC1 expression. Confocal microscopy images indicate the NKCC1 increase was pronounced in the initial axonal segment of granule cells. The blockage of these cotransporters during NEA induction with bumetanide suppressed the DC shift increase and diminished all parameters of NEA that were quantified for all groups treated with ethanol. Therefore, the increase in NKCC1 expression and the effective activity of this cotransporter, which were observed in the treated groups, suggest that drugs that act for block NKCC1 represent promising strategies for diminishing the effects of alcohol damage on the brain.
The generation of DNA modifications in cells is in most cases accidental and associated with detrimental consequences such as increased mutation rates and an elevated risk of malignant transformation. Accordingly, repair enzymes involved in the removal of the modifications have primarily a protective function. Among the well-established exceptions of this rule are 5-methylcytosine and uracil, which are generated in DNA enzymatically under controlled conditions and fulfill important regulatory functions in DNA as epigenetic marks and in antibody diversification, respectively. More recently, considerable evidence has been obtained that also 8-oxo-7,8-dihydroguanine (8-oxoG), a frequent pro-mutagenic DNA modification generated by endogenous or exogenous reactive oxygen species (ROS), has distinct roles in the regulation of both transcription and signal transduction. Thus, the activation of transcription by the estrogen receptor, NF-κB, MYC and other transcription factors was shown to depend on the presence of 8-oxoG in the promoter regions and its recognition by the DNA repair glycosylase OGG1. The lysine-specific histone demethylase LSD1, which produces H2O2 as a by-product, was indentified as a local generator of 8-oxoG in some of these cases. In addition, a complex of OGG1 with the excised free substrate base was demonstrated to act as a guanine nucleotide exchange factor (GEF) for small GTPases such as Ras, Rac and Rho, thus stimulating signal transduction. The various findings and intriguing novel mechanisms suggested will be described and compared in this review.
Precision medicine continues to be the benchmark to which we strive in cancer research. Seeking out actionable aberrations that can be selectively targeted by drug compounds promises to optimize treatment efficacy and minimize toxicity. Utilizing these different targeted agents in combination or in sequence may further delay resistance to treatments and prolong antitumor responses. Remarkable progress in the field of immunotherapy adds another layer of complexity to the management of cancer patients. Corresponding advances in companion biomarker development, novel methods of serial tumor assessments, and innovative trial designs act synergistically to further precision medicine. Ongoing hurdles such as clonal evolution, intra- and intertumor heterogeneity, and varied mechanisms of drug resistance continue to be challenges to overcome. Large-scale data-sharing and collaborative networks using next-generation sequencing (NGS) platforms promise to take us further into the cancer 'ome' than ever before, with the goal of achieving successful precision medicine.
A newly isolated endo-β-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and β-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and β-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to β-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and β-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials.
Health systems in low-income countries are often characterized by poor health outcomes. While many reasons have been advanced to explain the persistently poor outcomes, management of the system has been found to play a key role. According to a WHO framework, the management of health systems is central to its ability to deliver needed health services. In this study, we examined how district managers in a rural setting in Uganda perceived existing approaches to strengthening management so as to provide a pragmatic and synergistic model for improving management capacity building.
Twenty-two interviews were conducted with district level administrative and political managers, district level health managers and health facility managers to understand their perceptions and definitions of management and capacity building. Kathy Charmaz's constructive approach to grounded theory informed the data analysis process.
An interative, dynamic and complex model with three sub-process of building a competent health manager was developed. A competent manager was understood as one who knew his/her roles, was well informed and was empowered to execute management functions. Professionalizing health managers which was viewed as the foundation, the use of engaging learning approaches as the inside contents and having a supportive work environment the frame of the model were the sub-processes involved in the model. The sub-processes were interconnected although the respondents agreed that having a supportive work environment was more time and effort intensive relative to the other two sub-processes.
The model developed in our study makes four central contributions to enhance the WHO framework and the existing literature. First, it emphasizes management capacity building as an iterative, dynamic and complex process rather than a set of characteristics of competent managers. Second, our model suggests the need for professionalization of health managers at different levels of the health system. Third, our model underscores the benefits that could be accrued from the use of engaging learning approaches through prolonged and sustained processes that act in synergy. Lastly, our model postulates that different resource investments and a varied range of stakeholders could be required at each of the sub-processes.
Triple-negative breast cancer (TNBC) has a high risk of relapse and there are few chemotherapy options. Although 5-hydroxytryptamine (5-HT, serotonin) signaling pathways have been suggested as potential targets for anti-cancer drug development, the mechanism responsible for the action of 5-HT in TNBC remains unknown.
Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. Cell proliferation was measured using CellTiter 96 Aqueous One Solution. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Levels of 5-HT and vascular endothelial growth factor (VEGF) were measured using ELISA kits. Chick chorioallantoic membrane (CAM) assay and mouse tumor model were used to investigate the in vivo effects of SB269970, a 5-HT7 receptor antagonist, and BJ-1113, a novel synthetic compound.
TNBC cell lines (MDA-MB-231, HCC-1395, and Hs578T) expressed higher levels of tryptophan hydroxylase 1 (TPH1) than hormone-responsive breast cancer cell lines (MCF-7 and T47D). In MDA-MB-231 cells, 5-HT promoted invasion and proliferation via 5-HT7 receptor, and interestingly, the stimulatory effect of 5-HT on MDA-MB-231 cell invasion was stronger than its effect on proliferation. Likewise, downstream signaling pathways of 5-HT7 differed during invasion and proliferation, that is, Gα-activated cAMP and Gβγ-activated kinase signaling during invasion, and Gβγ-activated PI3K/Akt signaling during proliferation. Also, 5-HT increased the protein expressions of TPH1 and VEGF in MDA-MB-231 cells. These results provide insight of the stimulatory effect of 5-HT on breast cancer progression; 5-HT was found to act more strongly during the first stage of metastasis (during invasion and migration) than during the later proliferative phase after local invasion. Interestingly, these actions of 5-HT were inhibited by BJ-1113, a 6-amino-2,4,5-trimethylpyridin-3-ol analog. BJ-1113 blocked intracellular signaling pathways initiated by 5-HT7 receptor activation, and exhibited anti-proliferative and anti-invasive activities against MDA-MB-231 cells. Furthermore, the inhibitory effect of BJ-1113 against MDA-MB-231 tumor growth was greater than that of SB269970, a 5-HT7 receptor antagonist.
5-HT7 receptor which mediates 5-HT-induced cancer progression is a potential therapeutic target in TNBC, and BJ-1113 offers a novel scaffold for the development of anti-cancer agents against TNBC.
The proper spatial and temporal regulation of dorsal telencephalic progenitor behavior is a prerequisite for the formation of the highly-organized, six-layered cerebral cortex. Premature differentiation of cells, disruption of cell cycle timing, excessive apoptosis, and/or incorrect neuronal migration signals can have devastating effects, resulting in a number of neurodevelopmental disorders involving microcephaly and/or lissencephaly. Though genes encoding many key players in cortical development have been identified, our understanding remains incomplete. We show that the gene encoding Akirin2, a small nuclear protein, is expressed in the embryonic telencephalon. Converging evidence indicates that Akirin2 acts as a bridge between transcription factors (including Twist and NF-κB proteins) and the BAF (SWI/SNF) chromatin remodeling machinery to regulate patterns of gene expression. Constitutive knockout of Akirin2 is early embryonic lethal in mice, while restricted loss in B cells led to disrupted proliferation and cell survival.
We generated cortex-restricted Akirin2 knockouts by crossing mice harboring a floxed Akirin2 allele with the Emx1-Cre transgenic line and assessed the resulting embryos using in situ hybridization, EdU labeling, and immunohistochemistry.
The vast majority of Akirin2 mutants do not survive past birth, and exhibit extreme microcephaly, with little dorsal telencephalic tissue and no recognizable cortex. This is primarily due to massive cell death of early cortical progenitors, which begins at embryonic day (E)10, shortly after Emx1-Cre is active. Immunostaining and cell cycle analysis using EdU labeling indicate that Akirin2-null progenitors fail to proliferate normally, produce fewer neurons, and undergo extensive apoptosis. All of the neurons that are generated in Akirin2 mutants also undergo apoptosis by E12. In situ hybridization for Wnt3a and Wnt-responsive genes suggest defective formation and/or function of the cortical hem in Akirin2 null mice. Furthermore, the apical ventricular surface becomes disrupted, and Sox2-positive progenitors are found to "spill" into the lateral ventricle.
Our data demonstrate a previously-unsuspected role for Akirin2 in early cortical development and, given its known nuclear roles, suggest that it may act to regulate gene expression patterns critical for early progenitor cell behavior and cortical neuron production.
Cardiovascular disease (CVD) disproportionately affects disadvantaged people, but reliable quantitative evidence on socioeconomic variation in CVD incidence in Australia is lacking. This study aimed to quantify socioeconomic variation in rates of primary and secondary CVD events in mid-age and older Australians.
Baseline data (2006-2009) from the 45 and Up Study, an Australian cohort involving 267,153 men and women aged ≥ 45, were linked to hospital and death data (to December 2013). Outcomes comprised first event - death or hospital admission - for major CVD combined, as well as myocardial infarction and stroke, in those with and without prior CVD (secondary and primary events, respectively). Cox regression estimated hazard ratios (HRs) for each outcome in relation to education (and income and area-level disadvantage), separately by age group (45-64, 65-79, and ≥ 80 years), adjusting for age and sex, and additional sociodemographic factors.
There were 18,207 primary major CVD events over 1,144,845 years of follow-up (15.9/1000 person-years), and 20,048 secondary events over 260,357 years (77.0/1000 person-years). For both primary and secondary events, incidence increased with decreasing education, with the absolute difference between education groups largest for secondary events. Age-sex adjusted hazard ratios were highest in the 45-64 years group: for major CVDs, HR (no qualifications vs university degree) = 1.62 (95% CI: 1.49-1.77) for primary events, and HR = 1.49 (1.34-1.65) for secondary events; myocardial infarction HR = 2.31 (1.87-2.85) and HR = 2.57 (1.90-3.47) respectively; stroke HR = 1.48 (1.16-1.87) and HR = 1.97 (1.42-2.74) respectively. Similar but attenuated results were seen in older age groups, and with income. For area-level disadvantage, CVD gradients were weak and non-significant in older people (> 64 years).
Individual-level data are important for quantifying socioeconomic variation in CVD incidence, which is shown to be substantial among both those with and without prior CVD. Findings reinforce the opportunity for, and importance of, primary and secondary prevention and treatment in reducing socioeconomic variation in CVD and consequently the overall burden of CVD morbidity and mortality in Australia.
The act of detecting bodily changes is a pre-requisite for subsequent responses to symptoms, such as seeking medical help. This is the first study to explore associations between self-reported body vigilance and help-seeking in a community sample currently experiencing cancer 'alarm' symptoms.
Using a cross-sectional study design, a 'health survey' was mailed through primary care practices to 4913 UK adults (age ≥50 years, no cancer diagnosis), asking about symptom experiences and medical help-seeking over the previous three months. Body vigilance, cancer worry and current illness were assessed with a small number of self-report items derived from existing measures.
The response rate was 42% (N = 2042). Almost half the respondents (936/2042; 46%) experienced at least one cancer alarm symptom. Results from logistic regression analysis revealed that paying more attention to bodily changes was significantly associated with help-seeking for cancer symptoms (OR = 1.44; 1.06-1.97), after controlling for socio-demographics, current illness and cancer worry. Being more sensitive to bodily changes was not significantly associated with help-seeking.
Respondents who paid attention to their bodily changes were more likely to seek help for their symptoms. Although the use of a cross-sectional study design and the limited assessment of key variables preclude any firm conclusions, encouraging people to be body vigilant may contribute towards earlier cancer diagnosis. More needs to be understood about the impact this might have on cancer-related anxiety.
To analyse over 700,000 cross-sectional measurements from the Mine Safety and Health Administration (MHSA) and develop statistical models to predict noise exposure for a worker.
Descriptive statistics were used to summarise the data. Two linear regression models were used to predict noise exposure based on MSHA-permissible exposure limit (PEL) and action level (AL), respectively. Twofold cross validation was used to compare the exposure estimates from the models to actual measurement. The mean difference and t-statistic was calculated for each job title to determine whether the model predictions were significantly different from the actual data.
Measurements were acquired from MSHA through a Freedom of Information Act request.
From 1979 to 2014, noise exposure has decreased. Measurements taken before the implementation of MSHA's revised noise regulation in 2000 were on average 4.5 dBA higher than after the law was implemented. Both models produced exposure predictions that were less than 1 dBA different than the holdout data.
Overall noise levels in mines have been decreasing. However, this decrease has not been uniform across all mining sectors. The exposure predictions from the model will be useful to help predict hearing loss in workers in the mining industry.
Surface barrier technology is used to isolate radioactive waste and to reduce or eliminate recharge water to the waste zone for 1000 years or longer. However, the design and evaluation of such a barrier is challenging because of the extremely long design life. After establishing a set of design and performance objectives, a package of design solutions was developed for 1000-year surface barriers over nuclear waste sites. The Prototype Hanford Barrier (PHB) was then constructed in 1994 in the field over an existing waste site as a demonstration. The barrier was tested to evaluate surface-barrier design and performance at the field scale under conditions of enhanced and natural precipitation and of no vegetation. The monitoring data demonstrate that the barrier satisfied nearly all objectives in the past two decades. The PHB far exceeded the Resource Conservation and Recovery Act criteria, functioned in Hanford's semiarid climate, limited drainage to well below the 0.5 mm yr(-1) performance criterion, limited runoff, and minimized erosion and bio-intrusion. Given the two-decade record of successful performance and consideration of the processes and mechanisms that could affect barrier stability and hydrology in the future, the results suggest the PHB is very likely to perform for its 1000-year design life. This conclusion is based on two assumptions: (1) the exposed subgrade receives protection against erosion and (2) institutional controls prevent inadvertent human activity at the barrier. The PHB design can serve as the basis for site-specific barriers over waste sites containing underground nuclear waste, uranium mine tailings, and hazardous mine waste.
The first randomized trial of a smartphone application (app) for adult smoking cessation (SmartQuit 1.0) revealed key features that predict cessation. These findings guided the revision of this Acceptance & Commitment Therapy (ACT)-based application (SmartQuit 2.0), which was primarily tested to examine participant receptivity, short-term cessation and reduction, and the relationship between program completion, smoking cessation and reduction. Secondarily, outcomes were descriptively compared with the SmartQuit1.0 trial.
Adult participants (78% female, 25% with high school or less education, 30% unemployed) were recruited into the single-arm pilot trial (N=99) of SmartQuit 2.0 with a two-month follow-up (85% retention).
Regarding receptivity, 84% of participants were satisfied with SmartQuit 2.0 (vs. 59% for SmartQuit1.0), 73% would recommend it to a friend (vs. 48% for SmartQuit1.0), 81% found the ACT exercises useful for quitting (vs. 44% for SmartQuit1.0). At the 2-month follow-up, the quit rates were 21% for 7-day point prevalence (vs. 23% for SmartQuit1.0), 11% for 30-day point prevalence (vs. 13% for SmartQuit1.0), and 75% of participants reduced their smoking frequency (vs. 57% for SmartQuit1.0). Among program completers (24% of total sample), the quit rates were 33% for 7-day point prevalence, 28% for 30-day point prevalence, and 88% of participants reduced their smoking frequency.
The revised app had high user receptivity, modest quit rates, and high smoking reduction rates. Program completion may be key to boosting the app's effectiveness.
Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) receptor is the most prevalent FLT3 mutation accounting for 20% of acute myeloid leukemia (AML) patients. FLT3-ITD mutation results in ligand-independent constitutive activation of the receptor at the plasma membrane and 'impaired trafficking' of the receptor in compartments of the endomembrane system, such as the endoplasmic reticulum (ER). FLT3-ITD expressing cells have been shown to generate increased levels of reactive oxygen species (ROS), in particular NADPH oxidase (NOX)-generated ROS which act as pro-survival signals. The purpose of this study is to investigate FLT3-ITD production of ROS at the plasma membrane and ER in the FLT3-ITD expressing AML cell line MV4-11. Receptor trafficking inhibitors; Tunicamycin and Brefeldin A induce ER retention of FLT3-ITD, resulting in a decrease in protein expression of NOX4 and its partner protein p22(phox), thus demonstrating the critical importance of FLT3-ITD localization for the generation of pro-survival ROS. NOX-generated ROS contribute to total endogenous hydrogen peroxide (H2O2) in AML as quantified by flow cytometry using the cell-permeable H2O2-probe Peroxy Orange 1 (PO1). We found that PI3K/AKT signaling only occurs when FLT3-ITD is expressed at the plasma membrane and is required for the production of NOX-generated ROS. ER retention of FLT3-ITD resulted in NOX4 deglycosylation and p22(phox) protein degradation.
Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.
Two main pillars are implicated in nasal polyposis development: a severe imbalance in immunomodulation and a mechanical dysfunction because of an abnormal remodeling process. Dendritic cells play a crucial role in the link between innate and adaptive immune response and orchestrating the T-cell response and are implicated in the severe inflammatory process found in nasal polypoid tissue. This review summarizes the existent knowledge about dendritic cells in nasal polyposis.
Dendritic cells are found increased in nasal polyposis, regardless of subset. Of interest, plasmacytoid dendritic cells are decreased in patients with a more severe Th2 profile, suggesting an important role of the cytokines milieu in their functional response or that plasmacytoid dendritic cell could act mitigating the inflamed process found in polypoid tissue.
Understanding the dendritic cell subset expression in different environments, as well as the effect of these subsets on T-cell differentiation will greatly improve the development of new therapies in nasal polyposis.
A general theory of mammalian sexual differentiation is proposed. All biological sex differences are the result of the inequality in effects of the sex chromosomes, which are the only factors that differ in XX vs. XY zygotes. This inequality leads to male-specific effects of the Y chromosome, including expression of the testis-determining gene Sry that causes differentiation of testes. Thus, Sry sets up lifelong sex differences in effects of gonadal hormones. Y genes also act outside of the gonads to cause male-specific effects. Differences in the number of X chromosomes between XX and XY cells cause sex differences in expression (1) of Xist, (2) of X genes that escape inactivation, and (3) of parentally imprinted X genes. Sex differences in phenotype are ultimately the result of multiple, independent sex-biasing factors, hormonal and sex chromosomal. These factors act in parallel and in combination to induce sex differences. They also can offset each other to reduce sex differences. Other mechanisms, operating at the level of populations, cause groups of males to differ on average from groups of females. The theory frames questions for further study, and directs attention to inherent sex-biasing factors that operate in many tissues to cause sex differences, and to cause sex-biased protection from disease. © 2016 Wiley Periodicals, Inc.
Sex hormones act throughout the entire brain of both males and females via both genomic and nongenomic receptors. Sex hormones can act through many cellular and molecular processes that alter structure and function of neural systems and influence behavior as well as providing neuroprotection. Within neurons, sex hormone receptors are found in nuclei and are also located near membranes, where they are associated with presynaptic terminals, mitochondria, spine apparatus, and postsynaptic densities. Sex hormone receptors also are found in glial cells. Hormonal regulation of a variety of signaling pathways as well as direct and indirect effects on gene expression induce spine synapses, up- or downregulate and alter the distribution of neurotransmitter receptors, and regulate neuropeptide expression and cholinergic and GABAergic activity as well as calcium sequestration and oxidative stress. Many neural and behavioral functions are affected, including mood, cognitive function, blood pressure regulation, motor coordination, pain, and opioid sensitivity. Subtle sex differences exist for many of these functions that are developmentally programmed by hormones and by not yet precisely defined genetic factors, including the mitochondrial genome. These sex differences and responses to sex hormones in brain regions, which influence functions not previously regarded as subject to such differences, indicate that we are entering a new era of our ability to understand and appreciate the diversity of gender-related behaviors and brain functions. © 2016 Wiley Periodicals, Inc.
Studies using the nematode C. elegans have provided unique insights into the development and function of sex differences in the nervous system. Enabled by the relative simplicity of this species, comprehensive studies have solved the complete cellular neuroanatomy of both sexes as well as the complete neural connectomes of the entire adult hermaphrodite and the adult male tail. This work, together with detailed behavioral studies, has revealed three aspects of sex differences in the nervous system: sex-specific neurons and circuits; circuits with sexually dimorphic synaptic connectivity; and sex differences in the physiology and functions of shared neurons and circuits. At all of these levels, biological sex influences neural development and function through the activity of a well-defined genetic hierarchy that acts throughout the body to translate chromosomal sex into the state of a master autosomal regulator of sexual differentiation, the transcription factor TRA-1A. This Review focuses on the role of genetic sex in implementing sex differences in shared neurons and circuits, with an emphasis on linking the sexual modulation of specific neural properties to the specification and optimization of sexually divergent and dimorphic behaviors. An important and unexpected finding from these studies is that chemosensory neurons are a primary focus of sexual modulation, with genetic sex adaptively shaping chemosensory repertoire to guide behavioral choice. Importantly, hormone-independent functions of genetic sex are the principal drivers of all of these sex differences, making nematodes an excellent model for understanding similar but poorly understood mechanisms that likely act throughout the animal kingdom. © 2016 Wiley Periodicals, Inc.
The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival.
Vertebrate jaws and dentitions fit and function together, yet the genetic processes that coordinate cranial and dental morphogenesis and evolution remain poorly understood. Teeth but not jaws fail to form in the edentate p63(-/-) mouse mutant, which we used here to identify genes important to odontogenesis, but not jaw morphogenesis, and that may allow dentitions to change during development and evolution without necessarily affecting the jaw skeleton. With the working hypothesis that tooth and jaw development are autonomously controlled by discreet gene regulatory networks, using gene expression microarray assays validated by quantitative reverse-transcription PCR we contrasted expression in mandibular prominences at embryonic days (E) 10-13 of mice with normal lower jaw development but either normal (p63(+/-) , p63(+/+) ) or arrested (p63(-/-) ) tooth development. The p63(-/-) mice showed significantly different expression (fold change ≥2, ≤-2; P ≤ 0.05) of several genes. Some of these are known to help regulate odontogenesis (e.g., p63, Osr2, Cldn3/4) and/or to be targets of p63 (e.g., Jag1/2, Fgfr2); other genes have no previously reported roles in odontogenesis or the p63 pathway (e.g., Fermt1, Cbln1, Pltp, Krt8). As expected, from E10 to E13, few genes known to regulate mandible morphogenesis differed in expression between mouse strains. This study newly links several genes to odontogenesis and/or to the p63 signaling network. We propose that these genes act in a novel odontogenic network that is exclusive of lower jaw morphogenesis, and posit that this network evolved in oral, not pharyngeal, teeth.
Decades of ecological study have demonstrated the importance of top-down and bottom-up controls on food webs, yet few studies within this context have quantified the magnitude of energy and material fluxes at the whole-ecosystem scale. We examined top-down and bottom-up effects on food web fluxes using a field experiment that manipulated the presence of a consumer, the Trinidadian guppy Poecilia reticulata, and the production of basal resources by thinning the riparian forest canopy to increase incident light. To gauge the effects of these reach-scale manipulations on food web fluxes, we used a nitrogen ((15) N) stable isotope tracer to compare basal resource treatments (thinned canopy vs. control) and consumer treatments (guppy introduction vs. control). The thinned canopy stream had higher primary production than the natural canopy control, leading to increased N fluxes to invertebrates that feed on benthic biofilms (grazers), fine benthic organic matter (collector-gatherers), and organic particles suspended in the water column (filter feeders). Stream reaches with guppies also had higher primary productivity and higher N fluxes to grazers and filter feeders. In contrast, N fluxes to collector-gatherers were reduced in guppy introduction reaches relative to upstream controls. N fluxes to leaf-shredding invertebrates, predatory invertebrates, and the other fish species present (Hart's killifish, Anablepsoides hartii) did not differ across light or guppy treatments, suggesting that effects on detritus-based linkages and upper trophic levels were not as strong. Effect sizes of guppy and canopy treatments on N flux rates were similar for most taxa, though guppy effects were the strongest for filter feeding invertebrates while canopy effects were the strongest for collector-gatherer invertebrates. Combined, these results extend previous knowledge about top-down and bottom-up controls on ecosystems by providing experimental, reach-scale evidence that both pathways can act simultaneously and have equally strong influence on nutrient fluxes from inorganic pools through primary consumers.
The majority of humanity now lives in cities or towns, with this proportion expected to continue increasing for the foreseeable future. As novel ecosystems, urban areas offer an ideal opportunity to examine multi-scalar processes involved in community assembly as well as the role of human activities in modulating environmental drivers of biodiversity. Although ecologists have made great strides in recent decades at documenting ecological relationships in urban areas, much remains unknown, and we still need to identify the major ecological factors, aside from habitat loss, behind the persistence or extinction of species and guilds of species in cities. Given this paucity of knowledge, there is an immediate need to facilitate collaborative, interdisciplinary research on the patterns and drivers of biodiversity in cities at multiple spatial scales. In this review, we introduce a new conceptual framework for understanding the filtering processes that mold diversity of urban floras and faunas. We hypothesize that the following hierarchical series of filters influence species distributions in cities: (1) regional climatic and biogeographical factors; (2) human facilitation; (3) urban form and development history; (4) socioeconomic and cultural factors; and (5) species interactions. In addition to these filters, life history and functional traits of species are important in determining community assembly and act at multiple spatial scales. Using these filters as a conceptual framework can help frame future research needed to elucidate processes of community assembly in urban areas. Understanding how humans influence community structure and processes will aid in the management, design, and planning of our cities to best support biodiversity.
In pediatric chronic pain, research indicates a positive relation between parental psychological flexibility (i.e., the parent's willingness to experience distress related to the child's pain in the service of valued behavior) and level of functioning in the child. This points to the utility of targeting parental psychological flexibility in pediatric chronic pain. The Parent Psychological Flexibility Questionnaire (PPFQ) is currently the only instrument developed for this purpose, and two previous studies have indicated its reliability and validity. The current study sought to validate the Swedish version of the 17-item PPFQ (PPFQ-17) in a sample of parents (n = 263) of children with chronic pain. Factor structure and internal reliability were evaluated by means of principal component analysis (PCA) and Cronbach's alpha. Concurrent criterion validity was examined by hierarchical multiple regression analyses with parental anxiety and depression as outcomes. The PCA supported a three-factor solution with 10 items explaining 69.5% of the total variance. Cronbach's alpha (0.86) indicated good internal consistency. The 10-item PPFQ (PPFQ-10) further explained a significant amount of variance in anxiety (29%), and depression (35.6%), confirming concurrent validity. In conclusion, results support the reliability and validity of the PPFQ-10, and suggest its usefulness in assessing psychological flexibility in parents of children with chronic pain.
Glioblastoma (GBM) is the most common primary brain tumor with median survival of approximately one year. This dismal poor prognosis is due to resistance to currently available chemotherapeutics; therefore, new cytotoxic agents are urgently needed. In the present study, we reported the cytotoxicity of toosendanin (TSN) in the GBM U87 and C6 cell lines in vitro and in vivo. By using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, flow cytometry analysis, and Western blot, we found that TSN inhibited U87 and C6 cell proliferation and induced apoptosis at a concentration as low as 10 nM. Administration of TSN also reduced tumor burden in a xenograft model of athymic nude mice. Pharmacological and molecular studies suggested that estrogen receptor β (ERβ) and p53 were prominent targets for TSN. GBM cell apoptosis induced by TSN was a stepwise biological event involving the upregulation of ERβ and contextual activation of functional p53. Collectively, our study indicates, for the first time, that TSN is a candidate of novel anti-cancer drugs for GBM. Furthermore, ERβ and p53 could act as predictive biomarkers for the sensitivity of cancer to TSN.
Growth and development are key characteristics of childhood and sensitive markers of health and adequate nutrition. The first 1000 days of life-conception through 24 months of age-represent a fundamental period for development and thus the prevention of childhood obesity and its adverse consequences is mandatory. There are many growth drivers during this complex phase of life, such as nutrition, genetic and epigenetic factors, and hormonal regulation. The challenge thus involves maximizing the potential for normal growth without increasing the risk of associated disorders. The Mediterranean Nutrition Group (MeNu Group), a group of researchers of the Mediterranean Region, in this Special Issue titled "Prevent Obesity in the First 1000 Days", presented results that advanced the science of obesity risk factors in early life, coming both from animal model studies and studies in humans. In the future, early-life intervention designs for the prevention of pediatric obesity will need to look at different strategies, and the MeNu Group is available for guidance regarding an appropriate conceptual framework to accomplish either prevention or treatment strategies to tackle pediatric obesity.
The articular surfaces and menisci act with the anterior cruciate ligament (ACL) to stabilize the knee joint. Their role in resisting applied rotatory loads characteristic of instability events is unclear despite commonly observed damage to these intra-articular structures in the acute and chronic ACL injury settings.
Ten fresh-frozen human cadaveric knees were mounted to a robotic manipulator. Combined valgus and internal rotation torques were applied in the presence and absence of a 300-N compressive load. Forces carried by the individual menisci and via cartilage-to-cartilage contact on each femoral condyle in ACL-intact and ACL-sectioned states were measured using the principle of superposition.
In response to applied valgus and internal rotation torques in the absence of compression, sectioning of the ACL increased the net force carried by the lateral meniscus by at most 65.8 N (p < 0.001). Moreover, the anterior shear force carried by the lateral meniscus increased by 25.7 N (p < 0.001) and 36.5 N (p = 0.042) in the absence and presence of compression, respectively. In response to applied valgus and internal rotation torques, sectioning of the ACL increased the net force carried by cartilage-to-cartilage contact on the medial femoral condyle by at most 38.9 N (p = 0.006) and 46.7 N (p = 0.040) in the absence and presence of compression, respectively. Additionally, the lateral shear force carried by cartilage-to-cartilage contact on the medial femoral condyle increased by at most 21.0 N (p = 0.005) and by 28.0 N (p = 0.025) in the absence and presence of compression, respectively. Forces carried by the medial meniscus and by cartilage-to-cartilage contact on the lateral femoral condyle changed by <5 N as a result of ACL sectioning.
ACL sectioning increased the net forces carried by the lateral meniscus and medial femoral condyle-and the anterior shear and lateral shear forces, respectively-in response to multiplanar valgus and internal rotation torque.
These loading patterns provide a biomechanical rationale for clinical patterns of intra-articular derangement such as lateral meniscal injury and osseous remodeling of the medial compartment seen with ACL insufficiency.
1. The present study investigated the effects of encapsulated benzoic acid (BA) supplementation in broiler feed on performance and gastrointestinal microbiota. 2. Eighty broilers were randomly divided into two groups. Birds in the control group were fed on maize-soybean based diets. Birds in the treatment group were provided the same diet supplemented with 2 g/kg BA encapsulated in a vegetable oil matrix. 3. At the end of the trial (d 35), pH, bacterial composition and metabolites were determined in the crop, jejunum, ileum and caecum. 4. Growth performance variables and pH were not significantly different. 5. BA concentration decreased rapidly in the proximal gut. However, the treatment diet showed higher BA in the crop, jejunum, ileum and caecum. 6. Total lactate in the crop and D-lactate in the jejunum was higher in the BA treated group. Caecal total and branched chain fatty acids were decreased due to the treatment. 7. Lactobacilli populations were significantly altered by BA supplementation. A trend for increased lactobacilli was observed in the crop, while it became significant in the jejunum and ileum. Lactobacillus species responded differently to the treatment. Four of 5 measured Lactobacillus species, particularly in the ileum, followed the course observed for total lactobacilli; only L. salivarius was not modified. 8. Correlation analysis showed that BA modified the intestinal microbiota. Lactobacilli correlated negatively to all studied clostridial clusters and enterobacteria. Clostridial clusters IV and XIVa were significantly increased in the jejunum, whereas only clostridial cluster XIVa was increased in the caecum. 9. Encapsulated BA modified the intestinal microbiota which can lead to the conclusion, that the main beneficial mode of action of BA in the gut appears to be the enhancement of lactic acid bacteria, which in turn may act as a vanguard against pathogens.
The three-membered RUNX gene family includes RUNX1, a major mutational target in human leukemias, and displays hallmarks of both tumour suppressors and oncogenes. In mouse models the Runx genes appear to act as conditional oncogenes, as ectopic expression is growth suppressive in normal cells but drives lymphoma development potently when combined with over-expressed Myc or loss of p53. Clues to underlying mechanisms emerged previously from murine fibroblasts where ectopic expression of any of the Runx genes promotes survival through direct and indirect regulation of key enzymes in sphingolipid metabolism associated with a shift in the 'sphingolipid rheostat' from ceramide to sphingosine-1-phosphate (S1P). Testing of this relationship in lymphoma cells was therefore a high priority. We find that ectopic expression of Runx1 in lymphoma cells consistently perturbs the sphingolipid rheostat, while an essential physiological role for Runx1 is revealed by reduced S1P levels in normal spleen after partial Cre-mediated excision. Furthermore we show that ectopic Runx1 expression confers increased resistance of lymphoma cells to glucocorticoid-mediated apoptosis, and elucidate the mechanism of cross-talk between glucocorticoid and sphingolipid metabolism through Sgpp1. Dexamethasone potently induces expression of Sgpp1 in T-lymphoma cells and drives cell death which is reduced by partial knockdown of Sgpp1 with shRNA or direct transcriptional repression of Sgpp1 by ectopic Runx1. Together these data show that Runx1 plays a role in regulating the sphingolipid rheostat in normal development and that perturbation of this cell fate regulator contributes to Runx-driven lymphomagenesis. This article is protected by copyright. All rights reserved.
Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus.
Neurofeedback-guided motor imagery training (NF-MIT) has been suggested as a promising therapy for stroke-induced motor impairment. Whereas much NF-MIT research has aimed at signal processing optimization, the type of sensory feedback given to the participant has received less attention. Often the feedback signal is highly abstract and not inherently coupled to the mental act performed. In this study, we asked whether an embodied feedback signal is more efficient for neurofeedback operation than a non-embodiable feedback signal. Inspired by the rubber hand illusion, demonstrating that an artificial hand can be incorporated into one's own body scheme, we used an anthropomorphic robotic hand to visually guide the participants' motor imagery act and to deliver neurofeedback. Using two experimental manipulations, we investigated how a participant's neurofeedback performance and subjective experience were influenced by the embodiability of the robotic hand, and by the neurofeedback signal's validity. As pertains to embodiment, we found a promoting effect of robotic-hand embodiment in subjective, behavioral, electrophysiological and electrodermal measures. Regarding neurofeedback signal validity, we found some differences between real and sham neurofeedback in terms of subjective and electrodermal measures, but not in terms of behavioral and electrophysiological measures. This study motivates the further development of embodied feedback signals for NF-MIT.
Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.Oncogene advance online publication, 21 November 2016; doi:10.1038/onc.2016.388.
Determining and acting on thermo-physical properties at the nanoscale is essential for understanding/managing heat distribution in micro/nanostructured materials and miniaturized devices. Adequate thermal nano-characterization techniques are required to address thermal issues compromising device performance. Scanning thermal microscopy (SThM) is a probing and acting technique based on atomic force microscopy using a nano-probe designed to act as a thermometer and resistive heater, achieving high spatial resolution. Enabling direct observation and mapping of thermal properties such as thermal conductivity, SThM is becoming a powerful tool with a critical role in several fields, from material science to device thermal management. We present an overview of the different thermal probes, followed by the contribution of SThM in three currently significant research topics. First, in thermal conductivity contrast studies of graphene monolayers deposited on different substrates, SThM proves itself a reliable technique to clarify the intriguing thermal properties of graphene, which is considered an important contributor to improve the performance of downscaled devices and materials. Second, SThM's ability to perform sub-surface imaging is highlighted by thermal conductivity contrast analysis of polymeric composites. Finally, an approach to induce and study local structural transitions in ferromagnetic shape memory alloy Ni-Mn-Ga thin films using localized nano-thermal analysis is presented.
We carried out a single-center retrospective study to assess the predictive value of body mass index (BMI) in the outcome of Chinese patients with diffuse large B-cell lymphoma (DLBCL). 143 eligible patients were enrolled between January 2008 and May 2015. These patients were stratified into two groups, 74 patients in low BMI group (BMI <23.0 kg/m(2)) and 69 patients in high BMI group (BMI ≥23.0 kg/m(2)). We compared the baseline characteristics, primary response, and survival outcome in two groups. Well-known influence factors were similar between the two groups, while gender was not (p = .023) but did not act as a risk factor. No association between BMI and primary response was observed. Patients with high BMI were inclined to have better overall survival (OS) (p = .018), but we didn't find an association in progression-free survival (PFS) (p = .067). We also found a sex-dependent effect of BMI on OS, with high BMI increased OS in female patients (p = .027) but showed no correlation in male patients (p = .310).
Fungi of the Pucciniales order cause rust diseases, which altogether affect thousands of plant species worldwide and pose major threat to several crops. How rust effectors - virulence proteins delivered into infected tissues to modulate host functions - contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplars and its genome encodes 1,184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of GFP-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth. This article is protected by copyright. All rights reserved.
To examine how alcohol brands use sport in their communication activities on social media. Despite extensive research exploring alcohol advertising and sponsorship through sport, minimal attention has been given to digital platforms.
This study undertakes a qualitative content analysis to examine the social media activity of alcohol brands sponsoring the three largest spectator sports in Australia: Australian rules football, rugby league and cricket.
Four sport-related social media strategies are identified through which alcohol brands solicit interaction with consumers, often involving co-creation of content and social activation. These strategies act as 'calls to action' and through the association of sport and alcohol encourage consumers to engage in competition, collaboration, celebration and consumption. These strategies are further strengthened by communications which draw upon themes of identity and camaraderie to resonate with the consumer.
Sport-linked social media strategies utilised by alcohol brands extend beyond just promoting their product. They seek higher levels of engagement with the consumer to amplify and augment the connection between alcohol and the sport spectator experience. The discussion highlights the powerful combination of sport and social media as a mechanism by which these brands seek to interact with consumers and encourage them to both create and promote content to their social networks. These strategies allow alcohol brands to extend their marketing efforts in a manner which can elude alcohol codes and prove difficult for regulators to identify and control. [Westberg K, Stavros C, Smith ACT, Munro G, Argus K. An examination of how alcohol brands use sport to engage consumers on social media. Drug Alcohol Rev 2016;00:000-000].
To examine changes in children's albuterol use and out-of-pocket (OOP) costs in response to increased copayments after the Food and Drug Administration banned inhalers with chlorofluorocarbon (CFC) propellants.
Four health maintenance organizations (HMOs), two that increased copayments for albuterol inhalers that went from generic CFC-containing to branded CFC-free versions, and two that retained generic copayments for CFC-free inhalers (controls). We included children with asthma aged 4-17 years with commercial coverage from 2007 to 2010.
Interrupted time series with comparison series.
We obtained enrollee and plan characteristics from enrollment files, and utilization data from pharmacy and medical claims; OOP expenditures were extracted from pharmacy claims for two HMOs with cost data available.
There were no significant differences in albuterol use between the group with increased cost-sharing and controls with respect to changes after the policy change. There was a postpolicy increase of $6.11 OOP per month per child using albuterol among those with increased cost-sharing versus $0.36 in controls; the difference between groups was significant (p < .01).
Increased copayments for brand-name CFC-free albuterol after the CFC ban did not lead to a decrease in children's albuterol use, but it led to a modest increase in OOP costs.
Four new ruthenium arene PTA type complexes have been synthesized using substituted picolinamide derivatives as ancillary ligands and characterized by spectroscopic methods. In one of the complexes, the ancillary ligand has shown an unprecedented valence-bond tautomerization in the presence of an ammonium salt to act as a polar neutral donor ligand making the ligand more prone towards substitution. The same compound has shown remarkable antiproliferative activity against three cancer cell lines with GI50 values comparable to Adriamycin, a known therapeutic drug. Along with this it also strongly inhibits the action of thioredoxin reductase, which might be a probable reason for the enhanced proliferative action of the valence-bond tautomerized compound.
The corticostriatothalamic circuit regulates learning behaviors via dopamine neurotransmission. D2 long (D2L) receptors are an isoform of dopamine D2 receptors (D2Rs) and may act mainly at postsynaptic sites. It is well known that D2Rs influence high brain functions, but the roles of individual D2R isoforms are still unclear. To assess the influence of D2L receptors in visual discrimination learning, we performed visual discrimination and reversal tasks with D2L knockout mice using a touchscreen operant system. There were no significant differences in an operant conditioning task between genotypes. However, D2L knockout mice were impaired in both visual discrimination and reversal learning tasks. D2L knockout mice were also significantly slower than wild-type mice in collecting the reward in the visual discrimination task. These results indicate that D2L receptors play an important role in visual discrimination and reversal learning.
More than a decade, overlapping genes in RNA viruses became a subject of research which has explored various effect of gene overlapping on the evolution and function of viral genomes like genome size compaction. Additionally, overlapping regions (OVRs) are also reported to encode elevated degree of protein intrinsic disorder (PID) in unspliced RNA viruses. With the aim to explore the roles of OVRs in HIV-1 pathogenesis, we have carried out an in-depth analysis on the association of gene overlapping with PID in 35 HIV1- M subtypes. Our study reveals an over representation of PID in OVR of HIV-1 genomes. These disordered residues endure several vital, structural features like short linear motifs (SLiMs) and protein phosphorylation (PP) sites which are previously shown to be involved in massive host-virus interaction. Moreover, SLiMs in OVRs are noticed to be more functionally potential as compared to that of non-overlapping region. Although, density of experimentally verified SLiMs, resided in 9 HIV-1 genes, involved in host-virus interaction do not show any bias toward clustering into OVR, tat and rev two important proteins mediates host-pathogen interaction by their experimentally verified SLiMs, which are mostly localized in OVR. Finally, our analysis suggests that the acquisition of SLiMs in OVR is mutually exclusive of the occurrence of disordered residues, while the enrichment of PPs in OVR is solely dependent on PID and not on overlapping coding frames. Thus, OVRs of HIV-1 genomes could be demarcated as potential molecular recognition sites during host-virus interaction.
Prostaglandin reductase-1 (Ptgr1) is an alkenal/one oxidoreductase that is involved in the catabolism of eicosanoids and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Recently, we reported that Ptgr1 is overexpressed in human clinical and experimentally induced samples of hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated and its role in carcinogenesis are not yet known. Here, we studied parameters associated with antioxidant responses and the mechanisms underlying the induction of Ptgr1 expression by the activation of Nuclear Factor (erythroid-derived-2)-like-2 (NRF2). For these experiments, we used two protocols of induced hepatocarcinogenesis in rats. Furthermore, we determined the effect of PTGR1 on cell proliferation and resistance to oxidative stress in cell cultures of the epithelial liver cell line, C9. Ptgr1 was overexpressed during the early phase in altered hepatocyte foci, and this high level of expression was maintained in persistent nodules until tumors developed. Ptgr1 expression was regulated by NRF2, which bound to an antioxidant response element at -653bp in the rat Ptgr1 gene. The activation of NRF2 induced the activation of an antioxidant response that included effects on proteins such as glutamate-cysteine ligase, catalytic subunit, NAD(P)H dehydrogenase quinone-1 (NQO1) and glutathione-S-transferase-P (GSTP1). These effects may have produced a reduced status that was associated with a high proliferation rate in experimental tumors. Indeed, when Ptgr1 was stably expressed, we observed a reduction in the time required for proliferation and a protective effect against hydrogen peroxide- and 4-HNE-induced cell death. These data were consistent with data showing colocalization between PTGR1 and 4-HNE protein adducts in liver nodules. These findings suggest that Ptgr1 and antioxidant responses act as a metabolic adaptation and could contribute to proliferation and cell-death evasion in liver tumor cells. Furthermore, these data indicate that Ptgr1 could be used to design early diagnostic tools or targeted therapies for HCC.
Gene expression often requires interaction between promoters and distant enhancers, which occur within the context of highly organized topologically associating domains (TADs). Using a series of engineered chromosomal rearrangements at the Shh locus, we carried out an extensive fine-scale characterization of the factors that govern the long-range regulatory interactions controlling Shh expression. We show that Shh enhancers act pervasively, yet not uniformly, throughout the TAD. Importantly, changing intra-TAD distances had no impact on Shh expression. In contrast, inversions disrupting the TAD altered global folding of the region and prevented regulatory contacts in a distance-dependent manner. Our data indicate that the Shh TAD promotes distance-independent contacts between distant regions that would otherwise interact only sporadically, enabling functional communication between them. In large genomes where genomic distances per se can limit regulatory interactions, this function of TADs could be as essential for gene expression as the formation of insulated neighborhoods.
The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.
In February, 2016, WHO released a report for the development of national action plans to address the threat of antibiotic resistance, the catastrophic consequences of inaction, and the need for antibiotic stewardship. Antibiotic stewardship combined with infection prevention comprises a collaborative, multidisciplinary approach to optimise use of antibiotics. Efforts to mitigate overuse will be unsustainable without learning and coordinating activities globally. In this Personal View, we provide examples of international collaborations to address optimal prescribing, focusing on five countries that have developed different approaches to antibiotic stewardship-the USA, South Africa, Colombia, Australia, and the UK. Although each country's approach differed, when nurtured, individual efforts can positively affect local and national antimicrobial stewardship programmes. Government advocacy, national guidelines, collaborative research, online training programmes, mentoring programmes, and social media in stewardship all played a role. Personal relationships and willingness to learn from each other's successes and failures continues to foster collaboration. We recommend that antibiotic stewardship models need to evolve from infection specialist-based teams to develop and use cadres of health-care professionals, including pharmacists, nurses, and community health workers, to meet the needs of the global population. We also recommend that all health-care providers who prescribe antibiotics take ownership and understand the societal burden of suboptimal antibiotic use, providing examples of how countries can learn, act globally, and share best antibiotic stewardship practices.
Ex-vivo lung perfusion (EVLP) can be used to extend overall lung preservation time by splitting one long cold ischaemic time into two shorter ones and interposing an additional EVLP time. We assessed the outcomes after clinical transplantation of lungs with more than 12 h of preservation time.
For this retrospective study, we searched the Toronto Lung Transplant Program database for patients who had received at least one lung transplant between Jan 1, 2006, and April 30, 2015, at a single hospital in Toronto, Canada. We split the identified patients into those with a total preservation time of more than 12 h and those with a total preservation time of less than 12 h to act as the control group. Total preservation time was defined as the sum of first cold ischaemic time, EVLP time, and second cold ischaemic time. We excluded patients if they had received a heart-lung transplant or were younger than 18 years. In bilateral lung transplantations, we used the longer preservation time of the two lungs for analysis. Lung preservation was done according to present standards of care and EVLP was done according to the Toronto EVLP technique. The primary outcomes were survival and International Society for Heart and Lung Transplantation Primary Graft Dysfunction (PGD) grade at 72 h post-transplantation. We compared outcomes with our control group using univariable and multivariable models.
We identified 906 patients who met eligibility criteria and had sufficient data for analysis (<12 h group [n=809]; mean lung preservation time 400·8 min [SD 121·8] vs >12 h group [n=97]; 875·7 min [109·0]). Median hospital and intensive-care unit length of stay were similar between the less than 12 h group and the more than 12 h group (hospital stay: 23 days [16-42] vs 25·5 days [17-50·25], p=0·60; intensive-care unit stay: 4 days [2-14] vs 4 days [2-16], p=0·53). PGD grade was also not different between the two groups at 72 h post-transplantation (p=0·85). There was also no difference in survival between the two groups as shown on Kaplan-Meier survival curves (p=0·61). Multivariable survival analysis using Cox's model showed increasing recipient age to be a significant variable affecting survival.
Extension of graft preservation time beyond 12 h with EVLP does not negatively affect early lung transplantation outcomes. Extension of clinical lung preservation times might allow for more transplantations to be done as a result of improved facilitation and increased flexibility around timing of lung transplantation operations.
None.
Both patients with non-neuronopathic Gaucher disease (GD) and heterozygous GBA mutation carrier are at increased risk for Parkinson disease (PD). The risk for PD in these groups does not linearly increase with glucosylceramide (GC) accumulation or with acid β-glucocerebrosidase (GCase) activity. This observation, together with other clinical systemic observations raises the possibility that extra-cellular GC actually has beneficial, anti-inflammatory, properties. Based on this hypothesis, we suggest here that the administration of supplementary oral GC to GBA carriers at risk for PD may slow inflammatory-driven secondary neuronal death. Such a treatment may act synergistically in GBA carriers once given in combination with an agent that prevent the primary pathologic process that leads to cell death. Ambroxol hydrochloride, a pharmacological chaperone, which reduces endoplasmic reticulum (ER) stress induced by accumulation of mutant misfolded GCase could serve as such an agent. The efficacy of this combined therapy, derived from clinical observations, in vivo and in vitro studies, should be evaluated in clinical trials.
I discuss biologist Garrett Hardin's view of human nature, with examples from the background to his seminal 1968 essay "The Tragedy of the Commons" and his testimony before the US House of Representatives in the 1970s. Hardin saw the human species as being governed by deterministic laws of the same kind that controlled all other forms of life. Humans, as much as cattle and microbes, were in inevitable competition for space and resources. Equal parts Malthusian political economy and Cold War systems science, his view was the survival of the human race depended on obeying these iron laws. Human freedom was the recognition of-and obedience to-its nature. This determination for humanity to act within the strictures placed on itself by its own nature was what he called "lifeboat ethics." In order for the citizens of the rich countries to survive, many in the Third World would have to die. In this sense I characterize Hardin's sense of life as tragic, both as a play on the title of his famous essay, and to emphasize his view that the problem of human population growth had no 'win-win' solutions.
Myocardial bridging (MB) and a long recurrent wraparound left anterior descending artery (wrap-LAD) are coronary anatomic variants that have been recently suggested to be associated with takotsubo syndrome (TS). Until now, coronary artery tortuosity (CAT) has never been investigated in this setting. Our study sought to evaluate the prevalence of the aforementioned anatomic variants in a large population with TS. In this retrospective angiographic study, 109 patients with TS were compared with 109 age- and gender-matched subjects without coronary artery disease, valve heart disease, or cardiomyopathy. CAT was identified by ≥3 consecutive curvatures ≥90° (criteria 1) or by ≥2 consecutive curvatures ≥180° (criteria 2). Wrap-LAD was defined if any part of the vessel outreached the apex of the left ventricle and MB as the presence of a milking effect or a step-up and step-down phenomenon. An anatomic variant was found in 79 patients with TS (72%) and in 48 controls (44%) (p <0.001). CAT in at least 1 vessel (criteria 1: 49% vs 20%, p <0.001; criteria 2: 38% vs 13%, p <0.001), ≥2 vessels (criteria 2: 14% vs 3%, p = 0.005), and wrap-LAD (41% vs 27%, p = 0.02) were significantly more frequent in patients with TS than in controls. The prevalence of MB (9% vs 5%, p = 0.18) did not differ between groups. In conclusion, CAT and wrap-LAD have higher prevalence in patients with TS than in matched controls. These findings could support the hypothesis that anatomic variants might act as potential pathogenic substrates in TS.
A facile and green strategy is reported here to synthesize gold (Au), silver (Ag) and gold-silver (Au-Ag) alloy nanoparticles (NPs) through bio-reduction reactions of aqueous corresponding metal precursors mediated by extracts of aerial parts of R. hypocrateriformis, which act as both reducing and stabilizing agents, under microwave irradiation. UV-vis spectrophotometer, XRD, FT-IR, FESEM/TEM, TGA and EDAX analysis were used to characterize the obtained NPs. The formation of NPs is evident from their surface plasmon resonance peak observed at λmax=∼550, 450 and 500nm for Au, Ag and Au-Ag alloy NPs respectively. XRD pattern revealed that fcc structure, while FT-IR spectra signify the presence of phytochemicals adsorbed on NPs. Such a biofunctionalized NPs were characterized by their weight loss, 30% due to thermal degradation of plant phytochemicals observed in TG analysis. The spherical shape of Au, Ag and Au-Ag alloy NPs (∼10-50nm) is observed by FE-SEM/TEM images. EDAX analysis confirms the expected elemental composition. Moreover, these NPs showed enhanced antimicrobial, antioxidant, and anticancer activities, though it is more pronounced for Au-Ag alloy NPs, which is due to the combining effect of phytochemicals, Au and Ag metals. Thus, the biosynthesized NPs could be applied as effective growth inhibitors for various biomedical applications.
This article identifies concepts, trends, and policy gaps in the availability and service delivery of assistive technology utilized by older adults in disasters, as well as implications for emergency management planning and shelter administration. Definitions of types of assistive technology, as well as views of older adults using technology as at-risk individuals for emergency management service provision, are provided. An overview of peer-reviewed articles and gray literature is conducted, focusing on publications from 2001 to the present in the United States. Analytical frameworks used by emergency management organizations as well as regulations such as the Americans with Disabilities Act and recent court decisions on emergency shelter accessibility in disasters are reviewed. Research on the use of assistive technology by older adults during disasters is a neglected issue. The current and potential benefits of defining standards for provision and use of assistive technology for older adults during disasters has received limited recognition in emergency management planning. Older adults with disabilities utilize assistive technology to maintain their independence and dignity, and communities as well as emergency services managers need to become more aware of the needs and preferences of these older adults in their planning processes and drills as well as in service delivery during actual events. (Disaster Med Public Health Preparedness. 2016;page 1 of 5).
1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene. In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which 1,3-diphenylisobenzofuran reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is 1,2-dibenzoylbenzene. Only under the action of H2O2 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis and cyclic voltammetry. A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196 - 3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 μM, respectively. The kinetics of the reaction between DBBF and H2O2 was also studied.
The mechanism of Fe (II) - oxone conditioning to improve sludge dewaterability was investigated in this study. Five different types of sludge were tested, including raw sludge (Group 1: mixed primary and secondary sludge, waste activated sludge and anaerobic digested sludge) and pretreated sludge with prior solubilisation (Group 2: ultrasonic or thermal pretreated sludge). After Fe (II) - oxone conditioning, the concentrations of dissolved organic carbon, protein and polysaccharide of soluble extracellular polymeric substances (SB EPS) increased for Group 1, but decreased for Group 2. For all types of sludge investigated, the related organic compounds of loosely bound (LB) and tightly bound (TB) EPS decreased with Fe (II) - oxone conditioning, and increased sludge filterability showed strong and positive correlation with the removal of low molecular weight protein and neutrals in LB EPS. Fe (II) - oxone was very effective in disintegrating cell membrane and caused potential cell lysis, as indicated by increased percentage of damaged microbial cells. From this study, the mechanism of Fe (II) - oxone conditioning was proposed and can be divided into two steps: (1) Oxidation step - sulfate radicals degraded organic compounds in LB and TB EPS in sludge and transformed bound water to free water that was trapped in TB and LB EPS; It also damaged cells membrane and may help to release intracellular water content. Sludge flocs were broken into smaller particles; (2) Coagulation step - Fe (III), generated from the oxidation step can act as a coagulant to agglomerate smaller particles into larger ones and reduce the repulsive electrostatic interactions. Combined effects from above two steps can greatly improve sludge filterability.
Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis.
Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aβ(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA.
We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA.
HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis.
Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.
Inonotus obliquus polysaccharides (IOPs) are a potential drug for the prevention and treatment of cancer, cardiopathy, diabetes, AIDs, pancreatitis and other diseases. In this study, we found that IOP can act as a broad-spectrum antiviral drug against feline viruses in the in vitro experiment. Using cell models of feline calicivirus (FCV), we demonstrated that IOP treatment was capable of exhibiting anti-FCV strain F9 activity in cell-based assays and also showed low cytotoxicity. Investigation of the mechanism of action of the compound revealed that IOP treatment induces its inhibitory actions directly on virus particles through blocking viral binding/absorpting. The inhibitory activity against other FCV isolates from China was also identified. More importantly, we found that IOP exhibited broad-spectrum antiviral activity against the feline herpesvirus 1, feline influenza virus H3N2 and H5N6, feline panleukopenia virus and feline infectious peritonitis virus that can contribute to respiratory and gastrointestinal diseases in cats. These findings suggest that IOP may be a potential broad-spectrum antiviral drug against feline viruses.
Monitoring and updating temporal predictions are critical abilities for adaptive behavior. Here, we investigated whether neural oscillations are related to violation and updating of temporal predictions. Human participants performed an experiment in which they had to generate a target at an expected time point, by pressing a button while taking into account a variable delay between the act and the stimulus occurrence. Our behavioral results showed that participants quickly adapted their temporal predictions in face of an error. Concurrent electrophysiological (EEG) data showed that temporal errors elicited markers that are classically related to error coding. Furthermore, intertrial phase coherence of frontal theta oscillations was modulated by error magnitude, possibly indexing the degree of surprise. Finally, we found that delta phase at stimulus onset was correlated with future behavioral adjustments. Together, our findings suggest that low frequency oscillations play a key role in monitoring and in updating temporal predictions.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the "one-molecule-one-target" paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with β-secretase (BACE1) activity, Aβ peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aβ peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease.
Mousepox is caused by the orthopoxvirus ectromelia virus (ECTV), and is thought to be transmitted via skin abrasions. We studied the ECTV virulence factor N1 following subcutaneous infection of mousepox-susceptible BALB/c mice. In this model, ECTV lacking N1L gene was attenuated more than 1000-fold compared with wild-type virus and replication was profoundly reduced as early as four days after infection. However, in contrast to data from an intranasal model, N1 protein was not required for virus dissemination. Further, neither T cell nor cytokine responses were enhanced in the absence of N1. Together with the early timing of reduced virus titres, this suggests that in a cutaneous model, N1 exerts its function at the level of infected cells or in the inhibition of the very earliest effectors of innate immunity.
Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high risk B-progenitor ALL such as BCR-ABL1 positive (Ph+) and Ph-like ALL, and are associated with poor outcome, even in the era of contemporary chemotherapy incorporating tyrosine kinase inhibitors in the treatment of Ph+ ALL. Recent experimental mouse modeling of B-progenitor ALL has shown that IKZF1 alterations have multiple effects, including arresting differentiating, skewing lineage of leukemia from myeloid to lymphoid, and in Ph+ leukemia, conferring resistance to TKI therapy without abrogating ABL1 inhibition. These effects are in part mediated by acquisition of an aberrant hematopoietic stem cell like program accompanied by induction of cell surface expression of stem cell and adhesion molecules that mediate extravascular invasion and residence in the niche, and activation of integrin signaling pathways. These effects can be exploited therapeutically using several approaches. IKZF1 alterations also result in upregulation of RXRA that encodes part of the heterodimeric retinoic acid X receptor. Rexinoids, a synthetic class of retinoids that specifically bind to retinoid "X" receptors, such as bexarotene potently reverse aberrant adhesion and niche mislocalization in vivo, and induce differentiation and cell cycle arrest. Focal adhesion kinase inhibitors block the downstream integrin-mediated signaling, and also reverse adhesion and niche mislocalization. Both agents act synergistically with TKI to prolong survival of Ph+ ALL in mouse and human xenograft model, with long term remission induced by FAK inhibitors. Thus, these findings provide important new conceptual insights into the mechanisms by which IKZF1 alterations result in drug resistance, and indicate that therapeutic strategies directed against the pathways deregulated by mutation, rather than attempting to restore IKZF1 expression directly, represent promising therapeutic approaches in this disease.
We developed the first user-friendly, semi-quantitative, and quick-to-perform Radboud Centre for Mitochondrial Medicine Pediatric MRI score (RCMM-PMRIS), focusing on the six most commonly described neuroimaging abnormalities in the literature. The RCMM-PMRIS was validated through individual review of 30 sets of brain MRI studies in 24 patients with genetically confirmed mitochondrial disorders by six raters. The application of RCMM-PMRIS can help to define the extent of the brain involvement and therefore to assess the radiological mitochondrial disease severity, to monitor disease progression and consequently to act as an outcome measure for treatment effects in patients with mitochondrial disease.
Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead.
Supernumerary teats (SNT) are any abnormal teats found on a calf in addition to the usual and functional 4 teats. The presence of SNT has also been termed hyperthelia since the end of the 19th century. Supernumerary teats can act as an incubator for bacteria, infecting the whole udder, and can interfere with the positioning of the milking machine, and consequently, have economic relevance. Different types of SNT are observed at different positions on the udder. Caudal teats are in the rear, ramal teats are attached to another teat, and intercalary teats are found between 2 regular teats. Not all teats are equally developed; some are completely functional but most are rudimentary and not attached to any mammary gland tissue. Recently, different studies showed the poly/oligogenic character of these malformations in cattle as well as in other mammalian species. The objective of this study was to analyze the genetic architecture and incidence of hyperthelia in Swiss Brown Swiss cattle using both traditional genetic evaluation as well as imputed whole genome sequence variant information. First, phenotypes collected over the last 20 yr were used together with pedigree information for estimation of genetic variance. Second, breeding values of Brown Swiss bulls were estimated applying the BLUP algorithm. The BLUP-EBV were deregressed and used as phenotypes in genome-wide association studies. The gene LGR5 on chromosome 5 was identified as a candidate for the presence of SNT. Using alternative trait coding, genomic regions on chromosome 17 and 20 were also identified as being involved in the development of SNT with their own supernumerary mammary gland tissue. Implementing knowledge gained in this study as a routine application allows a more accurate evaluation of the trait and reduction of SNT prevalence in the Swiss Brown Swiss cattle population.
The EMPA-REG OUTCOME and the LEADER trials have revealed a new era in the management of type 2 diabetes. The SGLT2 inhibitor empagliflozin demonstrated a lower rate of the primary composite outcome of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke compared to placebo. Liraglutide, a GLP-1 analogue, succeeded to demonstrate reduction on a composite outcome including first occurrence of cardiovascular death, nonfatal myocardial infarction or non-fatal stroke. These two medications act through different mechanisms and has consequently shown different patterns of cardiovascular benefit. In one hand, empagliflozin showed an earlier effect compared to those observed using liraglutide. On the other hand, the difference between empagliflozin and placebo was driven by a significant reduction in death from cardiovascular causes, with and striking disconnect showing no significant between-group difference in the risk of myocardial infarction or stroke. In contrast, liraglutide reduced consistently all components of the composite endpoint. Based on the different temporal pattern of achieving clinical benefit one might flirt with the idea that liraglutide seems to provide a chronic "protection" that better fits in a longer metabolic effect with an impact in the progression of atherosclerosis, whilst empagliflozin provides an acute effect compatible with an immediate hemodynamic action. After years going from "bench to bedside" in order to discover the holy grail of cardioprotection, these 2 new studies suggest that we may have reached this state and it is time to go from "bed back to bench side" to understand the mechanisms of this potential paradigm shift.
Searching for prospective vanadium-based drugs for cancer treatment, a new series of structurally related [V(IV)O(L-2H)(NN)] compounds (1-8) was developed. They include a double deprotonated salicylaldimine Schiff base ligand (L-2H) and different NN-polypyridyl co-ligands having DNA intercalating capacity. Compounds were characterized in solid state and in solution. EPR spectroscopy suggests that the NN ligands act as bidentate and bind through both nitrogen donor atoms in an axial-equatorial mode. The cytotoxicity was evaluated in human tumoral cells (ovarian A2780, breast MCF7, prostate PC3). The cytotoxic activity was dependent on type of cell and incubation time. At 24h PC3 cells presented low sensitivity, but at 72h all complexes showed high cytotoxic activity in all cells. Human kidney HEK293 and ovarian cisplatin resistant A2780cisR cells were also included to evaluate selectivity towards cancer cells and potency to overcome cisplatin resistance, respectively. Most complexes showed no detectable interaction with plasmid DNA, except 2 and 7 which depicted low ability to induce single strand breaks in supercoiled DNA. Based on the overall cytotoxic profile, complexes with 2,2´-bipyridine and 1,10-phenanthroline ligands (1 and 2) were selected for further studies, which consisted on cellular distribution and ultrastructural analyses. In the A2780 cells both depicted different distribution profiles; the former accumulates mostly at the membrane and the latter in the cytoskeleton. Morphology of treated cells showed nuclear atypia and membrane alterations, more severe for 1. Complexes induce different cell death pathways, predominantly necrosis for 1 and apoptosis for 2. Complexes alternative mode of cell death motivates the possibility for further developments.
The low-volume hospital (LVH) payment adjustment established in the Patient Protection and Affordable Care Act (ACA) of 2010 is scheduled to sunset on October 1, 2017. The purpose of this analysis was: (1) to estimate the effect of the ACA LVH adjustment on qualifying hospitals' profitability margins; and (2) to examine hospital and market characteristics of the hospitals that would be most adversely affected by the loss of the ACA LVH adjustment.
2004-2015 data from the Hospital Cost Report Information System, Hospital Market Service Area File and Nielsen-Claritas Pop-Facts file were used to estimate difference-in-difference regression models with hospital-level random effects in order to determine whether the ACA LVH adjustment improved qualifying rural hospitals' profitability margins. Recycled predictions estimated the effect of losing the ACA LVH adjustment on profitability margins. Bivariate analyses explored associations between the predicted profitability margins and hospital and market characteristics.
The ACA LVH adjustment significantly improved Sole Community Hospitals' Medicare inpatient margins in the year they received the adjustment, and it had a large but statistically insignificant effect on the profitability margins of other rural hospitals. Hospitals that would be the most adversely affected by loss of the ACA LVH adjustment were more likely to be small, located in the South, and in high-poverty markets with higher proportions of black and uninsured individuals.
Elimination of the ACA LVH adjustment would have differential effects on subgroups of hospitals, and those located in markets serving historically underserved populations would be the most adversely affected.
Sunlight-driven dinitrogen fixation can lead to a novel concept for the production of ammonia under mild conditions. However, the efficient artificial photosynthesis of ammonia from ordinary air (instead of high pure N2 ) has never been implemented. Here, we report for the first time the intrinsic catalytic activity of Bi2 MoO6 catalyst for direct ammonia synthesis under light irradiation. The edge-exposed coordinatively unsaturated Mo atoms in an Mo-O coordination polyhedron can act as activation centers to achieve the chemisorption, activation, and photoreduction of dinitrogen efficiently. Using that insight as a starting point, through rational structure and defect engineering, the optimized Bi2 MoO6 sunlight-driven nitrogen fixation system, which simultaneously possesses robust nitrogen activation ability, excellent light-harvesting performance, and efficient charge transmission was successfully constructed. As a surprising achievement, this photocatalytic system demonstrated for the first time ultra-efficient (1.3 mmol g(-1)  h(-1) ) and stable sunlight-driven nitrogen fixation from air in the absence of any organic scavengers.
The asexual freshwater planarian is a constitutive adult, whose central nervous system (CNS) is in a state of constant homeostatic neurogenesis. However, very little is known about the extrinsic signals that act on planarian stem cells to modulate rates of neurogenesis. We have identified two planarian homeobox transcription factors, Smed-nkx2.1 and Smed-arx, which are required for the maintenance of cholinergic, GABAergic, and octopaminergic neurons in the planarian CNS. These very same neurons also produce the planarian hedgehog ligand (Smed-hh), which appears to communicate with brain-adjacent stem cells to promote normal levels of neurogenesis. Planarian stem cells nearby the brain express core hh signal transduction genes, and consistent hh signaling levels are required to maintain normal production of neural progenitor cells and new mature cholinergic neurons, revealing an important mitogenic role for the planarian hh signaling molecule in the adult CNS.
Plant hormones are for a long time known to act as chemical messengers in the regulation of physiological processes during a plant's life cycle, from germination to senescence. Furthermore, plant hormones simultaneously coordinate physiological responses to biotic and abiotic stresses. To study the hormonal regulation of physiological processes, three main approaches have been used (1) exogenous application of hormones, (2) correlative studies through measurements of endogenous hormone levels, and (3) use of transgenic and/or mutant plants altered in hormone metabolism or signaling. A plant hormone profiling method is useful to unravel cross talk between hormones and help unravel the hormonal regulation of physiological processes in studies using any of the aforementioned approaches. However, hormone profiling is still particularly challenging due to their very low abundance in plant tissues. In this chapter, a sensitive, rapid, and accurate method to quantify all the five "classic" classes of plant hormones plus other plant growth regulators, such as jasmonates, salicylic acid, melatonin, and brassinosteroids is described. The method includes a fast and simple extraction procedure without time consuming steps as purification or derivatization, followed by optimized ultrahigh-performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS) analysis. This protocol facilitates the high-throughput analysis of hormone profiling and is applicable to different plant tissues.
The first signaling peptide discovered and purified was insulin in 1921. However, it was not until 1991 that the first peptide signal, systemin, was discovered in plants. Since the discovery of systemin, peptides have emerged as a potent and diverse class of signaling molecules in plant systems. Peptides consist of small amino acid sequences, which often act as ligands of receptor kinases. However, not all peptides are created equal, and signaling peptides are grouped into several subgroups dependent on the type of post-translational processing they undergo. Here, we focus on the application of synthetic, post-translationally modified peptides (PTMPs) to plant systems, describing several methods appropriate for the use of peptides in Arabidopsis thaliana and crop models.
Clinical and experimental studies have highlighted the significance of inflammation in coordinating wound repair and regeneration. However, it remains challenging to control the inflammatory response and tolerance at systemic levels without causing toxicity to injured tissues. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties and facilitate tissue repair by releasing exosomes, which generate a suitable microenvironment for inflammatory resolution. Exosomes contain several effective bioactive molecules and act as a cell-cell communication vehicle to influence cellular activities in recipient cells. During this process, the horizontal transfer of exosomal microRNAs (miRNAs) to acceptor cells, where they regulate target gene expression, is of particular interest for understanding the basic biology of inflammation ablation, tissue homeostasis, and development of therapeutic approaches. In this review, we describe a signature of three specific miRNAs (miR-21, miR-146a, and miR-181) present in human umbilical cord MSC-derived exosomes (MSC-EXO) identified microarray chip analysis and focus on the inflammatory regulatory functions of these immune-related miRNAs. We also discuss the potential mechanisms contributing to the resolution of wound inflammation and tissue healing.
In Niger, malaria is a major public health problem, due to the high number of deaths that are attributable to it and because of its heavy weight and socioeconomic status. The objective of this study was to contribute to the inventory of medical practices to rationalize the use of medicines and minimize the resistance phenomenon. This is a prospective study, which took place from May 1 to July 31, 2009, in the pediatric ward of the National Hospital of Lamordé. It concerned children aged from 0 to 14 years hospitalized and having received one or more antimalarial drugs during at least 24 h. During the 3 months of the study, 1,248 children had been admitted in the department. Among them, 881 children received antimalarial treatment, i.e., 70.5% with prescriptions. Malaria was confirmed by microscopy in 410 children, i.e., 46.5% of the sick children received antimalarial treatment. Prescription control was deemed noncompliant in 258 patients, i.e., 29.3% of the total. Treatments based on microscopic diagnosis are one of the strategies that will help to streamline the use of antimalarial drugs to improve their effectiveness and efficiency and also to reduce the risk of emergence of resistance.
The most effective anti-inflammatory drugs used to treat patients with airways disease are topical glucocorticosteroids (GCs). These act on virtually all cells within the airway to suppress airway inflammation or prevent the recruitment of inflammatory cells into the airway. They also have profound effects on airway structural cells to reverse the effects of disease on their function. Glucorticosteroids act via specific receptors-the glucocorticosteroid receptor (GR)-which are a member of the nuclear receptor family. As such, many of the important actions of GCs are to modulate gene transcription through a number of distinct and complementary mechanisms. Targets genes include most inflammatory mediators such as chemokines, cytokines, growth factors and their receptors. GCs delivered by the inhaled route are very effective for most patients and have few systemic side effects. However, in some patients, even high doses of topical or even systemic GCs fail to control their disease. A number of mechanisms relating to inflammation have been reported to be responsible for the failure of these patients to respond correctly to GCs and these provide insight into GC actions within the airways. In these patients, the side-effect profile of GCs prevent continued use of high doses and new drugs are needed for these patients. Targeting the defective pathways associated with GC function in these patients may also reactivate GC responsiveness.
The effect of practice facilitation that provides onsite quality improvement (QI) and electronic health record (EHR) coaching on chronic care outcomes is unclear. This study evaluates the effectiveness of such a program-similar to an agricultural extension center model-that provides these services.
Through the Health Information Technology for Economic and Clinical Health (HITECH) portion of the American Recovery and Reinvestment Act, the North Carolina Area Health Education Centers program became the Regional Extension Center for Health Information Technology (REC) for North Carolina. The REC program provides onsite technical assistance to help small primary care practices achieve meaningful use of certified EHRs. While pursuing meaningful use functionality, practices were also offered complementary onsite advice regarding QI issues. We followed the first 50 primary care practices that utilized both EHR and QI advice targeting diabetes care.
The achievement of meaningful use of certified EHRs and performance of QI with onsite practice facilitation showed an absolute improvement of 19% in the proportion of patients who achieved excellent diabetes control (hemoglobin A1c < 7%) compared to baseline. In addition, the percentages of patients with poorly controlled diabetes (hemoglobin A1c > 9%) fell steeply in these practices.
No control group was available for comparison.
Practice facilitation that provided EHR and QI coaching support showed important improvements in diabetes outcomes in practices that achieved meaningful use of their EHR systems. This approach holds promise as a way to help small primary care practices achieve excellent patient outcomes.
Besides degrading aberrant mRNAs that harbor a premature translation termination codon (PTC), nonsense-mediated mRNA decay (NMD) also targets many seemingly "normal" mRNAs that encode for full-length proteins. To identify a bona fide set of such endogenous NMD targets in human cells, we applied a meta-analysis approach in which we combined transcriptome profiling of knockdowns and rescues of the three NMD factors UPF1, SMG6 and SMG7. We provide evidence that this combinatorial approach identifies NMD-targeted transcripts more reliably than previous attempts that focused on inactivation of single NMD factors. Our data revealed that SMG6 and SMG7 act on essentially the same transcripts, indicating extensive redundancy between the endo- and exonucleolytic decay routes. Besides mRNAs, we also identified as NMD targets many long non-coding RNAs as well as miRNA and snoRNA host genes. The NMD target feature with the most predictive value is an intron in the 3' UTR, followed by the presence of upstream open reading frames (uORFs) and long 3' UTRs. Furthermore, the 3' UTRs of NMD-targeted transcripts tend to have an increased GC content and to be phylogenetically less conserved when compared to 3' UTRs of NMD insensitive transcripts.
Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators have been identified that drive gene expression required for organelle biogenesis during MCC differentiation, which are activated by, and act downstream, of two related, small coiled-coil proteins, Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in the Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those observed in mutants in Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 is required to co-activate some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that while Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants compared to single mutants. Finally, in contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. Together these results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of cilia in large numbers.
Allosteric modulators of pentameric ligand gated ion channels (pLGICs) are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors (nAChRs) and the α4β2-selective positive modulators 17β-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pLGICs. Previous studies have shown that the binding site for potentiating βEST is in the C-terminal (post-M4 region) of the α4 subunit. Here, using homology modelling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top-half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and F170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Y309) and F167, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating βEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.
The Family Smoking Prevention and Tobacco Control Act (FSPTCA) of 2009 creates the first national system of pre-market regulation of tobacco products in American history. The FDA must now review and give marketing authorization to all new tobacco products, based upon a public health standard, before they can be legally marketed. Yet the law also contains an alternative pathway for market entry-the substantial equivalence (SE) clause-by which novel and altered tobacco products can be marketed by demonstrating their substantial equivalence to existing products. Over 99 percent of tobacco product applications sent to the FDA under the new law have used this mechanism, and loose application of the SE mechanism carries the risk of undoing the FDA's gatekeeping power under the law. We review the statutory and regulatory precedent for SE, examining the FSPTCA itself as well as regulatory precedent from drug and device regulation (from which the term "substantial equivalence" and much of the associated statutory language was derived). Our review of standards and scientific precedent demonstrates that exacting scrutiny under the public health standard should govern all SE reviews, and that clinical data incorporating social scientific evidence should be routinely required for SE claims by tobacco product sponsors.
Polycomb chromobox (CBX) proteins participate in the polycomb repressive complex (PRC1) that mediates epigenetic gene silencing and endows PRC1 with distinct oncogenic or tumor suppressor functions in a cell-type-dependent manner. In this study, we report that inhibition of cell migration, invasion, and metastasis in colorectal carcinoma requires CBX4-mediated repression of Runx2, a key transcription factor that promotes colorectal carcinoma metastasis. CBX4 inversely correlated with Runx2 expression in colorectal carcinoma tissues, and the combination of high CBX4 expression and low Runx2 expression significantly correlated with overall survival, more so than either CBX4 or Runx2 expression alone. Mechanistically, CBX4 maintained recruited histone deacetylase 3 (HDAC3) to the Runx2 promoter, which maintained a deacetylated histone H3K27 state to suppress Runx2 expression. This function of CBX4 was dependent on its interaction with HDAC3, but not on its SUMO E3 ligase, its chromodomain, or the PRC1 complex. Disrupting the CBX4-HDAC3 interaction abolished Runx2 inhibition as well as the inhibition of cell migration and invasion. Collectively, our data show that CBX4 may act as a tumor suppressor in colorectal carcinoma, and strategies that stabilize the interaction of CBX4 with HDAC3 may benefit the colorectal carcinoma patients with metastases. Cancer Res; 76(24); 1-13. ©2016 AACR.
There are two superoxide dismutases in yeast: cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in yeast ethanol tolerance. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p - one of the key stress-resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1-target genes, were decreased in the absence of Sod2p. The analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondria-to-nucleus signaling by Rtg-proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance.
Bakers yeast harbor multiple systems that ensure tolerance to high concentrations of ethanol. Still, a role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of mitochondria-to-nucleus signaling pathway. We also report a crosstalk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.
The copper-containing enzyme nitrous oxide reductase (N2OR) catalyzes the transformation of nitrous oxide (N2O) to dinitrogen (N2) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites CuA and CuZ, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N2O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg(2+) that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.
The medicinal plant generally known as monkey's comb (Amphilophium crucigerum) has been popularly described for the treatment of neuropathic and inflammatory pain, specially seeds preparations.
The goal of the present study was to evaluate the antinociceptive effect of the crude extract (Crd) and dichloromethane fraction (Dcm) of A. crucigerum seeds, and investigate the involvement of transient receptor potential vanilloid 1 (TRPV1) receptor in this effect.
Male Swiss mice were used in this study. The effects of Crd and Dcm was tested on capsaicin-induced Ca(2+) influx or the specific binding of [(3)H]-resiniferatoxin. Moreover, after treatment with Crd or Dcm, animals were exposed to acute pain (hot water tail-flick and capsaicin intraplantar test) or chronic pain models (injection of complete Freund's adjuvant or partial ligation of the sciatic nerve). Acute adverse effects were also noted: locomotor activity, corporal temperature, hepatic or renal damage, gastrointestinal transit alteration, and ulcerogenic activity.
The oral administration of Crd or Dcm resulted in an antinociceptive effect in the hot water tail-flick (48°C) and capsaicin intraplantar tests. Furthermore, these preparations exhibited antinociceptive and anti-inflammatory effects in a chronic inflammatory pain model, and antinociceptive effects in a neuropathic pain model. Moreover, Crd and Dcm reduced capsaicin-induced Ca(2+) influx and diminished the [(3)H]-resiniferatoxin specific binding to spinal cord membranes. Acute adverse events were not found with Crd or Dcm administration.
In conclusion, our results support the analgesic effect of A. crucigerum and suggest the presence of compounds that may act as TRPV1 antagonists.
Bone marrow-derived cells are thought to participate and enhance the healing process contributing to skin cells or releasing regulatory cytokines. Directional cell migration in a weak direct current electric field (DC-EF), known as electrotaxis, may be a way of cell recruitment to the wound site. Here we examined the influence of electric field on bone marrow adherent cells (BMACs) and its potential role as a factor attracting mesenchymal stem cells to cutaneous wounds. We observed that in an external EF, BMAC movement was accelerated and highly directed with distinction of two cell populations migrating toward opposite poles: mesenchymal stem cells migrated toward the cathode, whereas macrophages toward the anode. Analysis of intracellular pathways revealed that macrophage electrotaxis mostly depended on Rho family small GTPases and calcium ions, but interruption of PI3K and Arp2/3 had the most pronounced effect on electrotaxis of MSCs. However, in all cases we observed only a partial decrease in directionality of cell movement after inhibition of certain proteins. Additionally, although we noticed the accumulation of EGFR at the cathodal side of MSCs, it was not involved in electrotaxis. Moreover, the cell reaction to EF was very dynamic with first symptoms occurring within <1min. In conclusion, the physiological DC-EF may act as a factor positioning bone marrow cells within a wound bed and the opposite direction of MSC and macrophage movement did not result either from utilizing different signalling or redistribution of investigated cell surface receptors.
Clay-bacterial interaction can significantly influence the biodegradation of organic contaminants in the environment. A moderate heat treatment of palygorskite could alter the physicochemical properties of the clay mineral and thus support the growth and function of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria. By using (14)C-labelled phenanthrene and a model bacterium Burkholderia sartisoli, we studied the mineralization of phenanthrene on the surface of a moderately heat-treated (up to 400°C) palygorskite. The heat treatment at 400°C induced a reduction of binding sites (e.g., by the elimination of organic matter and/or channel shrinkage) in the palygorskite and thus imparted a weaker sequestration of phenanthrene on its surface and within the pores. As a result, a supplement with the thermally modified palygorskite (400°C) significantly increased (20-30%; p<0.05) the biomineralization of total phenanthrene in a simulated soil slurry system. These results are highly promising to develop a clay mineral based technology for the bioremediation of PAH contaminants in water and soil environments.
During the National Geochemical Survey of Australia over 1300 top (0-10cm depth) and bottom (~60-80cm depth) sediment samples (including ~10% field duplicates) were collected from the outlet of 1186 catchments covering 81% of the continent at an average sample density of 1 site/5200km(2). The <2mm fraction of these samples was analysed for 59 elements by ICP-MS following an aqua regia digestion. Results are used here to establish the geochemical background variation of these elements, including potentially toxic elements (PTEs), in Australian surface soil. Different methods of obtaining geochemical threshold values, which differentiate between background and those samples with unusually high element concentrations and requiring attention, are presented and compared to Western Australia's 'ecological investigation levels' (EILs) established for 14 PTEs. For Mn and V these EILs are so low that an unrealistically large proportion (~24%) of the sampled sites would need investigation in Australia. For the 12 remaining elements (As, Ba, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sn and Zn) few sample sites require investigation and as most of these are located far from human activity centres, they potentially suggest either minor local contamination or mineral exploration potential rather than pollution. No major diffuse source of contamination by PTEs affects Australian soil at the continental scale. Of the statistical methods used to establish geochemical threshold values, the most pertinent results come from identifying breaks in cumulative probability distributions, the Tukey inner fence and the 98th percentile. Geochemical threshold values for 59 elements, including emerging 'high-tech' critical elements such as lanthanides, Be, Ga or Ge, for which no EILs currently exist, are presented.
ABO, H, secretor and Lewis histo-blood system genes control the expression of part of the carbohydrate repertoire present in areas of the body occupied by microorganisms. These carbohydrates, besides having great structural diversity, act as potential receptors for pathogenic and non-pathogenic microorganisms influencing susceptibility and resistance to infection and illness. Despite the knowledge of some structural variability of these carbohydrate antigens and their polymorphic levels of expression in tissue and exocrine secretions, little is known about their biological importance and potential applications in medicine. This review highlights the structural diversity, the biological importance and potential applications of ABO, H, Lewis and secretor histo-blood carbohydrates.
The study aims to investigate the expression of SIRT1 in articular cartilage of patients with primary knee osteoarthritis (OA) and its relationship with disease severity.
Cartilage tissue samples were collected from 38 knee OA patients and 9 normal healthy controls and then ascribed to normal, mild, moderate, and severe groups on the basis of the improved Mankin grading system. The expression of SIRT1 in articular cartilage was detected by immunohistochemistry and western blots. The expression of p53 and acetylated p53 (Ac-p53) was also measured by western blots.
The mutual comparisons of the SIRT1 expression levels in all groups have statistical significance except the one between the mild and moderate groups. Moreover, western blot results showed that the SIRT1 was decreased and p53/Ac-p53 were increased in the OA group. The average gray level of SIRT1 increases with the improving grade of the improved Mankin grading system scorers.
The expression of SIRT1 in articular cartilage is negatively associated with severity of knee OA, indicating that SIRT1 may act as a monitoring indicator for determining development and progression of knee OA.
Recent data have shown that γδ T cells can act as mediators for immune defense against tumors. Our previous study has demonstrated that persisting clonally expanded TRDV4 T cells might be relatively beneficial for the outcome of patients with T cell acute lymphoblastic leukemia after hematopoietic stem cell transplantation (HSCT). However, little is known about the distribution and clonality of the TRDV repertoire in T cell receptor (TCR) of γδ T cells and their effects on the clinical outcome of patients with acute myeloid leukemia (AML). The aim of this study was to assess whether the oligoclonal expansion of TCR Vδ T cells could be used as an immune biomarker for AML outcome.
γδ T cells were sorted from the peripheral blood of 30 patients with untreated AML and 12 healthy donors. The complementarity-determining region 3 (CDR3) sizes of eight TCR Vδ subfamily genes (TRDV1 to TRDV8) were analyzed in sorted γδ T cells using RT-PCR and GeneScan. The most frequently expressed TRDV subfamilies in the AML patients were TRDV8 (86.67 %) and TRDV2 (83.33 %), and the frequencies for TRDV1, TRDV3, TRDV4, and TRDV6 were significantly lower than those in healthy individuals. The most frequent clonally expanded TRDV subfamilies in the AML patients included TRDV8 (56.67 %) and TRDV4 (40 %). The clonal expansion frequencies of the TRDV2 and TRDV4 T cells were significantly higher than those in healthy individuals, whereas a significantly lower TRDV1 clonal expansion frequency was observed in those with AML. Moreover, the oligoclones of TRDV4 and TRDV8 were independent protective factors for complete remission. Furthermore, the oligoclonal expansion frequencies of TRDV5 and TRDV6 in patients with relapse were significantly higher than those in non-recurrent cases.
To the best of our knowledge, we characterized for the first time a significant alteration in the distribution and clonality of the TRDV subfamily members in γδ T cells sorted from AML patients. Clonally expanded TRDV4 and TRDV8 T cells might contribute to the immune response directed against AML, while oligoclonal TRDV5 and TRDV6 might occur in patients who undergo relapse. While the function of such γδ T cell clones requires further investigation, TRDV γδ T cell clones might be potential immune biomarkers for AML outcome.
Introduction of direct acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral-targeting compounds, is limited since HBV requires only four viral genes for its efficient replication/infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell-based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, while it did not affect the NTCP-mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan-genotypic anti-HBV activity and was also effective against a clinically relevant nucleoside analog-resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by co-treatment. Moreover, derivative analysis could identify small molecules that demonstrated more potent anti-HBV activity over PAC.
PAC and its analogs present a new class of anti-HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well-tolerated, and broadly active inhibitor of HBV infection. This article is protected by copyright. All rights reserved.
We previously observed reduced YWHAE (14-3-3ε) protein expression in a small set of gastric cancer samples. YWHAE may act as a negative regulator of the cyclin CDC25B, which is a transcriptional target of MYC oncogene. The understanding of YWHAE role and its targets is important for the better knowledge of gastric carcinogenesis. Thus, we aimed to evaluate the relationship among YWHAE, CDC25B, and MYC in vitro and in vivo. For this, we analyzed the YWHAE, CDC25B, and MYC expression in YWHA-silenced, CDC25B-silenced, and MYC-silenced gastric cancer cell lines, as well as in gastric cancer and non-neoplastic gastric samples. In gastric cancer cell lines, YWHAE was able to inhibit the cell proliferation, invasion and migration through the reduction of MYC and CDC25B expression. Conversely, MYC induced the cell proliferation, invasion and migration through the induction of CDC25B and the reduction of YWHAE. Most of the tumors presented reduced YWHAE and increased CDC25B expression, which seems to be important for tumor development. Increased MYC expression was a common finding in gastric cancer and has a role in poor prognosis. In the tumor initiation, the opposite role of YWHAE and CDC25B in gastric carcinogenesis seems to be independent of MYC expression. However, the inversely correlation between YWHAE and MYC expression seems to be important for gastric cancer cells invasion and migration. The interaction between YWHAE and MYC and the activation of the pathways related to this interaction play a role in the metastasis process.
Identification of a potential gene signature for improved diagnosis in non-small cell lung cancer (NSCLC) patient is necessary. Here, we aim to establish and validate the prognostic efficacy of a gene set that can predict prognosis and benefits of adjuvant chemotherapy (ACT) in NSCLC patients from various ethnicities. An 8-gene signature was calculated from the gene expression of 181 patients using univariate Cox proportional hazard regression analysis. The prognostic value of the signature was robustly validated in 1,477 patients from five microarray independent data sets and one RNA-seq data set. The 8-gene signature was identified as an independent predictor of patient survival in the presence of clinical parameters in univariate and multivariate analyses [hazard ratio (HR): 2.84, 95% confidence interval CI (1.74-4.65), p=3.06e-05, [HR] 2.62, 95% CI (1.51-4.53), p=0.001], respectively. Subset analysis demonstrated that the 8-gene signature could identify high-risk patients in stage II-III with improved survival from ACT [(HR) 1.47, 95% CI (1.01-2.14), p=0.044]. The 8-gene signature also stratified risk groups in EGFR-mutated and wild-type patients. In conclusion, the 8-gene signature is a strong and independent predictor that can significantly stratify patients into low- and high-risk groups. Our gene signature also has the potential to predict patients in stage II-III that are likely to benefit from ACT.
Purpose Colorectal cancer is the third most common cancer and the third leading cause of cancer deaths in the United States. Lack of insurance coverage has been associated with more advanced disease at presentation, more emergent admissions at time of colectomy, and lower survival relative to privately insured patients. The 2006 Massachusetts health care reform serves as a unique natural experiment to assess the impact of insurance expansion on colorectal cancer care. Methods We used the Hospital Cost and Utilization Project State Inpatient Databases to identify patients with colorectal cancer with government-subsidized or self-pay (GSSP) or private insurance admitted to a hospital between 2001 and 2011 in Massachusetts (n = 17,499) and three control states (n = 144,253). Difference-in-differences models assessed the impact of the 2006 Massachusetts coverage expansion on resection of colorectal cancer, controlling for confounding factors and secular trends. Results Before the 2006 Massachusetts reform, government-subsidized or self-pay patients had significantly lower rates of resection for colorectal cancer compared with privately insured patients in both Massachusetts and the control states. The Massachusetts insurance expansion was associated with a 44% increased rate of resection (rate ratio = 1.44; 95% CI, 1.23 to 1.68; P < .001), a 6.21 percentage point decreased probability of emergent admission (95% CI, -11.88 to -0.54; P = .032), and an 8.13 percentage point increased probability of an elective admission (95% CI, 1.34 to 14.91; P = .019) compared with the control states. Conclusion The 2006 Massachusetts health care reform, a model for the Affordable Care Act, was associated with increased rates of resection and decreased probability of emergent resection for colorectal cancer. Our findings suggest that insurance expansion may help improve access to care for patients with colorectal cancer.
High-throughput sequencing methods promise to improve our ability to infer the evolutionary histories of lineages and to delimit species. These are exciting prospects for the study of Australian vertebrates, a group comprised of many globally unique lineages with a long history of isolation. The evolutionary relationships within many of these lineages have been difficult to resolve with small numbers of loci, and we now know that many lineages also exhibit substantial cryptic diversity. Here, we present a set of phylogenetically diverse transcriptome resources to enable exon-based sequence capture studies of Australian vertebrates, including transcriptome sequences for four species of birds, four frogs, seven lizards and seven mammals. We also use exon data from the marsupial transcriptomes we generated to examine an approach for choosing a moderate number (dozens or hundreds) of phylogenetically informative exons based on a single transcriptome sequence, and a relatively distant reference genome.
The design, synthesis, and photoelectrochemical characterization of Co3 (PO4 )2 , a hydrogen evolving catalyst modified with reduced graphene oxide (RGO), is reported. The 3 D flowerlike Co3 (PO4 )2 heterojunction system, consisting of 3 D flowerlike Co3 (PO4 )2 and RGO sheets, was synthesized by a one-pot in situ photoassisted method under visible-light irradiation, which was achieved without the addition of surfactant or a structure-directing reagent. For the first time, Co3 (PO4 )2 is demonstrated to act as a hydrogen evolving catalyst rather than being used as an oxygen evolving photoanode. In particular, 3 D flowerlike Co3 (PO4 )2 anchored to RGO nanosheets is shown to possess dramatically improved photocatalytic activity. This enhanced photoactivity is mainly due to the staggered type II heterojunction system, in which photoinduced electrons from 3 D flowerlike Co3 (PO4 )2 transfer to the RGO sheets and result in decreased charge recombination, as evidenced by photoluminescence spectroscopy. The band gap of Co3 (PO4 )2 was calculated to be 2.35 eV by the Kubelka-Munk method. Again, the Co3 (PO4 )2 semiconductor displays n-type behavior, as observed from Mott-Schottky measurements. These RGO-Co3 (PO4 )2 conjugates are active in the visible range of solar light for water splitting and textile dye degradation, and can be used towards the development of greener and cheaper photocatalysts by exploiting solar light.
Despite significant workflow reform to comply with the federally mandated National Emergency Access Target (NEAT), Australian public hospitals continue to face significant barriers in achieving good ED patient flow. This study was undertaken to identify and analyse the impact of individual waypoints on an ED patient's journey and identify which waypoints act as bottlenecks to a hospital's 4 h ED disposition performance.
This study involves retrospective analysis and simulation employing 2 years of ED administrative data from a sample of two major and two large metropolitan hospitals in Queensland, Australia. The main outcome measures included waypoint wait times (Treatment Delay and Departure Delay), ED length of stay (EDLOS) and compliance with the NEAT target, measured for all (overall NEAT) and admitted (Admitted NEAT) patients. Variations in outcome measures were analysed as functions of hour of day, day of week, departure status and triage category. Simulations identified the impact of potential ED workflow changes in the context of NEAT performance.
Departure Delay accounted for 60 and 20% of EDLOS across large and major metropolitan hospitals, respectively. Higher gains in NEAT compliance are associated with improvements in departure delay rather than treatment delay. Simulation identified that halving Departure Delay improves Admitted NEAT by up to 22 and 4% at large and major metropolitan hospitals, respectively.
The results reinforces the need for a whole-of-hospital effort to address flow bottlenecks, and identify moving a patient from emergency to inpatient care as the critical bottleneck in ED system performance.
The nematode Bursaphelenchus xylophilus and its insect vectors from the Monochamus genus are major global quarantine pests of timber products. Due to the phaseout of methyl bromide for plant quarantine and pre-shipment treatments, an alternative fumigant is essential. Based on preliminary laboratory studies on the efficacy of ethanedinitrile (C2 N2 ) to B. xylophilus and Monochamus alternatus, three quarantine trials were conducted at three dosages and three temperatures. Potential for inhalation exposure was assessed by monitoring atmospheric C2 N2 in relation to the threshold limit value.
Concentration × time products (Ct) of 398.6, 547.2 and 595.9 g h m(-3) were obtained for each trial. A100% mortality of B. xylophilus and M. alternatus larvae at 23 ± 4 °C and 10 ± 4 °C occurred with a load factor of pine logs of 46% and at 3 ± 1 °C with a load factor of 30%. During all fumigations, atmospheric levels of C2 N2 20 m downwind were below the TLV. During aeration, levels 10 m and 5 m downwind were below the TLV after 0.4 h and 1 h, respectively.
For the purpose of quarantine or phytosanitary treatment, specific doses of C2 N2 at the trial temperatures could control B. xylophilus and M. alternatus larvae without significant inhalation risk to workers.
The aim of the present study was to understand the meaning of resilience, as described by people with schizophrenia. Building resilience is a component of recovery-oriented mental health care, and yet almost no research has been conducted into the resilience of people who live with schizophrenia and who are routinely considered vulnerable. Establishing the meaning of resilience in the context of schizophrenia is an important first step in building understanding. van Kaam's psychophenomenological method was used to interpret 14 interviews with people with schizophrenia who are currently well and living in the community. Resilience is invoked in the tension between opposing forces of challenge and support and in the act of 'striving' to take control of schizophrenia. Striving includes repeated, seemingly backwards steps, and during this, the person takes risks and seeks out and uses supportive people and resources. Those same supportive people and resources can also be challenging. Resilience is an energy embedded in the process of recovery from schizophrenia, and is manifest in an attitude of striving. Taking on challenges and engaging in risk is important within treatment and recovery from schizophrenia.
Climate warming is causing rapid loss of glaciers and snowpack in mountainous regions worldwide. These changes are predicted to negatively impact the habitats of many range-restricted species, particularly endemic, mountaintop species dependent on the unique thermal and hydrologic conditions found only in glacier-fed and snowmelt-driven alpine streams. Though progress has been made, existing understanding of the status, distribution, and ecology of alpine aquatic species, particularly in North America, is lacking, thereby hindering conservation and management programs. Two aquatic insects - the meltwater stonefly Lednia tumana and the glacier stonefly Zapada glacier - were recently proposed for listing under the U.S. Endangered Species Act due to climate-change-induced habitat loss. Using a large dataset (272 streams, 482 total sites) with high-resolution climate and habitat information, we describe the distribution, status, and key environmental features that limit L. tumana and Z. glacier across the northern Rocky Mountains. Lednia tumana was detected in 113 streams (175 sites) within Glacier National Park (GNP) and surrounding areas. The probability of L. tumana occurrence increased with cold stream temperatures and close proximity to glaciers and permanent snowfields. Similarly, densities of L. tumana declined with increasing distance from stream source. Zapada glacier was only detected in 10 streams (20 sites), six in GNP and four in mountain ranges up to ~600 km southwest. Our results show that both L. tumana and Z. glacier inhabit an extremely narrow distribution, restricted to short sections of cold, alpine streams often below glaciers predicted to disappear over the next two decades. Climate warming-induced glacier and snow loss clearly imperils the persistence of L. tumana and Z. glacier throughout their ranges, highlighting the role of mountaintop aquatic invertebrates as sentinels of climate change in mid-latitude regions. This article is protected by copyright. All rights reserved.
When skin dysbiosis occurs as a result of skin disorders, probiotics can act as modulators, restoring microbial balance. Several properties of selected probiotics were evaluated so that their topical application could be considered.
Adhesion, antimicrobial, quorum sensing and antibiofilm assays were carried out with several probiotic strains and tested against selected skin pathogens. All tested strains displayed significant adhesion to keratin. All lactobacilli with the exception of L. delbrueckii, showed antimicrobial activity against skin pathogens, mainly due to organic acid production. Most of them also prevented biofilm formation, but only P. innocua was able to break down mature biofilms.
This study demonstrates that although all tested probiotics adhered to human keratin, they showed limited ability to prevent adhesion of some potential skin pathogens. Most of the tested probiotics successfully prevented biofilm formation, suggesting that they may be successfully used in the future as a complement to conventional therapies in the treatment of a range of skin disorders.
The topical use probiotics may be a natural, targeted treatment approach to several skin disorders and a complement to conventional therapies which present many undesirable side effects. This article is protected by copyright. All rights reserved.
Asthma control can be assessed with the Asthma Control Test (ACT) and a score of 20 or higher indicates good asthma control. Patients pay for their consultation and treatment in the fee-for-service primary healthcare system in Singapore. We hypothesized that achieving asthma control would result in lower asthma costs through reduced acute exacerbations, fewer physician consultations and lower lost productivity. The study compared the healthcare costs of patients who achieved asthma control and those with suboptimal asthma control based on ACT scores. Factors influencing asthma control and healthcare expenditure over time were also examined.
A total of 736 patients were enrolled into an asthma care programme in two polyclinics during 2008 and 2013. Direct costs of asthma management were derived from the frequency of polyclinic consultations, medication costs and hospitalization. Indirect costs were estimated from lost workdays due to exacerbations. The generalized estimating equation (GEE) approach was used to longitudinally model the factors associated with total healthcare expenditure.
Patients with asthma control spent S$48 (US$36) more per doctor visit on asthma drugs (P < 0.01) but incurred S$65 (US$48) less per doctor visit in total costs (P < 0.01) than those with suboptimal asthma control. The savings from achieving asthma control for obese patients were greater than for normal-weight patients (S$42 or the equivalent of US$31; P < 0.05).
Optimal asthma control was associated with reduced healthcare costs. An effective treatment regimen should also consider other modifiable factors such as weight control to achieve asthma control and eventually reduce asthma costs.
Crisis helplines are designed to provide short-term support to people in an immediate crisis. However, there is a group of users who call crisis helplines frequently over an extended period of time. The reasons for their ongoing use remain unclear. The aim of this study was to investigate the differences in the reasons for calling between frequent and other users of crisis helplines. This was achieved by examining the findings from a brief survey completed by callers to Lifeline Australia at the end of their call between February and July 2015. In the survey, callers reported on their socio-demographics, reasons for their current call and number of calls made in the past month. Survey respondents were categorised as frequent, episodic and one-off users, and analyses were conducted using ordered logistic regression. Three hundred and fifteen callers completed the survey, which represented 57% of eligible callers. Twenty-two per cent reported calling 20 times or more in the past month (frequent users), 51% reported calling between 2 and 19 times (episodic users) and 25% reported calling once (one-off users). Two per cent were unable to recall the number of calls they made in the past month. Frequent users reported similar reasons for calling as other users but they were more likely to call regularly to talk about their feelings [OR = 6.0; 95% CI: 3.7-9.8]. This pattern of service use is at odds with the current model of care offered by crisis helplines which is designed to provide one-off support. There is a need to investigate further the factors that drive frequent users to call crisis helplines regularly.
Over the last decade, the recognized host range of hantaviruses has expanded considerably with the discovery of distinct hantaviruses in shrews, moles and bats. Unfortunately, in-depth studies of these viruses have been limited. Here we describe a comprehensive analysis of the spatial distribution, genetic diversity and evolution of Nova virus, a hantavirus that has the European mole as its natural host. Our analysis demonstrated that Nova virus has a high prevalence and widespread distribution in Belgium. While Nova virus displayed relatively high nucleotide diversity in Belgium, amino acid changes were limited. The nucleocapsid protein was subjected to strong purifying selection, reflecting the strict evolutionary constraints placed upon Nova virus by its host. Spatio-temporal analysis using Bayesian evolutionary inference techniques demonstrated that Nova virus had efficiently spread in the European mole population in Belgium, forming two distinct clades, representing east and west of Belgium. The influence of landscape barriers, in the form of the main waterways, on the dispersal velocity of Nova virus was assessed using an analytical framework for comparing Bayesian viral phylogenies with environmental landscape data. We demonstrated that waterways did not act as an environmental resistance factor slowing down Nova virus diffusion in the mole population. With this study, we provide information about the spatial diffusion of Nova virus and contribute sequence information that can be applied in further functional studies.
Histones and their posttranslational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive feature of some autoimmune and viral diseases. Electrophoretically and immunologically homogeneous IgGs containing no canonical enzymes were isolated from sera of human immunodeficiency virus-infected patients by chromatography on several affinity sorbents. In contrast to canonical proteases (trypsin, chymotrypsin, and proteinase K), IgGs from human immunodeficiency virus-infected patients purified by affinity chromatography on Sepharose containing immobilized histones specifically recognized and hydrolyzed only histones but not many other tested globular proteins. Using matrix-assisted laser desorption/ionization mass spectrometry, the sites of H1 histone (193 amino acids [AAs]) cleavage by anti-H1 histone IgGs were determined for the first time. It was shown that 1 cluster of 2 major and 4 moderate sites of cleavage is located at the beginning (106-112 AAs) of the known antigenic determinants disposed at the long C-terminal sequence of H1. Two clusters of minor and very weak sites of the protein cleavage correspond to middle (8 sites, 138-158 AAs) and terminal (5 sites, 166-176 AAs) parts of the antigenic determinants. It was shown that in contrast to canonical proteases, N-terminal part of H1 histone (1-136 AAs) containing no antigenic determinants is an unpredictably very resistant against hydrolysis by abzymes, while it can be easily cleavage by canonical proteases. Because histones act as damage-associated molecules, abzymes against H1 and other histones can play important role in pathogenesis of acquired immune deficiency syndrome and probably other different diseases.
Comparing the phenotypes produced when cells are treated with the Hsp90 inhibitors AUY922 or 17-AAG (classical inhibitors) to cells with Hsp90alpha knocked down reveals that the knockdown phenotype is unique from that produced by the inhibitors. Pull-down assays using classical inhibitors verify that these molecules do not bind to Hsp90 as their primary target. Classical inhibitors induce similar protein markers as other anti-cancer therapies Cisplatin and bortezomib, suggesting that AUY922 and 17-AAG act on targets other than Hsp90 and kill cells through multiple mechanisms. Comparing these classical inhibitors to C-terminal Hsp90 modulators reveals that C-terminal modulators effectively bind to Hsp90, and induce phenotypic markers consistent with the CRISPR knockdown data. Our findings challenge the current interpretation of Hsp90 inhibitors suggest that a large body of literature describing the Hsp90 phenotype and inhibitors requires re-examination.
Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two-dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78-kDa glucose-regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta-1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.
Carotenoids are lipid-soluble pigments and important for a variety of physiological functions. They are major dietary vitamin A precursors and act as lipophilic antioxidants in a variety of tissues and are associated with important health benefits in humans and animals. All animals must acquire carotenoids from their diet, but to our knowledge, there are no studies investigating the intestinal carotenoid absorption and their blood concentrations in New World camelids. The present study aimed to assess the serum concentrations of selected carotenoids in llamas (n = 13) and alpacas (n = 27). Serum carotenoids as well as retinol (vitamin A) and α-tocopherol (vitamin E) were determined by high-performance liquid chromatography coupled with mass spectrometry and these were unable to detect any carotenoids (α- and β-carotene, α- and β-cryptoxanthin, lutein, zeaxanthin, lycopene) in the samples. The concentrations of retinol in alpacas (2.89 ± 1.13 μmol/l; mean ± SD) were higher (p = 0.024) than those found in llamas (2.05 ± 0.87 μmol/l); however, the concentrations of α-tocopherol were not significantly (p = 0.166) different (llamas: 3.98 ± 1.83 μmol/l; alpacas: 4.95 ± 2.14 μmol/l). The results show that both llamas and alpacas are not able to absorb intact carotenoids, but efficiently convert provitamin A carotenoids to retinol.
In managed natural resource systems, such as fisheries and rangelands, there is a recognized trade-off between managing for short-term benefits and managing for longer-term resilience. Management actions that stabilize ecological attributes or processes can improve productivity in the supply of ecosystems goods and services short-term, but erode system resilience at longer time scales. For example, fire suppression in rangelands can increase grass biomass short-term, but ultimately result in an undesirable, shrub-dominated system. Analyses of this phenomenon have focused largely on how management actions influence slow-changing biophysical system attributes (such as vegetation composition). Data on the frequency of management actions that reduce natural ecological variation on 66 Private Land Conservation Areas (PLCAs) in South Africa were used to investigate how management actions are influenced by manager decision-making approaches; a largely ignored part of the problem. The pathology of natural resource management was evident on some PLCAs: an increased focus on revenue-generation in decision-making resulted in an increased frequency of actions to stabilize short-term variation in large mammal populations, which led to increased revenues from ecotourism or hunting. On many PLCAs, these management actions corresponded with a reduced focus on ecological monitoring, and an increase in extralimital species and game overstocking. Positives in natural resource management also existed, however, with some managers monitoring slower-changing ecological attributes, resulting in less intensive management, fewer extralimital species and lower stocking rates. This study's unique, empirical investigation of monitoring-management relationships illustrates that management informed by revenue-monitoring versus ecological-monitoring can have opposing consequences for natural resource productivity, and the likelihood of unsustainable management practices. Promoting management actions that maintain resilience in natural resource systems therefore requires cognizance of why managers act the way they do and how these actions can gradually shift managers towards unsustainable strategies. This article is protected by copyright. All rights reserved.
Variation in avian bilateral symmetry can be an indicator of development instability in response to a variety of stressors, including environmental contaminants. We used composite measures of fluctuating asymmetry to examine the influence of mercury concentrations in two tissues on fluctuating asymmetry within four waterbird species. Fluctuating asymmetry increased with mercury concentrations in whole blood and breast feathers of Forster's terns (Sterna forsteri), a species with elevated mercury concentrations. Specifically, fluctuating asymmetry in rectrix feather number one was the most strongly correlated structural variable of those tested (wing chord, tarsus, primary feather number 10, rectrix feather number 6) with mercury concentrations in Forster's terns. However, for American avocets (Recurvirostra americana), black-necked stilts (Himantopus mexicanus), and Caspian terns (Hydroprogne caspia), we found no relationship between fluctuating asymmetry and either whole blood or breast feather mercury concentrations, even though these species had moderate to elevated mercury exposure. Our results indicate that mercury contamination may act as an environmental stressor during development and feather growth, and contribute to fluctuating asymmetry of some species of highly contaminated waterbirds. This article is protected by copyright. All rights reserved.
Clinical caring science will be described from a theory of science perspective.
The aim of this theoretical article to give a comprehensive overview of clinical caring science as a human science-based discipline grounded in a theory of science argumentation.
Clinical caring science seeks idiographic or specific variations of the ontology, concepts and theories, formulated by caring science. The rationale is the insight that the research questions do not change when they are addressed in different contexts. The academic subject contains a concept order with ethos concepts, core and basic concepts and practice concepts that unites systematic caring science with clinical caring science. In accordance with a hermeneutic tradition, the idea of the caring act is based on the degree to which the theory base is hermeneutically appropriated by the caregiver. The better the ethos, essential concepts and theories are understood, the better the caring act can be understood. In order to understand the concept order related to clinical caring science, an example is given from an ongoing project in a disaster context.
The concept order is an appropriate way of making sense of the essence of clinical caring science. The idea of the concept order is that concepts on all levels need to be united with each other. A research project in clinical caring science can start anywhere on the concept order, either in ethos, core concepts, basic concepts, practice concepts or in concrete clinical phenomena, as long as no parts are locked out of the concept order as an entity. If, for example, research on patient participation as a phenomenon is not related to core and basic concepts, there is a risqué that the research becomes meaningless.
I. II. III. IV. V. References SUMMARY: Meiosis is fundamental to sexual reproduction and creates genetic variation in progeny. During meiosis paired homologous chromosomes undergo recombination, which can result in reciprocal crossovers. This process can recombine independently arising mutations onto the same chromosome. Recombination locations are highly variable between meioses, although total crossover numbers are tightly regulated. In addition to the effect of meiosis on genetic variation, sequence polymorphisms between homologous chromosomes can feedback onto the recombination pathways. Here we review the major crossover pathways in plants and some of the known homeostatic mechanisms that act during meiotic recombination. We then examine how sequence polymorphisms between homologous chromosomes, that is, heterozygosity, can influence meiotic recombination pathways in cis and trans. Finally, we provide a brief perspective on the relevance of these interconnections for natural selection and adaptation in plants.
Optimising training and performance through nutrition strategies is central to supporting elite sportspeople, much of which has focused on manipulating the relative intake of carbohydrate and fat and their contributions as fuels for energy provision. The ketone bodies, namely acetoacetate, acetone and β-hydroxybutyrate (βHB), are produced in the liver during conditions of reduced carbohydrate availability and serve as an alternative fuel source for peripheral tissues including brain, heart and skeletal muscle. Ketone bodies are oxidised as a fuel source during exercise, are markedly elevated during the post-exercise recovery period, and the ability to utilise ketone bodies is higher in exercise-trained skeletal muscle. The metabolic actions of ketone bodies can alter fuel selection through attenuating glucose utilisation in peripheral tissues, anti-lipolytic effects on adipose tissue, and attenuation of proteolysis in skeletal muscle. Moreover, ketone bodies can act as signalling metabolites, with βHB acting as an inhibitor of histone deacetylases, an important regulator of the adaptive response to exercise in skeletal muscle. Recent development of ketone esters facilitates acute ingestion of βHB that results in nutritional ketosis without necessitating restrictive dietary practices. Initial reports suggest this strategy alters the metabolic response to exercise and improves exercise performance, while other lines of evidence suggest roles in recovery from exercise. The present review focuses on the physiology of ketone bodies during and after exercise and in response to training, with specific interest in exploring the physiological basis for exogenous ketone supplementation and potential benefits for performance and recovery in athletes.
Despite substantial evidence of the benefits of breastfeeding for both mothers and children, rates of sustained breastfeeding in the United States are quite low. This study examined whether mandated coverage of lactation support services under the Affordable Care Act (ACA) affects breastfeeding behavior.
We studied the census of U.S. births included in the National Vital Statistics System from 2009 to 2014.
We used regression-adjusted difference-in-differences (DD) to examine changes in breastfeeding rates for privately insured mothers relative to those covered by Medicaid. We adjusted for several health and sociodemographic measures. We also examined the extent to which the effect varied across vulnerable populations-by race/ethnicity, maternal education, WIC status, and mode of delivery.
Results suggest that the ACA mandate increased the probability of breastfeeding initiation by 2.5 percentage points, which translates into about 47,000 more infants for whom breastfeeding was initiated in 2014. We find larger effects for black, less educated, and unmarried mothers.
The Affordable Care Act-mandated coverage of lactation services increased breastfeeding initiation among privately insured mothers relative to mothers covered by Medicaid. The magnitude of the effect size varied with some evidence of certain groups being more likely to increase breastfeeding rates.
To examine the impact of the Affordable Care Act on dental care use among poor adults ages 21-64 in 2014.
2010-2014 Gallup-Healthways Wellbeing Index Survey.
Among poor adults with income at or below 138% of the Federal Poverty Level, a differences-in-differences analysis was used to compare the changes in dental care use in states with different Medicaid expansion and adult dental policies.
Relative to the pre-reform period and other states, in Medicaid expansion states with adult dental benefits, dental care use increased between 2 and 6 percent points in the second half of 2014, but most of these changes were not statistically significant.
Early evidence suggests that the Affordable Care Act may either not be having a substantial impact on dental care use or it is too early to assess the impact.
The Sustainable Groundwater Management Act (SGMA) aims to control, for the first time in California's history, the state's significant use and depletion of groundwater. SGMA gives local agencies a high degree of discretion in relation to a new permitting power, but the discretion is a double-edged sword: agencies gain maximum flexibility to tailor their regime to local conditions, yet the statute provides no direction on appropriate components of a groundwater permitting regime. We introduce SGMA and the broader legislative context to its permitting power, and we explain the continuing common law context in which the legislation operates. This information is used as the foundation for a comparative legal analysis of fundamental elements of permitting regimes. We compare a selection of six other south-western permitting regimes established in legislation for areas recognized as requiring intensive management through permitting: "special permitting areas" (SPAs). We find that permitting regimes in south-western SPAs share a structure containing several almost universal elements, although the policy settings that apply to those elements vary widely. The established permitting regimes in the other south-western states' SPAs may inform Californian agencies seeking to use their new permitting power for the first time, as well as water agencies further afield, as to important components of a permitting regime, and the different policy settings that could apply to those components. Californian local agencies, and its Department of Water Resources, which is charged with providing local agencies technical advice, should have regard to these permitting possibilities.
We designed this study to investigate whether cadmium induces caspase-independent apoptosis and to investigate the relationship between the caspase-dependent and caspase-independent apoptotic pathways. Cadmium (1.25-2.5 μM) induced oxidative stress in rat proximal tubular (rPT) cells, as seen in the reactive oxygen species levels; N-acetylcysteine prevented this. Cyclosporin A (CsA) prevented mitochondrial permeability transition pore opening and apoptosis; there was mitochondrial ultrastructural disruption, mitochondrial cytochrome c (cyt c) translocation to the cytoplasm, and subsequent caspase-9 and caspase-3 activation. Z-VAD-FMK prevented caspase-3 activation and apoptosis and decreased BNIP-3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression levels and apoptosis-inducing factor/endonuclease G (AIF/Endo G) translocation. Simultaneously, cadmium induced prominent BNIP-3 expression in the mitochondria and cytoplasmic AIF/Endo G translocation to the nucleus. BNIP-3 silencing significantly prevented AIF and Endo G translocation and decreased the apoptosis rate, cyt c release, and caspase-9 and caspase-3 activation. These results suggest that BNIP-3 is involved in the caspase-independent apoptotic pathway and is located upstream of AIF/Endo G; both the caspase-dependent and caspase-independent pathways are involved in cadmium-induced rPT cell apoptosis and act synergistically.
Fiddler crabs (Brachyura, Ocypodidae), like many other marine organisms, disperse via planktonic larvae. A lengthy pelagic larval duration is generally assumed to result in genetic connectivity even among distant populations. However, major river outflows, such as of the Amazon or Orinoco, or strong currents may act as phylogeographic barriers to ongoing gene flow. For example, the Mona Passage, located between Puerto Rico and Hispaniola, has been postulated to impair larval exchange of several species. In this study, Cox1 mtDNA data was used to analyze population genetic structure of two fiddler crab species from the western Atlantic, comparing the continental coastline and Caribbean islands. The results indicate genetic homogeneity in Minuca rapax among Atlantic (continental) populations (Suriname, Brazil), whereas Caribbean populations show significantly restricted gene flow among the constituent islands and towards continental populations. Our data support the hypothesis of the Mona Passage hindering larval exchange. Contrastingly, Caribbean Leptuca leptodactyla populations appear to be devoid of detectable variation, while Atlantic-continental (i.e. Brazilian) populations show much higher haplotype and nucleotide diversities and display slight genetic differentiation among populations within the Atlantic region, though not statistically significant. Both species show a pronounced divergence between regions, supporting the presence of a phylogeographic barrier.
Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection.
Scatter-hoarding rodents can act as both predators and dispersers for many large-seeded plants because they cache seeds for future use, but occasionally forget them in sites with high survival and establishment probabilities. The most important fruit or seed trait influencing rodent foraging behavior is seed size; rodents prefer large seeds because they have higher nutritional content, but this preference can be counterbalanced by the higher costs of handling larger seeds. We designed a cafeteria experiment to assess whether fruit and seed size of Myrcianthes coquimbensis, an endangered desert shrub, influence the decision-making process during foraging by three species of scatter-hoarding rodents differing in body size: Abrothrix olivaceus, Phyllotis darwini and Octodon degus. We found that the size of fruits and seeds influenced foraging behavior in the three rodent species; the probability of a fruit being harvested and hoarded was higher for larger fruits than for smaller ones. Patterns of fruit size preference were not affected by rodent size; all species were able to hoard fruits within the entire range of sizes offered. Finally, fruit and seed size had no effect on the probability of seed predation, rodents typically ate only the fleshy pulp of the fruits offered and discarded whole, intact seeds. In conclusion, our results reveal that larger M. coquimbensis fruits have higher probabilities of being harvested, and ultimately of its seeds being hoarded and dispersed by scatter-hoarding rodents. As this plant has no other dispersers, rodents play an important role in its recruitment dynamics.
Hematopoietic stem cells (HSCs) are multipotent progenitors that generate all vertebrate adult blood lineages. Recent analyses have highlighted the importance of somite-derived signaling factors in regulating HSC specification and emergence from dorsal aorta hemogenic endothelium. However, these factors remain largely uncharacterized. We provide evidence that the vitamin A derivative retinoic acid (RA) functions as an essential regulator of zebrafish HSC formation. Temporal analyses indicate that RA is required for HSC gene expression prior to dorsal aorta formation, at a time when the predominant RA synthesis enzyme, aldh1a2, is strongly expressed within the paraxial mesoderm and somites. Previous research implicated the Cxcl12 chemokine and Notch signaling pathways in HSC formation. Consequently, to understand how RA regulates HSC gene expression, we surveyed the expression of components of these pathways in RA-depleted zebrafish embryos. During somitogenesis, RA-depleted embryos exhibit altered expression of jam1a and jam2a, which potentiate Notch signaling within nascent endothelial cells. RA-depleted embryos also exhibit a severe reduction in the expression of cxcr4a, the predominant Cxcl12b receptor. Furthermore, pharmacological inhibitors of RA synthesis and Cxcr4 signaling act in concert to reduce HSC formation. Our analyses demonstrate that somite-derived RA functions to regulate components of the Notch and Cxcl12 chemokine signaling pathways during HSC formation.
Bicuspid aortic valve patients can develop thoracic aortic aneurysms and therefore require serial imaging to monitor aortic growth. This study investigates the reliability of contrast-enhanced magnetic resonance angiography (CEMRA) volumetry compared with 2-dimensional diameter measurements to identify thoracic aortic aneurysm growth.
A retrospective, institutional review board-approved, and Health Insurance Portability and Accountability Act-compliant study was conducted on 20 bicuspid aortic valve patients (45 ± 8.9 years, 20% women) who underwent serial CEMRA with a minimum imaging follow-up of 11 months. Magnetic resonance imaging was performed at 1.5 T with electrocardiogram-gated, time-resolved CEMRA. Independent observers measured the diameter at the sinuses of Valsalva (SOVs) and mid ascending aorta (MAA) as well as ascending aorta volume between the aortic valve annulus and innominate branch. Intraobserver/interobserver coefficient of variation (COV) and intraclass correlation coefficient (ICC) were computed to assess reliability. Growth rates were calculated and assessed by Student t test (P < 0.05, significant). The diameter of maximal growth (DMG), defined as the diameter at SOV or MAA with the faster growth rate, was recorded.
The mean time of follow-up was 2.6 ± 0.82 years. The intraobserver COV was 0.01 for SOV, 0.02 for MAA, and 0.02 for volume (interobserver COV: 0.02, 0.03, 0.04, respectively). The ICC was 0.83 for SOV, 0.86 for MAA, 0.90 for DMG, and 0.95 for volume. Average aortic measurements at baseline and (follow-up) were 42 ± 3 mm (42 ± 3 mm, P = 0.11) at SOV, 46 ± 4 mm (47 ± 4 mm, P < 0.05) at MAA, and 130 ± 23 mL (144 ± 24 mL, P < 0.05). Average size changes were 0.2 ± 0.6 mm/y (1% ± 2%) at SOV, 0.5 ± 0.8 mm/y (1% ± 2%) at MAA, 0.7 ± 0.7 mm/y (2% ± 2%) at DMG, and 6 ± 3 mL/y (4% ± 3%) with volumetry.
Three-dimensional CEMRA volumetry exhibited a larger effect when examining percentage growth, a better ICC, and a marginally lower COV. Volumetry may be more sensitive to growth and possibly less affected by error than diameter measurements.
The authors offer this as a 'brief guide' to the Mental Health Act and the content is commendable.
To evaluate the reliability and validity of MED ID, a novel survey assessing caregiver-perceived ability to identify inhaled asthma medications.
We analyzed baseline data from the School-Based Asthma Care for Teens (SB-ACT) trial in Rochester, NY. Caregivers of adolescents with persistent asthma named the inhaled medications used by their child, and identified medications on a pictorial chart. Accurate identification was defined as completed matches between listed names and selected images. Caregivers answered the MED ID survey of four scaled questions on perceived ability to identify inhaled medications. We determined internal consistency reliability using Cronbach's alpha; examined concurrent validity by comparing MED ID sum scores with accurate identification using bivariate and multivariate analyses; and assessed the diagnostic utility of MED ID through ROC analysis.
126 caregivers (76% of enrolled) reported >1 inhaled medication; 52% of caregivers accurately identified medications. Two MED ID questions were removed during analysis. The two remaining questions had a score range of 2-10 points; higher scores indicate greater caregiver-perceived ability to identify medications. The Cronbach's alpha was 0.603. Accurate identification of medications was associated with a higher mean MED ID score (8.6 vs 7.6, P = 0.01). Accuracy was most strongly associated with MED ID scores ≥8 points (88% vs 60%, P<0.001, Phi 0.32); findings were consistent in regression analysis. The greatest AUC was seen with MED ID scores ≥8 points (0.638).
The two item MED ID survey is a reliable and valid way to assess caregiver ability to identify inhaled asthma medications.
A long-standing hypothesis asserts that plant-feeding insects specialize on particular host plants because of negative interactions (trade-offs) between adaptations to alternative hosts, yet empirical evidence for such trade-offs is scarce. Most studies have looked for microevolutionary performance trade-offs within insect species, but host use could also be constrained by macroevolutionary trade-offs caused by epistasis and historical contingency. Here we used a phylogenetic approach to estimate the micro- and macroevolutionary correlations between use of alternative host-plant taxa within two major orders of plant-feeding insects: Lepidoptera (caterpillars) and Hemiptera (true bugs). Across 1,604 caterpillar species, we found both positive and negative pairwise correlations between use of 11 host-plant orders, with overall network patterns suggesting that different host-use constraints act over micro- and macroevolutionary timescales. In contrast, host-use patterns of 955 true bug species revealed uniformly positive correlations between use of the same 11 host plant orders over both timescales. The lack of consistent patterns across timescales and insect orders indicates that host-use trade-offs are historically contingent rather than universal constraints. Moreover, we observed few negative correlations overall despite the wide taxonomic and ecological diversity of the focal host-plant orders, suggesting that positive interactions between host-use adaptations, not trade-offs, dominate the long-term evolution of host use in plant-feeding insects.
Due to its physiological and clinical roles, carbonic anhydrase (CA) is one of the most interesting case studies. There are different classes of CAinhibitors including sulfonamides, polyamines, coumarins and dithiocarbamates (DTCs). However, many of them hardly act as a selective inhibitor against a specific isoform. Therefore, finding highly selective inhibitors for different isoforms of CA is still an ongoing project. Proteochemometrics modeling (PCM) is able to model the bioactivity of multiple compounds against different isoforms of a protein. Therefore, it would be extremely applicable when investigating the selectivity of different ligands towards different receptors. Given the facts, we applied PCM to investigate the interaction space and structural properties that lead to the selective inhibition of CA isoforms by some dithiocarbamates. Our models have provided interesting structural information that can be considered to design compounds capable of inhibiting different isoforms of CA in an improved selective manner. Validity and predictivity of the models were confirmed by both internal and external validation methods; while Y-scrambling approach was applied to assess the robustness of the models. To prove the reliability and the applicability of our findings, we showed how ligands-receptors selectivity can be affected by removing any of these critical findings from the modeling process.
WHAT IS KNOWN ON THE SUBJECT?: There is disagreement about the costs and benefits of asking women about their abuse experiences in practice and research settings. No known meta-syntheses have been conducted to evaluate the qualitative data that exist on the experiences of women being asked about their abuse. WHAT THIS PAPER ADDS TO THE EXISTING KNOWLEDGE?: This review adds robust qualitative evidence that interviewing women about their abuse experiences can be a beneficial and healing experience for them. Mental health nurses are in an ideal position to create a therapeutic environment to interview women about their abuse experiences. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Mental health nurses are strongly encouraged to ask about and document abuse in all of their interviews and assessments, as well as act as advocates and discuss reported abuse within their interdisciplinary teams, to provide individualized, trauma-informed care. The mental health nurse should listen attentively, avoid judgement, offer reassurance, make appropriate referrals and provide resources for women, including those with both past and current abuse histories. Nurses are strongly encouraged to ask about abuse in all settings in which they encounter women, including in psychiatric and mental health settings.
Introduction Collections of quantitative data exist outlining the costs and benefits of asking female research participants about their abuse experiences; however, no known meta-syntheses have been conducted to evaluate the qualitative data that exist on the experiences of women being asked about their abuse. Aim/Question The purpose of this qualitative systematic review was to analyse and interpret qualitative findings regarding asking women about their abuse experiences with the intention of understanding risks and benefits. Method The sample (N = 11) was derived from an expansive search of peer-reviewed literature using multidisciplinary electronic databases. Qualitative findings were extracted, coded and categorized. Reflective memos were developed, and themes emerged. Results While initially distressing, being interviewed about abuse is more beneficial than harmful for women, due to the therapeutic process of talking about abuse. Discussion To maximize the therapeutic impact of discussing abuse, women must maintain autonomy and feel they are in a safe and confidential environment. Within this supportive atmosphere, very few women report any regret after discussing abuse and are able to identify positive outcomes. Implications for practice Findings from this review support the need for mental health nurses and other clinicians to create an optimal environment for discussing abuse and offer relevant practice recommendations. Researchers are encouraged to include women in studies that involve asking about abuse experiences.
The incredible pollination mechanisms displayed by orchid flowers has inspired biologists over the centuries. Based on the intriguing flower structures, the relationship among orchid species and their pollinators has been frequently regarded as very specialised. Given that visits on flowers pollinated by oil-collecting bees are regularly rare, and in Oncidiinae the flowers frequently attractexclusively species that act as effective pollinators, the comparative reproductive biology and pollinator specificity of two sympatric Gomesa (G. varicosa and G. montana; Oncidiinae) were analysedbased on records of floral morphology, production of floral rewards, pollinators and pollination mechanisms. Furthermore, experimental pollinations were carried out in order to examine the breeding systems. The results have show that in the studied population, both Gomesa are visited by several bee species, but these orchids present a specific pollination system.Pollinaria are deposited on the head of Centridini (G. varicosa and G. montana) and Epicharitini (G. varicosa) bees when landed on the central callus of the labellumto collect lipoidal substances produced by glandular elaiophores on lateral lobes of the labellum. Both species are dependent on a biotic pollen vector to set fruits. Gomesamontana is completely self-incompatible, while G. varicosa is partially self-compatible. Our results indicate that although the occurrence of self-sterile species seems to be common in Oncidiinae, in partially self-incompatible species, as is the case of G. varicosa, self-compatibility has been considered as an important factor favouring reproductive assurance in populations with low visitation frequencies, despite occurrence of inbreeding depression.
Biting midges in the genus Culicoides act as vectors of arboviruses throughout the world and as vectors of filariasis in Latin America, the Caribbean, and parts of Africa. Although Culicoides spp. are currently not considered to be vectors of Leishmania protozoa, the high abundance of biting midges in areas with active cutaneous leishmaniasis transmission points to the possibility of Culicoides infection by these pathogens. We used PCR to test captured Culicoides species for natural infection with Leishmania spp. We tested 450 Culicoides females, divided into 30 pools of 15 individuals each, as follows: nine pools of C. foxi (135 specimens), seven pools of C. filariferus (105), seven pools of C. insignis (105), five pools of C. ignacioi (75), and two pools of C. flavivenula (30). PCR confirmed the presence of Leishmania braziliensis DNA in C. ignacioi (0.14%), C. insignis (0.14%), and C. foxi (0.11); and Le. amazonensis DNA in C. filariferus (0.14%) and C. flavivenula (0.50%). We conclude that these Culicoides species can be naturally infected, but vector competence and transmission capability must be confirmed in future studies. Our results warrant further investigation into the role of these biting midge species in the leishmaniasis epidemiological cycle.
Understanding of the extent of acclimation of light-saturated net photosynthesis (An ) to temperature (T), and associated underlying mechanisms, remains limited. This is a key knowledge gap given the importance of thermal acclimation for plant functioning, both under current and future higher temperatures, limiting the accuracy and realism of Earth System Model (ESM) predictions. Given this, we analysed and modelled T-dependent changes in photosynthetic capacity in 10 wet-forest tree species; six from temperate forests and four from tropical forests. Temperate and tropical species were each acclimated to three daytime growth temperatures (Tgrowth ): temperate - 15, 20 and 25°C; tropical - 25, 30 and 35°C. CO2 response curves of An were used to model maximal rates of RuBP (ribulose-1,5-bisphosphate) carboxylation (Vcmax ) and electron transport (Jmax ) at each treatment's respective Tgrowth , and at a common measurement T (25°C). SDS-PAGE gels were used to determine abundance of the CO2 -fixing enzyme, Rubisco. Leaf chlorophyll, nitrogen (N) and mass per unit leaf area (LMA) were also determined. For all species and Tgrowth , An at current atmospheric CO2 partial pressure was Rubisco-limited. Across all species, LMA decreased with increasing Tgrowth . Similarly, area-based rates of Vcmax at a measurement T of 25°C (Vcmax(25) ) linearly declined with increasing Tgrowth , linked to a concomitant decline in total leaf protein per unit leaf area and Rubisco as a percentage of leaf N. The decline in Rubisco constrained Vcmax and An for leaves developed at higher Tgrowth and resulted in poor predictions of photosynthesis by currently widely used models that do not account for Tgrowth -mediated changes in Rubisco abundance that underpin the thermal acclimation response of photosynthesis in wet-forest tree species. A new model is proposed that accounts for the effect of Tgrowth -mediated declines in Vcmax(25) on An , complementing current photosynthetic thermal acclimation models that do not account for T-sensitivity of Vcmax(25) . This article is protected by copyright. All rights reserved.
To compare a novel multicoil compressed sensing technique with flexible temporal resolution, golden-angle radial sparse parallel (GRASP), to conventional fat-suppressed spoiled three-dimensional (3D) gradient-echo (volumetric interpolated breath-hold examination, VIBE) MRI in evaluating the conspicuity of benign and malignant breast lesions.
Between March and August 2015, 121 women (24-84 years; mean, 49.7 years) with 180 biopsy-proven benign and malignant lesions were imaged consecutively at 3.0 Tesla in a dynamic contrast-enhanced (DCE) MRI exam using sagittal T1-weighted fat-suppressed 3D VIBE in this Health Insurance Portability and Accountability Act-compliant, retrospective study. Subjects underwent MRI-guided breast biopsy (mean, 13 days [1-95 days]) using GRASP DCE-MRI, a fat-suppressed radial "stack-of-stars" 3D FLASH sequence with golden-angle ordering. Three readers independently evaluated breast lesions on both sequences. Statistical analysis included mixed models with generalized estimating equations, kappa-weighted coefficients and Fisher's exact test.
All lesions demonstrated good conspicuity on VIBE and GRASP sequences (4.28 ± 0.81 versus 3.65 ± 1.22), with no significant difference in lesion detection (P = 0.248). VIBE had slightly higher lesion conspicuity than GRASP for all lesions, with VIBE 12.6% (0.63/5.0) more conspicuous (P < 0.001). Masses and nonmass enhancement (NME) were more conspicuous on VIBE (P < 0.001), with a larger difference for NME (14.2% versus 9.4% more conspicuous). Malignant lesions were more conspicuous than benign lesions (P < 0.001) on both sequences.
GRASP DCE-MRI, a multicoil compressed sensing technique with high spatial resolution and flexible temporal resolution, has near-comparable performance to conventional VIBE imaging for breast lesion evaluation.
3 J. Magn. Reson. Imaging 2016.
Detailed molecular profiling of Oryza sativa (rice) was carried out to uncover the features that are essential to germination and early seedling growth under anoxic conditions. Temporal analysis of the transcriptome and methylome from germination to young seedlings under aerobic and anaerobic conditions revealed 82% similarity in the transcriptome and no differences in the epigenome up to 24-h. Following germination, significant transcriptomic and DNA methylation changes were observed between 4-d aerobic and anaerobic grown coleoptiles. A link between the epigenomic state and cell division versus cell elongation is suggested, as no DNA methylation differences were observed between 24-h aerobic and anaerobic germinating embryos, when there is little cell division. After that, epigenetic changes appear to correlate with differences between cell elongation (anaerobic conditions) versus cell division (aerobic conditions) in the coleoptiles. Re-oxygenation of 3-d anaerobic grown seedlings resulted in rapid transcriptomic DNA methylation changes in these coleoptiles. Unlike the transcriptome, DNA methylation changes upon re-oxygenation did not reflect those seen in aerobic coleoptiles, but instead, reverted to a pattern similar to dry seeds. Reversion to the 'dry seed' state of DNA methylation upon re-oxygenation may act to "reset the clock" for the rapid molecular changes and cell division that result upon re-oxygenation. This article is protected by copyright. All rights reserved.
Our study aims to test the effectiveness of binding communication based interventions (vs classical persuasive communication based ones) inciting non-donors to act in favour of blood donation.
The implementation of effective communication interventions represents a major public health issue. Nevertheless, persuasive media campaigns appear to have little effect on behaviours. Even though non-donors hold a positive attitude towards blood donation, they are not inclined to donate. As an alternative to producing behavioural changes, many recent studies have shown the superiority of binding communication over persuasive communication.
All participants, non-donors, were randomly assigned to one of four experimental conditions of a 2 (type of communication: persuasive vs binding) × 2 (source credibility: low vs high) factorial design. Then, they were asked to report their intention to donate blood, and their intention to distribute leaflets regarding blood donation.
Binding communication is a more effective strategy for increasing intention towards blood donation compared with persuasive communication, especially when combined with high credibility source.
Accordingly this study calls for more consideration of knowledge of social psychology to design effective communication interventions and increase the number of donations.
Psychological essentialism is a folk theory characterized by the belief that a causal internal essence or force gives rise to the common outward behaviors or attributes of a category's members. In two studies, we investigated whether 4- to 7-year-old children evidenced essentialist reasoning about heart transplants by asking them to predict whether trading hearts with an individual would cause them to take on the donor's attributes. Control conditions asked children to consider the effects of trading money with an individual. Results indicated that children reasoned according to essentialism, predicting more transfer of attributes in the transplant condition versus the non-bodily money control. Children also endorsed essentialist transfer of attributes even when they did not believe that a transplant would change the recipient's category membership (e.g., endorsing the idea that a recipient of a pig's heart would act pig-like, but denying that the recipient would become a pig). This finding runs counter to predictions from a strong interpretation of the "minimalist" position, an alternative to essentialism.
Individuals with Down syndrome (DS) are diagnosed with autism spectrum disorder (ASD) at a significantly higher frequency than the typical population. The differentiation of ASD symptoms from those of severe intellectual disability presents diagnostic challenges, which have led to more refined methods in the clinical evaluation of ASD in DS. These improved phenotypic characterization methods not only provide better diagnosis of ASD in DS, but may also be useful in elucidating the etiology of the increased prevalence of ASD in DS. Because all individuals with the classic presentation of DS have trisomy 21, it is possible that those with co-occurring DS and ASD may have additional genetic variants which can act as modifiers of the phenotype, leading to the development of ASD. © 2016 John Wiley & Sons, Ltd.
The aim of this study was to identify the species of ked infesting dogs in the cities of central Poland. A total of 510 dogs were observed between June and September 2015. The presence of keds was noted in 182 (35.7%) animals. Keds were more prevalent in female (38.0%) than in male (33.2%) dogs, and were more frequently found in animals younger than 1 year (46.2%) and in long-haired dogs (36.6%). The body areas most heavily colonized by keds were the groin (35.4%) and neck (21.4%). A total of 904 keds were isolated from dogs, including Hippobosca equina (Diptera: Hippoboscidae) (17.2%), Lipoptena cervi (Diptera: Hippoboscidae) (32.0%), and two species not previously encountered in Poland: Hippobosca longipennis (45.0%) and Lipoptena fortisetosa (5.9%). Hippoboscidae may act as vectors of pathogens and any shifts in their geographic range may lead to the spread of new diseases affecting animals.
The ST18 gene was proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis was missing so far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 mRNA and protein were detectable neither in the normal liver nor in cultured hepatoblasts, but were readily expressed following subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as co-culture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors, caused rapid tumor involution, associated with pervasive morphological changes, proliferative arrest and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in the hepatoblasts. Finally, RNA-seq profiling of ST18-depleted tumors prior to involution revealed down-regulation of inflammatory response genes, pointing to the suppression of NF-kB-dependent transcription.
ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. This article is protected by copyright. All rights reserved.
Studies of the polymorphism of central metabolic genes as a source of fitness variation in natural populations date back to the discovery of allozymes in the 1960s. The unique features of these genes and their enzymes and our knowledge base greatly facilitates the systems-level study of this group. The expectation that pathway flux control is central to understanding the molecular evolution of genes is discussed, as well as studies that attempt to place gene-specific molecular evolution and polymorphism into a context of pathway and network architecture. There is an increasingly complex picture of the metabolic genes assuming additional roles beyond their textbook anabolic and catabolic reactions. In particular, this review emphasizes the potential role of these genes as part of the energy-sensing machinery. It is underscored that the concentrations of key cellular metabolites are the reflections of cellular energy status and nutritional input. These metabolites are the top-down signaling messengers that set signaling through signaling pathways that are involved in energy economy. I propose that the polymorphisms in central metabolic genes shift metabolite concentrations and in that fashion act as genetic modifiers of the energy-state coupling to the transcriptional networks that affect physiological trade-offs with significant fitness consequences.
Serine-arginine protein kinase 1 (SRPK1) phosphorylates proteins involved in the regulation of several mRNA processing pathways including alternative splicing. SRPK1 has been recently reported to be over-expressed in multiple cancers including prostate, breast, lung and glioma. Several studies have shown that inhibition of SRPK1 has anti-tumoural effects and consequently SRPK1 has become a new candidate for targeted therapies. Interestingly, in terms of molecular mechanism, SRPK1 seems to act heterogeneously and has been reported to affect several processes in different cancers, for example angiogenesis in prostate and colon cancer, apoptosis in breast and colon cancer and migration in breast cancer. A recent report adds to this puzzle, showing that the main effect of overexpression of SRPK1 in non-small-cell lung carcinoma is to stimulate a stem cell-like phenotype. This pleiotropy might be related to preferential activation of different downstream signaling pathways by SRPK1 in various cancers.
Following the complete eradication of the alien piscivorous perch Perca fluviatilis from a potable reservoir, the abundance of the endemic western minnow Galaxias occidentalis, which was previously undetectable prior to the initial eradication event, increased dramatically. The study reveals the potential of reservoirs to act as ecological refuges and has implications for understanding the relative effects of alien fishes v. habitat alteration on native freshwater fishes.
The global expansion of aquaculture has changed the structure of fish populations in coastal environments, with implications for disease dynamics. In Pacific Canada, farmed salmon act as reservoir hosts for parasites and pathogens, including sea lice (Lepeophtheirus salmonis and Caligus clemensi) that can transmit to migrating wild salmon. Assessing the impact of salmon farms on wild salmon requires regular monitoring of sea-louse infections on both farmed and wild fish. Since 2001, we have collected juvenile pink (Oncorhynchus gorbuscha) and chum (O. keta) salmon annually at three sites in the Broughton Archipelago in British Columbia, Canada, during the annual juvenile salmon migration from fresh water to the open ocean. From sampled fish, we recorded counts of parasitic copepodid-, chalimus-, and motile-stage sea lice. We report louse abundances as well as supplementary observations of fish size, development, and health.
The drivers of background tree mortality rates-the typical low rates of tree mortality found in forests in the absence of acute stresses like drought-are central to our understanding of forest dynamics, the effects of ongoing environmental changes on forests, and the causes and consequences of geographical gradients in the nature and strength of biotic interactions. To shed light on factors contributing to background tree mortality, we analyzed detailed pathological data from 200,668 tree-years of observation and 3,729 individual tree deaths, recorded over a 13-yr period in a network of old-growth forest plots in California's Sierra Nevada mountain range. We found that: (1) Biotic mortality factors (mostly insects and pathogens) dominated (58%), particularly in larger trees (86%). Bark beetles were the most prevalent (40%), even though there were no outbreaks during the study period; in contrast, the contribution of defoliators was negligible. (2) Relative occurrences of broad classes of mortality factors (biotic, 58%; suppression, 51%; and mechanical, 25%) are similar among tree taxa, but may vary with tree size and growth rate. (3) We found little evidence of distinct groups of mortality factors that predictably occur together on trees. Our results have at least three sets of implications. First, rather than being driven by abiotic factors such as lightning or windstorms, the "ambient" or "random" background mortality that many forest models presume to be independent of tree growth rate is instead dominated by biotic agents of tree mortality, with potentially critical implications for forecasting future mortality. Mechanistic models of background mortality, even for healthy, rapidly growing trees, must therefore include the insects and pathogens that kill trees. Second, the biotic agents of tree mortality, instead of occurring in a few predictable combinations, may generally act opportunistically and with a relatively large degree of independence from one another. Finally, beyond the current emphasis on folivory and leaf defenses, studies of broad-scale gradients in the nature and strength of biotic interactions should also include biotic attacks on, and defenses of, tree stems and roots.
Understanding how biotic and abiotic processes influence community assembly is a fundamental aim in ecology. Although spatial scales at which communities are studied may affect the relative importance of such assembly processes, spatial influences on community assembly have not been thoroughly addressed. We tested how spatial scale affects inferences of habitat filtering and competitive exclusion assembly processes in darter (Percidae: Etheostomatinae) assemblages across four temperate stream systems. We predicted competitive exclusion would influence assembly in fine-scale assemblages, and habitat filtering would be more influential as spatial scale increased. Moreover, we assumed that habitat heterogeneity would increase with scale, and consequently alleviate direct competitive exclusion acting at finer scales. Using a framework that incorporated genetic relatedness, morphological traits, and habitat use among co-occurring darter species, we identified ecological and evolutionary patterns of structure, which allowed us to elucidate processes of assembly. Based on phylogenetic structure, assemblages showed an increase in habitat filtering (i.e., increased phylogenetic clustering) as we scaled up from fine to intermediate assemblages; however, we found mixed signals for habitat filtering and competitive exclusion at the broadest spatial scale. While habitat filtering was found to have an overall high relative importance during assembly, we also found influence of competitive exclusion processes based on limited morphological similarity among co-occurring species. Our results generally support an increased influence of habitat filtering processes in broader scale assemblages. Moreover, we suggest that habitat filtering and competitive exclusion processes act simultaneously during assembly, although the relative influence of each process may be spatial-scale dependent.
Many organisms spend a significant portion of their life cycle as haploids and as diploids (a haploid-diploid life cycle). However, the evolutionary processes that could maintain this sort of life cycle are unclear. Most previous models of ploidy evolution have assumed that the fitness effects of new mutations are equal in haploids and homozygous diploids, however, this equivalency is not supported by empirical data. With different mutational effects, the overall (intrinsic) fitness of a haploid would not be equal to that of a diploid after a series of substitution events. Intrinsic fitness differences between haploids and diploids can also arise directly, for example because diploids tend to have larger cell sizes than haploids. Here, we incorporate intrinsic fitness differences into genetic models for the evolution of time spent in the haploid versus diploid phases, in which ploidy affects whether new mutations are masked. Life-cycle evolution can be affected by intrinsic fitness differences between phases, the masking of mutations, or a combination of both. We find parameter ranges where these two selective forces act and show that the balance between them can favor convergence on a haploid-diploid life cycle, which is not observed in the absence of intrinsic fitness differences.
Individuals from disadvantaged communities are among the millions of uninsured Americans gaining insurance under the Affordable Care Act. The extent to which health insurance can mitigate the effects of the social determinants of health on cancer care is unknown.
This study linked the Surveillance, Epidemiology, and End Results registries to US Census data to study patients diagnosed with the 4 leading causes of cancer deaths between 2007 and 2011. A county-level social determinant score was developed with 5 measures of wealth, education, and employment. Patients were stratified into quintiles, with the lowest quintile representing the most disadvantaged communities. Logistic regression and Cox proportional hazards models were used to estimate associations and cancer-specific survival.
A total of 364,507 patients aged 18 to 64 years were identified (134,105 with breast cancer, 106,914 with prostate cancer, 62,606 with lung cancer, and 60,882 with colorectal cancer). Overall, patients from the most disadvantaged communities (median household income, $42,885; patients below the poverty level, 22%; patients completing college, 17%) were more likely to present with distant disease (odds ratio, 1.6; P < .001) and were less likely to receive cancer-directed surgery (odds ratio, 0.8; P < .001) than the least disadvantaged communities (median income, $78,249; patients below the poverty level, 9%; patients completing college, 42%). The differences persisted across quintiles regardless of the insurance status. The effect of having insurance on cancer-specific survival was more pronounced in disadvantaged communities (relative benefit at 3 years, 40% vs 31%). However, it did not fully mitigate the effect of social determinants on mortality (hazard ratio, 0.75 vs 0.68; P < .001).
Cancer patients from disadvantaged communities benefit most from health insurance, and there is a reduction in disparities in outcome. However, the gap produced by social determinants of health cannot be bridged by insurance alone. Cancer 2016. © 2016 American Cancer Society.
Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.
The past decade has witnessed a rapid increase in the use of Social Networking Sites (SNSs) in health communication campaigns seeking to achieve an ambitious range of health-related impacts. This article provides a review of 40 studies and research protocols, with a focus on two key factors that differentiate SNSs from more traditional health communication approaches of the past. The first is the potential dualism between message sender and receiver, in which receivers become receiver-sources who forward and amplify the content and reach of health messages. The second is the potential dualism between message and message impact, in which the act of forwarding and modifying messages by receiver-sources itself becomes a measure of message impact. Each of these dualisms has implications for the design and evaluation of contemporary health communication campaigns. The review concludes with a series of observations and recommendations for future health communication research.
We evidence critical fluctuations in the strain rate of granular flows that are weakly vibrated. Strikingly, the critical point arises at finite values of the mean strain rate and vibration strength, far from the yielding critical point at a zero flow rate. We show that the global rheology, as well as the amplitude and correlation time of the fluctuations, are consistent with a mean-field, Landau-like description, where the strain rate and the stress act as conjugated variables. We introduce a general model which captures the observed phenomenology and argue that this type of critical behavior generically arises when self-fluidization competes with friction.
A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.
Actin cross-linking toxins are produced by Gram-negative bacteria from Vibrio and Aeromonas genera. The toxins were named actin cross-linking domains (ACD), since the first and most of the subsequently discovered ACDs were found as effector domains in larger MARTX and VgrG toxins. Among recognized human pathogens, ACD is produced by Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Upon delivery to the cytoplasm of a host cell, ACD covalently cross-links actin monomers into non-polymerizable actin oligomers of various lengths. Provided sufficient doses of toxin are delivered, most or all actin can be promptly cross-linked into non-functional oligomers, leading to cell rounding, detachment from the substrate and, in many cases, cell death. Recently, a deeper layer of ACD toxicity with a less obvious but more potent mechanism was discovered. According to this finding, low doses of the ACD-produced actin oligomers can actively disrupt the actin cytoskeleton by potently inhibiting essential actin assembly proteins, formins. The first layer of toxicity is direct (as actin is the immediate and the only target), passive (since ACD-cross-linked actin oligomers are toxic only because they are non-functional), and less potent (as bulk quantities of one of the most abundant cytoplasmic proteins, actin, have to be modified). The second mechanism is indirect (as major targets, formins, are not affected by ACD directly), active (because actin oligomers act as "secondary" toxins), and highly potent [as it affects scarce and essential actin-binding proteins (ABPs)].
Most passerine birds practice nest sanitation whereby they remove debris from their nest. Nest sanitation has been posited as a pre-adaptation for egg ejection by hosts of avian brood parasites. However, relatively few North American hosts of the brood parasitic brown-headed cowbird (Molothrus ater) eject cowbird eggs to the detriment of their fitness. In this study, I added either a piece of flagging tape or a pine cone bract scale along with an artificial cowbird egg to nests of the red-winged blackbird (Agelaius phoeniceus) to determine whether the act of nest sanitation would elicit egg ejection. All red-winged blackbirds removed the debris within 24 h, but all individuals also accepted the cowbird eggs and this rate of ejection did not differ from that in nests that only received a cowbird egg. While nest cleaning and egg ejection are similar mechanically, they differ cognitively and egg ejection is not elicited in red-winged blackbirds during the act of removing debris from their nests.
Brownian dynamics simulations are used to study the detachment of a particle from a substrate. Although the model is simple and generic, we attempt to map its energy, length and time scales onto a specific experimental system, namely a bead that is weakly bound to a cell and then removed by an optical tweezer. The external driving force arises from the combined optical tweezer and substrate potentials, and thermal fluctuations are taken into account by a Brownian force. The Jarzynski equality and Crooks fluctuation theorem are applied to obtain the equilibrium free energy difference between the final and initial states. To this end, we sample non-equilibrium work trajectories for various tweezer pulling rates. We argue that this methodology should also be feasible experimentally for the envisioned system. Furthermore, we outline how the measurement of a whole free energy profile would allow the experimentalist to retrieve the unknown substrate potential by means of a suitable deconvolution. The influence of the pulling rate on the accuracy of the results is investigated, and umbrella sampling is used to obtain the equilibrium probability of particle escape for a variety of trap potentials.
Bioisosteric ferrocenyl-containing quinolines and ferrocenylamines containing organosilanes and their carbon analogues, were prepared and fully characterised. The molecular structures of two ferrocenyl-containing quinolines, determined using single-crystal X-ray diffraction, revealed that the compounds crystallise in a folded conformation. The compounds were screened for their antiplasmodial activity against the chloroquine-sensitive (NF54) and CQ-resistant (Dd2) strains of P. falciparum, as well as for their cytotoxicity against Chinese Hamster Ovarian (CHO) cells. The ferrocenyl-containing quinolines displayed activities in the low nanomolar range (6-36 nM), and showed selectivity towards parasites. β-Haematin inhibition assays suggest that the compounds may in part act via the inhibition of haemozoin formation, while microsomal metabolic stability studies reveal that the ferrocenyl-containing quinolines are rapidly metabolised in liver microsomes. Further, antitrichomonal screening against the metronidazole-sensitive (G3) strain of the mucosal pathogen T. vaginalis revealed that the quinoline-based compounds displayed superior parasite growth inhibition when compared to the ferrocenylamines. The library was also tested E. coli and on Lactobacilli spp. found as part of the normal flora of the human microbiome and no effect on growth in vitro was observed, supporting the observation that these compounds are specific for eukaryotic pathogens.
Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations.
The septins are a conserved family of GTP-binding proteins present in all eukaryotic cells except plants. They were originally discovered in the baker's yeast Saccharomyces cerevisiae that serves until today as an important model organism for septin research. In yeast, the septins assemble into a highly ordered array of filaments at the mother bud neck. The septins are regulators of spatial compartmentalization in yeast and act as key players in cytokinesis. This minireview summarizes the recent findings about structural features and cell biology of the yeast septins.
Humans act as an intermediate host for Toxocara canis and Toxocara cati. Toxocara may be an important risk factor for asthma in humans. The aim of the present study was to evaluate immunoglobulin G (IgG) anti-Toxocara canis antibody, using enzyme-linked immunosorbent assay (ELISA) in asthmatic patients (aged 5-15 years), referring to a clinic of pulmonary diseases in Arak, Iran.
In this bi-group cross sectional study, serum samples were collected from 110 children with confirmed asthma and 70 children without asthma within one year. IgG anti-Toxocara antibody was detected viaELISA method. The collected data were analyzed, using SPSS.
The seroprevalence of antibodies against Toxocara species was estimated at 1.8% (two males) in asmathic children viaELISA method; however, no antibodies against Toxocara canis were detected in the control group. There was no significant correlation between the frequency of antibodies against Toxocara and variables such as age, gender, or place of residence (P>0.05). Moreover, the frequency of antibodies against Toxocara was not significantly correlated with contact with dogs, consumption of unwashed fruits and vegetables, or use of raw/undercooked sheep liver (P>0.05).
The present study showed anti-Toxocara antibody in 1.8% of asthmatic children and determined the seroprevalence of toxocariasis in asthmatic children and adolescents in Arak, Iran. Based on the findings, the low rate of infection with Toxocara among asthmatic children may be attributed to acceptable personal hygiene and religious considerations.
Heat treatment and cooking are common interventions for reducing the numbers of vegetative cells and eliminating pathogenic microorganisms in food. Current cooking method requires the internal temperature of beef patties to reach 71°C. However, some pathogenic Escherichia coli such as the beef isolate E. coli AW 1.7 are extremely heat resistant, questioning its inactivation by current heat interventions in beef processing. To optimize the conditions of heat treatment for effective decontaminations of pathogenic E. coli strains, sufficient estimations, and explanations are necessary on mechanisms of heat resistance of target strains. The heat resistance of E. coli depends on the variability of strains and properties of food formulations including salt and water activity. Heat induces alterations of E. coli cells including membrane, cytoplasm, ribosome and DNA, particularly on proteins including protein misfolding and aggregations. Resistant systems of E. coli act against these alterations, mainly through gene regulations of heat response including EvgA, heat shock proteins, σ(E) and σ(S), to re-fold of misfolded proteins, and achieve antagonism to heat stress. Heat resistance can also be increased by expression of key proteins of membrane and stabilization of membrane fluidity. In addition to the contributions of the outer membrane porin NmpC and overcome of osmotic stress from compatible solutes, the new identified genomic island locus of heat resistant performs a critical role to these highly heat resistant strains. This review aims to provide an overview of current knowledge on heat resistance of E. coli, to better understand its related mechanisms and explore more effective applications of heat interventions in food industry.
The relations of epilepsy and the sensory systems are bidirectional. Epilepsy may act on sensory systems by producing sensory seizure symptoms, by altering sensory performance, and by epilepsy treatment causing sensory side effects. Sensory system activity may have an important role in both generation and inhibition of seizures.
Approaching the convicted patient is a topical issue in terms of alignment with EU provisions and recommendations, more so in the context of year by year increase in the number of convicts and consequently, prison patients. The prison patient exhibits increased vulnerability in regard to the rest of the convicts due to his/her medical status overlapping personality changes induced, while coping with a new environment. This represents a challenge for the physicians involved in the expertise process, which must act objectively within the limits and by the principles of professional ethics, while confronting a patient influenced by the prison environment.
We studied the existing legal and ethical framework concerning the expertise in view of sentence postponement/interruption on medical grounds and made a comparison between the theoretical information available and the "real life" situations encountered in our experience at the Institute of Legal Medicine Cluj-Napoca. Following this step we tried to establish some principles needed to optimize health care in the penitentiary system by detecting and sanctioning situations of deceptive behavior, doubled or not by simulation and over-simulation.
Convicts present pathologies documented in medical records, but accuse new symptoms that could suggest a new pathological condition. During the expertise, convicted patients emphasize their symptoms and/or claim new symptoms unrelated to their documented medical condition. Convicts submit repeated requests for which treatment solutions within the NAP healthcare system had been already formulated.
The patient must be properly informed about the steps to be taken and duration expected in performing a legal medicine expertise in pursuit of sentence postponement or interruption for the treatment of a medical condition that cannot be properly addressed within the NAP sanitary system. Information should come from authorized sources. Efforts to determine unauthorized sources (mainly "experienced" detainees with records of unsubstantiated demands) are surely beneficial.
The control mechanisms of respiration as a vital function are complex: voluntary - cortical, and involuntary - metabolic, neural, emotional and endocrine. Hormones and hypothalamic neuropeptides (that act as neurotrasmitters and neuromodulators in the central nervous system) play a role in the regulation of respiration and in bronchopulmonary morphology. This article presents respiratory manifestations in adult endocrine diseases that evolve with hormone deficit or hypersecretion. In hyperthyroidism, patients develop ventilation disorders, obstructive and central sleep apnea, and pleural collection. The respiratory abnormalities in hyperthyroidism as a result of the hypermetabolic action of thyroid hormones are hyperventilation, myopathy and cardiovascular involvement; recent studies have reported pulmonary arterial hypertension in Graves' disease, as a result of the association of several mechanisms. Thyroid hypertrophy can induce through compression of the upper airways dyspnea, stridor, wheezing and cough. The respiratory disorders in acromegaly are ventilatory dysfunction and sleep apnea, which contribute to an unfavorable evolution of the disease. Respiratory changes in parathyroid, adrenal and reproductive system diseases have been described. Respiratory disorders should be recognized, investigated and monitored by medical practitioners of various specialties (family physicians, internists, endocrinologists, pneumologists, cardiologists). They are frequently severe, causing an unfavorable evolution of the associated endocrine and respiratory disease.
Warfarin-related intracranial haemorrhage (WRICH) is a life-threatening complication of warfarin use. Rapid and complete reversal of the coagulopathy is required. Reversal protocols which include prothrombin complex concentrates (PCC) are now recommended. We report on a quality improvement project to implement and refine such a protocol.
Retrospective and then prospective audits of all WRICH patients presenting to a single centre. The protocol development and subsequent refinements are described. Outcomes included times to scanning, treatment and overall door-needle times, as well as use of PCC.
Across the three cohorts, use of PCC increased over time from 15% to 100% of eligible patients (p<0.001). There were significant improvements in median time to scanning (1.9 to 1.5 to 1.3 hours, p=0.03) and median door-needle times (4.5 to 2.9 to 1.9 hours, p=0.018). Key steps in the change process included (1) identifying need for change, (2) utilising senior clinical opinion leaders, (3) using "Plan-do-study-act" cycles, (4) involvement of all relevant stakeholders, (5) having a broad implementation and education plan, (6) a "change friendly" environment and (7) collaborating across departments.
The introduction (and revisions) of an anticoagulation reversal strategy for WRICH has led to increased PCC use and reduced times to both diagnosis and treatment. Further work is required to improve door-needle times and monitoring.
Epigenetics refers to the study of heritable changes in gene expression not involving changes in DNA sequence and is presently an active area of research in biology and medicine. There is increasing evidence that epigenetics is involved in the pathogenesis of psychiatric disorders.
Several studies conducted to date have suggested that psychosocial factors act by modifying epigenetic mechanisms of gene expression in the brain in the pathogenesis of psychiatric disorders. Such studies have been conducted both on brain tissues and also using peripheral tissues as substitutes for brain tissues. This article reviews such studies.
Epigenetic mechanisms of gene expression in the brain appear to link one individual with another in the context of social psychiatry. Epigenetics appears to be of major importance to the field of social psychiatry.
Rapid progress in nanophotonics is driven by the ability of optically resonant nanostructures to enhance near-field effects controlling far-field scattering through intermodal interference. A majority of such effects are usually associated with plasmonic nanostructures. Recently, a new branch of nanophotonics has emerged that seeks to manipulate the strong, optically induced electric and magnetic Mie resonances in dielectric nanoparticles with high refractive index. In the design of optical nanoantennas and metasurfaces, dielectric nanoparticles offer the opportunity for reducing dissipative losses and achieving large resonant enhancement of both electric and magnetic fields. We review this rapidly developing field and demonstrate that the magnetic response of dielectric nanostructures can lead to novel physical effects and applications.
Fast radio bursts (FRBs) are millisecond-duration events thought to originate beyond the Milky Way galaxy. Uncertainty surrounding the burst sources, and their propagation through intervening plasma, has limited their use as cosmological probes. We report on a mildly dispersed (dispersion measure 266.5 ± 0.1 pc cm(-3)), exceptionally intense (120 ± 30 Jy), linearly polarized, scintillating burst (FRB 150807) that we directly localize to 9 arcmin(2) Based on a low Faraday rotation (12.0 ± 0.7 rad m(-2)), we infer negligible magnetization in the circum-burst plasma and constrain the net magnetization of the cosmic web along this sightline to <21 nG, parallel to the line-of-sight. The burst scintillation suggests weak turbulence in the ionized intergalactic medium.
For the individual market, 2014 was the first year Affordable Care Act medical claims experience data were available to set 2016 rates. Accessing Centers for Medicare and Medicaid Services rate data for 175 state insurers, this study compares projected medical claims with actual medical claims of 2014, as well as the cost and utilization of benefit categories for inpatient, outpatient, professional, and prescription drug spending. Actual costs per member per month (pmpm) were greater than projected in 2014 for inpatient, outpatient, and prescription spending but not for professional care. Overall, actual median medical cost was $443 pmpm, which was significantly higher by $41 than projected cost. Greater utilization of health care was primarily responsible for higher realized medical claims. In terms of the specific benefit categories-inpatient, outpatient, and prescription-actual costs pmpm were significantly higher than projected values. In terms of the drivers of inpatient costs, on an admission basis, higher costs and greater utilization of admissions resulted in higher inpatient costs. For outpatient costs pmpm, higher utilization rather than unit cost per visit drove increased costs. Higher than expected prescription drug costs were driven by both greater utilization and cost per prescription.
To test whether genetically decreased vitamin D levels are associated with Alzheimer disease (AD) using mendelian randomization (MR), a method that minimizes bias due to confounding or reverse causation.
We selected single nucleotide polymorphisms (SNPs) that are strongly associated with 25-hydroxyvitamin D (25OHD) levels (p < 5 × 10(-8)) from the Study of Underlying Genetic Determinants of Vitamin D and Highly Related Traits (SUNLIGHT) Consortium (N = 33,996) to act as instrumental variables for the MR study. We measured the effect of each of these SNPs on 25OHD levels in the Canadian Multicentre Osteoporosis Study (CaMos; N = 2,347) and obtained the corresponding effect estimates for each SNP on AD risk from the International Genomics of Alzheimer's Project (N = 17,008 AD cases and 37,154 controls). To produce MR estimates, we weighted the effect of each SNP on AD by its effect on 25OHD and meta-analyzed these estimates using a fixed-effects model to provide a summary effect estimate.
The SUNLIGHT Consortium identified 4 SNPs to be genome-wide significant for 25OHD, which described 2.44% of the variance in 25OHD in CaMos. All 4 SNPs map to genes within the vitamin D metabolic pathway. MR analyses demonstrated that a 1-SD decrease in natural log-transformed 25OHD increased AD risk by 25% (odds ratio 1.25, 95% confidence interval 1.03-1.51, p = 0.021). After sensitivity analysis in which we removed SNPs possibly influenced by pleiotropy and population stratification, the results were largely unchanged.
Our results provide evidence supporting 25OHD as a causal risk factor for AD. These findings provide further rationale to understand the effect of vitamin D supplementation on cognition and AD risk in randomized controlled trials.
To outline current understanding and recommended treatments for paraphilic or sexual deviant disorders in adolescents and youth.
An overview of the diagnosis, development and scope of paraphilic disorders. Evidence and an algorithm for pharmacological treatments in youth are outlined.
Paraphilic disorders are relatively common in adolescents and youth, with fantasies and urges preceding the onset of deviant behaviour by a handful of years. Research in youth is sparse, but the available evidence supports that paraphilic disorders respond favourably to psychological and pharmacological treatments.
The detection of paraphilic disorders in adolescents and youth presents a window of opportunity, where treatment may be provided before deviant behaviour occurs, potentially reducing the future incidence of sexual abuse.
Photons with a twisted phase front carry a quantized amount of orbital angular momentum (OAM) and have become important in various fields of optics, such as quantum and classical information science or optical tweezers. Because no upper limit on the OAM content per photon is known, they are also interesting systems to experimentally challenge quantum mechanical prediction for high quantum numbers. Here, we take advantage of a recently developed technique to imprint unprecedented high values of OAM, namely spiral phase mirrors, to generate photons with more than 10,000 quanta of OAM. Moreover, we demonstrate quantum entanglement between these large OAM quanta of one photon and the polarization of its partner photon. To our knowledge, this corresponds to entanglement with the largest quantum number that has been demonstrated in an experiment. The results may also open novel ways to couple single photons to massive objects, enhance angular resolution, and highlight OAM as a promising way to increase the information capacity of a single photon.
Every close relationship has a history, but how people manage their relational past varies and can have important implications in the present. The current research investigated the role of subjective representation of time: How feeling subjectively close (vs. distant) to a past relational transgression (vs. kind act) predicted "kitchen thinking"-the tendency to bring to mind relational past memories in new, unrelated contexts. We explored the role of attachment anxiety as a predictor of subjective time perception and kitchen thinking. We found support for our hypothesis that when negative memories felt subjectively closer relative to positive memories, people were more likely to kitchen think (Studies 1-3). Kitchen thinking, in turn, predicted negative relationship outcomes (Study 4). Furthermore, people high (vs. low) in attachment anxiety were less likely to perceive the timing of their relational memories adaptively, accounting for more kitchen thinking and in turn, maladaptive relational outcomes.
The SnRK1 (SNF1-related kinase 1) kinases are the plant cellular fuel gauges, activated in response to energy-depleting stress conditions to maintain energy homeostasis while also gatekeeping important developmental transitions for optimal growth and survival. Similar to their opisthokont counterparts (animal AMP-activated kinase, AMPK, and yeast Sucrose Non-Fermenting 1, SNF), they function as heterotrimeric complexes with a catalytic (kinase) α subunit and regulatory β and γ subunits. Although the overall configuration of the kinase complexes is well conserved, plant-specific structural modifications (including a unique hybrid βγ subunit) and associated differences in regulation reflect evolutionary divergence in response to fundamentally different lifestyles. While AMP is the key metabolic signal activating AMPK in animals, the plant kinases appear to be allosterically inhibited by sugar-phosphates. Their function is further fine-tuned by differential subunit expression, localization, and diverse post-translational modifications. The SnRK1 kinases act by direct phosphorylation of key metabolic enzymes and regulatory proteins, extensive transcriptional regulation (e.g. through bZIP transcription factors), and down-regulation of TOR (target of rapamycin) kinase signaling. Significant progress has been made in recent years. New tools and more directed approaches will help answer important fundamental questions regarding their structure, regulation, and function, as well as explore their potential as targets for selection and modification for improved plant performance in a changing environment.
Spermatozoa are one of the few mammalian cells types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single gene knockout mice is slow as generation of each mutant line can take 6-9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new non-surgical, non-viral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNAi against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at 5 sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that act as potent and highly specific male contraceptives.
Globally, forests represent highly productive ecosystems that act as carbon sinks where soil organic matter is formed from residuals after biomass decomposition as well as from rhizodeposited carbon. Forests exhibit a high level of spatial heterogeneity and the importance of trees, the dominant primary producers, for their structure and functioning. Fungi, bacteria and archaea inhabit various forest habitats: foliage, the wood of living trees, the bark surface, ground vegetation, roots and the rhizosphere, litter, soil, deadwood, rock surfaces, invertebrates, wetlands or the atmosphere, each of which has its own specific features, such as nutrient availability or temporal dynamicy and specific drivers that affect microbial abundance, the level of dominance of bacteria or fungi as well as the composition of their communities. However, several microorganisms, and in particular fungi, inhabit or even connect multiple habitats, and most ecosystem processes affect multiple habitats. Forests are dynamic on a broad temporal scale with processes ranging from short-term events over seasonal ecosystem dynamics to long-term stand development after disturbances such as fires or insect outbreaks. The understanding of these processes can be only achieved by the exploration of the complex 'ecosystem microbiome' and its functioning using focused, integrative microbiological and ecological research performed across multiple habitats.
Although Clonorchis sinensis lives in the bile duct, few studies have investigated the local immune response in the liver and bile duct. To investigate the local immune response to C. sinensis, we investigated the activation and recruitment of various immune cells and cytokine levels in the liver and bile duct lymph nodes (BLN) in FVB mice after primary infection and re-infection. Male 4-week-old FVB mice were divided into 6 experimental groups: uninfected controls, primary infection lasting 1week (PI 1w), primary infection lasting 4weeks (PI 4w), praziquantel treatment after PI 4w (Tx), re-infection lasting 1week after Tx (RI 1w), and re-infection lasting 4weeks after Tx (RI 4w). Recovery rates were 80.0% and 73.0% in PI 1w and PI 4w mice, respectively, but significantly decreased during re-infection to 26.6% in RI 1w and 13.3% in RI 4w. This result suggested that the mice were resistant to re-infection. In the liver, Kupffer cells were augmented 70-fold in PI 1w mice (P<0.001). Kupffer cells expressed Th2-related cytokines (IL-10 and IL-13) during primary infection. In addition, serum levels of C. sinensis-specific IgG1 and IgG2a strongly increased in RI 1w mice. Secretion of C. sinensis-specific IgE reached a plateau at 4weeks after primary infection, and remained elevated in all infected groups. In conclusion, during infection with C. sinensis, Kupffer cells likely act as antigen-presenting cells, stimulating the Th2 cytokine production system.
The beta genus comprises more than 50 beta human papillomavirus (HPV) types that are suspected to be involved, together with ultraviolet (UV) irradiation, in the development of non-melanoma skin cancer (NMSC), the most common form of human cancer. Two members of the genus beta, HPV5 and HPV8, were first identified in patients with a genetic disorder, epidermodysplasia verruciformis (EV), that confers high susceptibility to beta HPV infection and NMSC development. The fact that organ transplant recipients (OTRs) with an impaired immune system have an elevated risk of NMSC raised the hypothesis that beta HPV types may also be involved in skin carcinogenesis in non-EV patients. Epidemiological studies have shown that serological and viral DNA markers are weakly, but significantly, associated with history of NMSC in OTRs and the general population. Functional studies on mucosal high-risk (HR) HPV types have clearly demonstrated that the products of two early genes, E6 and E7, are the main viral oncoproteins, which are able to deregulate events closely linked to transformation, such as cell cycle progression and apoptosis. Studies on a small number of beta HPV types have shown that their E6 and E7 oncoproteins also have the ability to interfere with the regulation of key pathways/events associated with cellular transformation. However, the initial functional data indicate that the molecular mechanisms leading to cellular transformation are different from those of mucosal HR HPV types. Beta HPV types may act only at early stages of carcinogenesis, by potentiating the deleterious effects of other carcinogens, such as UV radiation.
Central to the basis of ecological immunology are the ideas of costs and trade-offs between immunity and life history traits. As a physical barrier, the insect cuticle provides a key resistance trait, and Tenebrio molitor shows phenotypic variation in cuticular colour that correlates with resistance to the entomopathogenic fungus Metarhizium anisopliae. Here we first examined whether there is a relationship between cuticular colour variation and two aspects of cuticular architecture that we hypothesised may influence resistance to fungal invasion through the cuticle: its thickness and its porosity. Second, we tested the hypothesis that tyrosine, a semi-essential amino acid required for immune defence and cuticular melanisation and sclerotisation, can act as a limiting resource by supplementing the larval diet and subsequently examining adult cuticular colouration and thickness. We found that stock beetles and beetles artificially selected for extremes of cuticular colour had thicker less porous cuticles when they were darker, and thinner more porous cuticles when they were lighter, showing that colour co-varies with two architectural cuticular features. Experimental supplementation of the larval diet with tyrosine led to the development of darker adult cuticle and affected thickness in a sex-specific manner. However, it did not affect two immune traits. The results of this study provide a mechanism for maintenance of cuticular colour variation in this species of beetle; darker cuticles are thicker, but their production is potentially limited by resource constraints and differential investments in resistance mechanisms between the sexes.
Managing ecosystems to provide ecosystem services in the face of global change is a pressing challenge for policy and science. Predicting how alternative management actions and changing future conditions will alter services is complicated by interactions among components in ecological and socioeconomic systems. Failure to understand those interactions can lead to detrimental outcomes from management decisions. Network theory that integrates ecological and socioeconomic systems may provide a path to meeting this challenge. While network theory offers promising approaches to examine ecosystem services, few studies have identified how to operationalize networks for managing and assessing diverse ecosystem services. We propose a framework for how to use networks to assess how drivers and management actions will directly and indirectly alter ecosystem services.
The combined-immunotherapy of adoptive cell therapy (ACT) and cyclophosphamide (CTX) is one of the most efficient treatments for melanoma patients. However, synergistic effects of CTX and ACT on the spatio-temporal dynamics of immunocytes in vivo have not been described. Here, we visualized key cell events of immunotherapy-elicited immunoreactions in a multicolor-coded tumor microenvironment, and then established an optimal strategy of metronomic combined-immunotherapy to enhance anti-tumor efficacy. Intravital imaging data indicated that regulatory T cells formed an 'immunosuppressive ring' around a solid tumor. The CTX-ACT combined-treatment elicited synergistic immunoreactions in tumor areas, which included relieving the immune suppression, triggering the transient activation of endogenous tumor-infiltrating immunocytes, increasing the accumulation of adoptive cytotoxic T lymphocytes, and accelerating the infiltration of dendritic cells. These insights into the spatio-temporal dynamics of immunocytes are beneficial for optimizing immunotherapy and provide new approaches for elucidating the mechanisms underlying the involvement of immunocytes in cancer immunotherapy.
In the fight against malaria, the discovery of chemical compounds with a novel mode of action and/or chemistry distinct from currently used drugs is vital to counteract the parasite's known ability to develop drug resistance. Another desirable aspect is efficacy against gametocytes, the sexual developmental stage of the parasite which enables the transmission through Anopheles vectors. Using a chemical rescue approach, we previously identified compounds targeting Plasmodium falciparum coenzyme A (CoA) synthesis or utilization, a promising target that has not yet been exploited in anti-malarial drug development.
We report on the outcomes of a series of biological tests that help to define the species- and stage-specificity, as well as the potential targets of these chemically diverse compounds. Compound activity against P. falciparum gametocytes was determined to assess stage-specificity and transmission-reducing potential. Against early stage gametocytes IC50 values ranging between 60 nM and 7.5 μM were obtained. With the exception of two compounds with sub-micromolar potencies across all intra-erythrocytic stages, activity against late stage gametocytes was lower. None of the compounds were specific pantothenate kinase inhibitors. Chemical rescue profiling with CoA pathway intermediates demonstrated that most compounds acted on either of the two final P. falciparum CoA synthesis enzymes, phosphopantetheine adenylyltransferase (PPAT) or dephospho CoA kinase (DPCK). The most active compound targeted either phosphopantothenoylcysteine synthetase (PPCS) or phosphopantothenoylcysteine decarboxylase (PPCDC). Species-specificity was evaluated against Trypanosoma cruzi and Trypanosoma brucei brucei. No specific activity against T. cruzi amastigotes was observed; however three compounds inhibited the viability of trypomastigotes with sub-micromolar potencies and were confirmed to act on T. b. brucei CoA synthesis.
Utilizing the compounds we previously identified as effective against asexual P. falciparum, we demonstrate for the first time that gametocytes, like the asexual stages, depend on CoA, with two compounds exhibiting sub-micromolar potencies across asexual forms and all gametocytes stages tested. Furthermore, three compounds inhibited the viability of T. cruzi and T. b. brucei trypomastigotes with sub-micromolar potencies and were confirmed to act on T. b. brucei CoA synthesis, indicating that the CoA synthesis pathway might represent a valuable new drug target in these parasite species.
Epilepsy is one of the most common neurological diseases characterized by excessive hyperexcitability of neurons. Molecular mechanisms of epilepsy are diverse and not really understood. All in common is the misregulation of proteins that determine excitability such as potassium and sodium channels as well as GABA receptors; which are all known as biomarkers for epilepsy. Two recently identified key pathways involve the kinases mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinases (MAPK). Interestingly, mRNAs coding for those biomarkers are found to be localized at or near synapses indicating a local misregulation of synthesis and activity.
Research in the last decade indicates that RNA-binding proteins (RBPs) responsible for mRNA localization, stability and translation mediate local expression control. Among others, they are affected by mTOR and MAPK to guide expression of epileptic factors. These results suggest that mTOR/MAPK act on RBPs to regulate the fate of mRNAs, indicating a misregulation of protein expression at synapses in epilepsy.
We propose that mTOR and MAPK regulate RBPs, thereby guiding the local expression of their target-mRNAs encoding for markers of epilepsy. Thus, misregulated mTOR/MAPK-RBP interplay may result in excessive local synthesis of ion channels and receptors thereby leading to hyperexcitability. Continuous stimulation of synapses further activates mTOR/MAPK pathway reinforcing their effect on RBP-mediated expression control establishing the basis for epilepsy. Here, we highlight findings showing the tight interplay between mTOR as well as MAPK with RBPs to control expression for epileptic biomarkers.
Global prioritization of single-disease eradication programs over improvements to basic diagnostic capacity in the Global South have left the world unprepared for epidemics of chikungunya, Ebola, Zika, and whatever lies on the horizon. The medical establishment is slowly realizing that in many parts of sub-Saharan Africa (SSA), particularly urban areas, up to a third of patients suffering from acute fever do not receive a correct diagnosis of their infection.
Malaria is the most common diagnosis for febrile patients in low-resource health care settings, and malaria misdiagnosis has soared due to the institutionalization of malaria as the primary febrile illness of SSA by international development organizations and national malaria control programs. This has inadvertently created a "malaria-industrial complex" and historically obstructed our complete understanding of the continent's complex communicable disease epidemiology, which is currently dominated by a mélange of undiagnosed febrile illnesses. We synthesize interdisciplinary literature from Ghana to highlight the complexity of communicable disease care in SSA from biomedical, social, and environmental perspectives, and suggest a way forward.
A socio-environmental approach to acute febrile illness etiology, diagnostics, and management would lead to substantial health gains in Africa, including more efficient malaria control. Such an approach would also improve global preparedness for future epidemics of emerging pathogens such as chikungunya, Ebola, and Zika, all of which originated in SSA with limited baseline understanding of their epidemiology despite clinical recognition of these viruses for many decades. Impending ACT resistance, new vaccine delays, and climate change all beckon our attention to proper diagnosis of fevers in order to maximize limited health care resources.
Dysregulation of histone methylation has emerged as a major driver of neurodevelopmental disorders including intellectual disabilities and autism spectrum disorders. Histone methyl writer and eraser enzymes generally act within multisubunit complexes rather than in isolation. However, it remains largely elusive how such complexes cooperate to achieve the precise spatiotemporal gene expression in the developing brain. Histone H3K4 methylation (H3K4me) is a chromatin signature associated with active gene-regulatory elements. We review a body of literature that supports a model in which the RAI1-containing H3K4me writer complex counterbalances the LSD1-containing H3K4me eraser complex to ensure normal brain development. This model predicts H3K4me as the nexus of previously unrelated neurodevelopmental disorders.
For lung adenocarcinoma (LUAD) patients receiving platinum-based adjuvant chemotherapy (ACT), predictive signatures extracted from survival data solely are not directly associated with platinum response. Another limitation of reported signatures, commonly based on risk scores summarised from gene expressions, is that they could not be applied directly to samples measured by different laboratories due to experimental batch effects.
Using 60 samples of LUAD patients receiving platinum-based ACT in TCGA, we pre-selected gene pairs whose within-samples relative expression orderings (REOs) were significantly associated with both pathological response and 5-year survival, from which we selected an optimal signature whose within-samples REOs could identify responders with improved 5-year survival rate.
A predictive signature consisting of three gene pairs was developed. In an independent data set integrated from five small data sets, the predicted responders had a significantly higher 5-year survival rate than the predicted non-responders if and only if they received platinum-based ACT (log-rank P=0.0006). The predicted responders showed a 22% absolute benefit of platinum-based ACT in 5-year survival rate compared with untreated patients (log-rank P=0.0019).
The REO-based signature can individually predict response to platinum-based ACT with concordant survival benefit directly for LUAD samples measured by different laboratories.
HMGA1 is a non-histone nuclear protein that regulates cellular proliferation, invasion and apoptosis and is overexpressed in many carcinomas. In this study we sought to explore the expression of HMGA1 in HCCs and cirrhotic tissues, and its effect in in vitro models.
We evaluated HMGA1 expression using gene expression microarrays (59 HCCs, of which 37 were matched with their corresponding cirrhotic tissue and 5 normal liver donors) and tissue microarray (192 HCCs, 108 cirrhotic tissues and 79 normal liver samples). HMGA1 expression was correlated with clinicopathologic features and patient outcome. Four liver cancer cell lines with stable induced or knockdown expression of HMGA1 were characterized using in vitro assays, including proliferation, migration and anchorage-independent growth.
HMGA1 expression increased monotonically from normal liver tissues to cirrhotic tissue to HCC (P<.01) and was associated with Edmondson grade (P<.01). Overall, 51% and 42% of HCCs and cirrhotic tissues expressed HMGA1, respectively. Patients with HMGA1-positive HCCs had earlier disease progression and worse overall survival. Forced expression of HMGA1 in liver cancer models resulted in increased cell growth and migration, and vice versa. Soft agar assay showed that forced expression of HMGA1 led to increased foci formation, suggesting an oncogenic role of HMGA1 in hepatocarcinogenesis.
HMGA1 is frequently expressed in cirrhotic tissues and HCCs and its expression is associated with high Edmondson grade and worse prognosis in HCC. Our results suggest that HMGA1 may act as oncogenic driver of progression, implicating it in tumor growth and migration potential in liver carcinogenesis.
Increasing production and use of graphene oxide nanoparticles (GONPs) boost their wide dissemination in the subsurface environments where biofilms occur ubiquitously, representative of the physical and chemical heterogeneities. This study aimed at investigating the influence of Gram-positive Bacillus subtilis (BS) and Gram-negative Pseudomonas putida (PP) biofilms on the transport of GONPs under different ionic strengths (0.1, 0.5, and 1.0 mM CaCl2) at neutral pH 7.2 in water-saturated porous media. Particularly, the X-ray micro-computed tomography was used to quantitatively characterize the pore structures of sand columns in the presence and absence of biofilms. Our results indicated that the presence of biofilms reduced the porosity and narrowed down the pore sizes of packed columns. Transport experiments in biofilm-coated sand showed that biofilms, irrespective of bacterial species, significantly inhibited the mobility of GONPs compared to that in cleaned sand. This could be due to the Ca(2+) complexation, increased surface roughness and charge heterogeneities of collectors, and particularly enhanced physical straining caused by biofilms. The two-site kinetic retention model-fitted value of maximum solid-phase concentration (Smax2) for GONPs was higher for biofilm-coated sand than for cleaned sand, demonstrating that biofilms act as favorable sites for GONPs retention. Our findings presented herein are important to deepen our current understanding on the nature of particle-collector interactions.
Pemphigus is an autoimmune disease that causes blistering and is life-threatening if left untreated. Nowadays, finding a promising treatment for pemphigus remains a serious challenge. Various treatments are currently recommended to treat this disease, but they rarely lead to complete and durable remission. Regulatory cells appear to have a critical role in numerous autoimmune diseases, so it is possible that promotion of these cells may induce remission. This study presents a new approach to treating pemphigus that has not been discussed to date. This approach introduces interleukin (IL)-35 as a new treatment for pemphigus. This cytokine could induce two different types of regulatory cell, including IL-35-that produces induced regulatory T cells and IL-35(+) regulatory B cells, which could suppress both effector T cells and effector B cells. It seems that IL-35 may act as an efficient therapeutic strategy for pemphigus. It probably limits progression of the disease and may even contribute to long-lasting remission. However, further study is required to evaluate the efficacy and safety of treating pemphigus with IL-35.
As US public health faces increasing threats from outbreaks, impending changes to the healthcare landscape in the United States may alter the way that health departments are able to detect and control some of the most common infectious diseases. The Patient Protection and Affordable Care Act (ACA) has made significant changes in the way health care is provided in the United States. While many of the clinical, economic, and policy implications of the ACA are well described, there has been limited analysis of changes, if any, in the surveillance and control of infectious diseases of public health importance-such as tuberculosis, sexually transmitted infections, and HIV-that are anticipated or occurring as the ACA is implemented across the United States. To address these questions, we reviewed the literature for evidence of changing trends and conducted 66 semi-structured, not-for-attribution interviews with 82 participants from healthcare systems; academia; federal, state, and local public health agencies; and professional and nongovernmental organizations across the United States. This analysis identifies several ways in which ACA implementation has not fully addressed the public health needs associated with diagnosis, treatment, surveillance, and control of infectious diseases of public health importance.
Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.
Using density functional theory (DFT) calculations we demonstrate how electron injection can facilitate the creation of Frenkel defects in amorphous (a)-SiO2. The precursor sites composed of wide O-Si-O bond angles in amorphous SiO2 act as deep electron traps and can accommodate up to two extra electrons. Trapping of two electrons at these intrinsic sites results in weakening of a Si-O bond and creates an efficient bond breaking pathway for producing neutral O vacancies and [Formula: see text] interstitial ions characterized by low transition barriers. The low barriers for the migration of [Formula: see text] ions of about 0.2 eV facilitate the separation of created defects. This mechanism may have important implications for our understanding of dielectric breakdown and resistance switching in a-SiO2 based electronic and memory devices.
The Care Act 2014 has placed a responsibility on local authorities in England to provide services that prevent deterioration and minimise the use of other health and social care services. Housing adaptations have been identified as 1 of the 10 most promising prevention services for older adults, with bathing adaptations being the most requested. However, many local authorities have lengthy waiting times which may increase costs, reduce effectiveness and reduce the preventive effect. There is no robust evidence of the effect of these adaptations on: health, well-being and functional ability.
This is a feasibility randomised controlled trial (RCT) with nested qualitative interview study. The RCT will recruit between 40 and 60 people who have been referred for an accessible showering facility, and their carers, from 1 local authority in England. They will be randomised to either usual adaptations (∼3-month wait) or immediate adaptations (no wait). The primary outcome is the feasibility of conducting a powered study. The outcomes assessed will be: health and social care-related quality of life, independence in activities of daily living and bathing, falls and use of health and social care services. Outcomes will be assessed at 3 and 6 months. Preliminary health economic feasibility will be established.
Favourable ethical opinion was provided by the Social Care Research Ethics Committee (reference number 16/IEC08/0017). The results of this study will lay the foundations for a further powered study. This would investigate the effect of bathing adaptations on quality of life and whether increased waiting times are associated with poorer outcomes and increased costs. The results have further potential to inform trials of other housing or social care interventions using the novel waiting list control method. Dissemination will include peer-reviewed publications and presentations at national and international conferences.
ISRCTN14876332; Pre-results.
Objective The aim of the present study was to explore the perspectives of older people following their recent participation in a 75+ Health Assessment (75+HA) and interrogate these perspectives using a person-centred lens.Methods A qualitative descriptive study design was used within a larger study funded by the Australian Primary Health Care Research Institute. Nineteen participants from four different general practices in one Australian state described their perceptions of the 75+HA in a face-to-face interview. Data were then analysed using a qualitative content analysis approach.Results The purpose of the 75+HA was not well understood by participants. Participant responses reveal that where, when, who and how a primary health professional conducted the 75+HA affected what older people talked about, the guidance they sought to deal with issues and, in turn, the actioning of issues that were discussed during the 75+HA.Conclusion To enable older people to make informed decisions about and successfully manage their own health and well being, and to choose when to invite others to act on their behalf, primary health professionals need to ask questions in the 75+HA within a person-centred mindset. The 75+HA is an opportunity to ensure older people know why they need support, which ones, and agree to, supports and services they require.What is known about the topic? The Australian Medicare Benefits Schedule includes the 75+HA, developed as a proactive primary care opportunity for general practitioners and practice nurses to identify issues affecting community-dwelling older people's health and well being. The aim of the 75+HA is to consider a broad range of factors that could affect physical, psychological and social functioning, which, in turn, affects overall health, and the capacity of older people to live independently in the community. Underlying the 75+HA is the importance of detecting early functional decline to enable healthy aging.What does this paper add? There is scant, if any, attention in the literature to the views of consumers who have completed a 75+HA, especially with regard to whether this opportunity is conducted with a person-centred mindset. This paper addresses this gap. Even after participating in the 75+HA, most participants were unclear as to the purpose of the assessment, what information had been recorded and what would happen from any concerns identified in the assessment. Comments about the 75+HA included that it did not ask people about their goals and what comprised their functionality to ensure their independent living.What are the implications for practitioners? A person-centred approach requires active collaboration between primary health professionals and older people who are living the process of, and planning for, aging-in-place. Assessments like the 75+HA can assist in identifying whether older people may be experiencing early signs of functional decline, even if older people self-report living without problems in their home. Practitioners need to ask questions of older people and respond to what they say with a person-centred mindset.
To examine the effects of the Affordable Care Act's (ACA's) Marketplace on Texas residents and determine which population subgroups benefited the most and which the least.
We analyzed insurance coverage rates among nonelderly Texas adults using the Health Reform Monitoring Survey-Texas from September 2013, just before the first open enrollment period in the Marketplace, through March 2016.
Texas has experienced a roughly 6-percentage-point increase in insurance coverage (from 74.7% to 80.6%; P = .012) after implementation of the major insurance provisions of the ACA. The 4 subgroups with the largest increases in adjusted insurance coverage between 2013 and 2016 were persons aged 50 to 64 years (12.1 percentage points; P = .002), Hispanics (10.9 percentage points; P = .002), persons reporting fair or poor health status (10.2 percentage points; P = .038), and those with a high school diploma as their highest educational attainment (9.2 percentage points; P = .023).
Many population subgroups have benefited from the ACA's Marketplace, but approximately 3 million Texas residents still lack health coverage. Adopting the ACA's Medicaid expansion is a means to address the lack of coverage.
The release of neutrophil extracellular traps (NETs) is a major immune mechanism intended to capture pathogens. These histone and protease-coated DNA structures are released by neutrophils in response to a variety of stimuli, including respiratory pathogens, and have been identified in the airways of patients with respiratory infection, cystic fibrosis, acute lung injury, primary graft dysfunction and COPD. NET production has been demonstrated in the lungs of mice infected with Staphylococcus aureus, Klebsiella pneumoniae and Aspergillus fumigatus. Since the discovery of NETs over a decade ago, evidence that "NET evasion" might act as a immune protection strategy among respiratory pathogens including group A Streptococcus, Bordetella pertussis, and Haemophilus influenzae has been growing, with the majority of these studies being published in the past two years. Evasion strategies fall into three main categories: inhibition of NETs release by down-regulating host inflammatory responses, degradation of NETs using pathogen-derived deoxyribonucleases, and resistance to the microbicidal components of NETs, which involves a variety of mechanisms including encapsulation. Hence, the evasion of NETs appears to be a widespread strategy to allow pathogen proliferation and dissemination, and is currently a topic of intense research interest. This article will outline the evidence supporting the three main strategies of NET evasion: inhibition, degradation, and resistance, with particular reference to common respiratory pathogens.
State licensing boards have obligations to protect the public from impaired professionals and to protect the rights of professionals applying for licensure. Competently functioning professionals who have or have had a mental health diagnosis or are being treated for a mental health condition should not be screened out, according to the Americans with Disabilities Act (ADA). A review of case law shows applicable precedents from discrimination among physicians and lawyers but not, to date, among psychologists. An examination of psychology licensure application materials from all 50 states and the District of Columbia revealed that some states, particularly Alaska, Arkansas, Colorado, Florida, Georgia, Kentucky, Missouri, Montana, and New Hampshire, include language that might screen out professionals with lived experience who are currently functioning competently. For comparison, we review a sample of licensure applications for physicians and lawyers and find a similar pattern. Five of the present authors offer ourselves and other published authors as examples of competent licensed psychologists who have lived with mental illnesses. We conclude with recommendations for more inclusive language and protection of confidentiality. (PsycINFO Database Record
More than a decade before the Civil Rights Act of 1964, as an African American teenager from Baltimore, Maryland, Esther McCready challenged the discriminatory admissions policies of the University of Maryland School of Nursing (UMSON). The article explores nurse advocacy and how Esther McCready advocated for herself and greater racial equity in nursing education during a time of civil rights turmoil. Her actions eventually resulted in the formation of numerous schools of nursing for African Americans across the south. This article recounts McCready’s early life experiences and the powerful impact her actions had on creating educational options for nurses during a time when they were severely limited for African American women, including discussion of her student days at UMSON and her journey after nursing school. A review of pertinent legal cases and policies related to segregation and integration of higher education in the mid-twentieth century is presented, along with details of McCready’s continued education and advocacy.
Pediatric chronic pain is common and can result in substantial long-term disability. Previous studies on acceptance and commitment therapy (ACT) have shown promising results in improving functioning in affected children, but more research is still urgently needed. In the current clinical pilot study, we evaluated an ACT-based interdisciplinary outpatient intervention (14 sessions), including a parent support program (four sessions). Adolescents were referred to the clinic if they experienced disabling chronic pain. They were then randomized, along with their parents, to receive group (n = 12) or individual (n = 18) treatment. Adolescent pain interference, pain reactivity, depression, functional disability, pain intensity and psychological flexibility, along with parent anxiety, depression, pain reactivity and psychological flexibility were assessed using self-reported questionnaires. There were no significant differences in outcomes between individual and group treatment. Analyses illustrated significant (p < 0.01) improvements (medium to large effects) in pain interference, depression, pain reactivity and psychological flexibility post-treatment. Additionally, analyses showed significant (p < 0.01) improvements (large effects) in parent pain reactivity and psychological flexibility post-treatment. On all significant outcomes, clinically-significant changes were observed for 21%-63% of the adolescents across the different outcome measures and in 54%-76% of the parents. These results support previous findings and thus warrant the need for larger, randomized clinical trials evaluating the relative utility of individual and group treatment and the effects of parental interventions.
Bariatric surgery has serious implications on metabolic health. The reasons for a failure of bariatric surgery, i.e., limited weight loss, are multifactorial and include psychological factors. We established a theoretical model of how impulsivity is related to weight loss outcome. We propose that depressive symptoms act as a mediator between impulsivity and pathological eating behavior, and that pathological eating behavior has a direct impact on weight loss outcome. We calculated excessive weight loss (%EWL) and assessed self-reported impulsivity (using the Baratt Impulsiveness Scale (BIS-15) total score), depressive symptoms (the Patient Health Questionnaire (PHQ-9) score), and pathological eating behavior (the Eating Disorder Inventory 2 (EDI-2) total score) in 65 patients four years after laparoscopic sleeve gastrectomy. Regression and mediation analyses were computed to validate the theoretical model. The BIS-15, PHQ-9, and EDI-2 have medium to high correlations between each other, and EDI-2 correlated with %EWL. The mediation analysis yielded that the PHQ-9 represents a significant mediator between BIS-15 and EDI-2. The regression model between EDI-2 and %EWL was also significant. These results support our theoretical model, i.e., suggest that impulsivity has an indirect impact on weight loss outcome after bariatric surgery, mediated by depression and transferred through pathological eating behavior. Thus, the underlying psychological factors should be addressed in post-operative care to optimize weight loss outcome.
BK virus (BKV)-associated nephropathy is the second leading cause of graft loss in kidney transplant recipients. Due to the high prevalence of persistent infection with BKV in the general population, it is possible that either the transplant recipient or donor may act as the source of virus resulting in viruria and viremia. Although several studies suggest a correlation between donor-recipient serostatus and the development of BK viremia, specific risk factors for BKV-related complications in the transplant setting remain to be established.
We retrospectively determined the pretransplant BKV neutralizing serostatus of 116 donors (D)-recipient (R) pairs using infectious BKV neutralization assays with representatives from the 4 major viral serotypes. The neutralizing serostatus of donors and recipients was then correlated with the incidence of BK viremia during the first year posttransplantation.
There were no significant differences in baseline demographics or transplant data among the 4 neutralizing serostatus groups, with the exception of calculated panel-reactive antibody which was lowest in the D+/R- group. Recipients of kidneys from donors with significant serum neutralizing activity (D+) had elevated risk for BK viremia, regardless of recipient serostatus (D+ versus D-: odd ratio, 5.0; 95% confidence interval, 1.9-12.7]; P = 0.0008). Furthermore, donor-recipient pairs with D+/R- neutralizing serostatus had the greatest risk for BK viremia (odds ratio, 4.9; 95% confidence interval, 1.7-14.6; P = 0.004).
Donor neutralizing serostatus correlates significantly with incidence of posttransplant BK viremia. Determination of donor-recipient neutralizing serostatus may be useful in assessing the risk of BKV infection in kidney transplant recipients.
The mechanistic target of rapamycin (mTOR) is an evolutionary conserved protein with a serine/threonine kinase activity that regulates cell growth, proliferation, motility, survival, protein synthesis, autophagy and transcription. It is embedded in 2 large protein complexes: mTORC1 and mTORC2. Regulation of specific mTOR pathway functions depends on multiple GTPases, that act either as regulators of mTOR protein complexes, coupling energy availability with mTORC1 activity, or as downstream effectors of both mTORC1 and mTORC2. In this commentary, we highlight the advantages of studying the mTOR pathway in C. elegans, including the subcellular localization of the signaling pathway components and the animal phenotypes following tissue specific protein over-expression or knockdown. One important regulator that is not limited to the mTOR pathway is RHEB. We discuss in vitro and in vivo data suggesting that RHEB can function as an inhibitor of mTOR when not bound to GTP. RHEB-1 itself is regulated by Rab GDP dissociation inhibitor β, which directly binds to ATX-2. We also highlight the roles of these proteins in dietary restriction-depended reduction in animal size and fat content.
Post-translational modifications of histones play essential roles in regulating chromatin structure and function. These are tightly regulated in vivo and there is an intricate cross-talk between different marks as they are recognized by specific reader modules present in a large number of nuclear factors. In order to precisely dissect these processes in vitro native reagents like purified chromatin and histone modifying/remodeling enzymes are required to more accurately reproduce physiological conditions. The vast majority of these enzymes need to be part of stable multiprotein complexes with cofactors enabling them to act on chromatin substrates and/or read specific histone marks. In the accompanying chapter, we have described the protocol for purification of native chromatin from yeast cells (Chapter 3 ). Here, we present the methods to obtain highly purified native chromatin modifying complexes from Saccharomyces cerevisiae, based on Tandem Affinity Purification (TAP). We also present possible applications and useful functional assays that can be performed using these yeast native reagents.
Purification of native biological material provides powerful tools for the functional analysis of enzymes and proteins in chromatin. In particular, histone proteins harbor numerous post-translational modifications, which may differ between species, tissues, and growth conditions and are lacking on recombinant histones. Moreover, the physiological substrate of most enzymes that modify histones is chromatin and the majority of these enzymes need to be part of a multiprotein assembly to be able to act on chromatin. For the yeast Saccharomyces cerevisiae different chromatin purification protocols are available but often result in poor yields or rely on genetic manipulation. We present a simple purification protocol that can yield up to 150 μg of pure native chromatin per liter of yeast culture. The purified material can be obtained from mutant cells lacking specific histone modifications and can be used in in vitro chromatin assembly for biochemical studies. Based on the extremely high degree of conservation throughout eukaryotes, this modifiable native chromatin can be used in studies with factors from other organisms including humans.
Matrix metalloproteinases-2 (MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2), may presumably have an important role on the invasion and metastatic spread of malignancies attributed to an uncontrolled degradation of the extracellular matrix (ECM). A retrospective chart analysis was carried out to study the expression of MMP-2 and TIMP-2 on the archival samples of oral squamous cell carcinoma (OSCC) (n = 30) and normal mucosa (n = 10) by immunohistochemistry and compared with the clinicopathologic parameters of cases. Both MMP-2 and TIMP-2 expressions showed a positive correlation with the grades, stages and metastatic capacities of tumors (Spearman's correlation, p < 0.05). Concomitant increase in the expression of TIMP-2 and MMP-2 suggested that the rate of MMP-2/TIMP-2 expression is a better marker for characterization of MMP-2 concentration. High expression and/or activity of MMP-2 were linked with poorer survival in OSCC cases, while TIMPs have been shown to apparently act as either growth-stimulating or suppressor factors for tumors. It was also revealed that MMP-2 and TIMP-2 were secreted by both tumor cells and stromal cells. A new concept, supposing the dynamic, anticancer partnership between the residual genome stabilizer machinery of tumor cells and defensive cells adjacent to tumors, may illuminate the controversial results. In conclusion, the stronger the infiltrative and metastatic capacity of cancers, the higher is the rate of MMP-2/TIMP-2 expression helping the arrival of humoral and cellular anticancer forces.
The psychostimulant, methylphenidate (MPD), is the first line treatment as a pharmacotherapy to treat behavioral disorders such as attention deficit hyperactivity disorder (ADHD). MPD is commonly misused in non-ADHD (normal) youth and young adults both as a recreational drug and for cognitive enhancing effects to improve their grades. MPD is known to act on the reward circuit; including the caudate nucleus (CN). The CN is comprised of medium spiny neurons containing largely dopamine (DA) D1 and D2 receptors. It has been widely shown that the DA system plays an important role in the response to MPD exposure. We investigated the role of both D1 and D2 DA receptors in the CN response to chronic MPD administration using specific D1 and D2 DA antagonist. Four groups of young adult, male SD rats were used: a saline (control) and three MPD dose groups (0.6, 2.5, and 10.0 mg/kg). The experiment lasted 11 consecutive days. Each MPD dose group was randomly divided into two subgroups to receive either a 0.4 mg/kg SCH-23390 selective D1 DA antagonist or a 0.3 mg/kg raclopride selective D2 DA antagonist prior to their final (repetitive) MPD rechallenge administration. It was observed that selective D1 DA antagonist (SCH-23390) given 30 min prior to the last MPD exposure at ED11 partially reduced or prevented the effect induced by MPD exposure in CN neuronal firing rates across all MPD doses. Selective D2 DA antagonist (raclopride) resulted in less obvious trends; some CN neuronal firing rates exhibited a slight increase in all MPD doses.
Photoacids on the basis of pyrenol have been extensively studied in the past 60 years. As their photophysical properties strongly depend on the substituents at the aromatic scaffold, we introduced two reactive moieties with different electronic coefficients thus creating multi-wavelength fluorescent probes. One probe is capable of monitoring two orthogonal transformations by four fluorescence colors, distinguishable even by the naked human eye. Another derivative can act as a three-color sensor for a wide range of different pH values. Both the presented compounds allow for mimicking of fundamental and advanced two-input logic operations due to the multi-wavelength emission. Furthermore, these compounds can process information in a logically reversible way (Feynman gate).
Adoptive T-cell therapy (ACT) is an emerging paradigm in which T cells are genetically modified to target cancer-associated antigens and eradicate tumors. However, challenges treating epithelial cancers with ACT reflect antigen targets that are not tumor-specific, permitting immune damage to normal tissues, and preclinical testing in artificial xenogeneic models, preventing prediction of toxicities in patients. In that context, mucosa-restricted antigens expressed by cancers exploit anatomical compartmentalization which shields mucosae from systemic antitumor immunity. This shielding may be amplified with ACT platforms employing antibody-based chimeric antigen receptors (CARs), which mediate MHC-independent recog-nition of antigens. GUCY2C is a cancer mucosa antigen expressed on the luminal surfaces of the intestinal mucosa in mice and humans, and universally overexpressed by colorectal tumors, suggesting its unique utility as an ACT target. T cells expressing CARs directed by a GUCY2C-specific antibody fragment recognized GUCY2C, quantified by expression of activation markers and cytokines. Further, GUCY2C CAR-T cells lysed GUCY2C-expressing, but not GUCY2C-deficient, mouse colorectal cancer cells. Moreover, GUCY2C CAR-T cells reduced tumor number and morbidity and improved survival in mice harboring GUCY2C-expressing colorectal cancer metastases. GUCY2C-directed T cell efficacy reflected CAR affinity and surface expression and was achieved without immune-mediated damage to normal tissues in syngeneic mice. These observations highlight the potential for therapeutic translation of GUCY2C-directed CAR-T cells to treat metastatic tumors, without collateral autoimmunity, in patients with metastatic colorectal cancer.
Investigating the factors regulating fish condition is crucial in ecology and the management of exploited fish populations. The body condition of cod (Gadus morhua) in the Baltic Sea has dramatically decreased during the past two decades, with large implications for the fishery relying on this resource. Here, we statistically investigated the potential drivers of the Baltic cod condition during the past 40 years using newly compiled fishery-independent biological data and hydrological observations. We evidenced a combination of different factors operating before and after the ecological regime shift that occurred in the Baltic Sea in the early 1990s. The changes in cod condition related to feeding opportunities, driven either by density-dependence or food limitation, along the whole period investigated and to the fivefold increase in the extent of hypoxic areas in the most recent 20 years. Hypoxic areas can act on cod condition through different mechanisms related directly to species physiology, or indirectly to behaviour and trophic interactions. Our analyses found statistical evidence for an effect of the hypoxia-induced habitat compression on cod condition possibly operating via crowding and density-dependent processes. These results furnish novel insights into the population dynamics of Baltic Sea cod that can aid the management of this currently threatened population.
Animals exhibit different degrees of preference toward various visual stimuli. In addition, it has been shown that strongly preferred stimuli can often act as a reward. The aim of the present study was to determine what features determine the strength of the preference for visual stimuli in order to examine neural mechanisms of preference judgment. We used 50 color photographs obtained from the Flickr Material Database (FMD) as original stimuli. Four macaque monkeys performed a simple choice task, in which two stimuli selected randomly from among the 50 stimuli were simultaneously presented on a monitor and monkeys were required to choose either stimulus by eye movements. We considered that the monkeys preferred the chosen stimulus if it continued to look at the stimulus for an additional 6 s and calculated a choice ratio for each stimulus. Each monkey exhibited a different choice ratio for each of the original 50 stimuli. They tended to select clear, colorful and in-focus stimuli. Complexity and clarity were stronger determinants of preference than colorfulness. Images that included greater amounts of spatial frequency components were selected more frequently. These results indicate that particular physical features of the stimulus can affect the strength of a monkey's preference and that the complexity, clarity and colorfulness of the stimulus are important determinants of this preference. Neurophysiological studies would be needed to examine whether these features of visual stimuli produce more activation in neurons that participate in this preference judgment.
Background and aims: The aim of the study was to evaluate and compare the surface microhardness and surface morphology of permanent tooth enamel after Er,Cr:YSGG laser irradiation and Fluoride application. Materials and methods: One hundred and twenty premolars extracted for orthodontic purpose were used in the study and randomly divided into 6 groups. Group A was not subjected to any treatment. Group B was subjected to Er,Cr:YSGG laser irradiation. Group C was subjected to Er,Cr:YSGG laser irradiation followed by application of 2% NaF gel for 4 minutes. Group D was subjected to laser irradiation and 1.23% APF gel for 4 minutes. Group E was subjected to 2% NaF gel pretreatment technique followed by laser irradiation. Group F was subjected to 1.23% APF gel pretreatment technique followed by laser irradiation. All the test groups were subjected to microhardness testing and scanning electron microscope evaluation at 500 X and 1500 X. Results: All the treated groups showed an increase in microhardness value in comparison to the control group. The highest increase in microhardness was seen in Group F. Increase in microhardness values of Group B and Group D was not statistically significant as compared to Group A. Scanning Electron Micrographs showed few craters and fine porosities for Group A. These craters and porosities increased in size and often showed glass like appearance after laser irradiation. Conclusions: It can be suggested by means of present study that Er,Cr:YSGG laser irradiation alone or in combination with fluoride gel is an effective tool to provide resistance against the caries. Significantly higher resistance (p< 0.05) was seen when APF gel was used prior to Er,Cr:YSGG laser irradiation and this combination can act as an efficient tool for prevention against dental caries.
Primary enterolithiasis is a rare surgical ailment. The underlying cause is intestinal stasis. Numerous anatomical and micro environmental factors such as enteritis, incarcerated hernia, malignancy, diverticula, blind loops, and enteroenterostomy predispose to clinically significant concretions. Enterolithiasis in tuberculosis can be due to the presence of strictures, intestinal bands, or interbowel/parietal adhesions, leading to intestinal stasis. Secondary enterolithiasis is generally caused by gallstones or renal stones migrating to the gastrointestinal tract due to fistula formation. During stasis, food particles act as a nidus and calcium salts are deposited over the food particles, leading to stone formation. A 57-year-old male patient presented to the Emergency Department of Jawaharlal Nehru Medical College, AMU, Aligarh, with features of intestinal obstruction. The patient underwent emergency laparotomy, revealing 2 strictures in the distal ileum with 15.24cm of the bowel between them containing a 2×2 cm enterolith. The strictured segment was resected, and end ileostomy and mucus fistula were created. The patient's postoperative recovery was fine, and he wasdischarged with ileostomy on antitubercular treatment (after histopathologicalconfirmation). Ileostomy closure wasplanned after 6 weeks. The incidence and prevalence of enterolithiasis has been on the rise recently because of advancement in radiological imaging studies. Endoscopic and surgical stone removal along with the treatment of the underlying pathology is recommended.
Tropomyosin receptor kinase C (TrkC) targeted ligand-photosensitizer construct, IYIY-diiodo-boron-dipyrromethene (IYIY-I2-BODIPY) and its scrambled counterpart YIYI-I2-BODIPY have been prepared. IYIY-I2-BODIPY binds TrkC similar to neurotrophin-3 (NT-3), and NT-3 has been reported to modulate immune responses. Moreover, it could be shown that photodynamic therapy (PDT) elevates antitumor immune responses. This prompted us to investigate the immunological impacts mediated by IYIY-I2-BODIPY in pre- and post-PDT conditions. We demonstrated that IYIY-I2-BODIPY (strong response) and YIYI-I2-BODIPY (weak response) at 10 mg/kg, but not I2-BODIPY control, increased the levels of IL-2, IL-4, IL-6 and IL-17, but decreased the levels of systemic immunoregulatory mediators TGF-β, myeloid-derived suppressor cells and regulatory T-cells. Only IYIY-I2-BODIPY enhanced the IFN-γ(+) and IL-17(+) T-lymphocytes, and delayed tumor growth (~20% smaller size) in mice when administrated daily for 5 days. All those effects were observed without irradiation; when irradiated (520 nm, 100 J/cm(2), 160 mW/cm(2)) to produce PDT effects (drug-light interval 1 h), IYIY-I2-BODIPY induced stronger responses. Moreover, photoirradiated IYIY-I2-BODIPY treated mice had high levels of effector T-cells compared to controls. Adoptive transfer of immune cells from IYIY-I2-BODIPY-treated survivor mice that were photoirradiated gave significantly delayed tumor growth (~40-50% smaller size) in recipient mice. IYIY-I2-BODIPY alone and in combination with PDT modulates the immune response in such a way that tumor growth is suppressed. Unlike immunosuppressive conventional chemotherapy, IYIY-I2-BODIPY can act as an immune-stimulatory chemotherapeutic agent with potential applications in clinical cancer treatment.
The Affordable Care Act requires nonprofit hospitals to collaborate with public health agencies and community stakeholders to identify and address community health needs. As a rural organization, Wabash County (Indiana) Hospital pursued new approaches to achieve these revised requirements of the community benefit mandate. Using a case study approach, the authors provide a historical review of governmental relationships with nonprofit community hospitals, offer a case study application for implementing legislative mandates and community benefit requirements, share the insights they garnered on their journey to meet the mandates, and conclude that drawing upon the existing resources in the community and using current community assets in novel ways can help conserve time, and also financial, material, and human resources in meeting legislative mandates.
Multiple factors act simultaneously on plants to establish complex interaction networks involving nutrients, elicitors and metabolites. Metabolomics offers a better understanding of complex biological systems, but evaluating the simultaneous impact of different parameters on metabolic pathways that have many components is a challenging task. We therefore developed a novel approach that combines experimental design, untargeted metabolic profiling based on multiple chromatography systems and ionization modes, and multiblock data analysis, facilitating the systematic analysis of metabolic changes in plants caused by different factors acting at the same time. Using this method, target geraniol compounds produced in transgenic tobacco cell cultures were grouped into clusters based on their response to different factors. We hypothesized that our novel approach may provide more robust data for process optimization in plant cell cultures producing any target secondary metabolite, based on the simultaneous exploration of multiple factors rather than varying one factor each time. The suitability of our approach was verified by confirming several previously reported examples of elicitor-metabolite crosstalk. However, unravelling all factor-metabolite networks remains challenging because it requires the identification of all biochemically significant metabolites in the metabolomics dataset.
Hippos transfer massive quantities of trophic resources from terrestrial to aquatic ecosystems through defecation. The ramifications of the latter for the functioning of benthic ecosystems are unknown, but are dependent ultimately on rates of utilisation relative to inputs. Low input and high utilisation can strengthen bottom-up pathways and enhance consumer biomass and abundance. However, if inputs exceed utilisation rates, dung can accumulate, leading to a decline in water quality, with important repercussions for resident assemblages. Here, we quantify the consequences of hippo dung inputs on benthic assemblages in an estuarine lake in South Africa. The system supports over a thousand hippos, and during recent drought periods (extending over a decade), hippo dung has been observed to form mats over benthic habitats. Enrichment of plots using exclusion/inclusion cages with dung at naturally occurring concentrations indicated a decline in benthic chl-a by roughly 50% and macrofaunal abundance, biomass and richness by up to 76, 56 and 27% respectively. Our findings suggest that persistent inputs of hippo dung can act as an important stressor of benthic systems, leading ultimately to a loss of productivity. Accumulation of hippo dung over benthic habitats is therefore an important mechanism by which hippos indirectly structure aquatic ecosystems.
The Acceptance and Commitment Therapy (ACT) is one of the modern, so-called third-wave behavioural therapies. Among them the most successful is ACT, both in the number of therapists and respective scientific research. ACT's theoretical and philosophical background is described explicitly and its therapeutic interventions were developed according to this philosophy. Its psychopathological model is based on the idea that mainly the person's regulatory efforts of their own thoughts and feelings lead to psychological problems. That is, the source of human suffering and various psychological problems is the so called psychological inflexibility: control attempts of private events instead of living a life based on personal values and long-term goals. Therefore, clinical work in ACT focuses on the acceptance and defusion of the unwanted inner experiences and on the development of a meaningful life. The present article aims to provide a comprehensive description of ACT in Hungarian: its theoretical background, clinical techniques, and efficacy. At the end of the article, the state of ACT in Hungary will also be briefly discussed.
Membrane localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis treated with bacterial flagellin. We identified multiple receptor-like protein kinases (RLKs) changing in abundance, including cysteine-rich receptor-like kinases (CRKs) that are upregulated upon perception of flagellin. CRKs possess extracellular cysteine-rich domains and comprise a gene family consisting of 46 members in Arabidopsis. Single T-DNA insertion lines in CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae. Expression of CRK28 in N. benthamiana induced cell death, which required intact extracellular cysteine residues and a conserved kinase active site. CRK28-mediated cell death required the common RLK co-receptor BAK1. CRK28 associated with BAK1 as well as the activated FLS2 immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses.
Mothers can shape the developmental trajectory of their offspring through the transmission of resources such as hormones, antioxidants or immunoglobulins. Over the last two decades, an abundant literature on maternal effects in birds has shown that several of these compounds (i.e. androgens, glucocorticoids and antioxidants) often influence the same offspring phenotypic traits (i.e. growth, immunity or oxidative stress levels), making interaction effects between egg components a likely scenario. However, the potential interactive effects of maternally transmitted compounds on offspring development and potential co-adjustment of these compounds within an egg are still poorly understood. Here, we report the results of an interspecific comparative analysis on birds' egg yolk composition (i.e. androgens and antioxidants) where we found that yolk carotenoid and vitamin E concentrations are positively associated, supporting the hypothesis that these two antioxidants act in synergy. The concentrations of vitamin E also increased with increasing concentrations of testosterone. This last result confirms the emerging idea that androgens and antioxidants are co-adjusted within eggs and that maternally transmitted antioxidants might limit the potential direct and indirect effects of prenatal exposure to high testosterone levels on oxidative stress.
Increasing evidence supports the involvement of inflammatory and immune processes in temporal lobe epilepsy (TLE). MicroRNAs (miRNA) represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression controlling different biological processes, including immune-system homeostasis and function. We investigated the expression and cellular distribution of miRNA-146a (miR-146a) in a rat model of TLE. Prominent upregulation of miR-146a activation was evident at 1 week after status epilepticus (SE) and persisted in the chronic phase. The predicted miR-146a's target complement factor H (CFH) mRNA and protein expression was also downregulated in TLE rat model. Furthermore, transfection of miR-146a mimics in neuronal and glial cells downregulated CFH mRNA and protein levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression via 3'-untranslated-region (UTR) pairing. Downregulating miR-146a by intracerebroventricularly injection of antagomir-146a enhanced the hippocampal expression of CFH in TLE model and decreased seizure susceptibility. These findings suggest that immunopathological deficits associated with TLE can in part be explained by a generalized miR-146a-mediated down-regulation of CFH that may contribute to epileptogenesis in a rat model of TLE.
Headaches and facial pain have been identified as the most prevalent form of pain among patients with glioblastoma multiforme, the most common malignant primary brain tumour. Despite this, minimal research has been undertaken investigating the direct and indirect impact these headaches have on their quality of life. Therefore, in this study, we aimed at gaining a personal insight into the importance and impact that these headaches have on the quality of life of patients with glioblastoma multiforme.
Exploratory study using face-to-face semistructured interviews. Interviews were audio-recorded, transcribed verbatim and then qualitatively analysed using thematic analysis.
Participants recruited from a tertiary referral hospital in Birmingham, UK.
Purposive sampling of 14 registered outpatients recently diagnosed with glioblastoma multiforme.
3 themes were identified: (1) an underlying attitude of determination and positivity; (2) impact of headache unpredictability on social interaction; (3) headaches found to act as a springboard onto thoughts regarding their disease and future.
While the quality of life of patients with glioblastoma multiforme is clearly multifactorial, headaches do indeed play a part for some. However, it is not the direct pain of the headache as one might expect that impacts on the quality of life of these patients, but the indirect effect of headaches through limiting patients' social lives and by serving as a painful psychological reminder of having a life-threatening illness. In clinical practice, using headache diaries for these patients may help provide a more comprehensive assessment and further aid management plans. Alongside acting as an important reminder of the potential secondary implications of this disease, suggestions for future research include quantitatively investigating whether headaches can act as a prognostic indicator for quality of life within this patient demographic and determining whether these conclusions also hold true for a wider spectrum of patients with brain tumour.
Up to 18% of adolescents will engage in an act of self-harm before young adulthood, with the majority of acts occurring in private. Mobile apps may offer a way of providing support for young people at times of distress to prevent self-harm.
This is a proof-of-concept study designed to explore the safety, acceptability, feasibility, and usability of a smartphone app, BlueIce, with young people who are self-harming.
In this phase I open trial we will evaluate BlueIce, a smartphone app developed and coproduced with young people with lived experience of self-harm. BlueIce includes a mood-monitoring diary, selection of mood-lifting techniques based on cognitive behavior therapy and dialectical behavior therapy, and direct access to emergency telephone numbers. We will recruit young people (n=50) attending specialist child and adolescent mental health services with a current or past history of self-harm to trial BlueIce as an adjunct to their usual care. Questionnaires and interviews will be completed at baseline, postfamiliarization (2 weeks), and at follow-up (12 weeks after baseline) to assess safety, app use, and acceptability. Interviews will be undertaken with clinicians to assess the feasibility of BlueIce within a clinical setting.
Recruitment occurred between May and November 2016. The recruitment target was 50, and by the beginning of November 54 young people had been referred.
This study is the first to evaluate an app specifically developed with young people for young people (under the age of 18 years) who self-harm. It will determine whether BlueIce is acceptable, how often it is used, and whether it is safe and does not have any unintentional adverse effects. This information will determine whether a feasibility trial to test recruitment, randomization, retention, and appropriate outcome measures should be pursued.
Behaviourally spontaneous confabulation denotes a distinct syndrome consisting of confabulations that patients act upon, disorientation, and amnesia. It corresponds to the stable form of the original Korsakoff syndrome. While the syndrome may also occur in confusional states and degenerative dementia, this article is about the syndrome as it occurs after acute and focal brain damage. The patients act according to ideas and obligations that can mostly be traced back to real experiences in their past, but which are not currently valid guides of thinking and behaviour. This inability to abandon behavioural guides (anticipations) that are currently not valid corresponds to a failure of behavioural extinction and to the inability to abandon a previously rewarded choice in reversal learning when the expected reward (outcome) fails to occur, that is, following extinction trials. This article describes evidence from human and animal experiments showing that the posterior medial orbitofrontal cortex (OFC), which is typically damaged in these patients, and connected structures of the reward system contain the neural apparatus to signal the non-occurrence of anticipated outcomes, thereby presumably synchronizing thought and behaviour with current reality. Failure of this function, which we call orbitofrontal reality filtering, is associated with behaviourally spontaneous confabulation and disorientation after acute and focal brain damage, but not with other forms of confabulation, and not with reality confusion in degenerative dementia. Potential links with psychosis and decision making will be discussed.
Current endocrine therapeutics for the estrogen-dependent disease endometriosis often lead to considerable side-effects as they act by reducing estrogen action systemically. A more recent approach takes advantage of the fact that the weak estrogen estrone (E1) which is abundant in the plasma, is activated in the target cell to the highly estrogenic estradiol (E2) by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). 17β-HSD1 is overexpressed in endometriosis and thus a promising target for the treatment of this disease, with the prospect of less target-associated side-effects. Potent inhibitors from the class of bicyclic substituted hydroxyphenylmethanones with sulfonamide moiety recently described by us suffered from high molecular weight and low selectivity over 17βHSD2, the physiological adversary of 17β-HSD1. We describe the structural optimizations leading to the discovery of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-yl)(2,6-difluoro-3-hydroxyphenyl)methanone 20, which displayed a sub-nanomolar IC50 towards 17β-HSD1 as well as high selectivity over the type 2 enzyme, the estrogen receptors α and β and a range of hepatic CYP enzymes. The compound did neither show cellular toxicity, nor PXR activation nor mutagenicity in the AMES II assay. Additional favourable pharmacokinetic properties (rat) make 20 a suitable candidate for proof-of-principle studies using xenotransplanted immunodeficient rats.
Physical activity interventions targeting children and adolescents (≤18 years) often focus on complex intra- and inter-personal behavioral constructs, social-ecological frameworks, or some combination of both. Recently published meta-analytical reviews and large-scale randomized controlled trials have demonstrated that these intervention approaches have largely produced minimal or no improvements in young people's physical activity levels.
In this paper, we propose that the main reason for previous studies' limited effects is that fundamental mechanisms that lead to change in youth physical activity have often been overlooked or misunderstood. Evidence from observational and experimental studies is presented to support the development of a new theory positing that the primary mechanisms of change in many youth physical activity interventions are approaches that fall into one of the following three categories: (a) the expansion of opportunities for youth to be active by the inclusion of a new occasion to be active, (b) the extension of an existing physical activity opportunity by increasing the amount of time allocated for that opportunity, and/or (c) the enhancement of existing physical activity opportunities through strategies designed to increase physical activity above routine practice. Their application and considerations for intervention design and interpretation are presented. The utility of these mechanisms, referred to as the Theory of Expanded, Extended, and Enhanced Opportunities (TEO), is demonstrated in their parsimony, logical appeal, support with empirical evidence, and the direct and immediate application to numerous settings and contexts. The TEO offers a new way to understand youth physical activity behaviors and provides a common taxonomy by which interventionists can identify appropriate targets for interventions across different settings and contexts. We believe the formalization of the TEO concepts will propel them to the forefront in the design of future intervention studies and through their use, lead to a greater impact on youth activity behaviors than what has been demonstrated in previous studies.
Despite advances in asthma treatment, severe asthma (SA) still results in high morbidity and use of health resources. Our hypothesis was that SA patients would achieve adequate control with a systematic protocol, including oral corticosteroids, budesonide/formoterol maintenance and reliever therapy and a multidisciplinary approach to improve adherence.
Non-controlled (NC) SA patients were enrolled to receive 2 weeks of oral corticosteroids and 12 weeks of formoterol + budesonide. Assessments included asthma control questionnaire (ACQ), asthma control test (ACT), daily symptom diary, lung function and health-related quality of life (HRQoL) questionnaires.
Of 51 patients, 13 (25.5%) achieved control. NC patients had higher utilization of health resources and higher exacerbation rates. Both controlled (C) and NC patients had significantly reduced ACQ scores after oral corticosteroid treatment. After 12 weeks, C patients continued improving. NC patients did not have significant changes. A similar pattern was found regarding lung function, use of rescue medication, and days free of symptoms. After 2 weeks of oral corticosteroids, an increase occurred in those who achieved the ACQ cut off; however, 53.8% of C patients had an ACQ < 1.57 versus 21.1% of NC patients (p = 0.03). Both groups had low HRQoL at baseline with improvement after intervention.
Despite rigorous, optimized follow-up treatment, 75% of SA patients did not achieve adequate symptom control and presented with impaired quality of life. Conversely, application of a low-cost, easy to implement systematic protocol can prevent up to 25% of SA patients from up-titrating to new and complex therapies, thus reducing costs and morbidity.
Retrospectively registered at ClinicalTrial.gov on 22 February 2010 ( NCT01089322 ).
Geographic variation in health care use has been demonstrated in many countries over many years. Such variation can be warranted - in response to patient need or preference for care - or unwarranted. Unwarranted variation raises concerns about equity and appropriateness of care. Recent analyses of health care provision in the Australian atlas of healthcare variation show that when routinely available Australian data are mapped by residence of patient, there are wide variations in rates of use of diagnostic tests, dispensing of prescriptions for a range of indications, surgical procedures and hospital admission rates. Despite the wealth of studies demonstrating variation in care internationally, there is relatively little research that explores the best ways of responding to unwarranted variation. Recommendations for action in the Australian Atlas focus on some approaches that could be used in Australia.
Groucho (Gro)/Transducin-like enhancer of split (TLE) family proteins act as co-repressors of many transcription factors, and are involved in key signaling pathways. TLE1 negatively regulates inflammation and has potential roles in various diseases, including cancer. Previous studies suggest TLE1 could be used as a diagnostic marker and is a possible therapeutic target in various malignancies. It is therefore important to elucidate the mechanisms underlying TLE1 function during cancer initiation and metastasis. In this review, we highlight the functions of TLE1 in cancer and explore targeted approaches for cancer diagnosis and treatment. In particular, we discuss the TLE1 function in pancreatic cancer.
The interactions between alkynylplatinum(II) terpyridine complexes 1-3 and the G-quadruplex DNA, including c-myc and telomeric quadruplex DNA, are investigated both in dilute solution and under molecular crowding conditions. The UV-vis absorption spectroscopy, circular dichroism and molecular docking studies suggest that 1-3 associate with telomeric and c-myc G-quadruplexes via groove binding, and electrostatic interactions. Experimental studies indicate that under molecular crowding conditions (in the presence of 40wt% PEG 200), 1-2 show weak affinity for c-myc, while 3 still displays high affinity and selectivity for c-myc. On the other hand, 1-3 act as efficient and selective ligand for telomeric quadruplex DNA under molecular crowding conditions. The complex 3 exhibits excellent cytotoxicity against A549, K562 and SGC-7901, with IC50 values that are 35.0-fold, 10.0-fold, and 12.1-fold lower than the values of cisplatin under the same conditions, respectively.
The behavior of biochemical reactions requiring repeated enzymatic substrate modification depends critically on whether the enzymes act processively or distributively. Whereas processive enzymes bind only once to a substrate before carrying out a sequence of modifications, distributive enzymes release the substrate after each modification and thus require repeated bindings. Recent experimental and computational studies have revealed that distributive enzymes can act processively due to rapid rebindings (so-called quasi-processivity). In this study, we derive an analytical estimate of the probability of rapid rebinding and show that well-mixed ordinary differential equation models can use this probability to quantitatively replicate the behavior of spatial models. Importantly, rebinding requires that connections be added to the well-mixed reaction network; merely modifying rate constants is insufficient. We then use these well-mixed models to suggest experiments to 1) detect quasi-processivity and 2) test the theory. Finally, we show that rapid rebindings drastically alter the reaction's Michaelis-Menten rate equations.
Contractile rings play critical roles in a number of biological processes, including oogenesis, wound healing, and cytokinesis. In many cases, the activity of motor proteins such as nonmuscle myosins is required for appropriate constriction of these contractile rings. In the gonad of the nematode worm Caenorhabditis elegans, ring channels are a specialized form of contractile ring that are maintained at a constant diameter before oogenesis. We propose a model of ring channel maintenance that explicitly incorporates force generation by motor proteins that can act normally or tangentially to the ring channel opening. We find that both modes of force generation are needed to maintain the ring channels. We demonstrate experimentally that the type II myosins NMY-1 and NMY-2 antagonize each other in the ring channels by producing force in perpendicular directions: the experimental depletion of NMY-1/theoretical decrease in orthogonal force allows premature ring constriction and cellularization, whereas the experimental depletion of NMY-2/theoretical decrease in tangential force opens the ring channels and prevents cellularization. Together, our experimental and theoretical results show that both forces, mediated by NMY-1 and NMY-2, are crucial for maintaining the appropriate ring channel diameter and dynamics throughout the gonad.
Magainin 2 (MAG2) and PGLa are two α-helical antimicrobial peptides found in the skin of the African frog Xenopus laevis. They act by permeabilizing bacterial membranes and exhibit an exemplary synergism. Here, we determined the detailed molecular alignment and dynamical behavior of MAG2 in oriented lipid bilayers by using (2)H-NMR on Ala-d3-labeled peptides, which yielded orientation-dependent quadrupolar splittings of the labels. The amphiphilic MAG2 helix was found to lie flat on the membrane surface in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), as expected, with a tilt angle close to 90°. This orientation fits well with all-atom molecular-dynamics simulations of MAG2 performed in DMPC and DMPC/DMPG. In the presence of an equimolar amount of PGLa, the NMR analysis showed that MAG2 becames tilted at an angle of 120°, and its azimuthal rotation angle also changes. Since this interaction was found to occur in a concentration range where the peptides per se do not interact with their own type, we propose that MAG2 forms a stable heterodimer with PGLa. Given that the PGLa molecules in the complex are known to be flipped into a fully upright orientation, with a helix tilt close to 180°, they must make up the actual transmembrane pore. We thus suggest that the two negative charges on the C-terminus of the obliquely tilted MAG2 peptides neutralize some of the cationic groups on the upright PGLa helices. This would stabilize the assembly of PGLa into a toroidal pore with an overall reduced charge density, which could explain the mechanism of synergy.
Across the entire distribution of a species, populations may have variable responses to environmental perturbations. Many bat species experience mortality in large portions of their range during hibernation and along migratory paths to and from wintering grounds, from White-nose syndrome (WNS) and wind energy development, respectively. In some areas, warm temperatures may allow bats to remain active through winter, thus decreasing their susceptibility to WNS and/or mortality associated with migration to wintering grounds. These areas could act as a refugia and be important for the persistence of local populations. To determine if warmer temperatures affect bat activity, we compared year-round activity of bat populations in the Coastal Plain and Piedmont of North Carolina, USA, two regions that differ in winter temperature. We established six recording stations, four along a 295-kilometer north-south transect in the Coastal Plain, and two in the Piedmont of North Carolina. We recorded bat activity over two years. We supplemented our recordings with mist-net data. Although bat activity was lower during winter at all sites, the odds of recording a bat during winter were higher at Coastal Plain sites when compared with Piedmont sites. Further, bats in the Piedmont had a lower level of winter activity compared to summer activity than bats in the Coastal Plain that had more similar levels of activity in the winter and summer. We found high bat species richness on the Coastal Plain in winter, with winter-active species including those known to hibernate throughout most of their range and others known to be long distance migrants. In particular, two species impacted by WNS, the northern long-eared bat (Myotis septentrionalis) and tricolored bat (Perimyotis subflavus), were present year round in the Coastal Plain. The tricolored bat was also present year-round in the Piedmont. In the Coastal Plain, the long distance migratory hoary bat (Lasiurus cinereus) was active in the winter but not present during the other seasons, and the long distance migratory silver-haired bat (Lasionycteris noctivagans) was active primarily in the winter, suggesting the Coastal Plain may be an overwintering ground for these two species. We suggest that the winter activity exhibited by populations of bats on the North Carolina Coastal Plain has important conservation implications and these populations should be carefully monitored and afforded protection.
We investigated the effect of structural interdependencies between groups (especially inequality), and interdependencies between individuals on ingroup favoritism in minimal group situations. Previous research has attempted to determine whether ingroup favoritism is produced by categorization or intragroup interdependencies (reciprocation expectations), but recent literature suggests that it is not possible to tease these influences apart. We report two studies that investigate how ingroup favoritism evolves over time in social interaction. The levels of ingroup favoritism were affected by categorization and inequality, and the level of ingroup favoritism changed over time, increasing or decreasing depending on the nature of the initial intergroup structure. We conclude by providing two explanations for this change: emergent norms, and changes to the intergroup situation produced by interaction. Our experiments confirm the value of studying the evolution of minimal group behavior, especially for explaining why low status groups act to preserve intergroup inequalities.
To determine how stakeholder opinions of treatments influence service user decisions to adhere to courses of actions necessary to treat metabolic conditions.
Qualitative open-ended interviews were conducted with 20 service providers, 25 service users, and 9 caregivers. Grounded theory was used to generate an understanding that linked preferences of care with adherence to follow-up treatments.
Participants spoke about several considerations when discussing adherence: Resource limitations were the predominant consideration. Social considerations such as stigma and support surfaced in caregiver and service-user interviews. The influence of symptoms, especially their absence could reduce adherence, and organizational considerations related to the opinions they had about the qualifications of professionals.
A rational patient model partially organizes our findings, but emotional components related to stigma and the opinion of service providers do not fit well into such a model. If service providers do not consider components of the decision making process which fall outside of the rational patient model, they may incorrectly be leveraging suboptimal values to bring about adherence to treatment plans. Being sensitive to the values of service users and their caregivers may allow service providers to better act on points that may bring about change in non-compliant service users with schizophrenia and metabolic comorbidities.
Multiple sources of data in combination are essential for species delimitation and classification of difficult taxonomic groups. Here we investigate a cicada taxon with unusual cryptic diversity and we attempt to resolve seemingly contradictory data sets. Cicada songs act as species-specific premating barriers and have been used extensively to reveal hidden taxonomic diversity in morphologically similar species. The Palaearctic Cicadetta montana species complex is an excellent example where distinct song patterns have disclosed multiple recently described species. Indeed, two taxa turned out to be especially diverse in that they form a "complex within the complex": the Cicadetta cerdaniensis song group (four species studied previously) and Cicadetta brevipennis (examined in details here). Based on acoustic, morphological, molecular, ecological and spatial data sampled throughout their broad European distribution, we find that Cicadetta brevipennis s. l. comprises five lineages. The most distinct lineage is identified as Cicadetta petryi Schumacher, 1924, which we re-assign to the species level. Cicadetta brevipennis litoralis Puissant & Hertach ssp. n. and Cicadetta brevipennis hippolaidica Hertach ssp. n. are new to science. The latter hybridizes with Cicadetta brevipennis brevipennis Fieber, 1876 at a zone inferred from intermediate song patterns. The fifth lineage requires additional investigation. The C. cerdaniensis and the C. brevipennis song groups exhibit characteristic, clearly distinct basic song patterns that act as reproductive barriers. However, they remain completely intermixed in the Bayesian and maximum likelihood COI and COII mitochondrial DNA phylogenies. The closest relative of each of the four cerdaniensis group species is a brevipennis group taxon. In our favoured scenario the phylogenetic pairs originated in common Pleistocene glacial refuges where the taxa speciated and experienced sporadic inter-group hybridization leading to extensive introgression and mitochondrial capture.
Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS-EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS-EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS-EcMutL-EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.
Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB's RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870-1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.
Congress authorized creation of the Extremity Trauma and Amputation Center of Excellence (EACE) as part of the 2009 National Defense Authorization Act. The legislation mandated the Department of Defense (DoD) and Department of Veterans Affairs (VA) to implement a comprehensive plan and strategy for the mitigation, treatment, and rehabilitation of traumatic extremity injuries and amputation. The EACE also was tasked with conducting clinically relevant research, fostering collaborations, and building partnerships across multidisciplinary international, federal, and academic networks to optimize the quality of life of service members and veterans who have sustained extremity trauma or amputations. To fulfill the mandate to conduct research, the EACE developed a Research and Surveillance Division that complements and collaborates with outstanding DoD, VA, and academic research programs across the globe. The EACE researchers have efforts in four key research focus areas relevant to extremity trauma and amputation: (1) Novel Rehabilitation Interventions, (2) Advanced Prosthetic and Orthotic Technologies, (3) Epidemiology and Surveillance, and (4) Medical and Surgical Innovations. This overview describes the EACE efforts to innovate, discover, and translate knowledge gleaned from collaborative research partnerships into clinical practice and policy.
The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas.
Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors.
Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair.
Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.
this study aims to discuss the use of Brazilian sign language as the first language for a deaf individual going to a bilingual dialogic clinic from dialogic activities.
This is a longitudinal study, including one deaf individual, called N, interacting with his family and speech therapists.
During the therapeutic process developed inside the bilingual dialogical clinic, N participated in interactive contexts and could constitute himself as author of his sign language texts. In addition, he started to act dialogically and use verbal and nonverbal signs.
Through interactive and dialogical situations developed inside the speech language therapy clinic, this deaf participant got control of his sign language, and started to get interest in and control of the Portuguese language, especially in the written form.
To describe how public prosecutors self-assess their communication approaches and how listeners react to them; to analyze how this relates to gender, age, and work experience.
Descriptive, transversal study. A questionnaire was developed and sent to 126 public prosecutors for completion. Thirty-three completed questionnaires were sent back. The independent variables were gender, age, and number of years of professional experience. The dependent variables were communication self-assessment throughout the years of work, communication parameters used, and listeners' reactions. A descriptive analyzis and Fisher's Exact Test was carried out.
the sample contained both male and female participants with a median age of 43 years and an average of 20 years of professional experience. Most of the respondents claimed they had experienced demotivation, insecurity, tension, and difficulty when trying to convince listeners. More women than men reported they felt that their communication had worsened throughout their careers. All the women reported they experienced insecurity when speaking in public. One third of the public prosecutors stated they suffered from disorders on their voice. Those respondents aged over 43, experienced greater proportion on voice change than younger ones. In contrast to their younger colleagues, the majority of public prosecutors with more than 20 years of professional experience revealed that they felt insecure when speaking in public.
the public prosecutors identified their strong and weak communication parameters. Gender, age, and work experience affect communicative performance.
Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors and very little is known about their functions. RpoE is a sigma factor which in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and high amounts of secretion of ACT. By comparing parental B. pertussis to a rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNAseq analysis was performed and 263 genes were differentially regulated by RpoE and surprisingly the rseA mutant strain where RpoE activity was elevated, expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high RpoE activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through, unidentified mechanisms, in response to conditions.
Electronic devices made from organic materials have the potential to support a more ecologically friendly and affordable future. However, the ability to fabricate devices with well-defined and reproducible electrical and optical properties is hindered by the sensitivity to the presence of chemical impurities. Oxygen in particular is an impurity that can trap electrons and modify conductive properties of some organic materials. Until now the 3-dimensional profiling of oxygen species in organic semiconductors has been elusive and the effect of oxygen remains disputed. In this study we map out high-spatial resolution 3-dimensional distributions of oxygen inclusions near the surface of single crystal rubrene, using Time of Flight Secondary Ion Mass Spectroscopy (ToF-SIMS). Channels of diffused oxygen are found extending from uniform oxygen inclusion layers at the surface. These channels extend to depths in excess of 1.8 μm and act as an entry point for oxygen to diffuse along the ab-plane of the crystal with at least some of the diffused oxygen molecularly binding to rubrene. Our investigation of surfaces at different stages of evolution reveals the extent of oxygen inclusion, which affects rubrene's optical and transport properties, and is consequently of importance for the reliability and longevity of devices.
Engulfment and cell motility 1 (ELMO1) functions as a guanine exchange factor for Rac1 and was recently found to protect endothelial cells from apoptosis. Genome wide association studies suggest that polymorphisms within human elmo1 act as a potential contributing factor for the development of diabetic nephropathy. Yet, the function of ELMO1 with respect to the glomerulus and how this protein contributes to renal pathology was unknown. Thus, this study aimed to identify the role played by ELMO1 in renal development in zebrafish, under hyperglycaemic conditions, and in diabetic nephropathy patients. In zebrafish, hyperglycaemia did not alter renal ELMO1 expression. However, hyperglycaemia leads to pathophysiological and functional alterations within the pronephros, which could be rescued via ELMO1 overexpression. Zebrafish ELMO1 crispants exhibited a renal pathophysiology due to increased apoptosis which could be rescued by the inhibition of apoptosis. In human samples, immunohistochemical staining of ELMO1 in nondiabetic, diabetic and polycystic kidneys localized ELMO1 in glomerular podocytes and in the tubules. However, ELMO1 was not specifically or distinctly regulated under either one of the disease conditions. Collectively, these results highlight ELMO1 as an important factor for glomerular protection and renal cell survival via decreasing apoptosis, especially under diabetic conditions.
Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors.
Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1((MRI61689/+)) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1((MRI61689/+)) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1((MRI61689/+)) erythrocytes were also more likely to be cleared from circulation during infection; the "bystander effect". This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1((MRI61689/+)) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.
The computational repertoire of neurons is enhanced by regenerative electrical signals initiated in dendrites. These events, referred to as dendritic spikes, can act as cell-intrinsic amplifiers of synaptic input. Among these signals, dendritic NMDA spikes are of interest in light of their correlation with synaptic LTP induction. Because it is not possible to block NMDA spikes pharmacologically while maintaining NMDA receptors available to initiate synaptic plasticity, it remains unclear whether NMDA spikes alone can trigger LTP. Here we use dendritic recordings and calcium imaging to analyse the role of NMDA spikes in associative LTP in CA3 pyramidal cells. We show that NMDA spikes produce regenerative branch-specific calcium transients. Decreasing the probability of NMDA spikes reduces LTP, whereas increasing their probability enhances LTP. NMDA spikes and LTP occur without back-propagating action potentials. However, action potentials can facilitate LTP induction by promoting NMDA spikes. Thus, NMDA spikes are necessary and sufficient to produce the critical postsynaptic depolarization required for associative LTP in CA3 pyramidal cells.
A fundamental function of the intestinal epithelium is to act as a barrier that limits interactions between luminal contents such as the intestinal microbiota, the underlying immune system and the remainder of the body, while supporting vectorial transport of nutrients, water and waste products. Epithelial barrier function requires a contiguous layer of cells as well as the junctions that seal the paracellular space between epithelial cells. Compromised intestinal barrier function has been associated with a number of disease states, both intestinal and systemic. Unfortunately, most current clinical data are correlative, making it difficult to separate cause from effect in interpreting the importance of barrier loss. Some data from experimental animal models suggest that compromised epithelial integrity might have a pathogenic role in specific gastrointestinal diseases, but no FDA-approved agents that target the epithelial barrier are presently available. To develop such therapies, a deeper understanding of both disease pathogenesis and mechanisms of barrier regulation must be reached. Here, we review and discuss mechanisms of intestinal barrier loss and the role of intestinal epithelial barrier function in pathogenesis of both intestinal and systemic diseases. We conclude with a discussion of potential strategies to restore the epithelial barrier.
Most HIV transmissions among men who have sex with men (MSM), the group that accounted for 67% of new US infections in 2014, occur via exposure to the rectal mucosa. However, it is unclear how the act of condomless receptive anal intercourse (CRAI) may alter the mucosal immune environment in HIV-negative MSM. Here, we performed a comprehensive characterization of the rectal mucosal immune environment for the phenotype and production of pro-inflammatory cytokines by CD4 and CD8 T cells, global transcriptomic analyses, and the composition of microbiota in HIV-negative MSM. Our results show that compared with men who had never engaged in anal intercourse, the rectal mucosa of MSM engaging in CRAI has a distinct phenotype characterized by higher levels of Th17 cells, greater CD8+ T cell proliferation and production of pro-inflammatory cytokines, molecular signatures associated with mucosal injury and repair likely mediated by innate immune cells, and a microbiota enriched for the Prevotellaceae family. These data provide a high-resolution model of the immunological, molecular, and microbiological perturbations induced by CRAI, will have direct utility in understanding rectal HIV transmission among MSM, and will enhance the design of future biomedical prevention interventions, including candidate HIV vaccines.Mucosal Immunology advance online publication 16 November 2016. doi:10.1038/mi.2016.97.
Von Willebrand disease (MW) is the most common constitutional bleeding disorders. It is caused by a quantitative or qualitative abnormality of the von Willebrand factor (VWF). The laboratory assessment of the disease combines a FVIII assay, and a determination of the antigen and activity of VWF. The analytical validation of VWF:Ag, VWF:Act, vWF:CB is reported in this work and demonstrates good test performance of all three assays, with a coefficient of variation lower than 10% for both the repeatability and reproducibility, stability with a deviation of less than 5% from the target value after six hours at room temperature. The dosages are linear through the following ranges: 2% to 346% for VWF:Ag, 3% to 170% for VWF:Act, and 0.07% to 259% for VWF:CB. The usual values determined on 32 control subjects are in the range of reference values published in the literature. However as the number of control samples tested is small, we will adopt the reference values of the literature of 50 to 200% in routine. The other functional test VWF:CB will be used in our daily practice to differentiate the type of 2M 2A type. However, given that the type 2M is extremely rare, we think it is rather aimed at specialized laboratories in which a large number of patients referred.
Out of 3 billion base pairs in human genome only ~2% code for proteins; and out of 180,000 transcripts in human cells, about 20,000 code for protein, remaining 160,000 are non-coding transcripts. Most of these transcripts are more than 200 base pairs and constitute a group of long non-coding RNA (lncRNA). Many of the lncRNA have its own promoter, and are well conserved in mammals. Accumulating evidence indicates that lncRNAs act as molecular switches in cellular differentiation, movement, apoptosis, and in the reprogramming of cell states by altering gene expression patterns. However, the role of this important group of molecules in angiogenesis is not well understood. Angiogenesis is a complex process and depends on precise regulation of gene expression. Dysregulation of transcription during this process may lead to several diseases including various cancers. As angiogenesis is an important process in cancer pathogenesis and treatment, lncRNA may be playing an important role in angiogenesis. In support of this, lncRNA microvascular invasion in hepatocellular carcinoma (MVIH) has been shown to activate angiogenesis. Furthermore, lncRNA-Meg3-knockout mouse showed increased expression of vascular endothelial growth factor pathway genes and increased cortical microvessel density. Overall, there is strong evidence that lncRNA is an important class of regulatory molecule, and a number of studies have demonstrated that these can be targeted to change cellular physiology and functions. In this review, we have attempted to summarize these studies and elucidate the potential of this novel regulatory molecule as a therapeutic target.
This is the first assessment of the Republic of Slovenia's efforts to synthesize and report physical activity (PA) standards for children and youth following the Active Healthy Kids Global Alliance grading system model.
The Republic of Slovenia Report Card relies on research findings published in peer-reviewed journals, data compiled from national databases, and government initiatives which have been monitoring physical fitness standards in schools for the past 34 years (SLOfit and ACDSi). The Report Card initiative has been jointly coordinated by the University of Primorska and the University of Ljubljana. A Research Work Group consisting of 12 representatives from various aspects of Slovenia's public, private, and government sectors convened to evaluate evidence and assign grades for each PA indicator.
Grades (A, highest, to F, lowest; INC, incomplete) for Slovenia are as follows: Overall Physical Activity (A-), Organized Sport Participation (B-), Active Play (D), Active Transportation (C), Sedentary Behaviors (B+), Family and Peers (INC), Schools (A), Community and the Built Environment (INC), and Government (B+).
This inclusive PA report indicates that overall physical activity minutes remain high in Slovenian children and youth; however, more research is needed to determine the effects of family life, peer influences, and the built environment on active play behaviors.
Physical inactivity in children and youth in India is a major public health problem. The 2016 Indian Report Card on Physical Activity for Children and Youth has been conceptualized to highlight this epidemic by appraising behaviors, contexts, strategies, and investments related to physical activity of Indian children and youth.
An international research collaboration resulted in the formation of a Research Working Group (RWG). RWG determined key indicators; identified, synthesized, and analyzed existing evidence; developed criteria for assigning grades; and, finally, assigned grades to indicators based on consensus.
Overall Physical Activity Levels were assigned a grade of C-. Active Transportation and Sedentary Behaviors were both assigned a grade of C. Government Strategies and Investments was assigned a grade of D. Six other indicators, including the country-specific indicator Physical Fitness, were graded as INC (incomplete) due to the lack of nationally representative evidence.
Based on existing evidence, it appears that most Indian children do not achieve recommended levels of physical activity and spend most of their day in sedentary pursuits. The report card identifies gaps in both investments and research that need to be addressed before understanding the complete picture of active living in children and youth in India.
Currently, there is limited evidence on estimates for physical activity (PA) behavior and sedentary behavior (SB) in Ghana. This report card (RC) is intended to increase awareness and sensitivity about issues surrounding PA and SB in Ghana.
Data were collected from peer-reviewed literature, graduate students' theses, physical education and sports documents, and a survey of opinions of stakeholders covering the 10 key RC indicators and benchmarks. The principal investigator harmonized all grades. A consensus meeting of the RC team was held to assign the final grades.
School and Community grades declined from a D in 2014 to an F in 2016. SB declined from B to D. Family and Active Play were not graded in 2014 and now received an F and a B, respectively. Family and Built Environment were graded F, Active Transportation received a C, and Government and Overall PA were graded D.
A conscious national investment effort can increase overall PA among children.
This article describes the procedures and development of the first Portuguese Report Card on Physical Activity in Children and Adolescents.
Comprehensive searches for data related to indicators of physical activity (PA) were completed by a committee of physical activity and sports specialists. Grades were assigned to each indicator consistent with the process and methodology outlined by the Active Healthy Kids Canada Report Card model.
Nine indicators of PA were graded. The following grades were assigned: Overall Physical Activity Levels, D; Organized Sport Participation, B; Active Play, D; Active Transportation, C; Sedentary Behaviors, D; Family and Peers, C; Schools, B; Community and the Built Environment, D; and Government, C.
Portuguese children and adolescents do not reach sufficient physical activity levels and spend larger amounts of time spent in sedentary behaviors compared with recommendations. Effective policies of PA promotion and implementation are needed in different domains of young people's daily lives.
The Report Card on Physical Activity for Children and Youth aims to consolidate existing evidence, encourage greater evidence-informed physical activity, and improve surveillance of physical activity.
The Japan report card followed the methodology of the Canadian and Scottish report cards, but was adapted to reflect the Japanese context. Nationally representative data were used to score each of the respective indicators.
The 2016 Japan Report Card on Physical Activity for Children and Youth consists of Health Behaviors and Outcomes (7 indicators), and Influences on Health Behaviors (4 indicators). Three Health Behaviors and Outcomes received C grades (Participation in Sport; Sedentary Behavior; Recreational Screen Time; Physical Fitness), while 2 indicators could not be graded (Overall Physical Activity, and Active Play). The indicators Active Transportation (B) and Weight Status were favorable (A). In the Influences domain, Family Influence and Community and the Built Environment were graded as D, while School and Government Strategies and Investments were favorable (B).
The Japan report card illustrated some favorable health behaviors, health outcomes, and influences. There is a need for more evidence especially on overall physical activity levels, active play, and community and the built environment.
The Venezuelan Report Card on Physical Activity for Children and Youth is the first assessment of information related to physical activity in Venezuela. It provides a compilation of existing information throughout the country and assesses how well it is doing at promoting opportunities for children and youth. The aim of this article is to summarize the information available.
Thirteen physical activity indicators were graded by a committee of experts using letters A to F (A, the highest, to F, the lowest) based on national surveys, peer review studies, and policy documents.
Some indicators report incomplete information or a lack of data. Overweight and Obesity were classified as A; Body Composition and Nongovernmental Organization Policies as B; Municipal Level Policies as C; and Overall Physical Activity Levels and National Level Policies as D.
63% of children and youth have low physical activity levels. Venezuela needs to undergo a process of articulation between the several existing initiatives, and for said purposes, political will and a methodological effort is required. Investments, infrastructure, and opportunities will be more equal for all children and youth if more cooperation between institutions is developed and communication strategies are applied.
Physical activity (PA) is a key performance indicator for policy documents in both the Republic of Ireland and Northern Ireland. Building on baseline grades set in 2014, Ireland's second Report Card on Physical Activity for Children and Youth allows for continued surveillance of indicators related to PA in children and youth.
Data and information were extracted and collated for 10 indicators and graded using an international standardized grading system.
Overall, 7 grades stayed the same, 2 increased, and 1 decreased. Grades were assigned as follows: Overall PA, D (an increase); Sedentary Behavior (TV), C-; Physical Education, D-; Active Play, Incomplete/Inconclusive (INC); Active Transportation, D; School, D (a decrease); Home (Family), INC; Community and the Built Environment, B+ (an increase); and Government, INC. Unlike 2014's report card, different grades for the Republic (C-) and Northern Ireland (C+) were assigned for Organized Sport Participation.
Although the grade for Overall PA levels increased to a D, this may reflect the increased quality and quantity of data available. The double burden of low PA and high sedentary levels are concerning and underscore the need for advocacy toward, and surveillance of, progress in achieving targets set by the new National Physical Activity Plan in the Republic and obesity and sport plans in the North.
The Active Healthy Kids 2016 United Arab Emirates (UAE) Report Card provides a systematic evaluation of how the UAE is performing in supporting and engaging physical activity (PA) in children and adolescents.
The Active Healthy Kids Global Alliance framework and standardized set of procedures were used to perform the systematic assessment of PA in UAE youth and children. Indicator grades were based on the proportion of children and youth achieving a defined benchmark: A = 81% to 100%; B = 61% to 80%; C = 41% to 60%; D = 21% to 40%; F = 0% to 20%; INC = incomplete data.
Overall Physical Activity Level and Active Transportation both received a grade of D-/F-. Sedentary Behavior and Family and Peers both received a C- minus grade and School was graded D. Minus grades indicate PA disparities related to age, gender, nationality, socioeconomic status, and geographic location. Government Strategies and Investments received a B+ grade. Sport Participation, Active Play, and Community and the Built Environment were graded INC due to a lack of nationally representative data for all 7 emirates.
The majority of UAE children are not achieving the daily recommended level of PA. The UAE leadership has invested significant resources into improving PA through school- and community-based PA interventions; however, inter- and intraemirate population-based strategies remain fragmented.
Kenya's 2016 report card aimed to highlight the health and well-being of Kenyan children and youth using the best available evidence on the physical activity of Kenyan children and youth. The report pointed at areas where Kenya was succeeding and areas where more action is required.
Inclusive analyses of available data sources on the core indicators related to physical activity and body weights of Kenyan children and youth (5 to 17 years) were conducted. These were assigned grades based on a set of specific criteria.
Results show that Active Play, Active Transportation, Overweight and Obesity, and Sedentary Behavior were favorable with a grade of B. Overall Physical Activity, Organized Sport Participation, and School (infrastructure, policies, and programs) each received a grade of C, while Family and Peers, Government and Nongovernment organizations, as well as the Community and the Built Environment were assigned grade D.
Over 72% of Kenyan children and youth use active transportation to and from school and in their daily lives. Although majority of the children and youth have normal body weight, there is need to ensure that they meet and maintain the physical activity levels recommended by the World Health Organization. More needs to be done especially in relation to the governmental and nongovernmental organizations, organized sports participation, as well as involvement of family and peers in promoting healthy active lifestyles among Kenyan children and youth. More representative data for all indicators are required in Kenya.
The 2016 Chilean Report Card on Physical Activity for Children and Youth is a review of the evidence across indicators of behaviors, settings, and sources of influence associated with physical activity (PA) of Chilean children and youth.
A Research Work Group reviewed available evidence from publications, surveys, government documents and datasets to assign a grade for 11 indicators for PA behavior based on the percentage of compliance for defined benchmarks. Grades were defined as follows: A, 81% to 100% of children accomplishing a given benchmark; B, 61% to 80%; C, 41% to 60%; D, 21% to 40%; F, 0% to 20%; INC, incomplete data available to assign score.
Grades assigned were for i) 'Behaviors that contribute to overall PA levels': Overall PA, F; Organized Sport Participation, D; Active Play, INC; and Active Transportation, C-; ii) 'Factors associated with cardiometabolic risk': Sedentary Behavior, D; Overweight and Obesity, F; Fitness, F; and iii) 'Factors that influence PA': Family and Peers, D; School, D; Community and Built Environment, C; Government Strategies and Investments, C.
Chile faces a major challenge as most PA indicators scored low. There were clear research and information gaps that need to be filled with the implementation of consistent and regular data collection methods.
The first Qatar Active Healthy Kids (QAHK) Report Card was developed in 2015-2016. It is a synthesis of the available evidence on physical activity in children and youth in the state of Qatar-an assessment of the state of the nation. The report card is important for future physical activity advocacy, policy, and program development.
The QAHK Report Card was inspired by the Active Healthy Kids Scotland 2013 Report Card. The methodology used in Scotland's report card was adapted for Qatar. A Working Group identified indicators for physical activity and related health behaviors, and evaluated the available data on these indicators. The card grades were determined by the percentage of children meeting guidelines or recommendations.
The 2016 QAHK Report Card consisted of 9 indicators: 6 Physical Activity and Health Behaviors and Outcomes, and 3 Settings and Influences on these health behaviors and outcomes. The indicator National Policy, Strategy, and Investment was assigned the highest grade (B). Four indicators were assigned D grades: Sedentary Behavior, Dietary Habits, Organized Sports Participation, and Family and Peer Influence. Physical Activity and Obesity were both graded F. Two indicators could not be graded due to insufficient data and/or absence of a recommendation: Active and Outdoor Play, and Community and School Influence.
The QAHK Report Card identified weaknesses and gaps in the evidence on physical activity and health in children and youth in Qatar. The quality of evidence was poor for some indicators, with some data collection methods of limited validity and reliability, or only available for a limited age range, so the grades are best estimates of the current situation in Qatar. Future surveys and research using objective physical activity measures will support the development of a second QAHK Report Card by 2018.
Regular physical activity improves physical and mental health, yet children's physical activity levels were low in England's 2014 Report Card. Within this paper, we update the 2014 Report Card to assess current information for the 9 indicators of physical activity.
A search for nationally representative data on 9 indicators of physical activity was conducted and the data were assessed by an expert panel. The panel assigned grades [ie, A, B, C, D, F, or INC (incomplete)] to each indicator based on whether children across England were achieving specific benchmarks. The 2016 Report Card was produced and disseminated.
The following grades were awarded: Overall Physical Activity Levels: D-; Organized Sport Participation: D; Active Play: INC; Active Transportation: C-; Sedentary Behaviors: INC; Family and Peers: INC; School: B+; Community and the Built Environment: B; Government Strategies and Investment: INC.
The grades have not improved since the 2014 Report Card and several gaps in the literature are still present. While children's physical activity levels remain low alongside competing sedentary choices, further national plans and investment with local actions are urgently needed to promote physical activity especially via active play, active transport, and family support.
The Active Healthy Kids Global Alliance organized the concurrent preparation of Report Cards on the physical activity of children and youth in 38 countries from 6 continents (representing 60% of the world's population). Nine common indicators were used (Overall Physical Activity, Organized Sport Participation, Active Play, Active Transportation, Sedentary Behavior, Family and Peers, School, Community and the Built Environment, and Government Strategies and Investments), and all Report Cards were generated through a harmonized development process and a standardized grading framework (from A = excellent, to F = failing). The 38 Report Cards were presented at the International Congress on Physical Activity and Public Health in Bangkok, Thailand on November 16, 2016. The consolidated findings are summarized in the form of a Global Matrix demonstrating substantial variation in grades both within and across countries. Countries that lead in certain indicators often lag in others. Average grades for both Overall Physical Activity and Sedentary Behavior around the world are D (low/poor). In contrast, the average grade for indicators related to supports for physical activity was C. Lower-income countries generally had better grades on Overall Physical Activity, Active Transportation, and Sedentary Behaviors compared with higher-income countries, yet worse grades for supports from Family and Peers, Community and the Built Environment, and Government Strategies and Investments. Average grades for all indicators combined were highest (best) in Denmark, Slovenia, and the Netherlands. Many surveillance and research gaps were apparent, especially for the Active Play and Family and Peers indicators. International cooperation and cross-fertilization is encouraged to address existing challenges, understand underlying determinants, conceive innovative solutions, and mitigate the global childhood inactivity crisis. The paradox of higher physical activity and lower sedentary behavior in countries reporting poorer infrastructure, and lower physical activity and higher sedentary behavior in countries reporting better infrastructure, suggests that autonomy to play, travel, or chore requirements and/or fewer attractive sedentary pursuits, rather than infrastructure and structured activities, may facilitate higher levels of physical activity.
Finland's 2016 Report Card on Physical Activity for Children and Youth gathers and translates research results and assesses the status and promotion of physical activity (PA) among Finnish children and youth less than 18 years of age. This article summarizes the results and provides grades for 9 indicators.
The working group evaluated the evidence and assigned grades of A (highest, 81% to 100%), B, C, D, or F (lowest, 0% to 20%) for 9 PA indicators using the Active Healthy Kids Canada Report Card development process.
The grades varied in Finland as follows: 1) Overall PA/fulfillment of recommendations = D, 2) Organized Sport Participation = C, 3) Active Play = C, 4) Active Transportation = B, 5) Sedentary Behaviors = D, 6) Family and Peers = C, 7) School = B, 8) Community and the Built Environment = B, 9) Government = B.
Despite good policies and programs to promote PA in Finland, children and youth overall PA levels are low, whereas their time spent sedentary is high. More effective interventions, operation models, concrete tools as well as environmental solutions are needed to support the work toward more physically active childhood and youth.
The Active Healthy Kids the Netherlands (AHKN) Report Card consolidates and translates research and assesses how the Netherlands is being responsible in providing physical activity (PA) opportunities for children and youth (<18 years). The primary aim of this article is to summarize the results of the 2016 AHKN Report Card.
Nine indicators were graded using the Active Healthy Kids Global Alliance report card development process, which includes a synthesis of the best available research, surveillance, policy and practice findings, and expert consensus.
Grades assigned were: Overall Physical Activity Levels, D; Organized Sport Participation, B; Active Play, B; Active Transportation, A; Sedentary Behaviors, C; Family and Peers, B; School, C; Community and the Built Environment, A; Government Strategies and Investments, INC.
Sedentary behavior and overall PA levels are not meeting current guidelines. However, the Dutch youth behaviors in sports, active transportation, and active play are satisfactory. Several modifiable factors of influence might be enhanced to improve these indicators or at least prevent regression. Although Dutch children accumulate a lot of daily PA through cycling, it is not enough to meet the current national PA guidelines of 60 minutes of moderate-to-vigorous PA per day.
Very few studies have comprehensively analyzed the physical activity of children and adolescents in Brazil. The purpose of this article is to show the methodology and summarize findings from the first Brazilian Report Card on Physical Activity for Children and Youth.
Three Brazilian research institutions coordinated the activities to develop the Brazilian 2016 Report Card. The data available were collected independently and then synthesized by the Research Work Group using the grade system developed for the First Global Matrix released in 2014, which included 9 indicators of physical activity. Where possible, grades were assigned based on the percentage of children and youth meeting each indicator: A is 81% to 100%; B is 61% to 80%; C is 41% to 60%; D is 21% to 40%; F is 0% to 20%; INC is incomplete data.
Among the 9 indicators, only 5 had sufficient data for grading. Overall Physical Activity received a C- grade, Active Transportation received a C+ grade, Sedentary Behavior received a D+ grade, and Government Strategies and Investments received a D grade.
The low grades observed highlight the need for continued efforts aimed at improving physical activity in Brazilian children.
Physical activity (PA) is recognized as one of the core modifiable risk factors of noncommunicable diseases. However, little is known about PA in the Thai population, particularly in children. The report card (RC) project provided Thailand with an opportunity to assess PA behaviors in children. This paper summarizes the methodology, grading process, and the final grades of the Thai RC.
A school-based survey was conducted to collect data from a nationally representative sample of children aged 6 to 17 years. Survey results provided the primary source for the RC. Nine indicators were graded using the Global Matrix 2.0 framework. Grading was undertaken by a national committee comprising experts from key stakeholders.
Grades ranged from F to B. Overall PA and Sedentary Behaviors both received the grade D-. Organized Sport Participation scored a C. Active Play scored the grade F. Active Transport and support from Family and Peers were both graded B. School, Community, and Government indicators were scored C.
In Thai children, participation in PA and active play is very low; conversely, sedentary behaviors are high. These first data on patterns of activity for the Thailand RC will serve to guide national actions and advocacy aimed at increasing PA in children.
The 2016 Report Card on Physical Activity for Children and Youth, the first of its kind, aims to set baseline physical activity (PA) indicators using the Active Healthy Kids Global Alliance grading system.
A research work group analyzed and selected data for the grade assignment meeting (GAM). During the GAM, 17 leading researchers and policy experts from Estonia assessed the data and assigned grades for each of the 9 PA indicators. In addition, recommendations were provided for further actions to improve the grades.
Grades from A (highest) to F (lowest) were assigned as follows: 1) Overall PA (F); 2) Organized Sport (C); 3) Active Play [incomplete data (INC)]; 4) Active Transportation (INC); 5) Sedentary Behaviors (F); 6) Family and Peers (C); 7) School (C); 8) Community and the Built Environment (B); and 9) Government (C). An indicator was marked as incomplete (INC) when there was a lack of representative quality data.
Evidence suggests that PA levels of Estonian children remain very low, despite moderately supportive social, environmental, and regulatory factors. There are many challenges to overcome in supporting and promoting PA of children and youth (eg, cross-sectional cooperation, implementing interventions, changing social norms, empowerment of parents and educational institutions).
Internationally comparable evidence is important to advocate for young people's physical activity. The aim of this article is to present the inaugural Shanghai (China) Report Card on Physical Activity for Children and Youth.
Since no national data are available, the working group developed the survey questionnaire and carried out the school surveys for students (n = 71,404), parents (n = 70,346), and school administrators and teachers (n = 1398). The grades of 9 report card indicators were assigned in accordance with the survey results against a defined benchmark: A is 81% to 100%; B is 61% to 80%; C is 41% to 60%, D is 21% to 40%; F is 0% to 20%.
The 9 indicators were graded as follows: Overall Physical Activity Levels (F), Organized Sport Participation (F), Active Play (D-), Active Transportation (C-), Sedentary Behavior (F), Family and Peers (B), School (B+), Community and the Built Environment (D+), and Government (D).
Levels of physical activity and sedentary behavior were low and below the respective recommended guidelines. Interventions and policies at the community level should be encouraged to promote physical activity and reduce sedentary behavior. Future national surveys should be encouraged to strengthen Shanghai's Report Card on Physical Activity for Children and Youth.
Hong Kong's 2016 Report Card on Physical Activity (PA) for Children and Youth is the first evidence-based synthesis of various indicators related to individual behaviors that contribute to overall PA levels, settings and sources of influence, and strategies and investments in Hong Kong.
Following a standardized protocol, currently best available data for Hong Kong youth were collated and evaluated by an expert consensus panel on 9 indicators (5 activity behaviors and 4 influences on these behaviors).
Less than half of the children and youth met the recommended PA level. As a result, a D grade was given for Overall PA levels. Organized Sport Participation and Active Transportation received grades of C- and B, respectively. Sedentary Behaviors and School scored a C grade. Community and the Built Environment scored a grade of B. Family Influence received as low a score as Overall PA (D). Active Play and Government were not graded due to incomplete data.
PA levels are low and sedentary behaviors are high for children and youth in Hong Kong. Promising policies exist in schools and features of community and the built environment are favorable. Increasing family support should be emphasized for future PA promotion.
Two years on from the inaugural Active Healthy Kids Australia (AHKA) Physical Activity Report Card, there has been little to no change with the majority of Australian children still insufficiently active.
The 2016 AHKA Report Card was developed using the best available national- and state-based physical activity data, which were evaluated by the AHKA Research Working Group using predetermined weighting criteria and benchmarks to assign letter grades to the 12 Report Card indicators.
In comparison with 2014, Overall Physical Activity Levels was again assigned a D- with Organized Sport and Physical Activity Participation increasing to a B (was B-) and Active Transport declining to a C- (was C). The settings and sources of influence again performed well (A- to a C+), however Government Strategies and Investments saw a decline (C+ to a D). The traits associated with physical activity were also graded poorly (C- to a D).
Australian youth are insufficiently active and engage in high levels of screen-based sedentary behaviors. While a range of support structures exist, Australia lacks an overarching National Physical Activity Plan that would unify the country and encourage the cultural shift needed to face the inactivity crisis head on.
In this article, we report the grades for the second New Zealand Report Card on Physical Activity for Children and Youth, which represents a synthesis of available New Zealand evidence across 9 core indicators.
An expert panel of physical activity (PA) researchers collated and reviewed available nationally representative survey data between March and May 2016. In the absence of new data, (2014-2016) regional level data were used to inform the direction of existing grades. Grades were assigned based on the percentage of children and youth meeting each indicator: A is 81% to 100%; B is 61% to 80%; C is 41% to 60%, D is 21% to 40%; F is 0% to 20%; INC is Incomplete data.
Overall PA, Active Play, and Government Initiatives were graded B-; Community Environments was graded B; Sport Participation and School Environment received a C+; Sedentary Behaviors and Family/Peer Support were graded C; and Active Travel was graded C-.
Overall PA participation was satisfactory for young children but not for youth. The grade for PA decreased slightly from the 2014 report card; however, there was an improvement in grades for built and school environments, which may support regional and national-level initiatives for promoting PA.
The first Danish Report Card on Physical Activity (PA) for Children and Youth describes Denmark's efforts in promoting and facilitating PA and PA opportunities for children and youth.
The report card relies primarily on a synthesis of the best available research and policy strategies identified by the Report Card Research Committee consisting of a wide presentation of researchers and experts within PA health behaviors and policy development. The work was coordinated by Research and Innovation Centre for Human Movement and Learning situated at the University of Southern Denmark and the University College Lillebaelt. Nine PA indicators were graded using the Active Healthy Kids Canada Report Card development process.
Grades from A (highest) to F (lowest) varied in Denmark as follows: 1) Overall Physical Activity (D+), 2) Organized Sport Participation (A), 3) Active Play (INC; incomplete), 4) Active Transportation (B), 5) Sedentary Behaviors (INC), 6) Family and Peers (INC), 7) School (B), 8) Community and the Built Environment (B+), and 9) Government strategies and investments (A-).
A large proportion of children in Denmark do not meet the recommendations for PA despite the favorable investments and intensions from the government to create good facilities and promote PA.
The report card was a synthesis of the best available evidence on the performance of Zimbabwean children and youth on key physical activity (PA) indicators. The aim of this article was to summarize the results from the 2016 Zimbabwe Report Card.
The Report Card Working Group gathered and synthesized the best available evidence, met, discussed and assigned grades to 10 indicators based on the Active Healthy Kids Global Alliance global matrix grading system.
The indicators were graded as follows: overall PA (C+), organized sport participation (B), active play (D+), active transportation (A-), sedentary behaviors (B), school (D), family and peers (Incomplete), community and the built environment (F), government (D) and nongovernmental organizations (Incomplete).
Although the majority of children used active transport, played organized sports and engaged in acceptable levels of PA, most of them did not meet the recommended hours of unstructured/unorganized play per day. At present, there are limited data to accurately inform the Zimbabwe Report Card therefore studies employing robust research designs with representative samples are needed. Zimbabwe also needs to prioritize policies and investments that promote greater and safe participation in PA among children and youth.
South Korea's 2016 Report Card on Physical Activity for Children and Youth is the first assessment of physical activity according to the indicators set by Active Healthy Kids Global Alliance.
National surveys were used as preferred sources of data. This was then supported by peer-reviewed papers and government reports identified by a systematic search of the literature written in English or Korean. A Research Working Group then graded indicators based on the collected evidence.
Each indicator was graded as follows: Overall Physical Activity, D-; Organized Sport and Physical Activity Participation, C-; Active Transport, C+; Sedentary Behavior, F; School, D; Government and Investment, C; Active Play, Physical Literacy, Family and Peers, and Community and Built Environment were graded INC (incomplete) due to lack of available evidence.
Though the final grades of key indicators for South Korean children and youth are not satisfactory, increasing interests and investments have been demonstrated at a national level. More evidence is required for comprehensive assessment on all indicators to better inform policy and practice. This should be accompanied by the use of consistent criteria to contribute to global efforts for active healthy kids.
Physical activity (PA) is vital to the holistic development of young people. Regular participation in PA is associated with substantial benefits for health, cognitive function, and social inclusion. Recognizing the potential of PA in the context of the current peace process in Colombia, the purpose of this article is to present the methodology and results of Colombia's second Report Card on PA for children and youth.
A group of experts on PA graded 14 PA indicators based on data from national surveys and policy documents.
National and departmental policy indicators received a grade of B, while organized sport participation, overweight, obesity, community influence, and nongovernment initiatives indicators received a grade of C. Overall PA levels, active transportation, sedentary behaviors, and school influence received a grade of D. Active play, low physical fitness, and family influence received an Incomplete grade.
PA levels are low and sedentary behaviors are high in Colombian children and youth, with notable geographic differences. A broad policy framework translated into specific actions could provide unique opportunities to bridge the gap between knowledge and practice, and contribute to social integration goals in a postconflict setting.
The 2016 Active Healthy Kids Scotland Report Card aims to improve surveillance of physical activity (PA), facilitate international comparisons, and encourage evidence-informed PA and health policy.
Active Healthy Kids Canada Report Card methodology was used: a search for data on child and adolescent PA and health published after the 2013 Scottish Report Card was carried out. Data sources were considered for grading if based on representative samples with prevalence estimates made using methods with low bias. Ten health behaviors/outcomes were graded on an A to F scale based on quintiles (prevalence meeting recommendations ≥80% graded A down to <20% graded F).
Three of the seven Health Behaviors and Outcomes received F or F- grades: Overall PA, Sedentary Behavior, and Obesity. Active and Outdoor Play and Organized Sport Participation could not be graded. Active Commuting to School was graded C, and Diet was graded D-. Family and Peer Influence was graded D-; Perceived Safety and Availability of Space for PA as well as the National Policy Environment were more favorable (both B).
Grades were identical to those in 2013. Scotland has a generally favorable environment for PA, but children and adolescents have low PA and high sedentary behavior. Gaps in surveillance included lack of objectively measured PA, no surveillance of moderate-to-vigorous PA in children, summary surveillance data not expressed in ways which match recommendations (eg, for PA in young children; for screen-time), and no surveillance of Sport Participation, Active and Outdoor Play, or Sitting. Scottish policy does not include sedentary behavior at present.
Poland's 2016 Report Card on Physical Activity for Children and Youth is the first assessment of child and youth physical activity (PA) in Poland using the Active Healthy Kids Global Alliance grading system. The main goal was to summarize and describe the current state of child and youth PA to increase awareness and surveillance.
The systematic methodology that underpins the Active Healthy Kids Canada Report Card was adapted and applied to the Polish report card. The best available data were consolidated, reviewed by a group of experts, and used to assign the letter grades to 9 core PA indicators on a scale ranging from A (highest) to F (lowest).
The 9 indicators were graded as such: 1) Overall Physical Activity (D), 2) Organized Sport Participation (C), 3) Active Play (INC), 4) Active Transportation (C), 5) Sedentary Behaviors (D), 6) Family and Peers (C), 7) School (B), 8) Community and the Built Environment (C), and 9) Government Strategies and Investments (C).
The final grades show a strong role of school in providing PA for children and youth in Poland. However, promotion of school-based sport participation appears to be insufficient by itself to sustainably promote PA in this group.
The 2016 Mexican Report Card on Physical Activity for Children and Youth aims to assess how Mexico is doing in terms of providing physical activity (PA) opportunities for Mexican children and youth. The purpose of this article is to summarize results from the Mexican 2016 Report Card.
A literature search was conducted in Spanish and English languages using major databases, and complemented with a review of government/nongovernment documents, websites, and national health surveys. Information on the 9 indicators outlined in the Global Matrix of Report Card Grades was extracted. A team of Mexican experts met to discuss and assign a grade on each indicator based on the best available evidence and established benchmarks.
Daily behaviors grades were Overall PA (C), Organized Sport Participation (D), Active Play (D-), Active Transportation (C), and Sedentary Behavior (D). For Settings and Sources of Influence, grades were Family and Peers (INC), School (D-), and Community and Environment (D). Strategies and Investments grades were Government Strategies (C) and Non-Government (F).
PA and sedentary behaviors among Mexican children and youth remain below the recommended levels. Government and communities are far from providing appropriate and sufficient physical activity opportunities for children and youth.
The ParticipACTION Report Card on Physical Activity for Children and Youth is the most comprehensive assessment of child and youth physical activity in Canada and provides an update or "state of the nation" that assesses how Canada is doing at promoting and facilitating physical activity opportunities for children and youth. The purpose of this paper is to summarize the results of the 2016 ParticipACTION Report Card.
Twelve physical activity indicators were graded by a committee of experts using a process that was informed by the best available evidence. Sources included national surveys, peer-reviewed literature, and gray literature such as government and nongovernment reports and online content.
Grades were assigned to Daily Behaviors (Overall Physical Activity: D-; Organized Sport and Physical Activity Participation: B; Active Play: D+; Active Transportation: D; Physical Literacy: D+; Sleep: B; Sedentary Behaviors: F), Settings and Sources of Influence (Family and Peers: C+; School: B; Community and Environment: A-), and Strategies and Investments (Government: B-; Nongovernment: A-).
Similar to previous years of the Report Card, Canada generally received good grades for indicators relating to investment, infrastructure, strategies, policies, and programming, and poor grades for behavioral indicators (eg, Overall Physical Activity, Sedentary Behaviors).
The 2016 United States (U.S.) Report Card on Physical Activity for Children and Youth provides a comprehensive evaluation of physical activity levels and factors influencing physical activity among children and youth.
The report card includes 10 indicators: Overall Physical Activity, Sedentary Behavior, Active Transportation, Organized Sport Participation, Active Play, Health-related Fitness, Family and Peers, School, Community and the Built Environment, and Government Strategies and Investments. Nationally representative data were used to evaluate the indicators using a standard grading rubric.
Sufficient data were available to assign grades to 7 of the indicators, and these ranged from B- for Community and the Built Environment to F for Active Transportation. Overall Physical Activity received a grade of D- due to the low prevalence of meeting physical activity guidelines. A grade of D was assigned to Health-related Fitness, reflecting the low prevalence of meeting cardiorespiratory fitness standards. Disparities across age, gender, racial/ethnic and socioeconomic groups were observed for several indicators.
Continued poor grades suggest that additional work is required to provide opportunities for U.S. children to be physically active. The observed disparities indicate that special attention should be given to girls, minorities, and those from lower socioeconomic groups when implementing intervention strategies.
We present results of the 2016 Healthy Active Kids South Africa (HAKSA) Report Card on the current status of physical activity (PA) and nutrition in South African youth. The context in which we interpret the findings is that participation in PA is a fundamental human right, along with the right to "attainment of the highest standard of health."
The HAKSA 2016 Writing Group was comprised of 33 authorities in physical education, exercise science, nutrition, public health, and journalism. The search strategy was based on peer-reviewed manuscripts, dissertations, and 'gray' literature. The core PA indicators are Overall Physical Activity Level; Organized Sport Participation; Active and Outdoor Play; Active Transportation; Sedentary Behaviors; Family and Peer Influences; School; Community and the Built Environment; and National Government Policy, Strategies, and Investment. In addition, we reported on Physical Fitness and Motor Proficiency separately. We also reported on nutrition indicators including Overweight and Under-nutrition along with certain key behaviors such as Fruit and Vegetable Intake, and policies and programs including School Nutrition Programs and Tuck Shops. Data were extracted and grades assigned after consensus was reached. Grades were assigned to each indicator ranging from an A, succeeding with a large majority of children and youth (81% to 100%); B, succeeding with well over half of children and youth (61% to 80%); C, succeeding with about half of children and youth (41% to 60%); D, succeeding with less than half but some children and youth (21% to 40%); and F, succeeding with very few children and youth (0% to 20%); INC is inconclusive.
Overall PA levels received a C grade, as we are succeeding with more than 50% of children meeting recommendations. Organized Sports Participation also received a C, and Government Policies remain promising, receiving a B. Screen time and sedentary behavior were a major concern. Under- and over-weight were highlighted and, as overweight is on the rise, received a D grade.
In particular, issues of food security, obesogenic environments, and access to activity-supportive environments should guide social mobilization downstream and policy upstream. There is an urgent need for practice-based evidence based on evaluation of existing, scaled up interventions.
This is the second Active Healthy Kids Wales Report Card. The 2016 version consolidates and translates research related to physical activity (PA) among children and youth in Wales, and aims to raise the awareness of children's engagement in PA and sedentary behaviors.
Ten PA indicators were graded using the Active Healthy Kids-Canada Report Card methodology involving a synthesis and expert consensus of the best available evidence.
Grades were assigned as follows: Overall PA, D+; Organized Sport Participation, C; Active and Outdoor Play, C; Active Transportation, C; Sedentary Behaviors, D-; Physical Literacy, INC; Family and Peer Influences, D+; School, B; Community and the Built Environment, C; and National Government Policy, Strategies, and Investments, B-.
Despite the existence of sound policies, programs, and infrastructure, PA levels of children and youth in Wales are one of the lowest and sedentary behavior one of the highest globally. From the 2014 Report Card, the Family and Peer Influences grade improved from D to D+, whereas Community and the Built Environment dropped from B to C. These results indicate that a concerted effort is required to increase PA and decrease sedentary time in children and young people in Wales.
This 2016 Belgium Report Card on Physical Activity for Children and Youth is the first systematic evaluation of physical activity (PA) behaviors, related health behaviors, health outcomes, and influences thereon, using the Active Healthy Kids Canada grading framework.
A research working group consisting of PA experts from both Flanders and Wallonia collaborated to determine the indicators to be graded, data sources to be used, and factors to be taken into account during the grading process. Grades were finalized after consensus was reached among the research working group and 2 stakeholder groups consisting of academic and policy experts in the fields of PA, sedentary behavior, and dietary behavior.
Eleven indicators were selected and assigned the following grades: Overall PA (F+), Organized Sport Participation (C-), Active Play (C+), Active Transportation (C-), Sedentary Behaviors (D-), School (B-), Government Strategies and Investment (C+), and Weight Status (D). Incomplete grades were assigned to Family and Peers, Community and the Built Environment, and Dietary Behaviors due to a lack of nationally representative data.
Despite moderately positive social and environmental influences, PA levels of Belgian children and youth are low while levels of sedentary behaviors are high.
This article describes the procedures and development of the 2016 Mozambican Report Card on Physical Activity for Children and Adolescents.
Following the procedures adopted in 2014 for that year's report card, comprehensive searches on new data related to indicators of physical activity (PA) were done. A committee composed of physical activity and sports specialists graded each indicator consistent with the process and methodology outlined by the Active Healthy Kids Canada Report Card model.
Nine indicators of PA were graded. Compared with 2014 there were several differences which were caused by changes in the country as well as a more effective evaluation from the committee. The following grades were assigned: Overall Physical Activity Levels, C; Organized Sport Participation, F; Active Play, D; Active Transportation, C; Schools, D; Community and the Built Environment, F; and Government, F. Sedentary Behaviors and Family and Peers were graded Incomplete due to the lack of available information.
The decline of the PA habits in urban centers reported in 2014 are accentuated and is influencing the rural areas in several ways. At present, there is no strategy or effective action from authorities to reverse this negative trend.
The 2016 Malaysia Active Healthy Kids Report Card aims to collect, assess, and grade current and comprehensive data on physical activity (PA) and associated factors in Malaysian children and adolescents aged 5 to 17 years.
This report card was developed following the Active Healthy Kids Canada Report Card protocol. The Research Working Group identified the core matrices, assessed the key data sources, and evaluated the evidence gathered for grade assignments. A grade was assigned to each indicator by comparing the best available evidence against relevant benchmark using a standardized grading scheme.
Overall Physical Activity, Active Transportation, and Sedentary Behavior were assigned the D grade. The lowest grade of F was assigned to Diet, while School and Government Strategies and Investments were graded higher with a B. Five indicators were assigned INC (incomplete) due to a lack of representative data.
The report card demonstrates that Malaysian children and adolescents are engaging in low levels of PA and active commuting, high levels of screen time, and have extremely low compliance with dietary recommendations. More efforts are needed to address the root causes of physical inactivity while increasing the opportunities for children and adolescents to be more physically active.
The Nigerian Report card on Physical Activity (PA) in Children and Youth was first developed in 2013 to inform practice and policy on healthy living and prevention of noncommunicable diseases among Nigerian children and youth. This article summarizes the results of the 2016 report card and provides updated evidence on the current situation in Nigeria.
A comprehensive review of literature was undertaken by the Report Card Working Group. Grades were assigned to 10 PA indicators based on the criteria used for the 2013 edition.
Grades assigned to the indicators were Overall PA, D; Active Play and Leisure, C; Active Transportation, B; Sedentary Behaviors (screen-based, F and nonscreen-based, D); Overweight and Obesity, A; PA in Schools, C-; Government/Nongovernment Organizations/Private Sector/Policy, B. The following indicators were graded as Incomplete: Organized Sport and PA, Community and Built Environment, and Family and Peers.
The overall PA levels of Nigerian children and youth seemed to be declining compared with the 2013 Report card but with slight improvement in active play and leisure, and PA in school settings. A substantial number of Nigerian children and youth still have high sedentary behaviors, overweight and obesity. Efforts are needed to promote PA among them.
The first Active Healthy Kids Spanish Report Card aims to gather the most robust information about physical activity (PA) and sedentary behavior of children and adolescents.
A Research Working Group of experts on PA and sport sciences was convened. A comprehensive data search, based on a review of the literature, dissertations, gray literature, and experts' nonpublished data, was conducted to identify the best sources to grade each indicator following the procedures and methodology outlined by the Active Healthy Kids Canada Report Card model.
Overall PA (based on objective and self-reported methods) was graded as D-, Organized Sports Participation as B, Active Play as C+, Active Transportation as C, Sedentary Behavior as D, School as C, and Family and Peers as Incomplete, Community and the Built Environment as Incomplete, and Government as Incomplete.
Spanish children and adolescents showed low levels of adherence to PA and sedentary behavior guidelines, especially females and adolescents. There is a need to achieve consensus and harmonize methods to evaluate PA and sedentary behavior to monitor changes over time and to evaluate the effectiveness of policies to promote PA.
The 2016 Swedish Report Card on Physical Activity (PA) for Children and Youth is a unique compilation of the existing physical and health related data in Sweden. The aim of this article is to summarize the procedure and results from the report card.
Nationally representative surveys and individual studies published between 2005-2015 were included. Eleven PA and health indicators were graded using the Active Healthy Kids Canada grading system. Grades were assigned based on the percentage of children/youth meeting a defined benchmark (A: 81% to 100%, B: 61% to 80%, C: 41% to 60%, D: 21% to 40%, F: 0% to 20%, or incomplete (INC).
The assigned grades were Overall Physical Activity, D; Organized Sport Participation, B+; Active Play, INC; Active Transportation, C+; Sedentary Behaviors, C; Family and Peers, INC; School, C+; Community and the Built Environment, B; Government Strategies and Investments, B; Diet, C-; and Obesity, D.
The included data provides some support that overall PA is too low and sedentary behavior is too high for almost all age groups in Sweden, even with the many national policies as well as an environment that is favorable to the promotion of PA.
'School nurses alone cannot fill the gap in sex and relationships education but they can act as advocates, providing one-to-one advice for pupils'.
Alzheimer's disease (AD), the most common neurodegenerative disorder associated with dementia, not only severely decreases the quality of life for its victims, but also brings a heavy economic burden to the family and society. Unfortunately, few chemical drugs designed for clinical applications have reached the expected preventive or therapeutic effect so far, and combined with their significant side-effects, there is therefore an urgent need for new strategies to be developed for AD treatment. Traditional Chinese Medicine has accumulated many experiences in the treatment of dementia during thousands of years of practice; modern pharmacological studies have confirmed the therapeutic effects of many active components derived from Chinese herbal medicines (CHM). Ginsenoside Rg1, extracted from Radix Ginseng, exerts a [Formula: see text]-secretase inhibitor effect so as to decrease A[Formula: see text] aggregation. It can also inhibit the apoptosis of neuron cells. Tanshinone IIA, extracted from Radix Salviae miltiorrhizae, and baicalin, extracted from Radix Scutellariae[Formula: see text] can inhibit the oxidative stress injury in neuronal cells. Icariin, extracted from Epimedium brevicornum, can decrease A[Formula: see text] levels and the hyperphosphorylation of tau protein, and can also inhibit oxidative stress and apoptosis. Huperzine A, extracted from Huperzia serrata, exerts a cholinesterase inhibitor effect. Evodiamine, extracted from Fructus Evodiae, and curcumin, extracted from Rhizoma Curcumae Longae, exert anti-inflammatory actions. Curcumin can act on A[Formula: see text] and tau too. Due to the advantages of multi-target effects and fewer side effects, Chinese medicine is more appropriate for long-term use. In this present review, the pharmacological effects of commonly used active components derived from Chinese herbal medicines in the treatment of AD are discussed.
As part of its Single Technology Appraisal (STA) process, the National Institute for Health and Care Excellence (NICE) invited the manufacturer of evolocumab (Amgen) to submit evidence on the clinical and cost effectiveness of evolocumab. The appraisal assessed evolocumab as monotherapy or in combination with a statin (HMG-CoA reductase inhibitor) with or without ezetimibe, or in combination with ezetimibe (without statin therapy), in adult patients with primary hypercholesterolaemia (which includes mixed dyslipidaemia), for whom statins do not provide optimal control of their low-density lipoprotein cholesterol (LDL-C) levels and/or for whom statins are contraindicated or not tolerated. The School of Health and Related Research Technology Appraisal Group at the University of Sheffield was commissioned to act as the independent Evidence Review Group (ERG). The ERG produced a critical review of the evidence for the clinical and cost effectiveness of the technology, based on the company's submission to NICE. The evidence was derived mainly from four randomised controlled trials comparing evolocumab either with ezetimibe or placebo in adults with primary familial or non-familial hypercholesterolaemia, who were either able to take statins or who were statin-intolerant. The clinical effectiveness review found that evolocumab is efficacious at lowering LDL-C but that there was uncertainty regarding its impact on cardiovascular disease outcomes. In response to the ERG's critique of the submitted health economic model, the company submitted an amended model, which also included a Patient Access Scheme (PAS). Based on this, the deterministic incremental cost-effectiveness ratios (ICERs) for evolocumab against ezetimibe were above £74,000 and £45,000 per quality-adjusted life-year (QALY) gained within the non-familial primary and secondary prevention population, respectively, whilst the ICERs within the heterozygous familial hypercholesterolaemia population were approximately £23,000 per QALY gained. The final determination was that evolocumab would be a clinically and cost effective use of UK National Health Service resources in certain patient subgroups.
Mobile health (mHealth) applications provide an excellent opportunity for collecting rich, fine-grained data necessary for understanding and predicting day-to-day health behavior change dynamics. A computational predictive model (ACT-R-DStress) is presented and fit to individual daily adherence in 28-day mHealth exercise programs. The ACT-R-DStress model refines the psychological construct of self-efficacy. To explain and predict the dynamics of self-efficacy and predict individual performance of targeted behaviors, the self-efficacy construct is implemented as a theory-based neurocognitive simulation of the interaction of behavioral goals, memories of past experiences, and behavioral performance.
Seeds of Securigera securidaca (Fabaceae) are used in Iranian folk medicine as an antidiabetic treatment. In this study, the antihyperglycemic activity of chloroform and methanol fractions (CF and MF) from S. securidaca seed extract was investigated and their bioactive constituents were identified. The antidiabetic effects of fractions were assessed by streptozocin-induced diabetic Naval Medical Research Institute mice. The hypoglycemic activity of MF at 100 mg/kg and CF at 400 mg/kg was comparable with glibenclamide (3 mg/kg). MF at 400 mg/kg and CF at 600 mg/kg showed equal hypoglycemic responses to 12.5 IU/kg insulin (P > 0.05). Three cardiac glycosides were isolated as active constituents responsible for the hypoglycemic activity. Securigenin-3- O -β-glucopyranosyl-(1 → 4)-β-xylopyranoside (1) was a major compound in seeds. Securigenin-3- O -inositol-(1 → 3)-β-glucopyranosyl-(1 → 4)-β-xylopyranoside (2) and securigenin-3- O -α-rhamnopyranosyl-(1 → 4)-α-glucopyranoside (3) were found as new natural products. When 1-3 were tested at 10 mg/kg there was a significant reduction of blood glucose levels in diabetic mice, comparable to that of 3 mg/kg glibenclamide (P > 0.05). The hypoglycemic effect was due to an increase in insulin secretion; the insulin levels in the diabetic mice significantly improved and were comparable with those in healthy animals (P > 0.05). Compounds responsible for the hypoglycemic properties of S. securidaca seeds were identified as cardiac glycosides and were found to act via an increase of insulin levels in a diabetic mouse model.
Partitioning of the niche space is a mechanism used to explain the coexistence of similar species. Ectotherms have variable body temperatures and their body temperatures influence performance and, ultimately, fitness. Therefore, many ectotherms use behavioral thermoregulation to avoid reduced capacities associated with body temperatures far from the optimal temperature for performance. Several authors have proposed that thermal niche partitioning in response to interspecific competition is a mechanism that allows the coexistence of similar species of ectotherms. We reviewed studies on thermal resource partitioning to evaluate the evidence for this hypothesis. In almost all studies, there was insufficient evidence to conclude unequivocally that thermal resource partitioning allowed species coexistence. Future studies should include sites where species are sympatric and sites where they are allopatric to rule out alternative mechanisms that cause differences in thermal traits between coexisting species. There is evidence of conservatism in the evolution of most thermal traits across a wide range of taxa, but thermal performance curves and preferred temperatures do respond to strong selection under laboratory conditions. Thus, there is potential for selection to act on thermal traits in response to interspecific competition. Nevertheless, more stringent tests of the thermal resource partitioning hypothesis are required before we can assess whether it is widespread in communities of ectotherms in nature.
Several biological processes as well as infectious agents, physiological or environmental stress, and perturbed antioxidant response can promote oxidative stress. Oxidative stress usually happens when cells are exposed to more electrically charged reactive oxygen species (ROS) such as H2O2 or O2(-). ROS are well known for being both beneficial and deleterious. Recent studies have indicated that ROS are deleterious to cells, leading to programmed cell death (PCD) at high concentrations. At low concentrations, however, ROS can act as signaling molecules in a variety of cellular processes. In this review, we present an update of our current understanding of the role and regulation of reactive oxygen species in various viral infections, cellular signaling pathways and immune responses. We then discuss how the antioxidant defense system acts as an antiviral effector to limit cell damage.
In the present work, we examine the possibility of a benzo-18-crown-6 ether of styrylquinoline molecule (1) in acetonitrile solvent to act as a sensor for the Ca(++) cation and as a molecular logical gate. DFT and TDDFT calculations are carried out using the M06-2X and the PBE0 functionals. The quinoline moiety is an electron donor and an H(+) receptor, while the crown ether is a Ca(++) receptor forming host-guest complexes with Ca(++). The calculations show that there are 8 thermally stable forms, i.e., trans and cis isomers of neutral (1), protonated (1H+), complexed with Ca(++) (1Ca++), and both protonated and Ca(++) complexed (1H+Ca++), with different absorption and emission spectra, and which can be interconverted from one form to another. The addition of H(+) and/or Ca(++) to 1 results in variation of the oscillator strength of the major absorption and emission peaks as well as in significant shifts of the major absorption and emission peaks including shifting from the vis spectral area to UV and vice versa. Consequently, 1 is a candidate for a sensor for the Ca(++) cation. Furthermore it is shown that 1 can act as a molecular optical switch owing to its ability to be reversibly protonated and/or Ca(++) complexed with substantial accompanying differences in the spectral properties. Similarly, 1 can be used as a sensor molecular logic gate, in which using H(+) and Ca(++) and irradiation as input, the emission output at 500, 470, 430, and 407 nm can be utilized as output to build AND, NOR, XOR, XNOR, INHIBIT, and IMPLICATION logic gates.
In deformation processes, the presence of grain boundaries has a crucial influence on dislocation behavior; these boundaries drastically change the mechanical properties of polycrystalline materials. It has been considered that grain boundaries act as effective barriers for dislocation glide, but the origin of this barrier-like behavior has been a matter of conjecture for many years. We directly observe how the motion of individual dislocations is impeded at well-defined high-angle and low-angle grain boundaries in SrTiO3, via in situ nanoindentation experiments inside a transmission electron microscope. Our in situ observations show that both the high-angle and low-angle grain boundaries impede dislocation glide across them and that the impediment of dislocation glide does not simply originate from the geometric effects; it arises as a result of the local structural stabilization effects at grain boundary cores as well, especially for low-angle grain boundaries. The present findings indicate that simultaneous consideration of both the geometric effects and the stabilization effects is necessary to quantitatively understand the dislocation impediment processes at grain boundaries.
We evaluated the feasibility of angiographic computed tomography (ACT) for visualizing stent material in patients who underwent intracranial or extracranial stent placement to treat atherosclerotic lesions or stent assisted coil embolization.
We performed intrarterial and intravenous ACT on biplane angiography system equipped with flat panel detectors (Axiom Arits dBA; Siemens Medical Solutions, Forchheim, Germany). Vistipaque 320 was injected for contrast medium, total 150 mL at flow rate of 5 mL/s through artery and 77 mL at flow rate of 3.5 mL/s through vein.
ACT is a new imaging modality that provides a clear visualization of stent strut.
Therefore this new application has potential to become the noninvasive option for follow-up after endovascular surgery using stents.
Two case reports describing a new technique of creating a repositionable piezoelectric bony window osteotomy during apicoectomy in order to preserve bone and act as an autologous graft for the surgical site are described. Endodontic microsurgery of anterior teeth with an intact cortical plate and large periapical lesion generally involves removal of a significant amount of healthy bone in order to enucleate the diseased tissue and manage root ends. In the reported cases, apicoectomy was performed on the lateral incisors of two patients. A piezoelectric device was used to create and elevate a bony window at the surgical site, instead of drilling and destroying bone while making an osteotomy with conventional burs. Routine microsurgical procedures - lesion enucleation, root-end resection, and filling - were carried out through this window preparation. The bony window was repositioned to the original site and the soft tissue sutured. The cases were re-evaluated clinically and radiographically after a period of 12 - 24 months. At follow-up, radiographic healing was observed. No additional grafting material was needed despite the extent of the lesions. The indication for this procedure is when teeth present with an intact or near-intact buccal cortical plate and a large apical lesion to preserve the bone and use it as an autologous graft.
An unavoidable reciprocal influence characterizes the mother-child dyad. Within this relationship, the presence of depression, somatization, hostility, paranoid ideation, and interpersonal sensitivity symptoms at a subclinical level and their possible input on infant motor competences has not been yet considered. Bearing in mind that motor abilities represent not only an indicator of the infant's health-status, but also the principal field to infer his/her needs, feelings and intentions, in this study the quality of infants' movements were assessed and analyzed in relationship with the maternal attitudes. The aim of this research was to investigate if/how maternal symptomatology may pilot infant's motor development during his/her first year of life by observing the characteristics of motor development in infants aged 0-11 months. Participants included 123 mothers and their infants (0-11 months-old). Mothers' symptomatology was screened with the Symptom Checklist-90-Revised (SCL-90-R), while infants were tested with the Peabody Developmental Motor Scale-Second Edition. All dyads belonged to a non-clinical population, however, on the basis of SCL-90-R scores, the mothers' sample was divided into two groups: normative and subclinical. Descriptive, t-test, correlational analysis between PDMS-2 scores and SCL-90-R results are reported, as well as regression models results. Both positive and negative correlations were found between maternal perceived symptomatology, Somatization (SOM), Interpersonal Sensitivity (IS), Depression (DEP), Hostility (HOS), and Paranoid Ideation (PAR) and infants' motor abilities. These results were further verified by applying regression models to predict the infant's motor outcomes on the basis of babies' age and maternal status. The presence of positive symptoms in the SCL-90-R questionnaire (subclinical group) predicted good visual-motor integration and stationary competences in the babies. In particular, depressive and hostility feelings in mothers seemed to induce an infant motor behavior characterized by a major control of the environmental space. When mothers perceived a higher level of hostility and somatization, their babies showed difficulties in sharing action space, such as required in the development of stationary positions and grasping abilities. In a completely different way, when infants can rely on a mother with low-perceived symptoms (normative group) his/her motor performances develop with a higher degree of freedom/independence. These findings suggest, for the first time, that even in a non-clinical sample, mother's perceived-symptoms can produce important consequences not in infant motor development as a whole, but in some specific areas, contributing to shape the infant's motor ability and his/her capability to act in the world.
Dopamine (DA) modulatory activity critically supports motivated behavior. This modulation operates at multiple timescales, but the functional roles of these distinct dynamics on cognition are still being characterized. Reward processing has been robustly linked to DA activity; thus, examining behavioral effects of reward anticipation at different timing intervals, corresponding to different putative dopaminergic dynamics, may help in characterizing the functional role of these dynamics. Towards this end, we present two research studies investigating reward motivation effects on cognitive control and episodic memory, converging in their manipulation of rapid vs. multi-second reward anticipation (consistent with timing profiles of phasic vs. ramping DA, respectively) on performance. Under prolonged reward anticipation, both control and memory performances were enhanced, specifically when combined with other experimental factors: task-informative cues (control task) and reward uncertainty (memory task). Given observations of ramping DA under uncertainty (Fiorillo et al., 2003) and arguments that uncertainty may act as a control signal increasing environmental monitoring (Mushtaq et al., 2011), we suggest that task information and reward uncertainty can both serve as "need for control" signals that facilitate learning via enhanced monitoring, and that this activity may be supported by a ramping profile of dopaminergic activity. Observations of rapid (i.e., phasic) reward on control and memory performance can be interpreted in line with prior evidence, but review indicates that contributions of different dopaminergic timescales in these processes are not well-understood. Future experimental work to clarify these dynamics and characterize a cross-domain role for reward motivation and DA in goal-directed behavior is suggested.
Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery.
Luteinizing hormone (LH) rises dramatically during and after menopause, and has been correlated with an increased incidence of Alzheimer's disease and decreased memory performance in humans and animal models. To test whether LH acts directly on the dorsal hippocampus to affect memory, ovariectomized female rats were infused with either the LH-homologue human chorionic gonadotropin (hCG) or the LH receptor antagonist deglycosylated-hCG (dg-hCG).Infusion of hCG into either the lateral ventricle or the dorsal hippocampus caused significant memory impairments in ovariectomized estradiol-treated females. Consistent with this, infusion of the LH antagonist dg-hCG into the dorsal hippocampus caused an amelioration of memory deficits in ovariectomized females. Furthermore, the gonadotropin-releasing hormone antagonist Antide, failed to act in the hippocampus to affect memory. These findings demonstrate a significant role for LH action in the dorsal hippocampus in spatial memory dysfunction.
Violations of safety rules and procedures are commonly identified as a causal factor in accidents in the oil and gas industry. Extensive knowledge on effective management practices related to improved compliance with safety procedures is therefore needed. Previous studies of the causal relationship between safety climate and safety compliance demonstrate that the propensity to act in accordance with prevailing rules and procedures is influenced to a large degree by workers' safety climate. Commonly, the climate measures employed differ from one study to another and identical measures of safety climate are seldom tested repeatedly over extended periods of time. This research gap is addressed in the present study.
The study is based on a survey conducted four times among sharp-end workers of the Norwegian oil and gas industry (N=31,350). This is done by performing multiple tests (regression analysis) over a period of 7years of the causal relationship between safety climate and safety compliance. The safety climate measure employed is identical across the 7-year period.
Taking all periods together, the employed safety climate model explained roughly 27% of the variance in safety compliance. The causal relationship was found to be stable across the period, thereby increasing the reliability and the predictive validity of the factor structure. The safety climate factor that had the most powerful effect on safety compliance was work pressure.
The factor structure employed shows high predictive validity and should therefore be relevant to organizations seeking to improve safety in the petroleum sector. The findings should also be relevant to other high-hazard industries where safety rules and procedures constitute a central part of the approach to managing safety.
Perceived coercion is a sense of pressure related to the experience of being referred to treatment. The sense of pressure arises from the patient's internal perception of coercion. The sources of coercion may be the legal system, the family, the health system, or self-criticism (internal sources). Here, we studied patients diagnosed with substance use disorders that were involuntarily admitted to hospital, pursuant to a social services act. We sought to determine whether these patients perceived coercion differently than patients that were admitted voluntarily.
This study included patients admitted to combined substance use disorder and psychiatry wards in three publicly funded treatment centres in Norway in the period 2009-2011. Participants included 63 patients that were admitted involuntarily, pursuant to the Norwegian Public Health Act, and 129 patients that were admitted voluntarily. All participants completed the Perceived Coercion Questionnaire. Sociodemographic variables were determined with the European Addiction Severity Index. The range of psychopathological symptoms was evaluated with the Symptom Checklist-90-R. Independent sample t-tests, the chi-squared test, and Fisher's exact test were used to detect statistically significant differences between groups.
Scores on the Perceived Coercion Questionnaire showed that patients admitted voluntarily and those admitted involuntarily experienced similar levels of perceived coercion. Those admitted voluntarily reported higher levels of perceived coercion from internal sources, and those admitted involuntarily perceived significantly higher coercion from legal sources. No differences between groups were found with the other tests.
Our results suggested that assumptions about involuntary admissions should be evaluated carefully to determine how best to alleviate counterproductive feelings of coercion when a coerced admission is planned. Informing and collaborating with the patient will most likely facilitate a better experience during admission and treatment. Moreover, the patient is more likely to experience a better recovery process.
Rab proteins are small monomeric enzymes which belong to the large Ras protein superfamily and allow hydrolysis of guanosine triphosphate (GTP) to guanosine (GDP). Up to now more than 60 proteins have been described that act primarily as regulators of intracellular transport. Rab GTPases are mostly located at the intracellular membranes, where they provide connections to motor proteins and to the cytoskeleton and control various steps of the traffic pathways including the formation and movement of vesicles or membrane fusion controlling secretion, endocytosis, recycling and degradation of proteins. Today, the deregulated expression of Rab protein is discussed in different types of malignancies. The number of identified diseases associated with mutations in Rab proteins or their cooperating partners increases and the evidence for the involvement of Rab to the human pathologies such as the immune failure, obesity and diabetes, Alzheimers disease or hereditary genetic diseases is growing. The malfunctions of Rab proteins caused by mutations or aberrant posttranslational modifications lead to changes in the protein and vesicle trafficking, which play a crucial role in the formation and development of cancer and the deregulation of Rab expression frequently influences the migration, invasion, proliferation and drug resistance of the tumor cells.
This article summarizes the main functions of Rab proteins in the cells, describes the mechanism of their activity and focuses on the current knowledge about the roles of these GTPases in carcinogenesis.Key words: Rab GTPases - protein transport - carcinogenesisThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 5. 2016Accepted: 31. 5. 2016.
Endoplasmic reticulum chaperones are stress induced proteins capable of translocation into cytosol, cell membrane or extracellular space. The chaperones are transported from the endoplasmic reticulum particularly under endoplasmic reticulum stress conditions, while their constitutive extracellular expression was found in many cancers. Cell surface or extracellular endoplasmic reticulum chaperones take up distinct functions compared to their endoplasmic reticulum resident variants because they act like multifunctional receptors and thus affect cell signaling and proliferation.
The presented review focuses primarily on endoplasmic reticulum chaperones expression on the cell surface of cancer cells and into extracellular space. The work describes possible mechanisms of chaperones translocation to the cancer cell surface, including KDEL transport mechanism and retrotranslocation and the influence of chaperone posttranslation modifications on their localization within the cell. Well described cancer cell surface endoplasmic reticulum chaperones include GRP78, GRP94, calreticulin and calnexin that are involved in cancer cell signaling in different ways. The attention is also paid to immunogenic properties of membrane-localized chaperones for their ability to participate in immune reactions. They can take part in innate and adaptive immune response through their interaction with toll-like receptors or during the antigen presentation as well as in tumor-specific immunity. The expression of endoplasmic reticulum chaperones on the cancer cells surface is potentially exploitable in specific antitumor therapy as well as vaccine therapy, thus the final part of this review is dedicated to this topic.Key words: endoplasmic reticulum - glucose-regulated proteins - molecular chaperones - KDEL sequence - immunobiologyThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 17. 5. 2016Accepted: 20. 7. 2016.
The overall survival rate of ovarian cancer patients is still poor because of the difficulties encountered in detection, diagnosis and treatment. Here, we aim to systematically identify the genetic factors causing ovarian cancer and find the accurate diagnostic and therapeutic targets for ovarian cancer.
We collected the known archived ovarian cancer-related genes from the databases used as the investigated targets and employed the minimum redundancy maximum relevance and random forest classification to identify the novel ovarian cancer-related genes in addition to the known ones. We further identified candidates as the markers for the detection of the ovarian cancer based on the gene expression data and then confirmed them by quantitative real-time PCR.
We found out the genetic terms to interpret the mechanism of ovarian cancer. Based on those terms, we predicted 860 novel related genes as candidates. These candidates can act as expression biomarkers for clinical detection and they achieved a 100% accuracy. We verified 10 of them as the optimal biomarkers for detection in the expression data.
We employed the features of achieved ovarian cancer-related genes to identify 860 novel ovarian cancer genes. We further validated 10 genes as biomarkers for detection of ovarian cancer.
Intestinal inflammation can impair mucosal healing, thereby establishing a vicious cycle leading to chronic inflammatory bowel disease (IBD). However, the signaling networks driving chronic inflammation remain unclear. Here we report that CD4(+) T cells isolated from patients with IBD produce high levels of interleukin-22 binding protein (IL-22BP), the endogenous inhibitor of the tissue-protective cytokine IL-22. Using mouse models, we demonstrate that IBD development requires T cell-derived IL-22BP. Lastly, intestinal CD4(+) T cells isolated from IBD patients responsive to treatment with antibodies against tumor necrosis factor-α (anti-TNF-α), the most effective known IBD therapy, exhibited reduced amounts of IL-22BP expression but still expressed IL-22. Our findings suggest that anti-TNF-α therapy may act at least in part by suppressing IL-22BP and point toward a more specific potential therapy for IBD.
Humans operate with a "theory of mind" with which they are able to understand that others' actions are driven not by reality but by beliefs about reality, even when those beliefs are false. Although great apes share with humans many social-cognitive skills, they have repeatedly failed experimental tests of such false-belief understanding. We use an anticipatory looking test (originally developed for human infants) to show that three species of great apes reliably look in anticipation of an agent acting on a location where he falsely believes an object to be, even though the apes themselves know that the object is no longer there. Our results suggest that great apes also operate, at least on an implicit level, with an understanding of false beliefs.
The rising prevalences of type 2 diabetes and obesity constitute major threats to human health globally. Powerful social and economic factors influence the distribution of these diseases among and within populations. These factors act on a substrate of individual predisposition derived from the composite effects of inherited DNA variation and a range of environmental exposures experienced throughout the life course. Although "Western" lifestyle represents a convenient catch-all culprit for such exposures, effective treatment and prevention will be informed by characterization of the most critical, causal environmental factors. In this Review, we examine how burgeoning understanding of the genetic basis of type 2 diabetes and obesity can highlight nongenetic exposures that drive development of these conditions.
The study represents synthesis, characterization and biological evaluation of redox responsive polymeric nanoparticles based on random multiblock copolymer for doxorubicin delivery in breast cancer. The random multiblock copolymer was synthesized via ring opening polymerization of lactide with polyethylene glycol to form triblock copolymer followed by isomerization polymerization of the triblock copolymer and 2-hydroxyethyl disulfide with the help of hexamethylene diisocynate in presence of dibutyltin dilaurate as a catalyst. Folic acid was conjugated to hydroxyl group from the multiblock polymer through DCC-NHS coupling. High drug loading content of ∼22% was achieved in the polymeric nanoparticles with size range of ∼110nm and polyethylene glycol fraction of ∼18% in the multiblock copolymer. Drug release profile confirmed the redox responsive behavior of polymeric nanoparticles with ∼72% drug release at pH 5.5 in presence of 10mM GSH as compared to ∼18% drug release at pH 7.4. In vitro cellular uptake studies showed ∼22% cellular uptake with dual (folic acid and trastuzumab) conjugated polymeric nanoparticles as compared to non-targeted polymeric nanoparticles. Fluorescence activated cell sorting (FACS) studies demonstrated higher apoptosis (∼80%) as compared to non-conjugated polymeric nanoparticles (20%) in MCF-7 cell line. In vivo studies showed 91% tumor regression in Ehrlich ascites tumor (EAT) as compared to free doxorubicin treated mice without showing any significant toxicity. Thus, it is envisaged that these redox responsive polymeric nanocarriers act as Trojan horses in cancer therapeutics.
bZIP transcription factors play key roles in plant growth, development, and stress signaling. A bZIP gene BnbZIP2 (GenBank accession number: KP642148) was cloned from ramie. BnbZIP2 has a 1416 base pair open reading frame, encoding a 471 amino acid protein containing a characteristic bZIP domain and a leucine zipper. BnbZIP2 shares high sequence similarity with bZIP factors from other plants. The BnbZIP2 protein is localized to both nuclei and cytoplasm. Transcripts of BnbZIP2 were found in various tissues in ramie, with significantly higher levels in female and male flowers. Its expression was induced by drought, high salinity, and abscisic acid treatments. Analysis of the cis-elements in promoters of BnbZIP2 identified cis-acting elements involved in growth, developmental processes, and a variety of stress responses. Transgenic Arabidopsis plants' overexpression of BnbZIP2 exhibited more sensitivity to drought and heavy metal Cd stress during seed germination, whereas more tolerance to high-salinity stress than the wild type during both seed germination and plant development. Thus, BnbZIP2 may act as a positive regulator in plants' response to high-salinity stress and be an important candidate gene for molecular breeding of salt-tolerant plants.
Infections caused by Mycobacterium tuberculosis and other mycobacteria are major challenges for global public health. Particularly worrisome are infections caused by multidrug-resistant bacteria, which are increasingly difficult to treat because of the loss of efficacy of the current antibacterial agents, a problem that continues to escalate worldwide. There has been a limited interest and investment on the development of new antibacterial agents in the past decades. This has led to the current situation, in which there is an urgent demand for innovative therapeutic alternatives to fight infections caused by multidrug-resistant pathogens, such as multidrug-resistant tuberculosis. The identification of compounds that can act as adjuvants in antimycobacterial therapeutic regimens is an appealing strategy to restore the efficacy lost by some of the antibiotics currently used and shorten the duration of the therapeutic regimen. In this work, by setting Mycobacterium smegmatis as a model organism, we have developed a methodological strategy to identify, in a fast and simple approach, compounds with antimycobacterial activity or with potential adjuvant properties, by either inhibition of efflux or other unrelated mechanisms. Such an approach may increase the rate of identification of promising molecules, to be further explored in pathogenic models for their potential use either as antimicrobials or as adjuvants, in combination with available therapeutic regimens for the treatment of mycobacterial infections. This method allowed us to identify a new molecule that shows promising activity as an efflux inhibitor in M. smegmatis.
In this paper, we investigate the secure transmission in wireless sensor networks (WSNs) consisting of one multiple-antenna base station (BS), multiple single-antenna legitimate users, one single-antenna eavesdropper and one multiple-antenna cooperative jammer. In an effort to reduce the scheduling complexity and extend the battery lifetime of the sensor nodes, the switch-and-stay combining (SSC) scheduling scheme is exploited over the sensor nodes. Meanwhile, transmit antenna selection (TAS) is employed at the BS and cooperative jamming (CJ) is adopted at the jammer node, aiming at achieving a satisfactory secrecy performance. Moreover, depending on whether the jammer node has the global channel state information (CSI) of both the legitimate channel and the eavesdropper's channel, it explores a zero-forcing beamforming (ZFB) scheme or a null-space artificial noise (NAN) scheme to confound the eavesdropper while avoiding the interference to the legitimate user. Building on this, we propose two novel hybrid secure transmission schemes, termed TAS-SSC-ZFB and TAS-SSC-NAN, for WSNs. We then derive the exact closed-form expressions for the secrecy outage probability and the effective secrecy throughput of both schemes to characterize the secrecy performance. Using these closed-form expressions, we further determine the optimal switching threshold and obtain the optimal power allocation factor between the BS and jammer node for both schemes to minimize the secrecy outage probability, while the optimal secrecy rate is decided to maximize the effective secrecy throughput for both schemes. Numerical results are provided to verify the theoretical analysis and illustrate the impact of key system parameters on the secrecy performance.
Existing implantable stimulators use powering approaches that result in stiff and bulky systems or result in systems incapable of producing the current magnitudes required for neuromuscular stimulation. This hampers their use in neuroprostheses for paralysis. We previously demonstrated an electrical stimulation method based on electronic rectification of high frequency (HF) current bursts. The implants act as rectifiers of HF current that flows through the tissues by galvanic coupling, transforming this current into low frequency current capable of performing neuromuscular stimulation. Here we developed 2 mm thick, semi-rigid, injectable and addressable stimulators made of off-the-shelf components and based on this method. The devices were tested in vitro to illustrate how they are powered by galvanic coupling. In addition they were tried in an animal model to demonstrate their ability to perform controlled electrical stimulation. The implants were deployed by injection into two antagonist muscles of an anesthetized rabbit and were addressed resulting in independent isometric contractions. Low frequency currents of 2 mA were delivered by the implants. The HF currents are safe in terms of unwanted electrostimulation and tissue heating according to standards. This indicates that the proposed electrical stimulation method will allow unprecedented levels of miniaturization for neuroprostheses.
To explore significant experiences of adolescents as next of kin that the general practitioner (GP) should identify and recognize.
Qualitative study with focus-group interviews.
Three focus-group interviews were conducted with a total of 15 Norwegian adolescents each with an ill or substance-abusing parent. The participants were recruited from existing support groups.
The adolescents' days were dominated by unpredictability in their family situation and their own exhausting efforts to keep up an ordinary youth life. Mostly, they consulted GPs for somatic complaints. In encounters with the GP, they wanted to be met both as a unique person and as a member of a family with burdens. Their expectations from the GP were partly negatively formed by their experiences. Some had experienced that both their own and their parent's health problems were not addressed properly. Others reported that the GP did not act when he or she should have been concerned about their adverse life situation. The GP may contribute to better long-term psychosocial outcomes by ensuring that the adolescents receive information about the parent's illness and have someone to talk to about their feelings and experiences. In addition, the GP may help by supporting their participation in relieving activities.
Burdened adolescents seek a GP most often for somatic complaints. The GP has a potential to support them by taking the initiative to talk about their life situation, and by recognizing their special efforts. Key points Little is known about how a general practitioner can support adolescents with ill or substance-abusing parents. Adolescents experience unpredictability in life and strive to find balance between their own needs and the restrictions caused by parental illness. In encounters with adolescents having ill parents, the GP should take the initiative to talk about their family situation. The GP may help them by recognizing their experiences and struggles, give information, offer talks and support coping strategies.
Heat shock proteins are a family of proteins that are produced by cells in response to exposure to stressful conditions. The best studied heat shock protein is HSP70, which is known to act as a molecular chaperone to maintain cellular homeostasis and inhibit protein aggregation in response to stress. While early animal studies suggested that increasing HSP70 in the heart (using a transgenic, gene transfer or pharmacological approach) provided cardiac protection against acute cardiac stress, recent studies have found no benefit of increasing HSP70 in mouse models of chronic cardiac stress. As HSP70 has been considered a potential therapeutic target, it is important to comprehensively assess HSP70 therapies in preclinical models of acute and chronic cardiac disease.
Reviews the book, Clinical Uncertainty in Primary Care: The Challenge of Collaborative Engagement edited by Lucia Siegel Sommers and John Lautner (see record 2013-30484-000). The bulk of this book comprises a wide array of different models for collaborative uncertainty work. From Balint groups that help participants to tolerate uncertainty to narrative-based supervision that poses challenging self-reflective questions, each chapter provides a description of group intervention design, theoretical context, the structure for facilitation, and outcome data on impact. The book is a powerfully thoughtful and detailed exploration of the often overlooked topic of not understanding or knowing how to act in the best interest of a patient. The beautiful and self-disclosing writing is a gift to the reader, and the testimonies of the utility of these practices are undeniably compelling. (PsycINFO Database Record
Federally Qualified Health Centers--commonly referred to as Community Health Centers (CHCs)--serve as a safety net for people who did not gain health insurance under the Affordable Care Act (ACA), including those immigrants not eligible for Medicaid or health insurance exchange coverage. ACA-driven changes in health insurance coverage, funding, and related policy have created new challenges for these safety net organizations. This policy brief reports the findings from analyses of the U.S. HRSA Uniform Data System and interviews conducted in 2014-16 with the leadership of 31 CHCs. The CHCs were located in communities with high concentrations of immigrants and uninsured residents, in states that either expanded Medicaid (California and New York) or that chose not to expand it (Georgia and Texas). The study found that most CHCs now see more patients, including significant numbers without insurance. The ACA has brought new resources to CHCs but has also reinforced challenges, including the need for stable revenue streams, sufficient staffing support, and assistance in leveraging new reimbursement mechanisms. Policy recommendations to address these challenges include continuing core federal funding, insuring the remaining uninsured, addressing workforce challenges, and preparing CHCs for alternative payment mechanisms.
In California, personal health care expenditures are estimated to total more than $367 billion in 2016. Approximately 71 percent of these expenditures will be paid for with public funds (i.e., taxpayer dollars). This estimated contribution of public funds to health care expenditures is much higher than estimates that include only major health insurance programs such as Medicare and Medicaid. Several additional public funding sources also contribute to health care expenditures in the state, including government spending for public employee health benefits, tax subsidies for employer-sponsored insurance and the Affordable Care Act (ACA) insurance exchange, and county health care expenditures. As health care reform continues to take effect, it will be important to monitor the public versus private contributions to state health care expenditures to ensure that funds are being distributed both efficiently and equitably.
An educational intervention for HAI prevention based on a combination of training, motivation and subsequent application in the current clinical practice in an Italian teaching hospital.
In 2015-2016 a pilot mandatory training on HAI targeted to HCWs was organized in the 450 bed teaching hospital Sant'Andrea in Rome. By adopting the "Impact/control matrix" prioritization tool, the relative level of impact (risk in causing or favoring HAI) and control (possibility for HCWs to prevent HAI) attributed by the participants to the issues associated to HAI during their working groups was evaluated.
Overall, 34 physicians, 43 nurses and 15 non clinical professionals participated actively in seven courses, identifying 58 different issues related to HAI, which were reported 128 times. Results showed frequently that, within the same type of issue, HCW referred various levels of impact (risk in causing or favoring HAI) and personal control (possibility for HCW to prevent HAI). Overall staff shortage was the most reported problem by HCW in our hospital. Also hand washing was regarded as a main problem, but HCW expressed the feeling that individuals could act more successfully on this issue (high or medium control). Results showed that staff frequently did not know how to handle correctly visitors, similarly many colleagues expressed some difficulty in communicating information to patients and relatives on HAI. Surprisingly, "antimicrobial therapy" and "excessive invasive procedures" were not particularly highlighted by the personnel. HCW expressed satisfaction for the course approac.
The study showed an overall good level of knowledge regarding the importance and principles of infection control in our teaching hospital HCW. However personnel perceived a variability in the impact of many issues on HAI and even more on the personal possibility to control their effect. In order to improve HCW compliance with HAI prevention programs, the "Adult Learning" model seems to be very useful.
Unexpected abscission of flowers or fruits is a major limiting factor for crop productivity. Key genes controlling abscission in plants, especially in popular fruit trees, are largely unknown. Here we identified a litchi (Litchi chinensis Sonn.) IDA-like (INFLORESCENCE DEFICIENT IN ABSCISSION-like) gene LcIDL1 as a potential key regulator of abscission. LcIDL1 encodes a peptide that shows the closest homology to Arabidopsis IDA, and is localized in cell membrane and cytoplasm. Real-time PCR analysis showed that the expression level of LcIDL1 accumulated gradually following flower abscission, and it was obviously induced by fruit abscission-promoting treatments. Transgenic plants expressing LcIDL1 in Arabidopsis revealed a role of LcIDL1 similar to IDA in promoting floral organ abscission. Moreover, ectopic expression of LcIDL1 in Arabidopsis activated the expression of abscission-related genes. Taken together, our findings provide evidence that LcIDL1 may act as a key regulator in control of abscission.
Neutrophils play significant regulatory roles within the tumor microenvironment by directly promoting tumor progression that leads to poor clinical outcomes. Identifying the tumor associated molecules that regulate neutrophil infiltration into tumors may provide new and specific therapeutic targets for cancer treatment. The a2-isoform of vacuolar ATPase (a2V) is uniquely and highly expressed on cancer cell plasma membrane. Cancer cells secrete a peptide from a2V (a2NTD) that promotes the pro-tumorigenic properties of neutrophils. This provides a2V the propensity to control neutrophil migration. Here, we report that the treatment of human neutrophils with recombinant a2NTD leads to neutrophil adherence and polarization. Moreover, a2NTD treatment activates surface adhesion receptors, as well as FAK and Src kinases that are essential regulators of the migration process in neutrophils. Functional analysis reveals that a2NTD can act as a chemo-attractant and promotes neutrophil migration. In addition, a2Neuɸ secrete high levels of IL-8 via NF-κB pathway activation. Confirmatory assays demonstrate that the promoted migration of a2Neuɸ was dependent on the autocrine secretion of IL-8 from a2Neuɸ. These findings demonstrate for the first time the direct regulatory role of cancer associated a2-isoform V-ATPase on neutrophil migration, suggesting a2V as a potential target for cancer therapy.
Antimicrobial peptides (AMPs) are short cationic host-defense molecules that provide the early stage of protection against invading microbes. They also have important modulatory roles and act as a bridge between innate and acquired immunity. The types and functions of oral AMPs were reviewed and experimental reports on the use of natural AMPs and their synthetic mimics in caries and pulpal infections were discussed. Natural AMPs in the oral cavity, predominantly defensins, cathelicidins and histatins, possess antimicrobial activities against oral pathogens and biofilms. Incomplete debridement of microorganisms in root canal space may precipitate an exacerbated immune response that results in periradicular bone resorption. Because of their immunomodulatory and wound healing potentials, AMPs stimulate pro-inflammatory cytokine production, recruit host defense cells and regulate immuno-inflammatory responses in the vicinity of the pulp and periapex. Recent rapid advances in the development of synthetic AMP mimics offer exciting opportunities for new therapeutic initiatives in root canal treatment and regenerative endodontics.
Identification of new therapeutic strategies to combat antibiotic-resistant pathogens and biofilm-associated infections continues to be one of the major challenges in modern medicine. Despite the presence of commercialization hurdles and scientific challenges, interests in using antimicrobial peptides as therapeutic alternatives and adjuvants to combat pathogenic biofilms have never been foreshortened. Not only do these cationic peptides possess rapid killing ability, their multi-modal mechanisms of action render them advantageous in targeting different biofilm sub-populations. These factors, together with adjunctive bioactive functions such as immunomodulation and wound healing enhancement, render AMPs or their synthetic mimics exciting candidates to be considered as adjuncts in the treatment of caries, infected pulps and root canals.
As people grow older, speech perception difficulties become highly prevalent, especially in noisy listening situations. Moreover, it is assumed that speech intelligibility is more affected in the event of background noises that induce a higher cognitive load, i.e., noises that result in informational versus energetic masking. There is ample evidence showing that speech perception problems in aging persons are partly due to hearing impairment and partly due to age-related declines in cognition and suprathreshold auditory processing. In order to develop effective rehabilitation strategies, it is indispensable to know how these different degrading factors act upon speech perception. This implies disentangling effects of hearing impairment versus age and examining the interplay between both factors in different background noises of everyday settings. To that end, we investigated open-set sentence identification in six participant groups: a young (20-30 years), middle-aged (50-60 years), and older cohort (70-80 years), each including persons who had normal audiometric thresholds up to at least 4 kHz, on the one hand, and persons who were diagnosed with elevated audiometric thresholds, on the other hand. All participants were screened for (mild) cognitive impairment. We applied stationary and amplitude modulated speech-weighted noise, which are two types of energetic maskers, and unintelligible speech, which causes informational masking in addition to energetic masking. By means of these different background noises, we could look into speech perception performance in listening situations with a low and high cognitive load, respectively. Our results indicate that, even when audiometric thresholds are within normal limits up to 4 kHz, irrespective of threshold elevations at higher frequencies, and there is no indication of even mild cognitive impairment, masked speech perception declines by middle age and decreases further on to older age. The impact of hearing impairment is as detrimental for young and middle-aged as it is for older adults. When the background noise becomes cognitively more demanding, there is a larger decline in speech perception, due to age or hearing impairment. Hearing impairment seems to be the main factor underlying speech perception problems in background noises that cause energetic masking. However, in the event of informational masking, which induces a higher cognitive load, age appears to explain a significant part of the communicative impairment as well. We suggest that the degrading effect of age is mediated by deficiencies in temporal processing and central executive functions. This study may contribute to the improvement of auditory rehabilitation programs aiming to prevent aging persons from missing out on conversations, which, in turn, will improve their quality of life.
The aim of the present study was to explore the usefulness of the alpha/theta (A/T) training in reducing Food Craving (FC) in a non-clinical sample. The modifications of electroencephalographic (EEG) power spectra associated with A/T training was also investigated. Fifty subjects were enrolled in the study and randomly assigned to receive ten sessions of A/T training [neurofeedback group (NFG)=25], or to act as controls [waiting list group (WLG)=25]. All participants were administered the Food Cravings Questionnaire-Trait, the Eating Disorder Examination Questionnaire and the Symptom Checklist-90-Revised. In the post training assessment, compared to the WLG, the NFG showed a significant reduction of intentions and plans to consume food (F1; 49=4.90; p=.033; d=0.626) and of craving as a physiological state (F1; 49=8.09; p=.007; d=803). In NFG, changes in FC persisted after 4months follow-up. Furthermore, A/T training was associated with significant a increase of resting EEG alpha power in several brain areas involved in FC (e.g., insula) and food cue reactivity (e.g., parahippocampal gyrus, inferior and superior temporal gyrus). Taken together, our results showed that ten sessions of A/T training are associated with a decrease of self-reported FC in a non-clinical sample. These findings suggest that this brain-directed intervention may be useful in the treatment of dysfunctional eating behaviors characterized by FC.
Various preclinical/clinical studies support the effectiveness of ketoprofen in periodontitis; however, the literature reveals that novel delivery systems have been less explored for the drug in periodontitis. The current investigation aims to explore the potential of a pro-vesicular approach-based proniosomal drug delivery of ketoprofen for its effectiveness and validation in experimental periodontal disease (EPD). Formulations were developed using I-optimal mixture design. Developed formulations were characterized for entrapment efficiency, vesicle size, and in vitro drug release. Selected proniosomal gels were evaluated for mucoadhesiveness, ex vivo drug permeation, and retention studies. Optimized proniosomal gel was evaluated for surface morphology, rheological behavior, texture studies, and pharmacodynamic activity in EPD. The results showed that ketoprofen-loaded proniosomal formulations formed a mucoadhesive hydrogel comprising spherical and flexible vesicles. Viscosity and texture studies showed good adhesion and smoothness, which are desired for enhanced permeation. The disease condition was improved with preserved bone resorption process, that too with intact cementum vis-à-vis marketed gel formulation, when evaluated in the EPD model. The results lead to the conclusion that proniosomes can act as a promising carrier and can be effectively used for improved ketoprofen delivery in periodontal pockets.
Here, we report the decrease of JA-sensitivity and enhancement of tolerance to salt and PEG stresses in Arabidopsis overexpressing apple MdJAZ2. As signalling molecules, jasmonates (JAs) play significant roles in plant development and stress responses. JAZ proteins are the targets of the SCF(COI1) complex and act as the negative regulators in JA signalling pathway. However, there are no reports regarding the biological function of apple JAZ genes. In this study, one JAZ gene, MdJAZ2 from apple, was functionally characterized in detail. The expression of MdJAZ2 was up-regulated by MeJA and wounding treatments. MdJAZ2-GFP fusion protein was observed in nucleus in transient expression assay. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that MdJAZ2 could form homo- and heteromers, and also interact with F-box protein MdCOI1. Overexpression of MdJAZ2 conferred impaired JA-sensitivity in transgenic Arabidopsis, including JA-mediated root growth inhibition, susceptibility to the bacterial pathogen Pst DC3000, and the expression of JA response genes. Additionally, MdJAZ2 overexpression also improved tolerance to NaCl and PEG treatments in transgenic Arabidopsis. Together, our findings suggest that apple MdJAZ2 was not only involved in the JA response but also played roles in stress tolerance.
Doped BiVO4 is a promising photoelectrochemical water splitting anode, whose activity is hampered by poor charge transport. Here we use a set of X-ray spectroscopic methods to probe the origin and nature of localized electron states in W:BiVO4. Furthermore, using the polarized nature of the X-rays, we probe variations in the electronic structure along the crystal axes. In this manner, we reveal aspects of the electronic structure related to electron localization and observations consistent with conductivity anisotropy between the ab-plane and c-axis. We verify that tungsten substitutes as W(6+) for V(5+) in BiVO4. This is shown to result in the presence of inter-band gap states related to electrons at V(4+) sites of e symmetry. The energetic position of the states in the band gap suggest that they are highly localized and may act as recombination centres. Polarization dependent X-ray absorption spectra reveal anisotropy in the electronic structure between the ab-plane and c-axis. Results show the superior hybridization between V 3d and O 2p states, higher V wavefunction overlap and broader conduction bands in the ab-plane than in the c-axis. These insights into the electronic structure are discussed in the context of existing experimental and theoretical reports regarding charge transport in BiVO4.
It is essential to develop predictive algorithms for Amyotrophic Lateral Sclerosis (ALS) disease progression to allow for efficient clinical trials and patient care. The best existing predictive models rely on several months of baseline data and have only been validated in clinical trial research datasets. We asked whether a model developed using clinical research patient data could be applied to the broader ALS population typically seen at a tertiary care ALS clinic.
Based on the PRO-ACT ALS database, we developed random forest (RF), pre-slope, and generalized linear (GLM) models to test whether accurate, unbiased models could be created using only baseline data. Secondly, we tested whether a model could be validated with a clinical patient dataset to demonstrate broader applicability.
We found that a random forest model using only baseline data could accurately predict disease progression for a clinical trial research dataset as well as a population of patients being treated at a tertiary care clinic. The RF Model outperformed a pre-slope model and was similar to a GLM model in terms of root mean square deviation at early time points. At later time points, the RF Model was far superior to either model. Finally, we found that only the RF Model was unbiased and was less subject to overfitting than either of the other two models when applied to a clinic population.
We conclude that the RF Model delivers superior predictions of ALS disease progression.
To describe spongiform scleropathy in a patient with oculodermal melanosis and without evidence of uveal melanoma.
Clinical-pathological correlation conducted in compliance with HIPPA (Health Insurance Privacy and Portability Act) regulations.
Melanoma-associated spongiform scleropathy was an incidental finding in an 87-year-old woman with oculodermal melanocytosis treated for primary orbital melanoma. All previously reported cases of this scleropathy have been associated with uveal melanoma.
The mechanism of scleral degeneration in melanoma-associated spongiform scleropathy is unknown, and its clinical and prognostic significance is speculative. This is the first case of a so-called melanoma-associated spongiform scleropathy reported in an eye without uveal melanoma.
Archwires act as gears to move teeth with light, continuous forces. However, the intraoral use of orthodontic archwires is liable to surface deposits which alter the mechanical properties of archwires, causing an increase in the friction coefficient.
To evaluate the surface changes of the stainless steel archwires after 6 weeks of intraoral use and its influence on frictional resistance during sliding mechanics.
As-received rectangular 0.019" × 0.025" stainless steel orthodontic archwires (control) were compared with the archwires retrieved after the final phase of leveling and alignment stage of orthodontic treatment collected after 6 weeks of intraoral exposure (test samples) from 10 patients undergoing treatment. The control and test samples were used to evaluate surface debris using Scanning Electron Microscopy, surface roughness was assessed using Atomic Force Microscope and frictional forces were measured using Instron Universal Testing Machine in the buccal inter-bracket region that slides through the molar tube for space closure. Unpaired t-test and Pearson correlation tests were used for statistical analysis (P < 0.05 level of significance).
Significant increase was observed in the level of debris (P = 0.0001), surface roughness (P = 0.0001), and friction resistance (P = 0.001) of orthodontic archwires after their intraoral exposure. Significant positive correlations (P < 0.05) were also observed between these three variables.
Stainless steel test archwires showed a significant increase in the degree of debris and surface roughness, increasing the frictional forces between the archwire-bracket interfaces which would considerably reduce the normal orthodontic forces. Thus, continuing the same archwire after levelling and alignment for space closure is not recommended.
Conducting medical research is not limited to academia and pharmaceutical industry but even multispeciality hospitals need to venture in this area along with patient care. To develop research culture among well-established non-acedemic hospital is always difficult and challenging task. This article attempts to evaluate the performance of the department in 'Research naïve' hospital in the last two years and review the strengths and challenges it faced at each step.
This was a retrospective document analysis study evaluating the steps towards setting and sustaining of Medical Research Department of Bhaktivedanta Hospital during the period of January 2013 to June 2015 (30 Months). The authors developed a checklist (along with performance indicators) to assess the Preparatory phase and Activity phase of the research department which were evaluated by Institute Quality Management Team. Each step of both phases was also reviewed in terms of strengths and challenges as perceived by the authors.
During 2 year journey of research naïve Hospital, Institute had witnessed Hospital initiated (n=24, 59%) and sponsored projects (n=17, 41%) in all specialties. HRC reviewed (n=2.13) projects per meeting for administrative consideration while IEC reviewed (n=2.15) projects for scientific and ethical review. Challenges during preparatory phases were circumvent by immense cooperation of hospital management for initial investment, sensitization through research workshops for consultants, established procedures and trained support manpower and constant encouragement by research coordinator.
Considering evaluation of 41 studies in very first 2 years in 'Research naive non academic institute demonstrated successful implementation of trio model of Hospital Research Committee for administrative review, IEC for scientific-ethical review, centralized MRD for coordinating all research projects under one roof which may act as role model for Research naive institutes.
The high mortality rate in Bangladesh is related to poverty, which results in protein malnutrition, essential fatty acid deficiency and lacks in adequate vitamins, minerals and calorie. Exploring new food items with improved dietary nutrition factors may, therefore, help to decrease the mortality rate in the poor countries like Bangladesh. Accordingly, the present study was a proximate composition and fatty acid analysis of L. purpureus seed-a legume seed which is given no careful attention locally, though it might be a good source of valuable nutrition factors for both animals and humans. The purpose of the study was, therefore, to generate awareness that L. purpureus could also act as a good source of food components essential for good health. Proximate analysis revealed that the seed powder contained 8.47 ± 0.52% moisture; 3.50 ± 0.0.07% ash; 1.02 ± 0.06% total fat; 23.95 ± 0.15% total protein; 1.21 ± 0.16% total dietary fiber; 61.86 ± 0.70% total carbohydrate and 352.4 ± 2.66 kcal/100 g energy. Phytic acid content (%) was 1.014 ± 0.048. Major fatty acid composition (%): the essential fatty acid linoleic acid (C18:2, ω-6) was 9.50 ± 0.68, while the linolenic acid (C18:3, ω-3) was 1.95 ± 0.18. Palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1) were, respectively, 2.96 ± 0.19, 0.77 ± 0.04 and 1.10 ± 0.06. Lignoceric acid (C24:0) was 0.11 ± 0.007%. Monounsaturated palmitoleic acid (0.006 ± 0.0), docosapentaenoic acid (DPA, C22:5, ω-3) and nervonic acid (0.002 ± 0.0) were present in trace amounts. Arachidonic acid (AA, C20:4, ω-6), eicosapentaenoic acid (C20:5, ω-3), and docosahexaenoic acid (C22:6, ω-3) were not detected. The fatty acid profile, thus, suggests that essential omega-6 fatty acid linoleic acid (C18:3, ω-6) and omega-3 linolenic acid (C18:3, ω-3) were the major polyunsaturated fatty acids (PUFA) present in L. purpureus seed. In addition, the seed contained high amount of proteins. Finally, these results suggest that L. purpureus seed could be used as a good source of quality food components, including protein and essential fatty acids.
Great rivers were generally looked at as the geographical barrier to gene flow for many taxonomic groups. The Yangtze River is the third largest river in the world, and flows across South China and into the East China Sea. Up until now, few studies have been carried out to evaluate its effect as a geographical barrier. In this study, we attempted to determine the barrier effect of the Yangtze River on the tufted deer (Elaphodus cephalophus) using the molecular ecology approach. Using mitochondrial DNA control region (CR) sequences and 13 nuclear microsatellite loci, we explored the genetic structure and gene flow in two adjacent tufted deer populations (Dabashan and Wulingshan populations), which are separated by the Yangtze River. Results indicated that there are high genetic diversity levels in the two populations, but no distinguishable haplotype group or potential genetic cluster was detected which corresponded to specific geographical population. At the same time, high gene flow was observed between Wulingshan and Dabashan populations. The tufted deer populations experienced population decrease from 0.3 to 0.09 Ma BP, then followed by a distinct population increase. A strong signal of recent population decline (T = 4,396 years) was detected in the Wulingshan population by a Markov-Switching Vector Autoregressions(MSVAR) process population demography analysis. The results indicated that the Yangtze River may not act as an effective barrier to gene flow in the tufted deer. Finally, we surmised that the population demography of the tufted deer was likely affected by Pleistocene climate fluctuations and ancient human activities.
Social media can act as an important platform for debating, discussing, and disseminating information about vaccines. Our objectives were to map and describe the roles played by web-based mainstream media and social media as platforms for vaccination-related public debates and discussions during the Polio crisis in Israel in 2013: where and how did the public debate and discuss the issue, and how can these debates and discussions be characterized?
Polio-related coverage was collected from May 28 to October 31, 2013, from seven online Hebrew media platforms and the Facebook groups discussing the Polio vaccination were mapped and described. In addition, 2,289 items from the Facebook group "Parents talk about Polio vaccination" were analyzed for socio-demographic and thematic characteristics.
The traditional media mainly echoed formal voices from the Ministry of Health. The comments on the Facebook vaccination opposition groups could be divided into four groups: comments with individualistic perceptions, comments that expressed concerns about the safety of the OPV, comments that expressed distrust in the Ministry of Health, and comments denying Polio as a disease. In the Facebook group "Parents talk about the Polio vaccination", an active group with various participants, 321 commentators submitted 2289 comments, with 64 % of the comments written by women. Most (92 %) people involved were parents. The comments were both personal (referring to specific situations) and general in nature (referring to symptoms or wide implications). A few (13 %) of the commentators were physicians (n = 44), who were responsible for 909 (40 %) of the items in the sample. Half the doctors and 6 % of the non-doctors wrote over 10 items each. This Facebook group formed a unique platform where unmediated debates and discussions between the public and medical experts took place.
The comments on the social media, as well as the socio-demographic profiles of the commentators, suggest that social media is an active and versatile debate and discussion-facilitating platform in the context of vaccinations. This paper presents public voices, which should be seen as authentic (i.e. unmediated by the media or other political actors) and useful for policy making purposes. The policy implications include identifying social media as a main channel of communication during health crises, and acknowledging the voices heard on social media as authentic and useful for policy making. Human and financial resources need to be devolved specifically to social media. Health officials and experts need to be accessible on social media, and be equipped to readily provide the information, support and advice the public is looking for.
Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals.
We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions.
These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.
Yin-deficiency-heat (YDH) syndrome is a concept in Traditional Chinese Medicine (TCM) for describing subhealth status. However, there are few efficient diagnostic methods available for confirming YDH syndrome. To explore the novel method for diagnosing YDH syndrome, we applied iTRAQ to observe the serum protein profiles in YDH syndrome rats and confirmed protein levels by ELISA. A total of 92 differentially expressed proteins (63 upregulated proteins and 29 downregulated proteins), which were mainly involved in complement and coagulation cascades and glucose metabolism pathway, were identified by the proteomic experiments. Kininogen 1 (KNG1) was significantly increased (p < 0.0001), while apolipoprotein C-III (APOC3, p < 0.005) and paraoxonase 1 (PON1, p < 0.001) were significantly decreased in the serum of YDH syndrome rats. The combination of KNG1, APOC3, and PON1 constituted a diagnostic model with 100.0% sensitivity and 85.0% specificity. The results indicated that KNG1, APOC3, and PON1 may act as potential biomarkers for diagnosing YDH syndrome. KNG1 may regulate cytokines and chemokines release in YDH syndrome, and the low levels of PON1 and APOC3 may increase oxidative stress and lipolysis in YDH syndrome, respectively. Our work provides a novel method for YDH syndrome diagnosis and also provides valuable experimental basis to understand the molecular mechanism of YDH syndrome.
Objective The basal ganglia are a group of structures that act as a cohesive functional unit. They are situated at the base of the forebrain and are strongly connected with the cerebral cortex and thalamus. Some speech disorders such as stuttering can resulted from disturbances in the circuits between the basal ganglia and the language motor area of the cerebral cortex. Stuttering consists of blocks, repetitive, prolongation or cessation of speech. We present a 7.5 -year-old male child with bilateral basal ganglia lesion in globus pallidus with unclear reason. The most obvious speech disorders in patient was stuttering, but also problems in swallowing, monotone voice, vocal tremor, hypersensitivity of gag reflex and laryngeal dystonia were seen. He has failed to respond to drug treatment, so he went on rehabilitation therapy when his problem progressed. In this survey, we investigate the possible causes of this type of childhood neurogenic stuttering.
Despite an extensive evaluation program, patients may remain diagnostically unresolved with regard to the etiology of their cognitive dysfunction. Cerebrospinal fluid neuroinflammation and Alzheimer disease (AD) biomarkers may act as indicators of neurodegenerative disorders in diagnostically unresolved patients.
Data on 348 patients were retrospectively evaluated. All participants had a standardized diagnostic workup and follow-up in a memory clinic.
Aβ42 levels and Aβ42/p-tau ratios were reduced and levels of t-tau and p-tau as well as the t-tau × p-tau/Aβ42 ratio were elevated in diagnostically unresolved patients who clinically progressed, compared to a stable group. No differences in neuroinflammatory parameters were found.
AD biomarkers - in particular the Aβ42/p-tau ratio, but not neuroinflammatory parameters - predicted clinical progression, regardless of etiology.
This article examines the current state of affairs of the health information management role in facility closure as well as the impact on revenue cycle operations. Health information management professionals are uniquely positioned to assist an organization in closure efforts because of their knowledge of revenue cycle operations, ability to work with the software products used to generate and store patient information, and solid understanding of the process of care and treatment of the patient. This information is integral to ensuring that patient information in a variety of formats and locations is properly secured and saved so that it will be available to patients and caregivers who need it after the facility closes. This article also goes into detail concerning some of the financial tasks required to be completed when a facility closes because the required data come from coded information or, in the case of the Worker Adjustment and Retraining Notification (WARN) Act, affect how employees will be compensated. This article provides a detailed look at the process to help guide other health information management professionals in a diverse set of care environments in the process of facility closure.
Healthcare regulators are directing attention to the pharmaceutical supply chain with the passage of the Drug Quality and Security Act (DQSA) and the Drug Supply Chain Security Act (DSCSA). Adoption of Radio-Frequency Identification (RFID) technology has the ability to improve compliance, reduce costs, and improve safety in the supply chain but its implementation has been limited; primarily because of hardware and tag costs. The purpose of this research study was to analyze the benefits to the pharmaceutical industry and healthcare system of the adoption of RFID technology as a result of newly implemented supply chain regulations. The methodology was a review following the steps of a systematic review with a total of 96 sources used. With the DSCSA, pharmaceutical companies must track and trace prescription drugs across the supply chain, and RFID can resolve many track-and-trace issues with manufacturer control of data. The practical implication of this study is that pharmaceutical companies must continue to have the potential to increase revenues, decrease associated costs, and increase compliance with new FDA regulations with RFID. Still, challenges related to regulatory statute wording, implementation of two-dimensional barcode technology, and the variety of interfaces within the pharmaceutical supply chain have delayed adoption and its full implementation.
Glioma patients have a poor overall survival; however, patients can show distinct clinical outcomes due to the high heterogeneity of the tumor, which may be indicated by certain clinicobiological parameters. Kinesin family member 20A (KIF20A), which participates in cytokinesis and intracellular transportation, has been recently reported to be upregulated in pancreatic cancer, breast cancer, and bladder cancer. In the current study, we investigated the expression of KIF20A in gliomas and its significance in predicting the prognosis after surgery. We found that KIF20A positive expression in glioma tissues correlated significantly with Ki67 protein expression and advanced World Health Organization grade. Univariate and multivariate analysis revealed that KIF20A can act as an independent prognostic factor for predicting the overall survival of glioma patients. Moreover, we demonstrated that KIF20A can positively regulate the expression of Ki67 in glioma cell lines. Correspondingly, overexpression of KIF20A can promote cell proliferation and invasion, whereas knockdown of KIF20A can inhibit cell viability and invasion capacity. In vitro study also showed that under the treatment of plumbagin, an anticancer drug, KIF20A expression decreased in a dose-dependent manner. In addition, the overexpression of KIF20A can also increase the drug resistance toward plumbagin, which provided the possibility that KIF20A may contribute to the chemotherapy resistance of gliomas.
MLL protein genes encode a family of crucial transcription factors that play a key role in multiple cancer development. The functions of different MLL proteins have not been definitively studied. MLL1 is a histone methyltransferase that mediates histone H3 lysine 4, and it has been found to have aberrant expression in several tumors. However, the function of MLL1 in cervical carcinoma is little known. We used tissue analysis, cell culture experiments, and molecular profiling to investigate the mechanism of MLL1 in cervical carcinoma development. We report here that MLL1 is overexpressed in cervical carcinoma tissues and cell lines, and its overexpression is correlated with the tumor grade. Through FACScan flow cytometry assay, we found that MLL1 promotes cell proliferation by promoting the G1/S transition through transcriptional activation of CCND1 in cervical carcinoma cells. Furthermore, we utilized co-immunoprecipitation and glutathione S-transferase pull-down assays to identify β-catenin as the transcription partner for MLL1 and demonstrated that MLL1 and β-catenin act in synergy in the transcriptional activation of CCND1 in cervical carcinoma cells. In addition, transwell assay and anchorage-independent cell growth assay also revealed that MLL1 promotes metastasis of cervical carcinoma cells through interaction with β-catenin. Our study not only demonstrated a role for MLL1 in the proliferation and metastasis of cervical carcinoma cells but also revealed the interaction of MLL1 with β-catenin to play a different role.
In recent decades, morbidity and mortality have been found to be significantly increased in patients with chronic obstructive pulmonary disease (COPD) complicated with pulmonary tuberculosis (PTB). Platelet-lymphocyte ratio (PLR) is an indicator for inflammatory diseases. This study aims to investigate whether PLR could act as a potential marker for patients with COPD complicated with PTB.
In this retrospective study, laboratory characteristics of 87 COPD patients complicated with PTB (determined by Mycobacterium tuberculosis positive culture from sputum or bronchial lavage fluid) and 83 COPD patients (as the control group, determined by M. tuberculosis culture negativity from sputum or bronchial lavage fluid) were investigated. Data obtained on the day of admission were analyzed.
PLR >216.82 was identified as the optimal cutoff value for discriminating COPD patients with PTB (sensitivity 92.4%, specificity 84.5%, positive-predictive value 91.6%, negative-predictive value 86.2%, and area under the curve [AUC] was 0.87) from patients with COPD alone. The AUC of PLR was significantly greater than that of neutrophil-lymphocyte count ratio (AUC, 0.74; 95% confidence interval, 0.67-0.81; P<0.01).
PLR could be developed as a valuable maker for identifying tuberculosis infection in COPD patients.
Safety climate evaluation is increasingly used by hospitals as part of quality improvement initiatives. Consequently, it is necessary to have validated tools to measure changes.
To evaluate the construct validity and internal consistency of a survey tool to measure Australian hospital pharmacy patient safety climate.
A 42 item cross-sectional survey was used to evaluate the patient safety climate of 607 Australian hospital pharmacy staff. Survey responses were initially mapped to the factor structure previously identified in European community pharmacy. However, as the data did not adequately fit the community pharmacy model, participants were randomly split into two groups with exploratory factor analysis performed on the first group (n = 302) and confirmatory factor analyses performed on the second group (n = 305).
Following exploratory factor analysis (59.3% variance explained) and confirmatory factor analysis, a 6-factor model containing 28 items was obtained with satisfactory model fit (χ(2) (335) = 664.61 p < 0.001, RMSEA = 0.06, CFI = 0.93, TLI = 0.92), internal reliability (α > 0.643) and model nesting between the groups (Δχ(2) (22) = 30.87, p = 0.10). Three factors (blame culture, organisational learning and working conditions) were similar to those identified in European community pharmacy and labelled identically. Three additional factors (preoccupation with improvement; comfort to question authority; and safety issues being swept under the carpet) highlight hierarchical issues present in hospital settings.
This study has demonstrated the validity of a survey to evaluate patient safety climate of Australian hospital pharmacy staff. Importantly, this validated factor structure may be used to evaluate changes in safety climate over time.
This paper focuses on the role of actors that operate outside formal health systems, but nevertheless have a vital, if often under-recognised, role in supporting public health. The specific example used is the 'social enterprise', an organisation that seeks, through trading, to maximise social returns, rather than the distribution of profits to shareholders or owners. In this paper we advance empirical and theoretical understanding of the causal pathways at work in social enterprises, by considering them as a particularly complex form of public health 'intervention'. Data were generated through qualitative, in depth, semi-structured interviews and a focus group discussion, with a purposive, maximum variation sample of social enterprise practitioners (n = 13) in an urban setting in the west of Scotland. A method of analysis inspired by critical realism - Causation Coding - enabled the identification of a range of explanatory mechanisms and potential pathways of causation between engagement in social enterprise-led activity and various outcomes, which have been grouped into physical health, mental health and social determinants. The findings then informed the construction of an empirically-informed conceptual model to act as a platform upon which to develop a future research agenda. The results of this work are considered to not only encourage a broader and more imaginative consideration of what actually constitutes a public health intervention, but also reinforces arguments that actors within the Third Sector have an important role to play in addressing contemporary and future public health challenges.
The regeneration of periodontal tissues lost as a consequence of destructive periodontal disease remains a challenge for clinicians. Guided tissue regeneration (GTR) has emerged as the most widely practiced regenerative procedure. Aim of this study was to electrospin chitosan (CH) membranes with a low or high degree of fiber orientation and examines their suitability for use as a surface layer in GTR membranes, which can ease integration with the periodontal tissue by controlling the direction of cell growth.
A solution of CH-doped with polyethylene oxide (PEO) (ratio 95:5) was prepared for electrospinning. Characterization was performed for biophysiochemical and mechanical properties by means of scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, swelling ratio, tensile testing and monitoring degradation using pH analysis, weight profile, ultraviolet-visible (UV-vis) spectroscopy and FTIR analysis. Obtained fibers were also assessed for viability and matrix deposition using human osteosarcoma (MG63) and human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells.
Random and aligned CH fibers were obtained. FTIR analysis showed neat CH spectral profile before and after electrospinning. Electropsun mats were conducive to cellular attachment and viability increased with time. The fibers supported matrix deposition by hES-MPs. Histological sections showed cellular infiltration as well.
The surface layer would act as seal to prevent junctional epithelium from falling into the defect site and hence maintain space for bone regeneration.
This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n=324) from Australian food-producing animals [cattle (n=169), porcine (n=114), poultry (n=32) and sheep (n=9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0-88.6%), ampicillin (9.4-71.1%), trimethoprim/sulfamethoxazole (11.1-67.5%) and streptomycin (21.9-69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.
Bisphosphonates are well established pharmaceutical drugs with wide applications in medicine. Nevertheless, the side chain and the nature of phosphorous groups could induce a poor aqueous solubility and act on their bioavailability. At the same time, cyclodextrins are cage molecules that facilitate transport of hydrophobic molecules to enhance the intestinal drug absorption of these molecules by forming inclusion complexes. Here we demonstrate that cyclodextrins could be used as a bisphosphonate carrier. The formation of cyclodextrins-bisphosphonate complexes was characterized by 1D and 2D NMR spectroscopy, Isothermal Titration Calorimetry and UV-vis spectroscopy. The results revealed that only the side chain of bisphosphonate was involved in the inclusion phenomenon and its length was a crucial parameter in the control of affinity. Findings from this study suggest that cyclodextrin will be a useful carrier for bisphosphonates.
Candida albicans is the most prevalent fungal pathogen in humans. Due to the development of drug resistance, there is today a need for new antifungal agents for the efficient management of C. albicans infections. Therefore, we reviewed antifungal activity, mechanisms of action, possible synergism with antifungal drugs of all natural substances experimented to be efficient against C. albicans for future.
An extensive and systematic review of the literature was undertaken and all relevant abstracts and full-text articles analyzed and included in the review.
A total of 111 documents were published and highlighted 142 anti-C. albicans natural products. These products are mostly are reported in Asia (44.37%) and America (28.17%). According to in vitro model criteria, from the 142 natural substances, antifungal activity can be considered as important for 40 (28.20%) and moderate for 24 (16.90%). Sixteen products have their antifungal activity confirmed by in vivo gold standard experimentation. Microbial natural products, source of antifungals, have their antifungal mechanism well described in the literature: interaction with ergosterol (polyenes), inhibition 1,3-β-d-glucan synthase (Echinocandins), inhibition of the synthesis of cell wall components (chitin and mannoproteins), inhibition of sphingolipid synthesis (serine palmitoyltransferase, ceramide synthase, inositol phosphoceramide synthase) and inhibition of protein synthesis (sordarins). Natural products from plants mostly exert their antifungal effects by membrane-active mechanism. Some substances from arthropods are also explored to act on the fungal membrane. Interestingly, synergistic effects were found between different classes of natural products as well as between natural products and azoles.
Search for anti-C. albicans new drugs is promising since the list of natural substances, which disclose activity against this yeast is today long. Investigations must be pursued not only to found more new anti-Candida compounds from plants and organisms but also to carried out details on molecules from already known anti-Candida compounds and to more elucidate mechanisms of action.
Amphetamine and methamphetamine use disorders are associated with severe health and social consequences. No pharmacological therapy has been approved for the treatment of these disorders. Psychostimulants can act as maintenance-like therapies for managing substance use among these patients. The aim of this study is to evaluate the literature examining the efficacy and safety of psychostimulant agents for increasing abstinence and treatment retention among patients with amphetamine and methamphetamine use disorders.
We searched MEDLINE, EMBASE, PsycInfo, Cochrane Central, and CINAHL from inception to August 2016. Selection of studies, data extraction, and risk of bias assessment were conducted independently by two reviewers. We conducted meta-analyses to provide a pooled summary estimate for included trials and report the review according to PRISMA guidelines.
We identified and selected 17 studies with 1387 participants. Outcome reporting across trials was inconsistent, and the overall quality of evidence was very low due to high risk of bias and indirectness. A meta-analysis of five trials (642 participants) found no effect of psychostimulants for end-of-study abstinence (odds ratio = 0.97, 95% confidence interval 0.65 to 1.45). Additionally, the pooled estimate from 14 studies (1184 participants) showed no effect of psychostimulants for treatment retention (odds ratio = 1.20, 95% confidence interval = 0.91 to 1.58). The incidence of serious adverse events did not differ between intervention and placebo groups based on qualitative reports from trials.
Quantitative analyses showed no effect of psychostimulants for sustained abstinence or treatment retention. We also identified the need for more rigorous studies in this research area with clinician and patient important outcomes.
Cooking skills are increasingly included in strategies to prevent and reduce chronic diet-related diseases and obesity. While cooking interventions target all age groups (Child, Teen and Adult), the optimal age for learning these skills on: 1) skills retention, 2) cooking practices, 3) cooking attitudes, 4) diet quality and 5) health is unknown. Similarly, although the source of learning cooking skills has been previously studied, the differences in learning from these different sources has not been considered. This research investigated the associations of the age and source of learning with the aforementioned five factors.
A nationally representative (Northern/Republic of Ireland) cross-sectional survey was undertaken with 1049 adults aged between 20-60 years. The survey included both measures developed and tested by the researchers as well as validated measures of cooking (e.g. chopping) and food skills (e.g. budgeting), cooking practices (e.g. food safety), cooking attitudes, diet quality and health. Respondents also stated when they learnt the majority of their skills and their sources of learning. The data was analysed using ANOVAs with post-hoc analysis and Chi(2) crosstabs with a significance level of 0.05.
Results showed that child (<12 years) and/or teen (13-18 years) learners had significantly greater numbers of, and confidence in, their cooking and food skills, cooking practices, cooking attitudes, diet quality (with the exception of fibre intake where adult learners were higher) and health. Mother was the primary source of learning and those who learnt only from this source had significantly better outcomes on 12 of the 23 measures.
This research highlights the importance of learning cooking skills at an early age for skill retention, confidence, cooking practices, cooking attitude and diet quality. Mother remained the primary source of learning, however, as there is a reported deskilling of domestic cooks, mothers may no longer have the ability to teach cooking skills to the next generation. A focus on alternative sources including practical cooking skills education starting at an early age is required. This study also highlights the need for further longitudinal research on the impact of age and source of learning on cooking skills.
Female genital mutilation (FGM) is a widespread practice mainly in Sub-Saharan Africa and is considered an affront on the dignity and health of women and young girls.
To establish a theoretical model, inspired by that of Madeleine Leininger, in order to examine the reasonings used to justify FGM.
Theorization through bibliographic review. Resuts and conclusions: The factors used to justify this act are diverse and convert the tradition into a form of cultural care. From this viewpoint, nurses might evaluate the supposed justifications via the Rising Sun Model in order to redirect such a practice through nursing interventions such as: research into propagating factors, sensitizing through hindering factors or health education, highlighting the contradictions existent in the justification of FGM.
We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.
Selective Serotonin Reuptake Inhibitors (SSRIs) and Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs) are widely prescribed for the treatment of depression and anxiety disorders. Consequently, these compounds are frequently identified in global waterways where they may pose a hazard to aquatic biota. Evidence demonstrates these compounds to be capable of influencing the behaviour of fish, but the relevance of many reported behavioural endpoints is unclear and the value of some findings has been questioned. Since these compounds act on neuroendocrine-mediated pathways in vertebrates, the present study explored how exposure to two representative SSRIs (fluoxetine and sertraline) and an SNRI (venlafaxine) affect circadian rhythms in fish. Male mosquitofish (Gambusia holbrooki) were exposed to 1, 10 and 100μg/L concentrations of these compounds individually and when present as a full mixture, for a period of one week. Neither fluoxetine nor sertraline had an impact on diurnal activity patterns when fish were exposed to these compounds alone at any concentration, whereas venlafaxine significantly disrupted normal circadian rhythmicity but only at 100μg/L. When fish were exposed to the full mixture, significantly altered diurnal activity patterns were rapidly observed at nominal concentrations of 1 and 100μg/L, but there was no effect at 10μg/L. This sort of non-monotonic dose relationship is not altogether unusual for fish exposed to antidepressants, but it poses a problem when attempting to evaluate potential risks to the aquatic environment. To evaluate the possibility for misinterpretation when collecting behavioural data over short temporal scales, the data for each day of the experiment was analysed separately. The outcomes demonstrate the importance of longer periods of data collection, which may be necessary to capture the full range of natural behavioural variability that exists both amongst and within individual fish. More importantly, these findings may help reveal why discrepancies are commonly being reported in the literature with regards behavioural effects in fish exposed to antidepressants. It is thus suggested that research be aimed at documenting behavioural variability in fish species used in toxicity testing, to establish guidelines for quality control and where possible inform the development of standardised methodologies so that behavioural analysis can be more appropriately applied to the broad field of aquatic toxicology.
A mammalian receptor for bacterial lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4), plays a beneficial role in controlling bacterial infections, but is also a main driver of aberrant inflammation in lethal sepsis. As a result, investigation of TLR4 signaling has been a major area of research. Despite this focus, our understanding of the mechanisms that regulate TLR4 activities remains primitive. Nowhere is our knowledge of TLR4 biology more lacking than at the receptor-proximal level, where many factors act in concert to regulate LPS signaling. Several recent studies have begun filling these gaps in our knowledge. In this review, we discuss the importance of these receptor proximal activities in the spatiotemporal regulation of TLR4 signaling, and suggest interesting areas for future research.
In the absence of congressional action to reinstate the federal ban on assault weapons, tort litigation offers an alternative strategy for regulating what have become the weapons of choice in mass shootings. However, opportunities to bring successful claims are limited. To prevail, plaintiffs must show that their suit fits within exceptions to the broad immunity from tort actions that Congress gave the firearm industry in the 2005 Protection of Lawful Commerce in Arms Act. In one particularly high-profile lawsuit, families of victims of the school shooting in Newtown, Connecticut, in 2012 sued the makers and sellers of the military-style rifle used in the attack, alleging negligence and deceptive marketing. The trial court dismissed the case on October 14, 2016, but the plaintiffs plan to appeal. We review the history of tort litigation against the firearm industry, outline the Newtown families' claims, and describe the decision.
Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.
This study projected the indirect costs of back problems through lost productive life years (PLYs) from the individual's perspective (lost disposable income), the governmental perspective (reduced taxation revenue, greater welfare spending), and the societal perspective (lost gross domestic product, GDP) from 2015 to 2030, using Health&WealthMOD2030-Australia's first microsimulation model on the long-term impacts of ill-health. Quantile regression analysis was used to examine differences in median weekly income, welfare payments, and taxes of people unable to work due to back problems with working full-time without back problems as comparator. National costs and lost GDP resulting from missing workers due to back problems were also projected. We projected that 90,000 people have lost PLYs due to back problems in 2015, increasing to 104,600 in 2030 (16.2% increase). People with lost PLYs due to back problems are projected to receive AU$340.91 less in total income and AU$339.77 more in welfare payments per week than full-time workers without back problems in 2030 and pay no income tax on average. National costs consisted of a loss of AU$2931 million in annual income in 2015, increasing to AU$4660 million in 2030 (60% increase). For government, extra annual welfare payments are projected to increase from AU$1462 million in 2015 to AU$1709 million in 2030 (16.9% increase), and lost annual taxation revenue to increase from AU$671 million in 2015 to $961 million in 2030 (43.2% increase). We projected losses in GDP of AU$10,543 million in 2015, increasing to AU$14,522 million in 2030 due to back problems.
From its beginning, the AIDS epidemic crystallized some of the major flaws of the American health care system. Most private health insurance was associated with employment, and job loss meant insurance loss. Private insurers refused new coverage for people with HIV infection. Medicaid, an important program for uninsured people with low income, was limited to only those in certain categories (eg, pregnant women or children), and although people who had progressed to AIDS were categorized as eligible (ie, "disabled"), those with early stage HIV disease were not. The Patient Protection and Affordable Care Act is a landmark change in health care law in general and for people with HIV infection in particular. Its provisions offer dramatic improvements in health coverage, although a Supreme Court ruling that limited the expansion of Medicaid poses ongoing problems in some states. This article summarizes a presentation by Timothy M. Westmoreland, JD, at the IAS-USA continuing education program, Improving the Management of HIV Disease, held in Washington, DC, in May 2015.
This review article discusses current knowledge on natural killer (NK) cells in asthma.
It is now well accepted that NK cell activities go beyond cancer immune surveillance and antiviral defense. Recent reports indicate that NK cells are activated in response to allergens in vivo. NK cells promote allergic sensitization, type-2 immune response, development of eosinophilic inflammation, and airway hyperresponsiveness. NK cells are activated by respiratory syncytial virus and other respiratory viruses. When infection occurs in the setting of active allergic inflammation, NK cells augment its magnitude and contribute to asthma exacerbations. Proasthma activities of NK cells can be programmed during embryogenesis through maternal exposure to environmental pollutants. Prenatally programmed NK cells produce type-2 and type-3 cytokines and mediate asthma predisposition. NK cells can also act as asthma antagonists. NK cells contribute to the resolution of inflammation through suppression of antigen-specific CD4+ T cells and type-3 immunity. When viral infection occurs in naïve mice prior to allergic sensitization, NK cells antagonize type-2 immunity and prevent development of asthma.
NK cells are nonredundant participants of allergic inflammation. The environmental context determines whether NK cells act as protagonists or antagonists.
Categorization has been associated with distributed networks of the primate brain, including the prefrontal cortex (PFC) and posterior parietal cortex (PPC). Although category-selective spiking in PFC and PPC has been established, the frequency-dependent dynamic interactions of frontoparietal networks are largely unexplored. We trained monkeys to perform a delayed-match-to-spatial-category task while recording spikes and local field potentials from the PFC and PPC with multiple electrodes. We found category-selective beta- and delta-band synchrony between and within the areas. However, in addition to the categories, delta synchrony and spiking activity also reflected irrelevant stimulus dimensions. By contrast, beta synchrony only conveyed information about the task-relevant categories. Further, category-selective PFC neurons were synchronized with PPC beta oscillations, while neurons that carried irrelevant information were not. These results suggest that long-range beta-band synchrony could act as a filter that only supports neural representations of the variables relevant to the task at hand.
The crystallization process of Hertzian spheres is studied by means of molecular dynamics simulations in an NPT ensemble where the total number of particles N, the pressure P, and the temperature T are kept constant. It has been observed that the bond orientational ordering rather than the translational ordering (density) plays a primary role. The crystal polymorphs are determined by the state points. Under the conditions of small supercooling, the system is likely to be nucleated into crystals that have a preference for the metastable bcc structure, which can be regarded as a manifestation of the Alexander-McTague mechanism. In contrast, small nuclei are found to have a preference for fcc symmetry under conditions of a high degree of supercooling. Prestructured precursors that act as seeds and wet on the nuclei during nucleation always have a high degree of bcc-like ordering, despite different state points. The results above may provide a clue to the understanding of the crystallization process in core-softened particles.
Members of the genus Candida, such as C. albicans and C. parapsilosis, are important human pathogens. Other members of this genus, previously believed to carry minimal disease risk, are increasingly recognised as important human pathogens, particularly because of variations in susceptibilities to widely used anti-fungal agents. Thus, rapid and accurate identification of clinical Candida isolates is fundamental in ensuring timely and effective treatments are delivered. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has previously been shown to provide a high-throughput platform for the rapid and accurate identification of bacterial and fungal isolates. In comparison to commercially available matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF), REIMS based methods require no preparative steps nor time-consuming cell extractions. Here, we report on the ability of REIMS-based analysis to rapidly and accurately identify 153 clinical Candida isolates to species level. Both handheld bipolar REIMS and high-throughput REIMS platforms showed high levels of species classification accuracy, with 96% and 100% of isolates classified correctly to species level respectively. In addition, significantly different (FDR corrected P value < 0.05) lipids within the 600 to 1000 m/z mass range were identified, which could act as species-specific biomarkers in complex microbial communities.
Lead-free relaxor ferroelectrics that feature a core-shell microstructure provide an excellent electromechanical response. They even have the potential to replace the environmentally hazardous lead-zirconia-titanate (PZT) in large strain actuation applications. Although the dielectric properties of core-shell ceramics have been extensively investigated, their piezoelectric properties are not yet well understood. To unravel the interfacial core-shell interaction, we studied the relaxation behaviour of field-induced ferroelectric domains in 0.75Bi1/2Na1/2TiO3-0.25SrTiO3 (BNT-25ST), as a typical core-shell bulk material, using a piezoresponse force microscope. We found that after poling, lateral domains emerged at the core-shell interface and propagated to the shell region. Phase field simulations showed that the increased electrical potential beneath the core is responsible for the in-plane domain evolution. Our results imply that the field-induced domains act as pivotal points at the coherent heterophase core-shell interface, reinforcing the phase transition in the non-polar shell and thus promoting the giant strain.
Valsalva maneuver is associated with diverse physiological changes. These changes are used in various diagnostic and therapeutic clinical settings. Valsalva maneuver is also employed during various phases of neurosurgical procedures to achieve specific targets and confirm intraoperative findings. In this article, we attempt to describe the various clinical applications of the Valsalva maneuver within the realms of clinical neurosurgery. The associated complications of this act have also been discussed.
This is the official guideline endorsed by the specialty associations involved in the care of head and neck cancer patients in the UK. It discusses the role of the clinical nurse specialist in the head and neck cancer patient journey and provides recommendations on the clinical nurse specialist led assessments and interventions for this group of patients receiving cancer care. Recommendations • All cancer patients should meet a clinical nurse specialist at the point of diagnosis. (R) • Clinical nurse specialists must act as gate keeper to the patients' cancer pathway to provide a seamless journey. (R) • Holistic needs assessment should be completed at different stages of the patient's pathway to reflect the changes of the patients' needs. (R) • Clinical nurse specialists to be part of local and national initiatives for health promotion and raising awareness in the public domain. (G) • Clinical nurse specialists should lead in redesigning of services and policies to ensure they are responsive to patient's needs for the future. (G) • Treatment summaries should become part of practice to provide good communication between primary and secondary care to enable continuity of care for the patient. (G).
At the turn of the century, several major efforts were initiated to combat HIV/AIDS and other major epidemics affecting low- and middle-income countries (LMICs). They were accompanied by initiatives to enable recipient countries to collect and use data to guide their public health programmes. These health information systems (HIS) typify systems in that they have multiple interacting components, and they are embedded within larger systems. Components of a larger system act as the context for all lower-level systems. Their effects can be pervasive, and thus be taken for granted or regarded as unchangeable. We identify four contextual factors that affect efforts to strengthen HIS: hierarchical roles, aid funding, corruption, and competing priorities. We provide examples of each as experienced by those working to strengthen HIS in LMICs. Each of these contextual factors can seriously diminish the effectiveness of HIS strengthening efforts and their long-term sustainability. We propose research questions about each that would enable those engaged in HIS strengthening to work effectively and sustainably.
Lived experience practitioners can contribute to improved outcomes for people with mental illness, supplementing traditional mental health services and reducing health care costs. However, lived experience practitioners frequently face stigma and discrimination within their work roles.
To understand the impact of stigma and discrimination on the effectiveness of lived experience roles from the perspective of lived experience practitioners.
In-depth interviews were conducted with 13 lived experience practitioners within a grounded theory study.
Issues of stigma and discrimination were identified as a core category of this study. Participants described stigma and discrimination so prevalent as to be considered a "normal" part of their working life. Professional isolation and attitudinal barriers from colleagues were seen to inhibit the effectiveness of lived experience roles.
Lived experience practitioners can provide a vital contribution to stigma reduction broadly, however, the stigma and discrimination they face within work roles must be addressed to allow this contribution to be effective.
Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot(®) technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot(®) assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot(®) assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot(®) assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.
Analgesic strategy of a single drug analgesia in bone cancer pain (BCP) has shifted to combined analgesia with different drugs which have different mechanism. After tumor cell inculation, the activation of signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) signaling pathway are involved in the development and maintenance of BCP, whereas a decrease in the expression of spinal STAT3 and ERK through using their specific blocker, lead to attenuation of BCP. Hence, in this study, we clarified that intrathecal (i.t.) injection of midazolam (MZL) and ropivacaine (Ropi) induces synergistic analgesia on BCP and is accompanied with different mechanisms of these analgesic effect. Hargreaves heat test was used to detect the analgesic effect of single dose of i.t. MZL, Ropi and their combination on the BCP rats. At consecutive daily administration experiment, thermal hyperalgesia was recorded, and immunohistochemical staining was used to detect the expression of c-Fos, spinal glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (IBA-1). Then, western blot analysis was used to examine spinal TSPO, GFAP, IBA-1, pERK/ERK and pSTAT3/STAT3 levels on day 14 after tumor cell inoculation. i.t. MZL or Ropi showed a short-term analgesia dose-dependently, and MZL displayed better effect on inhibition of pSTAT3 expression than pERK, but Ropi was just the reverse, then consecutive daily administrations of their combination acted synergistically to attenuate thermal hyperalgesia with downregulated spinal 'neuron-astrocytic activation' in the BCP rats. i.t. co-delivery of MZL and Ropi shows synergistic analgesia on the BCP with the inhibition of spinal 'neuron-astrocytic activation'. Spinal different signaling pathway inhibition for MZL and Ropi may be involved in this process.
Carbon nanotubes (CNTs) filled with iron sulfide nanoparticles (NPs) are prepared by inserting sulfur and ferrocene into the hollow core of CNTs followed by heat treatment. It is found that pyrrhotite-11T iron sulfide (Fe-S) NPs with an average size of ≈15 nm are encapsulated in the tubular cavity of the CNTs (Fe-S@CNTs), and each particle is a single crystal. When used as the anode material of lithium-ion batteries, the Fe-S@CNT material exhibits excellent electrochemical lithium storage performance in terms of high reversible capacity, good cyclic stability, and desirable rate capability. In situ transmission electron microscopy studies show that the CNTs not only play an essential role in accommodating the volume expansion of the Fe-S NPs but also provide a fast transport path for Li ions. The results demonstrate that CNTs act as a unique nanocontainer and reactor that permit the loading and formation of electrochemically active materials with desirable electrochemical lithium storage performance. CNTs with their superior structural stability and Li-ion transfer kinetics are responsible for the improved rate capability and cycling performance of Fe-S NPs in CNTs.
The group II chaperonin thermosome (THS) is a hollow protein nanoparticle that can encapsulate macromolecular guests. Two large pores grant access to the interior of the protein cage. Poly(amidoamine) (PAMAM) is conjugated into THS to act as an anchor for small interfering RNA (siRNA), allowing to load the THS with therapeutic payload. THS-PAMAM protects siRNA from degradation by RNase A and traffics KIF11 and GAPDH siRNA into U87 cancer cells. By modification of the protein cage with the cell-penetrating peptide TAT, RNA interference is also induced in PC-3 cells. THS-PAMAM protein-polymer conjugates are therefore promising siRNA transfection reagents and greatly expand the scope of protein cages in drug delivery applications.
The coordination polymer {[Cu(SiF6)(C10H8N2)2]·2C10H8N2·5H2O} n , systematic name: poly[[bis-(μ2-4,4'-bi-pyridine)(μ2-hexa-fluorido-silicato)copper(II)] 4,4'-bi-pyridine disolvate penta-hydrate], contains pores which are filled with water and 4,4'-bi-pyridine mol-ecules. As a result of the presence of these ordered species, the framework changes its symmetry from P4/mmm to P21/c. The 4,4'-bi-pyridine guest mol-ecules form chains inside the 6.5 × 6.9 Å pores parallel to [100] in which the mol-ecules inter-act through π-π stacking. Ordered water mol-ecules form infinite hydrogen-bonded chains inside a second pore system (1.6 × 5.3 Å free aperture) perpendicular to the 4,4'-bi-pyridine channels.
Crystals of bis-(tetra-butyl-ammonium) di-μ3-chlorido--tris-(μ2-4-iodo-pyrazolato-κ(2)N:N')tris-[chlorido-cuprate(II)] 1,4-dioxane hemisolvate, (C16H36N)2[Cu3(C3H2IN2)3Cl5]·0.5C4H8O or (Bu4N)2[Cu(II)3(μ3-Cl)2(μ-4-I-pz)3Cl3]·0.5C4H8O, were obtained by evaporating a solution of (Bu4N)2[{Cu(II)(μ-OH)(μ-4-I-pz)} n CO3] (n = 27-31) nanojars in chloro-form/1,4-dioxane. The decomposition of chloro-form in the presence of oxygen and moisture provides HCl, which leads to the breakdown of nanojars to the title trinuclear copper(II) pyrazolate complex, and possibly Cu(II) ions and free 4-iodo-pyrazole. Cu(II) ions, in turn, act as catalyst for the accelerated decomposition of chloro-form, ultimately leading to the complete breakdown of nanojars. The crystal structure presented here provides the first structural description of a trinuclear copper(II) pyrazolate complex with iodine-substituted pyrazoles. In contrast to related trinuclear complexes based on differently substituted 4-R-pyrazoles (R = H, Cl, Br, Me), the [Cu3(μ-4-I-pz)3Cl3] core in the title complex is nearly planar. This difference is likely a result of the presence of the iodine substituent, which provides a unique, novel feature in copper pyrazolate chemistry. Thus, the iodine atoms form halogen bonds with the terminal chlorido ligands of the surrounding complexes [mean length of I⋯Cl contacts = 3.48 (1) Å], leading to an extended two-dimensional, halogen-bonded network along (-110). The cavities within this framework are filled by centrosymmetric 1,4-dioxane solvent mol-ecules, which create further bridges via C-H⋯Cl hydrogen bonds with terminal chlorido ligands of the trinuclear complex not involved in halogen bonding.
An immersed boundary-lattice Boltzmann method is developed for fluid-structure interactions involving non-Newtonian fluids (e.g., power-law fluid). In this method, the flexible structure (e.g., capsule) dynamics and the fluid dynamics are coupled by using the immersed boundary method. The incompressible viscous power-law fluid motion is obtained by solving the lattice Boltzmann equation. The non-Newtonian rheology is achieved by using a shear rate-dependant relaxation time in the lattice Boltzmann method. The non-Newtonian flow solver is then validated by considering a power-law flow in a straight channel which is one of the benchmark problems to validate an in-house solver. The numerical results present a good agreement with the analytical solutions for various values of power-law index. Finally, we apply this method to study the deformation of a capsule in a power-law shear flow by varying the Reynolds number from 0.025 to 0.1, dimensionless shear rate from 0.004 to 0.1, and power-law index from 0.2 to 1.8. It is found that the deformation of the capsule increases with the power-law index for different Reynolds numbers and nondimensional shear rates. In addition, the Reynolds number does not have significant effect on the capsule deformation in the flow regime considered. Moreover, the power-law index effect is stronger for larger dimensionless shear rate compared to smaller values.
Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca(2+)-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca(2+) entry (ROCE). Although store-operated Ca(2+) entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca(2+) imaging. After depleting internal Ca(2+) stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca(2+)]i whereas lower concentrations (≤100 μM) also induced Ca(2+) oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca(2+) oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca(2+) signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca(2+) oscillations (P2X4) and sustained [Ca(2+)]i increases. We demonstrate specific drug effects on purinergic Ca(2+) pathways and provide new pharmacological insights into Ca(2+) oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca(2+)-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.
Viola odorata L. belongs to Violaceae family and is native to Iran. It is used in the form of Almond-Violet oil in traditional Persian medicine (TPM) since ancient times. Almond-Violet oil was used for the treatment of insomnia, headache, cough, and fever based on TPM textbooks. There are two methods for the preparation of Almond-Violet oil. The first is macerating voila flowers in sweet almond oil for several days under the sunlight. The second method is cold pressing of violet flowers and sweet almond.
In this study, after mixing the violet flowers with sweet almonds in 1:2 proportions, Almond-Violet oil was obtained under pressure. Fatty acid ingredients of Almond-Violet oil were analyzed by gas chromatography (GC) technique.
Analysis of Almond-Violet oil by GC method showed some major components such as oleic acid (70.54%), linoleic acid (Omega-6 fatty acids) (18.22%), palmitic acid (8.51%), stearic acid (1.58%), and palmitoleic acid (0.69%). Monounsaturated fat consumption has been considered to decrease low-density lipoprotein (LDL) cholesterol. Linoleic acid lipid radicals can also be used to act as an antioxidant agent in natural phenols. On the other hand, oleic acid may be responsible for the hypotensive (blood pressure reducing) effects. Palmitoleic acid is a beneficial fatty acid not only to increase insulin sensitivity by suppressing inflammation, but also to inhibit the destruction of insulin-secreting pancreatic beta cells.
In some aspects, the result of the present study does not fully match with the standards of the Europe Pharmacopoeia. This could be due to differences associated with the environment and cultivation of the plants. Such differences should be considered whilst studying native plants.
As English social care services reconstruct themselves in response to the personalization agenda, there is increased interest in the contribution of micro-providers - very small community-based organizations, which can work directly with individuals. These micro-providers are assumed to be able to cater for the 'seldom heard' groups which have been marginalized within mainstream social care services. This article reviews recent literature from the UK published in peer-reviewed journals from 2000 to 2013 on support provision for people with protected characteristics under the Equality Act 2010. It considers the marginalising dynamics in mainstream, statutory social care support provision, and how far local community, specialist or small-scale services are responding to unmet need for support and advice among marginalized groups. The review found that there is a tradition of compensatory self-organization, use of informal networks and a mobilization of social capital for all these groups in response to marginalization from mainstream, statutory services. This requires recognition and nurturing in ways that do not stifle its unique nature. Specialist and community-based micro-providers can contribute to a wider range of choices for people who feel larger, mainstream services are not suitable or accessible. However, the types of compensatory activity identified in the research need recognition and investment, and its existence does not imply that the mainstream should not address marginalization.
Oblongifolin C (OC) and guttiferone K (GUTK) are two anticancer compounds extracted from Garcinia yunnanensis Hu, but they act by different mechanisms. In this study we investigated whether a combination of OC and GUTK (1:1 molar ratio) could produce synergistic anticancer effects against human colorectal cancer cells in vitro. For comparison, we also examined the anticancer efficacy of ethanol extracts from G yunnanensis fruit, which contain OC and GUTK up to 5%. Compared to OC and GUTK alone, the combination of OC and GUTK as well as the ethanol extracts more potently inhibited the cancer cell growth with IC50 values of 3.4 μmol/L and 3.85 μg/mL, respectively. Furthermore, OC and GUTK displayed synergistic inhibition on HCT116 cells: co-treatment with OC and GUTK induced more prominent apoptosis than treatment with either drug alone. Moreover, the combination of OC and GUTK markedly increased cleavage of casapse-3 and PARP, and enhanced cellular ROS production and increased JNK protein phosphorylation. In addition, the combination of OC and GUTK exerted stronger effects under nutrient-deprived conditions than in complete medium, suggesting that autophagy played an essential role in regulating OC- and GUTK-mediated cell death. OC and GUTK are the main components that contribute to the anticancer activity of G yunnanensis and the compounds have apoptosis-inducing effects in HCT116 cells in vitro.
Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds preceding prolines. We investigated the protein-protein interaction between a 22-kDa PPIase (VaFKBP22, an FK506-binding protein) and the molecular chaperone DnaK derived from Vibrio anguillarum O1 (VaDnaK) using GST pull-down assays and a bacterial two-hybrid system for in vivo and in vitro studies, respectively. Furthermore, we analyzed the three-dimensional structure of the protein-protein interaction. Based on our results, VaFKBP22 appears to act as cochaperone of VaDnaK, and contribute to protein folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity.
Proton pump inhibitors (PPIs) were clinically introduced more than 25 years ago and have since proven to be invaluable, safe, and effective agents for the management of a variety of acid-related disorders. Although all members in this class act in a similar fashion, inhibiting active parietal cell acid secretion, there are slight differences among PPIs relating to their pharmacokinetic properties, metabolism, and Food and Drug Administration (FDA)-approved clinical indications. Nevertheless, each is effective in managing gastroesophageal reflux disease and uncomplicated or complicated peptic ulcer disease. Despite their overall efficacy, PPIs do have some limitations related to their short plasma half-lives and requirement for meal-associated dosing, which can lead to breakthrough symptoms in some individuals, especially at night. Longeracting PPIs and technology to prolong conventional PPI activity have been developed to specifically address these limitations and may improve clinical outcomes.
In the present study, the analytical study on blood flow containing nanoparticles through porous blood vessels is done in presence of magnetic field using Homotopy Perturbation Method (HPM). Blood is considered as the third grade non- Newtonian fluid containing nanoparticles. Viscosity of nanofluid is determined by Constant, Reynolds' and Vogel's models. Some efforts have been made to show the reliability and performance of the present method compared with the numerical method, Runge-Kutta fourth-order. The results reveal that the HPM can achieve suitable results in predicting the solution of these problems. Moreover, the influence of some physical parameters such as pressure gradient, Brownian motion parameter, thermophoresis parameter, magnetic filed intensity and Grashof number on temperature, velocity and nanoparticles concentration profiles is declared in this research. The results reveal that the increase in the pressure gradient and Thermophoresis parameter as well as decrease in the Brownian motion parameter cause the rise in the velocity profile. Furthermore, either increase in Thermophoresis or decrease in Brownian motion parameters results in enhancement in nanoparticle concentration. The highest value of velocity is observed when the Vogel's Model is used for viscosity.
Liposomes have been the centre of attention in research due to their potential to act as drug delivery systems. Although its versatility and manufacturing processes are still not scalable and reproducible. In this study, the microfluidic method for liposomes preparation is presented. DMPC and DSPC liposomes containing two different lipid/cholesterol ratios (1:1 and 2:1) are prepared. Results from this preparation process were compared with the film hydration method in order to understand benefits and drawbacks of microfluidics. Liposomes characterisation was evaluated through stability studies, encapsulation efficacy and drug release profiles of hydrophilic and lipophilic compounds. Stability tests were performed during 3 weeks and the liposomes properties of the most stable formulations were determined using Infrared Microscopy and Atomic Force Microscopy. Microfluidic allows loading of drugs and assembly in a quick single step and the chosen flow ratio for liposomes formulation plays a fundamental role for particle sizes. One hydrophilic and one lipophilic compounds were incorporated showing how formulation and physic-chemical characteristics can influence the drug release profile.
Quinine or alternative artemisinin-based combination treatment (ACT) is the recommended rescue treatment for uncomplicated malaria. However, patients are often re-treated with the same ACT though it is unclear whether this is the most suitable approach. We assessed the efficacy and safety of re-treating malaria patients with uncomplicated failures with the same ACT used for the primary episode, compared with other rescue treatments.
This was a bicentre, open-label, randomised, three-arm phase 3 trial done in Lisungi health centre in DR Congo, and Kazo health centre in Uganda in 2012-14. Children aged 12-60 months with recurrent malaria infection after treatment with the first-line ACT were randomly assigned to either re-treatment with the same first-line ACT, an alternative ACT, which were given for 3 days, or quinine-clindamycin (QnC), which was given for 5-7 days, following a 2:2:1 ratio. Randomisation was done by computer-generated randomisation list in a block design by country. The three treatment groups were assumed to have equivalent efficacy above 90%. Both the research team and parents or guardians were aware of treatment allocation. The primary outcome was the proportion of patients with an adequate clinical and parasitological response (ACPR) at day 28, in the per-protocol population. This trial was registered under the numbers NCT01374581 in ClinicalTrials.gov and PACTR201203000351114 in the Pan African Clinical Trials Registry.
From May 22, 2012, to Jan 31, 2014, 571 children were included in the trial. 240 children were randomly assigned to the re-treatment ACT group, 233 to the alternative ACT group, and 98 to the QnC group. 500 children were assessed for the primary outcome. 71 others were not included because they did not complete the follow-up or PCR genotyping result was not conclusive. The ACPR response was similar in the three groups: 91·4% (95% CI 87·5-95·2) for the re-treatment ACT, 91·3% (95% CI 87·4-95·1) for the alternative ACT, and 89·5% (95% CI 83·0-96·0) for QnC. The estimates for rates of malaria recrudescence in the three treatment groups were similar (log-rank test: χ(2)=0·22, p=0·894). Artemether-lumefantrine was better tolerated than QnC (p=0·0005) and artesunate-amodiaquine (p<0·0001) in the modified intention-to-treat analysis. No serious adverse events were observed. The most common adverse events reported in the re-treatment ACT group were anorexia (31 [13%] of 240 patients), asthenia (20 [8%]), coughing (16 [7%]), abnormal behaviour (13 [5%]), and diarrhoea (12 [5%]). Anorexia (13 [6%] of 233 patients) was the most frequently reported adverse event in the alternative ACT group. The most commonly reported adverse events in the QnC group were anorexia (12 [12%] of 98 patients), abnormal behaviour (6 [6%]), asthenia (6 [6%]), and pruritus (5 [5%]).
Re-treatment with the same ACT shows similar efficacy as recommended rescue treatments and could be considered for rescue treatment for Plasmodium falciparum malaria. However, the effect of this approach on the selection of resistant strains should be monitored to ensure that re-treatment with the same ACT does not contribute to P falciparum resistance.
Fonds Wetenschappelijk Onderzoek, Vlaamse Interuniversitaire Raad-Universitaire Ontwikkelings Samenwerking, European and Developing Countries Clinical Trials Partnership, and the Belgian Technical Cooperation-Programme d'Etudes et d'Expertises-in the Democratic Republic of Congo.
Creatine (Cr) is an important organic compound acting as intracellular high-energy phosphate shuttle and in energy storage. While located in most cells where it plays its main roles in energy metabolism and cytoprotection, Cr is highly concentrated in muscle and brain tissues, in which Cr also appears to act in osmoregulation and neurotransmission. This review discusses the basis of Cr metabolism, synthesis and transport within brain cells. The importance of Cr in brain function and the consequences of its impaired metabolism in primary and secondary Cr deficiencies are also discussed. Cr and phosphocreatine (PCr) in living systems can be well characterized using in vivo magnetic resonance spectroscopy (MRS). This review describes how (1)H MRS allows the measurement of Cr and PCr, and how (31)P MRS makes it possible to estimate the creatine kinase (CK) rate constant and so detect dynamic changes in the Cr/PCr/CK system. Absolute quantification by MRS using creatine as internal reference is also debated. The use of in vivo MRS to study brain Cr in a non-invasive way is presented, as well as its use in clinical and preclinical studies, including diagnosis and treatment follow-up in patients.
This work introduces a new electrochemical sensor based on polyvinyl pyrrolidone capped CoFe2O4@CdSe core-shell modified electrode for a rapid detection and highly sensitive determination of rifampicin (RIF) by square wave adsorptive stripping voltammetry. The new PVP capped CoFe2O4@CdSe with core-shell nanostructure was synthesized by a facile synthesis method for the first time. PVP can act as a capping and etching agent for protection of the outer surface nanoparticles and formation of a mesoporous shell, respectively. Another important feature of this work is the choice of the ligand (1,10-phenanthroline) for precursor cadmium complex that works as a chelating agent in order to increase optical and electrical properties and stability of prepared nanomaterial. The nanoparticles have been characterized by field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), UV-vis, photoluminescence (PL) spectroscopy, FT-IR, and cyclic voltammetry techniques. The PL spectroscopy study of CoFe2O4@CdSe has shown significant PL quenching by the formation of CoFe2O4 core inside CdSe, this shows that CoFe2O4 NPs are efficient electron acceptors with the CdSe. It is clearly observed that the biosensor can significantly enhance electrocatalytic activity towards the oxidation of RIF, under the optimal conditions. The novelty of this work arises from the new synthesis method for the core-shell of CoFe2O4@CdSe. Then, the novel electrochemical biosensor was fabricated for ultra-trace level determination of rifampicin with very low detection limit (4.55×10(-17)M) and a wide linear range from 1.0×10(-16) to 1.0×10(-7)M. The fabricated biosensor showed high sensitivity and selectivity, good reproducibility and stability. Therefore, it was successfully applied for the determination of ultra-trace RIF amounts in biological and pharmaceutical samples with satisfactory recovery data.
As modern humans dispersed from Africa throughout the world, they encountered and interbred with archaic hominins, including Neanderthals and Denisovans [1, 2]. Although genome-scale maps of introgressed sequences have been constructed [3-6], considerable gaps in knowledge remain about the functional, phenotypic, and evolutionary significance of archaic hominin DNA that persists in present-day individuals. Here, we describe a comprehensive set of analyses that identified 126 high-frequency archaic haplotypes as putative targets of adaptive introgression in geographically diverse populations. These loci are enriched for immune-related genes (such as OAS1/2/3, TLR1/6/10, and TNFAIP3) and also encompass genes (including OCA2 and BNC2) that influence skin pigmentation phenotypes. Furthermore, we leveraged existing and novel large-scale gene expression datasets to show many positively selected archaic haplotypes act as expression quantitative trait loci (eQTLs), suggesting that modulation of transcript abundance was a common mechanism facilitating adaptive introgression. Our results demonstrate that hybridization between modern and archaic hominins provided an important reservoir of advantageous alleles that enabled adaptation to out-of-Africa environments.
Ras-interacting protein 1 (Rasip1) is an endothelial-specific Rap1 and Ras effector, important for vascular development and angiogenesis. Here, we report the crystal structure of the Rasip1 RA domain (RRA) alone, revealing the basis of dimerization, and in complex with Rap1 at 2.8 Å resolution. In contrast to most RA domains, RRA formed a dimer that can bind two Rap1 (KD = 0.9 μM) or Ras (KD = 2.2 μM) molecules. We solved the Rap1-RRA complex and found that Rasip1 binds Rap1 in the Switch I region, and Rap1 binding induces few conformation changes to Rasip1 stabilizing a β strand and an unstructured loop. Our data explain how Rasip1 can act as a Rap1 and Ras effector and show that Rasip1 defines a subgroup of dimeric RA domains that could mediate cooperative binding to membrane-associated Ras superfamily members.
Lack of time is one of the most common reasons people give for not exercising or eating healthy food, yet few studies explicitly test its relationship with health behaviours.
Conceptualising time as a social determinant we estimate how scarcity - of income or time - generate barriers to health behaviours.
Using longitudinal, nationally-representative survey data on Australians aged 25-54 years, our design addresses endogeneity and reverse causation by considering how new episodes of scarcity are related to changes in healthy eating and physical activity. Regression models estimated how scarcity of income (low income or feeling poor) or time (heavy time commitments or feeling rushed for time) predicted change over two consecutive years.
We find that both income and time scarcity reduce physical activity and, in some cases, lead people to consume less fruit and vegetables, eat out more and eat more discretionary calories (food high in salt, sugar or fat). Further, income and time scarcity operate independently to constrain healthy choices, although for more than one in ten people they synergistically increase risk.
Because income and time scarcity are patterned by socio-economic status and gender, our results underline the need to address both if public health interventions are to be more effective and fair.
The evolutionary transition from an ape-like to human-like upper extremity occurred in the context of a behavioral shift from an upper limb predominantly involved in locomotion to one adapted for manipulation. Selection for overarm throwing and endurance running is thought to have further shaped modern human shoulder girdle morphology and its position about the thorax. Homo naledi (Dinaledi Chamber, Rising Star Cave, Cradle of Humankind, South Africa) combines an australopith-like cranial capacity with dental characteristics akin to early Homo. Although the hand, foot, and lower limb display many derived morphologies, the upper limb retains many primitive traits. Here, we describe the H. naledi upper extremity (excluding the hand) in detail and in a comparative context to evaluate the diversity of clavicular, scapular, humeral, radial, and ulnar morphology among early hominins and later Homo. Homo naledi had a scapula with a markedly cranially-oriented glenoid, a humerus with extremely low torsion, and an australopith-like clavicle. These traits indicate that the H. naledi scapula was situated superiorly and laterally on the thorax. This shoulder girdle configuration is more similar to that of Australopithecus and distinct from that of modern humans, whose scapulae are positioned low and dorsally about the thorax. Although early Homo erectus maintains many primitive clavicular and humeral features, its derived scapular morphology suggests a loss of climbing adaptations. In contrast, the H. naledi upper limb is markedly primitive, retaining morphology conducive to climbing while lacking many of the derived features related to effective throwing or running purported to characterize other members of early Homo.
Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.
The quality of fetal growth and development predicts the risk for a range of noncommunicable, chronic illnesses. These observations form the basis of the "developmental origins of health and disease" hypothesis, which suggests that the intrauterine signals that compromise fetal growth also act to "program" tissue differentiation in a manner that predisposes to later illness. Fetal growth also predicts the risk for later psychopathology. These findings parallel studies showing that antenatal maternal emotional well-being likewise predicts the risk for later psychopathology. Taken together, these findings form the basis for integrative models of fetal neurodevelopment, which propose that antenatal maternal adversity operates through the biological pathways associated with fetal growth to program neurodevelopment. The authors review the literature and find little support for such integrated models. Maternal anxiety, depression, and stress all influence neurodevelopment but show modest, weak, or no associations with known stress mediators (e.g., glucocorticoids) or with fetal growth. Rather, compromised fetal development appears to establish a "meta-plastic" state that increases sensitivity to postnatal influences. There also remain serious concerns that observational studies associating either fetal growth or maternal mental health with neurodevelopmental outcomes fail to account for underlying genetic factors. Finally, while the observed relation between fetal growth and adult health has garnered considerable attention, the clinical relevance of these associations remains to be determined. There are both considerable promise and important challenges for future studies of the fetal origins of mental health.
Distraction osteogenesis biologically resembles fracture healing with distinctive characteristics notably in the distraction phase of osteogenesis. In the latency phase of bone lengthening, like in the inflammatory phase of fracture repair, interleukines are released and act with growth factors released from platelets in the local haematoma, leading to attraction, proliferation and differentiation of mesenchymal stem cells into osteoblasts and other differentiated mesenchymal cells. These in turn produce matrix, collagen fibers and growth factors. A callus containing cells, collagen fibers, osteoid and cartilage matrix is formed. Provided stable fixation, distraction will trigger intramembranous bone formation. As distraction proceeds, the distraction gap develops five distinctive zones with unmineralized bone in the middle, remodelling bone peripherally, and mineralizing bone in between. During consolidation, the high concentration of anabolic growth factors in the regenerate diminishes with time as remodelling takes over to form mature cortical and cancellous bone. Systemic disease, congenital bone deficiencies, medications and substance abuse can influence the quality and quantity of regenerate bone, usually in a negative way. The regenerate bone can be manipulated when needed by using injection of mesenchymal stem cells and platelets, growth factors (BMP-2 and -7), and systemic medications (bisphosphonates and parathyroid hormone). Growth factors and systemic anabolic and antiresorptive drugs are prescribed on special indications, while distraction osteogenesis is not an authorized indication. To some extent, however, these compounds can be used off-label. Use in children presents special problems since growth factors and specific anabolic medications may involve a risk of inducing cancer.
Few studies have investigated the thyroid-disrupting effects of polybrominated diphenyl ethers (PBDEs) across multiple levels of biological organization in anurans, despite their suitability for the screening of thyroid disruptors. Therefore, the present study evaluated the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on development, thyroid histology and thyroid hormone-related gene expression in Xenopus laevis exposed to 0 (control), 50 (low), 500 (medium) or 5000μg BDE-47/g food (high) for 21days. Only the high dose of BDE-47 hindered growth and development; however, thyroid hormone-associated gene expression was downregulated in the brains of tadpoles regardless of dose. These results show that BDE-47 disrupts thyroid hormone signaling at the molecular and whole-organism levels and suggest that gene expression in the brain is a more sensitive endpoint than metamorphosis. Furthermore, the altered gene expression patterns among BDE-47-exposed tadpoles provide insight into the mechanisms of PBDE-induced thyroid disruption and highlight the potential for PBDEs to act as neurodevelopmental toxicants.
The purpose of this randomized non-inferiority trial was to compare video-teleconferencing (VTC) versus in-person (IP) delivery of an 8-week Acceptance and Commitment Therapy (ACT) intervention among veterans with chronic pain (N = 128) at posttreatment and 6-month follow-up. The primary outcome was the pain interference subscale of the Brief Pain Inventory. Secondary outcomes included measures of pain severity, mental and physical health-related quality of life, pain acceptance, activity level, depression, pain-related anxiety, and sleep quality. In intent-to-treat analyses using mixed linear-effects modeling, both groups exhibited significant improvements on primary and secondary outcomes, with the exception of sleep quality. Further, improvements in activity level at 6-month follow-up were significantly greater in the IP group. The non-inferiority hypothesis was supported for the primary outcome and several secondary outcomes. Treatment satisfaction was similar between groups; however, significantly more participants withdrew during treatment in the VTC group compared to the IP group, which was moderated by activity level at baseline. These findings generally suggest that ACT delivered via VTC can be as effective and acceptable as IP delivery for chronic pain. Future studies should examine the optimal delivery of ACT for patients with chronic pain who report low levels of activity. This trial was registered at ClinicalTrials.gov (NCT01055639).
This study suggests that Acceptance and Commitment Therapy for chronic pain can be implemented via video-teleconferencing with comparable reductions in pain interference as in-person delivery. This article contains potentially important information for clinicians using telehealth technology to deliver psychosocial interventions to individuals with chronic pain.
Formation of ruthenium(II) complexes of the type [RuH(CO)(PPh3)2(L)] (where L=N-Substituted 9-ethyl carbazole thiosemicarbazone ligands) has been described from the reactions of [RuHCl(CO)(PPh3)3] and substituted carbazole thiosemicarbazones in 1:1 equivalent respectively. The composition of the complexes was established by elemental analysis, IR, NMR ((1)H ,(13)C and (31)P) and UV-visible spectral methods. The solid state molecular structure of the ligands (L1-L3) and one of the complexes have been analysed by single-crystal X-ray studies, and found that the ruthenium(II) complexes possess a pseudo-octahedral geometry. The thiosemicarbazone ligand is coordinated to ruthenium as a monoanionic bidendate N,S-donor forming a four-membered chelate ring with a bite angle of 64.47(5)(°). The stability of the complexes in aqueous medium was confirmed by UV-visible and ESI-Mass spectral studies. The DNA binding interactions of the complexes with Calf thymus DNA have been investigated by absorption, emission, elctrochemical, circular dichromism and viscosity measurements revealed that the complexes could interact with DNA via intercalation. Further, their protein binding ability was monitored by the quenching of tryptophan emission using bovine serum albumin (BSA) as a model protein. The alterations in the secondary structure of BSA by the complexes were confirmed with synchronous and three-dimensional fluorescence spectral studies. The ability of complexes to cleave BSA varies from 3>2>1 in the presence of activator like H2O2, as revealed from SDS-PAGE is consistent with their strong hydrophobic interaction with the protein. Free-radical scavenging ability of all the complexes were also carried out against a panel of radicals such as DPPH, NO, OH, O2(-) and reducing power assay under in vitro experimental conditions. The potential of complexes to act as anticancer agents is thoroughly examined on human cervical cancer cell line HeLa, Osteosarcoma cell line MG-63 and a normal mouse embryonic fibroblasts cell line NIH-3T3 and screening shows the HeLa cell line exhibits maximum cytotoxicity. The correlation of cytotoxicity of these complexes to their hydrophobicity shows that an appropriate value of the hydrophobicity is essential for high antiproliferative activity. Further, the morphological changes and apoptosis have been evaluated by AO-EB staining techniques and flow cytometry analysis against HeLa cell line.
Cancer is characterized by cell heterogeneity and the development of 3D in vitro assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues in vitro and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.
The adult pancreas is only capable of limited regeneration. Unlike highly regenerative tissues such as the skin, intestinal crypts and hematopoietic system, no dedicated adult stem cells or stem cell niche have so far been identified within the adult pancreas. New β cells have been shown to form in the adult pancreas, in response to high physiological demand or experimental β-cell ablation, mostly by replication of existing β cells. The possibility that new β cells are formed from other sources is currently a point of major controversy. Under particular injury conditions, fully differentiated pancreatic duct and acinar cells have been shown to dedifferentiate into a progenitor-like state, however the extent, to which ductal, acinar or other endocrine cells contribute to restoring pancreatic β-cell mass remains to be resolved. In this review we focus on regenerative events in the pancreas with emphasis on the restoration of β-cell mass. We present an overview of regenerative responses noted within the different pancreatic lineages, following injury. We also highlight the intrinsic plasticity of the adult pancreas that allows for inter-conversion of fully differentiated pancreatic lineages through manipulation of few genes or growth factors. Taken together, evidence from a number of studies suggest that differentiated pancreatic lineages could act as facultative progenitor cells, but the extent to which these contribute to β-cell regeneration in vivo is still a matter of contention.
To analyze structural features of ω-Aga IVA, a gating modifier toxin from spider venom, we here investigated the NMR solution structure of ω-Aga IVA within DPC micelles. Under those conditions, the Cys-rich central region of ω-Aga IVA still retains the inhibitor Cys knot motif with three short antiparallel β-strands seen in water. However, (15)N HSQC spectra of ω-Aga IVA within micelles revealed that there are radical changes to the toxin's C-terminal tail and several loops upon binding to micelles. The C-terminal tail of ω-Aga IVA appears to assume a β-turn like conformation within micelles, though it is disordered in water. Whole-cell patch clamp studies with several ω-Aga IVA analogs indicate that both the hydrophobic C-terminal tail and an Arg patch in the core region of ω-Aga IVA are critical for Cav2.1 blockade. These results suggest that the membrane environment stabilizes the structure of the toxin, enabling it to act in a manner similar to other gating modifier toxins, though its mode of interaction with the membrane and the channel is unique.
Today overtreatment of indolent prostate cancers and undertreatment of aggressive prostate cancer are a major concern for patients, their families, and the health care system. New biomarkers distinguishing indolent and aggressive prostate cancer are needed to improve precision medicine. In prostate cancer, protein kinase A (PKA) is known to activate the androgen receptor and published data indicate that PKA subunits can act as predictive markers for response to radiation and chemotherapy. We have previously shown that the catalytic subunit, Cβ2, of PKA is up-regulated in prostate cancer and we would in this study investigate the potential of Cβ2 to become a prognostic biomarker in prostate cancer.
Data were sampled from a total of 241 patients from 3 independent cohorts. We measured and compared Cβ2 messenger RNA (mRNA) levels in prostate tumor and nontumor samples (n = 22), and exon levels in a cohort of 50 tumor samples, as well as acquiring mRNA data from the publicly available database The cancer genome atlas (n = 169).
Cβ2 mRNA was up-regulated in prostate cancer in all 3 cohorts, measured by 3 different methods. Furthermore, the relative Cβ2 mRNA expression levels were lower in prostate cancer samples with Gleason score 8 to 10 compared with samples with Gleason score<8 (P = 0.004). Finally, low expression of Cβ2 mRNA in prostate cancer biopsies correlated with poor survival (hazard ratio = 0.20; 95% CI: 0.048-0.86; P = 0.031), adjusted for risk group and age.
We suggest that Cβ2 mRNA expression may be used as a biomarker together with established prognostic markers to more precisely predict aggressiveness in patients diagnosed with prostate cancer.
Polycyclic aromatic hydrocarbons (PAHs) are a type of ubiquitous pollutant with the potential ability to cause endocrine disruption that would have an adverse health impact on the general population. To assess the maternal exposure to PAHs in neonates and evaluate the possible impact of PAHs on reproductive hormone levels, the concentration of PAHs and reproductive hormone levels in the umbilical cord serum of 98 mother-infant pairs in the Shengsi Islands were investigated. The median concentration of total PAHs was determined to be 164 (Inter-Quartile Range, IQR 93.6-267) ng g(-1) lipid, and 68% of the PAHs were lower-molecule congeners. The highest level was found for pyrene (PYR) and naphthalene (NAP), which contributed 54.6% of all the PAHs present in the samples. The exposure to PAHs negatively affected estradiol (E2) and Anti-Mullerian hormones (AMH) and positively affected FSH in the umbilical cord serum. The result expanded the database of the human burden of PAHs and suggested that PAHs can act as a type of Endocrine-Disrupting Chemical (EDC). These results may help to understand the complex pathways involved in disorders of human reproductive health associated with prenatal exposure to PAHs.
The 2008 economic crisis made the issue of good governance more present and visible, but the debate risks stalling in an academic and political discourse that barely exceeds the declarative. We acknowledge the existence of noteworthy documents from scientific medical societies and some institutional proposals that point towards promising changes. Viewing good governance as accountability, participation, transparency, intelligence and integrity, our objective is to identify the determinants of inaction and remove the barriers that prevent the adoption of rational and widely agreed-upon proposals. This led us: 1) to allocate the proposals to their appropriate governance level (macro, meso, micro and system environment) so as to more directly engage the agents of change; and 2) to highlight some decision nodes that can act as levers to catalyse selective transformations and to initiate the change processes. Taking into account the diversity of actors and scenarios, a top-down rational, integrated and reformist strategy for the whole Spanish National Health System does not seem likely or viable. Therefore, the purpose of this paper is to promote changes, setting a targeted and reasoned agenda to visualise key issues and to enable multilevel and multidimensional thinking and advocacy of health-sector and society stakeholders.
This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac(0)-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac(3)-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9μM(-1)min(-1) for BK and Toac(3)-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.
The immune system is important to protect the host from fungal infections. Diverse cell types belonging to the innate or adaptive branch of the immune system act in a tightly coordinated and tissue-specific manner. Experimental mouse models of fungal infections have proved essential for assessing the protective principles against different fungal pathogens. Besides pathological, histological, biochemical and molecular parameters, the analysis of phenotypic and functional aspects of immune cells in infected tissues is key for understanding the mechanisms of antifungal defense. In this chapter, we describe a method based on flow cytometry to assess innate and adaptive immune cells isolated from an in vivo context in a qualitative and quantitative manner.
Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular trafficking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein-protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.
The aim of this study is to investigate whether salaried and self-employed workers differ regarding factors relevant for return to work after being diagnosed with cancer. The possible mediators of an effect of self-employment on work ability were also investigated.
A total of 1115 cancer survivors (1027 salaried and 88 self-employed) of common invasive cancer types who were in work at the time of diagnosis completed a mailed questionnaire 15-39 months after diagnosis.
Twenty-four percent of self-employed cancer survivors reported that they had not returned to work at the time of the survey, and 18 % of those who were salaried had not. While 9 % of the self-employed had received disability or early retirement pension, only 5 % had received such a pension among salaried employees. Compared with the salaried workers, the self-employed people reported significantly more often reduced work hours (P < 0.001), negative cancer-related financial (P < 0.001), and occupational changes (P = 0.005) and low overall health (P = 0.02), quality of life (P = 0.04), and total work ability (P = 0.02). The negative effect of self-employment on total work ability seems to be mediated by reduced work hours and a negative cancer-related financial change.
Compared with salaried, self-employed workers in Norway, they seem to struggle with work after cancer. This may be because the two groups have different work tasks and because self-employed people have lower social support at work and less legal support from the Working Environment Act and public health insurance.
Self-employed people with cancer should be informed about the work-related challenges they may encounter and be advised to seek practical help from social workers who know about the legal rights of self-employed people.
Aim of this work was to assess the role of polymorphisms belonging to genes involved in the regulation of ionic homeostasis in Caucasian patients with Ménière Disease (MD). We recruited 155 patients with definite Ménière Disease and 186 controls (Control Group 1) without a lifetime history of vertigo, overlapping with patients for age and rate of hypertension. We validated the positive results on 413 Caucasian subjects selected from a European general population (Control Group 2). The clinical history for migraine and hypertension was collected; genomic DNA was characterized for a panel of 33 SNPs encoding proteins involved in ionic transport. We found a higher rate of migraineurs in MD subjects compared to Group 1 (46.8 vs 15.5%, p = 0.00005). Four SNPs displayed differences in MD patients compared to Group 1 controls: rs3746951 and rs2838301 in SIK1 gene, rs434082 and rs487119 in SLC8A1; the p values of Chi-squared test for genotype frequencies are 0.009, 0.023, 0.009 and 0.048, respectively. SLC8A1 gene encodes for Na(+)-Ca(++) exchanger, while SIK1 gene encodes for Salt Inducible Kinase 1, an enzyme associated with Na(+)-K(+) ATPase function. The validation with Control Group 2 displayed that only rs3746951 and rs487119 are strongly associated to MD (p = 0.001 and p = 0.0004, respectively). These data support the hypothesis that a genetically induced dysfunction of ionic transport may act as a predisposing factors to develop MD.
In this narrative review, the neurobiological mechanisms underlying substance abuse and addiction are discussed with a particular emphasis on the mechanisms that promote ongoing use and relapse. Addiction is estimated to affect 10-15% or more of the adult population, including physicians. Genetic predisposition, psychological and environmental risk factors, the timing of exposure to the substance, the type of substance used, and the frequency of use influence the individual's susceptibility to addiction. Abused substances act on the brain's reward system, a neural circuit that produces pleasurable feelings in response to stimuli that promote survival, thereby modifying future behavior to seek out similar stimuli. Endogenous activators include food, sex, and social interaction. Drugs of abuse hijack the reward circuit, producing intense activation. Repetitive exposure to substances leads to persistent, altered genetic expression and accumulation of ΔFos-B and corticotropin-releasing factor. High levels of these substances suppress the reward circuit and activate the endogenous stress response, resulting in a generalized state of discord. These changes are enduring and can trigger substance use relapse even after long periods of abstinence.
Readmissions after total joint arthroplasty have become a key quality measure in elective surgery in the United States. The Affordable Care Act includes the Hospital Readmission Reduction Program, which calls for reduced payments to hospitals with excessive readmissions. This policy uses a method to determine excess readmission ratios and calculate readmission payment adjustments to hospitals, however, it is unclear whether readmission rates are an effective quality metric. The reasons or conditions associated with readmission after elective THA have been well established but the extent to which readmissions can be prevented after THA remains unclear.
(1) Are unplanned readmissions after THA associated with orthopaedic or medical causes? (2) Are these readmissions preventable? (3) When during the course of aftercare are orthopaedic versus medical readmissions more likely to occur?
We retrospectively evaluated all 1096 elective THAs for osteoarthritis performed between January 1, 2011 and June 30, 2014 at a major academic medical center. Of those, 69 patients (6%) who met inclusion criteria were readmitted in our healthcare system within 90 days of discharge after the index procedure during the study period. Fifty patients were readmitted within 30 days of discharge after the index procedure (5%). We defined a readmission as any unplanned inpatient or observation status admission to the hospital spanning at least one midnight. A panel of physicians not involved in the care of these patients used available criteria and existing consensus guidelines to evaluate the medical records, radiographs, and operative reports to identify whether the underlying reason for readmission was orthopaedic versus medical. They subsequently were classified as either nonpreventable or potentially preventable readmissions, based on any care that may have occurred during the index hospitalization. To make such determinations, consensus specialty society guidelines were used whenever possible for each readmission diagnosis.
A total of 50 of 1096 patients (5% of those who underwent THA during the period in question) were readmitted within 30 days and 69 of 1096 (6%) were readmitted within 90 days of their index procedures. Thirty-one patients were readmitted for orthopaedic reasons (31/69; 45%) and 38 of 69 were readmitted for medical reasons (55%). Three readmissions (three of 69; 4%) were identified as potentially preventable. Of these potentially preventable readmissions, one was orthopaedic (hip dislocation) and two were medical. Thirty-day readmissions were more likely to be orthopaedic than 90-day readmissions (odds ratio, 4.06; 95% CI, 1.18-13.96; p = 0.026).
Using a panel of expert reviewers, available existing criteria, and consensus methodology, it appears only a small percentage of readmissions after THA are potentially preventable. Orthopaedic readmissions occur earlier during the postoperative course. Currently, existing policies and readmission penalties may not serve as valuable external quality metrics. The readmission rates in our study may represent the threshold for expected readmission rates after THA. Future studies should enroll larger numbers of patients and have independent review panels in efforts to refine criteria for what constitutes preventable readmissions.
Level III, therapeutic study.
We describe changes in emergency department (ED) visits and the proportion of patients with hospitalizations through the ED classified as Ambulatory Care Sensitive Hospitalization (ACSH) for the uninsured before (2011-2013) and after (2014-2015) Affordable Care Act (ACA) health insurance expansion in Illinois. Hospital administrative data from 201 non-federal Illinois hospitals for patients age 18-64 were used to analyze ED visits and hospitalizations through the ED. ACSH was defined using Agency for Healthcare Research and Quality (AHRQ) Prevention Quality Indicators (PQIs). Logistic regression was used to test the effect of time period on the odds of an ACSH for uninsured Illinois residents, controlling for patient sociodemographic characteristics, weekend visits and state region. Total ED visits increased 5.6% in Illinois after ACA implementation, with virtually no change in hospital admissions. Uninsured ED visits declined from 22.9% of all visits pre-ACA to 12.5% in 2014-2015, reflecting a 43% decline in average monthly ED visits and 54% in ED hospitalizations. The proportion of uninsured ED hospitalizations classified as ACSH increased from 15.4 to 15.5%, a non-significant difference. Older uninsured female, minority and downstate Illinois patients remained significantly more likely to experience ACSH throughout the study period. ED visits for the uninsured declined dramatically after ACA implementation in Illinois but over 12% of ED visits are for the remaining uninsured. The proportion of visits resulting in ACSH remained stable. Providing universal insurance with care coordination focused on improved access to home and ambulatory care could be highly cost effective.
Unfractionated heparin is the preferred anticoagulant in extracorporeal membrane oxygenation (ECMO) patients. However, there is a lack of consensus on its titration and monitoring. The objective of this study was to describe the efficacy and safety of a pharmacy managed heparin protocol utilizing activated partial thromboplastin time (aPTT) in comparison to our standard physician-managed activated clotting time (ACT)-based anticoagulation in ECMO patients.
Patients administered a heparin drip while on ECMO were included in the study. The primary endpoints were the incidence of hemorrhagic and thrombotic complications.
A total of 122 adult patients were identified who were on ECMO with heparin anticoagulation; sixty-one patients were managed with each of the physician-managed ACT and pharmacy managed aPTT protocols. No statistically significant difference was observed between the physician ACT and the pharmacy aPTT groups in overall hemorrhagic (69% vs 80%, p=0.145) or thrombotic complications (41% vs 39%, p=0.853).
There was a similar rate of thrombotic and bleeding events between the two study groups. A pharmacy managed heparin protocol utilizing aPTT monitoring appears to be a safe and effective method of providing anticoagulation in adult ECMO patients.
The root vascular tissues provide an excellent system for studying organ patterning, as the specification of these tissues signals a transition from radial symmetry to bisymmetric patterns. The patterning process is controlled by the combined action of hormonal signaling/transport pathways, transcription factors, and miRNA that operate through a series of non-linear pathways to drive pattern formation collectively. With the discovery of multiple components and feedback loops controlling patterning, it has become increasingly difficult to understand how these interactions act in unison to determine pattern formation in multicellular tissues. Three independent mathematical models of root vascular patterning have been formulated in the last few years, providing an excellent example of how theoretical approaches can complement experimental studies to provide new insights into complex systems. In many aspects these models support each other; however, each study also provides its own novel findings and unique viewpoints. Here we reconcile these models by identifying the commonalities and exploring the differences between them by testing how transferable findings are between models. New simulations herein support the hypothesis that an asymmetry in auxin input can direct the formation of vascular pattern. We show that the xylem axis can act as a sole source of cytokinin and specify the correct pattern, but also that broader patterns of cytokinin production are also able to pattern the root. By comparing the three modeling approaches, we gain further insight into vascular patterning and identify several key areas for experimental investigation.
Pain has been suggested to act as a stressor during aging, potentially accelerating declines in health and functioning. Our objective was to examine the longitudinal association between self-reported pain and the development, or worsening, of frailty among older men and women.
The study population consisted of 5,316 men and women living in private households in England, mean age 64.5 years, participating in the English Longitudinal Study of Ageing (ELSA). Data from Waves 2 and 6 of ELSA were used in this study with 8 years of follow-up. At Wave 2, participants were asked whether they were "often troubled with pain" and for those who reported yes, further information regarding the intensity of their pain (mild, moderate, or severe) was collected. Socioeconomic status (SES) was assessed using information about the current/most recent occupation and also net wealth. A frailty index (FI) was generated, with the presence of frailty defined as an FI >0.35. Among those without frailty at Wave 2, the association between pain at Wave 2 and frailty at Wave 6 was examined using logistic regression. We investigated whether pain predicted change in FI between Waves 2 and 6 using a negative binomial regression model. For both models adjustments were made for age, gender, lifestyle factors, depressive symptoms, and socioeconomic factors.
At Wave 2, 455 (19.7%) men and 856 (28.7%) women reported they often experienced moderate or severe pain. Of the 5,159 participants who were nonfrail at Wave 2, 328 (6.4%) were frail by Wave 6. The mean FI was 0.11 (standard deviation [SD] = 0.1) at Wave 2 and 0.15 (SD = 0.1) at Wave 6. After adjustment for age, gender, body mass index, lifestyle factors, and depressive symptoms, compared to participants reporting no pain at Wave 2 those reporting moderate (odds ratio [OR] = 3.08, 95% confidence interval [CI] = 2.28, 4.16) or severe pain (OR = 3.78, 95% CI = 2.51, 5.71) were significantly more likely to be frail at Wave 6. This association persisted after further adjustment for either occupational class and/or net wealth level. Compared to those without pain, those with mild, moderate, or severe pain were also more likely to develop worsening frailty, as assessed using the FI, and this association persisted after adjustment for SES. There was no evidence that the association between pain and frailty was influenced by gender.
Pain is associated with an increased risk and intensity of frailty in older men and women. Socioeconomic factors contribute to the occurrence of frailty; though in our study do not explain the relationship between pain and frailty.
The prevalence of a sedentary (SED) life style combined with calorically rich diets has spurred the rise in childhood obesity which, in turn, translates to adverse health effects in adulthood. Obesity and lack of active (ACT) lifestyle may increase susceptibility to air pollutants. We housed 22 day-old female Long-Evans rats in a cage without (SED) or with a running wheel (ACT). After 10 weeks the rats ran 310 ± 16.3 km (SEM). Responses of SED and ACT rats to whole-body O3 (0, 0.25, 0.5, or 1.0 ppm; 5 hr/day for 2 days) was assessed. Glucose tolerance (GTT) was performed following the first day of O3. ACT rats had less body fat and an improved glucose tolerance (GTT). Ventilatory function (plethysmography) of SED and ACT groups was similarly impaired by O3. Bronchoalveolar lavage fluid (BALF) was collected after the second O3 exposure. SED and ACT rats were hyperglycemic following 1.0 ppm O3. GTT was impaired by O3 in both groups; however, ACT rats exhibited improved recovery to 0.25 and 1.0 ppm O3. BALF cell neutrophils and total cells were similarly increased in ACT and SED groups exposed to 1.0 ppm O3. O3-induced increase in eosinophils was exacerbated in SED rats. Chronic exercise from post-weaning to adulthood improved some of the metabolic and pulmonary responses to O3 (GTT and eosinophils) but several other parameters were unaffected. The reduction in O3-induced rise in BALF eosinophils in ACT rats suggests a possible link between a SED lifestyle and incidence of asthma-related symptoms from O3.
Triple-negative breast cancer (TNBC) is characterized by high vascularity and frequent metastasis. Here, we found that activation of G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 can significantly inhibit interleukin 6 (IL-6) and vascular endothelial growth factor A (VEGF-A). TNBC tissue microarrays from 100 TNBC patients revealed GPER is negatively associated with IL-6 levels and higher grade and stage. Activation of GPER or anti-IL-6 antibody can inhibit both in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and migration of TNBC cells. While recombinant IL-6 supplementary can significantly reverse the inhibitory effects of G-1, suggesting the essential role of IL-6 in G-1 induced suppression of angiogenesis and invasiveness of TNBC cells. G-1 treatment decreased the phosphorylation, nuclear localization, transcriptional activities of NF-κB and suppressed its binding with IL-6 promoter. BAY11-7028, the inhibitor of NF-κB, can mimic the effect of G-1 to suppression of IL-6 and VEGF-A. While over expression of p65 can attenuate the inhibitory effects of G-1 on IL-6 and VEGF expression. The suppression of IL-6 by G-1 can further inhibit HIF-1α and STAT3 signals in TNBC cells by inhibition their expression, phosphorylation and/or nuclear localization. Moreover, G-1 also inhibited the in vivo NF-κB/IL-6 signals and angiogenesis and metastasis of MDA-MB-231 xenograft tumors. In conclusion, our study demonstrated that activation of GPER can suppress migration and angiogenesis of TNBC via inhibition of NF-κB/IL-6 signals, therefore it maybe act as an important target for TNBC treatment.
With a quantum yield of 0.66±0.03 the photoisomerization efficiency of the visual pigment rhodopsin (11-cis⇒all-trans chromophore) is exceptionally high. This is currently explained by coherent coupling of the excited state electronic wavepacket with local vibrational nuclear modes, facilitating efficient cross-over at a conical intersection onto the photoproduct energy surface. The 9-cis counterpart of rhodopsin, dubbed isorhodopsin, has a much lower quantum yield (0.26±0.03), which, however, can be markedly enhanced by modification of the retinal chromophore (7,8-dihydro and 9-cyclopropyl derivatives). The coherent coupling in the excited state is promoted by torsional skeletal and coupled HOOP vibrational modes, in combination with a twisted conformation around the isomerization region. Since such torsion will strongly enhance the infrared intensity of coupled HOOP modes, we investigated FTIR difference spectra of rhodopsin, isorhodopsin and several analog pigments in the spectral range of isolated and coupled HCCH wags. As a result we propose that the coupled HOOP signature in these retinal pigments correlates with the distribution of torsion over counteracting segments in the retinylidene polyene chain. As such the HOOP signature can act as an indicator for the photoisomerization efficiency, and can explain the higher quantum yield of the 7,8-dihydro and 9-cyclopropyl-isorhodopsin analogs.
The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum.
A novel three-dimensional DNA-AuNPs network structure amplification strategy was employed to design a lateral flow biosensor by introducing streptavidin coated gold nanoparticles (Au-SA) in this paper. They act as amplification probes which aggregate numerous gold nanoparticles (AuNPs) on test line by forming a three-dimensional DNA-AuNPs network structure in the presence of target. Sensitive detection of nucleic acid with point-of-care analysis is significant for infectious agent, early diagnosis and treatment of genetic diseases. The use of these particles in rapid ultrasensitive point of care (POC) lateral flow assays lead to a linear range from 0.1pM to 250nM with a limit of detection of 0.01 pM without polymerase chain reaction (PCR). The proposed method could increase the sensitivity by 4 orders of magnitudes than traditional sandwich assays labeled with AuNPs. Furthermore, the assay owns good reproducibility and stability, which will prove practical diagnostic applications.
Naringin, plant bioflavonoid extracted mainly from grapefruit and other related citrus species. This study was designed to assess the neuroprotective effect of naringin on ammonium chloride (NH4Cl) induced hyperammonemic rats. Experimental hyperammonemia was induced by intraperitonial injection (i.p) of NH4Cl (100mg/kg body weight (b.w.)) thrice a week for 8 consecutive weeks. Hyperammonemic rats were treated with naringin (80mg/kg b.w.) via oral gavage. Naringin administration drastically restored the levels of blood ammonia, plasma urea, nitric oxide (NO), glutamate, glutamine, lipid peroxidation, lipid profile, activities of liver marker enzymes, antioxidant status and sodium/potassium-ATPase (Na(+)/K(+)-ATPase). In addition, naringin supplementation reverted back the pathological changes of liver, brain and kidney tissues, the expressions of Glutamine synthetase (GS), Na(+)/K(+)-ATPase, neuronal nitric oxide (nNOS) and soluble guanylate cyclase (sGC) in hyperammonemic rats. Hence, this study suggested that nargingin exhibited their protective effect against NH4Cl induced toxicity via enhancing the activities of antioxidant enzymes and inhibiting the lipid peroxidation process. Take together, this study provides data that naingin effectively reduced neurotoxicity by attenuating hyperammonemia, suggesting that naringin act as a potential therapeutic agent to treat hyperammonemic rats.
Unconventional myosins are actin-based molecular motors that serve a multitude of roles within the cell. One group of myosin motors, the MyTH4-FERM myosins, play an integral part in building and maintaining finger-like protrusions, which allow cells to interact with their external environment. Suggested to act primarily as transporters, these motor proteins enrich adhesion molecules, actin-regulatory proteins and other factors at the tips of filopodia, microvilli, and stereocilia. Below we review data from biophysical, biochemical, and cell biological studies, which implicate these myosins as central players in the assembly, maintenance and function of actin-based protrusions.
The grey mullet usually occur in large numbers and biomass in the estuaries of all three South African biogeographic regions, thus making it an ideal family to use in terms of possibly acting as an environmental indicator of global warming. In this analysis the relative estuarine abundance of the dominant three groups of mugilids, namely tropical, warm-water and cool-water endemics, were related to sea surface coastal temperatures. The study suggests a strong link between temperature and the distribution and abundance of the three mullet groups within estuaries and indicates the potential of this family to act as an indicator for future climate change within these systems and adjacent coastal waters.
The emergency department is a unique practice environment in that the Emergency Medical Treatment and Active Labor Act (EMTALA), which mandates a medical screening examination for all presenting patients, effectively precludes any sort of patient volume control; staffing needs are therefore fluid and unpredictable. The purpose of this study is to explore emergency nurses' perceptions of factors involved in safe staffing levels and to identify factors that negatively and positively influence staffing levels and might lend themselves to more effective interventions and evaluations.
We used a qualitative exploratory design with focus group data from a sample of 26 emergency nurses. Themes were identified using a constructivist perspective and an inductive approach to content analysis.
Five themes were identified: (1) unsafe environment of care, (2) components of safety, (3) patient outcomes: risky care, (4) nursing outcomes: leaving the profession, and (5) possible solutions. Participants reported that staffing levels are determined by the number of beds in the department (as in inpatient units) but not by patient acuity or the number of patients waiting for treatment. Participants identified both absolute numbers of staff, as well as experience mix, as components of safe staffing. Inability to predict the acuity of patients waiting to be seen was a major component of nurses' perceptions of unsafe staffing.
Emergency nurses perceive staffing to be inadequate, and therefore unsafe, because of the potential for poor patient outcomes, including missed or delayed care, missed deterioration (failure to rescue), and additional ED visits resulting from ineffective discharge teaching. Both absolute numbers of staff, as well as skill and experience mix, should be considered to provide staffing levels that promote optimal patient and nurse outcomes.
The aim of this study was to determine practices, attitudes and experiences of UK prescribers of anthelmintics for horses and livestock. A questionnaire was sent by direct email to groups licenced to prescribe these medicines. These were veterinarians, Suitably Qualified Persons (SQPs, registered with the Animal Medicines Training Regulatory Authority) and veterinary pharmacists. The survey was also advertised through social media. It comprised questions relating to demographics, training experiences, current prescribing practices, as well as personal opinions on anthelmintic selection, diagnostics and anthelmintic resistance. A total of 193 veterinarians and 326 SQPs were included in final analysis. Pharmacists were excluded from detailed analysis due to the low numbers that responded (n=3). The results indicated that SQP participants were more likely to receive post-certification parasitology training than the veterinarians, and that both channels consulted similar sources for information about helminths and their control (paper articles in journals, online sources). The SQP participants stated a higher frequency of face-to-face interactions with clients/customers (96.1%) than the veterinarians (76.4%), who stated a higher frequency of telephone interactions (55.1% and 73.5%, respectively). Veterinarians were more likely to state that there were specific factors that limited interactions with their clients (54.1%) than SQPs (19.6%), such as competition from other suppliers. SQP participants considered a wider range of factors as important when deciding on which anthelmintic to recommend (i.e. knowledge of specific parasites, knowledge of specific anthelmintics, discussion of measures to avoid anthelmintic resistance and time to talk with clients/customers); however, the veterinarian participants were more likely to consider the results of diagnostic tests. While discussions about anthelmintic resistance were stated with similar frequency in both groups, less frequent were specific discussions about anthelmintic sensitivity testing. In-house faecal egg count analysis was more likely to be available from those that prescribed anthelmintics for equines alone, compared to prescribers who dispensed anthelmintics for livestock alone or livestock and equines. The SQP participants indicated that they felt a large number of organisations were responsible for ensuring that anthelmintics are used responsibly, whilst veterinarian participants were more likely to place responsibility on the prescribers alone. Taken together, these findings provide an insight into how prescribers of anthelmintics in the UK interact with their clients/customers before and at the point of sale and act as a unique source of information on how best practice advice pertaining to sustainable helminth control is disseminated by the various prescribing channels.
Vitamin D plays a key role in bone health. Consuming adequate vitamin D during young adulthood is important due to the development of peak bone mass; however, many Canadian young adults do not meet vitamin D recommendations. This study aimed to improve knowledge, perceptions, dietary intake and blood concentrations of vitamin D among a sample of young adults.
Using a pre-post design, 90 Ontario adults (38 men, 52 women; 18-25 years), were randomly assigned to intervention or control groups. Participants completed a socio-demographic survey, pre-post food frequency questionnaire, and a vitamin D knowledge questionnaire (3 time-points). The intervention group watched a video, received online information and tracked intake of vitamin D using a mobile application for 12 weeks. A sub-sample of participants completed pre-post blood 25(OH)D3 tests. Univariate ANOVA tested pre-post between-group differences in vitamin D intake and status. Repeated-measures ANOVA tested between-group differences in vitamin D knowledge and perceptions across 3 time-points.
Mean vitamin D intake in the sample increased significantly from pre-test (M = 407, SD = 460 IU) to post-test (M = 619, SD = 655 IU), t(88) = 5.37, p < 0.001. Mean intake increased significantly more in the intervention than control group after controlling for gender and education, F(1, 85) = 4.09, p = 0.046. Mean blood vitamin D3 was significantly higher among non-Caucasian than Caucasian participants at baseline, t(56.7) = 3.49, p = 0.001. Mean blood vitamin D3 increased significantly from pre-test (M = 28, SD = 16 nmol/L) to post-test (M = 43, SD = 29 nmol/L), t(53) = 11.36, p < 0.001, but did not differ significantly between groups. The increase in vitamin D knowledge from time 1-3 was significantly higher in the intervention than control group (t(88) = 2.26, p = 0.03). The intervention group (M = 3.52, SE = 0.13) had higher overall perceived importance of vitamin D supplementation than the control (M = 3.16, SE = 0.12), F(1, 88) = 4.38, p = 0.04, ηp(2) = 0.05.
Although recommendations suggest blood 25(OH)D3 concentrations of ≥50-75 nmol/L, vitamin D status was below national recommendations. While participating in an intervention did not improve vitamin D status, it led to increased vitamin D intake, knowledge and perceived importance of supplementation.
ClinicalTrails.gov registration #: NCT02118129 .
Preschools and childcare settings offer opportunities to promote adequate levels of physical activity. Research is needed to identify the key features of these settings to optimize young children's activity. The aims of this study were to determine if differences existed in preschool children's physical activity during care hours compared with outside care hours and to examine a comprehensive range of potential center-based correlates of physical activity for preschool boys and girls.
Data are from the Healthy Active Preschool and Primary Years study: 71 childcare centers, 65 preschools and 1002 preschool children. Percent of time in total (light- to vigorous-intensity) physical activity was measured using Actigraph GT1M accelerometers. Center physical environment characteristics, policies and practices were assessed by trained research staff using comprehensive audit tools. Data were collected in 2008/9 and were analyzed separately for boys and girls in Stata using multilevel mixed effects models.
Boys and girls were less active during care than outside care hours (51.1 % vs. 52.4 %, p = 0.01; 48.0 % vs. 51.5 %, p < 0.0001, respectively). In the final adjusted models, number of outdoor spaces with natural ground coverings was associated with boys' physical activity (coeff = 0.477, 95 % CI 0.089, 0.867) and the amount of time girls spent indoors before going outdoors was inversely associated with their physical activity (coeff = -0.035, 95 % CI -0.065, -0.004). The models explained 12 and 10 % of boys' and girls' physical activity during care hours, respectively.
This study identified that children are significantly less active during than outside care hours. Few center-based correlates of preschool children's physical activity were identified. Future research should explore other aspects of centers, such as what children actually do while they are outside, and broader potential influences on children's behaviours including social, cultural and policy contexts within which centers operate.
To systematically investigate the current status and methodology of health technology reassessment (HTR) in various countries to draw insights for the healthcare system in South Korea.
A systematic literature search was conducted on the articles published between January 2000 and February 2015 on Medline, EMBASE, the Cochrane Library, CINAHL, and PubMed. The titles and abstracts of retrieved records were screened and selected by two independent reviewers. Data related to HTR were extracted using a pre-standardised form. The review was conducted using narrative synthesis to understand and summarise the HTR process and policies.
Forty five studies, conducted in seven countries, including the United Kingdom, Australia, Canada, Spain, Sweden, Denmark, and the United States of America, fulfilled the inclusion criteria. Informed by the literature review, and complemented by informant interviews, we focused on HTR activities in four jurisdictions: the United Kingdom, Canada, Australia, and Spain. There were similarities in the HTR processes, namely the use of existing health technology assessment agencies, reassessment candidate technology identification and priority setting, stakeholder involvement, support for reimbursement coverage, and implementation strategies. Considering the findings of the systematic review in the context of the domestic healthcare environment in Korea, an appropriate HTR model was developed. This model included four stages, those of identification, prioritisation, reassessment and decision.
Disinvestment and reinvestment through the HTR was used to increase the efficiency and quality of care to help patients receive optimal treatment. Based on the lessons learnt from other countries' experiences, Korea should make efforts to establish an HTR process that optimises the National Healthcare Insurance system through revision of the existing Medical Service Act.
While the household context is important for lifestyle behavior interventions, few studies have examined parent-child associations for diet and physical activity (PA) changes over time in a rapidly urbanizing country. We aimed to investigate changes in diet, screen time, and PA behaviors over time in children and their parents living in the same household, and examine the parent-child association for these behaviors.
We studied dietary, screen time, and PA behaviors in 5,201 parent-child pairs (children aged 7-17y) using longitudinal data from the China Health and Nutrition Survey (1991, 1993, 1997, 2000, 2004, 2006, and 2009). We collected three-day 24-h recall diet data to generate percentages of energy from animal-source foods, away-from-home eating, and snacking from 1991-2009, which are known urbanization-related behaviors. We used a seven-day PA recall to collect screen time (hours/week) and leisure-time sports participation (yes/no) since 2004. We examined the changes in children's and parents' behaviors over time using random-effects negative binomial regression for diet and screen time, and random-effects logistic regression for leisure-time sports. We then regressed each of the behaviors of offspring on each of their parents' same behaviors to examine the parent-child association, using the same set of models.
We observed increases in energy from animal-source foods, eating away-from-home, and snacking, as well as screen time and leisure-time sports in parents and children over time, with different rates of change between children and their parents for some behaviors. We found positive parent-child associations for diet, screen time, and PA. When parental intakes increased by 10 % energy from each dietary behavior, children's increase in intakes ranged from 0.44 to 1.59 % total energy for animal-source foods, 0.17 % to 0.45 % for away-from-home eating, and 2.13 % to 7.21 % for snacking. Children were also more likely to participate in leisure-time sports if their parents participated in leisure-time sports.
Our findings support household-based health behavior interventions targeting both children and their parents. However, generation-specific intervention strategies may be needed for children and adults, especially for dietary behaviors, which changed differentially in children versus parents in this rapidly modernizing population.
Cachexia is defined as a complex metabolic syndrome associated with underlying illness and a loss of muscle with or without loss of fat mass. This disease is associated with high incidence with chronic diseases such as heart failure, cancer, chronic obstructive pulmonary disease, AIDS, among others. Since there is currently no effective treatment available, cachectic patients have a poor prognosis. Elucidation of the underlying mechanisms is therefore an important medical task. Recent Advances: There is accumulating evidence, that the diseased organs such as heart, lung, kidney or cancer tissue secrete soluble factors including Angiotensin II, myostatin (GDF8), GDF11, TGFβ, a.o., which act on skeletal muscle. There, they induce a set of genes called atrogenes which among others induce the ubiquitin-proteasome system, leading to protein degradation. Moreover, elevated ROS levels due to modulation of NADPH oxidases and mitochondrial function contribute to disease progression, which is characterized by loss of muscle mass, exercise resistance, and frailty.
With the identification of key components of the aberrant inter-organ communication leading to cachexia, studies in mice and men to inhibit cachexia demonstrated that inhibition of ROS formation, induction of anti-oxidative SOD´s, and upregulation of muscular NO formation either by pharmacological tools or by exercise are promising approaches to reduce the extent of skeletal muscle wasting.
Cachexia, which is frequently associated with chronic disease states, may represent an important target of anti-oxidative therapies, underscoring the need for specific tools to modulate cellular ROS formation.
Runt-related transcription factor 3 (RUNX3) is a tumor suppressor in many human solid tumors. In this study, renal cell carcinoma (RCC) microarray analysis showed that the level of RUNX3 expression was lower in RCC tissue than in adjacent normal renal tissues, and was correlated with depth of invasion (pT stage) (P<0.001) and Tumor Node Metastasis (TNM) stage (P<0.001). RUNX3 expression was negatively correlated with poor 5-year overall and disease-free patient survival. RUNX3 suppressed RCC metastasis and invasion and increased levels of E-cadherin, an important marker of epithelial-mesenchymal transition, in vitro and in vivo. RUNX3 also inhibited microRNA-6780a-5p, which directly targeted the E-cadherin 3'untranslated region and decreased its expression. We confirmed that miR-6780a-5p mimics abrogated RUNX3-mediated E-cadherin upregulation and RCC metastasis/invasion inhibition. Thus, RUNX3 targeted the miR-6780a-5p/E-cadherin/EMT signaling axis to suppress renal carcinoma cell migration and invasion. This pathway illustrates a new RUNX3 function and provides potential targets for the treatment of RUNX3 mutant and loss-of-function RCC tumors. RUNX3 may also act as an effective prognostic indicator in RCC.
Vascular calcification (VC) is a significant risk factor for cardiovascular morbidity and mortality. We recently reported that apocynin had benefits for preventing cardiovascular diseases. However, whether apocynin could attenuate VC is unknown. Here, we investigated the role of apocynin in VC and its underlying mechanisms. 163 participants with high or normal ankle-brachial index (ABI) were enrolled in this study for analyzing the demographic and biochemical data. In vitro, vascular smooth muscle cells (VSMCs) were exposed to calcification medium containing b-glycerophosphate and angiotensin II (Ang II) for 24 hours. The results showed that serum level of Ang II was significantly increased in patients with high ABI (P<0.05). In cultured VSMCs, Ang II significantly exacerbated osteogenic switching. The expression of osteogenic phenotype markers, including bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteopontin (OPN), were significantly upregulated, whereas contractile markers expression, including alpha smooth muscle actin (a-SMA) and smooth muscle 22 alpha (SM22a) were simultaneously downregulated. However, these effects were greatly attenuated by apocynin. Apocynin enhanced expression of a-SMA by 5.3%, and reduced expression of BMP2, Runx2, OPN by 3.37%, 0.61% and 3.07%, respectively. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was upregulated by Ang II, and this effect was also reversed by apocynin. Intriguingly, pretreatment with U0126, an inhibitor of ERK1/2, had similar effects with apocynin. Apocynin may act as a novel molecular candidate to protect against VSMCs osteogenic switching through suppressing ERK1/2 pathway.
Nitrogen-doped hierarchical porous carbons (NHPCs) with controllable nitrogen content were prepared via a template-free method by direct carbonization of melamine-resorcinol-terephthaldehyde networks. The synthetic approach is facile and gentle, resulting in a hierarchical pore structure with modest micropores and well-developed meso-/macropores, and allowing the easy adjusting of the nitrogen content in the carbon framework. The micropore structure was generated within the highly cross-linked networks of polymer chains, while the mesopore and macropore structure were formed from the interconnected 3D gel network. The as-prepared NHPC has a large specific surface area of 1150m(2)·g(-1), and a high nitrogen content of 14.5wt.%. CO2 adsorption performances were measured between 0°C and 75°C, and a high adsorption capacity of 3.96mmol·g(-1) was achieved at 1bar and 0°C. Moreover, these nitrogen-doped hierarchical porous carbons exhibit a great potential to act as electrode materials for supercapacitors, which could deliver high specific capacitance of 214.0F·g(-1) with an excellent rate capability of 74.7% from 0.1 to 10 A·g(-1). The appropriate nitrogen doping and well-developed hierarchical porosity could accelerate the ion diffusion and the frequency response for excellent capacitive performance. This kind of new nitrogen-doped hierarchical porous carbons with controllable hierarchical porosity and chemical composition may have a good potential in the future applications.
To determine what alternative pathways may act as mechanisms of bypass resistance to type 1 insulin-like growth factor receptor (IGF-1R) blockade in rhabdomyosarcoma (RMS), we compared expression of receptor tyrosine kinase activity in a number of IGF-1R antibody-resistant and -sensitive RMS cell lines. We found that platelet-derived growth factor receptor β (PDGFR-β) activity was upregulated in three xenograft-derived IGF-1R antibody-resistant cell lines that arose from a highly sensitive fusion-positive RMS cell line (Rh41). Furthermore, we identified four additional fusion-negative RMS cell lines that similarly upregulated PDGFR-β activity when selected for IGF-1R antibody resistance in vitro. In the seven cell lines described, we observed enhanced growth inhibition when cells were treated with dual IGF-1R and PDGFR-β inhibition in vitro. In vivo studies have confirmed the enhanced effect of targeting IGF-1R and PDGFR-β in several mouse xenograft models of fusion-negative RMS. These findings suggest that PDGFR-β acts as a bypass resistance pathway to IGF-1R inhibition in a subset of RMS. Therapy co-targeting these receptors may be a promising new strategy in RMS care.
In this work, hollow multiple-Ag-nanoclustes- C-shell nanocomposites (Ag@C) were synthesized by using silane coupling agent to graft carbon dots (CDs) with silver nanoparticles (AgNPs). CDs act as coating and stabilizing agent, protecting AgNPs from aggregation and oxidation. The resulting Ag@C nanocomposites demonstrate strong bactericidal effect against both gram-negative and gram-positive bacteria in the disk diffusion test. Cellular toxicity evaluation was performed using MTT assay. Meanwhile, the as-prepared Ag@C nanocomposites show a good biocompatibility.
This research aims to explore the neural correlates involved in altruistic punishment, parochial altruism and anti-social punishment, using the Third-Party Punishment (TPP) game. In particular, this study considered these punishment behaviors in in-group vs. out-group game settings, to compare how people behave with members of their own national group and with members of another national group. The results showed that participants act altruistically to protect in-group members. This study indicates that norm violation in in-group (but not in out-group) settings results in increased activity in the medial prefrontal cortex and temporo-parietal junction, brain regions involved in the mentalizing network, as the third-party attempts to understand or justify in-group members' behavior. Finally, exploratory analysis during anti-social punishment behavior showed brain activation recruitment of the ventromedial prefrontal cortex, an area associated with altered regulation of emotions.
The androgen receptor (AR) is not only a ligand-dependent transcription factor, but also functions as a licensing factor, a component of DNA replication, which is degraded during mitosis. Furthermore, the deregulation of AR activity is involved in the initiation of prostate cancer and contributes to castration resistant prostate cancer (CRPC). While AR degradation is known to occur primarily through a proteasome-mediated pathway, very little is known about how this process is regulated, especially in M phase. PC-1 is an androgen-responsive factor and expresses specificity in prostate cancer, with higher expression noted at G2/M. In this study, PC-1 was shown to interact with AR and E3 ligase CHIP (Carboxy-terminus of Hsc70 Interacting Protein) and to enhance AR/CHIP interactions, thereby decreasing AR stability. Moreover, PC-1 was found to act in conjunction with CHIP in the decreasing of AR via ubiquitination, with the subsequent degradation predominantly occurring during M phase. PC-1 was also found to repress AR transcriptional activity in androgen-dependent and androgen-independent prostate cancer cells and attenuate the growth inhibition of AR. In conclusion, these findings should provide new clues regarding the modulation of AR turnover and activity via PC-1 and reveals an essential role of PC-1 in AR signaling.
Long non-coding RNAs (lncRNAs) were shown to play critical roles in cancer biology. We investigated whether H. pylori infection could promote gastric cancer by regulating lncRNAs expression. Differentially expressed lncRNAs between H. pylori positive and negative tissues were identified by microarray and validated by qRT-PCR. Our results indicated that H. pylori positive tissues have a specific profile of lncRNAs. Cell biological assays with siRNA-mediated knockdown or lentivirus vector-mediated over-expression were performed to probe the functional relevance of the lncRNAs. We identified an lncRNA-AF147447 decreased expressed by H. pylori infection, which can inhibit GC proliferation and invasion in vitro and in vivo, act as a tumor suppressor in the development of H. pylori induced GC. LncRNA AF147447 could repress MUC2 expression by direct binding or increasing miR-34c expression. We also found that transcription factor E2F1 could be recruited to lncRNA AF147447 promoter by RNA immunoprecipatation and RNA pull down assays. These findings support a role of lncRNA AF147447 in tumor suppression. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by H. pylori infection and provides a rationale for the potential development of lncRNA-based targeted approaches for the treatment of H. pylori-related gastric cancer.
Resveratrol and curcumin are natural products with important therapeutic properties useful to treat several human diseases, including cancer. In the last years, the number of studies describing the effect of both polyphenols against cancer has increased; however, the mechanism of action in all of those cases is not completely comprehended. The unspecific effect and the ability to interfere in assays by both polyphenols make this challenge even more difficult. Herein, we analyzed the anticancer activity of resveratrol and curcumin reported in the literature in the last 11 years, in order to unravel the molecular mechanism of action of both compounds. Molecular targets and cellular pathways will be described. Furthermore, we also discussed the ability of these natural products act as chemopreventive and its use in association with other anticancer drugs.
Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences.
Viperin is an endoplasmic reticulum-associated antiviral responsive protein, which is highly up regulated in eukaryotic cells upon viral infection through both interferon-dependent and independent pathways. Viperin is predicted to be a radical S-adenosyl-L-methionine (SAM) enzyme, but it is unknown if viperin actually exploits radical SAM chemistry to exert its antiviral activity. We have investigated the interaction of viperin with its most firmly established cellular target, farnesyl pyrophosphate synthase (FPPS). Numerous enveloped viruses utilize cholesterol-rich lipid rafts to bud from the host cell membrane and it is thought that by inhibiting FPPS activity (and therefore cholesterol synthesis), viperin retards viral budding from infected cells. We demonstrate that, consistent with this hypothesis, over-expression of viperin in human embryonic kidney cells reduces the intracellular rate of accumulation of FPPS, but does not inhibit or inactivate FPPS. The endoplasmic reticulum-localizing, N-terminal amphipathic helix of viperin is specifically required for viperin to reduce cellular FPPS levels. However, although viperin reductively cleaves SAM to form 5-deoxyadenosine in a slow, uncoupled reaction characteristic of radical SAM enzymes, this cleavage reaction is independent of FPPS. Furthermore, mutation of key cysteinyl residues ligating the catalytic [Fe4S4] cluster in the radical SAM domain, surprisingly, does not abolish the inhibitory activity of viperin against FPPS; indeed some mutations potentiate viperin activity. These observations imply that viperin does not act as a radical SAM enzyme in regulating FPPS.
The small molecule metal-ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3-binding to CCR5, weak modulators of CCL4-binding, and as competitors for CCL5-binding. Here we describe their binding site using computational modeling, binding and functional studies on WT and mutated CCR5. The metal-ion Zn(2+) is anchored to the chemokine receptor-conserved E283(VII:06/7.39) Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the W248(VI:13/6.48) micro-switch, contributing to its 8-fold higher potency. The impact of W248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism, but maintained positive allosteric modulation of CCL3-binding. Despite a similar overall binding mode of all three metal-ion chelator complexes, the pyridine-ring of ZnClTerp blocks the conformational switch of W248 required for receptor activation, explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala-mutation of I116(III:16/3.40) - a residue that constrains the W248 micro-switch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site including residues Y37(I:07/1.39), W86(II:20/2.60) and F109(III:09/3.33) The small molecules and CCL3 approach this interface from opposite directions with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.
The goal of this paper is to demonstrate a novel approach that combines Empirical Mode Decomposition (EMD) with Notch filtering to remove the electrical stimulation (ES) artifact from surface electromyogram (EMG) data for interpretation of muscle responses during functional electrical stimulation (FES) experiments.
FES was applied to the rectus femoris (RF) muscle unilaterally of 6 able bodied (AB) and 1 individual with spinal cord injury (SCI). Each trial consisted of three repetitions of ES. We hypothesized that the EMD algorithm provides a suitable platform for decomposing the EMG signal into physically meaningful intrinsic mode functions (IMFs) which can be further used to isolate electrical stimulation (ES) artifact. A basic EMD algorithm was used to decompose the EMG signals collected during FES into IMFs for each repetition separately. IMFs most contaminated by ES were identified based on the standard deviation (SD) of each IMF. Each artifact IMF was Notch filtered to filter ES harmonics and added to remaining IMFs containing pure EMG data to get a version of a filtered EMG signal. Of all such versions of filtered signals generated from each artifact IMF, the one with maximum signal to noise ratio (SNR) was chosen as the final output. The validity of the filtered signal was assessed by quantitative metrics, a) root mean squared error (RMSE) and signal to noise (SNR) ratio values obtained by comparing a clean EMG and EMD-Notch filtered signal from the combination of simulated ES and clean EMG and, b) using EMG-force correlation analysis on the data collected from AB individuals. Finally, the potential applicability of this algorithm on a neurologically impaired population was shown by applying the algorithm on EMG data collected from an individual with SCI.
EMD combined with Notch filtering successfully extracted the EMG signal buried under ES artifact. Filtering performance was validated by smaller RMSE values and greater SNR post filtering. The amplitude values of the filtered EMG signal were seen to be consistent for three repetitions of ES and there was no significant difference among the repetition for all subjects. For the individual with a SCI the algorithm was shown to successfully isolate the underlying bursts of muscle activations during FES.
The data driven nature of EMD algorithm and its ability to act as a filter bank at different bandwidths make this method extremely suitable for dissecting ES induced EMG into IMFs. Such IMFs clearly show the presence of ES artifact at different intensities as well as pure artifact free EMG. This allows the application of Notch filters to IMFs containing ES artifact to further isolate the EMG. As a result of such stepwise approach, the extraction of EMG is achieved with minimal data loss.
This study provides a unique approach to dissect and interpret the EMG signal during FES applications.
Purpose In healthcare, organizational boundaries are often viewed as barriers to change. The purpose of this paper is to show how middle managers create inter-organizational change by doing boundary work: the dual act of redrawing boundaries and coordinating work in new ways. Design/methodology/approach Theoretically, the paper draws on the concept of boundary work from Science and Technology Studies. Empirically, the paper is based on an ethnographic investigation of middle managers that participate in a Dutch reform program across health, social care, and housing. Findings The findings show how middle managers create a sense of urgency for inter-organizational change by emphasizing "fragmented" service provision due to professional, sectoral, financial, and geographical boundaries. Rather than eradicating these boundaries, middle managers change the status quo gradually by redrawing composite boundaries. They use boundary objects and a boundary-transcending vocabulary emphasizing the need for societal gains that go beyond production targets of individual organizations. As a result, work is coordinated in new ways in neighborhood teams and professional expertise is being reconfigured. Research limitations/implications Since boundary workers create incremental change, it is necessary to follow their work for a longer period to assess whether boundary work contributes to paradigm change. Practical implications Organizations should pay attention to conditions for boundary work, such as legitimacy of boundary workers and the availability of boundary spaces that function as communities of practice. Originality/value By shifting the focus from boundaries to boundary work, this paper gives valuable insights into "how" boundaries are redrawn and embodied in objects and language.
Culturally appropriate, innovative strategies to increase physical activity (PA) in women of color are needed. This study examined whether participation in SALSA, an 8-week randomized, crossover pilot study to promote PA, led to improved psychosocial outcomes and whether these changes were associated with changes in PA over time. Women of color (N=50) completed internet-based questionnaires on PA, exercise self-efficacy, motivational readiness, stress and social support at three time points. Women reported high socioeconomic status and decreases in exercise self-efficacy and increases in motivational readiness for exercise and number of stressful events (p<.05); changes in motivational readiness for exercise varied by group (p=.043). Changes in psychosocial factors were associated with increases in PA. Latin dance improved motivational readiness for PA. Future studies are needed to determine whether Latin dance improves other psychological measures and quality of life in women of color in an effort to increase PA and reduce health disparities.
This secondary data analyses of a longitudinal study assessed whether self-efficacy for exercise (SEE) mediated an online intervention effects on exercise among older adults and whether age (50-64 vs. ≥ 65 years) moderated the mediation.
Data were from an online bone health intervention study. Eight hundred sixty-six older adults (≥ 50 years) were randomized to three arms: Bone Power (n = 301), Bone Power Plus (n = 302), or Control (n = 263). Parallel process latent growth curve modeling (LGCM) was used to jointly model growths in SEE and in exercise and to assess the mediating effect of SEE on the effect of intervention on exercise.
SEE was a significant mediator in 50- to 64-year-old adults (0.061, 95 BCI: 0.011, 0.163) but not in the ≥ 65 age group (-0.004, 95% BCI: -0.047, 0.025).
Promotion of SEE is critical to improve exercise among 50- to 64-year-olds.
Many older adults do not adhere to the recommended physical activity levels. This study examines the gait changes upon long-distance walking among healthy older adults. Gait tests of 24 adults aged 65 or more were conducted at the baseline, at the end of 30 and 60 minutes of treadmill walk. Spatial temporal, kinematic and kinetic gait data were computed. Perceived level of exertion was evaluated for each subject. Ten subjects (Group B) perceived higher exertion level than the remaining fourteen subjects (Group A). After walking, group B had significant reductions in dominant-side ankle joint range of motion and power, suggesting lower-leg muscle fatigue, which appeared to be compensated by significantly increased non-dominant side knee and hip motions. These changes were not observed in Group A. Differences in gait parameters between Group A and B implied that some biomechanical factors might contribute to the lack of walking of some older adults.
The purpose of this study was to determine the potential relationship between muscle power of the lower extremities and the physical activity in older men with chronic obstructive pulmonary disease (COPD). Forty-four men (70.3 ± 6.7 years old) with moderate-to-severe COPD completed the six-minute walk test (6MWT), BODE (body mass index, obstruction, dyspnea, and exercise), a one-repetition maximum strength of the quadriceps femoris (1RMQF), and muscle power at 50% and 70% 1RMQF. Physical activity was measured using an accelerometer. The 6MWT was associated with muscle power at 50% 1RMQF (r = 0.40; P = 0.013) but not muscle power at 70% 1RMQF (r = 0.24; P = 0.15) or 1RMQF (r = 0.13; P = 0.44). Light-intensity activity was positively correlated with muscle power at 50% 1RMQF (r = 0.52; P = 0.001). Lower limb muscle power is associated with the 6MWT and light-intensity activities in older men with COPD.
The FAT1 gene is involved in some cancers; however, its role in medulloblastoma is less clear. This study investigated the effects of FAT1 expression on the prognosis of medulloblastoma patients.
Whole exome sequencing was undertaken in 40 medulloblastoma patient samples. FAT1 mRNA and protein expression levels in normal and brain tumor tissues were determined by fluorescence quantitative PCR and immunohistochemistry, respectively. The association of FAT1 expression with overall survival (OS) was examined by Kaplan-Meier curve analysis with a log-rank test. Following lentiviral-mediated FAT1 knockdown using shRNA in Daoy cells, proliferation, Wnt signaling, and β-catenin protein expression were determined.
Eight FAT1 missense mutations were detected in 7 patients. FAT1 mRNA expression in tumors was significantly lower than in adjacent normal tissue (p = 0.043). The OS of patients with high FAT1 protein expression was significantly longer than that of patients with low FAT1 protein expression (median survival time: 24.3 vs 4.8 months, respectively; p = 0.002). shFAT1 cells had significantly higher proliferation rates than shControl cells (p≤0.028). Furthermore, the mRNA expression of LEF1, β-catenin, and cyclin D1 was significantly upregulated in shFAT1-Daoy cells (p≤0.018).
Low FAT1 expression was associated with poor prognosis in children with medulloblastoma. Furthermore, FAT1 may act on Wnt signaling pathway to exert its antitumor effect.
In many respects, employers are well positioned to take a leading role in helping create a culture of health. Employers have access to many programs that could be beneficial to their employees' health. The potential for financial gains related to health improvement may motivate employers to offer these programs, and if the gains are realized, they may help finance the programs. At the same time, employers' involvement in such programs may create substantial risks. Enthusiasm about the financial and health gains that wellness programs might yield coexists with concerns about health costs shouldered by employees, the possibility of employment discrimination, and the potential for employers' invasion of employees' privacy. A fragmented regulatory regime, including a recently issued final rule under the Americans with Disabilities Act, has been created to address these concerns. Whether the regime strikes the right balance between wellness program benefits and risks remains to be determined.
New care delivery models that hold providers more accountable for coordinated, high-quality care and the overall health of their patients have appeared in the US health care system, spurred by recent legislation such as the Affordable Care Act. These models support the integration of health care systems, but maximizing health and well-being for all individuals will require a broader conceptualization of health and more explicit connections between diverse partners. Integration of health services and systems constitutes the fourth Action Area in the Robert Wood Johnson Foundation's Culture of Health Action Framework, which is the subject of this article. This Action Area conceives of a strengthened health care system as one in which medical care, public health, and social services interact to produce a more effective, equitable, higher-value whole that maximizes the production of health and well-being for all individuals. Three critical drivers help define and advance this Action Area and identify gaps and needs that must be addressed to move forward. These drivers are access, balance and integration, and consumer experience and quality. This article discusses each driver and summarizes practice gaps that, if addressed, will help move the nation toward a stronger and more integrated health system.
VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa), and the shorter VirF21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21 The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.
Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF30 and VirF21, that are functionally distinct. The major form, VirF30, activates the genes necessary for virulence, whereas the minor VirF21, which shares the C-terminal two-thirds of VirF30, negatively autoregulates virF expression itself. VirF21 is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.
Piroxicam has been reported to be convertible to central nervous system (CNS) acting agents. It has serious depressant effects at high doses. In view of this structures of piroxicam metabolites were assessed for possible conversion to CNS depressants. Structural barbituric compounds, carboxamide, cyclohydrated, benzothiazone and carboxybenzothiazone metabolites which may act via dopamine and adrenergic receptors causing depression of CNS activities.
The periodontal therapies along with systemic antibiotic therapy aim at eliminating the subgingival microbiota to arrest the progression of periodontal diseases. The complete elimination is often difficult, and thus the probability of repopulation after periodontal therapy is also high. The objectives of the study are to develop in situ thermoreversible gelling system of green tea catechins suitable for periodontal pocket administration, which would act as an adjunct to mechanical periodontal therapy. Gel is prepared on a weight basis using a cold process. In vitro drug release pattern is observed through spectrophotometer analysis at 277 nm. The gel is subjected to serial dilution analysis to determine the minimum inhibitory concentration (MIC) and disc diffusion analysis to determine the in vitro antibacterial effectiveness. Release pattern studies showed a complete release of drug from gel occurred by 36 h. A volume of 1.25 mg/ml was determined as MIC required against the periodontal pathogens. Disc diffusion analysis showed a 14 mm zone of inhibition is present around the 75 µl well for all the four species and 12 mm zone of inhibition around the 50 µl well. The advantage of F-127 is its thermoreversible nature that used for in situ gel formulation. Pluronic gel proved to be a promising carrier for prolong and effective release of green tea catechin.
Maxillary segmental defects are a reconstructive challenge particularly when the anterior arch is involved. Missing bone, mucosa, and teeth should be replaced; fistulae closure should be guaranteed by bone and mucosa continuity; stable functional occlusion should be achieved; and facial aesthetics restored. These defects resulting from tumor excisions, if left untreated, will cause additional problems related to interposed scar and collapse of lip and nose. Immediate reconstruction should avoid these problems and should be considered when safe tumor-free margins are obtained. This study describes the treatment of such a defect resulting from a myxoma excision, with trifocal distraction requiring only one additional surgery. Internal distractors were fixed bilaterally for transport of two segmental discs created by inverted L osteotomies at the same surgical time of tumor excision. A second surgery was performed after central contact between transport discs was achieved, for device removal and creation of a dynamic system to regenerate molding and compression-focus development. This was accomplished by internal fixation of one side and central fixation of a full-open distractor to act as compression device when reversely activated. Central perfect adaptation between transported segments and good-quality attached mucosa lining the alveolar crest were determinant to obtain a stable functional and aesthetic result.
Long noncoding RNAs (lncRNAs) are a class of ubiquitous noncoding RNAs and have been found to act as tumor suppressors or oncogenes, which dramatically altered our understanding of cancer. Naked mole rat (NMR, Heterocephalus glaber) is an exceptionally long-lived and cancer-resistant rodent; however, whether lncRNAs play roles in cancer resistance in this seductive species remains unknown.
In this study, we developed a pipeline and identified a total of 4422 lncRNAs across the NMR genome based on 12 published transcriptomes. Systematic analysis revealed that NMR lncRNAs share many common characteristics with other vertebrate species, such as tissue specificity and low expression. BLASTN against with 1057 human cancer-related lncRNAs showed that only 5 NMR lncRNAs displayed homology, demonstrating the low sequence conservation between NMR lncRNAs and human cancer-related lncRNAs. Further correlation analysis of lncRNAs and protein-coding genes indicated that a total of 1295 lncRNAs were intensively coexpressed (r ≥ 0.9 or r ≤ -0.9, cP value ≤ 0.01) with potential tumor-suppressor genes in NMR, and 194 lncRNAs exhibited strong correlation (r ≥ 0.8 or r ≤ -0.8, cP value ≤ 0.01) with four high-molecular-mass hyaluronan related genes that were previously identified to play key roles in cancer resistance of NMR.
In this study, we provide the first comprehensive genome-wide analysis of NMR lncRNAs and their possible associations with cancer resistance. Our results suggest that lncRNAs may have important effects on anticancer mechanism in NMR.
Due to the impending depletion of fossil fuels, it has become important to identify alternative energy sources. The biofuel industry has proven to be a promising alternative. However, owing to the complex nature of plant biomass, hence the degradation, biofuel production remains a challenge. The copper-dependent Auxiliary Activity family 9 (AA9) proteins have been found to act synergistically with other cellulose-degrading enzymes resulting in an increased rate of cellulose breakdown. AA9 proteins are lytic polysaccharide monooxygenase (LPMO) enzymes, otherwise known as polysaccharide monooxygenases (PMOs). They are further classified as Type 1, 2 or 3 PMOs, depending on the different cleavage products formed. As AA9 proteins are known to exhibit low sequence conservation, the analysis of unique features of AA9 domains of these enzymes should provide insights for the better understanding of how different AA9 PMO types function.
Bioinformatics approaches were used to identify features specific to the catalytic AA9 domains of each type of AA9 PMO. Sequence analysis showed the N terminus to be highly variable with type-specific inserts evident in this region. Phylogenetic analysis was performed to cluster AA9 domains based on their types. Motif analysis enabled the identification of sub-groups within each AA9 PMO type with the majority of these motifs occurring within the highly variable N terminus of AA9 domains. AA9 domain structures were manually docked to crystalline cellulose and used to analyze both the type-specific inserts and motifs at a structural level. The results indicated that these regions influence the AA9 domain active site topology and may contribute to the regioselectivity displayed by different AA9 PMO types. Physicochemical property analysis was performed and detected significant differences in aromaticity, isoelectric point and instability index between certain AA9 PMO types.
In this study, a type-specific characterisation of AA9 domains was performed using various bioinformatics approaches. These highly variable proteins were found to have a greater degree of conservation within their respective types. Type-specific features were identified for AA9 domains, which could be observed at a sequence, structural and physicochemical level. This provides a basis under which to identify and group new AA9 LPMOs in future.
Large genome size and complexity hamper considerably the genomics research in relevant species. Rye (Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes.
Regret is an unpleasant feeling that may arise following decisions that ended poorly, and may affect the decision-maker's well-being and future decision making. Some studies show that a decision to act leads to greater regret than a decision not to act when both resulted in failure, because the latter is usually the norm. In some cases, when the norm is to act, this pattern is reversed. We suggest that the decision maker's regulatory focus, affects regret after action or inaction. Specifically, promotion-focused individuals, who tend to be more proactive, view action as more normal than prevention-focused individuals, and therefore experience regulatory fit when an action decision is made. Hence, we hypothesized that promotion-focused individuals will feel less regret than prevention-focused individuals when a decision to act ended poorly. In addition, we hypothesized that a trigger for change implied in the situation, decreases the level of regret following action. We tested our hypotheses on a sample of 330 participants enrolled in an online survey. The participants received six decision scenarios, in which they were asked to evaluate the level of regret following action and inaction. Individual regulatory focus was measured by two different scales. Promotion-focused individuals attributed less regret than prevention-focused individuals to action decisions. Regret following inaction was not affected by regulatory focus. In addition, a trigger for change decreases regret following action. Orthodox people tend to attribute more regret than non-orthodox to a person who made an action decision. The results contribute to the literature by showing that not only the situation but also the decision maker's orientation affects the regret after action vs. inaction.
Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αβ binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motor-impairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αβ or αβδ receptors lead to enhanced changes in the cerebellum-generated behavior.
Inflammatory bowel diseases (IBD) are chronic relapsing intestinal disorders characterized by up-regulation of pro-inflammatory cytokines followed by invasion of immune cells to the intestinal lamina propria. Standard therapies consist of anti-inflammatory or immunosuppressive drugs. Since clinical efficiency is not satisfactory and the established drugs have massive side effects, new strategies to treat IBD are required. Herein, we investigate the protective effect of the fixed combination herbal preparations STW5 and STW5-II and the contribution of the corresponding single components in an in vitro inflammation model. The normal human colon epithelial cell line, NCM460, was treated with STW5, STW5-II or their single components for 4 h followed by experimental conditions comparable to induction of colitis. A pro-inflammatory cytokine cocktail consisting of TNF-α, IL-β, and IFN-γ was used to simulate inflammatory stimuli normally caused by immune cells. The effects on NCM460 cells were investigated by enzyme-linked immunoassay and Proteome Profiler(®). Levels of IP-10, MCP-1, I-TAC, Groα, and IL-8 were elevated in chemokine-treated cells compared to untreated cells, but significantly reduced upon pretreatment with STW5 or STW5-II. However, the single compounds revealed only little effects on protein expression. Furthermore, we investigated the effect of both combination preparations on pro-inflammatory transcription factors of the STAT family using Western blot. In addition, we tested the effects on upstream MAPK p38. Both, STW5 and STW5-II did not show any effect on MAPK p38, but were effective in reducing phosphorylated levels of STAT1. In conclusion, both combination preparations act in an anti-inflammatory manner by influencing cytokine secretion via reduced activity of the JAK/STAT1 pathway. Relevant differences between STW5 and STW5-II were not found indicating similar efficacies.
Changes involving the health care economic landscape have affected physicians' workflow, productivity, compensation structures, and culture. Ongoing Federal legislation regarding regulatory documentation and imminent payment-changing methodologies have encouraged physician consolidation into larger practices, creating affiliations with hospitals, multidisciplinary medical specialties, and integrated delivery networks. As subspecialization and evolution of care models have accelerated, independent medical groups have broadened ancillary service lines by investing in enterprises that compete with hospital-based (academic and nonacademic) entities, as well as non-physician- owned multispecialty enterprises, for both outpatient and inpatient services. The looming and dramatic shift from volume- to value-based health care compensation will assuredly affect urology group compensation arrangements and productivity formulae. For groups that can implement change rapidly, efficiently, and harmoniously, there will be opportunities to achieve the Triple Aim goals of the Patient Protection and Affordable Care Act, while maintaining a successful medical-financial practice. In summary, implementing new payment algorithms alongside comprehensive care coordination will assist urology groups in addressing the health economic cost and quality challenges that have been historically encountered with fee-for-service systems. Urology group leadership and stakeholders will need to adjust internal processes, methods of care coordination, cultural dependency, and organizational structures in order to create better systems of care and management. In response, ancillary services and patient throughput will need to evolve in order to adequately align quality measurement and reporting systems across provider footprints and patient populations.
The Caribbean islands harbor rich biodiversity with high levels of single island endemism. Stretches of ocean between islands represent significant barriers to gene-flow. Yet some native species are widespread, indicating dispersal across oceans, even in wingless organisms like spiders. Argiope argentata (Fabricius, 1775) is a large, charismatic, and widespread species of orb-weaving spider ranging from the United States to Argentina and is well known to balloon. Here we explore the phylogeography of Argiope argentata in the Caribbean as a part of the multi-lineage CarBio project, through mtDNA haplotype and multi-locus phylogenetic analyses. The history of the Argiope argentata lineage in the Caribbean goes back 3-5 million years and is characterized by multiple dispersal events and isolation-by-distance. We find a highly genetically distinct lineage on Cuba which we describe as Argiope butchkosp. n. While the argentata lineage seems to readily balloon shorter distances, stretches of ocean still act as filters for among-island gene-flow as evidenced by distinct haplotypes on the more isolated islands, high F(ST) values, and strong correlation between intraspecific (but not interspecific) genetic and geographic distances. The new species described here is clearly genetically diagnosable, but morphologically cryptic, at least with reference to the genitalia that typically diagnose spider species. Our results are consistent with the intermediate dispersal model suggesting that good dispersers, such as our study species, limit the effect of oceanic barriers and thus diversification and endemism.
Nigeria's National Health Act 2014 (NHA 2014) was signed into law on October 31, 2014. It provides a legal framework for the regulation, development, and management of Nigeria's Health System. This study assessed the knowledge and perception of the NHA 2014 by health professionals.
This was a descriptive, cross-sectional, questionnaire-based study conducted in December 2015, in Ota, Ogun State, Nigeria. Data entry and analysis were done using the Statistical Package for the Social Sciences version 16 (IBM SPSS, Chicago, IL, USA) statistical software, with Pearson's Chi-square, which is used to determine the associations between variables. Statistical significance was set at a P < 0.05.
The study population comprised 130 health professionals (medical doctors/dentists, nurses, pharmacists, laboratory scientists, and other health-related professionals) in attendance at a medical conference. The respondents' age ranged from 21 to 75 years with a mean age of 44.53 ± 12.46 years. Medical practitioners accounted for 82.3% of the respondents. Although most (79.2%) respondents had a good perception of the NHA 2014 with majority (86.2%) claiming they were aware of the act, majority (73.8%) exhibited poor knowledge of the act. A little more than half (53.1%) of the respondents believed that the NHA 2014 will help to reduce strike actions in the health sector.
Although health professionals in Nigeria have good awareness and perception of the NHA 2014, their knowledge of the Act is poor.
Law governs the admission and management of involuntary admissions of mentally ill persons who are admitted under the provisions of the mental health act. The court directs the doctor to take charge of such persons. In the further dealings of such person the medical officer of the psychiatric facility comes across legal terms, which require understanding so that patients could be dealt with properly. Various terms such as accused, under police custody, judicial custody, remand prisoner, or under trial prisoner are used to denote their legal status. It is imperative for the medical officer to understand the nuances in the meanings of these terms. There are many times when the relevant section under which the admission is ordered is not found in the reception order. In these cases the terminology by which the patient is mentioned throws a light on the status of the patient. Towards this aim a study was carried out to assess the awareness and understanding of such terms by the faculty and post- graduates of a tertiary care hospital that deals with the admission and care of such patients.
They were administered a questionnaire containing these terms and asked to provide the meaning of these terms.
The results showed that nearly half the faculty and students were not having clarity in awareness or understanding of the terms.
Hence these terms and their meanings were gleaned from various judgments. The proper meaning of these terms and their use in judicial process and their importance is discussed.
Stephen Napier has argued against the soundness of what he calls the "Canon-Law argument" against the moral permissibility of a couple employing a condom for the sake of one spouse avoiding the contraction of HIV from the other spouse. Without an attempt to provide a full defense of the Canon-Law argument per se, this paper argues that Napier has not shown that argument to be inadequate. Napier's critique of that argument suffers from unsubstantiated counterexamples and from a failure to take into account analogous senses of "procreative end" in reference to the conjugal act. Using magisterial documents and canonical sources, this paper suggests that the distinction between validity and liceity can be usefully applied to conjugal acts. Lay Summary: Stephen Napier has argued in favor of the claim that there is no plausible argument for thinking that married couples who use a condom in order to prevent HIV transmission are necessarily doing something morally wrong. In responding to Napier by showing that his arguments in favor of his claim are inconclusive, this article introduces a distinction, frequently used in sacramental theology, between validity and liceity (lawfulness) and applies this distinction to marital actions. I argue that some modifications to martial actions make them merely unlawful, whereas other modifications-such as using an intact condom-make them both unlawful and "invalid."
Recent studies have identified that levonorgestrel administered orally in emergency contraception (LNG-EC) is only efficacious when taken before ovulation. However, the drug does not consistently prevent follicular rupture or impair sperm function.
The present systematic review is performed to analyze and more precisely define the extent to which pre-fertilization mechanisms of action may explain the drug's efficacy in pregnancy avoidance. We also examine the available evidence to determine if pre-ovulatory drug administration may be associated with post-fertilization effects.
The mechanism of action of LNG-EC is reviewed. The drug has no ability to alter sperm function at doses used in vivo and has limited ability to suppress ovulation. Our analysis estimates that the drug's ovulatory inhibition potential could prevent less than 15 percent of potential conceptions, thus making a pre-fertilization mechanism of action significantly less likely than previously thought. Luteal effects (such as decreased progesterone, altered glycodelin levels, and shortened luteal phase) present in the literature may suggest a pre-ovulatory induced post-fertilization drug effect.
Plan B is the most widely used emergency contraceptive available. It is important for patients and physicians to clearly understand the drug's mechanism of action (MOA). The drug was originally thought to work by preventing fertilization. Recent research has cast doubt on this. Our review of the research suggests that it could act in a pre-fertilization capacity, and we estimate that it could prevent ovulation in only 15 percent or less of cases. The drug has no ability to alter sperm function and limited ability to suppress ovulation. Further, data suggest that when administered pre-ovulation, it may have a post-fertilization MOA.
A number of long non-coding RNAs (lncRNAs) have been found to play critical roles in oncogenesis and tumor progression. We aimed to investigate whether lncRNAs could act as prognostic biomarkers for papillary thyroid cancer (PTC) that may assist us in evaluating disease status and prognosis for patients. We found 220 lncRNAs with expression alteration from the annotated 2773 lncRNAs approved by the HUGO gene nomenclature committee in The Cancer Genome Atlas (TCGA) dataset, of which FAM41C, CTBP1-AS2, LINC00271, HAR1A, LINC00310 and HAS2-AS1 were associated with recurrence. After adjusting classical clinicopathogical factors and BRAF(V600E) mutation, LINC00271 was found to be an independent risk factor for extrathyroidal extension, lymph node metastasis, advanced tumor stage III/IV and recurrence in multivariate analyses. Additionally, LINC00271 expression was significantly downregulated in PTCs versus adjacent normal tissues (P < 0.001). The Gene Set Enrichment Analysis (GSEA) revealed that genes associated with cell adhesion molecules, cell cycle, P53 signaling pathway and JAK/STAT signaling pathway were remarkably enriched in lower-LINC00271 versus higher-LINC00271 tumors. In conclusion, LINC00271 was identified as a possible suppressor gene in PTC in our study, and it may serve as a potential predictor of poor prognoses in PTC.
A big challenge is the discrimination of sulfhydryl-containing amino acids due to their structural similarity. We designed and synthesized a simple fluorescent probe 3 for specific detection of cysteine based on photo-induced electron transfer (PET). The acrylate and BODIPY moieties in probe 3 act as a reaction site and reporter group, respectively. So the synergistic effect of the substituent groups endows probe 3 very strong green fluorescence at 525nm (λex=500nm). The cleavage reaction induced by cysteine leads to acrylate hydrolysis, and thereby triggers PET on, which effectively quench the fluorescence of 3. Probe 3 exhibited a rapid response towards cysteine over homocysteine and glutathione. Probe 3 is successfully applied for sensing and imaging cysteine in vitro or in vivo cells with low cytotoxicity.
Theobromine, which is a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. Theobromine works as a phosphodiesterase (PDE) inhibitor to increase intracellular cyclic adenosine monophosphate (cAMP). cAMP activates the cAMP-response element-binding protein (CREB), which is involved in a large variety of brain processes, including the induction of the brain-derived neurotrophic factor (BDNF). BDNF supports cell survival and neuronal functions, including learning and memory. Thus, cAMP/CREB/BDNF pathways play an important role in learning and memory. Here, we investigated whether orally administered theobromine could act as a PDE inhibitor centrally and affect cAMP/CREB/BDNF pathways and learning behavior in mice. The mice were divided into two groups. The control group (CN) was fed a normal diet, whereas the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 30 days. We measured the levels of theobromine, phosphorylated vasodilator-stimulated phosphoprotein (p-VASP), phosphorylated CREB (p-CREB), and BDNF in the brain. p-VASP was used as an index of cAMP increases. Moreover, we analyzed the performance of the mice on a three-lever motor learning task. Theobromine was detectable in the brains of TB mice. The brain levels of p-VASP, p-CREB, and BDNF were higher in the TB mice compared with those in the CN mice. In addition, the TB mice performed better on the three-lever task than the CN mice did. These results strongly suggested that orally administered theobromine acted as a PDE inhibitor in the brain, and it augmented the cAMP/CREB/BDNF pathways and motor learning in mice.
Particular neutralizing monoclonal antibodies to certain cytokines act as agonists in vivo by protection of the cytokine's active site and prolongation of cytokine half-life. While this principle might be useful for targeted immunotherapy, its role in the pathogenesis of inflammation and autoimmunity is unclear.
We sought to determine whether slight, structurally non-relevant modifications of the prototypic pro-inflammatory cytokine interleukin-1β (IL-1β) during an immune response could elicit polyclonal anti-IL-1β antibody responses that modulated IL-1β's in vivo activity.
We engineered two different IL-1β variants, thereby mimicking the process of cytokine modification occurring during inflammation, and conjugated them to virus-like particles, followed by immunization of mice. The resulting polyclonal anti-IL-1β antibody responses were assessed using in vitro and in vivo assays as well as two relevant (auto-) inflammatory murine models.
While antibody responses generated to one variant were potently inhibiting IL-1β, antibody responses induced by the other variant even potentiated the in vivo effects of IL-1β; the latter led to enhanced morbidity in two different IL-1β-mediated mouse models, including a model of inflammatory bowel disease and an inflammatory arthritis model.
These data demonstrate that endogenous polyclonal anti-cytokine antibody responses can enhance the cytokine's activity in inflammatory and autoimmune diseases.
We studied the effects of extracellular ATP and Ca(2+) on uptake of bacteria (Staphylococcus aureus or Escherichia coli) and live yeast (Candida glabrata) by J774 macrophages to determine the role of endogenous P2X7 receptors in phagocytosis. Our findings show that phagocytosis of bio-particles coated with S. aureus or E. coli was blocked by ATP and the P2X7 receptor agonist BzATP, while yeast phagocytosis was not. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. To determine if P2X7-mediated Ca(2+) entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca(2+) with or without ATP. Ca(2+), in the absence of ATP, was required for engulfment of E. coli and C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus and E. coli regardless of Ca(2+), suggesting that Ca(2+) entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper-adherent C. glabrata mutants had an absolute requirement for extracellular Ca(2+) due to yeast adhesion to macrophages mediated by Ca(2+)-dependent adhesion proteins. We conclude that unstimulated P2X7 from J774 cells act as scavenger receptor for the uptake of S. aureus and E. coli but not of yeast; Ca(2+) entry via P2X7 receptors play no role in phagocytosis of S. aureus and E. coli; while the effect of Ca(2+) on C. glabrata phagocytosis was mediated by the adhesins Epa1, Epa6 and Epa7.
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs, also called PHDs and EglNs) are enzymes that act as cellular oxygen sensors. They are the main downregulators of the hypoxia-inducible factor (HIF). HIF-P4Hs can be targeted with small molecule inhibitors, which stabilize HIF under normoxia and initiate the hypoxia response. Such inhibitors are in phase 2 and 3 clinical trials for the treatment of anemia due to their ability to induce erythropoietin and iron metabolism genes. Recent data suggest that HIF-P4H inhibition has a therapeutic role beyond anemia in cardiac ischemia, obesity and metabolic dysfunction, and atherosclerosis. The molecular level mechanisms involved are HIF stabilization driven changes in gene expression that improve perfusion and endothelial function, reprogram metabolism to promote glucose intake and glycolysis over oxidative metabolism, reduce inflammation and beneficially modify innate immune system. This review discusses the recent findings in detail.
The principal mechanism by which bronchodilator β-adrenoceptor agonists act is to relax airways smooth muscle although they may also be anti-inflammatory. However, the extent of anti-inflammatory activity and the cell types affected by these agonists are uncertain. The purpose of this study was to evaluate whether β-adrenoceptor agonists prevent pro-inflammatory cytokine generation from activated human lung macrophages. Macrophages were isolated and purified from human lung. The cells were pre-treated with both short-acting (isoprenaline, salbutamol, terbutaline) and long-acting (formoterol, salmeterol, indacaterol) β-agonists before activation with lipopolysaccharide (LPS) to induce cytokine (TNFα, IL-6, IL-8 and IL-10) generation. The experiments showed that short-acting β-agonists were poor inhibitors of cytokine generation. Of the long-acting β-agonists studied, formoterol was also a weak inhibitor of cytokine generation whereas only indacaterol and salmeterol showed moderate inhibitory activity. Further experiments using the β2-adrenoceptor antagonist ICI-118,551 suggested that the effects of indacaterol were likely to be mediated by β2-adrenoceptors whereas those of salmeterol were not. These findings were corroborated by functional desensitization studies in which the inhibitory effects of indacaterol appeared to be receptor-mediated whereas those of salmeterol were not. Taken together, the data indicate that the anti-inflammatory effects of β-adrenoceptor agonists on human lung macrophages are modest.
Extremely hazardous substances can be released accidentally as a result of chemical spills, industrial explosions, fires, or accidents involving railroad cars and trucks transporting EHSs. Workers and residents in communities surrounding industrial facilities where these substances are manufactured, used, or stored and in communities along the nation's railways and highways are potentially at risk of being exposed to airborne EHSs during accidental releases or intentional releases by terrorists. Pursuant to the Superfund Amendments and Reauthorization Act of 1986, the U.S. Environmental Protection Agency (EPA) has identified approximately 400 EHSs on the basis of acute lethality data in rodents. Acute Exposure Guideline Levels for Selected Airborne Chemicals, Volume 20 reviews and updates the technical support document on acute exposure guideline levels (AEGLs) for selected chloroformates. This update focuses on establishing AEGL-3 values for n-propyl chloroformate and isopropyl chloroformate, but will also consider whether any new data are available that would affect the proposed values for the other 10 chloroformates. AEGLs represent threshold exposure limits (exposure levels below which adverse health effects are not likely to occur) for the general public and are applicable to emergency exposures ranging from 10 minutes (min) to 8 h. Three levels - AEGL-1, AEGL-2, and AEGL-3 - are developed for each of five exposure periods (10 min, 30 min, 1 h, 4 h, and 8 h) and are distinguished by varying degrees of severity of toxic effects. This report will inform planning, response, and prevention in the community, the workplace, transportation, the military, and the remediation of Superfund sites.
Carbapenems are beta-lactam antibiotics with a very broad spectrum of activity and act by disrupting the synthesis of bacterial cell walls. Others in the family of beta-lactam antibiotics are cephalosporins, which include cefepime and ceftazidime. In contrast, beta-lactamase inhibitors have little antibacterial activity alone. They include tazobactam and clavulanate. When used with beta-lactams however, the combination therapy provides enhanced and extended spectrum of activity against bacteria containing plasmid-mediated and chromosomal beta-lactamases. In the literature, the terminology “combination therapy” varies. For instance, the combination product piperacillin-tazobactam is often referred to as monotherapy unless combined with other agents such as amikacin or tobramycin. The growing problem of multi-drug resistance (MDR) worldwide has led to efforts in restricting the use of carbapenems. MDR and gram-negative bacterial infections are especially predominant in febrile neutropenia (FN), where over 50% of patients have an established or occult infection, and over 20% have bacteremia. If FN patients are not treated immediately, bacterial infections become rapidly fatal. In such patient populations, there are questions of how carbapenems fit in the context of care given the availability of alternative therapies such as beta-lactam/ beta-lactamase inhibitor combinations, and the different strategies of implementation such as escalation and de-escalation. Escalation begins with monotherapy and escalates to a regimen with a broader spectrum. De-escalation begins with a broad regimen and de-escalates to a regimen with a narrower spectrum after laboratory confirmation of the pathogen. The purpose of this report is to review evidence-based guidelines on the appropriate indications or circumstances for the use of carbapenems and other empiric therapies in the context of MDR infections and FN.
Bipolar disorder is a mental condition characterized by episodic mood swings between euphoric or irritable mania and hopeless depression which can affect social activities, functioning, and relationships. Episodes are typically followed by symptom-free periods referred to as euthymia. According to a survey conducted in 2002, one percent (1%) of Canadians 15 years and older demonstrated symptoms satisfying the criteria for bipolar disorder in the previous 12 months. Multiple types of bipolar disorders exist such as bipolar I, bipolar II and are defined in the Diagnostic and Statistical Manual (5(th) edition; DSM-V) from the American Psychiatric Association. Symptoms associated with mania can include increased creativity and productivity; however, mania can also lead to immediate hospitalization or involuntary committal under the Mental Health Act. Symptoms associated with depression can lead to increased rates of suicide and suicide ideation. Bipolar disorder with psychotic features refers to manic or depressive episodes including psychotic symptoms such as delusions or hallucinations. Psychotic features manifest in over 50% of manic episodes and are more common in the latter than in depressive episodes. Pharmacological treatment usually depends on the type of bipolar disorder (manic or depressive); however, the most common treatments include lithium and valproic acid. Antipsychotic medications are also used to treat bipolar disorder and can be classified as typical (first generation) or atypical (second generation). First generation antipsychotics mitigate bipolar disorder symptoms by antagonizing dopamine D2 receptors, while second generation antipsychotics have an affinity for serotonin 5-hydroxytryptamine receptors and adrenergic receptors in addition to being D2 receptor antagonists. Atypical antipsychotics, such as aripiprazole, olanzapine, quetiapine, risperidone and ziprasidone can be prescribed as monotherapy or in combination with mood stabilizers and antidepressants, as well as other treatment options. Although combinations of atypical antipsychotics have been used for the treatment of other disorders, such as schizophrenia, the effectiveness of combination therapy in bipolar disorder is unclear. This Rapid Response report aims to review the clinical and cost-effectiveness of combination atypical antipsychotics in adolescents or adults with bipolar disorder with psychotic features. Guidelines associated with the use of combination atypical antipsychotics in adolescents or adults with bipolar disorder with psychotic features will also be examined.
Recent health care payment reforms aim to improve the alignment of Medicare payment strategies with goals to improve the quality of care provided, patient experiences with health care, and health outcomes, while also controlling costs. These efforts move Medicare away from the volume-based payment of traditional fee-for-service models and toward value-based purchasing, in which cost control is an explicit goal in addition to clinical and quality goals. Specific payment strategies include pay-for-performance and other quality incentive programs that tie financial rewards and sanctions to the quality and efficiency of care provided and accountable care organizations in which health care providers are held accountable for both the quality and cost of the care they deliver. Accounting For Social Risk Factors in Medicare Payment: Data is the fourth in a series of five brief reports that aim to inform ASPE analyses that account for social risk factors in Medicare payment programs mandated through the IMPACT Act. This report provides guidance on data sources for and strategies to collect data on indicators of social risk factors that could be accounted for Medicare quality measurement and payment programs.
Gene expression control by microRNAs (miRs) is an important mechanism for maintenance of cellular homeostasis in physiological and pathological conditions as well as in response to different stimuli including nutritional factors and exercise. MiRs are involved in regulation of several processes such as growth and development, fuel metabolism, insulin secretion, immune function, miocardium remodeling, cell proliferation, differenciation, survival, and death. These molecules have also been proposed to be potential biomarkers and/or therapeutical targets in obesity, type 2 diabetes mellitus, cardiovascular diseases, metabolic syndrome, and cancer. MiRs are released by most cells and potentially act on intercellular communication to borderer or distant cells. Various studies have been performed to elucidate the involvement of miRs in exercise-induced effects. The aims of this review are: 1) to bring up the main advances for the comprehension of the mechanisms of action of miRs; 2) to present the main results on miR involvement in physical exercise; 3) to discuss the physiological effects of miRs modified by exercise. The state of the art and the perspectives on miRs associated with physical exercise will be presented. Thus, this review is important for updating recent advances and driving further strategies and studies on the exercise-related miR research.
Nicotine replacement therapy (NRT) is a common first-line treatment to prevent nicotine withdrawal in smokers. However, available literature reports conflicting results regarding its efficacy and safety in critically ill patients. The objective of this study was to evaluate the relationship between NRT in smokers in the intensive care unit (ICU) and outcomes. This case-control study was conducted in a university-affiliated tertiary hospital ICU. Over a period of five years, 126 active smokers who received transdermal NRT were matched to 126 active smokers who did not receive NRT. The groups were case-matched for sex, age and Acute Physiology and Chronic Health Evaluation II (APACHE II) score. The primary outcome was administration of antipsychotic medication. Secondary outcomes included use of physical restraints, 30-day mortality, and ventilation requirements. Antipsychotic medication was prescribed in 43 (34.1%) patients who received NRT compared to 14 (11.1%) in controls (<i>P</i> <0.01). Physical restraints were used in 37 (29.4%) patients who received NRT, compared to 12 (9.5%) of controls (<i>P</i> <0.01). The 30-day mortality and number of patients intubated was not statistically different between groups. Average length of intubation time was greater in the NRT group (2.56 days; standard deviation 4.16) compared to the control group (1.44 days; standard deviation 2.68) (<i>P</i>=0.012). The use of NRT to prevent nicotine withdrawal in ICU patients is associated with increased use of antipsychotic medication and physical restraint, and with prolonged mechanical ventilation.
Hematopoietic stem cell transplantation (HSCT) refers to therapies that aim to eliminate a patient's hematopoietic and immune system and replace it with his own (autologous) or someone else's (allogenic) system. The applications of this therapy are vast and growing, and include several malignant and benign diseases incurable by any other existing modalities. Pediatric patients constitute a minority of HSCT recipients with unique concerns. Despite substantial progress in the last two decades, limitations due to financial, infrastructural, manpower and research constraints act as barriers to fulfilling the large need for pediatric HSCT services in our country. Limited availability of unrelated donors and cord blood units is another constraint. Here in this oration, we discuss the current issues pertaining to pediatric HSCT in India and describe our experience with the same.
The use of parameters water equivalent diameter (D W ) and size-specific dose estimate (SSDE) are becoming increasingly established as a recognised method to relate patient dose from a CT examination to the dose indicator volume CT dose index (CTDIVOL). However, the role of the attenuation due to the patient table in these estimations requires careful consideration and is the subject of this study. The aim of this study is to investigate the impact of a minimal part of the patient table when calculating the D W and SSDE. We investigated 164 patients who had undergone CT examinations for the pelvis, abdomen, thorax and head. We subsequently calculated D W and SSDE using two methods: one using a small circular region of interest (ROI) including a minimal part of the patient table and the other using a ROI fitted to the patient border alone. The results showed that the water equivalent diameter calculated with the table included in the ROI (D W,t ) is greater, compared to that without the consideration of the patient table (D W,nt ), by 1.5-6.2% depending on the anatomy being imaged. On the other hand, the SSDE calculated with inclusion of the patient table (SSDEt) is smaller than otherwise (SSDEnt) by 1.0-5.5% again depending on the anatomy being imaged. The effect of the patient table on D W and SSDE in the thorax CT examination was statistically significant, but its effect on D W and SSDE in the other examinations of head, pelvis and abdomen was relatively small and not statistically significant.
In everyday communication, people often point. However, a pointing act is often misinterpreted as indicating a different spatial referent position than intended by the pointer. It has been suggested that this happens because pointers put the tip of the index finger close to the line joining the eye to the referent. However, the person interpreting the pointing act extrapolates the vector defined by the arm and index finger. As this line crosses the eye-referent line, it suggests a different referent position than the one that was meant. In this paper, we test this hypothesis by manipulating the geometry underlying the production and interpretation of pointing gestures. In Experiment 1, we compared naïve pointer-observed dyads with dyads in which the discrepancy between the vectors defining the production and interpretation of pointing acts has been reduced. As predicted, this reduced pointer-observer misunderstandings compared to the naïve control group. In Experiment 2, we tested whether pointers elevate their arms steeper than necessary to orient it toward the referent, because they visually steer their index finger tips onto the referents in their visual field. Misunderstandings between pointers and observers were smaller when pointers pointed without visual feedback. In sum, the results support the hypothesis that misunderstandings between (naïve) pointers and observers result from different spatial rules describing the production and interpretation of pointing gestures. Furthermore, we suggest that instructions that reduce the discrepancy between these spatial rules can improve communicating with pointing gestures.
Smoking is the most preventable and controllable health risk. Therefore, all health care professionals should give their utmost attention to and be more focused on the problem of smoking. Tobacco is a highly profitable product, because of its large-scale production and great number of consumers. Smoking control policies and treatment resources for smoking cessation have advanced in recent years, showing highly satisfactory results, particularly in Brazil. However, there is yet a long way to go before smoking can be considered a controlled disease from a public health standpoint. We can already perceive that the behavior of our society regarding smoking is changing, albeit slowly. Therefore, pulmonologists have a very promising area in which to work with their patients and the general population. We must act with greater impetus in support of health care policies and social living standards that directly contribute to improving health and quality of life. In this respect, pulmonologists can play a greater role as they get more involved in treating smokers, strengthening anti-smoking laws, and demanding health care policies related to lung diseases. RESUMO O tabagismo é o fator de risco mais prevenível e controlável em saúde e, por isso, precisa ter a máxima atenção e ser muito mais enfocado por todos os profissionais da saúde. O tabaco é um produto de alta rentabilidade pela sua grande produção e pelo elevado número de consumidores. As políticas de controle e os recursos terapêuticos para o tabagismo avançaram muito nos últimos anos e têm mostrado resultados altamente satisfatórios, particularmente no Brasil. Entretanto, ainda resta um longo caminho a ser percorrido para que se possa considerar o tabagismo como uma doença controlada sob o ponto de vista da saúde pública. Já se observam modificações do comportamento da sociedade com relação ao tabagismo, mas ainda em escala muito lenta, de modo que os pneumologistas têm nesse setor um campo muito promissor para atuar junto a seus pacientes e a população em geral. É preciso atuar com maior ímpeto em prol das políticas de saúde e das normas de convívio social que contribuem diretamente para melhorar a saúde e a vida. Nesse aspecto, os pneumologistas podem ter um papel de maior destaque na medida em que se envolvam com o tratamento dos fumantes, a aplicação da lei antifumo e as políticas de saúde relacionadas às doenças respiratórias.
Over the past few years, significant efforts have been made to decrease the effects of drought stress on plant productivity and quality. We propose that fullerenol nanoparticles (FNPs, molecular formula C60(OH)24) may help alleviate drought stress by serving as an additional intercellular water supply. Specifically, FNPs are able to penetrate plant leaf and root tissues, where they bind water in various cell compartments. This hydroscopic activity suggests that FNPs could be beneficial in plants. The aim of the present study was to analyse the influence of FNPs on sugar beet plants exposed to drought stress. Our results indicate that intracellular water metabolism can be modified by foliar application of FNPs in drought exposed plants. Drought stress induced a significant increase in the compatible osmolyte proline in both the leaves and roots of control plants, but not in FNP treated plants. These results indicate that FNPs could act as intracellular binders of water, creating an additional water reserve, and enabling adaptation to drought stress. Moreover, analysis of plant antioxidant enzyme activities (CAT, APx and GPx), MDA and GSH content indicate that fullerenol foliar application could have some beneficial effect on alleviating oxidative effects of drought stress, depending on the concentration of nanoparticles applied. Although further studies are necessary to elucidate the biochemical impact of FNPs on plants; the present results could directly impact agricultural practice, where available water supplies are often a limiting factor in plant bioproductivity.
BioEYES, a nonprofit outreach program using zebrafish to excite and educate K-12 students about science and how to think and act like scientists, has been integrated into hundreds of under-resourced schools since 2002. During the week-long experiments, students raise zebrafish embryos to learn principles of development and genetics. We have analyzed 19,463 participating students' pre- and post-tests within the program to examine their learning growth and attitude changes towards science. We found that at all grade levels, BioEYES effectively increased students' content knowledge and produced favorable shifts in students' attitudes about science. These outcomes were especially pronounced in younger students. Having served over 100,000 students, we find that our method for providing student-centered experiences and developing long-term partnerships with teachers is essential for the growth and sustainability of outreach and school collaborations.
Adopted in 2000, United Nations Millennium Development Goal 4 set a target to reduce child mortality by two thirds by 2015, with measles vaccination coverage as one of the progress indicators. In 2010, the World Health Assembly (WHA) set three milestones for measles control by 2015: 1) increase routine coverage with the first dose of measles-containing vaccine (MCV1) for children aged 1 year to ≥90% nationally and ≥80% in every district; 2) reduce global annual measles incidence to <5 cases per 1 million population; and 3) reduce global measles mortality by 95% from the 2000 estimate (1,2).* In 2012, WHA endorsed the Global Vaccine Action Plan(†) with the objective to eliminate measles in four World Health Organization (WHO) regions by 2015. Countries in all six WHO regions have adopted measles elimination goals. Measles elimination is the absence of endemic measles transmission in a region or other defined geographical area for ≥12 months in the presence of a well performing surveillance system. This report updates a previous report (3) and describes progress toward global measles control milestones and regional measles elimination goals during 2000-2015. During this period, annual reported measles incidence decreased 75%, from 146 to 36 cases per 1 million persons, and annual estimated measles deaths decreased 79%, from 651,600 to 134,200. However, none of the 2015 milestones or elimination goals were met. Countries and their partners need to act urgently to secure political commitment, raise the visibility of measles, increase vaccination coverage, strengthen surveillance, and mitigate the threat of decreasing resources for immunization once polio eradication is achieved.
We sought to identify factors associated with condom use during anal intercourse among self-identified human immunodeficiency virus-negative gay, bisexual, and other men who have sex with men (GBM) in Vancouver, Canada following "treatment as prevention" (TasP) scale-up in 2010.
Sexually active GBM were recruited using respondent-driven sampling from 2012 to 2014. We analyzed participants' most recent sexual encounter with up to their last 5 sexual partners within the past 6 months. In addition to individual- and event-level explanatory factors, we assessed potential associations with TasP awareness, TasP-related prevention practice (viral load sorting), and TasP-related attitudes (human immunodeficiency virus treatment optimism). Accounting for clustering at the respondent-driven sampling chain-level and participant-level, factors associated with event-level condom use versus nonuse were determined using a multivariable generalized linear mixed model built using backward selection and AIC minimization.
Of 513 participants, 436 GBM (85%) reported a total of 1196 anal sex events with 56% condom use. The proportion of condom-protected sexual events decreased monthly over the study period (odds ratio [OR], 0.95 per month, 95% confidence interval [CI], 0.92-0.98). The TasP practices and attitudes were significantly associated with lower odds of condom use at the univariate level, but were no longer significant at multivariate level. In the multivariable model, event-level partner methamphetamine use (adjusted OR [aOR], 0.18; 95% CI, 0.06-0.58), frequency of recent anal intercourse with that partner (aOR, 0.97 per act; 95% CI, 0.95-0.98) and time since first sex with that partner (aOR, 0.97 per 6 months; 95% CI, 0.95-0.99) were associated with lower odds of condom use, whereas event-level participant alcohol use (aOR, 1.41; 95% CI, 1.01-1.98) and no planned future sex with that partner (aOR, 1.56; 95% CI, 1.08-2.27) were associated with greater odds of condom use. Event-level receptive-only (aOR, 2.10; 95% CI, 1.38-3.20) or insertive-only (aOR, 2.53; 95% CI, 1.64-3.90) sexual positions were associated with greater odds of condom use compared with reporting both positions.
The TasP-related factors were not the most salient predictors of GBM's condom use. Health promotion must consider associations between condomless anal sex and substance use and relational factors.
Existing implantable stimulators use powering approaches that result in stiff and bulky systems or result in systems incapable of producing the current magnitudes required for neuromuscular stimulation. This hampers their use in neuroprostheses for paralysis. We previously demonstrated an electrical stimulation method based on electronic rectification of high frequency (HF) current bursts. The implants act as rectifiers of HF current that flows through the tissues by galvanic coupling, transforming this current into low frequency current capable of performing neuromuscular stimulation. Here we developed 2 mm thick, semi-rigid, injectable and addressable stimulators made of off-the-shelf components and based on this method. The devices were tested in vitro to illustrate how they are powered by galvanic coupling. In addition they were tried in an animal model to demonstrate their ability to perform controlled electrical stimulation. The implants were deployed by injection into two antagonist muscles of an anesthetized rabbit and were addressed resulting in independent isometric contractions. Low frequency currents of 2 mA were delivered by the implants. The HF currents are safe in terms of unwanted electrostimulation and tissue heating according to standards. This indicates that the proposed electrical stimulation method will allow unprecedented levels of miniaturization for neuroprostheses.
The quality of life (QOL) of caregivers of children with asthma may be related to children's responses to asthma management.
To evaluate change in QOL over time of caregivers of children with asthma through guideline-based management.
This was a 3-year prospective cohort study of children with asthma referred to our pediatric asthma center. Families completed Pediatric Asthma Caregiver's Quality of Life Questionnaire (PACQLQ), the Asthma Control Test™ (ACT), and reported the number of days/month of albuterol use and wheezing at each clinic visit.
We enrolled 143 children, ages 7-17 years (mean = 10.6±2.9), 56.6% male, 70.6% Caucasian. Patients were managed by the same MD (n = 65,45.5%) or APN (n = 78,54.5%) over time. The mean total PACQLQ significantly increased over the 3-year period (F = 67.418, p<.001). Total scores at the first visit were 4.8±1.6, which improved to 6.1±1 at the 3-month follow-up visit. This improvement was sustained at the 1, 2, and 3-year clinic visits. PACQLQ emotional function (F = 60.798, p<.001) and activity limitation (F = 41.517, p<.001) domains significantly improved as well. PACQLQ scores were significantly associated with improved ACT scores (r = .37 to .47, p<.05), fewer days/month of albuterol use (r = -.25 to -.36., p<.05), and wheezing (r = -.28 to -.33, p<.05). There were no significant differences in PACQLQ, or asthma clinical outcome measures between MD and APN providers.
Use of National Asthma Education and Prevention Program (NAEPP) guidelines significantly improved QOL of caregivers of children with asthma and in asthma-related symptoms. Improvements over time were independent of type of providers.
Viewed objects have been shown to afford suitable actions, even in the absence of any intention to act. However, little is known as to whether gaze behavior (i.e., the way we simply look at objects) is sensitive to action afforded by the seen object and how our actual motor possibilities affect this behavior. We recorded participants' eye movements during the observation of tools, graspable and ungraspable objects, while their hands were either freely resting on the table or tied behind their back. The effects of the observed object and hand posture on gaze behavior were measured by comparing the actual fixation distribution with that predicted by 2 widely supported models of visual attention, namely the Graph-Based Visual Saliency and the Adaptive Whitening Salience models. Results showed that saliency models did not accurately predict participants' fixation distributions for tools. Indeed, participants mostly fixated the action-related, functional part of the tools, regardless of its visual saliency. Critically, the restriction of the participants' action possibility led to a significant reduction of this effect and significantly improved the model prediction of the participants' gaze behavior. We suggest, first, that action-relevant object information at least in part guides gaze behavior. Second, postural information interacts with visual information to the generation of priority maps of fixation behavior. We support the view that the kind of information we access from the environment is constrained by our readiness to act. (PsycINFO Database Record
Research on stimulus-response (S-R) associations as the basis of behavioral automaticity has a long history. Traditionally, it was assumed that S-R associations are formed as a consequence of the (repeated) co-occurrence of stimulus and response, that is, when participants act upon stimuli. Here, we demonstrate that S-R associations can also be established in the absence of action. In an item-specific priming paradigm, participants either classified everyday objects by performing a left or right key press (task-set execution) or they were verbally presented with information regarding an object's class and associated action while they passively viewed the object (verbal coding). Both S-R associations created by task-set execution and by verbal coding led to the later retrieval of both the stimulus-action component and the stimulus-classification component of S-R associations. Furthermore, our data indicate that both associations created by execution and by verbal coding are temporally stable and rather resilient against overwriting. The automaticity of S-R associations formed in the absence of action reveals the striking adaptability of human action control. (PsycINFO Database Record
Sounds offer a rich source of information about events taking place in our physical and social environment. However, outside the domains of speech and music, little is known about whether humans can recognize and act upon the intentions of another agent's actions detected through auditory information alone. In this study we assessed whether intention can be inferred from the sound an action makes, and in turn, whether this information can be used to prospectively guide movement. In 2 experiments experienced and novice basketball players had to virtually intercept an attacker by listening to audio recordings of that player's movements. In the first experiment participants had to move a slider, while in the second one their body, to block the perceived passage of the attacker as they would in a real basketball game. Combinations of deceptive and nondeceptive movements were used to see if novice and/or experienced listeners could perceive the attacker's intentions through sound alone. We showed that basketball players were able to more accurately predict final running direction compared to nonplayers, particularly in the second experiment when the interceptive action was more basketball specific. We suggest that athletes present better action anticipation by being able to pick up and use the relevant kinematic features of deceptive movement from event-related sounds alone. This result suggests that action intention can be perceived through the sound a movement makes and that the ability to determine another person's action intention from the information conveyed through sound is honed through practice. (PsycINFO Database Record
Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized (35)S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be obtained. We also describe strategies to fortify the system with recombinant proteins to increase import yields and block specific parts of the machinery at a number of steps. The system recapitulates all the steps of the pathway, including mono-ubiquitination of PEX5L/S at the peroxisome membrane and its ATP-dependent export back into the cytosol by PEX1/PEX6. An in vitro import(/export) experiment can be completed in 24 h.
The present study investigates whether the chronic low-dose radiation exposure induces an in vivo radio-adaptive response in individuals from high-level natural radiation areas (HLNRA) of the Kerala coast. Peripheral blood samples from 54 adult male individuals aged between 26 and 65 years were collected for the study with written informed consent. Each of the whole blood sample was divided into three, one was sham irradiated, second and third was exposed to challenging doses of 1.0 and 2.0 Gy gamma radiation, respectively. Cytokinesis-block micronucleus (CBMN) assay was employed to study the radio-adaptive response. Seventeen individuals were from normal-level natural radiation area (NLNRA ≤1.5 mGy/year) and 37 from HLNRA (> 1.5 mGy/year). Based on the annual dose received, individuals from HLNRA were further classified into low-dose group (LDG, 1.51-5.0 mGy/year, N = 19) and high-dose group (HDG >5.0 mGy/year, N = 18). Basal frequency of micronucleus (MN) was comparable across the three dose groups (NLNRA, LDG and HDG, P = 0.64). Age of the individuals showed a significant effect on the frequency of MN after challenging dose exposures. The mean frequency of MN was significantly lower in elder (>40 years) individuals from HDG of HLNRA as compared to the young (≤40 years) individuals after 1.0 Gy (P < 0.001) and 2.0 Gy (P = 0.002) of challenging doses. However, young and elder individuals within NLNRA and LDG of HLNRA showed similar frequency of MN after the challenging dose exposures. Thus, increased level of chronic low-dose radiation (>5.0 mGy/year) seems to act as a priming dose resulting in the induction of an in vivo radio-adaptive response in elder individuals of the Kerala coast.
Alternative oxidase (AOX) is one of the terminal oxidases of the plant mitochondrial electron transport chain. AOX acts as a means to relax the highly coupled and tensed electron transport process in mitochondria thus providing and maintaining the much needed metabolic homeostasis by directly reducing oxygen to water. In the process AOX also act as facilitator for signalling molecules conveying the metabolic status of mitochondria to the nucleus and thus able to influence nuclear gene expression. Since AOX indirectly, is able to control the synthesis of important signalling molecules like hydrogen peroxide, superoxide, nitric oxide, thus it is also helping in stress signalling. AOX mediated signalling and metabolic activities are very much important for plant stress response. This include both biotic (fungal, bacterial, viral, etc.) and abiotic (drought, salinity, cold, heavy metal, etc.) stresses. The review provides a gist of regulation and functioning of AOX.
Ganoderma lucidum exerts antitumor activity, but the mechanism of G. lucidum polysaccharides on cancer is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduced tumor size and suppressed metastasis in vivo. Furthermore, FFLZ inhibited breast cancer cell migration and altered the epithelial-to-mesenchymal transition (EMT) phenotype. Transforming growth factor-β receptor (TGFR) pathways act as key mediators to promote tumor progression and metastasis. We found that FFLZ down-regulated TGFR and downstream signaling pathways, including the phosphorylation of Smad2/3 and the expression of Smad4. In an investigation of the underlying mechanisms, we found that FFLZ enhanced the Smurf2-dependent ubiquitination of TGFR by disrupting the balance of the lipid rafts, promoted the "re-localization" of the TGFR to the caveolae, and facilitated the degradation of TGFR. Together, our data indicated that FFLZ is associated with the inhibition of EMT and the prevention of metastasis by promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Moreover, the combination of FFLZ and trastuzumab synergistically inhibited the viability of certain trastuzumab-resistant human breast cancer cells. In summary, our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for cancer patients and that it functions via the caveolin-1/Smad7/Smurf2-dependent ubiquitin-mediated degradation of TGFR.
Monolayers of transition metal dichalcogenide materials emerged as a new material class to study excitonic effects in solid state, as they benefit from enormous Coulomb correlations between electrons and holes. Especially in WSe2, sharp emission features have been observed at cryogenic temperatures, which act as single photon sources. Tight exciton localization has been assumed to induce an anharmonic excitation spectrum; however, the evidence of the hypothesis, namely the demonstration of a localized biexciton, is elusive. Here we unambiguously demonstrate the existence of a localized biexciton in a monolayer of WSe2, which triggers an emission cascade of single photons. The biexciton is identified by its time-resolved photoluminescence, superlinearity and distinct polarization in micro-photoluminescence experiments. We evidence the cascaded nature of the emission process in a cross-correlation experiment, which yields a strong bunching behaviour. Our work paves the way to a new generation of quantum optics experiments with two-dimensional semiconductors.
Until recently, there was rather little interest in the structure-activity relationships (SARs) of cathinone analogs because so few agents were available and because they represented a relatively minor drug abuse problem. Most of the early SAR was formulated on the basis of behavioral (e.g., locomotor and drug discrimination) studies using rodents. With the emergence on the clandestine market in the last few years of a large number of new cathinone analogs, termed "synthetic cathinones", and the realization that they likely act at dopamine, norepinephrine, and/or serotonin transporters as releasing agents (i.e., as substrates) or reuptake inhibitors (i.e., as transport blockers), it has now become possible to better examine their SAR and even their quantitative SAR (QSAR), in a more effective and systematic manner. An SAR picture is beginning to emerge, and key structural features, such as the nature of the terminal amine, the size of the α-substituent, stereochemistry, and the presence and position of aromatic substituents, are being found to impact action (i.e., as releasing agents or reuptake inhibitors) and transporter selectivity.
For the purpose of clinical research of hepatocyte transplantation, procedures for isolation, cryopreservation, thawing, and functional assessment of hepatocytes are described. Although demands for human hepatocytes are increasing in not only cell therapy but also drug development, it is highly difficult to obtain good lots of hepatocytes from human liver tissue. This chapter describes essential issues such as alleviation of warm ischemia, prevention of shear stress, optimization of cryopreservation, and functional assessment, along with securement of quality. All procedures described here are compliant with good manufacturing procedure (GMP) in cell processing facility, approved by the act on measures to ensure safety of regenerative medicine and ethical regulations in Japan.
The tongue is a highly muscular organ, and the extrinsic muscles of the tongue overlap one another, which makes their configuration intricate. The aim was to clarify their spatial interrelationships.
The extrinsic muscles of the tongue were gross anatomically investigated in 42 cadavers.
The hyoglossus consisted of two parts arising from the hyoid body and the greater horn, respectively. They ascended to the tongue external to the genioglossus. The quadrilateral anterior part contained several parallel bundles, and the triangular posterior part comprised of three bundles converging to the insertion. The genioglossus consisted of many compartments. Although its fibers radiated from the mental spine to the tongue internal to the inferior longitudinal muscle, the lateral fibers of the inferior parts passed externally to it, and the most inferior part attached to the hyoid bone and the epiglottis. The chondroglossus arising from the lesser horn ascended internally to the lateral fibers of the inferior parts. The styloglossus arising from the styloid process coursed externally and internally to the hyoglossus and between its two parts. The external and internal bundles fused with the fibers of the palatoglossus and the superior pharyngeal constrictor, respectively. Three bundles were inserted into the apex with the inferior longitudinal muscle or joined the inferior parts of the genioglossus.
The extrinsic muscles are subdivided into small groups of fibers depending on their spatial interrelationships, suggesting that they contain many functional units. Some units act separately, and others cooperate across the traditional boundaries of the muscles.
To summarize recent research on unintended consequences associated with implementation and use of health information technology (health IT). Included in the review are original empirical investigations published in English between 2014 and 2015 that reported unintended effects introduced by adoption of digital interventions. Our analysis focuses on the trends of this steam of research, areas in which unintended consequences have continued to be reported, and common themes that emerge from the findings of these studies.
Most of the papers reviewed were retrieved by searching three literature databases: MEDLINE, Embase, and CINAHL. Two rounds of searches were performed: the first round used more restrictive search terms specific to unintended consequences; the second round lifted the restrictions to include more generic health IT evaluation studies. Each paper was independently screened by at least two authors; differences were resolved through consensus development.
The literature search identified 1,538 papers that were potentially relevant; 34 were deemed meeting our inclusion criteria after screening. Studies described in these 34 papers took place in a wide variety of care areas from emergency departments to ophthalmology clinics. Some papers reflected several previously unreported unintended consequences, such as staff attrition and patients' withholding of information due to privacy and security concerns. A majority of these studies (71%) were quantitative investigations based on analysis of objectively recorded data. Several of them employed longitudinal or time series designs to distinguish between unintended consequences that had only transient impact, versus those that had persisting impact. Most of these unintended consequences resulted in adverse outcomes, even though instances of beneficial impact were also noted. While care areas covered were heterogeneous, over half of the studies were conducted at academic medical centers or teaching hospitals.
Recent studies published in the past two years represent significant advancement of unintended consequences research by seeking to include more types of health IT applications and to quantify the impact using objectively recorded data and longitudinal or time series designs. However, more mixed-methods studies are needed to develop deeper insights into the observed unintended adverse outcomes, including their root causes and remedies. We also encourage future research to go beyond the paradigm of simply describing unintended consequences, and to develop and test solutions that can prevent or minimize their impact.
It is generally acknowledged that reactive oxygen species (ROS) play crucial roles in a variety of natural processes in cells. If increased to levels which cannot be neutralized by the defense mechanisms, they damage biological molecules, alter their functions, and also act as signaling molecules thus generating a spectrum of pathologies. In this review, we summarize current data on oxidative stress markers associated with human immunodeficiency virus type-1 (HIV-1) infection, analyze mechanisms by which this virus triggers massive ROS production, and describe the status of various defense mechanisms of the infected host cell. In addition, we have scrutinized scarce data on the effect of ROS on HIV-1 replication. Finally, we present current state of knowledge on the redox alterations as crucial factors of HIV-1 pathogenicity, such as neurotoxicity and dementia, exhaustion of CD4(+)/CD8(+) T-cells, predisposition to lung infections, and certain side effects of the antiretroviral therapy, and compare them to the pathologies associated with the nitrosative stress.
Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are both characterized by chronic low-grade inflammation. The role of Th17 and its related cytokines in T2DM and CAD is unclear. Here we investigated the serum levels of five Th17-related cytokines (IL-17, IL-22, MIP-3α, IL-9, and IL-27) in T2DM, CAD, and T2DM-CAD comorbidity patients. IL-22 was found to be elevated in all three conditions. Elevated serum IL-22 was independently associated with the incidence of T2DM and CAD. Conversely, IL-22 was found to protect endothelial cells from glucose- and lysophosphatidylcholine- (LPC-) induced injury, and IL-22R1 expression on endothelial cells was increased upon treatment with high glucose and LPC. Blocking of IL-22R1 with IL-22R1 antibody diminished the protective role of IL-22. Our results suggest that IL-22 functions as a double-edged sword in T2DM and CAD and that IL-22 may be used in the treatment of chronic inflammatory diseases such as T2DM and CAD.
Genetic polymorphisms in the endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 genes have been associated with several autoimmune diseases (AIDs) at a genome-wide significance level. In this study, we performed a cis expression quantitative trait locus (eQTL) screen to investigate whether seven fine-mapped AID single-nucleotide polymorphisms (SNPs) in the ERAP-region influence the gene-expression levels of ERAP1 and ERAP2 in thymus. After quality control, we identified six significant eQTLs. We further assessed the peak eQTL signals, and both genes showed highly significant and independent thymic eQTL signals (P=2.16 × 10(-15) and P=8.22 × 10(-23), respectively). Interestingly, the peak eQTL signal overlapped with the AID risk loci in ERAP2 (r(2)>0.94), but were distinct in ERAP1 (r(2)<0.4). Finally, among the SNPs showing the most significant eQTL associations with ERAP2 (P<3.4 × 10(-20)), six were located within transcription factor motifs in an enhancer region in thymus. Our study therefore reveals the fine-mapped AID risk variants that act as eQTLs with ERAP2 in thymus, and highlights the potential causal regulatory variants.
Fasting hypertriglyceridemia is positively associated with the morbidity of coronary heart disease (CHD), and postprandial (non-fasting) hypertriglyceridemia is also correlated with the risk status for CHD, which is related to the increase in chylomicron (CM) remnant lipoproteins produced from the intestine. CM remnant particles, as well as oxidized low density lipoprotein (LDL) or very low density lipoprotein (VLDL) remnants, are highly atherogenic and act by enhancing systemic inflammation, platelet activation, coagulation, thrombus formation, and macrophage foam cell formation. The cholesterol levels of remnant lipoproteins significantly correlate with small, dense LDL; impaired glucose tolerance (IGT) and CHD prevalence. We have developed an assay of apolipoprotein (apo)B-48 levels to evaluate the accumulation of CM remnants. Fasting apoB-48 levels correlate with the morbidity of postprandial hypertriglyceridemia, obesity, type III hyperlipoproteinemia, the metabolic syndrome, hypothyroidism, chronic kidney disease, and IGT. Fasting apoB-48 levels also correlate with carotid intima-media thickening and CHD prevalence, and a high apoB-48 level is a significant predictor of CHD risk, independent of the fasting TG level. Diet interventions, such as dietary fibers, polyphenols, medium-chain fatty acids, diacylglycerol, and long-chain n-3 polyunsaturated fatty acids (PUFA), ameliorate postprandial hypertriglyceridemia, moreover, drugs for dyslipidemia (n-3 PUFA, statins, fibrates or ezetimibe) and diabetes concerning incretins (dipeptidyl-peptidase IV inhibitor or glucagon like peptide-1 analogue) may improve postprandial hypertriglyceridemia. Since the accumulation of CM remnants correlates to impaired lipid and glucose metabolism and atherosclerotic cardiovascular events, further studies are required to investigate the characteristics, physiological activities, and functions of CM remnants for the development of new interventions to reduce atherogenicity.
Epidemiological and experimental data have indicated the beneficial and adverse effects of estrogenic replacement therapy. In the present study, we explored the effect of ethinylestradiol (EE) and 17β-estradiol (E2) on screening tests, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as the activity of coagulation factors (FVII, FX, FXI, and FXII) in male Wistar rats. Animals were injected subcutaneously during three consecutive days with EE or E2 (1, 3, 10, and 30 mg/kg) and propylene glycol (0.3 ml; vehicle, V). EE produced significant increments (P<0.05) on PT (8, 13, 15, and 10%) and APTT (32, 35, and 28%), whereas E2 did not show any effect. EE diminished the activity of factors VII (-10, -13, and -10%) and X (-10, -9, -15, and -14%; P<0.05), and E2 (1 mg/kg) produced a modest increment (8%; P<0.05) on FX only. E2 (10 mg/kg) showed a diminution of 9% (P<0.05), while EE did not produce any response on factor XII. EE diminished (-15, -14, -19, and -17%) but E2 augmented (10, 14, 24, and 24%) factor XI activity (P<0.05). Our findings suggest that EE and E2 produce different effects on coagulation and that EE seems to act across an inhibitory mechanism of coagulation factor activity in the present experimental model.
MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators. They are involved in the pathogenesis of different disorders including heart diseases. MiRNAs contribute to ischemia/reperfusion injury (I/RI) by altering numerous key signaling elements. Together with alterations in the various potential signaling pathways, modification in miRNA expression has been suggested as a part of the response network following ischemia/reperfusion (I/R). In addition, cardiac mitochondrial homeostasis is closely associated with cardiac function and impairment of mitochondrial activity occurred after ischemia/reperfusion injury. MiRNAs play a key role in the regulation of mitochondrial apoptotic pathway and signaling proteins. In this review, we summarize the knowledge currently available regarding the molecular mechanisms of miRNA-regulated mitochondrial functions during ischemia/reperfusion injury. This regulation occurs in different stages of mitochondrial apoptosis pathway.
Accumulating evidence has indicated that aberrantly expressed microRNAs (miRs) are extensively involved in cancer development and progression. MiR-639 has been reported to act as tumor promoter in various types of cancer. However, the biological function and underlying molecular mechanism of miR-639 in thyroid carcinoma (TC) have not been intensively investigated. Herein the present study aimed to investigate the functional role of miR-639 in TC. We found that miR-639 expression was upregulated in TC cells and clinical tissues. Overexpression of miR-639 promoted TC cell proliferation and cell cycle, with increased expression of CyclinE and c-myc, whereas miR-639-in reverses the function. Using prediction software and luciferase reporter assay, we found that CDKN1A was a target of miR-639. CDKN1A small interfering RNA (siRNA) abrogated the role of miR-639-in on cell proliferation of TC. In summary, our data demonstrated that miR-639 upregulation was associated with development of TC, miR-639 promoted cell proliferation and cell cycle by targeting CDKN1A in TC.
According to the US Affordable Care Act, restaurant chains are required to provide energy (calorie) and other nutrition information on their menu. The current study examined the impact of menu labelling containing calorie information and recommended daily calorie intake, along with subjective nutrition knowledge, on intention to select lower-calorie foods prior to the implementation of the Affordable Care Act.
Full factorial experimental design with participants exposed to four variants of a sample menu in a 2 (presence v. absence of calorie information) ×2 (presence v. absence of recommended daily calorie intake).
Large, public university in the Southwest USA.
Primarily undergraduate college students.
Majority of participants were 19-23 years of age (mean 21·8 (sd 3·6) years). Menu information about calorie content and respondents' subjective nutrition knowledge had a significantly positive impact on students' intention to select lower-calorie foods (β=0·24, P<0·001 and β=0·33, P<0·001, respectively); however, recommended daily calorie intake information on the menu board did not influence students' intention to select lower-calorie foods (β=0·10, P=0·105). Gender played a significant role on purchase intent for lower-calorie menu items, with females more affected by the calorie information than males (β=0·37, P<0·001).
Findings support the role menu labelling can play in encouraging a healthier lifestyle for college students. College students who are Generation Y desire healthier menu options and accept nutritional labels on restaurant menus as a way to easily and expediently obtain nutrition information.
The class Tremellomycete (Agaricomycotina) encompasses more than 380 fungi. Although there are a few edible Tremella spp., the only species with current biotechnological use is the astaxanthin-producing yeast Phaffia rhodozyma (Cystofilobasidiales). Besides astaxanthin, a carotenoid pigment with potent antioxidant activity and great value for aquaculture and pharmaceutical industries, P. rhodozyma possesses multiple exceptional traits of fundamental and applied interest. The aim of this study was to obtain, and analyze two new genome sequences of representative strains from the northern (CBS 7918(T), the type strain) and southern hemispheres (CRUB 1149) and compre them to a previously published genome sequence (strain CBS 6938). Photoprotection and antioxidant related genes, as well as genes involved in sexual reproduction were analyzed.
Both genomes had ca. 19 Mb and 6000 protein coding genes, similar to CBS 6938. Compared to other fungal genomes P. rhodozyma strains and other Cystofilobasidiales have the highest number of intron-containing genes and highest number of introns per gene. The Patagonian strain showed 4.4 % of nucleotide sequence divergence compared to the European strains which differed from each other by only 0.073 %. All known genes related to the synthesis of astaxanthin were annotated. A hitherto unknown gene cluster potentially responsible for photoprotection (mycosporines) was found in the newly sequenced P. rhodozyma strains but was absent in the non-mycosporinogenic strain CBS 6938. A broad battery of enzymes that act as scavengers of free radical oxygen species were detected, including catalases and superoxide dismutases (SODs). Additionally, genes involved in sexual reproduction were found and annotated.
A draft genome sequence of the type strain of P. rhodozyma is now available, and comparison with that of the Patagonian population suggests the latter deserves to be assigned to a distinct variety. An unexpected genetic trait regarding high occurrence of introns in P. rhodozyma and other Cystofilobasidiales was revealed. New genomic insights into fungal homothallism were also provided. The genetic basis of several additional photoprotective and antioxidant strategies were described, indicating that P. rhodozyma is one of the fungi most well-equipped to cope with environmental oxidative stress, a factor that has probably contributed to shaping its genome.
A basic task in drug discovery is to find new medication in the form of candidate compounds that act on a target protein. In other words, a drug has to interact with a target and such drug-target interaction (DTI) is not expected to be random. Significant and interesting patterns are expected to be hidden in them. If these patterns can be discovered, new drugs are expected to be more easily discoverable. Currently, a number of computational methods have been proposed to predict DTIs based on their similarity. However, such as approach does not allow biochemical features to be directly considered. As a result, some methods have been proposed to try to discover patterns in physicochemical interactions. Since the number of potential negative DTIs are very high both in absolute terms and in comparison to that of the known ones, these methods are rather computationally expensive and they can only rely on subsets, rather than the full set, of negative DTIs for training and validation. As there is always a relatively high chance for negative DTIs to be falsely identified and as only partial subset of such DTIs are considered, existing approaches can be further improved to better predict DTIs. In this paper, we present a novel approach, called ODT (one class drug target interaction prediction), for such purpose. One main task of ODT is to discover association patterns between interacting drugs and proteins from the chemical structure of the former and the protein sequence network of the latter. ODT does so in two phases. First, the DTI-network is transformed to a representation by structural properties. Second, it applies a one-class classification algorithm to build a prediction model based only on known positive interactions. Performance evaluation of ODT shows that it can be potentially useful. It confirms that predicting potential or missing DTIs based on the known interactions is a promising direction to solve problems related to the use of uncertain and unreliable negative samples and those related to the great demand in computational resources.
The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of β-thalassemia (β-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of β-thal major (β-TM) and moderate type of β-thal intermedia (β-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% β-TI patients, which was not the case in the β-TM patients or in the control group. Thus, the T allele is consequently associated with the β-TI group (p = 10(-3)). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10(-3)) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of β-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.
The basement membrane is crucial for cell polarity, adhesion, and motility, but how it is assembled on the cell surface remains unclear. Here, we find that ablation of glycosaminoglycan (GAG) side chains of proteoglycans in the neuroretina disrupts the retinal basement membrane, leading to arrested astrocyte migration and reduced angiogenesis. Using genetic deletion and time-lapse imaging, we show that retinal astrocytes require neuronal-derived PDGF as a chemoattractive cue and the retinal basement membrane as a migratory substrate. Genetic ablation of heparan sulfates does not produce the same defects as GAG null mutants. In contrast, enzymatic removal of heparan sulfates and chondroitin sulfates together inhibits de novo laminin network assembly. These results indicate that both heparan and chondroitin sulfate proteoglycans participate in retinal basement membrane assembly, thus promoting astrocyte migration and angiogenesis.
Hypothalamic oxytocin (OXT) and arginine vasopressin (AVP) are known to act oppositely on hypothalamic-pituitary-adrenal (HPA) axis, stress response and gastrointestinal (GI) motility. In rodents, exposure to restraint stress (RS) delays gastric emptying (GE), however, repeated exposure to the same stressor (chronic homotypic stress (CHS)), the delayed GE is restored to basal level, while hypothalamic OXT is upregulated. In contrast, when rats are exposed to chronic heterotypic stress (CHeS), these adaptive changes are not observed. Although the involvement of central OXT in gastric motor adaptation is partly investigated, the role of hypothalamic AVP in CHeS-induced maladaptive paradigm is poorly understood. Using in-vivo brain microdialysis in rats, the changes OXT and AVP release from hypothalamus were monitored under basal non-stressed (NS) conditions and in rats exposed to acute stress (AS), CHS and CHeS. To investigate the involvement of central endogenous OXT or AVP in CHS-induced habituation and CHeS-induced maladaptation, chronic central administration of selective OXT receptor antagonist L-371257 and selective AVP V1b receptor antagonist SSR-149415 was performed daily. OXT was measured higher in AS and CHS group, but not in CHeS-loaded rats, whereas AVP significantly increased in rats exposed to AS and CHeS. Additionally, the response of the hypothalamic OXT- and AVP-producing cells was amplified following CHS and CHeS, respectively. In rats exposed to AS for 90min solid GE significantly delayed. The delayed-GE was completely restored to the basal level following CHS, however, it remained delayed in CHeS-loaded rats. The CHS-induced restoration was prevented by L-371257, whereas SSR-149415 abolished the CHeS-induced impaired GE. A significant correlation was observed between GE and (i) OXT in CHS-loaded rats (rho=0.61, p<0.05, positively), (ii) AVP in CHeS-loaded rats (rho=0.69, p<0.05, negatively). Under long term stressed conditions, the release of AVP and OXT from hypothalamus may vary depending on the content of the stressors. Central AVP appears to act oppositely to OXT by mediating CHeS-induced gastric motor maladaptation. Long term central AVP antagonism might be a pharmacological approach for the treatment of stress-related gastric motility disorders.
Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.
We simulate the formation of spontaneous ruptures in supported phospholipid double bilayer membranes, using peridynamic modeling. Experiments performed on spreading double bilayers typically show two distinct kinds of ruptures, floral and fractal, which form spontaneously in the distal (upper) bilayer at late stages of double bilayer formation on high energy substrates. It is, however, currently unresolved which factors govern the occurrence of either rupture type. Variations in the distance between the two bilayers, and the occurrence of interconnections ("pinning sites") are suspected of contributing to the process. Our new simulations indicate that the pinned regions which form, presumably due to Ca2+ ions serving as bridging agent between the distal and the proximal bilayer, act as nucleation sites for the ruptures. Moreover, assuming that the pinning sites cause a non-zero shear modulus, our simulations also show that they change the rupture mode from floral to fractal. At zero shear modulus the pores appear to be circular, subsequently evolving into floral pores. With increasing shear modulus the pore edges start to branch, favoring fractal morphologies. We conclude that the pinning sites may indirectly determine the rupture morphology by contributing to shear stress in the distal membrane.
Western boundary currents-such as the Agulhas Current in the Indian Ocean-carry heat poleward, moderating Earth's climate and fuelling the mid-latitude storm tracks. They could exacerbate or mitigate warming and extreme weather events in the future, depending on their response to anthropogenic climate change. Climate models show an ongoing poleward expansion and intensification of the global wind systems, most robustly in the Southern Hemisphere, and linear dynamical theory suggests that western boundary currents will intensify and shift poleward as a result. Observational evidence of such changes comes from accelerated warming and air-sea heat flux rates within all western boundary currents, which are two or three times faster than global mean rates. Here we show that, despite these expectations, the Agulhas Current has not intensified since the early 1990s. Instead, we find that it has broadened as a result of more eddy activity. Recent analyses of other western boundary currents-the Kuroshio and East Australia currents-hint at similar trends. These results indicate that intensifying winds may be increasing the eddy kinetic energy of boundary currents, rather than their mean flow. This could act to decrease poleward heat transport and increase cross-frontal exchange of nutrients and pollutants between the coastal ocean and the deep ocean. Sustained in situ measurements are needed to properly understand the role of these current systems in a changing climate.
The End of Life Option Act in California, effective June 9, 2016, permits physicians to prescribe lethal medication to patients confirmed to be terminally ill and capable of independently making and carrying out a decision to ingest deadly medication. Medicine has traditionally excluded the provision of deadly medication from proper practice. Physicians reasonably may hold to that limit. However, honoring a repeated request from a capable, terminally ill patient to receive life-ending medication still can be considered to be a moral and permissible approach to relieve suffering. A physician choosing to expand his/her role within this narrowly defined context allows the patient to assume authority for a deeply personal decision that may belong to the patient more than to anyone else.
Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) lack robust diagnostics and prognostic biomarkers. Metabolomics is a postgenomics field that offers fresh insights for biomarkers of common complex as well as rare diseases. Using data on metabolite-disease associations published in the previous decade (2006-2016) in PubMed, ScienceDirect, Scopus, and Web of Science, we identified 101 metabolites as putative biomarkers for these three neurodegenerative diseases. Notably, uric acid, choline, creatine, L-glutamine, alanine, creatinine, and N-acetyl-L-aspartate were the shared metabolite signatures among the three diseases. The disease-metabolite-pathway associations pointed out the importance of membrane transport (through ATP binding cassette transporters), particularly of arginine and proline amino acids in all three neurodegenerative diseases. When disease-specific and common metabolic pathways were queried by using the pathway enrichment analyses, we found that alanine, aspartate, glutamate, and purine metabolism might act as alternative pathways to overcome inadequate glucose supply and energy crisis in neurodegeneration. These observations underscore the importance of metabolite-based biomarker research in deciphering the elusive pathophysiology of neurodegenerative diseases. Future research investments in metabolomics of complex diseases might provide new insights on AD, PD, and ALS that continue to place a significant burden on global health.
Synthetic surfactants represent a promising alternative to animal-derived preparations in the treatment of neonatal respiratory distress syndrome. The synthetic surfactant CHF5633 has proven biophysical effectiveness and, moreover, demonstrated anti-inflammatory effects in LPS-stimulated monocytes. With ureaplasmas being relevant pathogens in preterm lung inflammation, the present study addressed immunomodulatory features on Ureaplasma-induced monocyte cytokine responses.
Ureaplasma parvum-stimulated monocytes were exposed to CHF5633. TNF-α, IL-1β, IL-8, IL-10, TLR2 and TLR4 expression were analyzed using qPCR and flow cytometry.
CHF5633 did not induce pro-inflammation, and did not aggravate Ureaplasma-induced pro-inflammatory cytokine responses. It suppressed U. parvum-induced intracellular TNF-α (p < 0.05) and IL-1β (p < 0.05) in neonatal monocytes and inhibited Ureaplasma-induced TNF-α mRNA (p < 0.05), TNF-α protein (p < 0.001), and IL-1β (p = 0.05) in adult monocytes. Ureaplasma-modulated IL-8, IL-10, TLR2 and TLR4 were unaffected.
CHF5633 does neither act pro-apoptotic nor pro-inflammatory in native and Ureaplasma-infected monocytes. Suppression of Ureaplasma-induced TNF-α and IL-1β underlines anti-inflammatory features of CHF5633.
Numerous investigations, and especially in vitro studies, indicate that TGF-β1 may act as an important regulator of bone remodelling. Thus, it could be expected that disturbances of this cytokine production observed by several researchers might play a role in the mechanism leading to the development of osteoporosis in girls with anorexia nervosa (AN). The aim of the study was to determine whether 1) girls with AN exhibited a relationship between TGF-β1 and bone metabolism (as assessed based on serum OC and CTx concentrations) and 2) whether OPG and sRANKL might modify the possible relationship between TGF-β1 and bone metabolism.
Serum concentrations of TGF-β, OC, CTx, OPG, and its soluble ligand sRANKL were determined by ELISA in 60 girls with AN and in 20 healthy controls (C). All study participants were aged 13 to 17 years.
Body weight, BMI, BMI-SDS and the Cole index, serum TGF-β1, OC, CTx, and the OPG/sRANKL ratio were significantly reduced, while OPG and sRANKL levels were significantly increased, in girls with AN compared to healthy participants. BMI and the Cole index correlated negatively and significantly with serum CTx and OPG (AN group) or CTx only (groups C and C + AN). Girls with AN showed a positive and significant correlation between the Cole index and serum TGF-β1. The combination group (C + AN) showed a positive and significant correlation between BMI, the Cole index, and the OPG/sRANKL ratio and TGF-β1 concentration, while TGF-β1 correlated positively and significantly with OC concentrations and the OPG/sRANKL ratio. The Cole index and BMI were identified to be significant and independent predictors of CTx (C, AN, and C+AN groups) and OPG (AN group); the Cole index, BMI, and TGF-β1 independently predicted the OPG/sRANKL ratio (C, AN, and C + AN groups); TGF-β1 was found to be an independent predictor of OC (C + AN group).
Changes in bone markers, OPG, and/or OPG/sRANKL ratio observed in girls with AN are associated with changes in serum TGF-β1 concentrations. TGF-β1 suppression in girls with AN might lead to disturbances in the relationship between bone metabolism and the OPG/sRANKL system, which, in turn, might compromise the mechanism compensating for bone remodelling disturbances. (Endokrynol Pol 2016; 67 (5): 493-500).
We present a near-infrared (NIR) spectrum measurement method using a Schottky photodetector enhanced by surface plasmon resonance (SPR). An Au grating was fabricated on an n-type silicon wafer to form a Schottky barrier and act as an SPR coupler. The resulting photodetector provides wavelength-selective photodetection depending on the SPR coupling angle. A matrix was pre-calculated to describe this characteristic. The spectrum was obtained from this matrix and the measured photocurrents at various SPR coupling angles. Light with single and multiple wavelengths was tested. Comparative measurements showed that our method is able to detect spectra with a wavelength resolution comparable to that of a commercial spectrometer.
In this letter, we report on quantum light emission from bulk AlInAs grown on InP(111) substrates. We observe indium rich clusters in the bulk Al<sub>0.48</sub>In<sub>0.52</sub>As (AlInAs), resulting in quantum dot-like energetic traps for charge carriers, which are confirmed via cross-sectional scanning tunnelling microscopy (XSTM) measurements and 6-band k·p simulations. We observe quantum dot (QD)-like emission signals, which appear as sharp lines in our photoluminescence spectra at near infrared wavelengths around 860 nm, and with linewidths as narrow as 50 μeV. We demonstrate the capability of this new material system to act as an emitter of pure single photons as we extract g<sup>(2)</sup>-values as low as gcw(2)(0)=0.05-0.05+0.17 for continuous wave (cw) excitation and gpulsed,  corr.(2)=0.24±0.02 for pulsed excitation.
Nonmechanical beam steering is a rapidly growing branch of adaptive optics with applications such as light detection and ranging, imaging, optical communications, and atomic physics. Here, we present an innovative technique for one- and two-dimensional beam steering using multiple tunable liquid lenses. We use an approach in which one lens controls the spot divergence, and one to two decentered lenses act as prisms and steer the beam. Continuous 1D beam steering was demonstrated, achieving steering angles of ±39° using two tunable liquid lenses. The beam scanning angle was further enhanced to ±75° using a fisheye lens. By adding a third tunable liquid lens, we achieved 2D beam steering of ±75°. In this approach, the divergence of the scanning beam is controlled at all steering angles.
Reactive oxygen species (ROS) and electric currents modulate regeneration; however, the interplays between biochemical and biophysical signals during regeneration remain poorly understood. We investigate the interactions between redox and bioelectric activities during tail regeneration in Xenopus laevis tadpoles. We show that inhibition of NADPH oxidases-mediated production of ROS, or scavenging or blocking their diffusion into cells, impairs regeneration and consistently regulate the dynamics of membrane potential, transepithelial potential (TEP) and electric current densities (JI) during regeneration. Depletion of ROS mimics the altered TEP and JI observed in the non-regenerative refractory period. Short-term application of hydrogen peroxide (H2O2) rescues (from depleted ROS) and gains (from refractory period) regeneration, TEP increase and JI reversal. H2O2 is thereby necessary for and sufficient to induce regeneration and to regulate TEP and JI Epistasis assays show that voltage-gated Na(+) channels act downstream of H2O2 to modulate regeneration. Altogether, these results suggest a novel mechanism for regeneration via redox-bioelectric orchestration.
The obstacles to the development of therapeutic aptamers for systemic inflammatory diseases, such as nuclease degradation and renal clearance, have not been fully overcome. Here, we report a novel PEGylation method, sbC-PEGylation, which improves the pharmacokinetic properties of RNA aptamers that act against interleukin-17A (IL-17A) in mice and monkeys. sbC-PEGylated aptamers were synthesized by coupling the symmetrical branching molecule 2-cyanoethyl-N,N-diisopropyl phosphoroamidite to the 5' end of the aptamer, before conjugating two polyethylene glycol (PEG) molecules to the aptamer. Pharmacokinetic studies showed that compared with conventionally PEGylated aptamers, the sbC-PEGylated aptamer exhibited excellent stability in the blood circulation of mice and monkeys. In addition, one of the sbC-PEGylated aptamers, 17M-382, inhibited the interleukin-6 (IL-6) production induced by IL-17A in NIH3T3 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration of sbC-PEGylated 17M-382 was two times lower than that of non-PEGylated 17M-382. Furthermore, the intraperitoneal administration of sbC-PEGylated 17M-382 significantly inhibited the IL-6 production induced by IL-17A in a mouse air pouch model. Our findings suggest that the novel PEGylation method described in this study, sbC-PEGylation, could be used to develop anti-IL-17A aptamers as a therapeutic option for systemic inflammatory disease.
Long non-coding RNA (lncRNAs) play critical roles in the development of cancers. LncRNA GAS5 was identified to be involved in tumorigenesis of several cancers. However, its role and function in gastric cancer (GC) remains unknown.
The expression of GAS5, miR-23a and MT2A in 24 paired GC tissues was detected by qRT-PCR and subjected to correlation analysis. Bio-informatics analysis was performed by using DIANA Tools. Abnormal GAS5 expression was conducted in GC cells to analyze its regulation on miR-23a and MT2A via using qRT-PCR, western blot and luciferase reporter assay.
We showed that GAS5 expression was decreased in GC tissue and inversed correlated with up-regulated expression of miR-23a. GAS5 negatively regulated miR-23a expression in GC cells. The bio-informatics prediction showed putative miR-23a binding sites within GAS5 transcripts. Furthermore, our data indicated the positive regulation of GAS5 on the miR-23a target, MT2A, wherein GAS5 suppressed the negative regulation of miR-23a on MT2A by binding its 3'UTR. Additionally, the expression of MT2A was also decreased in GC tissues, showing a positive or negative correlation with GAS5 or miR-23a, respectively.
These observations suggested that GAS5 could act as a ceRNA play critical roles in GC pathogenesis and might serve as a potential therapeutic target for the treatment of gastric cancer.
MicroRNAs (miRNAs) play critical roles in the development and progression of human malignancy. MiR-143 as a tumor suppressor, is decreased in malignant tumors, including colorectal cancer (CRC). However, the potential mechanism of miR-143 in CRC remains largely unknown.
Target prediction programs and luciferase reporter assay was used to predict the targets of miR-143. Following overexpression of miR-143 in CRC cells, target gene matrix metallopeptidase 7(MMP7) expression was detected by quantitative real-time PCR (qRT-PCR) and western blot. In addition, the expression of MMP7 was quantified in CRC tissues and cell lines. Moreover, we determined the effect of MMP7 on CRC cell proliferation and invasion.
In the present study, Targetscan predicted that miR-143 could directly bind to 3'-UTR of MMP7 mRNA, and luciferase reporter assay further supportedthat MMP7 might act as a direct target gene of miR-143. Our data showed that increased expression of miR-143 repressed MMP7 expression in CRC cells both in mRNA and protein levels. Furthermore, qRT-PCR showed that the expression of MMP7 was increased in CRC tissues and cell lines, and inversely correlated with miR-143 expression in CRC tissues. Finally, our results indicated that increased expression of MMP7 reversed the potential influence of miR-143 on CRC cell proliferation and invasion ability.
Our results indicated that miR-143 might act as a tumor suppressor by targeting MMP7 during the development of CRC.
A tunable resistive pulse sensor, utilising a polyurethane nanopore, has been used to characterise nanoparticles as they traverse the pore opening. Herein we demonstrate that the translocation speed, conductive and resistive pulse magnitude, can be used to infer the surface charge of a nanoparticle, and act as a specific transduction signal for the binding of metal ions to ligands on the particle surface. Surfaces of silica nanoparticles were modified with a ligand to demonstrate the concept, and used to extract copper(ii) ions (Cu(2+)) from solution. By tuning the pH and ionic strength of the solution, a biphasic pulse, a conductive followed by a resistive pulse is recorded. Biphasic pulses are becoming a powerful means to characterise materials, and provide insight into the translocation mechanism, and herein we present their first use to detect the presence of metal ions in solution. We demonstrate how combinations of translocation speed and/or biphasic pulse behaviour are used to detect Cu(2+) with quantitative responses across a range of pH and ionic strengths. Using a generic ligand this assay allows a clear signal for Cu(2+) as low as 1 ppm with a short 5-minute incubation time, and is capable of measuring 10 ppm Cu(2+) in the presence of 5 other ions. The method has potential for monitoring heavy metals in biological and environmental samples.
Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment.
Issue: Without the cost-sharing reductions (CSRs) made available by the Affordable Care Act, health plans sold in the marketplaces may be unaffordable for many low-income people. CSRs are available to households earning between 100 percent and 250 percent of the federal poverty level that choose a silver-level marketplace plan. In 2016, about 7 million people received cost-sharing reductions that substantially lowered their deductibles, copayments, coinsurance, and out-of-pocket limits. Goal: To examine variations in consumer cost-sharing reductions between silver-level plans with CSRs to traditional marketplace plans and to employer-based insurance. Methods: Data analysis of 1,209 CSR-eligible plans sold in individual marketplaces in all 50 states and Washington, D.C. Key findings and conclusions: Cost-sharing amounts in silver plans with CSRs are much less than those in non-CSR base silver plans; silver plans with CSRs generally offer far better financial protection than those without. General annual deductibles range from $246 for CSR silver plans with a platinum-level actuarial value (94%) to as much as $3,063 for non-CSR silver plans. Out-of-pocket limits vary from $6,223 in base silver plans to $1,102 in silver plans with CSRs and a platinum-level actuarial level.
Adult flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), especially S. pullus Austen, 1909, S. uruma Shinonaga et Kano, 1966 and S. indicus Picard, 1908, are morphologically similar and sometimes difficult to distinguish when using external morphological characteristics. These species may act as vectors and/or potential vectors of many pathogens (virus, bacteria and protozoa). Their correct identification is important to target the vectors involved in the transmission of the pathogens and also helps in the fly control program.The aim of the present study was to distinguish three species which are difficult to separate using traditional diagnostic characters for species of Stomoxys such as colour patterns and body proportions. Modern morphometrics, both landmark and outline-based, was used to access wing geometry of S. pullus, S. uruma and S. indicus. A total of 198 and 190 wing pictures were analysed for landmark- and outline-based approaches, respectively. Wing shape was able to separate species and sexes of the three Stomoxys flies with highly significant difference of Mahalanobis distances. The cross-validated classification scores ranged from 76% to 100% for landmark and 77% to 96% for outline-based morphometrics. The geometry of wing features appears to be a very useful, low-cost tool to distinguish among the vectors S. pullus, S. uruma and S. indicus.
The aim of tumor-specific chemoradiotherapy is to achieve synergistic anticancer effects with clinically acceptable toxicity. Our previous studies showed that Pluronic P85 augments radiation cancer cell killing of (±)-gossypol in vitro. In this study, the radiosensitizing effect of (-)-gossypol, more potent Bcl protein inhibitor, with Pluronic P85 was investigated.
The inhibitory effect of (-)-gossypol solubilized Pluronic P85 with 0-8 Gy of radiation on clonogenic survival rate of A549 human lung adenocarcinoma cells was investigated in vitro. The anticancer effect of (-)-gossypol-solubilized Pluronic P85 with fractionated radiation of 15 Gy was assessed by A549 tumor-bearing mice.
(-)-Gossypol-loaded Pluronic P85 was found to be a more potent radiosensitizer in vitro. Pluronic P85 increased the anti-proliferative activity of (-)-gossypol against A549 cells (82 ± 42 versus 190 ± 60 nM). In addition, the combination of P85 and (-)-gossypol effectively reduced clonogenic survival of A549 cells: (11 ± 5%) compared to (-)-gossypol and P85 alone (62 ± 27% and 93 ± 13%, respectively), and enhanced radiation cancer cell killing. In vivo, P85 (200 mg/kg/day) and (-)-gossypol (15 mg/kg/day) could be safely injected intravenously over 5 days and enhanced radiation-related tumor control in an A549 xenograft model.
Pluronic P85 and (-)-gossypol act as a novel dual agent radiosensitizer and holds promise as a chemoradiotherapeutic strategy.
Mitotic exit requires the inactivation of cyclin-dependent kinase (Cdk) activity and reversal of Cdk-mediated phosphorylation events by protein phosphatases. In Saccharomyces cerevisiae the mitotic exit network (MEN) leads to activation and dispersal of the Cdc14 phosphatase throughout the cell following successful chromosome segregation. MEN-released Cdc14 is required for both full Cdk inactivation and dephosphorylation of Cdk substrates. While Cdc14 originally was thought to act broadly on mitotic Cdk substrates, recent biochemical studies revealed that Cdc14 possesses a strong preference for a subset of Cdk phosphorylation sites. This intrinsic specificity appears well conserved across fungi and animals. Identifying the direct physiological substrates of Cdc14 is an important step in fully understanding its biological functions, both in yeast and other species. Despite its strict specificity for phosphoserine Cdk sites, Cdc14 is structurally and mechanistically related to protein tyrosine phosphatases (PTPs). Like other PTPs, mutation of catalytic residues in the Cdc14 active site creates an inactive enzyme that retains high affinity substrate binding. Here we describe a protocol for using such "substrate trap" variants to biochemically isolate and detect direct substrates by co-immunopurification. The protocol is written for use in S. cerevisiae, but should be easily adaptable to other research organisms.
The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways.
In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies. Importantly, this gene body methylation is frequently associated with active transcription, and studies in the honeybee have shown that there are strong links between gene body methylation and alternative splicing. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. Here we discuss the current knowledge in this emerging field and highlight both similarities and differences between DNA methylation systems in mammals and invertebrates. Finally, we argue that the relationship between genetic variation, differential DNA methylation, other epigenetic modifications and the transcriptome must be further explored to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.
The interleukin (IL)-1 family is the largest family of interleukins. Eleven members of the IL-1 family of ligands are intracellular molecules, except a single isoform of an IL-1 receptor antagonist (IL-1Ra; also known as IL-1RN), which contains a signal peptide at the N-terminus for effective secretion. The inflammasome is a complex of intracellular molecules that is responsible for the processing of IL-1β and IL-18, whereas the remaining IL-1 family members, including IL-1α, are processed in an inflammasome caspase-1-independent pathway. Among the eleven members of the IL-1 family ligands, precursor IL-1α, IL-1β, and IL-33 have comparatively long pro-peptides of approximately 110 amino acid residues at the N-terminus. However, the other IL-1 members, except for IL-37 (also known as IL-1F7), have relatively short propeptides with fewer than 40 amino acid residues at the N-terminus. Most cytokines, including interferons and interleukins, possess a hydrophobic signal sequence for secretion. Therefore, soluble cytokines readily act on cell surface receptors immediately after their release from cells. Unlike other cytokine families, IL-1 family ligands exhibit two-step regulation: transcriptional induction at the mRNA level and post-translational modification at the protein level because of the lack of a hydrophobic signal sequence at the N-terminus. Various processing enzymes involved in the activation of intracellular IL-1 family cytokines likely provide effective immune regulation to protect the host from infections. In this review, we describe all eleven IL-1 family ligand processing enzymes, mature ligand functions, and mode of receptor conformation.
The family of interferon regulatory factors, which includes nine mammalian members (IRF1-IRF9), acts as transcription factors for interferons and thus exerts regulatory functions in the immune system and in oncogenesis. Among these members, IRF4 expression is restricted to immune cells such as T and B lymphocytes, macrophages, and dendritic cells where it is a key factor in the regulation of differentiation and is required during the immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Consequently, dysregulation of IRF4 is associated with many lymphoid malignancies. Recent studies have demonstrated that depending on the context and stage of hematopoietic cell differentiation in which its expression is dysregulated, IRF4 may act as either an oncogene or a tumor-suppressor-like factor. In addition, it has been shown that IRF4 plays a pivotal role in the development and function of several autoimmune-associated cells. Various genetic and functional studies have also pointed to IRF4 as a master regulator for autoimmunity. In this review, the roles of IRF4 in the immune response are briefly summarized and discussed, with particular focus on its essential and distinct functions in immune cell development.
Enalapril is used to treat hypertension and congestive heart failure in infants. However, enalapril is not labeled for neonates, and safety data in infants are sparse. To evaluate the safety of enalapril in young infants, we conducted a retrospective cohort study of infants who were exposed to enalapril in the first 120 days of life and were cared for in 348 neonatal intensive care units from 1997 to 2012. We determined the proportion of exposed infants who developed adverse events, including death, hypotension requiring pressors, hyperkalemia, and elevated serum creatinine. Using multivariable logistic regression, we examined risk factors for adverse events, including postnatal age at first exposure, exposure duration, gestational age group, small for gestational age status, race, sex, 5-min Apgar score, and inborn status. Of a cohort of 887,910 infants, 662 infants (0.07%) were exposed to enalapril. Among exposed infants, 142 infants (21%) suffered an adverse event. The most common adverse event was hyperkalemia (13%), followed by elevated serum creatinine (5%), hypotension (4%), and death (0.5%). Significant risk factors for adverse events included postnatal age <30 days at first exposure and longer exposure duration. This study is the largest to date examining the safety of enalapril in young term and preterm infants without significant structural cardiac disease.
Molecular richness of snake venoms is an important source of proteins and toxins with potent effects on the cardiovascular system. The alteration of the vascular system in the victim after a venomous snake bite is usually expressed by a significant decrease in blood pressure. Therefore, exploring snake venom to extract and characterize its biomolecules is of considerable medical interest, and formed the basis of this study. We assessed the potential of the venom of Montivipera bornmuelleri, a viper from Lebanon, to induce relaxant effect on isolated Wistar rat aorta via several mechanisms of action. The overall hypotensive effect of Montivipera bornmuelleri venom results from its synergetic action on different channels for the reduction of blood pressure. By actions of its metalloproteinases and phospholipase A2, the venom may induce the production of nitric oxide acting accordingly a vasodilator effect. It could act on the voltage-dependent potassium channels and/or the L-type calcium channels, inhibiting angiotensin converting enzyme and/or inhibiting the α1-adrenoceptors. This work demonstrates vasorelaxant effect of the Montivipera bornmuelleri venom acting on different pathways, reducing blood pressure.
In real-world social interactions, social status influences responses to resource distribution. However, the way in which one's own social status interacts with another's status to influence responses to resource distribution is far from clear. In the current study, we dynamically manipulated participants' social status and then asked participants to act as recipients in the ultimatum game (UG) along with proposers whose social status was made known to the participants. Experiment 1 used a between-participants design in which the participants were assigned as being of either high or low status according to their performance in a math competition (i.e., rank-inducing task). In Experiment 2, social status was manipulated within-subjects using the same rank-inducing task, with rounds of UG interleaved between rank-inducing sessions. Findings from the two experiments showed that both self-status and other-status influenced responses to UG offers, as participants were more likely to accept low offers from high status than low status proposers; this effect was particularly robust for low status participants when compared with high status participants. These findings suggest that, in comparison with individuals in high status, individuals in low status are more willing to accept low offers during resource distribution and are more affected by other-status considerations.
The "ant in the labyrinth" problem describes spatial constraints upon a moving agent in a disordered medium. In contrast with an animal-like agent (an "ant"), a clonal plant can stay in a place and move at the same time: some parts develop roots, while others continue moving by horizontal growth and branching. Hereby we present a spatially explicit, dynamic model for the study of percolation by plant growth rules in lattices that consist of open and closed sites. Growth always starts from a single seed in an open percolation cluster (patch). By increasing the proportion of open sites (p), we describe a new kind of threshold (the "tracking threshold", approximately pt=0.73), which is higher than the site percolation threshold (pc=0.5 in this lattice). At pc<p<pt the habitat contains a giant component, but the plant cannot spread successfully, because the pathways are too narrow compared to the scale of growth. We demonstrate this by varying the grain of the habitat pattern relative to the distance between two branching points. We conclude that fine-grained habitats can act as "labyrinths" for the plant within a broad range of p values. Within this range, the plant individual is likely to utilize only a small fraction of the available resources, leaving gaps open for colonization by other individuals. Therefore, the "labyrinth effect" is a considerable factor in the self-organization of plant communities.
Few data are available to assist clinicians with decisions regarding long-term use of asthma therapies, including omalizumab.
To evaluate the benefit and persistency of response in subjects continuing or withdrawing from long-term omalizumab treatment.
Evaluating the Xolair® Persistency Of Response After Long-Term Therapy (XPORT) was a randomized, double-blind, placebo-controlled withdrawal study that included subjects with moderate-to-severe persistent asthma receiving long-term omalizumab. Subjects were randomized using a hierarchical dynamic randomization scheme, to continue their same dose of omalizumab or withdraw to placebo and were then followed every 4 weeks for 1 year.
any protocol-defined severe asthma exacerbation. Secondary outcome: time to first protocol-defined severe asthma exacerbation. Exploratory outcomes included changes in Asthma Control Questionnaire (ACQ) and Asthma Control Test (ACT) scores.
Significantly more subjects in the omalizumab group (67%) had no protocol-defined exacerbation than in the placebo group (47.7%); absolute difference of 19.3% (95% CI: 5.0%, 33.6%) represents a 40.1% relative difference. Time to first protocol-defined exacerbation analysis revealed a significantly different between-group exacerbation pattern that was consistent with the primary analysis. Subjects continuing omalizumab had significantly better asthma control (mean [SD] change from baseline to Week 52: ACT, -1.16 [4.14] vs placebo, -2.88 [5.38]; P = .0188 and ACQ, 0.22 [0.66] vs placebo, 0.63 [1.13]); P = .0039. Discontinuation of omalizumab was associated with an increase in free IgE and an increase in basophil expression of the high-affinity IgE receptor. No safety concerns were noted.
Continuation of omalizumab following long-term treatment results in continued benefit as evidenced by improved symptom control and reduced exacerbation risk.
Borderline Personality Disorder (BPD) is characterized by severe and persistent impairments in interpersonal functioning. Given the complexity of social interactions, studying the interactive behavior of BPD patients is challenging. One way to implement both tight experimental control and realistic, externally valid settings is to use game-theoretical experiments. This review discusses findings from economic exchange studies in BPD against the background of game-theoretical literature. BPD patients do not seem to derive utility from mutual cooperation with others and appear not to "forgive" a partner's unfairness. By pursuing a strategy of negative reciprocity, BPD patients seem to act mostly "rationally" and in their own self-interest. Their "grim trigger strategy" resembles the theoretical ideal of the rational and self-interested agent homo economicus. Finally, we summarize how research findings from economics and clinical psychiatry may be mutually enriching and propose new research ideas in this fascinating field.
Vitamin D deficiency is common in children with asthma, and it associates with poor asthma control, reduced forced expiratory volume in one second (FEV1) and increased requirement for inhaled corticosteroids (ICS). Cross-sectional studies investigating the prevalence, determinants and clinical correlates of vitamin D deficiency in adults with asthma are lacking. We conducted a multi-centre cross-sectional study in 297 adults with a medical record diagnosis of ICS-treated asthma living in London, UK. Details of potential environmental determinants of vitamin D status, asthma control and medication use were collected by questionnaire; blood samples were taken for analysis of serum 25(OH)D concentration and DNA extraction, and participants underwent measurement of weight, height and fractional exhaled nitric oxide concentration (FeNO), spirometry and sputum induction for determination of lower airway eosinophil counts (n=35 sub-group). Thirty-five single nucleotide polymorphisms (SNP) in 11 vitamin D pathway genes (DBP, DHCR7, RXRA, CYP2R1, CYP27B1, CYP24A1, CYP3A4 CYP27A1, LRP2, CUBN, VDR) were typed using Taqman allelic discrimination assays. Linear regression was used to identify environmental and genetic factors independently associated with serum 25(OH)D concentration, and to determine whether vitamin D status was independently associated with Asthma Control Test (ACT) score, ICS dose, FeNO, forced vital capacity (FVC), FEV1 or lower airway eosinophilia. Mean serum 25(OH)D concentration was 50.6nmol/L (SD 24.9); 162/297 (54.5%) participants were vitamin D deficient (serum 25(OH)D concentration <50nmol/L). Lower vitamin D status was associated with higher body mass index (P=0.014), non-White ethnicity (P=0.036), unemployment (P for trend=0.012), lack of vitamin D supplement use (P<0.001), sampling in Winter or Spring (P for trend <0.001) and lack of a recent sunny holiday abroad (P=0.030), but not with potential genetic determinants. Vitamin D status was not found to associate with any marker of asthma control investigated. Vitamin D deficiency is common among UK adults with ICS-treated asthma, and classical environmental determinants of serum 25(OH)D operate in this population. However, in contrast to studies conducted in children, we found no association between vitamin D status and markers of asthma severity or control.
Following spinal cord injury (SCI), most axons fail to regenerate and instead form large, swollen endings generically called 'retraction bulbs.' These endings form and persist after SCI even under experimental therapeutic conditions where significant CNS regeneration occurs. Although retraction bulbs can arise from either activation of degenerative processes or deficits in regenerative processes, they are typically grouped as a single type of axonal ending. To facilitate the targeting of axonal endings for SCI repair, this review focuses on dissecting the different types of axonal endings present following injury by examining them in the context of the temporal, degenerative and regenerative changes that occur following injury. The stages of axonal dieback (also known as axonal retraction) and the steps necessary for successful axonal regeneration are outlined. The types of axonal endings that can arise as an axon successfully or unsuccessfully mounts a regenerative response are examined, with an emphasis on retraction bulbs, growth cones, and collapsed growth cones. Retraction bulbs are subdivided into those that arise from a failure to form a growth cone (endbulbs) and those that stall in response to inhibitory gradients (dystrophic axonal endings). The current understanding of the mechanisms that lead to the development of different types of axonal endings, how different experimental therapeutic interventions may act on different types of axonal endings, the current gaps in understanding the sites of action of some pro-regenerative therapies, and some of the methodological challenges to studying different types of axonal endings are discussed.
The fundamental mechanism behind the action of local anesthetics is still not clearly understood. Phenylethanol (PEtOH) is a constituent of essential oils with a pleasant odor and can act as a local anesthetic. In this work, we have explored the effect of PEtOH on the function of the hippocampal serotonin1A receptor, a representative neurotransmitter receptor belonging to the G protein-coupled receptor (GPCR) family. Our results show that PEtOH induces reduction in ligand binding to the serotonin1A receptor due to lowering of binding affinity, along with a concomitant decrease in the degree of G-protein coupling. Analysis of membrane order using the environment-sensitive fluorescent probe DPH revealed decrease in membrane order with increasing PEtOH concentration, as evident from reduction in rotational correlation time of the probe. Analysis of results obtained shows that the action of local anesthetics could be attributed to the combined effects of specific interaction of the receptor with anesthetics and alteration of membrane properties (such as membrane order). These results assume relevance in the perspective of anesthetic action and could be helpful to achieve a better understanding of the possible role of anesthetics in the function of membrane receptors.
Worldwide, consistent survival benefit for chemotherapy in hepatocellular carcinoma (HCC) is a golden goal for concerned researchers. Nexavar(®) (sorafenib) is the only approved agent that achieved touchable successes in this regard. Thus, there is a pressing medical need for new promising drugs to improve HCC therapy.
our designed lactosaminated albumin conjugate of doxorubicin (L-HSA-DOXO) that rapidly and preferentially accumulates in the liver is compared, for the first time at its MTD, with doxorubicin and sorafenib, not only for antitumor efficacy but also for overall survival.
HCC was induced in male Wistar rats with N-nitrosodiethylamine added to drinking water (100mg/L) for 8 weeks. Endpoints were antitumor efficacy, tolerability and overall survival.
L-HSA-DOXO proved to be superior at least over doxorubicin in the majority of assessed endpoints. Circulating AFP-L3% was diminished in L-HSA-DOXO (14.5%) and sorafenib (18.4%) groups compared to DENA (31.1%) and doxorubicin (29.5%) groups. This superiority was further confirmed by Western blot analyses of some novel HCC biomarkers. Survival study reinforced consistent benefits of both L-HSA-DOXO and sorafenib.
L-HSA-DOXO shows at least comparable activity to sorafenib which clinically achieves only ∼3 months overall survival benefit. Combination of these two agents could act beneficially or synergistically via two different modes of action to fight HCC.
Oncolytic viruses (OV) represent an encouraging new therapeutic concept for treatment of human cancers. OVs specifically replicate in tumor cells and initiate cell lysis whilst tumor cells act as endogenous bioreactors for virus amplification. This complex bidirectional interaction between tumor and oncolytic virus hampers the establishment of a straight dose-concentration-effect relation. We aimed to develop a generic mathematical pharmacokinetic/pharmacodynamics (PK/PD) model to characterize the relationship between tumor cell growth and kinetics of different OVs. U87 glioblastoma cell growth and titer of Newcastle disease virus (NDV), reovirus (RV) and parvovirus (PV) were systematically determined in vitro. PK/PD analyses were performed using non-linear mixed effects modeling. A viral dynamic model (VDM) with a common structure for the three different OVs was developed which simultaneously described tumor growth and virus replication. Virus specific parameters enabled a comparison of the kinetics and tumor killing efficacy of each OV. The long-term interactions of tumor cells with NDV and RV were simulated to predict tumor reoccurrence. Various treatment scenarios (single and multiple dosing with same OV, co-infection with different OVs and combination with hypothetical cytotoxic compounds) were simulated and ranked for efficacy using a newly developed treatment rating score. The developed VDM serves as flexible tool for the systematic cross-characterization of tumor-virus relationships and supports preselection of the most promising treatment regimens for follow-up in vivo analyses.
Multiple oxysterols (OHCs) have demonstrated the ability to act as agonists or antagonists of the hedgehog (Hh) signaling pathway, a developmental signaling pathway that has been implicated as a potential therapeutic target in a variety of human diseases. These OHCs are known to modulate Hh signaling through direct binding interactions with the N-terminal cysteine rich domain (CRD) of Smoothened, a key regulator of Hh signal transduction.
Homology modeling, molecular dynamics simulations, and MM/GBSA energy calculations were utilized to explore binding interactions between the OHC scaffold and the human Smoothened CRD. Follow-up cellular assays explored the in vitro activity of potential Hh pathway modulators.
Structural features that govern key molecular interactions between the Smoothened CRD and the OHC scaffold were identified. Orientation of the iso-octyl side chain as well as the overall entropy of the OHC-CRD complex are important for determining activity against the Hh pathway. OHC 9, which was previously thought to be inactive because it was not an Hh agonist, was identified as an inhibitor of Hh signal transmission.
Calculated MM/GBSA binding energies for OHCs in complex with the CRD of Smoothened correlate well with in vitro Hh modulatory activity. Compounds with high affinity stabilize Smoothened and are antagonists, whereas compounds with reduced affinity allow a conformational change in Smoothened that results in pathway activation.
Computational modeling and molecular dynamics simulations can be used to predict whether a small molecule that binds the Smoothened CRD will be an agonist or antagonist of the pathway.
Acetylsalicylic acid (ASA) is mainly recognized as painkiller or anti-inflammatory drug. However, ASA causes serious side effects towards gastrointestinal (GI) tract which limits its usefulness. Carbon monoxide (CO) and hydrogen sulfide (H2S) have been described to act as important endogenous messengers and mediators of gastroprotection but whether they can interact in gastroprotection against acute ASA-induced gastric damage remains unknown. In this study male Wistar rats were pretreated with 1) vehicle (saline, i.g.), 2) tricarbonyldichlororuthenium (II) dimer (CORM-2, 5mg/kg i.g.), 3) sodium hydrosulfide (NaHS, 5mg/kg i.g.), 4) zinc protoporphyrin (ZnPP, 10mg/kg i.p.), 5) D,L-propargylglycine (PAG, 30mg/kg i.g.), 6) ZnPP combined with NaHS, 7) PAG combined with CORM-2 or 8) 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10mg/kg i.p.) combined with CORM-2 or NaHS and 30min later ASA was administered i.g. in a single dose of 125mg/kg. After 1h, gastric blood flow (GBF) was determined by H2 gas clearance technique and gastric lesions were assessed by planimetry and histology. CO content in gastric mucosa and COHb concentration in blood were determined by gas chromatography and H2S production was assessed in gastric mucosa using methylene blue method. Protein and/or mRNA expression for cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), heme oxygenase (HO)-1, HO-2, hypoxia inducible factor-alpha (HIF)-1α, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX)-1 and COX-2, inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β were determined by Western blot or real-time PCR, respectively. ASA caused hemorrhagic gastric mucosal damage and significantly decreased GBF, H2S production, CO content, mRNA or protein expression for CSE, 3-MST, HO-2 and increased mRNA and/or protein expression for CBS, HO-1, Nrf-2, HIF-1α, iNOS, IL-1β, COX-2 in gastric mucosa and COHb concentration in blood. Pretreatment with CORM-2 or NaHS but not with PAG decreased ASA-damage and increased GBF. ZnPP reversed protective and hyperemic effect of NaHS but PAG failed to affect CORM-2-induced gastroprotection. CORM-2 elevated CO content, mRNA or protein expression for HO-1, Nrf-2, and decreased expression of CBS, HIF-1α, COX-2, IL-1β, iNOS, the H2S production in gastric mucosa and COHb concentration in blood. NaHS raised mRNA or protein expression for CSE, COX-1 and decreased mRNA expression for IL-1β and COHb level in blood. We conclude that CO is involved in gastroprotection induced by H2S while beneficial protective action of CO released from CORM-2 in gastric mucosa seems to be H2S-independent. In contrast to H2S, CO ameliorates hypoxia, regulates Nrf-2 expression but similarly to H2S acts on sGC-dependent manner to restore gastric microcirculation and exhibit anti-inflammatory activity in gastric mucosa compromised by ASA.
RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (β-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that β-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and β-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.
Rhipicephalus sanguineus sensu lato (s.l.) is a very common ectoparasite of domestic dogs able to transmit several pathogens of human and veterinary importance. Tick infestations and tick-borne diseases (TBDs) remain a serious and persistent problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to control are pursued. Whilst vaccination is recognised as a potential control method to reduce tick infestation, no anti-R. sanguineus vaccine is available. Ticks depend on their blood meals to obtain nutrients and to achieve sexual maturity, which exposes them to vast amounts of iron. Although an essential molecule for several biological processes, its excess can lead to oxidative stress. Iron homeostasis is achieved with the help of iron-binding proteins called ferritins, among others, present in several tick tissues and developmental stages. These evolutionarily conserved proteins regulate iron homeostasis by storing and releasing iron in a controlled manner. In this study the R. sanguineus ferritin 1 gene was silenced through RNA interference (RNAi) in adult females exposed to an experimental infection with Ehrlichia canis. The purpose of this study was to assess the role of this protein in tick feeding, ovary development, oogenesis, and pathogen acquisition. Our data has shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphologic and histochemical changes in the ovaries (OV) and oocytes. Furthermore, our data revealed that no E. canis DNA was found in either experimental group. Determining the function of molecules that act in key biological processes, such as blood digestion or reproduction, and that could be considered potential tick antigens will contribute towards the improvement of current control measures against these ectoparasites and the pathogens they vector.
Behavior, while complex and dynamic, is among the most diverse, derived, and rapidly evolving traits in animals. The highly labile nature of heritable behavioral change is observed in such evolutionary phenomena as the emergence of converged behaviors in domesticated animals, the rapid evolution of preferences, and the routine development of ethological isolation between diverging populations and species. In fact, it is believed that nervous system development and its potential to evolve a seemingly infinite array of behavioral innovations played a major role in the successful diversification of metazoans, including our own human lineage. However, unlike other rapidly evolving functional systems such as sperm-egg interactions and immune defense, the genetic basis of rapid behavioral change remains elusive.
Here we propose that the rapid divergence and widespread novelty of innate and adaptive behavior is primarily a function of its genomic architecture. Specifically, we hypothesize that the broad diversity of behavioral phenotypes present at micro- and macroevolutionary scales is promoted by a disproportionately large mutational target of neurogenic genes. We present evidence that these large neuro-behavioral targets are significant and ubiquitous in animal genomes and suggest that behavior's novelty and rapid emergence are driven by a number of factors including more selection on a larger pool of variants, a greater role of phenotypic plasticity, and/or unique molecular features present in large genes. We briefly discuss the origins of these large neurogenic genes, as they relate to the remarkable diversity of metazoan behaviors, and highlight key consequences on both behavioral traits and neurogenic disease across, respectively, evolutionary and ontogenetic time scales.
Current approaches to studying the genetic mechanisms underlying rapid phenotypic change primarily focus on identifying signatures of Darwinian selection in protein-coding regions. In contrast, the large mutational target hypothesis places genomic architecture and a larger allelic pool at the forefront of rapid evolutionary change, particularly in genetic systems that are polygenic and regulatory in nature. Genomic data from brain and neural tissues in mammals as well as a preliminary survey of neurogenic genes from comparative genomic data support this hypothesis while rejecting both positive and relaxed selection on proteins or higher mutation rates. In mammals and invertebrates, neurogenic genes harbor larger protein-coding regions and possess a richer regulatory repertoire of miRNA targets and transcription factor binding sites. Overall, neurogenic genes cover a disproportionately large genomic fraction, providing a sizeable substrate for evolutionary, genetic, and molecular mechanisms to act upon. Readily available comparative and functional genomic data provide unexplored opportunities to test whether a distinct neurogenomic architecture can promote rapid behavioral change via several mechanisms unique to large genes, and which components of this large footprint are uniquely metazoan.
The large mutational target hypothesis highlights the eminent roles of mutation and functional genomic architecture in generating rapid developmental and evolutionary change. It has broad implications on our understanding of the genetics of complex adaptive traits such as behavior by focusing on the importance of mutational input, from SNPs to alternative transcripts to transposable elements, on driving evolutionary rates of functional systems. Such functional divergence has important implications in promoting behavioral isolation across short- and long-term timescales. Due to genome-scaled polygenic adaptation, the large target effect also contributes to our inability to identify adapted behavioral candidate genes. The presence of large neurogenic genes, particularly in the mammalian brain and other neural tissues, further offers emerging insight into the etiology of neurodevelopmental and neurodegenerative diseases. The well-known correlation between neurological spectrum disorders in children and paternal age may simply be a direct result of aging fathers accumulating mutations across these large neurodevelopmental genes. The large mutational target hypothesis can also explain the rapid evolution of other functional systems covering a large genomic fraction such as male fertility and its preferential association with hybrid male sterility among closely related taxa. Overall, a focus on mutational potential may increase our power in understanding the genetic basis of complex phenotypes such as behavior while filling a general gap in understanding their evolution.
Human inflicted bruises in slaughter pigs are hampering animal welfare, are an infringement of the animal protection act, and are a focus of public attention. The aim of the present study was to evaluate the gross appearance of human inflicted bruises in slaughter pigs and to compare the inflammatory changes in two lesions as a basis for estimating the age of lesions in the same pig. Pigs with human inflicted bruises slaughtered at two major slaughterhouses in Denmark from November 2013 to May 2014 were evaluated. After slaughter, the bruises were examined grossly and skin and underlying muscle tissue from two similar but separate bruises (a and b) on each pig were sampled for histology.
Skin and muscle tissue from 101 slaughter pigs were subjected to gross evaluation. Eighty-one of these were also subjected to histological evaluation. Most frequently (51 out of 101 pigs, 50 %), bruises had a tram-line pattern due to blunt trauma inflicted with long objects such as sticks. Other bruises reflected the use of tattoo-hammers, plastic paddles, double U profiles and chains. Histological evaluation of two bruises from a pig with multiple lesions was found insufficient to assess the overall age of the lesions as substantial variation in the inflammatory response between bruises was present.
Grossly, the pattern of bruises often reflected the shape of the object used for inflicting the lesions. When determining the age of multiple bruises on a pig more than two lesions should be evaluated histologically.
Resistance to imatinib has been recognized as a major challenge for the treatment of chronic myeloid leukemia (CML). Aberrant expression of miR-451 has been reported to participate in anticancer drug resistance. However, the role of miR-451 in imatinib resistance has not been investigated. The present study was undertaken to determine the expression of miR-451 in order to find a possible association between the expression of this miRNA and imatinib resistance in Tunisian CML patients.
First, real-time RT-PCR was performed to identify the expression of miR-451 in peripheral leukocytes of 59 CML patients treated with imatinib. Then, bioinformatics analysis was carried out to understand the regulatory roles of miR-451 in imatinib-resistant process.
Downregulated miR-451 was observed in imatinib-resistant CML cases. In silico analysis identified MYC as a potential target of miR-451. We further revealed the existence of an MYC-binding site in MiR-451 promoter region. On the other hand, increased level of MYC was detected in imatinib-resistant CML cases which may explain the causative role of MYC in CML cases and the downregulation of miR-451.
Taken together, our findings suggest that miR-451 and MYC form together a regulatory loop which may act as a potential therapeutic target, and disruption of suggested regulatory loop could help to improve CML therapy.
Reactive astrogliosis has been occurred after intracerebral hemorrhage (ICH). Leukemia inhibitory factor (LIF) can act as a modulator for glial gene expression. Signal transducer and activator of transcription3 (STAT3) is a critical regulator of reactive astrogliosis. The present study tested whether endogenous leukemia inhibitory factor (LIF) acted on intracerebral hemorrhage (ICH)-induced reactive astrogliosis via STAT3 signaling pathway. Rats were divided into three parts of experiments: (1) Rats received either an ICH or a needle insertion (sham). (2) Rats received 100 ng LIF or an equal volume of phosphate buffered solution (PBS) by direct infusion into the lateral ventricle (LV) after ICH. (3) AG490 (0.25 mg/kg) was injected into the LV to block STAT3 signaling. Brains were perfused to identify proliferating cell nuclear antigen (PCNA)+/glial fibrillary acidic protein (GFAP)+nuclei. The expression of GFAP, LIF, leukemia inhibitory factor receptor (LIFR), glycoprotein130 (gp130) and p-STAT3 was evaluated by immunohistochemistry and western blot, respectively. After ICH, the number of the PCNA+/GFAP+ nuclei and the expression of GFAP, LIF, LIFR, gp130 and p-STAT3 were increased. Moreover, LIF increased the number of PCNA+/GFAP+ nuclei and the expression of GFAP, LIFR, gp130, and p-STAT3. The number of PCNA+/ GFAP+ nuclei and GFAP protein levels were attenuated markedly after inhibition of p-STAT3. Together, these data suggest that LIF contributes to ICH-related reactive astrogliosis via activation of STAT3 signaling.
Autophagy is a conserved cellular self-digestion pathway for maintenance of homeostasis under basal and stressed conditions. Autophagy plays pivotal roles in the pathogenesis of many diseases, such as aging-related diseases, autoimmune diseases, cardiovascular diseases, and cancers. Of special note is that accumulating data suggest an intimate relationship between autophagy and ovarian carcinoma. Autophagy is well identified to act as either as a tumor-suppressor or as a tumor-promoter in ovarian carcinoma. The exact function of autophagy in ovarian carcinoma is highly dependent on the circumstances of cancer including hypoxic, nutrient-deficient, chemotherapy and so on. However, the mechanism underlying autophagy associated with ovarian carcinoma remains elusive, the precise role of autophagy in ovarian carcinoma also remains undetermined. In this review, we tried to sum up and discuss recent research achievements of autophagy in ovarian cancer. Moreover, waves of novel therapies ways for ovarian carcinoma based on the functions of autophagy were collected.
The antimicrobial property of silver is associated to the quantity of silver and the grade of silver released. The ionized silver is extremely sensitive, as it binds to tissue proteins and gets operational alterations in the bacterial cell wall and nuclear membrane leading to cell modification and death.Silver nanoparticles have the talent to anchor to the bacterial cell wall and consequently infiltrate it, so causing physical modifications in the cell membrane like the absorptivity of the cell membrane and death of the cell. There are numerous concepts on the act of silver nanoparticle on bacteria to reason the microbicidal influence.
Glucocorticoids are invaluable in the therapy of chronic-inflammatory diseases, like rheumatoid arthritis (RA). They act fast and efficient to suppress inflammation and serve to bridge the gap until disease modifying drugs (DMARD) show effect. However, the value of glucocorticoids with regard to their cost / benefit ratio in long term RA therapy is still controvers. In this short review, the main aspects favoring glucocorticoids as DMARD in long term RA therapy will be discussed. It becomes apparent, that at low dosage (prednisolone ≤ 5 mg / d), careful selection and monitoring of patients, and osteoporosis prophylaxis according to guidelines, long term therapy with glucocorticoids is an option and, with a favorable cost / benefit ratio, contributes to inflammation control and prevention of structural damage.
To cope with heterogeneous environments and resource distributions, filamentous fungi have evolved a spatially extensive growth enabling their hyphae to penetrate air-water interfaces and pass through air-filled pores. Such mycelia are also known to act as dispersal networks for the mobilisation of bacteria ('fungal highways') and connection of microbial microhabitats. Hitherto, however, nothing is known about the effect of mycelia-based dispersal on interactions between bacterial predators and their prey and concomitant effects on biomass formation. We here hypothesise that mycelia enable the contact between predators and their prey and shape a prey's population. We investigated the impact of predation by Bdellovibrio bacteriovorus 109J on the growth of its potential prey Pseudomonas fluorescens LP6a in the presence of mycelia. Our data give evidence that hyphae increase the accessibility of the prey to B. bacteriovorus 109J and, hence, allow for efficient foraging and shaping of prey populations not seen in the absence of mycelia. To test our hypothesis tailored microbial landscapes were used for better reduction of emerging properties in complex systems. Our data suggest that mycelia have substantial influence on prey-predator relationship and hereby may promote the structure of prey and predator populations and, hence, may be a determinant for biomass formation in heterogeneous environments.The ISME Journal advance online publication, 8 November 2016; doi:10.1038/ismej.2016.135.
The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer.
Familial hypercholesterolemia (FH) is a life-threatening genetic disorder characterized by elevated levels of plasma low-density lipoprotein cholesterol (LDL-cholesterol). Current attempts at gene therapy for FH have been limited by the use of strong heterologous promoters which lack genomic DNA elements essential for regulated expression. Here, we have combined a mini-gene vector expressing the human LDLR cDNA from a 10 kb native human LDLR locus genomic DNA promoter element, with an efficient miRNA targeting 3-hydroxy-3-methylgutaryl-coenzyme A reductase (Hmgcr), to further enhance LDLR expression. We show that the combined vector suppresses endogenous Hmgcr transcripts in vivo, leading to an increase in LDLR transgene expression. In a diet-induced Ldlr(-/-) mouse model of FH, we show that administration of the combined vector reduces atherogenic plasma lipids by ~32%. Finally, we demonstrate that our episomal nonviral vectors are able to reduce atherosclerosis by ~40% after 12 weeks in vivo. Taken together, the vector system we describe exploits the normal cellular regulation of the LDLR to provide prolonged expression of LDLR through targeted knockdown of Hmgcr. This novel gene therapy system could act alone, or in synergy with current therapies that modulate intracellular cholesterol, such as statins, greatly enhancing its therapeutic application for FH.
Background A recent clinical trial has shown a beneficial effect of the antioxidant agent selenium in Graves' orbitopathy (GO). In order to shed light on the cellular mechanisms on which selenium may act, here we investigated its effects in cultured orbital fibroblasts. Methods Primary cultures of orbital fibroblasts from 6 GO patients and 6 control subjects were established. Cells were treated with H2O2 to induce oxidative stress, after pre-incubation with selenium-(Methyl)selenocysteine (SeMCys). The following assays were performed: glutathione disulfide (GSSG), as a measure of oxidative stress, glutathione peroxidase (GPX) activity, cell proliferation, hyaluronic acid (HA) and pro-inflammatory cytokines. Results H2O2 induced an increase in cell GSSG and fibroblast proliferation, which were reduced by SeMCys. Incubation of H2O2-treated cells with SeMCys was followed by an increase in glutathione peroxidase activity, one of the antioxidant enzymes into which selenium is incorporated. At the concentrations used (5 mcM), H2O2 did not affect significantly HA release, which was however reduced by SeMCys. H2O2 determined an increase in endogenous cytokines involved in the response to oxidative stress and GO pathogenesis, namely TNF-alpha, IL1-beta and IFN-gamma. The increases in TNF-alpha and IFN-gamma were rescued by SeMCys. Whereas the effects of SeMCys were similar in GO and control fibroblasts concerning oxidative stress and cytokines, they were exclusive to GO fibroblasts concerning proliferation and HA. Conclusions Selenium, in the form of SeMCys, rescues from some of the effects of oxidative stress in orbital fibroblasts, namely increased proliferation and pro-inflammatory cytokines. SeMCys reduces HA release in GO fibroblasts in a manner that seems at least in part independent from H2O2-induced oxidative stress. Some effects of SeMCys are specific for GO fibroblasts. Our findings reveal some cellular mechanisms by which selenium may act in patients with GO.
Marine sponges are filter feeding porous animals and usually harbor a remarkable array of microorganisms in their mesohyl tissues as transient and resident endosymbionts. The marine sponge-microbial interactions are highly complex and, in some cases, the relationships are thought to be truly symbiotic or mutualistic rather than temporary associations resulting from sponge filter-feeding activity. The marine sponge-associated bacteria are fascinating source for various biomolecules that are of potential interest to several biotechnological industries. In recent times, a particular attention has been devoted to bacterial biopolymer (polyesters) such as intracellular polyhydroxyalkanoates (PHAs) produced by sponge-associated bacteria. Bacterial PHAs act as an internal reserve for carbon and energy and also are a tremendous alternative for fossil fuel-based polymers mainly due to their eco-friendliness. In addition, PHAs are produced when the microorganisms are under stressful conditions and this biopolymer synthesis might be exhibited as one of the survival mechanisms of sponge-associated or endosymbiotic bacteria which exist in a highly competitive and stressful sponge-mesohyl microenvironment. In this review, we have emphasized the industrial prospects of marine bacteria for the commercial production of PHAs and special importance has been given to marine sponge-associated bacteria as a potential resource for PHAs.
In the late 1930s, Karl von Frisch reported that semiochemicals released upon injury, act as alarm substances (Schreckstoff) in fish. In Ostariophysi species, club cells in the epidermis are believed to contain cues related to alarm substance; however, the function of club cells, primarily as reservoirs of alarm substance has been debated. Here, I describe an alarm response in the Japanese rice fish Oryzias latipes (medaka), a member of the order Beloniformes. The response to alarm substance (Schreckreaction) in medaka is characterized by bouts of immobility and an increase in cortisol levels within minutes of exposure to conspecific skin extract. Histological analysis, however, suggests that club cells are either rare or absent in the medaka epidermis. In addition to describing an uncharacterized behavior in a vertebrate popular for genetic and developmental studies, these results support the hypothesis that the primary function of epidermal club cells may be unrelated to a role as alarm substance cells. The existence of similar behavioral responses in two evolutionarily distant but well established laboratory models, the zebrafish and the medaka, offers the possibility of comparative analyses of neural circuits encoding innate fear.
Plasmodium falciparum extensively modifies its chosen host cell, the mature human erythrocyte. This remodelling is carried out by parasite-encoded proteins that are exported into the host cell. To gain access to the human red blood cell, these proteins must cross the parasitophorous vacuole, a membrane bound compartment surrounding the parasite that is generated during the invasion process. Many exported proteins carry a so-called PEXEL/HT signal that directs their transport. We recently reported the unexpected finding of a species-restricted parasite-encoded Hsp70, termed PfHsp70x, which is exported into the host erythrocyte cytosol. PfHsp70x lacks a classical PEXEL/HT motif, and its transport appears to be mediated by a 7 amino acid motif directly following the hydrophobic N-terminal secretory signal. In this report, we analyse this short targeting sequence in detail. Surprisingly, both a reversed and scrambled version of the motif retained the capacity to confer protein export. Site directed mutagenesis of glutamate residues within this region leads to a block of protein trafficking within the lumen of the PV. In contrast to PEXEL-containing proteins, the targeting signal is not cleaved, but appears to be acetylated. Furthermore we show that, like other exported proteins, trafficking of PfHsp70x requires the vacuolar translocon, PTEX.
In this study, we observed the peak splitting of absorption spectra for two-dimensional sheets of silver nanoparticles due to the electromagnetically induced transparency (EIT) effect. This unique optical phenomenon was observed for the multilayered nanosheets up to 20 layers on a metal substrate, while this phenomenon was not observed on a transparent substrate. The wavelength and intensities of the split peaks depend on the number of layers, and the experimental results were well reproduced by the calculation of the Transfer-Matrix method by employing the effective medium approximation. The Ag nanosheets used in this study can act as a plasmonic metamaterial light absorber, which has a such large oscillator strength. This phenomenon is a fundamental optical property of a thin film on a metal substrate but has never been observed because native materials do not have a large oscillator strength. This new type of EIT effect using a plasmonic metamaterial light absorber presents the potential for the development of future optic and photonic technologies.
Propofol and etomidate are the most important intravenous general anesthetics in the current clinical use and that mediate gamma-aminobutyric acid's (GABAergic) synaptic transmission. However, their long-term effects on GABAergic synaptic transmission induced by neonatal propofol or etomidate exposure remain unclear. We investigated the long-term GABAergic neurotransmission alterations, following neonatal propofol and etomidate administration.
Sprague-Dawley rat pups at postnatal days 4-6 were underwent 6-h-long propofol-induced or 5-h-long etomidate-induced anesthesia. We performed whole-cell patch-clamp recording from pyramidal cells in the cornus ammonis 1 area of acute hippocampal slices of postnatal 80-90 days. Spontaneous and miniature inhibitory GABAergic currents (spontaneous inhibitory postsynaptic currents [sIPSCs] and miniature inhibitory postsynaptic currents [mIPSCs]) and their kinetic characters were measured. The glutamatergic tonic effect on inhibitory transmission and the effect of bumetanide on neonatal propofol exposure were also examined.
Neonatal propofol exposure significantly increased the frequency of mIPSCs (from 1.87 ± 0.35 Hz to 3.43 ± 0.51 Hz, P< 0.05) and did not affect the amplitude of mIPSCs and sIPSCs. Both propofol and etomidate slowed the decay time of mIPSCs kinetics (168.39 ± 27.91 ms and 267.02 ± 100.08 ms vs. 68.18 ± 12.43 ms; P< 0.05). Bumetanide significantly blocked the frequency increase and reversed the kinetic alteration of mIPSCs induced by neonatal propofol exposure (3.01 ± 0.45 Hz and 94.30 ± 32.56 ms).
Neonatal propofol and etomidate exposure has long-term effects on inhibitory GABAergic transmission. Propofol might act at pre- and post-synaptic GABA receptor A (GABAA) receptors within GABAergic synapses and impairs the glutamatergic tonic input to GABAergic synapses; etomidate might act at the postsynaptic site.
The striped stem borer, Chilo suppressalis, is a major target pest of transgenic rice expressing the Cry1Ab protein from the bacterium Bacillus thuringiensis (Bt) in China. Evolution of resistance in this pest is a major threat to the durability of Bt rice. Since Bt exerts its activity through binding to specific receptors in the midgut of target insects, identification of functional Cry1Ab receptors in the midgut of C. suppressalis larvae is crucial to evaluate potential resistance mechanisms and develop effective strategies for delaying insect resistance. In this work, we identified the putative Cry1Ab toxin-binding protein, aminopeptidase-N (APN), in the midgut of C. suppressalis by ligand blot and mass spectrometry. After cloning the full-length cDNAs encoding APN isoforms from the C. suppressalis larval midgut, we studied their spatiotemporal expression in different gut tissues and developmental stages. Furthermore, RNA interference (RNAi) against C. suppressalis aminopeptidases (CsAPNs) was employed to illustrate a functional role for CsAPNs in Cry1Ab toxicity to C. suppressalis larvae using injection and oral delivery of Stealth™ siRNA. Down-regulating the expression of CsAPNs by RNAi was closely associated with reduced susceptibility of C. suppressalis to Cry1Ab. These data provide the first direct evidence that CsAPNs participate in the mode of Cry1Ab action and may act as the functional receptor of Cry1A in C. suppressalis larvae.
It is commonly accepted that the renin-angiotensin-aldosterone system (RAAS) is a cardiovascular circulating hormonal system that plays also an important role in the modulation of several patterns in the brain. The pathway of the RAAS can be divided into two classes: the traditional pathway of RAAS, also named classic RAAS, and the non-classic RAAS. Both pathways play a role in both cardiovascular and neurological diseases through a peripheral or central control. In this regard, renewed interest is growing in the last years for the consideration that the brain RAAS could represent a new important therapeutic target to regulate not only the blood pressure via central nervous control, but also neurological diseases. However, the development of compounds able to cross the blood-brain barrier and to act on the brain RAAS is challenging, especially if the metabolic stability and the half-life are taken into consideration. To date, two drug classes (aminopeptidase type A inhibitors and angiotensin IV analogues) acting on the brain RAAS are in development in pre-clinical or clinical stages. In this article, we will present an overview of the biological functions played by peripheral and brain classic and non-classic pathways of the RAAS in several clinical conditions, focusing on the brain RAAS and on the new pharmacological targets of the RAAS.
The objective was to compare quality of diabetes and cardiovascular disease (CVD) care between advanced practice providers (APPs) and physicians in a primary care setting.
We identified diabetes (n=1,022,588) and CVD (n=1,187,035) patients receiving primary care between October 2013 and September 2014 in 130 Veterans Affairs facilities. We compared glycemic control (hemoglobin A1c <7%) in diabetic patients, blood pressure (BP) <140/90 mmHg in diabetic or CVD patients, cholesterol control (low-density lipoprotein cholesterol<100 mg/dL, receiving a statin) in diabetic or CVD patients, and those receiving a β-blocker (with history of myocardial infarction in the last 2 years) among patients receiving care from physicians and APPs. We also compared the proportion meeting composite measure (glycemic, BP, and cholesterol control in diabetic patients; BP, cholesterol control, and receipt of β-blocker among eligible CVD patients).
Diabetic patients receiving care from APPs were statistically more likely to have glycemic (50% vs 51.4%, odds ratio [OR] 1.06 [1.05-1.08]) and BP control (77.5% vs 78.4%, OR 1.04 [1.03-1.06]), whereas patients receiving care from physicians were more likely to have cholesterol control (receipt of statin 68% vs 66.5%, OR 0.94 [0.93-0.95]) in adjusted models, although these differences are not clinically significant. Similar results were seen in CVD patients. Few patients met the composite measure (27.1% and 27.6% of diabetic and 54.0% and 54.8% of CVD patients receiving care from physicians and APPs, respectively).
Diabetes and CVD care quality was comparable between physicians and APPs with clinically insignificant differences. Regardless of provider type, there is a need to improve performance on eligible measures in diabetes or CVD patients.
Neuroinflammation plays a vital role in Alzheimer's disease (AD) and other neurodegenerative conditions. Sophora alopecuroides is widely used in traditional Uighur's medicine for the treatment of inflammation. Sophoraflavanone G (SG), a major flavonoid found in the S. alopecuroides, has also been reported to exhibit anti-inflammatory activity both in vitro and in vivo. However, the effect of S. alopecuroides and SG on microglia-mediated neuroinflammation has not been investigated.
The present study was designed to evaluate the anti-neuroinflammatory effect of S. alopecuroides and SG against lipopolysaccharide (LPS)-activated BV2 microglial cells and to explore the underlying mechanisms.
We measured the production of pro-inflammatory mediators and cytokines, and analyzed relevant mRNA and protein expressions by qRT-PCR and Western Blot.
S. alopecuroides extract (SAE) and SG inhibited the LPS-induced release of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Additionally, SG reduced gene expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-6 and IL-1β, and further decreased the protein expressions of iNOS and COX-2. Mechanism studies found that SG down-regulated phosphorylated mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/AKT and Janus kinase/signal transducer and activator of transcription (JAK/STAT), and up-regulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). In addition, SG inhibited the cytotoxicity of conditioned medium prepared by LPS-activated BV2 microglia to neuronal PC12 cells and improved cell viability.
S. alopecuroides and SG displayed anti-neuroinflammatory activity in LPS-activated BV2 microglia. SG was able to inhibit the neuroinflammation by MAPKs, PI3K/AKT, JAK/STAT and Nrf2/HO-1 signaling pathways and might act as a natural therapeutic agent to be further developed for the treatment of various neuroinflammatory conditions.
Although it is generally accepted that selenium (Se) is important for life, it is not well known which forms of organic and/or inorganic Se compound are the most biologically active. In nature Se exists mostly in two forms, namely as selenite with fourvalent and selenate with sixvalent cations, from which all other inorganic and organic species are derived. Despite a small difference in their electronic structure, these two inorganic parent compounds differ significantly in their redox properties. Hence, only selenite can act as an oxidant, particularly in the reaction with free and/or protein-bound sulhydryl (SH) groups. For example, selenite was shown to inhibit the hydroxyl radical-induced reduction and scrambled reoxidation of disulfides in human fibrinogen thus preventing the formation of highly hydrophobic polymer, termed parafibrin. Such a polymer, when deposited within peripheral and/or cerebral circulation, may cause irreversible damage resulting in the development of cardiovascular, neurological and other degenerative diseases. In addition, parafibrin deposited around tumor cells produces a protease-resistant coat protecting them against immune recognition and elimination. On the other hand, parafibrin generated by Ebola's protein disulfide isomerase can form a hydrophobic 'spike' that facilitates virus attachment and entry to the host cell. In view of these specific properties of selenite this compound is a potential candidate as an inexpensive and readily available food supplement in the prevention and/or treatment of cardiovascular, neoplastic, neurological and infectious diseases.
Aimed at approved mental health professionals and others working in this area, this updated issue provides a quick and easy guide to mental health act assessments and surrounding legal frameworks.
Despite extensive insights on the interaction between hematopoietic stem cells (HSCs) and the supporting bone marrow (BM) stroma in hematopoietic homeostasis there remains unanswered questions on HSC regulation. We report on the mechanism by which HSCs attain cycling quiescence by addressing a role for inducible cyclic AMP early repressor (ICER). ICER negatively transcriptional regulators of cAMP activators such as CREM and CREB. These activators can be induced by hematopoietic stimulators such as cytokines. We isolated subsets of hematopoietic cells from ten healthy donors: CD34(+)CD38(-)/c-kit (+) (primitive progenitor), CD34(+)CD38(+)/c-kit(low) (mature progenitor) and CD34(-)CD38(+/-)/c-kit(low/-) (differentiated lineage-). The relative maturity of the progenitors were verified in long-term culture initiating assay. Immunoprecipitation indicated the highest level of ICER in the nuclear extracts of CD34(+)/CD38(-) cells. Phospho (p)-CREM was also present suggesting a balance between ICER and p-CREM in HSC. ICER seems to be responsible for decrease in G1 transition, based on reduced Cdk4 protein, decreased proliferation and functional studies with propidium iodide. There were no marked changes in the cycling inhibitors, p15 and p-Rb, suggesting that ICER may act independently of other cycling inhibitors. The major effects of ICER were validated with BM mononuclear cells (BMNCs) in which ICER was ectopically expressed, and with BMNCs resistant to 5-fluorouracil- or cyclophosphamide. In total, this study ascribes a novel role for ICER in G1 checkpoint regulation in HSCs. These findings are relevant to gene therapy that require engineering of HSCs, age-related disorders that are associated with hematopoietic dysfunction and other hematological disorders.
Emulsion-fusion PCR recovers long-range sequence information by combining products in cis from individual genomic DNA molecules. Emulsion droplets act as very numerous small reaction chambers in which different PCR products from a single genomic DNA molecule are condensed into short joint products, to unite sequences in cis from widely separated genomic sites. These products can therefore provide information about the arrangement of sequences and variants at a larger scale than established long-read sequencing methods. The method has been useful in defining the phase of variants in haplotypes, the typing of inversions, and determining the configuration of sequence variants in multiallelic CNVs. In this description we outline the rationale for the application of emulsion-fusion PCR methods to the analysis of multiallelic CNVs, and give practical details for our own implementation of the method in that context.
Cognitive control processes enable us to act flexibly in a world posing ever-changing demands on our cognitive system. To study cognitive control, conflict tasks and especially congruency sequence effects have been regarded as a fruitful tool. However, for the last decade a dispute has arisen whether or not congruency sequence effects are indeed a valid measure of cognitive control processes. This debate has led to the development of increasingly complex paradigms involving numerous, intricately designed experimental conditions which are aimed at excluding low-level, associative learning mechanisms like feature binding as an alternative explanation for the emergence of congruency sequence effects. Here, we try to go beyond this all-or-nothing thinking by investigating the assumption that both cognitive control processes as well as feature binding mechanisms occur within trials of the same task. Based on a theoretical dual-route-model of behavior under conflict, we show that both classes of cognitive mechanisms should affect behavior at different points of the decision process. By comparing these predictions to continuous mouse movements from an adapted Simon task, we find evidence that control processes and feature binding mechanisms do indeed coexist within the task but that they follow distinct timing patterns. We argue that this dynamic approach to cognitive processing opens up new ways to investigate the diversity of co-existing processes that contribute to the selection of behavior.
Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica.
A developmental program of epigenetic repression prepares each mammalian olfactory sensory neuron (OSN) to strongly express one allele from just one of hundreds of odorant receptor (OR) genes, but what completes this process of OR gene choice by driving the expression of this allele is incompletely understood. Conditional deletion experiments in mice demonstrate that Lhx2 is necessary for normal expression frequencies of nearly all ORs and all trace amine-associated receptors, irrespective of whether the deletion of Lhx2 is initiated in immature or mature OSNs. Given previous evidence that Lhx2 binds OR gene control elements, these findings indicate that Lhx2 is directly involved in driving OR expression. The data also support the conclusion that OR expression is necessary to allow immature OSNs to complete differentiation and become mature. In contrast to the robust effects of conditional deletion of Lhx2, the loss of Emx2 has much smaller effects and more often causes increased expression frequencies. Lhx2:Emx2 double mutants show opposing effects on Olfr15 expression that reveal independent effects of these two transcription factors. While Lhx2 is necessary for OR expression that supports OR gene choice, Emx2 can act differently; perhaps by helping to control the availability of OR genes for expression.
Calcium-binding proteins (CaBPs) form a subfamily of calmodulin-like proteins that were cloned from the retina. CaBP4 and CaBP5 have been shown to be important for normal visual function. Although CaBP1/caldendrin and CaBP2 have been shown to modulate various targets in vitro, it is not known whether they contribute to the transmission of light responses through the retina. Therefore, we generated mice that lack CaBP2 or CaBP1/caldendrin (Cabp2(-/-) and Cabp1(-/-) ) to test whether these CaBPs are essential for normal retinal function. By immunohistochemistry, the overall morphology of Cabp1(-/-) and Cabp2(-/-) retinas and the number of synaptic ribbons appear normal; transmission electron microscopy shows normal tethered ribbon synapses and synaptic vesicles as in wild-type retinas. However, whole-cell patch clamp recordings showed that light responses of retinal ganglion cells of Cabp2(-/-) and Cabp1(-/-) mice differ in amplitude and kinetics from those of wild-type mice. We conclude that CaBP1/caldendrin and CaBP2 are not required for normal gross retinal and synapse morphology but are necessary for the proper transmission of light responses through the retina; like other CaBPs, CaBP1/caldendrin and CaBP2 likely act by modulating presynaptic Ca(2+)-dependent signaling mechanisms.
Remarkable clinical responses have been seen in patients with metastatic melanoma with targeted therapy (BRAFi vemurafenib, MEKi) and with modern immune cell-based approaches such as TCR engineered adoptive cell transfer (ACT) and earlier experiences with high-dose IL-2. The proximal mediators of these immune therapies are tumor-reactive CTL. Various mechanisms of resistance to immune-mediated apoptotic signals have been described, including phenotypic changes, effector cell exhaustion, functional tolerance, deficiencies in Ag processing and presentation, and mutation or down-regulation of antigenic epitopes. The immune system and drugs eradicate tumors via apoptosis. Therefore, tumors' resistance to apoptosis may be a determining factor that limits the efficacy of immunotherapies. It is predicted that these therapies have limited efficacy in patients whose melanomas have developed resistance to targeted therapy such as vemurafenib. Upregulation of the immune checkpoint molecule CTLA-4 on activated T cells and its interaction with CD80/86 blocks T cell activation. The fully humanized mAb ipilimumab blocks this interaction, resulting in sustained T cell stimulation. Likewise, the programmed death receptor 1 (PD-1) is another member of the B7:CD28 family of costimulatory molecules that regulates T cell activation, whose ligand (PD-L1) is expressed on melanomas. The human anti-PD-1 mAb, Pembrolizumab, overcomes tolerance, has a favorable pharmacokinetics profile with minimal undesired toxic side effects and has shown remarkable improvement in melanoma therapy. This review focuses on recent advances in the development of various anti-PD-1 checkpoint blockade antibodies and will summarize recent clinical data using immune checkpoint blocking antibodies.
Based on clinical trial data patients with heart failure (HF) and evidence of iron deficiency should be offered intravenous (iv) iron with the aim of improving exercise capacity and symptoms. Baseline measurement in outpatient HF clinics demonstrated that only 50% of patients who may be eligible for iv iron were investigated with iron studies. Our aim was to make sure that 90% of the patients attending our heart failure clinics who were symptomatic and had an ejection fraction (EF) ≤45% should have their iron studies checked within the last six months. In an effort to increase the proportion of suitable patients in whom iron studies are requested, we carried out three plan-do-study-act (PDSA) cycles each with a different intervention. These interventions included a presentation of the clinical trial evidence at a HF multidisciplinary meeting, email reminders prior to clinic and stickers in the patient notes (repeated twice). The effect of each intervention was measured with the outcome being the proportion of eligible patients in whom iron studies were documented within the previous 6 months. The interventions increased the number of suitable patients who had iron studies checked, to as high as 100%, however this effect was not sustained. Root cause analysis revealed that clinicians were unenthusiastic to continue performing iron studies due to inefficiency in the process of admitting patients and giving them iv iron. For example median in-hospital stay of seven hours for an infusion that is given over 15 minutes. In an attempt to improve patient and physician satisfaction we piloted an ambulatory outpatient service to deliver iv iron. We demonstrated that this service was feasible and more efficient as less time was required waiting for a bed or spent in hospital and was less costly. In summary we have demonstrated interventions which can increase the identification of patients who would benefit from iv iron and piloted a new time and cost efficient system of administration of iv iron.
[This corrects the article DOI: 10.1186/s13017-016-0082-5.].
Despite the great breakthroughs we have witnessed in the last 50 years in the prevention, diagnosis, and treatment of hepatitis B, we are still far from eradicating or even curing the disease. Achieving further progress in controlling this disease will not be possible without discovering the exact pathogenesis behind it. One prime suspect in the pathogenesis of various diseases is oxidative stress. This review will exclusively explore hepatitis B in the context of oxidative stress to obtain a more comprehensive clinical perspective on its pathogenesis and eventual medical therapy.
We systematically searched PubMed, Google Scholar, Web of Science, EMBASE, and Scopus using an extensive list of keywords in the following three categories: 1) Hepatitis B and oxidation 2) Hepatitis B and antioxidant system 3) Effects of approved anti-hepatitis B drugs on redox status. All relevant articles were obtained and reviewed carefully after the exclusion criteria were deployed.
There is great evidence indicating extensive oxidative stress occurs in hepatitis B. This oxidative stress takes place on multiple levels, including lipid peroxidation, DNA oxidation, protein oxidation, and reactive oxygen and nitrogen species production. However, there are also conflicting results with regard to antioxidant therapy and antioxidant status in hepatitis B, some of which may be explained by the concept of "compensatory gaps." Nevertheless, further studies are indicated to reach a more thorough judgment.
Despite the presence of vast oxidative stress in hepatitis B, antioxidant therapy is not always effective as a treatment strategy, especially considering that antioxidants can act as "double-edged swords" or antioxidants; if not used at the right time or place or in the right combination, these substances can easily become pro-oxidants. Therefore, several studies will be needed to determine suitable antioxidant therapies. We propose the "2-step Combined Antioxidant Adjuvant Therapy for hepatitis B (2CAAT Hep B)" as a new strategy for antioxidant adjuvant therapy. We also suggest developing an international platform and database for antioxidant adjuvant therapy in hepatitis B (IPAATH and IDAATH) to canalize this field of research in a standardized direction, especially when complexity is a problem.
Key message "We identified both quantitative and quantitative resistance loci to Leptosphaeria maculans, a fungal pathogen, causing blackleg disease in canola. Several genome-wide significant associations were detected at known and new loci for blackleg resistance. We further validated statistically significant associations in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance in canola." Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola (Brassica napus). This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 694 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07, and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of Arabidopsis thaliana and Brassica napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola.
Ancestral sequence reconstruction has been widely used to test evolution-based hypotheses. The genome of the European tick vector, Ixodes ricinus, encodes for defensin peptides with diverse antimicrobial activities against distantly related pathogens. These pathogens include fungi, Gram-negative, and Gram-positive bacteria, i.e., a wide antimicrobial spectrum. Ticks do not transmit these pathogens, suggesting that these defensins may act against a wide range of microbes encountered by ticks during blood feeding or off-host periods. As demonstrated here, these I. ricinus defensins are also effective against the apicomplexan parasite Plasmodium falciparum. To study the general evolution of antimicrobial activity in tick defensins, the ancestral amino acid sequence of chelicerate defensins, which existed approximately 444 million years ago, was reconstructed using publicly available scorpion and tick defensin sequences (named Scorpions-Ticks Defensins Ancestor, STiDA). The activity of STiDA was tested against P. falciparum and the same Gram-negative and Gram-positive bacteria that were used for the I. ricinus defensins. While some extant tick defensins exhibit a wide antimicrobial spectrum, the ancestral defensin showed moderate activity against one of the tested microbes, P. falciparum. This study suggests that amino acid variability and defensin family expansion increased the antimicrobial spectrum of ancestral tick defensins.
In 2001, the National Health Interview Survey (NHIS) commenced in Taiwan. This survey, conducted on a sample of the whole Taiwanese population, is nationally representative and has a high response rate (>80 %). As a result, the four already completed surveys from 2001 to 2013 can be used to investigate the time trend of smoking prevalence, the rate of cessation, and exposure to secondhand smoking.
There were 72918 adults combined from the 2001, 2005, 2009 and 2013 National Health Interview Surveys (NHIS). Smoking status, exposure to secondhand smoking, and smoking cessation were asked, as well as demographic characteristics and other variables. Statistical analyses with sampling weights were carried out using SAS and SUDAAN.
In males, the prevalence of smoking significantly decreased (rates in 4 surveys were 44.4 %, 44.6 %, 38.9 %, and 34.2 %, respectively). Since 2005 the rate of smoking cessation increased significantly (p = 0.033). The odd ratio (OR) exposure of secondhand among non-smokes (OR) in 2009 and 2013 were 0.96 (CI = 0.85-1.08) and 0.78 (CI = 0.70-0.88) comparing to 2005. In females, the prevalence of smoking was stable over time. The rate of smoking cessation only appeared significantly high in the older age group. The OR for exposure to secondhand smoking were 0.81 (CI = 0.74-0.89) and 0.68 (CI = 0.62-0.74), for 2009 and 2013 comparing to 2005, respectively.
Early anti-smoking legislation in Taiwan might have raised the awareness of the harm of smoking. However, the implementation of the Tobacco Hazards Prevention Act (THPA) in 2009 had great contribution to the reduction of smoking rate, especially in males.
To monitor and improve nation-wide surgical outcome after groin hernia repair based on scientific evidence-based surgical strategies for the national and international surgical community.
Patients ≥18 years operated for groin hernia.
Type and size of hernia, primary or recurrent, type of surgical repair procedure, mesh and mesh fixation methods.
According to the Danish National Health Act, surgeons are obliged to register all hernia repairs immediately after surgery (3 minute registration time). All institutions have continuous access to their own data stratified on individual surgeons. Registrations are based on a closed, protected Internet system requiring personal codes also identifying the operating institution. A national steering committee consisting of 13 voluntary and dedicated surgeons, 11 of whom are unpaid, handles the medical management of the database.
The Danish Inguinal Hernia Database comprises intraoperative data from >130,000 repairs (May 2015). A total of 49 peer-reviewed national and international publications have been published from the database (June 2015).
The Danish Inguinal Hernia Database is fully active monitoring surgical quality and contributes to the national and international surgical society to improve outcome after groin hernia repair.
The purpose of this study was to examine current attitude of hand surgeons toward the Affordable Care Act (ACA). An electronic survey was sent to members of American Society for Surgery of the Hand (ASSH) to examine their attitude toward the Affordable Care Act. 974 ASSH members responded to the survey (33% response rate). The majority of respondents were male (89%), trained in orthopedic surgery (81%), and in private practice (75%). 41% of respondents rated their knowledge of the ACA as average. Respondents disagreed that the ACA would improve healthcare in the United States (median 2, mean 2.06, scale 1-5), while agreeing that the ACA would decrease reimbursements specific to hand surgery (median 4, mean 4.11).
Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type.
The fates of "transboundary" environmental systems depend on how nation states interact with one another. In the absence of a hegemon willing and able to coerce other states into avoiding a "tragedy of the commons," shared environments will be safeguarded if international cooperation succeeds and degraded or even destroyed if it fails. Treaties and related institutions of international law give form to these efforts to cooperate. Often, they implore states to act in their collective (as opposed to their national) interests. Sometimes, they impel cooperating states to punish free riders. A few agreements coordinate states' behavior. Here, I present simple game-theoretic models showing whether and how treaties and related institutions can change incentives, aligning states' self-interests with their collective interests. I show that, as a general matter, states struggle to cooperate voluntarily and enforce agreements to cooperate but that they find it relatively easy to coordinate actions. In some cases, the need for coordination is manifest. In other cases, it requires strategic thinking. Coordination may fall short of supporting an ideal outcome, but it nearly always works better than the alternatives.
Signal transduction pathways activated by chemoattractants have been extensively studied, but little is known about the events mediating responses to mechanical stimuli. We discovered that acute mechanical perturbation of cells triggered transient activation of all tested components of the chemotactic signal transduction network, as well as actin polymerization. Similarly to chemoattractants, the shear flow-induced signal transduction events displayed features of excitability, including the ability to mount a full response irrespective of the length of the stimulation and a refractory period that is shared with that generated by chemoattractants. Loss of G protein subunits, inhibition of multiple signal transduction events, or disruption of calcium signaling attenuated the response to acute mechanical stimulation. Unlike the response to chemoattractants, an intact actin cytoskeleton was essential for reacting to mechanical perturbation. These results taken together suggest that chemotactic and mechanical stimuli trigger activation of a common signal transduction network that integrates external cues to regulate cytoskeletal activity and drive cell migration.
Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of growth factors. The identity of these factors, however, remains largely unknown, and we hypothesized here that the secreted C1q-TNF-related protein-9 (CTRP9) might act as endothelial derived protein to modulate heart remodeling in response to pressure overload.
To examine the source of cardiac CTRP9 and its function during pressure overload.
CTRP9 was mainly derived from myocardial capillary endothelial cells. CTRP9 mRNA expression was enhanced in hypertrophic human hearts and in mouse hearts after transverse aortic constriction (TAC). CTRP9 protein was more abundant in the serum of patients with severe aortic stenosis and in murine hearts after TAC. Interestingly, heterozygous and especially homozygous (KO) C1qtnf9 gene-deleted (CTRP9 knock-out) mice were protected from the development of cardiac hypertrophy, left ventricular dilatation and dysfunction during TAC. CTRP9 overexpression, in turn, promoted hypertrophic cardiac remodeling and dysfunction after TAC in mice and induced hypertrophy in isolated adult cardiomyocytes. Mechanistically, CTRP9 knock-out mice showed strongly reduced levels of activated prohypertrophic ERK5 during TAC compared to wild-type mice, while CTRP9 overexpression entailed increased ERK5 activation in response to pressure overload. Inhibition of ERK5 by a dominant negative MEK5 mutant or by the ERK5/MEK5 inhibitor BIX02189 blunted CTRP9 triggered hypertrophy in isolated adult cardiomyocytes in vitro and attenuated mouse cardiomyocyte hypertrophy and cardiac dysfunction in vivo, respectively. Downstream of ERK5, we identified the prohypertrophic transcription factor GATA4, which was directly activated through ERK5 dependent phosphorylation.
The upregulation of CTRP9 during hypertrophic heart disease facilitates maladaptive cardiac remodeling and left ventricular dysfunction and might constitute a therapeutic target in the future.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated deaths worldwide. Given the efficacy of membrane proteins as therapeutic targets in human malignancies, we examined cell-surface receptors that may act as drivers of lung tumorigenesis. Here we report that the PROTOCADHERIN PCDH7 is overexpressed frequently in NSCLC tumors where this event is associated with poor clinical outcome. PCDH7 overexpression synergized with EGFR and KRAS to induce MAPK signaling and tumorigenesis. Conversely, PCDH7 depletion suppressed ERK activation, sensitized cells to MEK inhibitors and reduced tumor growth. PCDH7 potentiated ERK signaling by facilitating interaction of protein phosphatase PP2A with its potent inhibitor, the SET oncoprotein. By establishing an oncogenic role for PCDH7 in lung tumorigenesis, our results provide a rationale to develop novel PCDH7 targeting therapies which act at the cell surface of NSCLC cells to compromise their growth.
Depression and anxiety often emerge for the first time during youth. The school environment provides an ideal context to deliver prevention programs, with potential to offset the trajectory towards disorder. The aim of this review was to provide a comprehensive evaluation of randomised-controlled trials of psychological programs, designed to prevent depression and/or anxiety in children and adolescents delivered in school settings. Medline, PsycINFO and the Cochrane Library were systematically searched for articles published until February 2015. Eighty-one unique studies comprising 31,794 school students met inclusion criteria. Small effect sizes for both depression (g=0.23) and anxiety (g=0.20) prevention programs immediately post-intervention were detected. Small effects were evident after 12-month follow-up for both depression (g=0.11) and anxiety (g=0.13). Overall, the quality of the included studies was poor, and heterogeneity was moderate. Subgroup analyses suggested that universal depression prevention programs had smaller effect sizes at post-test relative to targeted programs. For anxiety, effect sizes were comparable for universal and targeted programs. There was some evidence that externally-delivered interventions were superior to those delivered by school staff for depression, but not anxiety. Meta-regression confirmed that targeted programs predicted larger effect sizes for the prevention of depression. These results suggest that the refinement of school-based prevention programs have the potential to reduce mental health burden and advance public health outcomes.
Estimates of the burden of antimicrobial resistance (AMR) are needed to ascertain AMR impact, to evaluate interventions, and to allocate resources efficiently. Recent studies have estimated health, cost, and economic burden relating to AMR, with outcomes of interest ranging from drug-bug resistance impact on mortality in a hospital setting to total economic impact of AMR on the global economy. However, recent collation of this information has been largely informal, with no formal quality assessment of the current evidence base (e.g. with predefined checklists). This review therefore aims to establish what perspectives and resulting methodologies have been used in establishing the burden of AMR, whilst also ascertaining the quality of these studies.
The literature review will identify relevant literature using a systematic review methodology. MEDLINE, EMBASE, Scopus and EconLit will be searched utilising a predefined search string. Grey literature will be identified by searching within a predefined list of organisational websites. Independent screening of retrievals will be performed in a two-stage process (abstracts and full texts), utilising a pre-defined inclusion and exclusion criteria. Data will be extracted into a data extraction table and descriptive examination will be performed. Study quality will be assessed using the Newcastle-Ottawa scales and the Philips checklists where appropriate. A narrative synthesis of the results will be presented.
This review will provide an overview of previous health, cost and economic definitions of burden and the resultant impact of these different definitions on the burden of AMR estimated. The review will also explore the methods that have been used to calculate this burden and discuss resulting study quality. This review can therefore act as a guide to methods for future research in this area.
PROSPERO CRD42016037510.
Approximately 29 million individuals are expected to enroll in health insurance using the Patient Protection and Affordable Care Act (ACA) Marketplace by 2022. Those seeking health insurance struggle to understand insurance options and choose a plan that best suits their needs.
We interviewed stakeholders to identify the challenges associated with the ACA Marketplace health insurance enrollment and elicited feedback about what to include in health insurance decision support tools. Interviews were transcribed and themes were identified using inductive thematic analysis.
Stakeholders stated that consumers felt frustrated by unclear terminology, high plan costs, and complex calculations required to assess costs. Consumers felt anxious about making the wrong choice and being unable to change plans within a calendar year. Stakeholders recommended using plain language tables defining complex terms, grouping information, and using engaging graphics to communicate information about health insurance. Stakeholders thought that narratives of how others made decisions about insurance might be helpful to consumers, but recommended that they be tailored to the needs of specific consumers.
Strategies that clarify health insurance terms using plain language and graphics, acknowledge concern associated with making the wrong choice, calculate and enable cost comparison, and tailor information to consumers' unique needs could benefit those enrolling in ACA Marketplace plans, Narratives developed should be simple and inclusive enough for diverse populations.
The internal C:N balance must be tightly controlled for the normal growth and development of plants. However, the underlying mechanisms, by which plants sense and balance the intracellular C:N status correspondingly to exogenous C:N availabilities remain elusive. In this study, we use comparative gene expression analysis to identify genes that are responsive to imbalanced C:N treatments in the aerial parts of rice seedlings. Transcripts of rice seedlings treated with four C:N availabilities (1:1, 1:60, 60:1 and 60:60) were compared and two groups of genes were classified: high C:low N responsive genes and low C:high N responsive genes. Our analysis identified several functional correlated genes including chalcone synthase (CHS), chlorophyll a-b binding protein (CAB) and other genes that are implicated in C:N balancing mechanism, such as alternative oxidase 1B (OsAOX1B), malate dehydrogenase (OsMDH) and lysine and histidine specific transporter 1 (OsLHT1). Additionally, six jasmonate synthetic genes and key regulatory genes involved in abiotic and biotic stresses, such as OsMYB4, autoinhibited calcium ATPase 3 (OsACA3) and pleiotropic drug resistance 9 (OsPDR9), were differentially expressed under high C:low N treatment. Gene ontology analysis showed that high C:low N up-regulated genes were primarily enriched in fatty acid biosynthesis and defense responses. Coexpression network analysis of these genes identified eight jasmonate ZIM domain protein (OsJAZ) genes and several defense response related regulators, suggesting that high C:low N status may act as a stress condition, which induces defense responses mediated by jasmonate signaling pathway. Our transcriptome analysis shed new light on the C:N balancing mechanisms and revealed several important regulators of C:N status in rice seedlings.
The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.
It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.
The 2014 United States Preventive Services Task Force systematic review found abdominal aortic aneurysm (AAA) screening decreased related mortality by close to half. Despite the simplicity of screening, research suggests poor adherence to the recommended AAA screening guidelines. Using the quality improvement plan-study-do-act cycle, we retrospectively established poor adherence to AAA screening and poor documentation of smoking history in our resident clinic. An electronic reminder was prospectively implemented into our electronic medical record (EMR) with the goal of improving screening rates. After 1 year, a retrospective chart review was conducted. Comparisons of the pre- and post-electronic reminder intervention data were made using chi-square tests and odds ratios (OR). The purposeful AAA screening rate improved 27.8% during the intervention, 40.3% (95% confidence interval [CI]: 28.6-52.0%) versus 12.5% (95% CI: 3.1-21.9%), p = .002, suggesting patients were more likely to be screened as a result of the electronic reminder, OR = 4.73 (95% CI: 1.77-12.65). This improvement translates to a large effect size, Cohen's d = 0.86 (95% CI: 0.31-1.40). Electronic reminders are a simple EMR addition that can provide evidence-based education while improving adherence rates with preventive health screening measures.
The Affordable Care Act established policy mechanisms to increase health insurance coverage in the United States. While insurance coverage has increased, 10%-15% of the US population remains uninsured.
To assess whether health insurance literacy and financial literacy predict being uninsured, covered by Medicaid, or covered by Marketplace insurance, holding demographic characteristics, attitudes toward risk, and political affiliation constant.
Analysis of longitudinal data from fall 2013 and spring 2015 including financial and health insurance literacy and key covariates collected in 2013.
A total of 2742 US residents ages 18-64, 525 uninsured in fall 2013, participating in the RAND American Life Panel, a nationally representative internet panel.
Self-reported health insurance status and type as of spring 2015.
Among the uninsured in 2013, higher financial and health insurance literacy were associated with greater probability of being insured in 2015. For a typical uninsured individual in 2013, the probability of being insured in 2015 was 8.3 percentage points higher with high compared with low financial literacy, and 9.2 percentage points higher with high compared with low health insurance literacy. For the general population, those with high financial and health insurance literacy were more likely to obtain insurance through Medicaid or the Marketplaces compared with being uninsured. The magnitude of coefficients for these predictors was similar to that of commonly used demographic covariates.
A lack of understanding about health insurance concepts and financial illiteracy predict who remains uninsured. Outreach and consumer-education programs should consider these characteristics.
Co-inhibitory and co-stimulatory receptors act in concert to regulate adaptive immune cell function, and the balance of these receptors is essential for the maintenance of immune homeostasis. Tumors constitute highly suppressive microenvironments in which elevated expression of co-inhibitory receptors on tumor-infiltrating lymphocytes (TILs) is often found. Functional blockade of the co-inhibitory receptors such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) have yielded encouraging outcomes in tumors, generating substantial interest in seeking for additional co-inhibitory molecules that may act as potential interfering targets. T-cell Ig and ITIM domain (TIGIT) is a newly identified co-inhibitory receptor expressed by regulatory T cells (Tregs), activated T cells, and natural killer (NK) cells. Several groups reported consistently that TIGIT expression was elevated on CD8(+) TILs and Tregs in a variety of tumors. Moreover, TIGIT blockade has exhibited therapeutic benefits in animal models of different tumors. Therefore, TIGIT upregulation plays an important role in tumor immunity and may serve as a promising therapeutic target for tumor management.
The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNA(Asp), we showed that this natural Dnmt2 substrate could be engineered to employ RNAs that act like guide RNAs in vitro. The 5'-half of tRNA(Asp) was able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.
Bilevel optimization, as the name reflects, deals with optimization at two interconnected hierarchical levels. The aim is to identify the optimum of an upper level leader problem, subject to the optimality of a lower level follower problem. Several problems from the domain of engineering, logistics, economics and transportation have inherent nested structure which requires them to be modeled as bilevel optimization problems. Increasing size and complexity of such problems has prompted active theoretical and practical interest in the design of efficient algorithms for bilevel optimization. Given the nested nature of bilevel problems, the computational effort (number of function evaluations) required to solve them is often quite high. In this paper, we explore the use of a Memetic Algorithm (MA) to solve bilevel optimization problems. WhileMAs have been quite successful in solving single level optimization problems, there have been relatively few studies exploring their potential for solving bilevel optimization problems. MAs essentially attempt to combine advantages of global and local search strategies to identify optimum solutions with low computational cost (function evaluations). The approach introduced in this paper is a nested Bilevel Memetic Algorithm (BLMA). At both upper and lower levels, either a global or a local search method is used during different phases of the search. The performance of BLMA is presented on twenty five standard test problems and two real-life applications. The results are compared with other established algorithms to demonstrate the efficacy of the proposed approach.
The aim of this study was to compare responses to single-agent chemotherapies and evaluate the predictive factors of resistance in low risk (LR) gestational trophoblastic disease (GTD). The chemotherapy agents included methotrexate (MTX) and actinomycin D (ACT-D).
We conducted a retrospective study of 126 patients with GTD who were treated between 2000 and 2013. A total of 71 patients with LR GTD were treated with MTX (8-day regimen or weekly regimen, n=53) or ACT-D (bi-weekly pulsed regimen or 5-day regimen, n=18). The successful treatment group and the failed treatment group were compared and analyzed to identify prognostic factors.
The complete response rates were 83.3% for ACT-D and 62.2% for MTX, with no statistically significant difference. There was no severe adverse effect reported for either group. Longer interval durations from the index pregnancy (>2 months, p=0.040) and larger tumor size (>3 cm, p=0.020) were more common in non-responders than in responders; these results were statistically significant.
Based on our results, ACT-D may be a better option than MTX as a first-line single chemotherapy agent for LR GTD. The bi-weekly pulsed ACT-D regimen had minimal, or at least the same, toxicities compared with MTX. However, due to the lack of strong supporting evidence, it cannot be conclusively stated that this is the best single agent for first-line chemotherapy in LR GTD patients. Further larger controlled trials will be necessary to establish the best guidelines for GTD treatment.
Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP is widely distributed in the nervous system, particularly at anatomical areas thought to be involved with migraine pathophysiology, including the trigeminovascular nociceptive system. Over the past two decades, a convergence of basic and clinical evidence has established the CGRP as a key player in migraine. CGRP enhances sensitivity to sensory input at multiple levels in both the periphery and central nervous system. Within the brain, the wide distribution of CGRP and CGRP receptors provides numerous possible targets for CGRP to act as a neuromodulator. Now, CGRP has emerged as a promising therapeutic target for a number of novel treatments for migraine. This review discusses the evidence behind the role of CGRP in migraine and the state of CGRP-based mechanism treatment development.
GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.
Immunization rates among medicine and nursing students -and among health professional in general- during hospital training are low. It is necessary to investigate the causes for these low immunization rates. The objective of this study was to design and validate a questionnaire for exploring the attitudes and behaviours of medicine and nursing students toward immunization of vaccine-preventable diseases.
An instrument validation study. The sample included 646 nursing and medicine students at University of Oviedo, Spain. It was a non-ramdom sampling. After the content validation process, a 24-item questionnaire was designed to assess attitudes and behaviours/behavioural intentions. Reliability (ordinal alpha), internal validity (exploratory factor analysis by parellel analysis), ANOVA and mediational model tests were performed.
Exploratory factor analysis yielded two factors which accounted for 48.8% of total variance. Ordinal alpha for the total score was 0.92. Differences were observed across academic years in the dimensions of attitudes (F5.447=3.728) and knowledge (F5.448=65.59), but not in behaviours/behavioural intentions (F5.461=1.680). Attitudes demonstrated to be a moderating variable of knowledge and attitudes/behavioural attitudes (Indirect effect B=0.15; SD=0.3; 95% CI:0.09-0.19).
We developed a questionnaie based on sufficient evidence of reliability and internal validity. Scores on attitudes and knowledge increase with the academic year. Attitudes act as a moderating variable between knowledge and behaviours/behavioural intentions.
Quantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔC t method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.
Due to undesirable hazardous interactions with biological systems, this investigation was undertaken to evaluate the effect of chronic exposure to silver on certain biochemical and some oxidative stress parameters with histopathological examination of brain, as well as the possible protective role of selenium and/or vitamin E as nutritional supplements. Thirty six male rats were divided into six groups of six each: the first group used as a control group. Group II given both vitamin E (400 mg/kg) of diet and selenium (Se) (1 mg/L) in their drinking water. Group III given silver as silver nitrate (AgNO3) (20 mg/L). Group IV given vitamin E and AgNO3. Group V given both AgNO3 and selenium. Group VI given AgNO3, vitamin E and Se. The animals were in the same exposure conditions for 3 months. According to the results which have been obtained; there was an increase in serum lactate dehydrogenase (LDH), lipase activities and cholesterol level, a decrease in serum total protein, calcium and alkaline phosphatase (ALP) activity in Ag-intoxicated rats. Moreover, the findings showed that Ag(+) ions affected antioxidant defense system by decreasing superoxide dismutase (SOD) activity and increasing vitamin E concentration with a high level of malondialdehyde (MDA) in brain tissue. The histological examination also exhibited some nervous tissue alterations including hemorrhage and cytoplasm vacuolization. However, the co-administration of selenium and/or vitamin E ameliorated the biochemical parameters and restored the histological alterations. In conclusion, this study indicated that silver could cause harmful effects in animal body and these effects can be more toxic in high concentrations or prolonged time exposure to this metal. However, selenium and vitamin E act as powerful antioxidants which may exercise adverse effect against the toxicity of this metal.
A defined balance between the generation and scavenging of reactive oxygen species (ROS) is essential to utilize ROS as an adaptive defense response of plants under biotic and abiotic stress conditions. Moreover, ROS are not only a major determinant of stress response but also act as signaling molecule that regulates various cellular processes including plant-microbe interaction. In particular, rhizosphere constitutes the biologically dynamic zone for plant-microbe interactions which forms a mutual link leading to reciprocal signaling in both the partners. Among plant-microbe interactions, symbiotic associations of arbuscular mycorrhizal fungi (AMF) and arbuscular mycorrhizal-like fungus especially Piriformospora indica with plants are well known to improve plant growth by alleviating the stress-impacts and consequently enhance the plant fitness. AMF and P. indica colonization mainly enhances ROS-metabolism, maintains ROS-homeostasis, and thereby averts higher ROS-level accrued inhibition in plant cellular processes and plant growth and survival under stressful environments. This article summarizes the major outcomes of the recent reports on the ROS-generation, scavenging and signaling in biotic-abiotic stressed plants with AMF and P. indica colonization. Overall, a detailed exploration of ROS-signature kinetics during plant-AMF/P. indica interaction can help in designing innovative strategies for improving plant health and productivity under stress conditions.
Acute graft-versus-host disease (GvHD) causes high mortality in patients undergoing allogeneic hematopoietic cell transplantation. An early event in the classical pathogenesis of acute GvHD is tissue damage caused by the conditioning treatment or infection that consecutively leads to translocation of bacterial products [pathogen-associated molecular patterns (PAMPs)] into blood or lymphoid tissue, as well as danger-associated molecular patterns (DAMPs), mostly intracellular components that act as pro-inflammatory agents, once they are released into the extracellular space. A subtype of DAMPs is nucleotides, such as adenosine triphosphate released from dying cells that can activate the innate and adaptive immune system by binding to purinergic receptors. Binding to certain purinergic receptors leads to a pro-inflammatory microenvironment and promotes allogeneic T cell priming. After priming, T cells migrate to the acute GvHD target organs, mainly skin, liver, and the gastrointestinal tract and induce cell damage that further amplifies the release of intracellular components. This review summarizes the role of different purinergic receptors in particular P2X7 and P2Y2 as well as nucleotides in the pathogenesis of GvHD.
Virtually all multicellular organisms host a community of symbionts composed of mutualistic, commensal, and pathogenic microbes, i.e., their microbiome. The mechanism of selection on host-microbe assemblages remains contentious, particularly regarding whether selection acts differently on hosts and their microbial symbionts. Here, we attempt to reconcile these viewpoints using a model that describes how hosts and their microbial symbionts alter each other's niche and thereby fitness. We describe how host-microbe interactions might change the shape of the host niche and/or reproductive rates within it, which are directly related to host fitness. A host may also alter the niche of a symbiotic microbe, although this depends on the extent to which that microbe is dependent on the host for reproduction. Finally, we provide a mathematical model to test whether interactions between hosts and microbes are necessary to describe the niche of either partner. Our synthesis highlights the phenotypic effects of host-microbe interactions while respecting the unique lifestyles of each partner, and thereby provides a unified framework to describe how selection might act on a host that is associated with its microbiome.
Objective There is little evidence showing that inhabitants of urban areas engage in more physical activity than do rural ones, although accumulating evidence concerning the best neighborhood environments conducive to physical activity supports the idea. This study sought to fill the research gap by examining the association between city scale and daily steps using data from Japan's National Health and Nutrition Survey (NHNS).Methods We analyzed data from 15,763 men and 18,479 women aged 20 years and older who participated in a one-day pedometer measurement during any part of the NHNS between 2006 and 2010. The data obtained for these years were combined into a single data set. City scale was categorized into 5 groups based on population: 1) 12 large cities and 23 wards, 2) population greater than 150,000, 3) population 50,000-150,000, 4) population less than 50,000, and 5) towns and villages. Differences in daily steps among city scale groups were analyzed using an ANCOVA, adjusting for age by gender. The Bonferroni method was employed for multiple comparisons, and linear regression was used to test for linear trends. Subgroup analyses were performed by age (20-39, 40-64, older than 64), and job status. The study was approved by the Tokyo Medical University ethics committee, and use of these data complied with the Statistics Act of Japan.Results The steps per day for men after adjusting for age were Group 1 : 7,494±4,429 (mean±SD), Group 2 : 7,407±4,428, Group 3 : 7,206±4,428, Group 4 : 6,911±4,428, and Group 5 : 6,715±4,429. Women's daily steps according to city scale group were 6,767±3,648, 6,386±3,647, 6,062±3,646, 6,069±3,649, and 6,070±3,649 for Groups 1 through 5, respectively. There were overall statistically significant differences (ANCOVA, P<0.001) between both genders. The larger the city scale, the more mean daily steps that were taken by both men and women (P values for both trends <0.001). Subgroup analyses revealed significant differences in the mean daily steps by city scale, regardless of age or job status for both genders. There were no significant differences among Groups 3, 4, or 5 among unemployed men, men older than 64, or overall women, which differed from the results for men with jobs, who took fewer steps in smaller cities.Conclusions Our study showed that men and women living in larger cities took more steps compared to those living in smaller cities. Subgroup analyses further revealed that the associations between city scale and physical activity differed according to gender, age, and job status.
Intestinal fibrosis is an intractable complication of Crohn's disease (CD), and, when occurring excessively, causes severe intestinal obstruction that often necessitates surgical resection. The fibrosis is characterized by an imbalance in the turnover of extracellular matrix (ECM) components, where intestinal fibroblasts/myofibroblasts play active roles in ECM production, fibrogenesis and tissue remodeling, which eventually leads to the formation of stenotic lesions. There is however a great paucity of knowledge about how intestinal fibrosis initiates and progresses, which hampers the development of effective pharmacotherapies against CD. Recently, we explored the potential implications of transient receptor potential (TRP) channels in the pathogenesis of intestinal fibrosis, since they are known to act as cellular stress sensors/transducers affecting intracellular Ca(2+) homeostasis/dynamics, and are involved in a broad spectrum of cell pathophysiology including inflammation and tissue remodeling. In this review, we will place a particular emphasis on the intestinal fibroblast/myofibroblast TRPC6 channel to discuss its modulatory effects on fibrotic responses and therapeutic potential for anti-fibrotic treatment against CD-related stenosis.
Rural populations in the U.S. face numerous barriers to health care access. The Patient Protection and Affordable Care Act (PPACA) was developed in part to reduce health care access barriers. We report rural women's access barriers and the PPACA elements that address these barriers as well as potential gaps.
For this qualitative study, we analyzed two datasets using a common framework. We used content analysis to understand rural, focus group participants' access barriers prior to PPACA implementation. Subsequently, we analyzed the PPACA text.
Participants described health care access barriers in two domains: availability and eligibility. The PPACA proposes solutions within each domain, including health care workforce training, Medicaid expansion, and employer-based health care provisions. However, in rural settings, access barriers likely persist.
While elements of the PPACA address some health care access barriers, additional research and policy development are needed to comprehensively and equitably address persistent access barriers for rural women.
Farmworkers are a unique population within rural communities and are often overlooked and undercounted. They face significant disparities in health and health care access compared with the general rural population. One goal of the Patient Protection and Affordable Care Act (ACA) is to increase access to health care and health insurance for the country's most vulnerable and underserved populations. Farmworkers' numerous barriers to health care and health insurance remain, despite the ACA's progress. Apart from anecdotal accounts, we lack the necessary data to assess the ACA's impact on farmworker communities. This commentary imparts information about farmworker enrollment in and barriers to accessing health insurance, collected through individual conversations, focus groups, interviews, and informal surveys. Based on identified challenges and limitations, we make policy recommendations to assess and improve the implementation and relevance of the ACA in farmworker communities.
Children who are deafblind are one of the lowest-incidence yet most diverse groups receiving services mandated by the Individuals With Disabilities Education Improvement Act. Despite this population's diversity, the development of communication skills is critical for all children who are deafblind, and is the foundation on which good transition planning can be built. The authors describe key research findings and other professional literature on transition planning and services guided by the quality of life principle. The role of the individualized education program and case law in transition planning is discussed. Through a person-centered approach to transition planning, a coordinated set of activities designed to support the young adult in moving from school to postschool settings and activities is identified. The authors conclude that effective transition efforts will involve extensive collaboration among school and agency professionals, families, and the young adult who is deafblind.
The study of neuropathological markers in patients affected by mental/psychiatric disorders is relevant for the comprehension of the pathogenesis and the correlation with the clinical symptomatology. The neuropathology of Alzheimer's disease (AD) recognizes intraneuronal and extracellular neurofibrillary formation responsible for neuronal degeneration. Immunohistochemical studies discovered many interesting results for a better interpretation of the AD pathogenesis, while the "metal hypothesis" supports that metal ions might differentially influence the formation of amyloid aggregates. The most relevant pathological findings reported in schizophrenia originate from computer assisted tomography (CT), Magnetic Resonance Imaging (MRI) studies and Diffusion Tensor Imaging (DTI), suggesting the brain abnormalities involved in the pathophysiology of schizophrenia. The theory of fetal programming illustrates the epigenetic factors that may act during the intrauterine life on brain development, with relevant consequences on the susceptibility to develop AD or schizophrenia later in life. The neuropathological interpretation of AD and schizophrenia shows that the presence of severe neuropathological changes is not always associated with severe cognitive impairment. A better dialogue between psychiatrics and pathologists might help to halt insurgence and progression of neurodegenerative diseases.
Pluripotent cells emerging at very early stages of development are the founders of differentiated cells. It has been established in mouse that the LIF/Jak/Stat-Nanog axis acts as a positive regulator to support the pluripotent state of cells whereas Fgf/Erk signaling acts as a negative regulator to direct cells to enter the differentiating state. In chicken, although Fgf/Erk signaling is known to act as a negative regulator, positive regulators remained unknown. Here, to identify positive regulator(s) of chicken pluripotency, we selected Jak1/Stat3 signaling as a candidate based on transcriptome analyses. Jak1/Stat3 signaling was activated specifically at stages before gastrulation: Stat3 protein was localized in nuclei at blastodermal stages, but translocated to cytoplasm after gastrulation. We conducted pharmacological and gene transfection analyses in the blastoderm-derived colony formation assay, in which Nanog-positive dense colonies represent a hallmark of the undifferentiated state, and found that Jak1/Stat3 signaling supports pluripotency in chicken early embryos. Jak1 inhibition abolished the formation of dense colonies, but the colony formation was restored when Stat3ER was artificially activated. We propose that the molecular mechanisms regulating pluripotency are conserved at the signaling network level between mouse and chicken, and possibly among a wider range of species.
The basic mechanisms underlying noxious cold perception are not well understood. We developed Drosophila assays for noxious cold responses. Larvae respond to near-freezing temperatures via a mutually exclusive set of singular behaviors-in particular, a full-body contraction (CT). Class III (CIII) multidendritic sensory neurons are specifically activated by cold and optogenetic activation of these neurons elicits CT. Blocking synaptic transmission in CIII neurons inhibits CT. Genetically, the transient receptor potential (TRP) channels Trpm, NompC, and Polycystic kidney disease 2 (Pkd2) are expressed in CIII neurons, where each is required for CT. Misexpression of Pkd2 is sufficient to confer cold responsiveness. The optogenetic activation level of multimodal CIII neurons determines behavioral output, and visualization of neuronal activity supports this conclusion. Coactivation of cold- and heat-responsive sensory neurons suggests that the cold-evoked response circuitry is dominant. Our Drosophila model will enable a sophisticated molecular genetic dissection of cold nociceptive genes and circuits.
Every spring, deer cast their old antlers and initiate a regeneration process, which yields a new set of antlers of up to 1m in length. Over the course of three months, branches of the trigeminal nerve, originating from the frontal skull, innervate velvet, a modified skin that covers the regenerating antler. The rate of growth of these axons reaches up to 2cm per day making them the fastest regenerating axons in adult mammals. Here, we aim to identify the factors secreted by velvet that promote such high speed axon growth. Our experiments with cultures of adult rat trigeminal neurons demonstrate that conditioned medium harvested from velvet organotypic cultures has greater axon growth-promoting properties than a medium conditioned by normal skin. The axon growth-promoting effects of velvet act synergistically with the extracellular matrix (ECM) protein laminin, a component of the basal lamina present in the deer antler. Our proteomic analyses identified several axon growth promoters in the velvet-conditioned medium (VCM), including soluble proteins such as nerve growth factor (NGF) and apolipoprotein A-1, as well as matrix extracellular proteins, such as periostin and SPARC. Additional in vitro analyses allowed us to determine that a synergic relationship between periostin and NGF may contribute to neurite growth-promoting effects of velvet secretome. A combinatorial approach using these factors may promote regeneration at high speeds in patients with peripheral neuropathies.
a variety of services to support women to undertake weight management behaviours during pregnancy have recently been implemented as a means to reduce the risks to mother and infant. In the UK, midwives lead the care of the majority of pregnant women and are seen as the ideal source of referral into antenatal services. However, midwives have reported concerns regarding raising the topic of weight with obese women and negative referral experiences have been cited as a reason not to engage with a service. This study explored midwives' experiences of referring women to one of two antenatal weight management services.
qualitative, cross-sectional interview and focus group study, with data analysed thematically.
midwifery teams in the West Midlands, England.
midwives responsible for referring to either a home-based, one to one service (N=12), or a community-based, group service (N=11).
four themes emerged from the data. Participants generally had a positive View of the service, but their Information needs were not fully met, as they wanted more detail about the service and feedback regarding the women they had referred. Approaches to referral differed, with some participants referring all women who met the eligibility criteria, and some offering women a choice to be referred or not. Occasionally the topic was not raised at all when a negative reception was anticipated. Reasons for poor uptake of the services included pragmatic barriers, and their perception of women's lack of interest in weight management.
midwives' differing views on choice and gaining agreement to refer means referral practices vary, which could increase the risk that obese women have inequitable access to weight management services. However, midwives' confidence in the services on offer may be increased with more detailed information about the service and feedback on referrals, which would additionally act as prompts to refer.
weight management services need to improve communication with their referral agents and try to overcome practical and psychosocial barriers to uptake. It would be beneficial to develop a shared understanding of the concept of 'informed choice' specifically regarding referral to health promotion services among midwives. Training which demonstrates effective methods of sensitively introducing a weight management service to obese women may increase midwives' confidence to consistently include this in their practice. These measures may improve women's engagement with services which have the potential to reduce the risks associated with maternal obesity.
Pharmaceutical products (PhP) are one of the most alarming emergent pollutants in the environment. Therefore, it is of extreme importance to investigate efficient PhP removal processes. Biologic synthesis of platinum nanoparticles (Bio-Pt) has been reported, but their catalytic activity was never investigated. In this work, we explored the potential of cell-supported platinum (Bio-Pt) and palladium (Bio-Pd) nanoparticles synthesized with Desulfovibrio vulgaris as biocatalysts for removal of four PhP: ciprofloxacin, sulfamethoxazole, ibuprofen and 17β-estradiol. The catalytic activity of the biological nanoparticles was compared with the PhP removal efficiency of D. vulgaris whole-cells. In contrast with Bio-Pd, Bio-Pt has a high catalytic activity in PhP removal, with 94, 85 and 70% removal of 17β-estradiol, sulfamethoxazole and ciprofloxacin, respectively. In addition, the estrogenic activity of 17β-estradiol was strongly reduced after the reaction with Bio-Pt, showing that this biocatalyst produces less toxic effluents. Bio-Pt or Bio-Pd did not act on ibuprofen, but this could be completely removed by D. vulgaris whole-cells, demonstrating that sulfate-reducing bacteria are among the microorganisms capable of biotransformation of ibuprofen in anaerobic environments. This study demonstrates for the first time that Bio-Pt has a high catalytic activity, and is a promising catalyst to be used in water treatment processes for the removal of antibiotics and endocrine disrupting compounds, the most problematic PhP.
Depressive symptoms occur in approximately half of trauma patients, negatively impacting on functional outcome and quality of life following severe head injury. Pontine noradrenaline has been shown to increase upon trauma and associated β-adrenergic receptor activation appears to consolidate memory formation of traumatic events. Blocking adrenergic activity reduces physiological stress responses during recall of traumatic memories and impairs memory, implying a potential therapeutic role of β-blockers. This study examines the effect of pre-admission β-blockade on post-traumatic depression.
All adult trauma patients (≥18 years) with severe, isolated traumatic brain injury (intracranial Abbreviated Injury Scale score (AIS) ≥3 and extracranial AIS <3) were recruited from the trauma registry of an urban university hospital between 2007 and 2011. Exclusion criteria were in-hospital deaths and prescription of antidepressants up to one year prior to admission. Pre- and post-admission β-blocker and antidepressant therapy data was requested from the national drugs registry. Post-traumatic depression was defined as the prescription of antidepressants within one year of trauma. Patients with and without pre-admission β-blockers were matched 1:1 by age, gender, Glasgow Coma Scale, Injury Severity Score and head AIS. Analysis was carried out using McNemar's and Student's t-test for categorical and continuous data, respectively.
A total of 545 patients met the study criteria. Of these, 15% (n=80) were prescribed β-blockers. After propensity matching, 80 matched pairs were analyzed. 33% (n=26) of non β-blocked patients developed post-traumatic depression, compared to only 18% (n=14) in the β-blocked group (p=0.04). There were no significant differences in ICU (mean days: 5.8 (SD 10.5) vs. 5.6 (SD 7.2), p=0.85) or hospital length of stay (mean days: 21 (SD 21) vs. 21 (SD 20), p=0.94) between cohorts.
β-blockade appears to act prophylactically and significantly reduces the risk of post-traumatic depression in patients suffering from isolated severe traumatic brain injuries. Further prospective randomized studies are warranted to validate this finding.
The literature reports a significant association between various mental disorders and asthma, in particular depression and/or anxiety, with some more robust data regarding anxiety disorders. However, the nature of this association remains largely unclear.
(1) To test the hypothesis of a specific association of anxiety and depressive disorder (according to the DSM-IV) with asthma and (2) to test the bidirectional hypothesis of causality between asthma and psychiatric disorders.
Ninety-six adults were compared with 96 control subjects matched according to main socio-demographic variables (i.e., gender, age, marital status, cohabiting/non-cohabiting, and BMI). Subjects with asthma were divided according to GINA and ACT classifications. All subjects underwent Structured Clinical Interviews for DSM-IV Axis I (SCID-I) diagnosis.
Significant association between asthma and lifetime anxiety disorders emerged (OR 3.03; p = 0.003); no significant association with other psychiatric diagnosis emerged. Moreover, lifetime and current anxiety were associated with asthma severity levels (p < 0.01 and p = 0.001 based on age). Asthma preceded anxiety in 48% of cases; in 52% of cases, anxiety preceded asthma, without significant group differences. The risk of asthma, particularly of severe, uncontrolled forms (p < 0.01), resulted higher in lifetime anxiety disorder patients (p = 0.003 and p = 0.001 based on age at onset). Current anxiety increased the risk of asthma, and that of an uncontrolled form (p < 0.05). Asthma increased the risk of lifetime anxiety disorders (p = 0.002 and p = 0.018 using ages). Intermittent asthma increased the risk of lifetime and current anxiety disorders (p < 0.01).
Anxiety disorders, in particular Lifetime Anxiety Disorders, represent the only psychiatric disorder significantly associated with asthma, with a possible bidirectional, anxiety-asthma relationship, each of which can be caused or result from the other.
DIX domain containing 1 (DIXDC1), the human homolog of coiled-coil-DIX1 (Ccd1), is a positive regulator of Wnt signaling pathway. Recently, it was found to act as a candidate oncogene in colon cancer, non-small-cell lung cancer, and gastric cancer. In this study, we aimed to investigate the clinical significance of DIXDC1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that DIXDC1 was overexpressed in glioma tissues and glioma cell lines. The expression level of DIXDC1 was evidently linked to glioma pathological grade and Ki-67 expression. Kaplan-Meier curve showed that high expression of DIXDC1 may lead to poor outcome of glioma patients. Serum starvation and refeeding assay indicated that the expression of DIXDC1 was associated with cell cycle. To determine whether DIXDC1 could regulate the proliferation and migration of glioma cells, we transfected glioma cells with interfering RNA-targeting DIXDC1; investigated cell proliferation with Cell Counting Kit (CCK)-8, flow cytometry assays, and colony formation analyses; and investigated cell migration with wound healing assays and transwell assays. According to our data, knockdown of DIXDC1 significantly inhibited proliferation and migration of glioma cells. These data implied that DIXDC1 might participate in the development of glioma, suggesting that DIXDC1 can become a potential therapeutic strategy for glioma.
We previously reported that the injection of nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. Our previous study established a severely anemic mouse model that was treated with a combination of NBP with phenylhydrazine (PHZ), which induced newly discovered hematopoietic organs in the omentum. No reports have shown that new hematopoietic organs form under any condition. We characterized the structures and factors related to the formation of these new organs. Splenectomized mice were treated with NBP to inhibit erythropoiesis in the BM and then injected with PHZ to induce hemolytic anemia. The mice showed severe anemia and wine-colored structures appeared in the omentum. Some hematopoietic cells, including megakaryocytes, and well-developed sinuses were observed in these structures. Numerous TER119-positive erythroblasts were located with cells positive for PCNA, a cell proliferation marker. C-kit-positive cells were detected and mRNAs related to hematopoiesis were expressed in these structures. Moreover, TER119-positive erythroblasts emerged and formed clusters and hematopoiesis-related factors were detected in the omentum of mice treated with NBP and PHZ. The levels of G-CSF in the serum and hematopoietic progenitor cells (HPCs) in the peripheral blood were increased upon treatment with both NBP and PHZ. These results suggest that the induced hematopoietic structures act as the sites of erythropoiesis and that NBP-induced G-CSF production causes HPC mobilization, homing and colonization in the omentum because they constitutively express some factors, including SDF-1; thus, the newly discovered hematopoietic structure in this study might be formed.
The human respiratory syncytial virus (hRSV) is the major cause of lower respiratory tract infection in children and elderly people worldwide. Its genome encodes 11 proteins including SH protein, whose functions are not well known. Studies show that SH protein increases RSV virulence degree and permeability to small compounds, suggesting it is involved in the formation of ion channels. The knowledge of SH structure and function is fundamental for a better understanding of its infection mechanism. The aim of this study was to model, characterize, and analyze the structural behavior of SH protein in the phospholipids bilayer environment. Molecular modeling of SH pentameric structure was performed, followed by traditional molecular dynamics (MD) simulations of the protein immersed in the lipid bilayer. Molecular dynamics with excited normal modes (MDeNM) was applied in the resulting system in order to investigate long time scale pore dynamics. MD simulations support that SH protein is stable in its pentameric form. Simulations also showed the presence of water molecules within the bilayer by density distribution, thus confirming that SH protein is a viroporin. This water transport was also observed in MDeNM studies with histidine residues of five chains (His22 and His51), playing a key role in pore permeability. The combination of traditional MD and MDeNM was a very efficient protocol to investigate functional conformational changes of transmembrane proteins that act as molecular channels. This protocol can support future investigations of drug candidates by acting on SH protein to inhibit viral infection. Graphical Abstract The ion channel of the human respiratory syncytial virus (hRSV) small hydrophobic protein (SH) transmembrane domainᅟ.
Mutations in the bone morphogenetic protein receptor (BMPR2) gene have been observed in 70 % of patients with heritable pulmonary arterial hypertension (HPAH) and in 11-40 % with idiopathic PAH (IPAH). However, carriers of a BMPR2 mutation have only 20 % risk of developing PAH. Since inflammatory mediators are increased and predict survival in PAH, they could act as a second hit inducing the development of pulmonary hypertension in BMPR2 mutation carriers. Our specific aim was to determine whether inflammatory mediators could contribute to pulmonary vascular cell dysfunction in PAH patients with and without a BMPR2 mutation. Pulmonary microvascular endothelial cells (PMEC) and arterial smooth muscle cells (PASMC) were isolated from lung parenchyma of transplanted PAH patients, carriers of a BMPR2 mutation or not, and from lobectomy patients or lung donors. The effects of CRP and TNFα on mitogenic activity, adhesiveness capacity, and expression of adhesion molecules were investigated in PMECs and PASMCs. PMECs from BMPR2 mutation carriers induced an increase in PASMC mitogenic activity; moreover, endothelin-1 secretion by PMECs from carriers was higher than by PMECs from non-carriers. Recruitment of monocytes by PMECs isolated from carriers was higher compared to PMECs from non-carriers and from controls, with an elevated ICAM-1 expression. CRP increased adhesion of monocytes to PMECs in carriers and non-carriers, and TNFα only in carriers. PMEC from BMPR2 mutation carriers have enhanced adhesiveness for monocytes in response to inflammatory mediators, suggesting that BMPR2 mutation could generate susceptibility to an inflammatory insult in PAH.
Comorbidities associated with obesity have become a worldwide public health concern. Obesity-associated hepatic steatosis is not benign, and the risk of developing severe liver disease is high. Currently, biopsy is the only clinical tool available for the diagnosis of pathological alterations in the liver. However, the procedure is painful and not without risk. As such, there is a need to identify non-invasive biomarkers of steatosis. There has been considerable progress in this area, but research appears to be limited to measurements of levels of certain parameters in patients with liver impairment relative to those of healthy controls. The clinically relevant aim should be to distinguish, at an early stage, those obese individuals with liver steatosis from those obese individuals without it. Plasma constituents that act as surrogates of altered hepatic energy metabolism in response to food intake are likely candidates. Targeted metabolomics, combined with quantitation of the metabolites involved, has been shown to be an efficient measurement tool. Indeed, the evaluation of exhaled volatile compounds might be sufficient, while other rapid, sensitive, and reproducible methods have been validated in preliminary studies in various clinical settings. Metabolomics methods are promising but require considerable expertise and sophisticated (and expensive) equipment not readily available in all centers. The challenge is to adapt this newly acquired, expanding knowledge to current, reasonably equipped clinical laboratories, while substantially reducing costs. Good outcomes are urgently required if effective prevention programs are to be developed to decrease the prevalence of liver disease.
In forensics, DNA profiling is used for the identification of the donor of a trace, while messenger RNA (mRNA) profiling can be applied to identify the cellular origin such as body fluids or organ tissues. The presence of male cell material can be readily assessed by the incorporation of Y-chromosomal markers in quantitation or STR profiling systems. However, no forensic marker exists to positively identify female cell material; merely the presence of female DNA is deduced from the absence of a Y peak, or unbalanced X-Y signals at the Amelogenin locus or unbalanced response of the total and Y-specific quantifier. The presence of two X-chromosomes in female cells invokes dosage compensation, which is achieved through inactivation of one of the X-chromosomes in females. Since this process involves specific RNA molecules, identification of female cellular material may be possible through RNA profiling. Additionally, male material may be identified through RNAs expressed from the Y-chromosome. RNAs preferentially expressed in either sex were assessed for their potential to act as sex markers in forensic RNA assays. To confirm sex-specificity, body fluids and organ tissues of multiple donors of either sex were tested. Additionally, sensitivity of the markers and the suitability of positively identifying male-female mixtures were assessed and degraded samples were used to assess performance of the markers in forensic settings. The addition of sex-specific markers is of added informative value in any RNA profiling system and both markers were incorporated into existing RNA assays that either target body fluids or organs. These are the first forensic assays that enable positive identification of female cellular material.
Cell cycle phase specific oscillation of gene transcription has long been recognized as an underlying principle for ordered processes during cell proliferation. The G1/S-specific and G2/M-specific cohorts of genes in plants are regulated by the E2F and the MYB3R transcription factors. Mutant analysis suggests that activator E2F functions might not be fully required for cell cycle entry. In contrast, the two activator-type MYB3Rs are part of positive feedback loops to drive the burst of mitotic gene expression, which is necessary at least to accomplish cytokinesis. Repressor MYB3Rs act outside the mitotic time window during cell cycle progression, and are important for the shutdown of mitotic genes to impose quiescence in mature organs. The two distinct classes of E2Fs and MYB3Rs together with the RETINOBLATOMA RELATED are part of multiprotein complexes that may be evolutionary related to what is known as DREAM complex in animals. In plants, there are multiple such complexes with distinct compositions and functions that may be involved in the coordinated cell cycle and developmental regulation of E2F targets and mitotic genes.
Epicardial adipose tissue (EAT) is a source of a number of cytokines which could act in the pathogenesis of coronary artery disease (CAD). The potential relationship between known cardiovascular risk factors, such as smoking, dyslipidaemia or diabetes mellitus and EAT humoral signalling, has not been fully elucidated. Therefore, we designed and conducted a cross-sectional study to determine whether selected cardiovascular risk factors are linked to levels of cytokines in epicardial and subcutaneous adipose tissue (SAT).
Samples of SAT and EAT were collected from consecutive patients undergoing scheduled cardiac surgery. Tissue concentrations of tumour necrosis factor-ɑ (TNF-α), interleukin-6 (IL-6), adipocyte fatty acid-binding protein, leptin, and adiponectin were determined by ELISA.
We enrolled 140 patients. TNF-α and IL-6 concentrations in EAT and SAT were significantly higher in current smokers (CS) than in never smokers (NS) and former smokers (FS). There were no differences between FS and NS. No other clinical variables were associated with cytokine concentrations in a regression analysis.
Smoking was independently associated with higher TNF-α and IL-6 concentrations in EAT and SAT. A novel observation that pro-inflammatory cytokines are elevated in EAT in smokers could contribute to identify potential mechanisms involved in the pathogenesis of adverse effects of tobacco smoking. There were no differences between EAT cytokine production in NS and FS, which support the importance of smoking cessation for cardiovascular risk reduction.
Activators of soluble guanylyl cyclase (sGC) act preferentially in conditions of enzyme oxidation or haem group removal. This study was designed to investigate the effects of the sGC activator BAY 60-2770 in murine airways inflammation and human eosinophil chemotaxis.
C57Bl/6 mice treated or not with BAY 60-2770 (1 mg/kg/day, 14 days) were intranasally challenged with ovalbumin (OVA). At 48 h, bronchoalveolar lavage fluid (BALF) was performed, and circulating blood, bone marrow and lungs were obtained. Human eosinophils purified from peripheral blood were used to evaluate the cell chemotaxis.
OVA-challenge promoted marked increases in eosinophil number in BAL, lung tissue, circulating blood and bone marrow, all of which were significantly reduced by BAY 60-2770. The IL-4 and IL-5 levels in BALF were significantly reduced by BAY 60-2770. Increased protein expression of iNOS, along with decreases of expression of sGC (α1 and β1 subunits) and cGMP levels were detected in lung tissue of OVA-challenged mice. BAY 60-2770 fully restored to baseline the iNOS and sGC subunit expressions, and cGMP levels. In human isolated eosinophils, BAY 60-2770 (1-5 μM) had no effects on the cGMP levels and eotaxin-induced chemotaxis; however, prior incubation with ODQ (10 μM) markedly elevated the BAY 60-2770-induced cyclic GMP production, further inhibiting the eosinophil chemotaxis.
BAY 60-2770 reduces airway eosinophilic inflammation and rescue the sGC levels. In human eosinophils under oxidized conditions, BAY 60-2770 elevates the cGMP levels causing cell chemotaxis inhibition. BAY 60-2770 may reveal a novel therapeutic target for asthma treatment.
Dysregulation of glucose metabolism is a primary hallmark of metabolic disease (i.e., diabetes, obesity, etc.). Complementary nonpharmaceutical strategies are needed to prevent and/or ameliorate dysregulation of glucose metabolism and prevent progression from normoglycemia to prediabetes and type 2 diabetes across the lifespan. Cocoa compounds, particularly the procyanidins, have shown promise for improving insulin sensitivity and blood glucose homeostasis. However, the molecular mechanisms by which cocoa procyanidins exert these functions remain poorly understood. Furthermore, cocoa procyanidins exhibit size diversity, and evidence suggests that procyanidin bioactivity and size may be related. Here, we show that a procyanidin-rich cocoa extract elicits an antidiabetic effect by stimulating glycogen synthesis and glucose uptake, independent of insulin. Cocoa procyanidins did not appear to act via stimulation of AMPK or CaMKII activities. Additionally, in the presence of insulin, glycogen synthesis and AKT phosphorylation were affected. These mechanisms of action are most pronounced in response to oligomeric and polymeric procyanidins. These results demonstrate (1) specific mechanisms by which cocoa procyanidins improve glucose utilization in skeletal muscle and (2) that larger procyanidins appear to possess enhanced activities. These mechanistic insights suggest specific strategies and biological contexts that may be exploited to maximize the antidiabetic benefits of cocoa procyanidins.
Genetic insect control, such as self-limiting RIDL(2) (Release of Insects Carrying a Dominant Lethal) technology, is a development of the sterile insect technique which is proposed to suppress wild populations of a number of major agricultural and public health insect pests. This is achieved by mass rearing and releasing male insects that are homozygous for a repressible dominant lethal genetic construct, which causes death in progeny when inherited. The released genetically engineered ('GE') insects compete for mates with wild individuals, resulting in population suppression. A previous study modelled the evolution of a hypothetical resistance to the lethal construct using a frequency-dependent population genetic and population dynamic approach. This found that proliferation of resistance is possible but can be diluted by the introgression of susceptible alleles from the released homozygous-susceptible GE males. We develop this approach within a spatial context by modelling the spread of a lethal construct and resistance trait, and the effect on population control, in a two deme metapopulation, with GE release in one deme. Results show that spatial effects can drive an increased or decreased evolution of resistance in both the target and non-target demes, depending on the effectiveness and associated costs of the resistant trait, and on the rate of dispersal. A recurrent theme is the potential for the non-target deme to act as a source of resistant or susceptible alleles for the target deme through dispersal. This can in turn have a major impact on the effectiveness of insect population control.
This work demonstrates the use of quantum dots (QDs) with photocatalytic activity as a sensitive, inexpensive and rapid colorimetric platform for Cu(2+) sensing. Based on the simple thiol compound mediated QDs growing method, CdS QDs can be quickly formed in situ, which possess excellent photocatalytic ability for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce a colored product under light irradiation. Cu(2+) can catalyze the oxidation of sulfhydryl groups in glutathione (GSH) which act as a stabilizer for CdS QDs. In the presence of Cu(2+), GSH is oxidized and loss the stabilization ability for the growth of CdS QDs, thus resulting in the decrease of the absorbance. Under optimum conditions, as low as 5.3nM Cu(2+) can be detected. This sensing system is simple, reliable and holds great potential to provide a new general platform for ultrasensitive monitoring of a variety of analytes.
A collaborative patient-pharmacist interaction is fundamental to greater patient satisfaction with pharmacy care and improved medication adherence. Effective pharmacist-patient communication occurs when both pharmacist and patient are able to successfully attend to not only the typical tasks and goals of the interaction but also basic face needs that underlie all social interaction; autonomy, competence or esteem, and fellowship. Addressing face needs occurs through conventional and strategic communication strategies that respond to the emerging needs throughout an interaction. Pharmacist-patient interactions are not just about transfer of information and medications. Both parties assess the situation, the others' intentions within the context of their own goals and this influences how they choose to act throughout the interaction. Face-work Theory provides a framework to understand these interaction processes in pharmacist-patient communication.
The aim of this study was to determine face needs, threats and the strategic communication strategies used to address these within community pharmacist-patient interactions.
This exploratory descriptive study drew upon principles of ethology to first describe naturally occurring behaviour and then to interpret this behaviour within the context of Face-work theory. Twenty-five audio-recorded community pharmacist-patient interactions were collected and analyzed. The average length of these interactions was 3:67 min with a range of 0.39 s-9:35 min.
Multiple face needs for both pharmacist and patient were evident in most interactions. Autonomy, competence and fellowship face needs were negotiated in the following contexts: participative relationships, concordant role expectations, sensitive topics, and negotiating expertise and knowledge. Competence face needs for both parties were the most dominant need found in negotiating role expectations. The most common communication strategies used to support face were solidarity based strategies while indirect and depersonalized questions were commonly employed to mitigate face threat.
Face-work Theory is a novel approach to understand processes and outcomes of patient-pharmacist interactions in community pharmacies. Linking speech acts with face needs and threats may help to elucidate how pharmacist-patient interactions achieve both task oriented and interpersonal goals.
To assess the impact of an accidental release of volatilized acrylonitrile on pharmacy services in Blount County, Tennessee.
A paper questionnaire was mailed to all pharmacies located in Blount County, Tennessee. The questionnaire assessed 5 domains relevant to pharmacy services after the derailment disaster: (1) disaster preparedness, (2) disaster response, (3) disaster information source awareness, (4) Pharmacy Practice Act amendment preference, and (5) pharmacy impact. Continuous (ratio-level), binary, and free-text data were collected and evaluated with the use of multiple correspondence analysis, tetrachoric correlations, and multiple regression modeling.
The data suggested that geographic proximity to and duration of the disaster and the number of patients presenting for 72-hour emergency prescription refills significantly influenced the overall impact on pharmacy service delivery. The increase in 72-hour prescription refill requests was strongly correlated with blood pressure and diabetes medications, with all respondents in favor of amending state law to allow pharmacists to dispense 30-day prescription refills in times of disaster. Pharmacy respondents are also largely unaware of online disaster-related information sources.
Disasters have the potential to affect all pharmacies in a locality but appear to have a significant impact on those located close to the scene. The number of patients presenting to pharmacies and the total duration of the disaster also appear to influence the overall impact of disasters on pharmacies. Pharmacists would be benefited by an awareness of current disaster information sources and legal supports pertinent to pharmacy practice in times of disaster.
Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel.
The genus Rosa (roses) has long been used in traditional or folk medicine worldwide for the treatment of various types of arthritis including rheumatoid arthritis and osteoarthritis. The active constituents of Rosa spp., such as flavonoids, triterpenoids, and phytosterols, could act on different targets in the NF-κB signalling pathway, inhibit pro-inflammatory enzymes (e.g. MMPs and COX-2), lower the production of inflammatory cytokines and chemokines (e.g. TNF-α, IL-1β, IL-6, CCL5), and reduce oxidative stress, which in turn suppress inflammatory processes. Preclinical and clinical studies have demonstrated that these species possess analgesic, anti-arthritic, anti-inflammatory, anti-oxidative and bone-preserving activities. This review presents comprehensive overview of the mode and mechanism of action of various extracts, preparations, and active constituents from this genus. The dynamic beneficial effects of the products prepared from this genus in arthritis management are summarized. The Rosa genus is a treasure waiting for further exploration by researchers interested in the development of safe and effective anti-arthritic agents.
As a part of the Chemicals Management Plan launched in 2006, the Government of Canada is assessing and managing, where appropriate, the potential health and ecological risks associated with approximately 4300 substances under the Canadian Environmental Protection Act (1999). Since that time, nearly 3000 substances have been assessed, with human biomonitoring (HBM) data playing an increasingly important role for some substances. Case studies are presented, including both inorganic and organic substances (i.e., selenium, triclosan, phthalates), which highlight the impact and overall role HBM has had in regulatory decision making in Canada for these three substances as well as criteria used in the application of HBM data in human health risk assessment. An overview of its limitations in terms of how and when HBM data can be applied, when assessing human health in a regulatory setting, is discussed as well as the role HBM data can play in priority setting.
Heterogeneity in radiation therapy (RT)-induced normal tissue toxicity is observed in 10% of cancer patients, limiting the therapeutic outcomes. In addition to treatment-related factors, normal tissue adverse reactions also manifest from genetic alterations in distinct pathways majorly involving DNA damage-repair genes, inflammatory cytokine genes, cell cycle regulation, and antioxidant response. Therefore, the common sequence variants in these radioresponsive genes might modify the severity of normal tissue toxicity, and the identification of the same could have clinical relevance as a predictive biomarker.
The present study was conducted in a cohort of patients with breast cancer to evaluate the possible associations between genetic variants in radioresponsive genes described previously and the risk of developing RT-induced acute skin adverse reactions. We tested 22 genetic variants reported in 18 genes (ie, NFE2L2, OGG1, NEIL3, RAD17, PTTG1, REV3L, ALAD, CD44, RAD9A, TGFβR3, MAD2L2, MAP3K7, MAT1A, RPS6KB2, ZNF830, SH3GL1, BAX, and XRCC1) using TaqMan assay-based real-time polymerase chain reaction. At the end of RT, the severity of skin damage was scored, and the subjects were dichotomized as nonoverresponders (Radiation Therapy Oncology Group grade <2) and overresponders (Radiation Therapy Oncology Group grade ≥2) for analysis.
Of the 22 single nucleotide polymorphisms studied, the rs8193 polymorphism lying in the micro-RNA binding site of 3'-UTR of CD44 was significantly (P=.0270) associated with RT-induced adverse skin reactions. Generalized multifactor dimensionality reduction analysis showed significant (P=.0107) gene-gene interactions between MAT1A and CD44. Furthermore, an increase in the total number of risk alleles was associated with increasing occurrence of overresponses (P=.0302).
The genetic polymorphisms in radioresponsive genes act as genetic modifiers of acute normal tissue toxicity outcomes after RT by acting individually (rs8193), by gene-gene interactions (MAT1A and CD44), and/or by the additive effects of risk alleles.
Gynecologic Oncology Group (GOG) 0174 compared weekly intramuscular methotrexate (MTX) with biweekly pulsed intravenous dactinomycin (Act-D) as single-agent chemotherapy for low-risk gestational trophoblastic neoplasia (GTN). Act-D had a higher rate of initial complete response (CR) (70% vs. 53%, p=0.01), but multi-day regimens of MTX have higher historic success rates. We assessed the cost-effectiveness of Act-D vs. MTX per GOG 0174 and explored multi-day MTX regimens.
A cost effectiveness decision model was constructed with data from GOG 0174. Outcome was cost per first-line treatment success expressed in terms of incremental cost-effectiveness ratio (ICER). Front-line failures were assumed to receive cross-over single agent therapy, second line failures; multi-agent chemotherapy. GOG 0174 had no quality of life (QOL) evaluation, so equal QOL (utility 1.0) was assumed but varied in sensitivity analysis. A second exploratory model included 5-day and 8-day MTX regimens.
Act-D ($18,505) was more expensive compared to weekly MTX ($8950) with an ICER of $56,215 per first-line treatment success compared to weekly MTX. Small decreases in QOL dramatically increased the ICER during sensitivity analysis. Models with multi-day MTX regimens were also more cost-effective than Act-D. If effectiveness was redefined as avoidance of multi-agent chemotherapy, weekly MTX was more effective.
With a complete cure rate for low-risk GTN regardless of initial agent, our model supports provider hesitation toward first line Act-D for low risk GTN. While Act-D is more effective for first line treatment success, it is more costly, and does not decrease rate of multi-agent chemotherapy use.
Development of science-based interventions in raw milk cheese production is challenging due to the large diversity of production procedures and final products. Without an agreed upon categorization scheme, science-based food safety evaluations and validation of preventive controls would have to be completed separately on each individual cheese product, which is not feasible considering the large diversity of products and the typically small scale of production. Thus, a need exists to systematically group raw milk cheeses into logically agreed upon categories to be used for food safety evaluations. This paper proposes and outlines one such categorization scheme that provides for 30 general categories of cheese. As a base for this systematization and categorization of raw milk cheese, we used Table B of the US Food and Drug Administration's 2013 Food Code, which represents the interaction of pH and water activity for control of vegetative cells and spores in non-heat-treated food. Building on this table, we defined a set of more granular pH and water activity categories to better represent the pH and water activity range of different raw milk cheeses. The resulting categorization scheme was effectively validated using pH and water activity values determined for 273 different cheese samples collected in the marketplace throughout New York State, indicating the distribution of commercially available cheeses among the categories proposed here. This consensus categorization of cheese provides a foundation for a feasible approach to developing science-based solutions to assure compliance of the cheese processors with food safety regulations, such as those required by the US Food Safety Modernization Act. The key purpose of the cheese categorization proposed here is to facilitate product assessment for food safety risks and provide scientifically validated guidance on effective interventions for general cheese categories. Once preventive controls for a given category have been defined, these categories would represent safe havens for cheesemakers, which would allow cheesemakers to safely and legally produce raw milk cheeses that meet appropriate science-based safety requirements (e.g., risk to human health equivalent to pasteurized milk cheeses).
Obstetric transport is a specialized medical transport for maternal, fetal, and neonatal concerns. Perinatal regionalization of care provides a broader geographic availability of obstetric services with defined levels of maternal and neonatal care so that women can be transported to centers with increased resources and capabilities to reduce morbidity and mortality. The Emergency Medical Treatment and Active Labor Act provides regulatory guidance for care of laboring women who require transfer to a higher level of care. The Situation, Background, Assessment, and Recommendation communication can identify key pieces of medical information with recommendations given for mutual expectations of next steps.
2-Amino-2-deoxy-d-glucose (d-glucosamine) is among the most abundant monosaccharides found in natural products. This constituent, recognized for its ubiquity, is presented in most instances as its N-acetyl derivative 2-acetamido-2-deoxy-d-glucopyranose (N-acetylglucosamine, GlcNAc, NAG). It occurs as the β-linked pyranosyl group in polysaccharides and oligosaccharides, and sometimes as the monosaccharide itself, either in its native state or as a glycoconjugate. The compound's acylation profile and other aspects of its structure are important elements in determining the variety of reactivities and functions of the molecule as a whole. Methods elaborated to investigate these challenges have been intensively reviewed; however, a relatively more comprehensive reviewing of this subject is introduced here to cover some aspects that have not been sufficiently covered. This might enable those who are beginners in this field to be aware of the subject in a more comprehensive context. 2-Amino-2-deoxy-d-glucosylation strategies demand robust amino-protecting groups that survive under a variety of chemical conditions, yet provide groups that can be deprotected under relatively mild conditions. At the end of this review, a table that includes all the N-protecting groups that have been used for glucosamine is provided to introduce them at a glance to aid in constructing building blocks that will act as useful 2-amino-2-deoxy-d-glucosyl donors.
Obesity is a chronic condition with significant health and economic consequences that requires more effective management in Australia. General practitioners (GPs) currently act as care co-ordinators in line with national guidelines for overweight and obesity. Australian patients indicate that they would appreciate more involvement from their GP in the management of obesity, and this is in line with international findings. Not all patients have access to specialist obesity services or affordable allied health care because of location, cost and time, particularly in rural and remote areas where there is a greater prevalence of obesity. Empowering GPs to use their skills as expert generalists to manage obesity is an option that should be explored to improve access for all individuals. GPs will require evidence-based tools to assist them in structuring obesity management within their own general practice environment.
Gingerols and shogaols are the primary non-volatile actives within ginger (Zingiber officinale). These compounds have demonstrated in vitro to exert 5-HT3 receptor antagonism which could benefit chemotherapy-induced nausea and vomiting (CINV). The site and mechanism of action by which these compounds interact with the 5-HT3 receptor is not fully understood although research indicates they may bind to a currently unidentified allosteric binding site. Using in silico techniques, such as molecular docking and GRID analysis, we have characterized the recently available murine 5-HT3 receptor by identifying sites of strong interaction with particular functional groups at both the orthogonal (serotonin) site and a proposed allosteric binding site situated at the interface between the transmembrane region and the extracellular domain. These were assessed concurrently with the top-scoring poses of the docked ligands and included key active gingerols, shogaols and dehydroshogaols as well as competitive antagonists (e.g. setron class of pharmacologically active drugs), serotonin and its structural analogues, curcumin and capsaicin, non-competitive antagonists and decoys. Unexpectedly, we found that the ginger compounds and their structural analogs generally outscored other ligands at both sites. Our results correlated well with previous site-directed mutagenesis studies in identifying key binding site residues. We have identified new residues important for binding the ginger compounds. Overall, the results suggest that the ginger compounds and their structural analogues possess a high binding affinity to both sites. Notwithstanding the limitations of such theoretical analyses, these results suggest that the ginger compounds could act both competitively or non-competitively as has been shown for palonosetron and other modulators of CYS loop receptors.
Highly anisotropic protein dynamics in equilibrium can be observed experimentally or through structural bioinformatics and molecular simulations. This anisotropic nature causes a response, to an external perturbation, along a small number of intrinsic large-amplitude directions as expected from the fluctuation-dissipation theorem. It is also key for controlling specific reactions as stochastic processes in macromolecular crowded environments. Protein anisotropy can be exploited for the calculation of physical properties, such as entropy, which can be employed for binding affinity studies. Energy frustration along soft modes including both global large-amplitude and localized small-amplitude movements is another key feature, as conformational transitions along soft modes, triggered by external perturbations such as the binding of other molecules, can act as a switch to control function.
We discuss the recent epidemiologic literature regarding health effects of uranium exposure in drinking water focusing on the chemical characteristics of uranium. While there is strong toxicologic evidence for renal and reproductive effects as well as DNA damage, the epidemiologic evidence for these effects in people exposed to uranium in drinking water is limited. Further, epidemiologic evidence is lacking for cardiovascular and oncogenic effects. One challenge in characterizing health effects of uranium in drinking water is the paucity of long-term cohort studies with individual level exposure assessment. Nevertheless, there are environmental justice concerns due to the substantial exposures for certain populations. For example, we present original data suggesting that individuals living in the Navajo Nation are exposed to high levels of uranium in unregulated well water used for drinking. In 10 out of 185 samples (5.4 %), concentrations of uranium exceeded standards under the Safe Drinking Water Act. Therefore, efforts to mitigate exposure to toxic elements in drinking water are warranted and should be prioritized.
Purpose To evaluate effects on somatic and mental health of a multicomponent inpatient occupational rehabilitation program compared to a less comprehensive outpatient program in individuals on sick leave for musculoskeletal complaints or mental health disorders. Methods A randomized clinical trial with parallel groups. Participants were individuals on sick-leave for 2-12 months with a sick-leave diagnosis within the musculoskeletal, psychological or general and unspecified chapters of ICPC-2. Potential participants were identified in the Social Security System Registry. The multicomponent inpatient program (4 + 4 days) consisted of Acceptance and Commitment Therapy, physical training and work-related problem-solving including creating a return to work plan and a workplace visit if considered relevant. The comparative outpatient program consisted primarily of ACT (6 sessions during 6 weeks). Self-reported health-related quality of life, subjective health complaints, pain and anxiety and depression symptoms were assessed up to 12 months after the program. Results 168 individuals were randomized to the multicomponent inpatient program (n = 92) or the outpatient program (n = 76). Linear mixed models showed no statistically significant differences between the programs, except for slightly more reduced pain after the outpatient program. Conclusions This study presents no support that a 4 + 4 days multicomponent inpatient rehabilitation program is superior to a less comprehensive outpatient program, in improving health outcomes.
Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4(+)Foxp3(+) regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.
The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B cell culture is the capacity to support mature B cell proliferation. We developed a culture method to support the efficient activation and proliferation of naive and memory human B cells. This culture supports extensive B cell proliferation, with ∼10(3)-fold increases following 8 d in culture and 10(6)-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naive B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved and, when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHC class II, CD80, and CD86. CD B cells act as APCs and present alloantigens and microbial Ags to T cells. We are able to activate and expand Ag-specific memory B cells; these cultured cells are highly effective in presenting Ag to T cells. We characterized the TCR repertoire of rare Ag-specific CD4(+) T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4(+) T cells differs from resting and unspecifically activated CD4(+) T cells. Moreover, we found that TT-specific TCR Vβ usage by CD4(+) T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.
γδ T cells act as a first line of defense against invading pathogens. However, despite their abundance in mucosal tissue, little information is available about their functionality in this compartment in the context of HIV/SIV infection. In this study, we evaluated the frequency, phenotype, and functionality of Vδ1 and Vδ2 T cells from blood, rectum, and the female reproductive tract (FRT) of rhesus macaques to determine whether these cells contribute to control of SIV infection. No alteration in the peripheral Vδ1/Vδ2 ratio in SIV-infected macaques was observed. However, CD8(+) and CD4(+)CD8(+) Vδ1 T cells were expanded along with upregulation of NKG2D, CD107, and granzyme B, suggesting cytotoxic function. In contrast, Vδ2 T cells showed a reduced ability to produce the inflammatory cytokine IFN-γ. In the FRT of SIV(+) macaques, Vδ1 and Vδ2 showed comparable levels across vaginal, ectocervical, and endocervical tissues; however, endocervical Vδ2 T cells showed higher inflammatory profiles than the two other regions. No sex difference was seen in the rectal Vδ1/Vδ2 ratio. Several peripheral Vδ1 and/or Vδ2 T cell subpopulations expressing IFN-γ and/or NKG2D were positively correlated with decreased plasma viremia. Notably, Vδ2 CD8(+) T cells of the endocervix were negatively correlated with chronic viremia. Overall, our results suggest that a robust Vδ1 and Vδ2 T cell response in blood and the FRT of SIV-infected macaques contribute to control of viremia.
In normal development, the order and synchrony of diverse developmental events must be explicitly controlled. In the nematode Caenorhabditis elegans, the timing of larval events is regulated by hierarchy of proteins and microRNAs known as the heterochronic pathway. These regulators are organized in feedforward and feedback interactions to form a robust mechanism for specifying the timing and execution of cell fates at successive stages. One member of this pathway is the RNA binding protein LIN-28, which promotes pluripotency and cell fate decisions in successive stages. Two genetic circuits control LIN-28 abundance: it is negatively regulated by the microRNA lin-4, and positively regulated by the transcription factor LIN-14 through a mechanism that was previously unknown. In this report we used animals that lack lin-4 to elucidate LIN-14's activity in this circuit. We demonstrate that three let-7 family microRNAs-miR-48, miR-84, and miR-241-inhibit lin-28 expression. Furthermore, we show genetically that these microRNAs act between lin-14 and lin-28, and that they comprise the pathway by which lin-14 positively regulates lin-28 We also show that the lin-4 family member mir-237, also regulates early cell fates. Finally, we show that the expression of these microRNAs is directly inhibited by lin-14 activity, making them the first known targets of lin-14 that act in the heterochronic pathway.
The pond snail Lymnaea learns and remembers not to respond to a food substance that is called conditioned taste aversion (CTA). The possible relationship between how well snails learn following taste-aversion training and brain dopamine contents is not known. We examined this relationship and found the followings. First, snails in the act of eating just before the commencement of training had the poor learning and had the highest dopamine contents in the brain. Second, snails, which had an ad libitum access to food but were not eating just before training, achieved the average grades and had the lower dopamine contents. Third, snails food-deprived for one day before training earned the best grades and had the significantly lower contents of dopamine. There was a negative correlation between the CTA grades and the brain dopamine contents in these 3 cohorts. Fourth, snails food-deprived for 5 days before training showed the poor grades and had the higher dopamine contents. That is, severe hunger increased the dopamine content in the brain. Because dopamine functions as a reward transmitter, CTA in the severely deprived snails (i.e., the forth cohort) was thought to be mitigated by a high dopamine content.
The concept of a morphogen - a molecule that specifies two or more cell fates in a concentration-dependent manner - is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate CNS, including the mouse dorsal telencephalic midline (DTM). Bone Morphogenetic Proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate - that of choroid plexus epithelial cells (CPECs) - in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal-Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.
Tripartite motif 16 (TRIM16), has been demonstrated to act as a tumor suppressor through affecting cell proliferation and migration or tumorigenicity in carcigenesis. However, the roles of TRIM16 in immune response were unknown up to now. Here, we cloned a TRIM16-like gene (TRIM16L) from orange spotted grouper (EcTRIM16L) and investigated its roles in response to virus infection. EcTRIM16L encoded a 478 amino acid peptide which showed 72% and 29% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Sequence alignments indicated that EcTRIM16L shared the different gene structures with human TRIM16, evidenced by the presence of RING domain, but absence of the B-box domain. In transfected grouper cells, the green fluorescence mainly distributed in cytoplasm, and the deletion of SPRY domain affected the accurate localization of EcTRIM16L. In response to different stimuli, including infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis (RGNNV), and transfection with b-DNA or poly I:C, the transcript of EcTRIM16L were differently regulated in grouper spleen cells. After incubation with SGIV, the ectopic expression of EcTRIM16L significantly enhanced the viral replication, demonstrated by the increase of cytopathic effect (CPE) severity and viral gene transcriptions. Simultaneously, we also found that overexpression of EcTIRM16L in vitro significantly weakened the expression of interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, and melanoma differentiation-associated protein 5 (MDA5). Moreover, the ectopic expression of EcTRIM16L significantly decreased both MDA5-and mediator of IRF3 activation (MITA)-induced interferon immune responses. Further studies showed that the RING domain played more important roles in the molecular action of EcTIRM16L during grouper virus infection. Our data, for the first time, demonstrated that fish TRIM16L exerted negative regulation on the interferon immune response against DNA virus infection.
Prolactin-like protein (PRL-L; LOC417800) is a homolog of PRL in non-mammalian vertebrates and can act as a functional ligand of PRL receptor (PRLR). Despite its widespread expression in extrapituitary tissues, mechanisms of regulation of PRL-L in the chicken ovary remain unknown. In this study, we first examined PRL-L expression in chicken ovarian developing follicles. PRL-L transcript levels were highest (P<0.05) in follicular walls of <2mm follicles and progressively declined during follicle maturation. Undifferentiated granulosa cells of 6-8mm follicles had higher (P<0.05) PRL-L mRNA levels than differentiated granulosa cells of F3, F2 or F1 follicles. In cultured undifferentiated granulosa cells, levels of PRL-L transcript were increased (P<0.05) by follicle stimulating hormone (FSH) treatment while were not altered by the addition of luteinizing hormone (LH). In addition, 10ng/ml non-glycosylated (NG-) and 1ng/ml glycosylated (G-) PRL increased (P<0.05) but at higher levels (100 or 1000ng/ml) both showed no effects on PRL-L expression. Furthermore, 100ng/ml NG-PRL enhanced (P<0.05) FSH-induced PRL-L expression, whereas the effects of G-PRL were not significant. These results suggest that PRL-L mRNA is differentially expressed in the follicular hierarchy and its high abundance in undifferentiated granulosa cells is under the regulation of FSH or PRL variants independently or in combination. Moreover, in undifferentiated granulosa cells we also provide evidence for a positive role for PKA, PKC and PI3K signaling while a negative role for ERK2 in mediating FSH stimulation of PRL-L transcription.
Environmental sensitivity varies across developmental phases in flowering plants. In the juvenile phase, microRNA156 (miR156)-mediated repression of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factors renders Arabidopsis plants incompetent to floral inductive signals, including long-day (LD) photoperiod. During the vegetative phase transition, which accompanies a reduction of miR156 and a concomitant elevation of its targets, plants acquire reproductive competence such that LD signals promote flowering. However, it remains largely unknown how developmental signals are associated with photoperiodic flowering. Here, we show that SPL3, SPL4, and SPL5 (SPL3/4/5) potentiate the FLOWERING LOCUS T (FT)-FD module in photoperiodic flowering. SPL3/4/5 function as transcriptional activators through the interaction with FD, a basic leucine zipper transcription factor which plays a critical role in photoperiodic flowering. SPL3/4/5 can directly bind to the promoters of APETALA1, LEAFY, and FRUITFULL, thus mediating their activation by the FT-FD complex. Our findings demonstrate that SPL3/4/5 act synergistically with the FT-FD module to induce flowering under LDs, providing a long-sought molecular knob that links developmental aging and photoperiodic flowering.
Some commercially important vinyl derivatives are produced by the decarboxylation of phenolic acids. Enzymatically, this process can be achieved by phenolic acid decarboxylases (PADs), which are able to act on phenolic acid substrates such as ferulic and p-coumaric acid. Although many microbial PADs have been characterized, little is known regarding their plant homologs. Transcriptome sequencing in the liverworts has identified seven putative PADs, which share a measure of sequence identity with microbial PADs, but are typically much longer proteins. Here, a PAD-encoding gene was isolated from the liverwort species Conocephalum japonicum. The 1197 nt CjPAD cDNA sequence was predicted to be translated into a 398 residue protein. When the gene was heterologously expressed in Escherichia coli, its product exhibited a high level of PAD activity when provided with either p-coumaric or ferulic acid as substrate, along with the conversion of caffeic acid and sinapic acid to their corresponding decarboxylated products. Both N- and C-terminal truncation derivatives were non-functional. The transient expression in tobacco of a GFP/CjPAD fusion gene demonstrated that the CjPAD protein is expressed in the cytoplasm. It is first time a PAD was characterized from plants and the present investigation provided a candidate gene for catalyzing the formation of volatile phenols.
Carybdea marsupialis is a widely distributed box jellyfish found in the Mediterranean and in the tropical waters of the Caribbean Sea. Its venom is a complex mixture of biologically active compounds that are used to catch prey. In order to evaluate the activity of the neurotoxins in the venom, bioassays were carried out using the marine crab Ocypode quadrata. The proteins with neurotoxic effect were partially purified using low-pressure liquid chromatography techniques. Gel filtration (Sephadex G-50M) was used as the first step and the active fraction in crabs was passed through a QAE Sephadex A-25 column. Finally, the active fraction was run onto a Fractogel EMD SO3(-) column. No further purification step could be carried out due to the loss of neurotoxic activity. The Fractogel EMD SO3(-) fraction was analyzed electrophysiologically using the voltage-clamp technique in Xenopus laevis oocytes expressing membrane proteins from rat brain through mRNA injection. The crude venom and a fraction were observed to affect crustaceans and showed at least two types of bioactivity in oocytes expressing brain proteins. The effects were dose-dependent and completely reversible. These results evidence the presence of neurotoxins in Carybdea marsupialis venom that act on membrane proteins of the vertebrate nervous system.
The voluntary use and abuse of alcohol and inhalants is a recognized health problem throughout the world. Previous studies have shown that these agents affect brain function in a variety of ways including direct inhibition of key ion channels that regulate neuronal excitability. Among these, the N-methyl-d-aspartate (NMDA) receptor is particularly important given its key role in glutamatergic synaptic transmission, neuronal plasticity and learning and memory. Previous studies from this laboratory and others have identified key residues within transmembrane (TM) domains of the NMDA receptor that appear to regulate its sensitivity to alcohol and anesthetics. In this study, we extend these findings and examine the role of a TM4 residue in modulating sensitivity of recombinant NMDA receptors to ethanol and toluene. HEK293 cells were transfected with GluN1-1a and either wild-type or tryptophan-substituted GluN2(A-D) subunits and whole-cell currents were recorded using patch-clamp electrophysiology in the absence or presence of ethanol or toluene. Both ethanol (100 mM) and toluene (1 or 3 mM) reversibly inhibited glutamate-activated currents from wild-type NMDARs with GluN2B containing receptors showing heightened sensitivity to either agent. Substitution of tryptophan (W) at positions 825, 826, 823 or 850 in the TM4 domain of GluN2A, GluN2B, GluN2C or GluN2D subunits; respectively, significantly reduced the degree of inhibition by ethanol. In contrast, toluene inhibition of glutamate-activated currents in cells expressing the TM4-W mutants was not different from that of the wild-type controls. These data suggest that despite similarities in their action on NMDARs, ethanol and toluene may act at different sites to reduce ion flux through NMDA receptors.
The American College of Cardiology Foundation /American Heart Association guidelines recommend a weight-based dose of unfractionated heparin (UFH) for primary percutaneous coronary intervention (PCI). However, it is convention to administer a fixed-bolus dose of 5,000 units of UFH. It is unclear if 5,000 units are sufficient to achieve a therapeutic first activated clotting time (ACT). We conducted a retrospective cohort study to determine the proportion of therapeutic first ACT in patients who received 5,000 units of UFH before primary PCI. We examined the association of therapeutic first ACT with clinical outcomes, including post-PCI Thombolysis in Myocardial Infarction (TIMI) grade flow, myocardial infarction, bleeding, and mortality. Among the 269 included patients, 74.7% were men, and 61.4% were overweight or obese. The mean first ACT was 243.4 (SD = 61.5) seconds. Most patients (56.1%) had an infratherapeutic first ACT, 21.9% had a therapeutic first ACT, and 21.9% had a supratherapeutic first ACT. Furthermore, 44.6% of patients who achieved the American College of Cardiology Foundation/American Heart Association target weight-based dosing had an infratherapeutic ACT. The proportion of patients with post-PCI TIMI grade flow 0 to 2 was 14.6% among those with a first ACT that was infratherapeutic versus 6.8% among those with a first ACT that was not infratherapeutic (relative risk 2.15, 95% CI 0.99 to 4.65). In conclusion, over half of patients with ST-elevation myocardial infarction administered 5,000 units of UFH have an infratherapeutic first ACT and the high rate of poor TIMI grade flow in patients with an infratherapeutic ACT is concerning.
Malaria continues to be a serious public health problem particularly in Africa. Many people infected with malaria do not access effective treatment due to high price. At the same time many individuals receiving malaria drugs do not suffer from malaria because of the common practice of presumptive diagnosis. A global subsidy on artemisinin-based combination therapy (ACT) has recently been suggested to increase access to the most effective malaria treatment.
Following the recommendation by World Health Organization that parasitological testing should be performed before treatment and ACT prescribed to confirmed cases only, it is investigated in this paper if a subsidy on malaria rapid diagnostic tests (RDTs) should be incorporated. A model is developed consisting of a representative individual with fever suspected to be malaria, seeking care at a specialized drug shop where RDTs, ACT medicines, and cheap, less effective anti-malarials are sold. Assuming that the individual has certain beliefs of the accuracy of the RDT and the probability that the fever is malaria, the model predicts the diagnosis-treatment behaviour of the individual. Subsidies on RDTs and ACT are introduced to incentivize appropriate behaviour: choose an RDT before treatment and purchase ACT only if the test is positive.
Solving the model numerically suggests that a combined subsidy on both RDT and ACT is cost minimizing and improves diagnosis-treatment behaviour of individuals. For certain beliefs, such as low trust in RDT accuracy and strong belief that a fever is malaria, subsidization is not sufficient to incentivize appropriate behaviour.
A combined subsidy on both RDT and ACT rather than a single subsidy is likely required to improve diagnosis-treatment behaviour among individuals seeking care for malaria in the private sector.
Office workers engage in high levels of sitting time. Effective, context-specific, and scalable strategies are needed to support widespread sitting reduction. This study aimed to evaluate organisational-support strategies alone or in combination with an activity tracker to reduce sitting in office workers.
From one organisation, 153 desk-based office workers were cluster-randomised (by team) to organisational support only (e.g., manager support, emails; 'Group ORG', 9 teams, 87 participants), or organisational support plus LUMOback activity tracker ('Group ORG + Tracker', 9 teams, 66 participants). The waist-worn tracker provided real-time feedback and prompts on sitting and posture. ActivPAL3 monitors were used to ascertain primary outcomes (sitting time during work- and overall hours) and other activity outcomes: prolonged sitting time (≥30 min bouts), time between sitting bouts, standing time, stepping time, and number of steps. Health and work outcomes were assessed by questionnaire. Changes within each group (three- and 12 months) and differences between groups were analysed by linear mixed models. Missing data were multiply imputed.
At baseline, participants (46 % women, 23-58 years) spent (mean ± SD) 74.3 ± 9.7 % of their workday sitting, 17.5 ± 8.3 % standing and 8.1 ± 2.7 % stepping. Significant (p < 0.05) reductions in sitting time (both work and overall) were observed within both groups, but only at 12 months. For secondary activity outcomes, Group ORG significantly improved in work prolonged sitting, time between sitting bouts and standing time, and overall prolonged sitting time (12 months), and in overall standing time (three- and 12 months); while Group ORG + Tracker, significantly improved in work prolonged sitting, standing, stepping and overall standing time (12 months). Adjusted for confounders, the only significant between-group differences were a greater stepping time and step count for Group ORG + Tracker relative to Group ORG (+20.6 min/16 h day, 95 % CI: 3.1, 38.1, p = 0.021; +846.5steps/16 h day, 95 % CI: 67.8, 1625.2, p = 0.033) at 12 months. Observed changes in health and work outcomes were small and not statistically significant.
Organisational-support strategies with or without an activity tracker resulted in improvements in sitting, prolonged sitting and standing; adding a tracker enhanced stepping changes. Improvements were most evident at 12 months, suggesting the organisational-support strategies may have taken time to embed within the organisation.
Australian New Zealand Clinical Trial Registry: ACTRN12614000252617 . Registered 10 March 2014.
The importance of the Wnt signaling cascade in the fields of developmental biology, regenerative medicine, cancer genetics, and neurobiology has fueled a wide search for potent pharmacological agents capable of controlling Wnt signaling. Numerous fields of study have lent assistance to this endeavor, yielding both natural and synthetic compounds that are capable of inducing or inhibiting Wnt at multiple stages within the pathway. Further, there is a large of body research which has investigated endogenous Wnt inducers and inhibitors, namely the secreted Wnts, Dickkof proteins, secreted Frizzled-Related Proteins, and Wnt Inhibitory Factor-1, along with others which may act via indirect means to stimulate or inhibit Wnt (e.g. the Smads, bone morphogenetic proteins, and Hedgehog proteins). This review will summarize the research surrounding currently available small molecules used to target Wnt signaling. These compounds will be classified based upon their ability to stimulate or inhibit Wnt, their derivation (natural or synthetic), and their specific mechanism of action.
Tartary buckwheat is a strongly abiotic, resistant coarse cereal, but its tolerance mechanisms for stress are largely unknown. MYB transcription factors play key roles in various physiological, biochemical and molecular responses, which can both positively and negatively regulate the stress tolerance of plants. In this study, we report that the expression of FtMYB10, a R2R3-MYB gene from Tartary buckwheat, was induced significantly by ABA and drought treatments. A seed germination test under ABA treatment indicated that transgenic lines were less sensitive to ABA. The overexpression of FtMYB10 in Arabidopsis reduced drought and salt tolerance. Further studies showed that the proline contents in the transgenic plants are markedly decreased associated with reduced expression of the P5CS1 gene under both normal and stress conditions. Furthermore, the expression of some stress-responsive genes, including DREB1/CBFs, RD29B, RD22, and several genes of the DRE/CRT class, decreased in response to FtMYB10 overexpression in Arabidopsis. These results suggest that FtMYB10 may play a key role in ABA signaling feedback regulation and act as a novel negative regulator of salt and drought stress tolerance in plants.
The prevalence of obesity is increasing worldwide and has tripled in men of reproductive age since the 1970s. Concerningly, obesity is not only comorbid with other chronic diseases, but there is mounting evidence that it increases the non-communicable disease load in their children (eg mortality, obesity, autism). Animal studies have demonstrated that paternal obesity increases the risk of metabolic (eg glucose metabolism defects, obesity) and reproductive disorders in offspring. Epigenetic changes within sperm are clear mechanistic candidates that are associated with both changes to the father's environment and offspring phenotype. Specifically there is emerging evidence that a father's sperm microRNA content both responds to paternal environmental cues and alters the gene expression profile and subsequent development of the early embryo. We used a mouse model of high fat diet (HFD) induced obesity to investigate whether male obesity could modulate sperm microRNA content. We also investigated whether this alteration to a father's sperm microRNA content lead to a similar change in the sperm of male offspring. Our investigations were initially guided by a Taqman PCR array, which indicated the differential abundance of 28 sperm borne microRNAs in HFD mice. qPCR confirmation in a much larger cohort of founder males demonstrated that 13 of these microRNAs were differentially abundant (11 up-regulated; 2 down-regulated) due to HFD feeding. Despite metabolic and reproductive phenotypes also being observed in grand-offspring fathered via the male offspring lineage, there was no evidence that any of the 13 microRNAs were also dysregulated in male offspring sperm. This was presumably due to the variation seen within both groups of offspring and suggests other mechanisms might act between offspring and grand-offspring. Thus 13 sperm borne microRNAs are modulated by a father's HFD and the presumed transfer of this altered microRNA payload to the embryo at fertilisation potentially acts to alter the embryonic molecular makeup post-fertilisation, altering its growth trajectory, ultimately affecting adult offspring phenotype and may contribute to paternal programming.
Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.
microRNAs (miRNAs) plays an important role in tumor development and progression and act as oncogenes or tumor suppressor genes in the carcinogenesis process. miRNA is stable in serum, and recent studies have demonstrated the feasibility of using circulating miRNA as biomarkers in cancer patients. However, currently, no serum biomarkers for the early diagnosis and prognosis of renal cell carcinoma (RCC) have been reported. Therefore, a new molecular marker for early diagnosis and evaluation of recurrence after surgery is required. Our purpose was to identify miRNA signatures that could distinguish the serum of RCC patients from matched healthy controls and validate identified miRNAs as potential biomarkers for RCC.
Serum samples from 30 RCC patients were collected before and 1 month after surgery. 30 cancer-free blood donor volunteers with no history of any cancer were recruited from the same institute. miR-21 and miR-106a expression levels were determined by real-time PCR.
The serum miR-21 level was significantly higher in RCC patients (median, 8.34) than in healthy control individuals (median, 0.70; p= 0.001). A month after surgery, serum miR-21 levels (median, 0.69) were significantly reduced (p= 0.032). The serum miR-106a level was higher in RCC patients (median, 8.99) compared with controls (median, 0.96; p= 0.000), while miR-106a levels (median, 1.01) were reduced a month after surgery (p= 0.028). The expression level of miR-21 and miR-106 a in RCC patients increased significantly, while miR-21 and miR-106a decreased after surgery. This outcome suggests that serum miR-21 and miR-106a expression level was closely related with kidney cancer tissue.
We conclude that serum miR-21 and miR106a are expected to be molecular markers for RCC.
Diffusion tensor imaging (DTI) shows great advantage in the diagnosis of brain diseases, including cervical spinal cord (CSC) disease. This study aims to obtain the normal values of the DTI parameters for a healthy population and to establish a baseline for CSC disease diagnosis using DTI.
A total of 36 healthy adults were subjected to magnetic resonance imaging (MRI) for the entire CSC using the Siemens 3.0 T MR System. Sagittal DTI acquisition was carried out with a single-shot spin-echo echo-planar imaging (EPI) sequence along 12 non-collinear directions. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values were determined at different cervical levels using a region of interest (ROI) method, following which they were correlated with parameters, like age and sex. Further, diffusion tensor tracking (DTT) was carried out to reconstruct the white matter fiber bundles of the CSC.
The full and complete fiber bundle structure of a normal CSC was confirmed in both the T2-weighted and DTI images. The FA and ADC values were significantly negatively correlated with each other and showed strongly negative and positive correlations with age, respectively, but not with sex. Additionally, there was no significant difference between the FA and the ADC values at different cervical levels.
The DTI technique can act as an important supplement to the conventional MRI technique for CSC observation. Moreover, the FA and ADC values can be used as sensitive parameters in the DTI study on the CSC by taking the effects of age into consideration.
With the passage of the Biologics Price Competition and Innovation Act of 2009, the US Food and Drug Administration established an abbreviated pathway for developing and licensing biosimilar and interchangeable biological products. The regulatory framework and the technical requirements of the US biosimilars program involve a stepwise approach that relies heavily on analytical methods to demonstrate through a "totality of the evidence" that a proposed product is biosimilar to its reference product. By integrating analytical, pharmacological, and clinical data, each of which has limitations, a high level of confidence can be reached regarding clinical performance. Although questions and concerns about the biosimilars pathway remain and may slow uptake, a robust scientific program has been put in place. With three biosimilars already licensed and numerous development programs under way, clinicians can expect to see many new biosimilars come onto the US market in the coming decade. Expected final online publication date for the Annual Review of Medicine Volume 68 is January 14, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Adequate protein intake is critical for health and development. Generally, protein of animal origin is of higher quality for humans owing to its amino acid pattern and good digestibility. When administered in mixtures it can enhance the quality of plant proteins, but it is often scarce in low-income communities, especially in young children, the elderly, and pregnant and lactating women, who have increased requirements and in whom high-quality protein also stimulates bone growth and maintenance. Although high protein intake was associated with increased type 2 diabetes mellitus risk, milk and seafood are good sources of branched chain amino acids and taurine, which act beneficially on glucose metabolism and blood pressure. However, high consumption of protein-rich animal food is also associated with adverse health effects and higher risk for noncommunicable diseases, partly related to other components of these foods, like saturated fatty acids and potential carcinogens in processed meat but also the atherogenic methionine metabolite homocysteine. In moderation, however, animal proteins are especially important for health maintenance in vulnerable persons. Expected final online publication date for the Annual Review of Animal Biosciences Volume 5 is February 15, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Although self-regulated learning (SRL) is considered a fundamental skill that must be developed in physician training, many programs of SRL utilize learning goals that are generated only at the beginning of learning experiences or are widely spaced apart in time. These goals are often not formally shared with those actually working with the learner in the clinical setting.
We developed a program of written, student-generated weekly learning goals in which students focused on processes of becoming better doctors for their patients. These goals were shared at the beginning of each week with students' clinical teams for feedback and incorporation into the work.
The weekly learning goals program was developed and implemented as part of a required 3rd-year neurology clerkship. At the end of each 4-week clerkship, students were asked to evaluate the program through an anonymous electronic survey utilizing quantitative and open-ended qualitative questions.
Seventy-six of 82 students completed the evaluation survey (93% response rate). Eighty-six percent reported that the weekly learning goals increased their awareness of their thoughts and actions. Seventy-eight percent reported that the learning goals helped to improve their clinical performance to some degree, and 57% reported that the learning goals increased their focus on patient care. Students described a greater sense of focus on self-assessment and accountability from their goals. Students often commented that engagement from attendings and residents regarding their goals was a key element for successful learning from their goals.
Student-generated weekly learning goals on a neurology clerkship appear to be an effective method to operationalize SRL. For most students, the frequency of the goals allowed for close self-monitoring, and the act of sharing goals with the team opened a new avenue for dialogue between students and their supervisors.
In Tunisia, potato virus Y (PVY) currently presents a significant threat to potato production, reducing tuber yield and quality. Three hundred and eighty-five potato samples (six different cultivars) collected in autumn 2007 from nine regions in Tunisia were tested for PVY infection by DAS-ELISA. The virus was detected in all regions surveyed, with an average incidence of 80.26%. Subsequently, a panel of 82 Tunisian PVY isolates (PVY-TN) was subjected to systematic biological, serological and molecular typing using immunocapture reverse-transcription polymerase chain reaction and a series of PVY(OC)- and PVY(N)-specific monoclonal antibodies. Combined analyses revealed ~67% of PVY(NTN) variants of which 17 were sequenced in the 5'NTR-P1 region to assess the genetic diversity and phylogenetic relationship of PVY-TN against other worldwide PVY isolates. To investigate whether selective constraints could act on viral genomic RNA, synonymous and non-synonymous substitution rates and their ratio were analyzed. Averages of all pairwise comparisons obtained in the 5'NTR-P1 region allowed more synonymous changes, suggesting selective constraint acting in this region. Selective neutrality test was significantly negative, suggesting a rapid expansion of PVY isolates. Pairwise mismatch distribution gave a bimodal pattern and pointed to an eventually early evolution characterizing these sequences. Genetic haplotype network topology provided evidence of the existence of a distinct geographical structure. This is the first report of such genetic analyses conducted on PVY isolates from Tunisia.
The translationally controlled tumor protein (TCTP) is upregulated in a range of cancer cell types, in part, by the activation of the mechanistic target of rapamycin (mTOR). Recently, TCTP has also been proposed to act as an indirect activator of mTOR. While it is known that mTOR plays a major role in the regulation of skeletal muscle mass, very little is known about the role and regulation of TCTP in this post-mitotic tissue. This study shows that muscle TCTP and mTOR signaling are upregulated in a range of mouse models (mdx mouse, mechanical load-induced hypertrophy, and denervation- and immobilization-induced atrophy). Furthermore, the increase in TCTP observed in the hypertrophic and atrophic conditions occurred, in part, via a rapamycin-sensitive mTOR-dependent mechanism. However, the overexpression of TCTP was not sufficient to activate mTOR signaling (or increase protein synthesis) and is thus unlikely to take part in a recently proposed positive feedback loop with mTOR. Nonetheless, TCTP overexpression was sufficient to induce muscle fiber hypertrophy. Finally, TCTP overexpression inhibited the promoter activity of the muscle-specific ubiquitin proteasome E3-ligase, MuRF1, suggesting that TCTP may play a role in inhibiting protein degradation. These findings provide novel data on the role and regulation of TCTP in skeletal muscle in vivo.
In recent work, we demonstrated that it is possible to obtain approximate representations of high-dimensional free energy surfaces with variationally enhanced sampling ( Shaffer, P.; Valsson, O.; Parrinello, M. Proc. Natl. Acad. Sci. , 2016 , 113 , 17 ). The high-dimensional spaces considered in that work were the set of backbone dihedral angles of a small peptide, Chignolin, and the high-dimensional free energy surface was approximated as the sum of many two-dimensional terms plus an additional term which represents an initial estimate. In this paper, we build on that work and demonstrate that we can calculate high-dimensional free energy surfaces of very high accuracy by incorporating additional terms. The additional terms apply to a set of collective variables which are more coarse than the base set of collective variables. In this way, it is possible to build hierarchical free energy surfaces, which are composed of terms that act on different length scales. We test the accuracy of these free energy landscapes for the proteins Chignolin and Trp-cage by constructing simple coarse-grained models and comparing results from the coarse-grained model to results from atomistic simulations. The approach described in this paper is ideally suited for problems in which the free energy surface has important features on different length scales or in which there is some natural hierarchy.
Osteogenesis imperfecta (OI) is a connective tissue disorder characterized by bone fragility, low bone mass, and bone deformities. The majority of cases are caused by autosomal dominant pathogenic variants in the COL1A1 and COL1A2 genes that encode type I collagen, the major component of the bone matrix. The remaining cases are caused by autosomal recessively or dominantly inherited mutations in genes that are involved in the post-translational modification of type I collagen, act as type I collagen chaperones, or are members of the signaling pathways that regulate bone homeostasis. The main goals of treatment in OI are to decrease fracture incidence, relieve bone pain, and promote mobility and growth. This requires a multi-disciplinary approach, utilizing pharmacological interventions, physical therapy, orthopedic surgery, and monitoring nutrition with appropriate calcium and vitamin D supplementation. Bisphosphonate therapy, which has become the mainstay of treatment in OI, has proven beneficial in increasing bone mass, and to some extent reducing fracture risk. However, the response to treatment is not as robust as is seen in osteoporosis, and it seems less effective in certain types of OI, and in adult OI patients as compared to most pediatric cases. New pharmacological treatments are currently being developed, including anti-resorptive agents, anabolic treatment, and gene- and cell-therapy approaches. These therapies are under different stages of investigation from the bench-side, to pre-clinical and clinical trials. In this review, we will summarize the recent findings regarding the pharmacological and biological strategies for the treatment of patients with OI. © 2016 Wiley Periodicals, Inc.
Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible.
Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay.
ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity.
These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin.
The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.
Reversible phosphorylation of thylakoid light harvesting proteins is a mechanism to compensate for unbalanced excitation of PSI vs PSII under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high-light response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in high light, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P-CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo-damaged PSII, are also responsible for the high light-dependent reversible phosphorylation of the inner antenna CP29. This article is protected by copyright. All rights reserved.
Mesoionic carbenes (MICs) are currently hugely popular as ligands and triazolylidenes are arguably the most prominent classes of such MICs. We present here mesoionic carbenes with ferrocenyl substituents that can act as metalloligands for the generation of heteromultimetallic iridium(I) and gold(I) complexes. The ferrocenyl substituents allow for reversible oxidation of these heteromultimetallic complexes and these oxidation steps have a strong influence on the donor properties of the MICs. Tolman electronic parameters (TEP) determined from analysis of the iridium-carbonyl complexes show that the neutral ferrocenyl-MIC ligands are stronger donors than the imidazolylidene based carbenes, the one-electron oxidized ferrocenyl MICs are in the range of the tricyclohexyl phosphines and the two-electron oxidized forms which are electron-poor lie in the range of triphenyl phosphines. Taking advantage of the generation of these electron-poor MICs, we show their gold(I) complexes are potent catalysts for the synthesis of oxazolines, with complexes of the oxidized MIC ligands, without any additional additive, outperforming their neutral counterparts by almost a factor of ten. These results thus present the first examples of MIC ligands that are reversibly electronically tunable, and show the potential of the oxidized MIC ligands in types of catalysis where electron-poor ligands are necessary. Furthermore, we also show the potential of MICs for molecular electroactive materials.
In yeast, SNF1 protein kinase is the orthologue of mammalian AMPK complex. It is a trimeric complex composed of Snf1 protein kinase (orthologue of AMPKα catalytic subunit), Snf4 (orthologue of AMPKγ regulatory subunit), and a member of the Gal83/Sip1/Sip2 family of proteins (orthologues of AMPKβ subunit) that act as scaffolds and also regulate the subcellular localization of the complex. In this chapter, we review the recent literature on the characteristics of SNF1 complex subunits, the structure and regulation of the activity of the SNF1 complex, its role at the level of transcriptional regulation of relevant target genes and also at the level of posttranslational modification of targeted substrates. We also review the crosstalk of SNF1 complex activity with other key protein kinase pathways such as cAMP-PKA, TORC1, and PAS kinase.
Rice fields are an important source of nitrous oxide (N2O), where rice plants could act as a key factor controlling N2O fluxes during the flooding-drying process; however, the microbial driving mechanisms are unclear. In this study, specially designed equipment was used to grow rice plants and collect emitted N2O from the root-growing zone (zone A), root-free zones (zones B, C, and D) independently, at tillering and booting stages under flooding and drying conditions. Soil samples from the four zones were also taken separately. Nitrifying and denitrifying community abundances were detected using quantitative polymerase chain reaction (qPCR). The N2O emission increased significantly along with drying, but the N2O emission capabilities varied among the four zones under drying, while zone B possessed the highest N2O fluxes that were 2.7~4.5 times higher than those from zones C and D. However, zone A showed N2O consumption potential. Notably, zone B also harbored the highest numbers of narG-containing denitrifiers and amoA-containing nitrifiers under drying at both tillering and booting stages. This study demonstrates that drying caused significant increase in N2O emission from rhizosphere soil, in which the higher abundance of AOB would help to produce more nitrate and significantly higher narG-containing microbes would drive more N2O production and emission.
The mechanistic target of rapamycin (mTOR) is a central regulator in cell growth, activation, proliferation, and survival. Activation of the mTOR pathway underlies the pathogenesis of systemic lupus erythematosus (SLE). While mTOR activation and its therapeutic reversal were originally discovered in T cells, recent investigations have also uncovered roles in other cell subsets including B cells, macrophages, and "non-immune" organs such as the liver and the kidney. Activation of mTOR complex 1 (mTORC1) precedes the onset of SLE and associated co-morbidities, such as anti-phospholipid syndrome (APS), and may act as an early marker of disease pathogenesis. Six case reports have now been published that document the development of SLE in patients with genetic activation of mTORC1. Targeting mTORC1 over-activation with N-acetylcysteine, rapamycin, and rapalogs provides an opportunity to supplant current therapies with severe side effect profiles such as prednisone or cyclophosphamide. In the present review, we will discuss the recent explosion of findings in support for a central role for mTOR activation in SLE.
The Gene Ontology (GO) is a framework designed to represent biological knowledge about gene products' biological roles and the cellular location in which they act. Biocuration is a complex process: the body of scientific literature is large and selection of appropriate GO terms can be challenging. Both these issues are compounded by the fact that our understanding of biology is still incomplete; hence it is important to appreciate that GO is inherently an evolving model. In this chapter, we describe how biocurators create GO annotations from experimental findings from research articles. We describe the current best practices for high-quality literature curation and how GO curators succeed in modeling biology using a relatively simple framework. We also highlight a number of difficulties when translating experimental assays into GO annotations.
Both miR-212 and miR-132 are usually downregulated in ovarian cancer and act as tumor suppressors. However, the mechanism of their downregulation in ovarian cancer is not clear. In this study, we investigated the regulative effects of miR-212 and miR-132 on SOX4 expression in ovarian cancer cells and also studied whether there is a feedback regulation between miR-212/miR-132 and SOX4 via an epigenetic mechanism. The results showed that both EZH2 and SOX4 overexpressions significantly repressed miR-212 and miR-132 expressions in SKOV3 and OV2008 cells. Immunoprecipitation assay showed that there are interactions among SOX4, EZH2, and H3K27me3, and ChIP assay confirmed significant enrichment of EZH2 and H3K27me3 in the promoter region of miR-212/132. Both pri-miR-212 and pri-miR-132 expressions decreased after enforced EZH2 or SOX4 expression. Western blot and dual-luciferase assay confirmed that miR-212 and miR-132 can target the same sites in the 3'UTR of SOX4 mRNA and suppress its expression in ovarian cancer cells. MiR-132 or miR-212 overexpression or knockdown of endogenous SOX4 reduced epithelial-mesenchymal transition (EMT)-like properties. Therefore, we infer that the SOX4/EZH2 complex can silence miR-212 and miR-132 expressions via binding to the promoter region and promoting H3K27me3, while miR-212 and miR-132 can directly bind to the 3'UTR of SOX4 and suppress its expression. This forms a MiR-132/212-SOX4/EZH2-H3K27me3 feedback loop in ovarian cancer cells. Functionally, SOX4 is a downstream effector of miR-212/132 modulating EMT of ovarian cancer cells.
Avascular necrosis (AVN) is a significant and potentially devastating complication following the treatment of developmental dysplasia of the hip (DDH). The reported rate of AVN following closed reduction for DDH ranges from 4 to 60%, and the resultant influence on hip development remains unclear.
A systematic review of the literature was undertaken to evaluate the frequency of AVN after more than 5 years of follow-up in children that underwent closed reduction at younger than 2-years of age for DDH.
The search strategy was formulated with key-concepts and keywords identified using the patient problem, intervention, comparison and outcome process. Searches were undertaken using Pubmed, Scopus and Web of Science up to and including May, 2016 to identify potential studies.
A total of seven papers met the a priori inclusion and exclusion criteria of this review. The overall rate of significant AVN in 441 patients (538 hips) was 10% at a mean length of follow-up of 7.6 years (5-18.8) following closed reduction. This finding can be used to inform the feasibility of future intervention studies, and act as a baseline for which surgeons to compare their results to a 'standard'.
Adoptive T cell transfer has been shown to be an effective method used to boost tumor-specific immune responses in several types of malignancies. In this study, we set out to optimize the ACT protocol for the experimental treatment of prostate cancer. The protocol includes a pre-stimulation step whereby T cells were primed with autologous dendritic cells loaded with the high hydrostatic pressure-treated prostate cancer cell line, LNCaP. Primed T cells were further expanded in vitro with anti-CD3/CD28 Dynabeads in the WAVE bioreactor 2/10 system and tested for cytotoxicity. Our data indicates that the combination of pre-stimulation and expansion steps resulted in the induction and enrichment of tumor-responsive CD4(+) and CD8(+) T cells at clinically relevant numbers. The majority of both CD4(+) and CD8(+) IFN-γ producing cells were CD62L, CCR7 and CD57 negative but CD28 and CD27 positive, indicating an early antigen experienced phenotype in non-terminal differentiation phase. Expanded T cells showed significantly greater cytotoxicity against LNCaP cells compared to the control SKOV-3, an ovarian cancer line. In summary, our results suggest that the ACT approach together with LNCaP-loaded dendritic cells provides a viable way to generate prostate cancer reactive T cell effectors that are capable of mounting efficient and targeted antitumor responses and can be thus considered for further testing in a clinical setting.
Preclinical models and studies in the metastatic and neoadjuvant settings suggest that single nucleotide polymorphisms in FCGR3A and FCGR2A may be associated with differential response to trastuzumab in the treatment of ERBB2/HER2-positive breast cancer, by modulating antibody-dependent cell-mediated cytotoxic effects.
To evaluate the effect of FCGR2A and FCGR3A polymorphisms on trastuzumab efficacy in the adjuvant treatment of ERBB2/HER2-positive breast cancer.
This is a retrospective analysis of patients enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 trial, a phase 3 cooperative group study conducted between 2000 and 2005. The NSABP B-31 trial randomized 2119 women with surgically resected node-positive, ERBB2/HER2-positive breast cancer to treatment with doxorubicin and cyclophosphamide followed by paclitaxel or the same regimen with the addition of 1 year of weekly trastuzumab. Patients were accrued at cooperative group sites across the United States and Canada. This analysis was performed between 2013 and 2016.
Doxorubicin and cyclophosphamide followed by paclitaxel or the same regimen with the addition of 1 year of weekly trastuzumab.
Disease-free survival.
The genotyped cohort (N = 1251) resembled the entire B-31 cohort based on clinical variables and the degree of benefit from trastuzumab. Median follow-up time was 8.2 years in the genotyped samples. The disease-free survival probability at 3, 5, and 8 years was 74% (95% CI, 71%-79%), 66% (95% CI, 62%-71%), and 58% (95% CI, 54%-63%) in patients who received ACT and 86% (95% CI, 83%-89%), 82% (95% CI, 79%-85%), and 78% (95% CI, 74%-81%) in patients who received ACTH. Addition of trastuzumab significantly improved patient outcome (hazard ratio [HR], 0.46; 95% CI, 0.37-0.57; P < .001). The expected trend for interaction between polymorphisms and trastuzumab was observed for both genes, but only FCGR3A-158 polymorphism reached statistical significance for interaction (P < .001). As hypothesized, patients with genotypes FCB3A-158V/V or FCB3A-158V/F received greater benefit from trastuzumab (HR, 0.31; 95% CI, 0.22-0.43; P < .001) than patients who were homozygous for the low-affinity allele (HR, 0.71; 95% CI, 0.51-1.01; P = .05).
The FCGR3A-158 polymorphism is predictive of trastuzumab efficacy in this cohort of patients with early ERBB2/HER2-positive breast cancer. Patients who are homozygous for phenylalanine at this position represent a considerable proportion of the population and, in contrast to previously reported analyses from similarly designed trials, our results indicate that trastuzumab may be less efficacious in these patients.
clinicaltrials.gov Identifier for NSABP B-31: NCT00004067.
Road-killed wild animals host zoonotic pathogens such as Toxoplasma gondii, offering a new opportunity for the epidemiological study of these infectious organisms.
This investigation aimed to determine the presence of T. gondii and other apicomplexan parasites in tissue samples of 64 road-killed wild animals, using polymerase chain reaction (PCR). Positive samples were then typed by PCR-restriction fragment length polymorphism (RFLP) using 7 markers: SAG1, 5'-3'SAG2, SAG3, BTUB, c29-6, PK1, and Apico. PCR-RFLP targeting 18S ribosomal RNA (rRNA) genes was also performed on all samples to detect other apicomplexan parasites.
T. gondii DNA was detected in 16 tissue samples from 8 individual animals, as follows: 1 Cerdocyon thous (crab-eating fox), 1 Didelphis albiventris (white-eared opossum), 1 Lutreolina crassicaudata (lutrine opossum), 2 Myrmecophaga tridactyla (giant anteater), 1 Procyon cancrivorus (crab-eating raccoon), and 2 Sphiggurus spinosus (Paraguay hairy dwarf porcupine). Seven different T. gondii genotypes were identified, 6 of which were novel. Typing by 18S rRNA verified these 16 T. gondii-infected samples, and identified 1 Sarcocystis spp.-infected animal [Dasypus novemcinctus (nine-banded armadillo)]. The amplified T. gondii (GenBank accession No. L37415.1) and Sarcocystis spp. 18S rRNA products were confirmed by sequencing.
Our results indicate that T. gondii is commonly present in wild mammals, which act as sources of infection for humans and animals, including other wild species. The approach employed herein proved useful for detecting T. gondii and Sarcocystis spp. in the environment and identifying their natural reservoirs, contributing to our understanding of host-parasite interactions.
The Indigenous Tobacco Control Initiative and Tackling Indigenous Smoking Measure were both announced by the Australian Government at a time when its rhetoric around the importance of evidence-based policy making was strong. This article will (1) examine how the Rudd Government used evidence in Indigenous tobacco control policy making and (2) explore the facilitators of and barriers to the use of evidence.
Data were collected through (1) a review of primary documents largely obtained under the Freedom of Information Act 1982 (Commonwealth of Australia) and (2) interviews with senior politicians, senior bureaucrats, government advisors, Indigenous health advocates, and academics. Through the Freedom of Information Act process, 24 previously undisclosed government documents relevant to the making of Indigenous tobacco control policies were identified. Interviewees (n = 31, response rate 62%) were identified through both purposive and snowball recruitment strategies. The Framework Analysis method was used to analyze documentary and interview data.
Government policy design was heavily influenced by the recommendations presented in government authored/commissioned literature reviews. Resulting policies were led by equivocal evidence for improved tobacco control outcomes among Indigenous Australians. Many of the cited studies had methodological limitations. In the absence of high-quality evidence, some policy makers supported policy recommendations that were perceived to be popular among the Indigenous community. Other policy makers recognized that there were barriers to accumulating rigorous, generalizable evidence; in the absence of such evidence, the policy makers considered that the "need for action" could be combined with the "need for research" by introducing innovative strategies and evaluating them.
Despite the absence of high-quality evidence, the formulation and adoption of Indigenous tobacco policy was neither irrational nor reckless. The decision to adopt an innovate and evaluate strategy was justifiable given (a) the potential for the gap between Indigenous and non-Indigenous health outcomes to worsen in the absence of an imminent policy response; (b) the existence of circumstances, which made it difficult to obtain high-quality evidence to guide policy; and (c) the need for policy solutions to reflect community preferences, given sociohistorical sensitivities.
Molybdenum disulfide (MoS2) has attracted a great attention as an excellent 2D material for future optoelectronic devices. Here, a novel MoS2 tribotronic phototransistor is developed by a conjunction of a MoS2 phototransistor and a triboelectric nanogenerator (TENG) in sliding mode. When an external friction layer produces a relative sliding on the device, the induced positive charges on the back gate of the MoS2 phototransistor act as a "gate" to increase the channel conductivity as the traditional back gate voltage does. With the sliding distance increases, the photoresponsivity of the device is drastically enhanced from 221.0 to 727.8 A W(-1) at the 100 mW cm(-2) UV excitation intensity and 1 V bias voltage. This work has extended the emerging tribotronics to the field of photodetection based on 2D material, and demonstrated a new way to realize the adjustable photoelectric devices with high photoresponsivity via human interfacing.
Ephemeral rivers act as linear oases in drylands providing key resources to people and wildlife. However, not much is known about these rivers' sensitivities to human activities. We investigated the landscape-level determinants of riparian tree dieback along the Swakop River, a dammed ephemeral river in Namibia, focusing on the native ana tree (Faidherbia albida) and the invasive mesquite (Prosopis spp.). We surveyed over 1,900 individual trees distributed across 24 sites along a 250 km stretch of the river. General linear mixed models were used to test five hypotheses relating to three anthropogenic threats: river flow disruption from damming, human settlement and invasive species. We found widespread dieback in both tree populations: 51% mortality in ana tree, with surviving trees exhibiting 18% canopy death (median); and 26% mortality in mesquite, with surviving trees exhibiting 10% canopy death. Dieback in the ana tree was most severe where trees grew on drier stretches of the river, where tributary flow was absent and where mesquite grew more abundantly. Dieback in the mesquite, a more drought-tolerant taxon, did not show any such patterns. Our findings suggest that dieback in the ana tree is primarily driven by changes in river flow resulting from upstream dam creation and that tributary flows provide a local buffer against this loss of main channel flow. The hypothesis that the invasive mesquite may contribute to ana tree dieback was also supported. Our findings suggest that large dams along the main channels of ephemeral rivers have the ability to cause widespread mortality in downstream riparian trees. To mitigate such impacts, management might focus on the maintenance of natural tributary flows to buffer local tree populations from the disruption to main channel flow.
Altruism toward strangers is considered a defining feature of humans. However, manifestation of this behaviour is contingent on the costliness of the selfless act. The extent of altruistic tendencies also varies cross-culturally, being more common in societies with higher levels of market integration. However, the existence of local variation in selfless behaviour within populations has received relatively little empirical attention. Using a 'lost letter' design, we dropped 300 letters (half of them stamped, half of them unstamped) in 15 residential suburbs of the greater Perth area that differ markedly in socioeconomic status. The number of returned letters was used as evidence of altruistic behaviour. Costliness was assessed by comparing return rates for stamped vs. unstamped letters. We predicted that there is a positive association between suburb socioeconomic status and number of letters returned and that altruistic acts decrease in frequency when costs increase, even minimally. Both predictions were solidly supported and demonstrate that socioeconomic deprivation and elevated performance costs independently impinge on the universality of altruistic behaviour in humans.
Rates of hybridization and introgression are increasing dramatically worldwide because of translocations, restocking of organisms and habitat modifications; thus, determining whether hybridization is occuring after reintroducing extirpated congeneric species is commensurately important for conservation. Restocking programs are sometimes criticized because of the genetic consequences of hatchery-bred fish breeding with wild populations. These concerns are important to conservation restocking programs, including those from the Australian freshwater fish family, Percichthyidae. Two of the better known Australian Percichthyidae are the Murray Cod, Maccullochella peelii and Trout Cod, Maccullochella macquariensis which were formerly widespread over the Murray Darling Basin. In much of the Murrumbidgee River, Trout Cod and Murray Cod were sympatric until the late 1970s when Trout Cod were extirpated. Here we use genetic single nucleotide polymorphism (SNP) data together with mitochondrial sequences to examine hybridization and introgression between Murray Cod and Trout Cod in the upper Murrumbidgee River and consider implications for restocking programs. We have confirmed restocked riverine Trout Cod reproducing, but only as inter-specific matings, in the wild. We detected hybrid Trout Cod-Murray Cod in the Upper Murrumbidgee, recording the first hybrid larvae in the wild. Although hybrid larvae, juveniles and adults have been recorded in hatcheries and impoundments, and hybrid adults have been recorded in rivers previously, this is the first time fertile F1 have been recorded in a wild riverine population. The F1 backcrosses with Murray cod have also been found to be fertile. All backcrosses noted were with pure Murray Cod. Such introgression has not been recorded previously in these two species, and the imbalance in hybridization direction may have important implications for restocking programs.
The "common variant-common disease" hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased vs. matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the "common variant-common disease" hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics. Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14 bp-indel) frequencies are significantly increased by more than 5% over control populations (Table 2). The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2). Three activating KIR genes: 3DS1, 2DS1, and 2DS2 have increased frequencies of 15, 22, and 14% in autism populations, respectively. There is a 6% increase in total activating KIR genes in autism over control subjects. And, more importantly there is a 12% increase in activating KIR genes and their cognate HLA alleles over control populations (Torres et al., 2012a). These data suggest the interaction of HLA ligand/KIR receptor pairs encoded on two different chromosomes is more significant as a ligand/receptor complex than separately in autism.
Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.
Unprincipled modeling decisions in large-domain ontologies, such as SNOMED CT, are problematic and might act as a barrier for their quality assurance and successful use in electronic health records. Most previous work has focused on clustering problematic concepts, which is helpful for quality control but faces difficulties in pinpointing the origin of those modeling problems. In this study, we examined the underlying structural patterns in SNOMED CT's data model as such patterns directly reflect the modeling strategies of editors. Our results showed that 92% of all structural patterns found accumulated in the Procedure and Clinical finding sub-hierarchies, and pattern reuse was low; over 30% of patterns were only used once. A qualitative analysis of a sample of 50 such singleton patterns revealed modeling problems, including redundancy, omission, and inconsistency. The problems detected in the sample suggest that the analysis of structural patterns is a valuable technique for revealing problematic areas of SNOMED CT and modeling the styles of terminology editors. Furthermore, the patterns that describe the modeling of a large number of concepts could provide insights for template creation and refinement in SNOMED CT.
Tungsten diselenide (WSe2) film was obtained by rapid selenization of magnetron sputtered tungsten (W) film. To prevent WSe2 film peeling off from the substrate during selenization, the W film was designed with a double-layer structure. The first layer was deposited at a high sputtering-gas pressure to form a loose structure, which can act as a buffer layer to release stresses caused by WSe2 growth. The second layer was deposited naturally on the first layer to react with selenium vapour in the next step. The effect of the W film deposition parameters(such as sputtering time, sputtering-gas pressure and substrate bias voltage)on the texture and surface morphology of the WSe2 film was studied. Shortening the sputtering time, increasing the sputtering-gas pressure or decreasing the substrate bias voltage can help synthesize WSe2 films with more platelets embedded vertically in the matrix. The stress state of the W film influences the WSe2 film texture. Based on the stress state of the W film, a model for growth of the WSe2 films with different textures was proposed. The insertion direction of the van der Waals gap is a key factor for the anisotropic formation of WSe2 film.
The effect of agricultural land use change on soil microbial community composition and biomass remains a widely debated topic. Here, we investigated soil microbial community composition and biomass [e.g., bacteria (B), fungi (F), Arbuscular mycorrhizal fungi (AMF) and Actinomycete (ACT)] using phospholipid fatty acids (PLFAs) analysis, and basal microbial respiration in afforested, cropland and adjacent uncultivated soils in central China. We also investigated soil organic carbon and nitrogen (SOC and SON), labile carbon and nitrogen (LC and LN), recalcitrant carbon and nitrogen (RC and RN), pH, moisture, and temperature. Afforestation averaged higher microbial PLFA biomass compared with cropland and uncultivated soils with higher values in top soils than deep soils. The microbial PLFA biomass was strongly correlated with SON and LC. Higher SOC, SON, LC, LN, moisture and lower pH in afforested soils could be explained approximately 87.3% of total variation of higher total PLFAs. Afforestation also enhanced the F: B ratios compared with cropland. The basal microbial respiration was higher while the basal microbial respiration on a per-unit-PLFA basis was lower in afforested land than adjacent cropland and uncultivated land, suggesting afforestation may increase soil C utilization efficiency and decrease respiration loss in afforested soils.
The genus Dacus is one of the most economically important tephritid fruit flies. The first complete mitochondrial genome (mitogenome) of Dacus species - D. longicornis was sequenced by next-generation sequencing in order to develop the mitogenome data for this genus. The circular 16,253 bp mitogenome is the typical set and arrangement of 37 genes present in the ancestral insect. The mitogenome data of D. longicornis was compared to all the published homologous sequences of other tephritid species. We discovered the subgenera Bactrocera, Daculus and Tetradacus differed from the subgenus Zeugodacus, the genera Dacus, Ceratitis and Procecidochares in the possession of TA instead of TAA stop codon for COI gene. There is a possibility that the TA stop codon in COI is the synapomorphy in Bactrocera group in the genus Bactrocera comparing with other Tephritidae species. Phylogenetic analyses based on the mitogenome data from Tephritidae were inferred by Bayesian and Maximum-likelihood methods, strongly supported the sister relationship between Zeugodacus and Dacus.
Fretting is a significant cause for the failure of orthopedic implants. Currently, since magnesium and its alloys have been developed as promising biodegradable implant materials, the fretting behavior of the Mg alloys is of great research significance. In this study, a Mg-Nd-Zn-Zr alloy (hereafter, denoted as JDBM alloy) was selected as experimental material, and its fretting behaviors were evaluated under 5 N, 10 N and 20 N normal loads with a displacement of 200 μm under the frequency of 10 Hz at 37 °C in air and in Hank's solution, respectively. The results indicated that while the friction coefficient decreased with the increment of the normal load, the wear volume of the alloy increased with the increment of the normal load both in air and in Hank's solution. Both the friction coefficients and the wear volume of the fretting in Hank's solution were much lower than those in air environment. The evolution trend of friction coefficients with time had different performance in air environment and the Hank's solution group. Although oxidation occurred during the fretting tests in Hank's solution, the damage of JDBM alloy was still reduced due to the lubrication effects of Hank's solution. Moreover, the addition of Fetal bovine serum (FBS) could act as lubrication and result in the reduction of the fretting damage.
Horizontal gene transfer (HGT) is a main mechanism of bacterial evolution endowing bacteria with new genetic traits. The transfer of mobile genetic elements such as plasmids (conjugation) requires the close proximity of cells. HGT between genetically distinct bacteria largely depends on cell movement in water films, which are typically discontinuous in natural systems like soil. Using laboratory microcosms, a bacterial reporter system and flow cytometry, we here investigated if and to which degree mycelial networks facilitate contact of and HGT between spatially separated bacteria. Our study shows that the network structures of mycelia promote bacterial HGT by providing continuous liquid films in which bacterial migration and contacts are favoured. This finding was confirmed by individual-based simulations, revealing that the tendency of migrating bacteria to concentrate in the liquid film around hyphae is a key factor for improved HGT along mycelial networks. Given their ubiquity, we propose that hyphae can act as focal point for HGT and genetic adaptation in soil.
The Rose-fronted Parakeet Pyrrhura roseifrons (Gray, 1859) is a poorly known species that occurs in lowland forest of western Amazonia, from eastern Peru to western Brazil and northern Bolivia (Collar 1997; Forshaw 2010). Like many Pyrrhura species, it is mostly green, and has a pale auricular patch, red tail and belly, but presents a bright red head, distinct from any other close relatives within the genus. It was long considered a subspecies of P. picta (Statius Miller, 1776) (Arndt 1983; Arndt 1996; Collar 1997; Juniper & Parr, 1998) and only recently has its specific rank been restored (Joseph 2000, 2002; Ribas et al. 2006) and accepted in the literature (Dickinson & Remsen 2013; del Hoyo & Collar 2014; Remsen et al. 2015).
The Australian species of the grapholitine genera Cryptophlebia Walsingham, 1900, Thaumatotibia Zacher, 1915 and Archiphlebia Komai & Horak, 2006, are revised, described and illustrated. A key to species is provided. Five named species of Cryptophlebia, C. ombrodelta (Lower), C. iridosoma (Meyrick), C. rhynchias (Meyrick) and C. pallifimbriana Bradley, are redescribed and three new species, C. wraggae, sp. nov., C. stigmata, sp. nov., and C. caulicola, sp. nov., are described. Cryptophlebia amblyopa Clarke, described from Micronesia, is synonymised with C. iridosoma. Cryptophlebia caulicola, sp. nov., is a borer in twigs of Acacia mangium Willd. in northern Queensland. Thaumatotibia aclyta (Turner) and T. zophophanes (Turner) are redescribed and the new species T. maculata, sp. nov., is described. Fruit of Acronychia spp. (Rutaceae) have been identified as native hosts of T. zophophanes, a pest species which damages macadamia (Proteaceae) and avocado (Lauraceae) on the Atherton Tableland. Archiphlebia endophaga (Meyrick) and A. rutilescens (Turner) are redescribed, and the new species A. gilva, sp. nov., is described.
The discovery of new vertebrate species in developed countries is still occurring at surprising rates for some taxonomic groups, especially the amphibians and reptiles. While this most often occurs in under-explored areas, it occasionally still happens in well-inhabited regions. We report such a case with the discovery and description of U. mahonyi sp. nov., a new species of frog from a highly populated region of New South Wales, Australia. We provide details of its morphology, calls, embryos and tadpoles, and phylogenetic relationships to other species of eastern Uperoleia. We also provide the results of targeted surveys to establish its distribution and provide observations of its habitat associations. As a consequence of these surveys, we comment on the likely restricted nature of the species' distribution and habitat, and place this in the context of a preliminary assessment of its putative conservation status, which should be assessed for listing under the IUCN's red list. We note this species, which is morphologically distinct, has gone unnoticed for many decades despite numerous ecological surveys for local development applications.
Porphyrin assemblies display interesting photophysical properties and a relatively high thermal stability. Moreover, meso-functionalized porphyrins with virtually fourfold symmetry can be relatively readily synthesized from pyrrole and the appropriate aldehyde. A number of metallo derivatives of 5,10,15,20-tetrakis(4-cyanophenyl)porphyrin, where the N atom of the linear cyano group can act both as a donor for coordination bonds or as an acceptor for hydrogen bonds, have been structurally characterized by single-crystal X-ray analysis. The supramolecular and structural chemistry of the corresponding 2- and 3-cyanophenyl isomers of the parent porphyrin, however, has remained largely unexplored. The crystal structure of [5,10,15,20-tetrakis(3-cyanophenyl)porphyrinato]copper(II) (CuTCNPP) nitrobenzene trisolvate, [Cu(C48H24N8)]·3C6H5NO2, has been determined at 80 K by synchrotron single-crystal X-ray diffraction. CuTCNPP exhibits a C2h-symmetric ααββ conformation, despite an unsymmetrical crystal environment, and is situated on a crystallographic centre of symmetry. The Cu(II) ion adopts a genuine square-planar coordination by the four pyrrole N atoms. The 24-membered porphyrin ring system shows no marked deviation from planarity. In the crystal, the CuTCNPP molecules and two nitrobenzene molecules are face-to-face stacked in an alternating fashion, resulting in corrugated layers. The remaining nitrobenzene guest molecule per CuTCNPP resides in the region between four neighbouring columnar stacks of CuTCNPP and sandwiched nitrobenzene molecules, and is disordered over four positions about a centre of symmetry.
Polynuclear complexes and coordination polymers of 3d metals have attracted significant interest evoked by a number of their unique properties. One of the most common approaches to the directed synthesis of coordination polymers is the linking of pre-prepared discrete coordination units by polydentate ligands. The formation of polynuclear complexes is usually a spontaneous process and precise prediction of the products of such reactions is virtually impossible in most cases. Tris(pyrazolyl)borates (Tp) act as tripodal `capping' ligands which form stable complexes with 3d metal ions. In such 1:1 compounds, three metal-ion coordination sites are occupied by N atoms from a Tp anion. This limits the number of remaining coordination sites, and thus the number of additional ligands which may coordinate, and opens an attractive approach for the directed design of desirable structures by exploiting ligands with appropriate composition and topology. In the present study, Tp anions with neopentyl [Tp(Np), tris(3-neopentylpyrazolyl)borate] and cyclohexyl [Tp(Cy), tris(3-cyclohexylpyrazolyl)borate] substituents were used as `capping' ligands and the dianion of tetraacetylethane (3,4-diacetylhexa-2,4-diene-2,5-diolate, tae(2-)) was employed as a bridge. The dinuclear complexes (μ-3,4-diacetylhexa-2,4-diene-2,5-diolato-κ(4)O(2),O(3):O(4),O(5))bis{[tris(3-cyclohexyl-1H-pyrazol-1-yl-κN(2))borato]cobalt(II)} acetonitrile disolvate, [Co2(C27H40BN6)2(C10H12O4)]·2CH3CN, (I)·2CH3CN, and (μ-3,4-diacetylhexa-2,4-diene-2,5-diolato-κ(4)O(2),O(3):O(4),O(5))bis{[tris(3-neopentyl-1H-pyrazol-1-yl-κN(2))borato]nickel(II)}, [Ni2(C24H40BN6)2(C10H12O4)], (II), were synthesized by the reaction of the mononuclear complexes Tp(Cy)CoCl or Tp(Np)NiCl with H2tae (3,4-diacetylhexane-2,5-dione or tetraacetylethane) in the presence of NEt3 as base. Compounds (I) and (II) were characterized by mass spectrometry, elemental analysis, and X-ray crystallography. They possess similar molecular structures, X-ray diffraction revealing them to be dinuclear in nature and composed of discrete Tp-M units in which two metal ions are linked by a tae(2-) dianion. Each metal ion possesses a five-coordinate square-pyramidal environment. The interplanar angles between the acetylacetonate fragments are significantly smaller than the near-90° values commonly observed.
The operation of the thoracic spiracular valves was analysed using anatomical and physiological techniques. Dense spiracular filter trichomes impede a diffusive gas exchange. However, the hinged posterior filter flap of the metathoracic spiracle (Sp2) opens passively during upstroke of the wings and closes by the suction of the sub-atmospheric tracheal pressure during the down stroke, which supports a unidirectional respiratory airflow. The action of the interior spiracular valve lids was recorded by photocell-sensors oriented above the enlarged spiracles and projected onto the screen of a video camera. The thoracic spiracles opened much quicker (approximately 0.1 s) than they closed (1 s) suggesting that the spiracular muscles are openers, confirmed by experimental induction of muscle contraction. Simultaneous photocell measurement revealed that the first and second thoracic spiracles act concordantly. At rest the spiracles were mostly closed or only slightly open (below 1%). During intermittent short flights, the valves opened wide at the start of the flight for a short time, and in many cases opened again after the flight ended. Often the opening was wider after the flight ended than during the preceding flight itself. During long spontaneous continuous flight phases (up to two hours) the valves were only slightly open (below 5%), widening shortly after transient increases of wing stroke intensity. It is an amazing paradox that the spiracles were only slightly open most of the time during sustained flight. The advantage of generating sub-atmospheric pressure, supporting a unidirectional airflow with a PO2 increase above the resting level, is discussed.
Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA3 ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA3, indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests.
This study aimed to understand the molecular mechanisms of nitrogen dioxide (NO2)-induced toxicity and cell death in plants. Exposure of Arabidopsis to high concentrations of NO2 induced cell death in a dose-dependent manner. No leaf symptoms were visible after fumigation for 1 h with 10 parts per million (ppm) NO2 However, 20 ppm NO2 caused necrotic lesion formation and 30 ppm NO2 complete leaf collapse, which had already started during the 1 h fumigation period. NO2 fumigation resulted in a massive accumulation of nitrite and in protein modifications by S-nitrosylation and tyrosine nitration. Nitric oxide (NO) at 30 ppm did not trigger leaf damage or any of the effects observed after NO2 fumigation. The onset of NO2-induced cell death correlated with NO and hydrogen peroxide (H2O2) signaling and a decrease in antioxidants. NO- and H2O2-accumulating mutants were more sensitive to NO2 than wild-type plants. Accordingly, experiments with specific scavengers confirmed that NO and H2O2 are essential promoters of NO2-induced cell death. Leaf injection of 100 mM nitrite caused an increase in S-nitrosylation, NO, H2O2, and cell death suggesting that nitrite functioned as a mediator of NO2-induced effects. A targeted screening of phytohormone mutants revealed a protective role of salicylic acid (SA) signaling in response to NO2 It was also shown that phytohormones were modulators rather than inducers of NO2-induced cell death. The established experimental set-up is a suitable system to investigate NO2 and cell death signaling in large-scale mutant screens.
Age at onset (AAO) in multiple sclerosis (MS) is an important marker of disease severity and may have prognostic significance. Understanding what factors can influence AAO may shed light on the aetiology of this complex disease, and have applications in the diagnostic process.
The study cohort of 22 162 eligible patients from 21 countries was extracted from the MSBase registry. Only patients with MS aged ≥16 years were included. To reduce heterogeneity, only centres of largely European descent were included for analysis. AAO was defined as the year of the first symptom suggestive of inflammatory central nervous system demyelination. Predictors of AAO were evaluated by linear regression.
Compared with those living in lower latitudes (19.0-39.9°), onset of symptoms was 1.9 years earlier for those at higher latitudes (50.0-56.0°) (p=3.83×10(-23)). A reciprocal relationship was seen for ambient ultraviolet radiation (UVR), with a significantly increasing AAO for patients with MS per each quartile increment of ambient UVR (p=1.56×10(-17)). We found that the AAO of female patients was ∼5 months earlier than male patients (p=0.002). AAO of progressive-onset patients with MS were ∼9 years later than relapsing-onset patients (p=1.40×10(-265)).
An earlier AAO in higher latitude regions was found in this worldwide European-descent cohort and correlated inversely with variation in latitudinal UVR. These results suggest that environmental factors which act at the population level may significantly influence disease severity characteristics in genetically susceptible populations.
Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the upregulation of NMDA-NR1 remains unexplored. We hypothesized that hypoxia via hypoxia-inducible factor 1α (HIF-1α) might contribute to the augmentation of the NMDA-NR1-mediated sympathoexcitatory responses from the PVN in CHF.
Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible factor 1α were upregulated within the PVN of left coronary artery-ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA and protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR1 mRNA, protein levels, and glutamate-induced Ca(+) influx. Chromatin immunoprecipitation assay identified HIF-1α binding to NMDA-NR1 promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR1, suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR1. Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone and sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF.
These results uncover a critical role for HIF-1 in the upregulation of NMDA-NR1 to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF.
Tyrosinase is involved in the production of melanin through the hydroxylation of monophenols to o-diphenols. The role of this enzyme was extensively studied in order to identify new therapeutics preventing skin pigmentation and melanoma. In this work we initially identified the 3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one (1a) as promising mushroom tyrosinase inhibitor (IC50 = 252 μM). Then, several chemical modifications were performed and new analogues related to compound 1a were synthesized. Biochemical assays demonstrated that several obtained compounds proved to be effective inhibitors showing IC50 values lower both than "lead compound" 1a and reference inhibitor kojic acid, as a well-known tyrosinase inhibitor. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that compounds 2 a-c and 10b act as non-competitive inhibitors while the most active inhibitor 2d (IC50 = 7.56 μM) is a mixed-type inhibitor. Furthermore, experimental and computational structural studies were performed in order to clarify the binding mode of the derivative 2d.
Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.
Spore-forming bacteria are able to grow under a wide range of environmental conditions, to form biofilms and to differentiate into resistant forms: spores. This resistant form allows their dissemination in the environment; consequently, they may contaminate raw materials. Sporulation can occur all along the food chain, in raw materials, but also in food processes, leading to an increase in food contamination. However, the problem of sporulation during food processing is poorly addressed and sporulation niches are difficult to identify from the farm to the fork. Sporulation is a survival strategy. Some environmental factors are required to trigger this differentiation process and others act by modulating it. The efficiency of sporulation is the result of the combined effects of these two types of factors on vegetative cell metabolism. This paper aims to explain and help identify sporulation niches in the food chain, based on features of spore-former physiology.
Serotonergic hallucinogens produce alterations of perceptions, mood, and cognition, and have anxiolytic, antidepressant, and antiaddictive properties. These drugs act as agonists of frontocortical 5-HT2A receptors, but the neural basis of their effects are not well understood. Thus, we conducted a systematic review of neuroimaging studies analyzing the effects of serotonergic hallucinogens in man. Studies published in the PubMed, Lilacs, and SciELO databases until 12 April 2016 were included using the following keywords: "ayahuasca", "DMT", "psilocybin", "LSD", "mescaline" crossed one by one with the terms "mri", "fmri", "pet", "spect", "imaging" and "neuroimaging". Of 279 studies identified, 25 were included. Acute effects included excitation of frontolateral/frontomedial cortex, medial temporal lobe, and occipital cortex, and inhibition of the default mode network. Long-term use was associated with thinning of the posterior cingulate cortex, thickening of the anterior cingulate cortex, and decreased neocortical 5-HT2A receptor binding. Despite the high methodological heterogeneity and the small sample sizes, the results suggest that hallucinogens increase introspection and positive mood by modulating brain activity in the fronto-temporo-parieto-occipital cortex.
A series of monomeric and dimeric Fe(III) complexes with O,O-; O,N-; O,S-coordination motifs has been prepared and characterized by standard analytical methods in order to elucidate their potential to act as model compounds for aquatic humic acids. Due to the postulated reduction of iron in humic acids and following uptake by microorganisms, the redox behavior of the models was investigated with cyclic voltammetry. Most of the investigated compounds showed iron reduction potentials accessible to biological reducing agents. Additionally, observed reduction processes were predominantly irreversible, suggesting that subsequent reactions can take place after reduction of the iron center. Also the stability of the synthesized complexes in pure water and artificial seawater was monitored from 24h up to 21days by means of UV-Vis spectrometry. Several complexes remained stable even after 21days, showing only partially precipitation but some of them showed changes in UV-Vis spectra already after 24h which were connected to protonation/deprotonation processes as well as redox processes and degradation of the complexes. The ability to act as an iron source for primary producers was tested in algal growth experiments with two marine algae species Chlorella salina and Prymnesium parvum. Some of the compounds showed effects on the algal cultures, which are comparable with natural humic acids and better as for the samples kept under ideal conditions. Those findings help to understand which functional groups of humic acids could be responsible for the reversible iron binding and transport in aquatic humic substances.
Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3 have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a3 propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3 only protonation of one heme a3 propionates was observed. Our data supports the postulated scenario where the heme a3 propionates are involved in the proton pathway.
Values can be useful for identifying what is important to individuals and communities, yet there is currently not a coherent way to conceptualize, identify, and organize the breadth of values that can be affected by a natural disaster. This research proposes a conceptual framework for how to conceptualize, identify, and organize values, and proposes a concrete, tangible value called the valued entity. The framework is applied in two studies of bushfire in Victoria, Australia: 112 submissions from individuals to the 2009 Victorian Bushfires Royal Commission and interviews with 30 members of the public in bushfire risk landscapes. Our findings suggest that: what people value ranges from abstract to concrete; prevalent abstract values include benevolence and universalism; prevalent mid-level valued attributes include natural attributes of landscapes and human life and welfare; prevalent valued entities are people and properties close to the person. Comparison between the two studies suggests people with more recent experience with bushfire refer less to the importance of natural places and natural attributes. The conceptual framework can act as a boundary object to facilitate researchers and policy-makers understanding the breadth of values affected by natural disaster events and management actions and how governance can better consider values at different scales.
Obesity-related lifestyle behaviors usually co-exist but few studies have examined their simultaneous relation with body weight. This study aimed to identify the hierarchy of lifestyle-related behaviors associated with being overweight in adults, and to examine subgroups so identified.
Data were obtained from a cross-sectional survey conducted across 60 urban neighborhoods in 5 European urban regions between February and September 2014. Data on socio-demographics, physical activity, sedentary behaviors, eating habits, smoking, alcohol consumption, and sleep duration were collected by questionnaire. Participants also reported their weight and height. A recursive partitioning tree approach (CART) was applied to identify both main correlates of overweight and lifestyle subgroups.
In 5295 adults, mean (SD) body mass index (BMI) was 25.2 (4.5) kg/m(2), and 46.0 % were overweight (BMI ≥25 kg/m(2)). CART analysis showed that among all lifestyle-related behaviors examined, the first identified correlate was sitting time while watching television, followed by smoking status. Different combinations of lifestyle-related behaviors (prolonged daily television viewing, former smoking, short sleep, lower vegetable consumption, and lower physical activity) were associated with a higher likelihood of being overweight, revealing 10 subgroups. Members of four subgroups with overweight prevalence >50 % were mainly males, older adults, with lower education, and living in greener neighborhoods with low residential density.
Sedentary behavior while watching television was identified as the most important correlate of being overweight. Delineating the hierarchy of correlates provides a better understanding of lifestyle-related behavior combinations which may assist in targeting preventative strategies aimed at tackling obesity.
Malaria is a major cause of morbidity and mortality in many African countries and parts of Asia and South America. Novel approaches to combating the disease have emerged in recent years and several drug candidates are now being tested clinically. However, it is long before these novel drugs can hit the market, especially due to a scarcity of safety and efficacy data.To reduce the malaria burden, the Medicines for Malaria Venture (MMV) was established in 1999 to develop novel medicines through industry and academic partners' collaboration. However, no reviews were focused following various preclinical and clinical studies published since the MMV initiation (2000) to till date.We identify promising approaches in the global portfolio of antimalarial medicines, and highlight challenges and patient specific concerns of these novel molecules. We discuss different clinical studies focusing on the evaluation of novel drugs against malaria in different human trials over the past five years.The drugs KAE609 and DDD107498 are still being evaluated in Phase I trials and preclinical developmental studies. Both the safety and efficacy of novel compounds such as KAF156 and DSM265 need to be assessed further, especially for use in pregnant women. Synthetic non-artemisinin ozonides such as OZ277 raised concerns in terms of its insufficient efficacy against high parasitic loads. Aminoquinoline-based scaffolds such as ferroquine are promising but should be combined with good partner drugs for enhanced efficacy. AQ-13 induced electrocardiac events, which led to prolonged QTc intervals. Tafenoquine, the only new anti-relapse scaffold for patients with a glucose-6-phosphate dehydrogenase deficiency, has raised significant concerns due to its hemolytic activity. Other compounds, including methylene blue (potential transmission blocker) and fosmidomycin (DXP reductoisomerase inhibitor), are available but cannot be used in children.At this stage, we are unable to identify a single magic bullet against malaria. Future studies should focus on effective single-dose molecules that can act against all stages of malaria in order to prevent transmission. Newer medicines have also raised concerns in terms of efficacy and safety. Overall, more evidence is needed to effectively reduce the current malaria burden. Treatment strategies that target the blood stage with transmission-blocking properties are needed to prevent future drug resistance.
While physical activity has been shown to improve cognitive performance and well-being, office workers are essentially sedentary. We compared the effects of physical activity performed as (i) one bout in the morning or (ii) as microbouts spread out across the day to (iii) a day spent sitting, on mood and energy levels and cognitive function.
In a randomized crossover trial, 30 sedentary adults completed each of three conditions: 6 h of uninterrupted sitting (SIT), SIT plus 30 min of moderate-intensity treadmill walking in the morning (ONE), and SIT plus six hourly 5-min microbouts of moderate-intensity treadmill walking (MICRO). Self-perceived energy, mood, and appetite were assessed with visual analog scales. Vigor and fatigue were assessed with the Profile of Mood State questionnaire. Cognitive function was measured using a flanker task and the Comprehensive Trail Making Test. Intervention effects were tested using linear mixed models.
Both ONE and MICRO increased self-perceived energy and vigor compared to SIT (p < 0.05 for all). MICRO, but not ONE, improved mood, decreased levels of fatigue and reduced food cravings at the end of the day compared to SIT (p < 0.05 for all). Cognitive function was not significantly affected by condition.
In addition to the beneficial impact of physical activity on levels of energy and vigor, spreading out physical activity throughout the day improved mood, decreased feelings of fatigue and affected appetite. Introducing short bouts of activity during the workday of sedentary office workers is a promising approach to improve overall well-being at work without negatively impacting cognitive performance.
NCT02717377 , registered 22 March 2016.
Artemisinin combination therapy (ACT) is used worldwide as the first-line treatment against uncomplicated Plasmodium falciparum malaria. Despite the success of ACT in reducing the global burden of malaria, the emerging of resistance to artemisinin threatens its use.
This report describes the first case of failure of dihydroartemisinin-piperaquine (DHA-PPQ) for the treatment of P. falciparum malaria diagnosed in Europe. It occurred in an Italian tourist returned from Ethiopia. She completely recovered after the DHA-PPQ treatment but 32 days after the end of therapy she had a recrudescence. The retrospective analysis indicated a correct DHA-PPQ absorption and genotyping demonstrated that the same P. falciparum strain was responsible for the both episodes.
In consideration of the growing number of cases of resistance to ACT, it is important to consider a possible recrudescence, that can manifest also several weeks after treatment.
Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets.
ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function.
Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu(2+), Mg(2+), Ni(+2) or Zn(+2)) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors.
Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.
Existing studies report a positive association between inadequate health literacy and immigrant's adverse health outcomes. Despite substantial research on this topic among immigrants, little is known about the level of health literacy among Somali women in Europe, and particularly in Norway.
A cross sectional study using respondent driven sampling was conducted in Oslo, Norway. A sample of 302 Somali women, 25 years and older, was interviewed using the short version of the European Health Literacy Questionnaire. Data was analysed using logistic regression.
Findings revealed that 71 % of Somali women in Oslo lack the ability to obtain, understand and act upon health information and services, and to make appropriate health decisions. Being unemployed (OR 3.66, CI 1.08-12.3) and socially less integrated (OR 8.17, CI 1.21-54.8) were independent predictors of an inadequate health literacy among Somali women.
Enhanced health literacy will most likely increase the chance to better health outcomes for immigrants, thereby moving towards health equity in the Norwegian society. Therefore, policies and programs are required to focus and improve health literacy of immigrant communities.
Body plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. We screened recently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods.
Our study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore we found GAF orthologs in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection.
Our results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, our study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.
Criminal cases involving human immunodeficiency virus transmission or exposure require that courts correctly comprehend the rapidly evolving science of HIV transmission and the impact of an HIV diagnosis. This consensus statement, written by leading HIV clinicians and scientists, provides current scientific evidence to facilitate just outcomes in Australian criminal cases involving HIV.Main recommendations: Caution should be exercised when considering charges or prosecutions regarding HIV transmission or exposure because:Scientific evidence shows that the risk of HIV transmission during sex between partners of different HIV serostatus can be low, negligible or too low to quantify, even when the HIV-positive partner is not taking effective antiretroviral therapy, depending on the nature of the sexual act, the viral load of the partner with HIV, and whether a condom or pre-exposure prophylaxis is employed to reduce risk.The use of phylogenetic analysis in cases of suspected HIV transmission requires careful consideration of its limited probative value as evidence of causation of HIV infection, although such an approach may provide valuable information, particularly in relation to excluding HIV transmission between individuals.Most people recently infected with HIV are able to commence simple treatment providing them a normal and healthy life expectancy, largely comparable with their HIV-negative peers. Among people who have been diagnosed and are receiving treatment, HIV is rarely life threatening. People with HIV can conceive children with negligible risk to their partner and low risk to their child.Changes in management as result of the consensus statement: Given the limited risk of HIV transmission per sexual act and the limited long term harms experienced by most people recently diagnosed with HIV, appropriate care should be taken before HIV prosecutions are pursued. Careful attention should be paid to the best scientific evidence on HIV risk and harms, with consideration given to alternatives to prosecution, including public health management.
There is no convincing evidence that classic Lyme disease occurs in Australia, nor is there evidence that the causative agent, Borrelia burgdorferi, is found in Australian animals or ticks. Lyme disease, however, can be acquired overseas but diagnosed in Australia; most people presenting with laboratory-confirmed Lyme disease in Australia were infected in Europe. Despite the lack of evidence that Lyme disease can be acquired in Australia, growing numbers of patients, their supporters, and some politicians demand diagnoses and treatment according to the protocols of the "chronic Lyme disease" school of thought. Antibiotic therapy for chronic "Lyme disease-like illness" can cause harm to both the individual (eg, cannula-related intravenous sepsis) and the broader community (increased antimicrobial resistance rates). Until there is strong evidence from well performed clinical studies that bacteria present in Australia cause a chronic debilitating illness that responds to prolonged antibiotics, treating patients with "Lyme disease-like illness" with prolonged antibiotic therapy is unjustified, and is likely to do much more harm than good.
Self-association of α-synuclein (αS) into pathogenic oligomeric species and subsequent formation of highly ordered amyloid fibrils is linked to the Parkinson's disease. So most of the recent studies are now focused on the development of potential therapeutic strategies against this debilitating disease. β-synuclein (βS), a presynaptic protein that co-localizes with αS has been recently reported to act as an inhibitor of αS self-assembly. But the specificity of molecular interaction, nature and location between αS/βS is not known despite the potential importance of βS as an inhibitor of αS. We used molecular dynamics and potential of mean force (PMF) to study association of αS/βS and αS/αS. The calculated PMF indicates that contact wells are significantly deeper and presence of a minimum at αS/βS separation of 13.5 Å with a free energy barrier of 40 kcal/mol. We observed the dissociation energy barrier to be two times higher for the hetero-dimer (αS/βS) than the homo-dimer (αS/αS). We also carried out umbrella samplings involving two degrees of freedom (one being the distance between the monomeric units and the other angle between the long axes of the two monomeric chains) and observed similar PMF profile. We noticed relatively stronger range of transient interactions between the monomeric units in hetero-dimer (αS/βS) than homo-dimer (αS/αS). So our findings suggest that αS readily combines with βS to form hetero-dimer than combining with itself in forming homo-dimer. Hence we see predominant transient interactions between αS and βS can be used to drive inhibition of αS aggregation.
Little is known about the physical activity (PA) and sedentary time (ST) habits of adolescents from superdiverse communities in the UK. The objectives of this study are to examine and report the patterns of PA/ST among adolescents in East London living in superdiverse communities, to identify opportunities/barriers to PA and inform policy/practice.
A total of 1260 young people (aged 11-13 years) from seven secondary schools in East London completed a questionnaire on PA/ST over the past seven days as part of the Newham's Every Child a Sports Person (NECaSP) intervention. Socio-demographic and anthropometric data were obtained. Significance tests were conducted to determine differences between socio-demographic and anthropometric predictors and PA/ST. Multinomial logit regression was used to explore the effects of ethnicity, sex, and body mass index (BMI) on PA levels.
Males were significantly more likely to engage in PA at least five times during school in the past week (U = 5.07, z = -11.76, p < .05). Obese participants were less likely to report engaging in PA five times in the past week (U = 4.11, z =-1.17, p < .05). Black Caribbean girls (U = 5.08, z = -1.92, p < .05) were significantly more likely to report engaging in no activity. Multinomial logit regression analyses revealed that girls with higher BMI were less likely to engage in PA at least four times after school in the last week than boys (b = .11, Wald X(2)(1) = 9.81, p < .01). Walking (36.4%), jogging/running (29.9%), and football (28%) were the most frequently reported activities.
Engaging girls in PA during and after school is important and making sports clubs and activities available and attractive to this target group may help increase engagement in PA and reduce ST. Findings support the need for more sex-specific and culturally responsive pedagogy in schools with curricula that respects diversity and individuality and has meaning and value amongst superdiverse young people. Finally, we need to extend current work presented and provide substantial evidence of the ways young people from minority ethnic groups process and act on the public health policy and the ways they understand and enact PA.
Electrode materials based on conversion reactions with lithium ions generally show much higher energy density. One of the main challenges in the design of these electrode materials is to improve initial Coulombic efficiency and alleviate the volume changes during the lithiation-delithiation processes. Here, we achieve fully reversible conversion in MoO3 as an anode for lithium ion batteries by the hybridization of CoMoO4. The porous MoO3-CoMoO4 microspheres are constructed by homogeneously dispersed MoO3 and CoMoO4 subunits and their lithiation/delithiation processes were studied by ex situ TEM to reveal the mechanism of the reversible conversion reaction. Co nanoparticles are in situ formed from CoMoO4 during the lithiation process, which then act as the catalyst to guarantee the reversible decomposition of Li2O, thus effectively improving the reversible specific capacity and initial Coulombic efficiency. Moreover, the pores in MoO3-CoMoO4 microspheres also greatly enhance their mechanical strength and provide enough cavity to alleviate volume changes during repeated cycling. Such a design concept makes MoO3 to be a potential promising anode in practical applications. The full cell (LiFePO4 cathode/MoO3-CoMoO4 anode) displays a high capacity up to 155.7 mAh g(-1) at 0.1 C and an initial Coulombic efficiency as high as 97.35%. This work provides impetus for further development in electrochemical charge storage devices.
The present study investigated the effects of (-)-sesamin on motor and memory deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) with l-3,4-dihydroxyphenylalanine (l-DOPA). MPTP-lesioned (30mg/kg/day, 5days) mice showed deficits in memory including habit learning memory and spatial memory, which were further aggravated by daily treatment with 25mg/kg l-DOPA for 21days. However, daily treatment with (-)-sesamin (25 and 50mg/kg) for 21days ameliorated memory deficits in an MPTP-lesioned mouse model of PD treated with l-DOPA (25mg/kg). Both (-)-sesamin doses reduced decreases in the retention latency time in the passive avoidance test, latency to fall of rotarod test and distance traveled in the open field test, and attenuated decreases in tyrosine hydroxylase (TH)-immunopositive cells, dopamine, and its metabolites in the substantia nigra-striatum. (-)-Sesamin reduced increases in the retention transfer latency time in the elevated plus-maze test and N-methyl-d-aspartate receptor (NMDAR) expression and reduced decreases in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus. In contrast, daily treatment with 10mg/kg l-DOPA for 21days ameliorated memory deficits in MPTP-lesioned mice, and this effect was further improved by treatment with (-)-sesamin (25 and 50mg/kg). These results suggest that (-)-sesamin protects against habit learning memory deficits by activating the dopamine neuronal system, while spatial memory deficits are decreased by its modulatory effects on the NMDAR-ERK1/2-CREB system. Accordingly, (-)-sesamin may act as an adjuvant phytonutrient for motor and memory deficits in patients with PD receiving l-DOPA.
Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults, including drug-induced liver injury.
Multidirectional interactions among the immune, endocrine, and nervous systems have been demonstrated in humans and non-human animal models for many decades by the biomedical community, but ecological and evolutionary perspectives are lacking. Neuroendocrine-immune interactions can be conceptualized using a series of feedback loops, which culminate into distinct neuroendocrine-immune phenotypes. Behavior can exert profound influences on these phenotypes, which can in turn reciprocally modulate behavior. For example, the behavioral aspects of reproduction, including courtship, aggression, mate selection and parental behaviors can impinge upon neuroendocrine-immune interactions. One classic example is the immunocompetence handicap hypothesis (ICHH), which proposes that steroid hormones act as mediators of traits important for female choice while suppressing the immune system. Reciprocally, neuroendocrine-immune pathways can promote the development of altered behavioral states, such as sickness behavior. Understanding the energetic signals that mediate neuroendocrine-immune crosstalk is an active area of research. Although the field of psychoneuroimmunology (PNI) has begun to explore this crosstalk from a biomedical standpoint, the neuroendocrine-immune-behavior nexus has been relatively underappreciated in comparative species. The field of ecoimmunology, while traditionally emphasizing the study of non-model systems from an ecological evolutionary perspective, often under natural conditions, has focused less on the physiological mechanisms underlying behavioral responses. This review summarizes neuroendocrine-immune interactions using a comparative framework to understand the ecological and evolutionary forces that shape these complex physiological interactions.
One of the major goals in speciation research is to understand which isolation mechanisms form the first barriers to gene flow. This requires examining lineages that are still in the process of divergence or incipient species. Here, we investigate the presence of behavioral and several cryptic barriers between the sympatric willow and birch host races of Lochmaea capreae. Behavioral isolation did not have any profound effect on preventing gene flow. Yet despite pairs mating indiscriminately, no offspring were produced from the heterospecific matings between birch females and willow males due to the inability of males to transfer sperm to females. We found evidence for differences in genital morphology that may contribute to failed insemination attempts during copulation. The heterospecific matings between willow females and birch males resulted in viable offspring. Yet fecundity and hatchability was remarkably reduced, which is likely the result of lower efficiency in sperm transportation and storage and lower survival of sperm in the foreign reproductive tract. Our results provide evidence for the contribution of several postmating-prezygotic barriers that predate behavioral isolation and act as primary inhibitors of gene flow in this system. This is a surprising, yet perhaps often overlooked feature of barriers acting early in sympatric speciation process.
The present study aimed to investigate the effects of berberine (BRB) on spatial and learning memory, anxiety, acetylcholinesterase activity and cell death in an experimental model of intracerebroventricular streptozotocin (ICV-STZ) induced sporadic Alzheimer's-like dementia. Sixty male Wistar rats were randomly divided into six groups: control (CTR), BRB 50mg/kg (BRB 50), BRB 100mg/kg (BRB 100), streptozotocin (STZ), streptozotocin plus BRB 50mg/kg (STZ+BRB 50), and streptozotocin plus BRB 100mg/kg (STZ+BRB 100). Rats were injected with ICV-STZ (3mg/kg) or saline, and daily oral BRB treatment began on day 4 for a period of 21days. Behavioral tests were carried out on day 17, and rats were euthanized on day 24. Cell death analysis and determination of acetylcholinesterase activity was performed on the cerebral cortex and hippocampus of the brain. Administration of BRB prevented the memory loss, anxiogenic behavior, increased acetylcholinesterase activity and cell death induced by ICV-STZ. This may be explained, in part, by a protective effect of BRB on ameliorating the progression of neurodegenerative diseases, including Alzheimer's disease, and the results of this study provide a better understanding of the effect of BRB on the brain. Thus, BRB may act as a potential neuroprotective agent.
Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by β-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca(2+) homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca(2+) response induced by low concentrations of human amylin (hAmylin). However, the Ca(2+) response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca(2+). Additionally, blockade of L-type Ca(2+) channels partially reduced hAmylin-induced Ca(2+) response. In whole-cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin-induced increase in Ca(2+). Single-cell RT-PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin-responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin-evoked Ca(2+) response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons.
The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-β, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling.
The Australian plague locust, Chortoicetes terminifera (Walker), is an important agricultural pest that oviposits into soil across vast semi-arid and arid regions. This study aimed to determine whether gravid female locusts can discriminate among substrates of increasing salinity (0, 4, 8, 12, 16, 20, 24, and 28ppt NaCl) when attempting oviposition, and quantify the effects of saline substrate on direct developing egg viability, and subsequent hatchling nymph body weight and survival. Gravid female locusts increasingly excavated and withdrew prior to completing oviposition in substrates of increasing salinity, but similar numbers of completed egg pods were observed across treatments. Egg weight at 50% total development time and successful egg development to nymph emergence decreased with increasing substrate salinity. Water balance equilibrium between the egg and the substrate occurred at approximately 12ppt NaCl corresponding to a water activity of ∼0.995. Eggs oviposited into sand containing ⩽12ppt NaCl weighed ⩾6.26±0.91mg and had ⩾76.8% successful development to nymph emergence. Eggs oviposited into sand containing >12ppt NaCl weighed ⩽5.16±1.27mg and had ⩽45.6% successful development to nymph emergence. Hatchling nymph body weight and survival to second instar also decreased with increasing substrate salinity. Nymphs that hatched and emerged from sand containing ⩽12ppt NaCl weighed ⩾5.55±0.43mg at emergence and had ⩾68.9% survival. Nymphs that hatched and emerged from sand containing >12ppt NaCl weighed ⩽5.28±0.67mg at emergence and had ⩽52.0% survival. These results indicate that C. terminifera is sufficiently resilient to develop and survive in saline substrates over most of their range.
Stabilized borylenes (L2 BH:) with weakly π-accepting substituents L, such as phosphines, were previously believed to be unstable. In the current manuscript, we describe a series of complexes formally containing a phosphine-stabilized borylene or boryl anion. In contrast to common trivalent boron compounds, the boron-based ligands in this study act as electron-donating ligands. The reported iron hydride complexes exhibit a unique reactivity pattern, undergoing a reversible B-H reductive elimination concomitant with oxidation of the boron(I) center.
Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.
Interleukin-2 inducible T-cell kinase (ITK) is expressed in T cells, and plays an important role in autoimmune inflammatory diseases through regulating the balance of Th17/Treg. However, its role in human systemic lupus erythematosus (SLE) remains unclear. The present study aims to measure the activation status of ITK in T cells from SLE patients and healthy controls, and identify its possible correlation to disease severity. We also discuss the serum levels of Th17, Treg related cytokines including IL-17, IL-21, IL-22, IL-10, analyzing correlation between ITK and Th17/Treg related cytokines.
Peripheral blood samples were drawn from 42 patients with SLE and 43 healthy blood donors, and the phosphorylation of ITK protein was studied in T cells using flow cytometry. In addition, serum levels of Th17/Treg related cytokines were studied with enzyme-linked immunosorbent assay (ELISA).
Percentages of CD4(+)pITK(+) T cells, CD8(+)pITK(+) T cells were higher in SLE patients compared with controls, and were positively related to disease activity, some clinical and laboratory parameters. Percentages of CD4(+)pITK(+) T cells, CD8(+)pITK(+) T cells were more prominent in active SLE patients compared with less active patients. Serum levels of Th17 and Treg related cytokines were higher in patients compared with controls. CD4(+)pITK(+) T cells were related to levels of IL-17, IL-21.
These data indicate that increased ITK expression could act as a disease activity marker and as a risk factor for involvement in SLE, but it still needs further study to confirm.
Subjective responses to meals are altered by shortened sleep time and anxiety state, but this effect has been poorly studied in shift workers - who act as a typical model concerning sleep restriction and present high levels of anxiety. The objective of this study was to compare subjective perceptions of meals and the levels of anxiety in the same subjects after working night shifts and after taking a nocturnal sleep, and to investigate associations between the responses to meals and the levels of anxiety under these two conditions. The study evaluated 34 male permanent night-shift workers who worked a 12-h shift followed by a 36-h rest period. Evaluations included: sleep pattern (on three days after working night shifts and after sleeping at night); hunger, enjoyment of eating foods and satiety after a meal (evaluated by visual analogue scales on three non-consecutive days after working night shifts and after nocturnal sleeps); and state of anxiety (on a day after working a night shift and a day after a nocturnal sleep). In the days following a night shift, workers had higher mean hunger scores before lunch and higher anxiety scores than when they had slept at night (p = 0.007 and 0.001, respectively). Linear regression indicated that, after a night shift, anxiety scores were negatively associated with hunger before breakfast (p = 0.04) and lunch (p = 0.03), the enjoyment of eating foods (p = 0.03) and the number of meals eaten during the course of the 24 h (p = 0.03). It is concluded that night shifts increase mean hunger and anxiety scores. Anxiety levels seem to interfere with the responses associated with food consumption.
We investigated neurophysiological mechanisms of subthalamic nucleus involvement in verbal fluency through a verbal generation task. The subthalamic nucleus is thought to act as a behavioural go/no-go instance by means of oscillatory communication in the theta band with the prefrontal cortex. Because subthalamic alpha-theta frequency stimulation has been shown to exert beneficial effects on verbal fluency in Parkinson's disease, we hypothesized that an alpha-theta oscillatory network involving the subthalamic nucleus underlies verbal generation task performance as a gating instance for speech execution. Postoperative subthalamic local field potential recordings were performed during a verbal generation compared to a control task. Time-frequency analysis revealed a significant alpha-theta power increase and enhanced alpha-theta coherence between the subthalamic nucleus and the frontal surface EEG during the verbal generation task. Beta and gamma oscillations were not significantly modulated by the task. Power increase significantly correlated with verbal generation performance. Our results provide experimental evidence for local alpha-theta oscillatory activity in the subthalamic nucleus and coherence to frontal associative areas as a neurophysiological mechanism underlying a verbal generation task. Thus, verbal fluency improvement during subthalamic alpha-theta stimulation in Parkinson's disease is likely due to an enhancement of alpha-theta oscillatory network activity. Alpha-theta oscillations can be interpreted as the rhythmic gating signature in a speech executing subthalamic-prefrontal network.
The conversion of 7-dehydrocholesterol to cholesterol, the final step of cholesterol synthesis in the Kandutsch-Russell pathway, is catalyzed by the enzyme 7-dehydrocholesterol reductase (DHCR7). Homozygous or compound heterozygous mutations in DHCR7 lead to the developmental disease Smith-Lemli-Opitz syndrome, which can also result in fetal mortality, highlighting the importance of this enzyme in human development and survival. Besides serving as a substrate for DHCR7, 7-dehydrocholesterol is also a precursor of vitamin D via the action of ultraviolet light on the skin. Thus, DHCR7 exerts complex biological effects, involved in both cholesterol and vitamin D production. Indeed, we argue that DHCR7 can act as a switch between cholesterol and vitamin D synthesis. This review summarizes current knowledge about the critical enzyme DHCR7, highlighting recent findings regarding its structure, transcriptional and post-transcriptional regulation, and its links to vitamin D synthesis. Greater understanding about DHCR7 function, regulation and its place within cellular metabolism will provide important insights into its biological roles.
Exogenous ciliary neurotrophic factor (CNTF) administration promotes the survival of motor neurons in a wide range of models. It also increases the expression of the critical neurotransmitter enzyme, choline acetyltransferase (ChAT), by in vitro motor neurons, likely independent of its effects on their survival. We have used the adult mouse facial nerve crush model and adult onset conditional disruption of the CNTF receptor α (CNTFRα) gene to directly examine the in vivo roles played by endogenous CNTF receptors in adult motor neuron survival and ChAT maintenance, independent of developmental functions. We have previously shown that adult activation of the CreER gene construct in floxed CNTFRα mice depletes this essential receptor subunit in a large subset of motor neurons (and all skeletal muscle, as shown here) but has no effect on the survival of intact or lesioned motor neurons, thereby indicating that these adult CNTF receptors play no essential survival role in this model, in contrast to their essential role during embryonic development. We show here that this same CNTFRα depletion does not affect ChAT labeling in non-lesioned motor neurons, but it significantly increases the loss of ChAT following nerve crush. The data suggest that while neither motor neuron nor muscle CNTF receptors play a significant, non-redundant role in the maintenance of ChAT in intact adult motor neurons, the receptors become essential for ChAT maintenance when the motor neurons are challenged by nerve crush. Therefore, the data suggest that the receptors act as a critical component of an endogenous neuroprotective mechanism. This article is protected by copyright. All rights reserved.
The existence of hard tissue pulleys that act to change the direction of a muscle insertion tendon is well known in the human body. These include (1) the trochlea for the extraocular obliquus superior muscle, (2) the pterygoid hamulus for the tensor veli palatini muscle, (3) the deep sulcus on the plantar aspect of the cuboid bone for the peroneus longus tendon, (4) the lesser sciatic notch for the obturator internus muscle, and (5) the bony trochleariformis process for the tensor tympani muscle tendon. In addition, (6) the stapedius muscle tendon shows a lesser or greater angulation at the pyramidal eminence of the temporal bone. Our recent studies have shown that the development of pulleys Nos. 1 and 2 can be explained by a change in the topographical relationship between the pulley and the tendon, that of pulley No. 3 by the rapidly growing calcaneus pushing the tendon, and that of pulley No. 4 by migration of the insertion along the sciatic nerve and gluteus medius tendon. Therefore, in Nos. 1-4, an initially direct tendon curves secondarily and obtains an attachment to the pulley. In case No. 6, the terminal part of the stapedius tendon originates secondarily from the interzone mesenchymal tissue of the incudostapedial joint. In the case of pulley No. 5, we newly demonstrated that its initial phase of development was similar to No. 6, but the tensor tympani tendon achieved a right-angled turn under guidance by a specific fibrous tissue and it migrated along the growing malleus manubrium.
Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (INa) amplitude, causing a hyperpolarizing shift in the inactivation curve of the INa, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (INaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.
Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4β-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.
Internationally, there is increasing recognition of the importance of multilevel policies and actions that address healthy and environmentally friendly food behaviours. However it is not yet clear which actions are most suitable to support consumers to adopt both behaviours concurrently. To this end, we undertook a qualitative study to assess consumer perceptions, experiences and attitudes towards healthy and environmentally friendly foods and four target behaviours: reducing overconsumption of food beyond energy needs, reducing consumption of low-nutrient energy dense foods, eating less animal- and more plant-derived foods, and reducing food waste. Online in-depth interviews were held with 29 Australian food shoppers representing different levels of involvement with health and environment in daily food choices. The results indicate that compared to health, the relationship between food and the environment is rarely considered by consumers. The four target food behaviours were primarily associated and motivated by an impact on health, except for not wasting foods. Participants had the most positive attitude and highest motivation for eating less processed and packaged foods, mostly to avoid excessive packaging and 'chemicals' in foods. This was followed by the behaviours reducing food waste and overconsumption. Conversely, there was a predominantly negative attitude towards, and low motivation for, eating less animal-derived products and more plant based foods. Overall, consumers found a joined concept of healthy and environmentally friendly foods an acceptable idea. We recommend that health should remain the overarching principle for policies and actions concerned with shifting consumer behaviours, as this personal benefit appears to have a greater potential to support behaviour change. Future consumer focused work could pay attention to framing behavioural messages, providing intermediate behavioural goals, and a multiple target approach to change habitual behaviours.
The recent finding that hydropersulfides (RSSH) are biologically prevalent in mammalian systems has prompted further investigation of their chemical properties in order to provide a basis for understanding their potential functions, if any. Hydropersulfides have been touted as hyper-reactive thiol-like species that possess increased nucleophilicity and reducing capabilities compared to their thiol counterparts. Herein, using persulfide generating model systems, the ability of RSSH species to act as one-electron reductants has been examined. Not unexpectedly, RSSH is relatively easily oxidized, compared to thiols, by weak oxidants to generate the perthiyl radical (RSS·). Somewhat surprisingly, however, RSS· was found to be stable in the presence of both O2 and NO and only appears to dimerize. Thus, the RSSH/RSS· redox couple is readily accessible under biological conditions and since dimerization of RSS· may be a rare event due to low concentrations and/or sequestration within a protein, it is speculated that the general lack of reactivity of individual RSS· species may allow this couple to be utilized as a redox component in biological systems.
Pluripotent stem cells act as an excellent cell source for disease therapy because of its specific characteristics of self-renewal and differentiation. Pluripotent stem cells are heterogeneous, consisting of naive stem cells as well as primed epiblast stem cells. However, the strategies and mechanisms of maintaining naive pluripotent stem cells remain unclear. In this study, we found that folic acid (FA) sustained mouse embryonic stem cell (ESC) pluripotency and enabled long-term maintenance of the naive state of ESCs under CHIR99021 conditions. Mechanistic experiments showed that STAT3 pathway partially mediated the effect of FA after which the interaction between STAT3 and importin α5 was enhanced. Meanwhile, MEK/ERK signaling also acted downstream of FA in maintaining ESC pluripotency. Furthermore, FA significantly promoted mouse somatic cell reprogramming. Overall, our study identified an effective chemical condition for maintaining homogeneous ESCs and highlighted the important roles of LIF/STAT3 and MEK/ERK signaling in naive ESC pluripotency.
Th17 cells are key players in defense against pathogens and maintaining tissue homeostasis, but also act as critical drivers of autoimmune diseases. Based on single-cell RNA-seq profiling of pathogenic versus nonpathogenic Th17 cells, we identified protein C receptor (PROCR) as a cell surface molecule expressed in covariance with the regulatory module of Th17 cells. Although PROCR expression in T cells was controlled by the cooperative action of the Th17 lineage-specific transcription factors RORγt, IRF4, and STAT3, PROCR negatively regulated Th17 differentiation. CD4(+) T cells from PROCR low expressor mutant mice readily differentiated into Th17 cells, whereas addition of the PROCR ligand, activated protein C, inhibited Th17 differentiation in vitro. In addition, PROCR acted as a negative regulator of Th17 pathogenicity in that it down-regulated expression of several pathogenic signature genes, including IL-1 and IL-23 receptors. Furthermore, T cell-specific deficiency of PROCR resulted in the exacerbation of experimental autoimmune encephalomyelitis (EAE) and higher frequencies of Th17 cell in vivo, indicating that PROCR also inhibits pathogenicity of Th17 cells in vivo. PROCR thus does not globally inhibit Th17 responses but could be targeted to selectively inhibit proinflammatory Th17 cells.
A bilayered electrospun membrane was produced in this study, using the electrospinning technique, to be applied as a skin substitute. The upper layer of the membrane was comprised by hyaluronic acid and polycaprolactone in order to provide mechanical support and also to act as a physical barrier against external threats. Chitosan and zein were used to produce the bottom layer that was loaded with salicylic acid, in order to confer anti-inflammatory and antimicrobial activity to this layer. The physicochemical properties of the membranes were determined and the obtained results showed that the produced electrospun membrane display an ideal porosity, appropriate mechanical properties, controlled water loss and a suitable salicylic acid release profile. In addition, membranes did not exhibit any toxic effects for human fibroblast cells, since cells were able to adhere, spread and proliferate. Furthermore, no biofilm formation was noticed on membranes' surface along the experiments. In conclusion, the gathered data reveal that this electrospun membrane has suitable properties to be used as a wound dressing.
Fusarium head blight (FHB) resistance in wheat is considered to be polygenic in nature. Cell wall fortification is one of the best resistance mechanisms in wheat against Fusarium graminearum which causes FHB. Metabolomics approach in our study led to the identification of a wide array of resistance-related (RR) metabolites, among which hydroxycinnamic acid amides (HCAAs), such as coumaroylagmatine and coumaroylputrescine, were the highest fold change RR metabolites in the rachis of a resistant near-isogenic line (NIL-R) upon F. graminearum infection. Placement of these metabolites in the secondary metabolic pathway led to the identification of a gene encoding agmatine coumaroyl transferase, herein referred to as TaACT, as a candidate gene. Based on wheat survey sequence, TaACT was located within a FHB quantitative trait loci on chromosome 2DL (FHB QTL-2DL) between the flanking markers WMC245 and GWM608. Phylogenetic analysis suggested that TaACT shared closest phylogenetic relationship with an ACT ortholog in barley. Sequence analysis of TaACT in resistant and susceptible NILs, with contrasting levels of resistance to FHB, led to the identification of several single nucleotide polymorphisms (SNPs) and two inversions that may be important for gene function. Further, a role for TaACT in FHB resistance was functionally validated by virus-induced gene silencing (VIGS) in wheat NIL-R and based on complementation studies in Arabidopsis with act mutant background. The disease severity, fungal biomass and RR metabolite analysis confirmed TaACT as an important gene in wheat FHB QTL-2DL, conferring resistance to F. graminearum.
The purpose of this study was to evaluate themes related to patients' experience in undergoing mammography, as expressed on Twitter.
A total of 464 tweets from July to December 2015 containing the hashtag #mammogram and relating to a patient's experience in undergoing mammography were reviewed.
Of the tweets, 45.5% occurred before the mammogram compared to 49.6% that occurred afterward (remainder of tweets indeterminate). However, in patients undergoing their first mammogram, 32.8% occurred before the examination, whereas in those undergoing follow-up mammogram, 53.0% occurred before the examination. Identified themes included breast compression (24.4%), advising other patients to undergo screening (23.9%), recognition of the health importance of the examination (18.8%), the act of waiting (10.1%), relief regarding results (9.7%), reflection that the examination was not that bad (9.1%), generalized apprehension regarding the examination (8.2%), interactions with staff (8.0%), the gown (5.0%), examination costs or access (3.4%), offering or reaching out for online support from other patients (3.2%), perception of screening as a sign of aging (2.4%), and the waiting room or waiting room amenities (1.3%). Of the tweets, 31.9% contained humor, of which 56.1% related to compression. Themes that were more common in patients undergoing their first, rather than follow-up, mammogram included breast compression (16.4% vs 9.1%, respectively) and that the test was not that bad (26.2% vs 7.6%, respectively).
Online social media provides a platform for women to share their experiences and reactions in undergoing mammography, including humor, positive reflections, and encouragement of others to undergo the examination. Social media thus warrants further evaluation as a potential tool to help foster greater adherence to screening guidelines.
Recently we have shown that capsaicin attenuates the strength of LTP in the lateral amygdala (LA) and demonstrated that this effect is mediated by the transient receptor potential (TRP) channel TRPV1. Here we further show that capsaicin, which is thought to act primarily through TRPV1, modifies long term depression (LTD) in the LA. Yet the application of various TRPV1 antagonists does not reverse this effect and it remains in TRPV1-deficient mice. In addition, voltage gated calcium channels, nitric oxide and CB1 receptors are not involved. Using pharmacology and TRPM1(-/-) mice, our electrophysiological data indicate that capsaicin-induced activation of TRPM1 channels contribute to the induction of LA-LTD. Whereas LA-LTD in general depends on the acitvation of NMDA receptors- and group II metabotropic glutamate receptors (mGluR), the modifying effect of capsaicin on LA-LTD via TRPM1 appears to be specifically mediated by group I mGluRs and in interaction with another member of the TRP family, TRPC5. Additionally, intact GABAergic transmission is required for the capsaicin-effect to take place. This is the first documentation that beside their function in the retina TRPM1 proteins are expressed in the brain and have a functional relevance in modifying synaptic plasticity.
In the synaptic cleft between type I hair cells and calyceal afferents, K(+) ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft. High-fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance. Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion. Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential. With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K(+) ]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs.
Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch-clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High-fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA-dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high-speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells.
Polymeric nanoparticles (NPs) with two-photon (TP) activity were prepared by grafting a naphthalimide fluorophore onto poly(acrylic acid) to yield an amphiphilic polymer, which self-assembled in water. The NPs were characterized using various analytical techniques such as transmission electron microscopy, dynamic light scattering and spectroscopic measurements. The in vitro and in vivo biocompatibilities of the NPs were assessed by a cytotoxicity assay using HeLa cells and a feeding assay using Caenorhabditis elegans (C. elegans) as a small animal model, respectively. Finally, TP fluorescence imaging (FI) of living cells and C. elegans labelled with the NPs were observed by TP confocal microscopy. The experimental outcomes demonstrated that the NPs had sufficient water-dispersity and biocompatibility, had TP fluorescence activity, were resistant to pH variation and illumination, and were physically stable. TP FI revealed that the NPs could enter living cells and were primarily located in the cytoplasm. In addition, the NPs were ingested by C. elegans during the feeding process and were recognized and taken up by the active transport system of the intestinal cells. These findings indicated the feasibility of using the developed NPs as a nanolabel for TP FI. Moreover, with numerous modifiable carboxyl groups on its surface, the NPs could act as a platform to build multifunctional probes for potential applications in biosensing and assay labeling.
N-acyl-homoserine lactones (AHL) are the quorum-sensing (QS) signal molecules used by many gram-negative bacteria to coordinate their collective behavior in a population. Recent evidence demonstrates their roles in plant root growth and defense responses. AtMYB44 is a multifaceted transcriptional factor that functions in many physiological processes in plants but whether AtMYB44 modulates the plant response to AHL with aspects of primary root elongation remains unknown. Here, we show that the expression of AtMYB44 was upregulated upon treatment with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). The stimulatory effect of 3OC6-HSL on primary root elongation was abolished in the AtMYB44 functional-deficiency mutant atmby44. In contrast, an enhanced promoting-impact of 3OC6-HSL on primary root growth was observed in AtMYB44-overexpressing plant MYB44OTA. Cellular analysis indicated that the prolonged primary root elicited by 3OC6-HSL is the consequence of increased cell division in the meristem zone and enhanced cell elongation in the elongation zone, and AtMYB44 may act as a positive regulator in this process. Furthermore, we demonstrated that AtMYB44 might participate in the 3OC6-HSL-mediated primary root growth via regulating the expression of cytokinin- and auxin-related genes. The data establish a genetic connection between the regulatory role of AtMYB44 in phytohormones-related gene expression and plant response to the bacterial QS signal.
The environmental coupling of the phycobiliprotein antenna complex PE555 and its excitonic energy transfer mechanisms are studied in detail. Molecular dynamics simulations were performed followed by calculations of the vertical transition energies along the classical ground-state trajectory. To this end, the distributions of energy levels for the PE555 complex were found to be similar to those of the PE545 complex despite the clear differences in the respective protein structures. In the PE555 complex the two αβ monomers are rotated by ∼73° compared to the PE545 structure leading to a water filled channel. Moreover, the connections between the bilins, which act as pigments in these aggregates, and the protein show clear differences in the two structures. Analyzing the coupling of the individual chromophores to the protein environment, however, yielded similar spectral densities in the two protein complexes. In addition, the partial transition charges of the involved bilins have been determined in order to calculate the electronic couplings using the transition charges from electrostatic potentials (TrEsp) method. For comparison purposes, the couplings have been extracted using the point-dipole approximation as well. On average the coupling values predicted by the dipole approximation are slightly larger than those from the TrEsp method leading to enhanced population decay rates as tested in ensemble-averaged wave packet dynamics. Moreover, the exciton dynamics in the PE555 structure is significantly slower than in the PE545 complex due to the smaller coupling values induced by the dissimilar arrangements of the monomers.
The effect of smoking cessation on centrilobular emphysema (CLE) and centrilobular nodularity (CN), two manifestations of smoking-related lung injury on computed tomography (CT) images, has not been clarified. The objective of this study is to leverage texture analysis to investigate differences in extent of CLE and CN between current and former smokers.
Chest CT scans from 350 current smokers, 401 former smokers, and 25 control subjects were obtained from the multicenter COPDGene Study, a Health Insurance Portability and Accountability Act-compliant study approved by the institutional review board of each participating clinical study center. Additionally, for 215 of these subjects, a follow-up CT scan was obtained approximately 5 years later. For each CT scan, 5000 circular regions of interest (ROIs) of 35-pixel diameter were randomly selected throughout the lungs. The patterns present in each ROI were summarized by 50 computer-extracted texture features. A logistic regression classifier was leveraged to classify each ROI as normal lung, CLE, or CN, and differences in the percentages of normal lung, CLE, and CN by study group were assessed.
Former smokers had significantly more CLE (P <0.01) but less CN (P <0.001) than did current smokers, even after adjustment for important covariates such as patient age, GOLD stage, smoking history, forced expiratory volume in 1 second, gas trapping, and scanner model. Among patients with longitudinal CT scans, continued smoking led to a slight increase in CLE (P = 0.13), whereas sustained abstinence from smoking led to further reduction in CN (P <0.05).
The proposed texture-based approach quantifies the extent of CN and CLE with high precision. Differences in smoking-related lung disease between longitudinal scans of current smokers and longitudinal scans of former smokers suggest that CN may be reversible on smoking cessation.
Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesia's depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.
Fuhrman nuclear grade is the most important histological parameter to predict prognosis in a patient of renal cell carcinoma (RCC). However, it suffers from inter-observer and intra-observer variation giving rise to need of a parameter that not only correlates with nuclear grade but is also objective and reproducible. Proliferation is the measure of aggressiveness of a tumour and it is strongly correlated with Fuhrman nuclear grade, clinical survival and recurrence in RCC. Ki-67 is conventionally used to assess proliferation. Mini-chromosome maintenance 2 (MCM-2) is a lesser known marker of proliferation and identifies a greater proliferation faction. This study was designed to assess the prognostic significance of MCM-2 by comparing it with Fuhrman nuclear grade and Ki-67.
n=50 cases of various ages, stages, histological subtypes and grades of RCC were selected for this study. Immunohistochemical staining using Ki-67(MIB-1, Mouse monoclonal antibody, Dako) and MCM-2 (Mouse monoclonal antibody, Thermo) was performed on the paraffin embedded blocks in the department of Morbid anatomy and Histopathology, University of Health Sciences, Lahore. Labeling indices (LI) were determined by two pathologists independently using quantitative and semi-quantitative analysis. Statistical analysis was carried out using SPSS 20.0. Kruskall-Wallis test was used to determine a correlation of proliferation markers with grade, and Pearson's correlate was used to determine correlation between the two proliferation markers.
Labeling index of MCM-2 (median=24.29%) was found to be much higher than Ki-67(median=13.05%). Both markers were significantly related with grade (p=0.00; Kruskall-Wallis test). LI of MCM-2 was found to correlate significantly with LI of Ki-67(r=0.0934;p=0.01 with Pearson's correlate). Results of semi-quantitative analysis correlated well with quantitative analysis.
Both Ki-67 and MCM-2 are markers of proliferation which are closely linked to grade. Therefore, they can act as surrogate markers for grade in a manner that is more objective and reproducible.
Studies have shown that advanced care planning improves communication and reduces suffering for patients and their bereaved caregivers. Despite this knowledge, the rates of advance care plans are low and physicians, as the primary gatekeepers, have made little progress in improving their rates. Through the lens of critical social theory, we examine these forces and identify the ideologies, assumptions, and social structures that curtail completion of advanced care plans such as Preserving Life, Ageism, Paternalism, and Market-Driven Healthcare System. A critical discourse provides suggestions to eliminate oppressive ideologies that act as barriers to advanced care planning.
Novel silver(I) complexes of coumarin oxyacetate ligands and their phenanthroline adducts have been prepared and characterised by microanalytical data and spectroscopic techniques (IR, (1)H, (13)C NMR, UV-Vis). The crystal structure of one Ag(I) complex was determined by X-ray diffraction analysis. The experimental spectroscopic data have been interpreted on the basis of molecular structure modeling and subsequent spectra simulation with density functional theory method. The binding modes of the coumarins and phenanthroline ligands (monodentate, bidentate, bridging) to Ag(I) have been theoretically modelled and discussed as to the most probable ligand binding in the series of complexes studied. The antimicrobial and antifungal activities have been determined and the complexes were found to have mostly moderate antibacterial activity but some of the phenanthroline adducts were found to have antifungal activity against the clinically important fungus C. albicans, comparable to that of the commercial agents, Amphotericin B and Ketoconazole. Preliminary investigations into the possible mechanism of action of the silver complexes indicated that they did not interact with DNA via nuclease activity or intercalation but the ability to act as a superoxide dismutase mimetic may be related to their antimicrobial activity.
Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4(+) T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4(+) Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.
Reductive cyclization of the 2,4,5-trisubstituted cyclohexenone 16 using dihydogen in the presence of Raney cobalt afforded compound 17 (60%) that could be elaborated over a further five steps, including one involving a cationic cyclization process, into the racemic modification of the unusual uleine alkaloid gilbertine. Single-crystal X-ray analyses of compounds (±)-1, 16, and a derivative of 17 are reported.
Naturally occurring anticancer agents and their derivatives act on multiple pathways to inhibit carcinogenesis and their inhibition of migration, invasion, growth, survival, and metastasis is associated with downregulation of genes associated with these responses. Several phytochemical-derived anticancer drugs including curcumin, betulinic acid, phenethylisothiocyanate and celastrol, and many others induce reactive oxygen species, and their effects on gene regulation show some overlap in various cancer cell lines. We hypothesize that reactive oxygen species-inducing anticancer agents and many other natural products target a common pathway in cancer cells, which initially involves downregulation of specificity protein 1 (Sp1), Sp3, and Sp4, which are highly expressed in tumors/cell lines derived from solid tumors. This hypothesis is supported by several published reports showing that a large number of phytochemical-derived anticancer agents downregulate Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes involved in cell growth (cyclin D1 and growth factor receptors), survival (bcl-2 and survivin), angiogenesis and migration (MMP-9, vascular endothelial growth factor and its receptors), and inflammation (NF-kB). The contribution of this pathway to the anticancer activity of drugs such as curcumin, celastrol, betulinic acid, and phenethylisothiocyanate must be determined in order to optimize clinical applications of drug combinations containing these compounds. Copyright © 2016 John Wiley & Sons, Ltd.
Long-wavelength solar UV radiation is implicated in photodamage to the human eye. The human lens contains multiple tryptophan-derived compounds that have significant absorbance bands in the UVA region (λ 315-400 nm) that act as efficient physical filters for these wavelengths. The concentrations of many of these UV filter compounds decrease with increase in age, resulting in diminished protection, increased oxidative damage and the accumulation of modified proteins implicated in nuclear cataract formation. This damage may arise via the formation of α,β-unsaturated carbonyls from the UV filter compounds, adduction to lens proteins and subsequent action as photosensitizers, and/or via the reactions of redox-active transition metal ions that accumulate in aged human lenses. The latter may promote the oxidation of free, or protein-bound, o-aminophenols, such as the UV filter compounds 3-hydroxykynurenine (3OHKyn) and 3-hydroxyanthranilic acid (3OHAA). It is shown here that Cu(II), and to a lesser extent Fe(III), enhance oxidation of free 3OHKyn, 3OHAA and 3OHKyn bound to specific amino acids and lens proteins, with this resulting in increased cross-linking of lens proteins. These data indicate that elevated levels of transition metal ions in aging lenses can enhance the loss of protective UV filter compounds, and contribute to the formation of high-molecular-mass dysfunctional crystallin proteins in a light-independent manner. These reactions may contribute to the formation of lens cataracts in humans.
In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.
Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled (3)H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25°C during a 30min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population.
Suicidal behavior comprises a diverse set of behaviors with significant differences among several behavioral categories. One noteworthy category includes individuals who have made serious suicide attempts, epidemiologically very similar to those completing suicide. This behavioral category is important, since interviewing survivors of a potentially lethal incident of self-harm enables a detailed investigation of the psychological process leading to the suicidal act.
To achieve a consensus definition and operational criteria of serious suicide attempts.
We reviewed studies that included the term serious suicide attempt or related terms (e.g., highly lethal), with a focus on the variety of operational criteria employed across studies.
More than 60 papers addressing various types of serious suicide attempt were explored. We found a large variety of operational definitions, reflecting the lack of consensus regarding terminology and criteria related to the term.
We undertook the challenge of developing an integrative and comprehensive set of criteria of serious suicide attempt and suggest a definition comprising three key dimensions: medical lethality, potential lethality of the method used, and severity of the objective circumstances of the suicide intent. Clinicians and researchers are strongly encouraged to consider using the term serious suicide attempt with its attendant components.
More than half the patients with rectal cancer present with locally advanced rectal disease at diagnosis with a high risk of recurrence. Preoperative chemoradiotherapy and standardized radical surgery with total mesorectal excision have been established as the 'gold standard' for treating these patients. Pathological staging using the ypTNM classification system to decide on adjuvant chemotherapy (ACT) is widely used in clinical practice, but the delivery of ACT is still controversial, as many discrepancies persist in the conclusions of different trials, due to heterogeneity of the inclusion criteria between studies, lack of statistical power, and variations in preoperative and adjuvant regimens. In 2014, a meta-analysis of four randomized phase-III trials (EORTC 22921, I-CNR-RT, PROCTOR-SCRIPT, CHRONICLE) failed to demonstrate any statistical efficacy of fluorouracil (5FU)-based ACT. Three recent randomized trials aimed to compare 5FU with 5FU plus oxaliplatin-based chemotherapy. Two of them (ADORE, CAO/ARO/AIO-04) appeared to find a disease-free survival benefit for patients treated with the combination therapy. Thus, while awaiting new data, it can be said that, as of 2015, patients with yp stage I tumors or histological complete response derived no benefit from adjuvant therapy. On the other hand, the FOLFOX chemotherapy regimen should be proposed for yp stage III patients, and may be considered for yp stage II tumors in fit patients with high-risk factors. Nevertheless, well-designed and sufficiently powered clinical trials dedicated to adjuvant treatments for rectal cancer remain justified in future to achieve a high level of proof in keeping with evidence-based medical standards.
The U.S. Endangered Species Act (ESA) requires that the "best available scientific and commercial data" be used to protect imperiled species from extinction and preserve biodiversity. However, it does not provide specific guidance on how to apply this mandate. Scientific data can be uncertain and controversial, particularly regarding species delineation and hybridization issues. The U.S. Fish and Wildlife Service (FWS) had an evolving hybrid policy to guide protection decisions for individuals of hybrid origin. Currently, this policy is in limbo because it resulted in several controversial conservation decisions in the past. Biologists from FWS must interpret and apply the best available science to their recommendations and likely use considerable discretion in making recommendations for what species to list, how to define those species, and how to recover them. We used semistructured interviews to collect data on FWS biologists' use of discretion to make recommendations for listed species with hybridization issues. These biologists had a large amount of discretion to determine the best available science and how to interpret it but generally deferred to the scientific consensus on the taxonomic status of an organism. Respondents viewed hybridization primarily as a problem in the context of the ESA, although biologists who had experience with hybridization issues were more likely to describe it in more nuanced terms. Many interviewees expressed a desire to continue the current case-by-case approach for handling hybridization issues, but some wanted more guidance on procedures (i.e., a "flexible" hybrid policy). Field-level information can provide critical insight into which policies are working (or not working) and why. The FWS biologists' we interviewed had a high level of discretion, which greatly influenced ESA implementation, particularly in the context of hybridization.
Many argue that monitoring conducted exclusively by scientists is insufficient to address ongoing environmental challenges. One solution entails the use of mobile digital devices in participatory monitoring (PM) programs. But how digital data entry affects programs with varying levels of stakeholder participation, from nonscientists collecting field data to nonscientists administering every step of a monitoring program, remains unclear. We reviewed the successes, in terms of management interventions and sustainability, of 107 monitoring programs described in the literature (hereafter programs) and compared these with case studies from our PM experiences in Australia, Canada, Ethiopia, Ghana, Greenland, and Vietnam (hereafter cases). Our literature review showed that participatory programs were less likely to use digital devices, and 2 of our 3 more participatory cases were also slow to adopt digital data entry. Programs that were participatory and used digital devices were more likely to report management actions, which was consistent with cases in Ethiopia, Greenland, and Australia. Programs engaging volunteers were more frequently reported as ongoing, but those involving digital data entry were less often sustained when data collectors were volunteers. For the Vietnamese and Canadian cases, sustainability was undermined by a mismatch in stakeholder objectives. In the Ghanaian case, complex field protocols diminished monitoring sustainability. Innovative technologies attract interest, but the foundation of effective participatory adaptive monitoring depends more on collaboratively defined questions, objectives, conceptual models, and monitoring approaches. When this foundation is built through effective partnerships, digital data entry can enable the collection of more data of higher quality. Without this foundation, or when implemented ineffectively or unnecessarily, digital data entry can be an additional expense that distracts from core monitoring objectives and undermines project sustainability. The appropriate role of digital data entry in PM likely depends more on the context in which it is used and less on the technology itself.
Recovery planning for species listed under the U.S. Endangered Species Act has been hampered by a lack of consistency and transparency, which can be improved by implementing a standardized approach for evaluating species status and developing measurable recovery criteria. However, managers lack an assessment method that integrates threat abatement and can be used when demographic data are limited. To help meet these needs, we demonstrated an approach for evaluating species status based on habitat configuration data. We applied 3 established persistence measures (patch occupancy, metapopulation capacity, and proportion of population lost) to compare 2 conservation strategies (critical habitat designated by the U.S. Fish and Wildlife Service and the Forest Service's Carbonate Habitat Management Strategy) and 2 threat scenarios (maximum limestone mining, removal of all habitat in areas with mining claims; minimum mining, removal of habitat only in areas with existing operations and high-quality ore) against a baseline of existing habitat for 3 federally listed plant species. Protecting all area within the designated critical habitat maintained a similar level (83.9-99.9%) of species persistence as the baseline, whereas maximum mining greatly reduced persistence (0.51-38.4% maintained). The 3 persistence measures provided complementary insights reflecting different aspects of habitat availability (total area, number of patches, patch size, and connectivity). These measures can be used to link recovery criteria developed following the 3 R principles (representation, redundancy, and resilience) to the resulting improvements in species viability. By focusing on amount and distribution of habitat, our method provides a means of assessing the status of data-poor species to inform decision making under the Endangered Species Act.
Diagnosis of developmental or congenital prosopagnosia (CP) involves self-report of everyday face recognition difficulties, which are corroborated with poor performance on behavioural tests. This approach requires accurate self-evaluation. We examine the extent to which typical adults have insight into their face recognition abilities across four experiments involving nearly 300 participants. The experiments used five tests of face recognition ability: two that tap into the ability to learn and recognize previously unfamiliar faces [the Cambridge Face Memory Test, CFMT; Duchaine, B., & Nakayama, K. (2006). The Cambridge Face Memory Test: Results for neurologically intact individuals and an investigation of its validity using inverted face stimuli and prosopagnosic participants. Neuropsychologia, 44(4), 576-585. doi:10.1016/j.neuropsychologia.2005.07.001; and a newly devised test based on the CFMT but where the study phases involve watching short movies rather than viewing static faces-the CFMT-Films] and three that tap face matching [Benton Facial Recognition Test, BFRT; Benton, A., Sivan, A., Hamsher, K., Varney, N., & Spreen, O. (1983). Contribution to neuropsychological assessment. New York: Oxford University Press; and two recently devised sequential face matching tests]. Self-reported ability was measured with the 15-item Kennerknecht et al. questionnaire [Kennerknecht, I., Ho, N. Y., & Wong, V. C. (2008). Prevalence of hereditary prosopagnosia (HPA) in Hong Kong Chinese population. American Journal of Medical Genetics Part A, 146A(22), 2863-2870. doi:10.1002/ajmg.a.32552]; two single-item questions assessing face recognition ability; and a new 77-item meta-cognition questionnaire. Overall, we find that adults with typical face recognition abilities have only modest insight into their ability to recognize faces on behavioural tests. In a fifth experiment, we assess self-reported face recognition ability in people with CP and find that some people who expect to perform poorly on behavioural tests of face recognition do indeed perform poorly. However, it is not yet clear whether individuals within this group of poor performers have greater levels of insight (i.e., into their degree of impairment) than those with more typical levels of performance.
Irritable bowel syndrome (IBS) is the most prevalent functional gastrointestinal (GI) disorder, which presents with abdominal pain and changes in the bowel habits. Although the exact cause of IBS remains uncertain, some studies have shown that the inflammation and cytokine imbalance may act as potential etiological factors. The aim of our study is to compare the serum levels of interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor-alpha (TNF-α) in patients with IBS with the healthy controls. The other aim of this study is to evaluate possible association between above-mentioned cytokines and IBS subtypes.
Seventy-four IBS patients diagnosed based on Rome III criteria and 75 gender and age-matched healthy controls were included in this study. Cytokines were measured in the serum using enzyme-linked immunosorbent assays (ELISA).
Patients were classified into groups of IBS with diarrhea (IBS-D): 34, IBS with constipation (IBS-C): 29, and IBS with mixed symptoms (IBS-M): 11. The serum levels of IL-6, IL-8 and TNF-α were significantly higher in patients with IBS as compared to controls (P<0.001). There was no difference in serum levels of cytokines based on IBS subtypes.
Higher serum level of IL-6, IL-8 and TNF-α in IBS suggests an important role of cytokines as immune mediators in the pathogenesis of this functional GI disorder. To understand any association between cytokines and IBS subtypes, further investigations with larger sample sizes are desired.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important mediators of signaling pathways in the endothelium. Specific members of the transient receptor potential (TRP) superfamily of cation channels act as important Ca(2+) influx pathways in endothelial cells, and are involved in endothelium-dependent vasodilation, regulation of barrier permeability, and angiogenesis. ROS and RNS can modulate the activity of certain TRP channels mainly by modifying specific cysteine residues or by stimulating the production of second messengers. In this review we highlight the recent literature describing in redox regulation of TRP channel activity in endothelial cells as well as the physiological importance of these pathways and implication for cardiovascular diseases. This article is protected by copyright. All rights reserved.
Solvothermal reactions between copper(I) halides and 4-mercaptophenol give rise to the formation of three coordination polymers with general formula [Cu3 X(HT)2 ]n (X=Cl, 1; Br, 2; and I, 3). The structures of these coordination polymers have been determined by X-ray diffraction at both room- and low temperature (110 K), showing a general shortening in Cu-S, Cu-X and Cu-Cu bond lengths at low temperatures. 1 and 2 are isostructural, consisting of layers in which the halogen ligands act as μ3 -bridges joining two Cu1 and one Cu2 atoms whereas in 3 the iodine ligands is as μ4 -mode but the layers are quasi-isostructural with 1 or 2. These compounds show a reversible thermochromic luminescence, with strong orange emission for 1 and 2, but weaker for 3 at room temperature, whereas upon cooling at 77 K 1 and 2 show stronger yellow emission, and 3 displays stronger green emission. DFT calculations have been used to rationalize these observations. These results suggest a high potential for this novel and promising stimuli-responsive materials.
How does emotion explain the relationship between social rejection and aggression? Rejection reliably damages mood, leaving individuals motivated to repair their negatively valenced affective state. Retaliatory aggression is often a pleasant experience. Rejected individuals may then harness revenge's associated positive affect to repair their mood. Across 6 studies (total N = 1,516), we tested the prediction that the rejection-aggression link is motivated by expected and actual mood repair. Further, we predicted that this mood repair would occur through the positive affect of retaliatory aggression. Supporting these predictions, naturally occurring (Studies 1 and 2) and experimentally manipulated (Studies 3 and 4) motives to repair mood via aggression moderated the rejection-aggression link. These effects were mediated by sadistic impulses toward finding aggression pleasant (Studies 2 and 4). Suggesting the occurrence of actual mood repair, rejected participants' affective states were equivalent to their accepted counterparts after an act of aggression (Studies 5 and 6). This mood repair occurred through a dynamic interplay between preaggression affect and aggression itself, and was driven by increases in positive affect (Studies 5 and 6). Together, these findings suggest that the rejection-aggression link is driven, in part, by the desire to return to affective homeostasis. Additionally, these findings implicate aggression's rewarding nature as an incentive for rejected individuals' violent tendencies. (PsycINFO Database Record
Our experiment is aimed at understanding how employee reactions to negative feedback are received by the feedback provider and how employee gender may play a role in the process. We focus specifically on the act of crying and, based on role congruity theory, argue that a male employee crying in response to negative performance feedback will be seen as atypical behavior by the feedback provider, which will bias evaluations of the employee on a number of different outcome variables, including performance evaluations, assessments of leadership capability, and written recommendations. That is, we expect an interactive effect between gender and crying on our outcomes, an effect that will be mediated by perceived typicality. We find support for our moderated mediation model in a sample of 169 adults, indicating that men who cry in response to negative performance feedback will experience biased evaluations from the feedback provider. Theoretical and practical implications are discussed. (PsycINFO Database Record
Three Ru-CO complexes, [Ru(pbn)2(CO)2](2+), [Ru(pbn)2(CO)(COOH)](+), [Ru(pbn)2(CO)(COO)](0) [pbn = 2-(pyridin-2-yl)benzo[b]-1,5-naphthyridine], exist as equilibrium mixtures in aqueous solutions. Thermal decarboxylation of [Ru(pbn)2(CO)(COOH)](+) and/or [Ru(pbn)2(CO)(COO)](0) induces a two-electron reduction of pbn to form [Ru(pbn)(pbnHH)(CO)(OH2)](2+) [pbnHH = 2-(pyridin-2-yl)-5,10-dihydrobenzo[b]-1,5-naphthyridine] in H2O.
The seven members of the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the TNF-R superfamily. Functionally, TRAFs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogen-activated protein kinases (MAPKs) and transcription factors of nuclear factor-κB family (NF-κB). Given the wide variety of stimuli intracellularly conveyed by TRAF proteins, they are physiologically involved in multiple biological processes, including embryonic development, tissue homeostasis and regulation of innate and adaptive immune responses. In the last few years, it has become increasingly evident the involvement of TRAF7, the last member of the TRAF family to be discovered, in the genesis and progression of several human cancers, placing TRAF7 in the spotlight as a novel tumor suppressor protein. In this paper, we review and discuss the literature recently produced on this subject. This article is protected by copyright. All rights reserved.
The interaction of silicon (Si) nanospheres (NSs) with a thin metal film is investigated numerically and experimentally by characterizing their forward scattering properties. A sharp resonant mode and a zero-scattering dip are found to be introduced in the forward scattering spectrum of a Si NS by putting it on a 50-nm-thick gold film. It is revealed that the sharp resonant mode arises from a new magnetic dipole induced by the electric dipole and its mirror image while the zero-scattering dip originates from the destructive interference between the new magnetic dipole and the original one together with its mirror image. A significant enhancement in both electric and magnetic fields is achieved at the contact point between the Si NS and the metal film. More interestingly, the use of a thin silver film can lead to vivid scattering light with different color indices. It is demonstrated that a small change in the surrounding environment of Si NSs results in the broadening of the resonant mode and the disappearance of the zero-scattering dip. Our findings indicate that dielectric-metal hybrid systems composed of semiconductor NSs and thin metal films act as attractive platforms on which novel nanoscale plasmonic devices can be realized.
Solar H2 generation from water has been intensively investigated as a clean method to convert solar energy into hydrogen fuel. During the past few decades, many studies have demonstrated that metal complexes can act as efficient photoactive materials for photocatalytic H2 production. Here, we review the recent progress in the application of metal-complex chromophores to solar-to-H2 conversion, including metal-complex photosensitizers and supramolecular photocatalysts. A brief overview of the fundamental principles of photocatalytic H2 production is given. Then, different metal-complex photosensitizers and supramolecular photocatalysts are introduced in detail, and the most important factors that strictly determine their photocatalytic performance are also discussed. Finally, we illustrate some challenges and opportunities for future research in this promising area.
Preserving global public goods, such as the planet's ecosystem, depends on large-scale cooperation, which is difficult to achieve because the standard reciprocity mechanisms weaken in large groups. Here we demonstrate a method by which reciprocity can maintain cooperation in a large-scale public goods game (PGG). In a first experiment, participants in groups of on average 39 people play one round of a Prisoner's Dilemma (PD) with their two nearest neighbours on a cyclic network after each PGG round. We observe that people engage in "local-to-global" reciprocity, leveraging local interactions to enforce global cooperation: Participants reduce PD cooperation with neighbours who contribute little in the PGG. In response, low PGG contributors increase their contributions if both neighbours defect in the PD. In a control condition, participants do not know their neighbours' PGG contribution and thus cannot link play in the PD to the PGG. In the control we observe a sharp decline of cooperation in the PGG, while in the treatment condition global cooperation is maintained. In a second experiment, we demonstrate the scalability of this effect: in a 1,000-person PGG, participants in the treatment condition successfully sustain public contributions. Our findings suggest that this simple "local-to-global" intervention facilitates large-scale cooperation.
α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.
A long-standing paradigm in drug discovery has been the concept of designing maximally selective drugs to act on individual targets considered to underlie a disease of interest. Nonetheless, although some drugs have proven to be successful, many more potential drugs identified by the "one gene, one drug, one disease" approach have been found to be less effective than expected or to cause notable side effects. Advances in systems biology and high-throughput in-depth genomic profiling technologies along with an analysis of the successful and failed drugs uncovered that the prominent factor to determine drug sensitivity is the intrinsic robustness of the response of biological systems in the face of perturbations. The complexity of the molecular and cellular bases of systems responses to drug interventions has fostered an increased interest in systems-oriented approaches to drug discovery. Consonant with this knowledge of the multifactorial mechanistic basis of drug sensitivity and resistance is the application of network-based approaches for the identification of molecular (multi-)feature signatures associated with desired (multi-)drug phenotypic profiles. This chapter illustrates the principal network analysis and inference techniques which have found application in systems-oriented drug design and considers their benefits and drawbacks in relation to the nature of the data produced by network pharmacology.
Stem cell therapy (SCT) raises the hope for cardiac regeneration in ischemic hearts. However, underlying molecular mechanisms for repair of dead myocardium by SCT in the ischemic heart is poorly understood. Growing evidences suggest that cardiac matrix stiffness and differential expressions of miRNAs play a crucial role in stem cell survival and differentiation. However, their roles on transplanted stem cells, for myocardial repair of the ischemic heart, remain unclear. Transplanted stem cells may act in an autocrine and/or paracrine manner to regenerate the dead myocardium. Paracrine mediators such as stem cell-derived exosomes are emerging as a novel therapeutic strategy to overcome some of the limitations of SCT. These exosomes carry microRNAs (miRNAs) that may regulate stem cell differentiation into a specific lineage. MicroRNAs may also contribute to stiffness of surrounding matrix by regulating extracellular matrix (ECM) turnover. The survival of transplanted stem cell depends on its autophagic process that maintains cellular homeostasis. Therefore, exosomes, miRNAs, extracellular matrix turnover, and autophagy may have an integral role in improving the efficacy of SCT. This review elaborates the specific roles of these regulatory components on cardiac regeneration in the ischemic heart during SCT.
For a successful nitrogen removal, Anammox process needs to be established in line with a stable partial nitritation pretreatment unit since wastewater influent is mostly unsuitable for direct treatment by Anammox. Partial nitritation is, however, a critical bottleneck for the nitrogen removal since it is often difficult to maintain the right proportions of NO2-N and NH4-N during long periods of time for Anammox process. This study investigated the potential of Anammox-zeolite biofilter to buffer inequalities in nitrite and ammonium nitrogen in the influent feed. Anammox-zeolite biofilter combines the ion-exchange property of zeolite with the biological removal by Anammox process. Continuous-flow biofilter was operated for 570 days to test the response of Anammox-zeolite system for irregular ammonium and nitrite nitrogen entries. The reactor demonstrated stable and high nitrogen removal efficiencies (approximately 95 %) even when the influent NO2-N to NH4-N ratios were far from the stoichiometric ratio for Anammox reaction (i.e. NO2-N to NH4-N ranging from 0 to infinity). This is achieved by the sorption of surplus NH4-N by zeolite particles in case ammonium rich influent came in excess with respect to Anammox stoichiometry. Similarly, when ammonium-poor influent is fed to the reactor, ammonium desorption took place due to shifts in ion-exchange equilibrium and deficient amount were supplied by previously sorbed NH4-N. Here, zeolite acted as a preserving reservoir of ammonium where both sorption and desorption took place when needed and this caused the Anammox-zeolite system to act as a buffer system to generate a stable effluent.
Real-world fall events objectively measured by body-worn sensors can improve the understanding of fall events in older people. However, these events are rare and hence challenging to capture. Therefore, the FARSEEING (FAll Repository for the design of Smart and sElf-adaptive Environments prolonging Independent livinG) consortium and associated partners started to build up a meta-database of real-world falls.
Between January 2012 and December 2015 more than 300 real-world fall events have been recorded. This is currently the largest collection of real-world fall data recorded with inertial sensors. A signal processing and fall verification procedure has been developed and applied to the data. Since the end of 2015, 208 verified real-world fall events are available for analyses. The fall events have been recorded within several studies, with different methods, and in different populations. All sensor signals include at least accelerometer measurements and 58 % additionally include gyroscope and magnetometer measurements. The collection of data is ongoing and open to further partners contributing with fall signals. The FARSEEING consortium also aims to share the collected real-world falls data with other researchers on request.
The FARSEEING meta-database will help to improve the understanding of falls and enable new approaches in fall risk assessment, fall prevention, and fall detection in both aging and disease.
Tissue organization and patterning are critical during development when genetically identical cells take on different fates. Lateral signalling plays an important role in this process by helping to generate self-organized spatial patterns in an otherwise uniform collection of cells. Recent data suggest that lateral signalling can be mediated both by junctional contacts between neighbouring cells and via cellular protrusions that allow non-neighbouring cells to interact with one another at a distance. However, it remains unclear precisely how signalling mediated by these distinct types of cell-cell contact can physically contribute to the generation of complex patterns without the assistance of diffusible morphogens or pre-patterns. To explore this question, in this work we develop a model of lateral signalling based on a single receptor/ligand pair as exemplified by Notch and Delta. We show that allowing the signalling kinetics to differ at junctional versus protrusion-mediated contacts, an assumption inspired by recent data which show that the cleavage of Notch in several systems requires both Delta binding and the application of mechanical force, permits individual cells to act to promote both lateral activation and lateral inhibition. Strikingly, under this model, in which Delta can sequester Notch, a variety of patterns resembling those typical of reaction-diffusion systems is observed, together with more unusual patterns that arise when we consider changes in signalling kinetics, and in the length and distribution of protrusions. Importantly, these patterns are self-organizing-so that local interactions drive tissue-scale patterning. Together, these data show that protrusions can, in principle, generate different types of patterns in addition to contributing to long-range signalling and to pattern refinement.
The hormone fluctuations that an animal experiences during ovulation can have lifelong effects on developing offspring. These hormones may act as an adaptive mechanism, allowing offspring to be 'pre-programmed' to survive in an unstable environment. Here, we used a transgenerational approach to examine the effects of elevated maternal corticosterone (CORT) on the future reproductive success of female offspring. We show that female zebra finches (Taeniopygia guttata) exposed to embryonic CORT produce daughters that have equal reproductive success (clutch sizes, fertility, hatching success) compared with the daughters produced from untreated mothers, but their offspring had accelerated post-hatching growth rates and were significantly heavier by nutritional independence. Although there was no significant effect on primary offspring sex ratio, females from CORT-treated mothers produced significantly female-biased clutches by nutritional independence. To the best of our knowledge, this is the first record of a transgenerational sex ratio bias in response to elevated maternal CORT in any avian species.
Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Post-translational modifications (PTM) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event of a conserved tyrosine (Y) in the bovine papillomavirus type 1 (BPV-1) E2 protein at amino acid 102. While its phospho-deficient mutant phenylalanine (F) activated both transcription and replication in luciferase reporter assays, the mutant that may act as a phospho-mimetic, Y102 to glutamate (E), lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1, however in contrast, Y102E associated with only a subset and was unable to bind to E1. While Y102F fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.
Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understanding the viral replicative cycle - and potential therapies to target it - is examining post-translational modification of viral proteins and its effect on function. Here we have discovered that the viral transcription and replication regulator bovine papillomavirus type 1 (BPV-1) E2 is phosphorylated at tyrosine residue Y102. While a phospho-deficient mutant at this site was fully functional, a phospho-mimetic mutant displayed impaired transcription and replication activity as well as lack of association with certain E2-binding proteins. This study highlights the influence of post-translational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.
The RIG-I signaling pathway is essential for the recognition of viruses and the initiation of host IFN-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds MAVS, leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway, through the use of Bimolecular Fluorescence Complementation (BiFC) and super resolution microscopy. Dimers of RIG-I, TRIM25 and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25 mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus (HCV) redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. By contrast, the influenza A virus (IAV) NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm, and inhibits the formation of TRIM25 homocomplexes, but not of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25 and MAVS, in the presence and absence of two viral IFN antagonistic proteins.
The first line of defense against viral infections is the innate immune response. Viruses are recognized by Pathogen Recognition Receptors (PRR), like the RIG-I like receptor (RLR) family, that activate a signaling cascade that induces IFN production. In the present study we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal leading to an efficient antiviral state. In addition, this work presents a new means by how, when and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.
Neutrophils act as a first line of defense against bacterial and fungal infections, but they are also important effectors of acute and chronic inflammation. Genome-wide association studies have established that the gene encoding the protein tyrosine phosphatase nonreceptor 22 (PTPN22) makes an important contribution to susceptibility to autoimmune disease, notably rheumatoid arthritis. Although PTPN22 is most highly expressed in neutrophils, its function in these cells remains poorly characterized. We show in this article that neutrophil effector functions, including adhesion, production of reactive oxygen species, and degranulation induced by immobilized immune complexes, were reduced in Ptpn22(-/-) neutrophils. Tyrosine phosphorylation of Lyn and Syk was altered in Ptpn22(-/-) neutrophils. On stimulation with immobilized immune complexes, Ptpn22(-/-) neutrophils manifested reduced activation of key signaling intermediates. Ptpn22(-/-) mice were protected from immune complex-mediated arthritis, induced by the transfer of arthritogenic serum. In contrast, in vivo neutrophil recruitment following thioglycollate-induced peritonitis and in vitro chemotaxis were not affected by lack of PTPN22. Our data suggest an important role for PTPN22-dependent dephosphorylation events, which are required to enable full FcγR-induced activation, pointing to an important role for this molecule in neutrophil function.
The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis.
Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT.
Clostridium difficile infection (CDI) is becoming less exclusively a healthcare-associated (HA) infection. Community-associated (CA)-CDI has increased over the past decades. It has been postulated that asymptomatic toxigenic C. difficile (TCD)-colonised patients may play a role in the transfer of C. difficile between the hospital setting and the community. Thus, to investigate the relatedness of C. difficile across the hospital and community settings, we compared host and pathogen characteristics among symptomatic and asymptomatic patients in these two settings over a 3-year period. Two studies were simultaneously conducted, the first study enrolled symptomatic CDI patients from two tertiary hospitals and the community in two Australian states; while the second study enrolled asymptomatic TCD-colonised patients from the same tertiary hospitals. A total of 324 patients (96 HA-CDI, 152 CA-CDI and 76 TCD-colonised) were enrolled. The predominant C. difficile ribotypes isolated in the hospital setting corresponded with those isolated in the community, 79% of the C. difficile isolates from the hospitals had a matching ribotype isolated from the community, suggesting that transmission between these two settings is occurring. Toxigenic C. difficile strains causing symptomatic infection were similar to those causing asymptomatic infection and patients exposed to antimicrobials prior to admission were more likely to develop symptomatic infection (OR 2.94; 95%CI 1.20-7.14). Our findings suggest that development of CDI symptoms in a setting without establishment of hospital epidemics with binary toxin producing C. difficile strains may be driven mainly by host susceptibility and exposure to antimicrobials, rather than by C. difficile strain characteristics.
There are profound sex differences in the incidence of many psychiatric disorders. Although these disorders are frequently linked to social stress and to deficits in social engagement, little is known about sex differences in the neural mechanisms that underlie these phenomena. Phenotypes characterized by dominance, competitive aggression, and active coping strategies appear to be more resilient to psychiatric disorders such as posttraumatic stress disorder (PTSD) compared with those characterized by subordinate status and the lack of aggressiveness. Here, we report that serotonin (5-HT) and arginine-vasopressin (AVP) act in opposite ways in the hypothalamus to regulate dominance and aggression in females and males. Hypothalamic injection of a 5-HT1a agonist stimulated aggression in female hamsters and inhibited aggression in males, whereas injection of AVP inhibited aggression in females and stimulated aggression in males. Striking sex differences were also identified in the neural mechanisms regulating dominance. Acquisition of dominance was associated with activation of 5-HT neurons within the dorsal raphe in females and activation of hypothalamic AVP neurons in males. These data strongly indicate that there are fundamental sex differences in the neural regulation of dominance and aggression. Further, because systemically administered fluoxetine increased aggression in females and substantially reduced aggression in males, there may be substantial gender differences in the clinical efficacy of commonly prescribed 5-HT-active drugs such as selective 5-HT reuptake inhibitors. These data suggest that the treatment of psychiatric disorders such as PTSD may be more effective with the use of 5-HT-targeted drugs in females and AVP-targeted drugs in males.
Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. Here, we discover that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo, a gene required in SHH-receiving cells, we found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg), to induce dermal adipogenesis. Our study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages.
Whether it is the result of a tragic news story, a thoughtful commentary, or a segment on the entertainment networks, patient privacy rights are never far from the top of our minds. The Privacy and Security Rules contained in the Health Insurance Portability and Accountability Act of 1996 (HIPAA) represent a concerted effort to protect the privacy and security of the volumes of patient data generated by the health care system. However, the last twenty years has seen innovations and advancements in health information technology that were unimaginable at that time. It is time for innovation to the Privacy and Security Rules. We offer a common and relatable scenario as proof that certain Privacy and Security Rules can tie the hands of educators and innovators and need to be transformed.
Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure-activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β-α',β'-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.
A child is a developing person with evolving capacities that include autonomy, mental (decisional) capacity and capacity to assume responsibility. Hence, children are entitled to participatory (autonomy) rights in South Africa as observed in the Children's Act 38 of 2005. According to section 129 of the Act a child may consent to his or her own medical treatment provided that he or she is over the age of 12 years and is of sufficient maturity and decisional capacity to understand the various implications of the treatment including the risks and benefits thereof. However, the Act does not provide a definition for what qualifies as 'sufficient maturity' nor does it stipulate how health professionals ought to assess the decisional capacity of a child. In addition, South Africa is a culturally diverse country. The Western liberal notion of autonomy may not necessarily find equal prominence in the mores of people with a different worldview. Hence we demonstrate a few salient comparisons between legal liberal moral theory and African communitarianism as pertinent to the autonomy of the child.
Children are rights-holders by virtue of their humanity. Their dignity as individual human persons affords them the entitlement to human rights as contemplated under the Constitution of the Republic of South Africa. However, contrary to the traditional Western notion of individual autonomous persons African societies hold a communalistic notion of person hence there is less regard for individual autonomy and rights with more emphasis on the communal good and maintaining the continuity of relationships and interdependencies shared within a community. A child considered in this view is not regarded as a full person. This implies that decisions concerning the child, including consent to medical treatment are discussed and determined by the community to which the child belongs. Lastly, in this article, we draw on the notion of capacity for responsibility to produce a pragmatic definition of sufficient maturity.
It seems reasonable to suggest a move away from a general legal age of consent for medical treatment toward more individualised, context-specific approaches in determining the maturity of a child patient to consent to medical treatment. Perhaps, decision-making with respect to consent to the medical treatment of a child belonging to a traditional African community where the notion of a person is embedded in communitarianism ought to involve the child's parents/guardians/caregivers where possible provided that the best interests of the child are awarded priority.
Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.
The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.
Nanowire networks act as self-healing smart materials, whose sheet resistance can be tuned via an externally applied voltage stimulus. This memristive response occurs due to modification of junction resistances to form a connectivity path across the lowest barrier junctions in the network. While most network studies have been performed on expensive noble metal nanowires like silver, networks of inexpensive nickel nanowires with a nickel oxide coating can also demonstrate resistive switching, a common feature of metal oxides with filamentary conduction. However, networks made from solely nickel nanowires have high operation voltages which prohibit large-scale material applications. Here we show, using both experiment and simulation, that a heterogeneous network of nickel and silver nanowires allows optimization of the activation voltage, as well as tuning of the conduction behavior to be either resistive switching, memristive, or a combination of both. Small percentages of silver nanowires, below the percolation threshold, induce these changes in electrical behaviour, even for low area coverage and hence very transparent films. Silver nanowires act as current concentrators, amplifying conductivity locally as shown in our computational dynamical activation framework for networks of junctions. These results demonstrate that a heterogeneous nanowire network can act as a cost-effective adaptive material with minimal use of noble metal nanowires, without losing memristive behaviour that is essential for smart sensing and neuromorphic applications.
Targeted delivery of cytotoxic drugs to tumour tissue has great importance for successful chemotherapy. Legumain is an asparaginyl endopeptidase that is highly up-regulated in a number of solid tumours. The aim of this work was to prepare a novel hyaluronic acid (HA) based legumain sensitive nanogel for the delivery of doxorubicin with a high targeting efficiency both in vitro and in vivo. The legumain sensitive property is achieved by the conjugation of doxorubicin with HA via a legumain substrate peptide bridge. This HA derivative is further crosslinked in a water/oil solvent system to form a polygonal nanogel. Doxorubicin released in the tumour tissue is sustained thanks to the combined action of legumain and hyaluronidase, which are both overexpressed in tumour tissues. Hyaluronic acid could act as a targeting agent to CD44 (HA receptor), which further improved the in vivo target effect and enhanced in vitro cellular uptake. The developed nanogel exhibited a high therapeutic index that improved tumour inhibition effects and reduced system toxicity in a lung cancer mice model. These results highlighted the advantages of using this multi-functional material for a successful delivery of doxorubicin against cancer.
Preclinical Research With the almost global availability of the Internet comes the expectation of universal accessibility to knowledge, including scientific knowledge-particularly that generated by public funding. Currently this is not the case. In this Commentary we discuss access to this knowledge, the politics that govern peer review and publication, and the role of this knowledge as a public good in medicine.  Gutenberg's invention of the printing press in 1440 opened an avenue for the distribution of scholarly information to the entire world. The scientific literature first appeared in 1665 with Le Journal des Sçavans followed in the same year by Philosophical Transactions. Today there are more than 5000 scientific publishing companies, 25,000 journals and 1.5 million articles published/year generating revenue of $25 billion USD.  The European Union and the Organization for Economic Cooperation and Development have argued for open access (OA) to scientific data for all publicly funded research by 2020 with a similar initiative in the USA via the Fair Access to Science and Technology Research Act (FASTR). However, OA to published science is but one step in this odyssey. If the products of science are not openly available then it can be argued that the norms of science as defined by Merton including "universalism" and "communalism" have yet to be accomplished. Nowhere is this more apparent than in the delivery of medicines to the poor and for rare diseases, the attempts to privatize human genetic information and, not least, dealing with the challenges of antibiotic resistance and new disease pandemics exacerbated by climate change.
Measures such as storm water ponds, constructed wetlands and buffer strips along streams are used to reduce diffuse phosphorus (P) loading to surface waters. These systems often retain particulate P well, whereas the retention of dissolved P is less efficient and might require addition of P adsorbents. In this study, we screened waterwork ochre sludge (WWS) originating from groundwater treatment and ochre sludge from ochre precipitation basins along streams for their applicability as P adsorbents at ambient P concentrations. We compared with a commercial ferric hydroxide (CFH 12™) for which adsorption properties is well described. The adsorption capacity of 9 products was measured over 24 h at different P concentrations (5-2000 µg L(-1)), a range that covers Danish drainage water and stormwater. WWS desorbed phosphate at concentrations below 50-200 µg P L(-1) and should only be considered for use in systems with a constantly high load of dissolved P. High affinity combined with little or no desorption characterized the commercial product and the ochre sludge from the precipitation basins, rendering these useful for treating drainage water and storm water. The study underlines that waste products may act as potentially effective P adsorbers at environmentally relevant P levels.
In the spring of 2015, 11 years after a mentally ill young man named Dan Markingson stabbed himself to death in an industry-sponsored drug study, officials at the University of Minnesota suspended recruitment of subjects into drug trials in its Department of Psychiatry. University officials agreed to act only after a scathing investigation by Minnesota Office of the Legislative Auditor found damning evidence of coerced recruitment, inadequate clinical care, superficial research oversight, a web of serious, disturbing conflicts of interest, and a pattern of misleading public statements by university officials aimed at deflecting scrutiny. In this article, I examine the larger institutional factors leading up to Markingson's suicide and prevented corrective action for so long.
Interaction of advanced glycation end products (AGEs) with its cell-bound receptor (RAGE) results in cell dysfunction through activation of nuclear factor kappa-B, increase in expression and release of inflammatory cytokines, and generation of oxygen radicals. Circulating soluble receptors, soluble receptor (sRAGE), endogenous secretory receptor (esRAGE) and cleaved receptor (cRGAE) act as decoy for RAGE ligands and thus have cytoprotective effects. Low levels of sRAGE and esRAGE have been proposed as biomarkers for many diseases. However sRAGE and esRAGE levels are elevated in diabetes and chronic renal diseases and still tissue injury occurs. It is possible that increases in levels of AGEs are greater than increases in the levels of soluble receptors in these two diseases. Some new parameters have to be used which could be an universal biomarkers for cell dysfunction. It is hypothesized that increases in serum levels of AGEs are greater than the increases in the soluble receptors, and that the levels of AGEs is correlated with soluble receptors and that the ratios of AGEs/sRAGE, AGEs/esRAGE and AGEs/cRAGE are elevated in patients with end-stage renal disease (ESRD) and would serve as an universal risk marker for ESRD. The study subject comprised of 88 patients with ESRD and 20 healthy controls. AGEs, sRAGE and esRAGE were measured using commercially available enzyme linked immune assay kits. cRAGE was calculated by subtracting esRAGE from sRAGE. The data show that the serum levels of AGEs, sRAGE, cRAGE are elevated and that the elevation of AGEs was greater than those of soluble receptors. The ratios of AGEs/sRAGE, AGEs/esRAGE and AGEs/cRAGE were elevated and the elevation was similar in AGEs/sRAGE and AGEs/cRAGE but greater than AGEs/esRAGE. The sensitivity, specificity, accuracy, and positive and negative predictive value of AGEs/sRAGE and AGEs/cRAGE were 86.36 and 84.88 %, 86.36 and 80.95 %, 0.98 and 0.905, 96.2 and 94.8 %, and 61.29 and 56.67 % respectively. There was a positive correlation of sRAGE with esRAGE and cRAGE, and AGEs with esRAGE; and negative correlation between sRAGE and AGEs/sRAGE, esRAGE and AGES/esRAGE, and cRAGE and AGES/cRAGE. In conclusion, AGEs/sRAGE, AGEs/cRAGE and AGEs/esRAGE may serve as universal risk biomarkers for ESRD and that AGEs/sRAGE and AGEs/cRAGE are better risk biomarkers than AGEs/esRAGE.
Brown adipose tissue (BAT) is the main site of adaptive thermogenesis and experimental studies have associated BAT activity with protection against obesity and metabolic diseases, such as type 2 diabetes mellitus and dyslipidaemia. Active BAT is present in adult humans and its activity is impaired in patients with obesity. The ability of BAT to protect against chronic metabolic disease has traditionally been attributed to its capacity to utilize glucose and lipids for thermogenesis. However, BAT might also have a secretory role, which could contribute to the systemic consequences of BAT activity. Several BAT-derived molecules that act in a paracrine or autocrine manner have been identified. Most of these factors promote hypertrophy and hyperplasia of BAT, vascularization, innervation and blood flow, processes that are all associated with BAT recruitment when thermogenic activity is enhanced. Additionally, BAT can release regulatory molecules that act on other tissues and organs. This secretory capacity of BAT is thought to be involved in the beneficial effects of BAT transplantation in rodents. Fibroblast growth factor 21, IL-6 and neuregulin 4 are among the first BAT-derived endocrine factors to be identified. In this Review, we discuss the current understanding of the regulatory molecules (the so-called brown adipokines or batokines) that are released by BAT that influence systemic metabolism and convey the beneficial metabolic effects of BAT activation. The identification of such adipokines might also direct drug discovery approaches for managing obesity and its associated chronic metabolic diseases.
The life of all animals is dominated by alternating feelings of hunger and satiety - the main involuntary motivations for feeding-related behaviour. Gut bacteria depend fully on their host for providing the nutrients necessary for their growth. The intrinsic ability of bacteria to regulate their growth and to maintain their population within the gut suggests that gut bacteria can interfere with molecular pathways controlling energy balance in the host. The current model of appetite control is based mainly on gut-brain signalling and the animal's own needs to maintain energy homeostasis; an alternative model might also involve bacteria-host communications. Several bacterial components and metabolites have been shown to stimulate intestinal satiety pathways; at the same time, their production depends on bacterial growth cycles. This short-term bacterial growth-linked modulation of intestinal satiety can be coupled with long-term regulation of appetite, controlled by the neuropeptidergic circuitry in the hypothalamus. Indeed, several bacterial products are detected in the systemic circulation, which might act directly on hypothalamic neurons. This Review analyses the data relevant to possible involvement of the gut bacteria in the regulation of host appetite and proposes an integrative homeostatic model of appetite control that includes energy needs of both the host and its gut bacteria.
Chromatin modifications shape cell heterogeneity by activating and repressing defined sets of genes involved in cell proliferation, differentiation and development. Polycomb-repressive complexes (PRCs) act synergistically during development and differentiation by maintaining transcriptional repression of common genes. PRC2 exerts this activity by catalysing H3K27 trimethylation. Here, we show that in the intestinal epithelium PRC2 is required to sustain progenitor cell proliferation and the correct balance between secretory and absorptive lineage differentiation programs. Using genetic models, we show that PRC2 activity is largely dispensable for intestinal stem cell maintenance but is strictly required for radiation-induced regeneration by preventing Cdkn2a transcription. Combining these models with genomewide molecular analysis, we further demonstrate that preferential accumulation of secretory cells does not result from impaired proliferation of progenitor cells induced by Cdkn2a activation but rather from direct regulation of transcription factors responsible for secretory lineage commitment. Overall, our data uncover a dual role of PRC2 in intestinal homeostasis highlighting the importance of this repressive layer in controlling cell plasticity and lineage choices in adult tissues.
Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.
In the eastern United States, human cases of West Nile virus (WNV) result from spillover from urban epizootic transmission between passerine birds and Culex mosquitoes. In Atlanta, GA, substantial WNV presence in hosts and vectors has not resulted in the human disease burden observed in cities with similar infection pressure. Our study goal was to investigate extrinsic ecological conditions that potentially contribute to these reduced transmission rates. We conducted WNV surveillance among hosts and vectors in urban Atlanta and recorded an overall avian seroprevalence of nearly 30%, which was significantly higher among northern cardinals, blue jays, and members of the mimid family, and notably low among American robins. Examination of temporal Culex feeding patterns showed a marked feeding shift from American robins in the early season to northern cardinals in the late season. We therefore rule out American robins as superspreaders in the Atlanta area and suggest instead that northern cardinals and mimids act as WNV "supersuppressor" species, which slow WNV transmission by drawing many infectious bites during the critical virus amplification period, yet failing to amplify transmission due to low host competencies. Of particular interest, urban forest patches provide spillover protection by increasing the WNV amplification fraction on supersuppressor species.
The testes are one of the most delicate organs in the male body and highly susceptible to the exogenous influences capable of inducing cell damage. Cancer therapies are well known to negatively affect the male reproductive tract with a severe impairment of spermatogenesis and infertility. The present work aimed to systematically review the available information about the different endogenous factors (hormonal and nonhormonal) that may have protective or advantageous properties on the recovery of male reproductive function after gonadal injury. Furthermore, the perspective that these endogenous molecules could act as cryoprotectants to improve the quality of cryopreserved semen samples was also discussed. The knowledge reviewed herein allowed to identify promising factors able to mitigate the male fertility problems arising either from oncological treatments or other gonadal damage, and opened new possibilities to ameliorate the recovery of spermatogenesis or to preserve fertility.
Previous studies have demonstrated that melatonin administration improves spatial learning and memory and hippocampal long-term potentiation in the adult Ts65Dn (TS) mouse, a model of Down syndrome (DS). This functional benefit of melatonin was accompanied by protection from cholinergic neurodegeneration and the attenuation of several hippocampal neuromorphological alterations in TS mice. Because oxidative stress contributes to the progression of cognitive deficits and neurodegeneration in DS, this study evaluates the antioxidant effects of melatonin in the brains of TS mice. Melatonin was administered to TS and control mice from 6 to 12 months of age and its effects on the oxidative state and levels of cellular senescence were evaluated. Melatonin treatment induced antioxidant and antiaging effects in the hippocampus of adult TS mice. Although melatonin administration did not regulate the activities of the main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) in the cortex or hippocampus, melatonin decreased protein and lipid oxidative damage by reducing the thiobarbituric acid reactive substances (TBARS) and protein carbonyls (PC) levels in the TS hippocampus due to its ability to act as a free radical scavenger. Consistent with this reduction in oxidative stress, melatonin also decreased hippocampal senescence in TS animals by normalizing the density of senescence-associated β-galactosidase positive cells in the hippocampus. These results showed that this treatment attenuated the oxidative damage and cellular senescence in the brain of TS mice and support the use of melatonin as a potential therapeutic agent for age-related cognitive deficits and neurodegeneration in adults with DS.
Viral contamination along the production chain is a significant concern in both food safety and livestock health. Pigs have been reported to act as a reservoir for zoonotic viruses, sometimes emerging ones, and epidemiological studies have shown direct links between the consumption of uncooked pork offal and cases of hepatitis caused by the hepatitis E virus (HEV) genotype 3 in humans. The presence of HEV in swine herds has been reported, but its dissemination in pork production environments is still unknown. To investigate viral contamination sources in the swine industry, 452 environment and fecal samples, including samples from livestock transportation vehicles, were collected over a period of 11 months from ten farms and one slaughterhouse that together represent a single production network. Hepatitis E virus RNA was detected by nested RT-PCR in 32 samples from both inside and outside farm buildings, on trucks, and, mostly, from fomites collected in the slaughterhouse yard, such as on a utility vehicle. Phylogenetic analysis showed a wide diversity of HEV genotype 3 strains, similar to human and swine strains previously found. According to the results of this study, the movements of trucks and utility vehicles might play an important role in HEV dissemination on a slaughterhouse site and throughout an entire network.
Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture.
To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change.
Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation.
This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.
This article discusses Geiger's review of empathy, expressed in a lecture at the IV German Congress of Experimental Psychology in 1910. It deals with the key psychological question of how it is possible to know the minds of others. This question is complex and needs clarification, so Geiger divided it into 3 basic questions: The first is phenomenological (what is the conscious experience of empathy?); the second relates to the psychological function performed by the empathic act; and the third question asks whether and how empathy is acquired during personal development. Finally, Geiger introduces a distinction between basic empathy and reliving. Geiger's conceptual clarification is discussed and its relevance for the psychology and philosophy of his time is considered, as well as its possible influence on Jaspers' General Psychopathology. Finally, the current debate in the neurocognitive science of empathy is examined in light of Geiger's conceptualization. (PsycINFO Database Record
The plasma membrane P-glycoprotein (Pgp) is an efflux transporter involved in multidrug resistance and in the onset of neurodegenerative disease. Its function and most mechanisms of action are still under investigation. We developed a C-11-labeled 2-arylethylphenylamine-([(11)C]AEPH) derivative for positron emission tomography (PET), as a novel probe to better understand the activity and the function of Pgp in vivo.
The synthetic procedure and the quality control of the selected lead compound, [(11)C]AEPH-1, were set up and optimized. The biodistribution and the dynamic extraction in target organs of [(11)C]AEPH-1 were studied in vivo by PET in healthy rats at baseline and after pre-treatment with a Pgp inhibitor (tariquidar).
In vivo dynamic imaging was consistent with the results of ex vivo extraction on explanted organs. An adequate stability for in vivo studies, as well as a high activity of [(11)C]AEPH-1 in intestine and barrier tissues, has been demonstrated. Results of the blockade study showed a decrease of uptake after the pre-treatment, indicating a behavior attributable to a Pgp ligand.
The suitable pharmacokinetics and the specificity tested in the pre-treated animals have indicated the potentiality of this AEPH derivative to act as Pgp ligand, providing new opportunities for further studies on expression and function of this important efflux transporter in the fields of neurology and oncology.
The main purpose of this study was to investigate the effects of a 12-week, clinician-referred, community-based exercise training program with supervised and unsupervised sessions for men with prostate cancer. The secondary purpose was to determine whether androgen deprivation therapy (ADT) modified responses to exercise training.
Secondary analysis was undertaken on data from a multicentre cluster randomised controlled trial in which 15 clinicians were randomly assigned to refer eligible patients to an exercise training intervention (n = 8) or to provide usual care (n = 7). Data from 119 patients (intervention n = 53, control n = 66) were available for this analysis. Outcome measures included fitness and physical function, anthropometrics, resting heart rate, and blood pressure.
Compared to the control condition, men in the intervention significantly improved their 6-min walk distance (M diff = 49.98 m, p adj = 0.001), leg strength (M diff = 21.82 kg, p adj = 0.001), chest strength (M diff = 6.91 kg, p adj = 0.001), 30-s sit-to-stand result (M diff = 3.38 reps, p adj = 0.001), and reach distance (M diff = 4.8 cm, p adj = 0.024). A significant difference (unadjusted for multiplicity) in favour of men in the intervention was also found for resting heart rate (M diff = -3.76 beats/min, p = 0.034). ADT did not modify responses to exercise training.
Men with prostate cancer who act upon clinician referrals to community-based exercise training programs can improve their strength, physical functioning, and, potentially, cardiovascular health, irrespective of whether or not they are treated with ADT.
Clinicians should inform men with prostate cancer about the benefits of exercise and refer them to appropriately qualified exercise practitioners and suitable community-based programs.
Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12610000609055.
The present study was aimed at testing the locus of word frequency effects in spelling to dictation: Are they located at the level of spoken word recognition (Chua & Rickard Liow, 2014) or at the level of the orthographic output lexicon (Delattre, Bonin, & Barry, 2006)? Words that varied on objective word frequency and on phonological neighborhood density were orally presented to adults who had to write them down. Following the additive factors logic (Sternberg, 1969, 2001), if word frequency in spelling to dictation influences a processing level, that is, the orthographic output level, different from that influenced by phonological neighborhood density, that is, spoken word recognition, the impact of the 2 factors should be additive. In contrast, their influence should be overadditive if they act at the same processing level in spelling to dictation, namely the spoken word recognition level. We found that both factors had a reliable influence on the spelling latencies but did not interact. This finding is in line with an orthographic output locus hypothesis of word frequency effects in spelling to dictation. (PsycINFO Database Record
Health care cost is consuming a large portion of the nation's gross domestic product while placing added economic burdens on physicians and their patients. With total joint replacement being one of the early-targeted procedures in the evolving health care environment, knee surgeons will benefit from developing a critical knowledge on health care reforms and their financial implications. The Medicare Access and Children's Health Insurance Program Reauthorization Act represents a cohesive movement toward value-based payment reform and contains several unchartered rulings that require detailed attention by knee surgeons. In this article, we provide a contextual framework of health care legislation that has led to the formation of the current health policy, and present a comprehensive summary and update on the Merit-Based Incentive Payment Systems and Alternative Payment Models reimbursement models.
The spinal glioblastoma (GBM) represents the one of most common tumors in humans. However, the biological processes and molecular mechanisms of spinal GBM are still unclear. It is known that miR-143 participates in the development of various tumor progressions. The present study was to evaluate the level of miR-143 in spinal GBM tissues and cells. We further investigated that the molecular mechanisms of miR-143 in U87 and U251 cell lines. Here, our data showed that the expression levels of miR-143 were significantly reduced in spinal GBM tissues and cell lines. Accordingly, the expression levels of the extracellular signal regulated kinase 5 (ERK5) were significantly increased in spinal GBM tissues and cell lines. Ectopic expression of miR-143 in U87 and U251 cells resulted in decreased cell growth and enhanced cell apoptosis, and inhibited the expression of epithelial-mesenchymal transition (EMT) biomarkers. Further study characterized the 3' untranslated region (3'-UTR) of ERK5 gene as a direct target of miR-143 in U87 and U251 cells as determined by luciferase reporter assays. In addition, the ectopic expression of miR-143 led to the down-regulation of epidermal growth factor receptor (EGFR), while the over-expression of ERK5 reversed the miR-143-inhibited EGFR expression, and promoted cell growth and the expression of EMT biomarkers. In conclusion, this study demonstrated that miR-143 plays a crucial role in regulating the EMT of GBM by directly targeting ERK5, and miR-143 may act as a potential therapeutic target in spinal GBM patients.
Hepatocellular carcinoma (HCC) treatment remains lack of effective chemotherapeutic drugs, therefore, discovering novel anti-HCC drugs is a very attractive and urgent task. In this study, we reported VOSL (volatile oil from Saussurea lappa root) exhibits potent therapeutic effect on SMMC-7721 xenografts without obvious side effects. In the in vitro experiments, VOSL inhibited HCC cell proliferation by arresting cell cycle at S and G2/M phases, and induced HCC cell apoptosis by activating the Caspase3 pathway. VOSL also decreased the capability of HCC cell migration and invasion through MMP-9 depression. Moreover, mechanistic study indicated that VOSL can act as an epithelial growth factor receptor (EGFR) inhibitor to suppress EGFR activation and then to suppress its downstream MEK/P38 and PI3-K/Akt pathways. These results suggested that VOSL may be a novel anti-HCC drug candidate.
Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors.
Uncontrolled hemorrhage is a leading cause of mortality following trauma accounting for up to 40% of deaths. Massive transfusion protocols (MTPs) offer a proven benefit in resuscitation of these patients. Recently, the superiority of thrombelastography (TEG)-guided resuscitation over strategies guided by conventional clotting assays (CCA) has been established. We seek to determine optimal thresholds for r-TEG driven resuscitation.
R-TEG data were reviewed for 190 patients presenting to our Level 1 Trauma Center from 2010 to 2015. Criteria for inclusion were highest level trauma activation in patients ≥ 18 years of age with hypotension presumed due to acute blood loss. Exclusion criteria included: isolated gun-shot wound to the head, pregnancy and chronic liver disease. Receiver operating characteristic (ROC) analysis was performed to test the predictive performance of r-TEG for massive transfusion requirement defined by need for 1) >10 units of RBCs total or death in the first six hours or 2) >4 units of RBCs in any hour within the first 6 hours. Cut-point analysis was then performed to determine optimal thresholds for TEG-based resuscitation.
ROC analysis of r-TEG yielded areas under the curve (AUC) greater than 70% for all outputs with respect to both transfusion thresholds considered, with exception of activated clotting time (ACT) and lysis at 30 minutes (LY30) for > 4U RBC in any hour in the first 6 hours. Optimal cut-point analysis of the resultant ROC curves was performed and for each value, the most sensitive cut-point was identified, respectively ACT ≥ 128 sec, angle (α) ≤ 65, maximum amplitude (MA) ≤ 55 mm and LY30 ≥ 5%.
Through ROC analysis of prospective TEG data, we have identified optimal thresholds to guide hemostatic resuscitation. These thresholds should be validated in a prospective multicenter trial.
Prognostic, level III.
As a result of the Affordable Care Act, increases in aging populations, and advances in technologies, there is a critical need for highly qualified, competent home healthcare nurses. According to the Joint Commission's Human Resource Standard, home healthcare nurses must demonstrate their competency at least every 3 years by participating in in-service education (). Unfortunately, not all home healthcare agencies (HHA) are accredited by the Joint Commissions. A review of several states healthcare regulatory rules-specifically California, New York, Nevada, and Virginia, reveals no mandates for home healthcare nurses to demonstrate competencies for skills specific for home healthcare nursing. There are many competency programs that focus on general nursing knowledge and skills; however, there is a gap in the literature regarding home healthcare nursing competencies. This article will discuss how a simulation tool kit can be the format by which HHA can enhance the knowledge and skills of their nurses, and promote effective health outcomes and protect the public from harm.
This article deals with compulsory licensing scenarios in India, provides a background of relevant provisions in the Patents Act and examines how these provisions are Trade-Related Aspects of Intellectual Property Rights compliant. This article further discusses the procedure followed by India in granting a compulsory license, provides an overview of compulsory license applications filed in India to date and judicial precedence regarding the same. This article also highlights how compulsory licensing is a great safeguard that balances the interests of the innovators and the public at large.
The Adverse Outcome Pathway (AOP) concept is expected to guide risk assessors in their work to use all existing information on the effects of chemicals on humans and wildlife, and to target the generation of additional information to the regulatory objective. AOPs will therefore be used in the Organisation for Economic Co-operation and Development (OECD) chemical safety programme, as underlying scientific rationales for the development of alternative methods for hazard assessment, such as read-across, in vitro test methods or the development of integrated testing strategies that have the potential to replace animal tests. As a proof-of-concept, the OECD has developed an AOP for skin sensitisation, and as a follow-up has: a) implemented the AOP into the OECD QSAR Toolbox, so that information related to the Key Events (KEs) in the AOP can be used to group chemicals that are expected to act by the same mechanism and hence have the same skin sensitisation potential; b) developed alternative test methods for the KEs, so that ultimately chemicals can be tested for skin sensitisation without the use of animal tests. The development of integrated testing strategies based on the AOP is ongoing. Building on this proof-of-concept, the OECD has launched an AOP development programme with a first batch of AOPs published in 2016. A number of IT tools, which together form an AOP Knowledge Base, are at various stages of development, and support the construction of AOPs and their use in the development of integrated approaches for testing and assessment. Following the publication of the first batch of AOPs, OECD member countries will decide on priorities for their use in supporting the development of tools for regulatory use.
Given the high mortality experienced by patients who deteriorate outside the intensive care unit, issues related to patient preferences around escalation of care are common. However, the literature on early warning systems (EWSs) provides limited information on how respecting patient preferences can be incorporated into clinical workflows. In this report, we describe how we developed workflows for integrating supportive care with an automated EWS in the context of a 2-hospital pilot. We used the Institute for Healthcare Improvement's Plan-Do-Study-Act approach to achieve consensus with clinicians and administrators. The workflows will serve as the basis for dissemination to an additional 19 hospitals. We were successful in integrating an automated EWS with supportive care. Our workflows take local resource availability into account and have been well received by hospitalists, nurses, and families. Our work demonstrates that one can achieve integration of proactive supportive care into the operation of an EWS. Creation of a palliative care response arm that is complementary to a clinical rescue arm ensures that patient preferences are respected. Journal of Hospital Medicine 2016;11:S40-S47. © 2016 Society of Hospital Medicine.
Efforts to improve outcomes of patients who deteriorate outside the intensive care unit have included the use of rapid response teams (RRTs) as well as manual and automated prognostic scores. Although automated early warning systems (EWSs) are starting to enter clinical practice, there are few reports describing implementation and the processes required to integrate early warning approaches into hospitalists' workflows. We describe the implementation process at 2 community hospitals that deployed an EWS. We employed the Institute for Healthcare Improvement's iterative Plan-Do-Study-Act approach. Our basic workflow, which relies on having an RRT nurse and the EWS's 12-hour outcome time frame, has been accepted by clinicians and has not been associated with patient complaints. Whereas our main objective was to develop a set of workflows for integrating the electronic medical record EWS into clinical practice, we also uncovered issues that must be addressed prior to disseminating this intervention to other hospitals. One problematic area is that of documentation following an alert. Other areas that must be addressed prior to disseminating the intervention include the need for educating clinicians on the rationale for deploying the EWS, careful consideration of interdepartment service agreements, clear definition of clinician responsibilities, pragmatic documentation standards, and how to communicate with patients. In addition to the deployment of the EWS to other hospitals, a future direction for our teams will be to characterize process-outcomes relationships in the clinical response itself. Journal of Hospital Medicine 2016;11:S25-S31. © 2016 Society of Hospital Medicine.
Sepsis is a leading cause of in-hospital death, and evidence suggests a higher mortality in patients presenting with sepsis on the ward compared to those presenting to the emergency department. Ward patients who develop severe sepsis may have poor outcomes for a variety of reasons, including delayed diagnosis, lack of readily available staffing, and delayed treatment. We report on a multihospital quality improvement program for early detection and treatment of sepsis on general medical-surgical wards. We describe a multipronged approach to improve severe sepsis outcomes using the Institute for Healthcare Improvement's Plan-Do-Study-Act model. Sixty sites engaged in a collaborative implementation process that aligned people, process, and technology. Based on our experience, we recommend a stepwise approach to implement such a program: (1) both administrative and clinical leadership commit to a common goal; (2) appoint clinical champions and give them authority to engage other clinicians to improve timeliness of interventions; (3) map workflows and processes to rely heavily on the nursing staff's ability to evaluate and report severe sepsis screening results; (4) if available, design and deploy technology with the assistance of clinical informaticians (eg, to enable electronic health records-based continuous screening); (5) to determine success, consider tracking screening compliance and process, and outcome measures such as length of stay and mortality. Journal of Hospital Medicine 2016;S11:32-S39. © 2016 Society of Hospital Medicine.
We demonstrate a random distributed feedback fiber laser at 2.1 μm. A high-power pulsed Tm-doped fiber laser operating at 1.94 μm with a temporal duty ratio of 30% was employed as a pump laser to increase the equivalent incident pump power. A piece of 150 m highly GeO<sub>2</sub>-doped silica fiber that provides a strong Raman gain and random distributed feedbacks was used to act as the gain medium. The maximum output power reached 0.5 W with the optical efficiency of 9%, which could be further improved by more pump power and optimized fiber length. To the best of our knowledge, this is the first demonstration of random distributed feedback fiber laser at 2 μm band based on Raman gain.
Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.
Data from the National Health Interview Survey, 2013-2015 •From 2013 through 2015, the percentage of adults aged 18-64 who were uninsured at the time of interview decreased for poor (40.0% to 26.2%), near-poor (37.8% to 23.9%), and not-poor (11.7% to 7.7%) adults. •The percentage of adults aged 18-64 who had a usual place to go for medical care increased for poor (66.9% to 73.6%) and near-poor (71.1% to 75.9%) adults. •The percentage of adults aged 18-64 who had seen or talked to a health professional in the past 12 months increased for poor (73.2% to 75.8%) and near-poor (71.9% to 75.9%) adults. •The percentage of adults aged 18-64 who did not obtain needed medical care due to cost at some time during the past 12 months decreased for poor (16.8% to 12.4%), near-poor (14.6% to 11.0%), and not-poor (4.9% to 3.8%) adults. In 2014, U.S. adults could purchase a private health insurance plan through the Health Insurance Marketplace or state-based exchanges established as part of the Affordable Care Act (ACA). Additionally, under ACA some states opted to expand Medicaid coverage to low-income adults. Individuals living in or near poverty may have benefited disproportionately from these changes given their lower rates of health insurance coverage (1). Data from the 2013-2015 National Health Interview Survey (NHIS) are used to describe recent changes in health insurance coverage and selected measures of health care access and utilization for adults aged 18-64 by family poverty level.
Data from the National Electronic Health Records Survey •In 2015, the percentage of physicians who had electronically sent patient health information ranged from 19.4% in Idaho to 56.3% in Arizona. •In 2015, the percentage of physicians who had electronically received patient health information ranged from 23.6% in Louisiana and Mississippi to 65.5% in Wisconsin. •In 2015, the percentage of physicians who had electronically integrated patient health information from other providers ranged from 18.4% in Alaska to 49.3% in Delaware. •In 2015, the percentage of physicians who had electronically searched for patient health information ranged from 15.1% in the District of Columbia to 61.2% in Oregon. The Health Information Technology for Economic and Clinical Health Act (HITECH) provides financial incentives to eligible providers using a certified electronic health record (EHR) system (1,2). In 2015, 77.9% of office-based physicians had a certified EHR system, up from 74.1% in 2014 (3-5). A federal plan to enhance the nation's health information technology infrastructure was published in 2015 to support information sharing (6,7). Therefore, this report uses the 2015 National Electronic Health Records Survey (NEHRS) to describe the extent to which physicians can electronically send, receive, integrate, and search for patient health information.
The Dutch Embryos Act (2000) contains a temporary ban on the creation of embryos for research, meaning that, at present, only research using "spare" IVF embryos is allowed. Recently, the government has announced a plan to lift this ban. This is in line with the original intention of the Act, which already contains conditions for research with specially created embryos that will come into force after the lifting of the ban, including the restriction that the research must be expected to yield new insights in the domains of infertility, assisted reproduction, hereditary or congenital disorders, or transplantation medicine. The government plans announced allow research only in the first three of these domains, adding the further criterion that the research must be 'directly relevant for clinical application'. According to the government, the reason for these additional restrictions was the need to protect 'human dignity'. The authors of this paper are not convinced.
In solid-organ transplant, cell therapy is used as an immunomodulation therapy or as a functioning graft (bioengineering medicine). Before such treatment can be more accepted among transplant societies, some uncertainties should be clarified. These include: why is such therapy mandatory? What are the indications for this therapy, and what are the mechanism(s) of actions? What types of cells are involved and what are their routes of actions? Finally, what is known about the safety? In general, the risks associated with intravenous administration of immunoregulatory cell products are similar to those encountered with conventional blood transfusions. The adverse effects of immunosuppressive drugs, such as infections, cardiovascular disease, metabolic complications, and malignancies, would be decreased with cell therapy. Immunoregulatory cells act when necessary and through multiple mechanisms through different targets. Immunoregulatory cells are not only passive inhibitors such as drugs, but they have active functions. Treatment would only be once or perhaps a few times but not indefinitely, which is in contrast to immunosuppressive drugs; therefore, complications involving immunosuppressive drugs are no longer present. Cell therapy in solid-organ transplant is indicated for treatment of ischemic-reperfusion injury, prevention of chronic allograft nephropathy, minimization of immune suppression, and induction of long-term allograft tolerance. Many cell types have being investigated as potential cell-based immunotherapies for use in solid-organ transplant, including mesenchymal stromal cells, regulatory macrophages, tolerogenic dendritic cells, regulatory T cells, and regulatory B cells. Efficacy and safety of each group of cells should be clarified before widespread clinical use. The landscape of transplant science would be at least partially, if not totally, changed with cell therapy.
Statins induce apoptosis of tumor cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanisms remain poorly understood. The present study showed that the induction of apoptosis by statins in four different human head and neck squamous cell carcinoma (HNSCC) cell lines, HSC-3, HEp-2, Ca9-22, and SAS cells was mediated by increased caspase-3 activity. Statins induced apoptosis by the suppression of geranylgeranyl pyrophosphate biosynthesis. Furthermore, statins decreased the levels of phosphorylated ERK and mTOR by inhibiting the membrane localization of Ras and enhancing Bim expression in HSC-3 and HEp-2 cells. We also found that in all the cell types analyzed, the IC50 values for fluvastatin and simvastatin were highest in HEp-2 cells. In addition, HSC-3, Ca9-22, and SAS cells had higher Ras expression and membrane localization, higher activation of ERK1/2 and mTOR, and lower levels of Bim expression than HEp-2 cells. Our results indicate that statins induce apoptosis by increasing the activation of caspase-3 and by enhancing Bim expression through inhibition of the Ras/ERK and Ras/mTOR pathways. Furthermore, the sensitivity of HNSCC cells to statin treatment was closely related to Ras expression and prenylation levels, indicating that statins may act more effectively against tumors with high Ras expression and Ras-variability. Therefore, our findings support the use of statins as potential anticancer agents. This article is protected by copyright. All rights reserved.
To examine the relationship between anti-Müllerian hormone (AMH) and the severity of the phenotype of patients with polycystic ovary syndrome (PCOS) and whether AMH can act as a diagnostic marker for PCOS?
A prospective diagnostic utility study of AMH as a marker of PCOS.
A consecutive series of women presenting to a tertiary infertility clinic (n = 164) plus a second series of women prepared for assisted conception treatments (n = 89) recruited between June 2012 and May 2013.
Polycystic ovary syndrome was diagnosed using the Rotterdam criteria. AMH was measured using the Generation II assay (Beckman Coulter). The diagnostic utility of AMH was established using receiver operator characteristic (ROC) curves. Cut-off values for the individual features of PCOS are proposed.
There was a significant difference in serum AMH concentration in women with normal ovaries (13·2 pmol/l), polycystic ovary morphology (PCOM) alone (37·8 pmol/l) and PCOS (53·2 pmol/l). Follicle number, increasing cycle length and evidence of hyperandrogenism were all independently associated with serum AMH concentration (P < 0·01). AMH was significantly affected by the different phenotypic presentations of PCOS with those with all components (PCOM, HA and OA) having the highest mean value [72·7 pmol/l (P < 0·01)].
Serum AMH has the capacity to act as a diagnostic test for PCOS. Moreover, since its value rises with the more marked phenotypes, different cut-off values need to be used to differentiate those patients with polycystic ovarian morphology (PCOM), hyperandrogenism (HA) and oligoanovulation (OA).
A facile approach has been developed to prepare well-designed MoS2-based flexible anisotropic actuators with tunable thermo- and photo-responses, in which MoS2 nanosheets act as the photothermal transduction agents and enable remote and precise control of the actuator locomotion. Taking advantage of the dual-responsive behaviors, programmable locomotion, reversible deformation, good mechanical properties and biocompatibility, the MoS2-based flexible anisotropic actuators are very promising in soft robotics for future intelligent applications.
Patients with eating disorders (EDs) frequently report a history of childhood trauma (CT). We investigated whether certain subtypes of CT are associated with more severe features of EDs, independently of psychiatric comorbidity, and whether they act additively. One hundred and ninety-two patients with DSM-V-defined EDs were consecutively recruited. Five clinical characteristics were assessed: restraint, eating, shape and weight concerns on the EDE-Q, and daily functioning. CT was assessed by the childhood traumatism questionnaire. The clinical features were associated with at least one CT subtype (emotional, sexual or physical abuse, emotional neglect). Multivariate analyses adjusted for lifetime comorbid psychiatric disorders revealed that emotional abuse independently predicted higher eating, shape and weight concerns and lower daily functioning, whereas sexual and physical abuse independently predicted higher eating concern. A dose-effect relationship characterised the number of CT subtypes and the severity of the clinical features, suggesting a consistent and partly independent association between CT and more severe clinical and functional characteristics in EDs. Emotional abuse seems to have the most specific impact on ED symptoms. Last, not all CT subtypes have the same impact but they do act additively.
Snake venoms are natural sources of biologically active molecules that are able to act selectively and specifically on different cellular targets, modulating physiological functions. Thus, these mixtures, composed mainly of proteins and peptides, provide ample and challenging opportunities and a diversified molecular architecture to design and develop tools and agents of scientific and therapeutic interest. Among these components, peptides and small proteins play diverse roles in numerous physiological processes, exerting a wide range of pharmacological activities, such as antimicrobial, antihypertensive, analgesic, antitumor, analgesic, among others. The pharmaceutical and cosmetic industries have recognized the huge potential of these privileged frameworks and believe them to be a promising alternative to contemporary drugs. A number of natural or synthetic peptides from snake venoms have already found preclinical or clinical applications for the treatment of pain, hypertension, cardiovascular diseases and aging skin. A well-known example is captopril, whose natural peptide precursor was isolated from Bothrops jararaca snake venom, which is a peptide-based drug that inhibits the angiotensin-converting enzyme, producing an anti-hypertensive effect. The present review looks at the main peptides (natriuretic peptides, bradykinin-potentiating peptides and sarafotoxins) and low mass proteins (crotamine, disintegrins and three-Finger toxins) from snake venoms, as well as synthetic peptides inspired by them, describing their biochemical, structural and physiological features, as well as their applications as research tools and therapeutic agents.
Heat shock proteins (HSPs) are families of molecular chaperones that play important homeostatic functions in the central nervous system (CNS) by preventing protein misfolding, promoting degradation of improperly folded proteins, and protecting against apoptosis and inflammatory damage especially during hyperthermia, hypoxia, or oxidative stress. Under stress conditions, HSPs are upregulated to protect cells from damage that accumulates during ageing as well as pathological conditions. An important, yet frequently overlooked function of some HSPs is their ability to function as extracellular messengers (also termed chaperokines) that modulate immune responses within the CNS. Given the strong association between protein aggregation, innate immune cell activation and neurodegeneration, the expression and roles of HSPs in the CNS is attracting attention in many neurodegenerative disorders including inflammatory diseases such as multiple sclerosis, protein folding diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, and genetic white matter diseases. This is especially so since several studies show that HSPs act therapeutically by modulating innate immune activation and may thus serve as neuroprotective agents. Here we review the evidence linking HSPs with neurodegenerative disorders in humans and the experimental animal models of these disorders. We discuss the mechanisms by which HSP protect cells, and how the knowledge of their endogenous functions can be exploited to treat disorders of the CNS.
A highly regio- and diastereoselective cross-coupling of allyl/propargyl ethers and δ-ketoesters, mediated by SmCp(R)2 reagents, delivers decorated δ-lactones. Screening of the Cp ligands on Sm(ii) was employed to achieve high regio and diastereocontrol in some cases. Crucially, SmI2 gave unsatisfactory results in the transformation. The process has been exploited in a telescoped approach to complex cycloheptanols in which two Sm(ii) reagents act in turn on the simple starting materials.
Diabetes mellitus- (DM-) related vascular diseases attract increased attention due to their high morbidity and mortality. The incidence of obesity, atherosclerosis, coronary heart disease, hypertension, and dyslipidemia is significantly higher in DM patients, with an earlier onset and faster progression compared with non-DM patients. DM-related vascular diseases including macrovascular and microvascular complications are characterized by endothelial dysfunction. Therefore, a better understanding of the etiology and mechanisms of endothelial dysfunction is important for the diagnosis and treatment of DM. Endothelial microparticles (EMPs) are new diagnostic and therapeutic targets and biomarkers in DM-related vascular disease. Circulating EMPs containing biologically active substances act as intercellular signals under physiological and pathological conditions. They serve as biological markers of altered vascular endothelium and reflect the pathological progression and diminished endothelial function of blood vessels. Recent evidence suggests that the plasma level of EMPs is significantly higher in DM patients than in healthy population and is significantly correlated with DM-related complications. These observations have prompted speculation that EMPs play a crucial role in the pathophysiology of DM. This review summarizes the known and potential roles of EMPs in the diagnosis, staging, treatment, and clinical prognosis of DM and related vascular diseases.
Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all species of warm-blooded animals. As any chemical-based drugs could not act against the tissue cyst stage of T. gondii, vaccination may be one of the ideal control strategies. In the present study, two new vaccine candidates, named TgENO2 and TgTrxLp, were purified from Escherichia coli with pET-30a(+) expression system and then were injected into BALB/c mice to evaluate the protective efficacy against acute and chronic toxoplasmosis. The results showed that both the recombinant proteins, either alone or in combination, could elicit strong humoral and cellular immune responses with a higher level of IgG antibodies, IFN-γ, IL-2, CD4(+), and CD8(+) T cells as compared to those in mice from control groups. After acute challenge with tachyzoites of the GJS strain, mice immunized with rTgTrxLp (8 ± 2.77 d), rTgENO2 (7.4 ± 1.81 d), and rTgTrxLp + rTgENO2 (8.38 ± 4.57 d) proteins showed significantly longer survival time than those that received Freund's adjuvant (6.78 ± 2.08 d) and PBS (6.38 ± 4.65 d) (χ(2) = 9.687, df = 4, P = 0.046). The protective immunity of rTgTrxLp, rTgENO2, and rTgTrxLp + rTgENO2 proteins against chronic T. gondii infection showed 69.77%, 58.14%, and 20.93% brain cyst reduction as compared to mice that received PBS. The present study suggested that both TgENO2 and TgTrxLp were potential candidates for the development of multicomponent vaccines against toxoplasmosis.
Oral dichloroacetate sodium (DCA) has been investigated as a novel metabolic therapy for various cancers since 2007, based on data from Bonnet et al that DCA can trigger apoptosis of human lung, breast and brain cancer cells. Response to therapy in human studies is measured by standard RECIST definitions, which define "response" by the degree of tumour reduction, or tumour disappearance on imaging. However, Blackburn et al have demonstrated that DCA can also act as a cytostatic agent in vitro and in vivo, without causing apoptosis (programmed cell death). A case is presented in which oral DCA therapy resulted in tumour stabilization of stage 4 colon cancer in a 57 years old female for a period of nearly 4 years, with no serious toxicity. Since the natural history of stage 4 colon cancer consists of steady progression leading to disability and death, this case highlights a novel use of DCA as a cytostatic agent with a potential to maintain long-term stability of advanced-stage cancer.
Growth factors such as bone morphogenetic proteins 6, 7, 15, and two isoforms of transforming growth factor-beta (BMP6, BMP7, BMP15, TGFB1, and TGFB2), and insulin-like growth factor system act as local regulators of ovarian follicular development. To elucidate if these factors as well as others candidate genes, such as estrogen receptor 1 (ESR1), growth differentiation factor 9 (GDF9), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), bone morphogenetic protein receptor, type 2 (BMPR2), type 1 insulin-like growth factor receptor (IGFR1), and key steroidogenic enzymes cytochrome P450 aromatase and 3-β-hydroxysteroid dehydrogenase (CYP19A1 and HSD3B1) could modulate or influence diestrus on the onset of puberty in Brahman heifers, their ovarian mRNA expression was measured before and after puberty (luteal phase). Six postpubertal (POST) heifers were euthanized on the luteal phase of their second cycle, confirmed by corpus luteum observation, and six prepubertal (PRE) heifers were euthanized in the same day. Quantitative real-time PCR analysis showed that the expression of FSHR, BMP7, CYP19A1, IGF1, and IGFR1 mRNA was greater in PRE heifers, when contrasted to POST heifers. The expression of LHR and HSD3B1 was lower in PRE heifers. Differential expression of ovarian genes could be associated with changes in follicular dynamics and different cell populations that have emerged as consequence of puberty and the luteal phase. The emerging hypothesis is that BMP7 and IGF1 are co-expressed and may modulate the expression of FSHR, LHR and IGFR1, and CYP19A1. BMP7 could influence the downregulation of LHR and upregulation of FSHR and CYP19A1, which mediates the follicular dynamics in heifer ovaries. Upregulation of IGF1 expression prepuberty, compared to postpuberty diestrus, correlates with increased levels FSHR and CYP19A1. Thus, BMP7 and IGF1 may play synergic roles and were predicted to interact, from the expression data (P = 0.07, r = 0.84). The role of these co-expressed genes in puberty and heifers luteal phase merits further research.
The heart provides the body with oxygen and nutrients and assists in the removal of metabolic waste through the blood vessels of the circulatory system. It is the first organ to form during embryonic morphogenesis. FGFs with diverse functions in development, health, and disease are signaling proteins, mostly as paracrine growth factors or endocrine hormones. The human/mouse FGF family comprises 22 members. Findings obtained from mouse models and human diseases with FGF signaling disorders have indicated that several FGFs are involved in heart development, health, and disease. Paracrine FGFs including FGF8, FGF9, FGF10, and FGF16 act as paracrine signals in embryonic heart development. In addition, paracrine FGFs including FGF2, FGF9, FGF10, and FGF16 play roles as paracrine signals in postnatal heart pathophysiology. Although FGF15/19, FGF21, and FGF23 are typical endocrine FGFs, they mainly function as paracrine signals in heart development or pathophysiology. In heart diseases, serum FGF15/19 levels or FGF21 and FGF23 levels decrease or increase, respectively, indicating their possible roles in heart pathophysiology. FGF2 and FGF10 also stimulate the cardiac differentiation of cultured stem cells and cardiac reprogramming of cultured fibroblasts. These findings provide new insights into the roles of FGF signaling in the heart and potential therapeutic strategies for cardiac disorders.
Increased prevalent use of methamphetamine is a global public challenge. Information on drug use can be helpful in preventing high-risk behavior related to drug abuse.
This study aims to investigate the sexual function changes related to methamphetamine use in the male clients of public and private addiction treatment centers.
In this qualitative study, 45 men (35 methamphetamine users, 5 family members of the users, and 5 psychiatrists or physicians who were famous for treating or researching addiction) are involved. An in-depth interview was done with therapists and key individuals.
The results show that the effects of methamphetamine on sexual function are not identical. The first usage is concomitant with the increased duration of sex, an increase in the quality and quantity of sexual pleasure, a delighted orgasm, and feeling more control of the sex act. These effects gradually decrease. A decreased libido and various sexual dysfunctions such as erectile dysfunction, premature ejaculation, and losing control during the sex act will appear over time.
There are differences in the libido and sexual functions of methamphetamine users. Personal perceptions of one's sexual function may be affected by cognitive changes resultant from the drug. Drug-use prevention, addiction treatments, appropriate sexual behavior education, and harm reduction are priorities.
Oncofertility is gaining importance because of increasing cancer incidence, high survivorship, the need to provide a good quality of life to survivors and the desire of patients to preserve their fertility. Disseminating information about the effect of cancer and cancer treatment on fertility and the availability and effectiveness of fertility preservation techniques is critical. Gynaecologists in India act as family physicians and are in a unique position to guide cancer patients on issues of fertility and fertility preservation. Their contribution in oncofertility is vital to improve the quality of life of many young survivors. This paper presents the result of a survey done with Indian gynaecologists. The aim of this survey was to ascertain awareness and knowledge of reproductive damage by cancer therapy, knowledge of fertility preservation techniques and an understanding of the barriers to fertility preservation. This information would assist in planning programs to improve oncofertility care and counselling.
Studies have shown that over 50% of end-of-life discussions take place for the first time in the hospital and that terminally ill patients often have unrealistic views regarding the possible scope of treatment. The Palliative Care information Act (PCIA) was passed in an attempt to address the lack of access for terminally ill patients to palliative care services. A multi-database systematic review was performed on published studies from 2010 to present, and there were none found measuring the effectiveness of the PCIA.
We aimed to study the effect of the PCIA on access to palliative care services.
We conducted a retrospective chart review of all terminally ill patients who died at Kingsbrook Jewish Medical Center from January 2010 to August 2013 in relation to passing of the PCIA.
Prelaw (prior to the law passing), 12.3% of the terminal patients received palliative care consults, 25% during the transition period (time between passing of law and when it came into effect) and 37.7% postlaw (after coming into effect) (P < 0.001).
Legislation can have a significant effect on terminally ill patient's access to palliative care services and can change the culture of a hospital to be more pro-palliative for the appropriate populations.
The aim of this study was to analyse the effect of altered viewing perspectives on the measurement of the glenopolar angle (GPA) and the differences between these measurements made on 3D CT reconstructions and anteroposterior (AP) scapular view radiographs.
The influence of the viewing perspective on the GPA was assessed, as were the differences in the measurements of the GPA between 3D CT reconstructions and AP scapular view radiographs in 68 cadaveric scapulae.
The median GPA in 3D reconstructions and AP scapular views were 42.7° (95% confidence intervals (CI), 42.0° to 43.5°) and 41.3° (95% CI 40.4° to 42.0°) respectively (p < 0.001). All but five of 20 malpositions demonstrated a significant difference in GPA compared with the respective AP scapular view (p ≤ 0.005). The GPA was most susceptible to malposition in retroversion/anteversion. Inter- and intra-observer reliability for all measurements of the GPA was excellent for 3D CT reconstructions (intraclass correlation (ICC) 0.93 (95% CI 0.87 to 0.96) and 0.94 (95% CI 0.89 to 0.97), respectively) and higher than on AP scapular radiographs (p < 0.001). The intra- and inter-observer reliability was excellent in AP scapular views and malpositions in extension/flexion (ICC ≥ 0.84) but tended to decrease with increasing viewing angle in retroversion/anteversion.
These data suggest that 3D reconstructions are more reproducible than AP scapular radiographs in the assessment of the GPA and should be used to compare data in different studies, to predict outcome, define malunion, and act as an indication for surgery in patients with a scapular fracture. Cite this article: Bone Joint J 2016;98-B:1510-16.
Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.
Because proteins are the main mediators of most cellular processes they are also prime therapeutic targets. Identifying physical links among proteins and between drugs and their protein targets is essential in order to understand the mechanisms through which both proteins themselves and the molecules they are targeted with act. Thus, there is a strong need for sensitive methods that enable mapping out these biomolecular interactions. Here we present a robust and sensitive approach to screen proteome-scale collections of proteins for binding to proteins or small molecules using the well validated MAPPIT (Mammalian Protein-Protein Interaction Trap) and MASPIT (Mammalian Small Molecule-Protein Interaction Trap) assays. Using high-density reverse transfected cell microarrays, a close to proteome-wide collection of human ORF clones can be screened for interactors at high throughput. The versatility of the platform is demonstrated through several examples. With MAPPIT, we screened a 15k ORF library for binding partners of RNF41, an E3 ubiquitin protein ligase implicated in receptor sorting, identifying known and novel interacting proteins. The potential related to the fact that MAPPIT operates in living human cells is illustrated in a screen where the protein collection is scanned for interactions with the glucocorticoid receptor (GR) in its unliganded versus dexamethasone-induced activated state. Several proteins were identified the interaction of which is modulated upon ligand binding to the GR, including a number of previously reported GR interactors. Finally, the screening technology also enables detecting small molecule target proteins, which in many drug discovery programs represents an important hurdle. We show the efficiency of MASPIT-based target profiling through screening with tamoxifen, a first-line breast cancer drug, and reversine, an investigational drug with interesting dedifferentiation and antitumor activity. In both cases, cell microarray screens yielded known and new potential drug targets highlighting the utility of the technology beyond fundamental biology.
The purpose of this study was to evaluate the impact of Medicare Part D on reducing the financial burden of prescription drugs in older adults with diabetes.
Using Medical Expenditure Panel Survey data (2000-2011), interrupted time series and difference-in-difference analyses were used to examine out-of-pocket costs for prescription drugs in 4,664 Medicare beneficiaries (≥65 years of age) compared with 2,938 younger, non-Medicare adults (50-60 years) with diabetes and to estimate causal effects of Medicare Part D.
Part D enrollment of Medicare beneficiaries with diabetes gradually increased from 45.7% (2006) to 52.4% (2011). Compared with years 2000-2005, out-of-pocket pharmacy costs decreased by 13.5% (SE 2.1) for all Medicare beneficiaries with diabetes following Part D implementation; on average, Part D beneficiaries had 5.3% (0.8) lower costs compared with those without Part D. Compared with a younger group with diabetes, out-of-pocket pharmacy costs decreased by 19.4% (1.7) for Medicare beneficiaries after Part D. Part D beneficiaries with diabetes who experienced the coverage gap decreased from 60.1% (2006) to 40.9% (2011) over this period.
These findings demonstrate that although Medicare Part D has been effective in reducing the out-of-pocket cost burden of prescription drugs, approximately two out of five Part D beneficiaries with diabetes experienced the coverage gap in 2011. Future research is needed to examine the impact of Affordable Care Act provisions to close the coverage gap on cost burden of prescription drugs for Medicare beneficiaries with diabetes.
Targeted therapies elicit seemingly paradoxical and poorly understood effects on tumor immunity. Here, we show that the MEK inhibitor trametinib abrogates cytokine-driven expansion of monocytic myeloid-derived suppressor cells (mMDSC) from human or mouse myeloid progenitors. MEK inhibition also reduced the production of the mMDSC chemotactic factor osteopontin by tumor cells. Together, these effects reduced mMDSC accumulation in tumor-bearing hosts, limiting the outgrowth of KRas-driven breast tumors, even though trametinib largely failed to directly inhibit tumor cell proliferation. Accordingly, trametinib impeded tumor progression in vivo through a mechanism requiring CD8(+) T cells, which was paradoxical given the drug's reported ability to inhibit effector lymphocytes. Confirming our observations, adoptive transfer of tumor-derived mMDSC reversed the ability of trametinib to control tumor growth. Overall, our work showed how the effects of trametinib on immune cells could partly explain its effectiveness, distinct from its activity on tumor cells themselves. More broadly, by providing a more incisive view into how MEK inhibitors may act against tumors, our findings expand their potential uses to generally block mMDSC expansion, which occurs widely in cancers to drive their growth and progression. Cancer Res; 76(21); 6253-65. ©2016 AACR.
The aim of this qualitative meta-synthesis was to search and then synthesise family caregivers' experiences of providing care to individuals with Parkinson's disease (PD).
A systematic search resulted in the identification of 11 qualitative studies. Noblit and Hare's seven-stage approach was used to provide a higher-order interpretation of how family caregivers' experienced the effects of taking on a caregiving role.
The process of reciprocal translation resulted in four overarching themes: (1) the need to carry on as usual - 'the caregiver must continue with his life'; (2) the importance of support in facilitating coping - 'I'm still going back to the support group'; (3) the difficult balancing act between caregiving and caregiver needs - 'I cannot get sick because I'm a caregiver'; (4) conflicts in seeking information and knowledge - 'maybe better not to know'.
The themes reflected different aspects of family caregivers' lives that were affected as a result of caring for a relative diagnosed with PD and these raise challenges for more simplistic theories of family caring and appropriate support structures. The findings also highlight several recommendations for clinical practice.
Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations.
Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.
Pt1ML/Pd3Al, which comprises a Pd3Al core protected by a Pt monolayer, may experience Al dealloying because of the strong affinity of Al toward O. To circumvent this issue, the Pt2ML/Os/Pd3Al catalyst has been designed to suppress the migration of Al by inserting an Os monolayer at the interface between the Pd3Al core and two Pt monolayers. On the basis of segregation energies, Al leaching from the core to the 1st layer is determined to be endothermic even under O coverage, indicating an energetic preference for Al to reside in the core structure. The Pt2ML/Os/Pd3Al catalyst benefits from the energetic disadvantage of the inward movement of Os and the presence of the 2 ML Pt layer. As an ORR electrocatalyst, the relatively weak adsorption ability of Pt2ML/Os/Pd3Al suggests improved ORR activity. Finally, a representative OOH association mechanism with low reaction barriers of 0.46, 0.31, 0.38 and 0.41 eV for the OOH formation, OOH dissociation, OH formation and H2O formation steps suggests that the catalyst can effectively activate the O-O bond and eliminate OH, which can act as a catalytic poison. These findings suggest the design of stable sandwich catalysts as potential candidates for ORR electrocatalysis.
Nanostructured ferritic alloys (NFAs) are prime candidates for structural and first wall components of fission and fusion reactors. The main reason for this is their ability to effectively withstand high concentrations of the transmutation product helium. A high number density of oxide nanoclusters dispersed throughout a BCC Fe matrix act as trapping sites for helium and prevent its eventual delivery to high risk nucleation sites. The current study uses density functional theory to investigate the helium trapping mechanisms at the boundary between BCC iron and Y2Ti2O7, a common stoichiometry of the oxide nanoclusters in NFAs. The investigation is carried out on a structure matched oxide nanocluster that is embedded within a BCC Fe supercell. Investigation of the electronic structure and a mapping of the potential energy landscape reveals that the localized iono-covalent bonds present within the oxides create a potential energy-well within the metallically bonded BCC Fe matrix, so that trapping of helium at the oxide nanocluster is thermodynamically and kinetically favorable.
The energetics and electronic structures of native defects in anatase TiO2 are comprehensively studied using hybrid density functional calculations. We demonstrate that oxygen vacancies (VO) and titanium interstitials (Tii) act as shallow donors, and can form at substantial concentrations, giving rise to free electrons with carrier densities from 10(11) to 10(19) cm(-3) under oxygen-rich and oxygen-poor conditions, respectively. The titanium vacancies (VTi), identified as deep acceptors and induced hole carriers, are incapable of fully compensating for the free electrons originating from the donor-type defects at any oxygen chemical potential. Even under extreme oxygen-rich conditions, the Fermi level, which is determined from the charge neutrality condition among charge defects, electron and hole carriers, is located 2.34 eV above the valence band maximum, indicating that p-type conductivity can never be realized under any growth conditions without external doping. This is consistent with common observations of intrinsic n-type conductivity of TiO2. At a typical annealing temperature and under a typical oxygen partial pressure, the carrier concentration is found to be approximately 5 × 10(13) cm(-3).
The spliceosomal B complex-specific protein Prp38 forms a complex with the intrinsically unstructured proteins MFAP1 and Snu23. Our binding and crystal structure analyses show that MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α helices, which have previously been recognized only as rigid connectors or force springs between protein domains. A variant of the Prp38-binding single α helix of MFAP1, in which ER/K motifs not involved in Prp38 binding were mutated, was less α-helical in isolation and showed a reduced Prp38 affinity, with opposing tendencies in interaction enthalpy and entropy. Our results indicate that the strengths of single α helix-based interactions can be tuned by the degree of helix stabilization in the unbound state. MFAP1, Snu23, and several other spliceosomal proteins contain multiple regions that likely form single α helices via which they might tether several binding partners and act as intermittent scaffolds that facilitate remodeling steps during assembly of an active spliceosome.
The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pKa of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.
TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.
The process model proposes that the ego depletion effect is due to (a) an increase in motivation toward indulgence, and (b) a decrease in motivation to control behaviour following an initial act of self-control. In contrast, the reflective-impulsive model predicts that ego depletion results in behaviour that is more consistent with desires, and less consistent with motivations, rather than influencing the strength of desires and motivations. The current study sought to test these alternative accounts of the relationships between ego depletion, motivation, desire, and self-control.
One hundred and fifty-six undergraduate women were randomised to complete a depleting e-crossing task or a non-depleting task, followed by a lab-based measure of snack intake, and self-report measures of motivation and desire strength.
In partial support of the process model, ego depletion was related to higher intake, but only indirectly via the influence of lowered motivation. Motivation was more strongly predictive of intake for those in the non-depletion condition, providing partial support for the reflective-impulsive model. Ego depletion did not affect desire, nor did depletion moderate the effect of desire on intake, indicating that desire may be an appropriate target for reducing unhealthy behaviour across situations where self-control resources vary.
Electrochemically splitting water for hydrogen evolution has attracted a lot attention and developed into a promising approach to produce hydrogen energy. Searching for high-activity and economical electrocatalysts to replace Pt-based catalysts remains a great challenge. In this paper, we reported a concise and effective strategy to fabricate the novel MoWS2 composite for use as the electrocatalyst through a hydrothermal method for the first time. The final obtained MoWS2 composite demonstrated a well-defined hierarchical structure and established that its densely stacked nanopetals act as the active sites in the corresponding hydrogen evolution reaction (HER) processes. Experimental results show that the composites can deliver a clearly promoted HER activity and are superior to the pure structure. In order to give a more in-depth explanation, we also performed a first-principles calculation to further survey the electronic properties, compound form, and HER mechanism of different structures. The charge distribution of MoWS2 composite indicates that electrons can directionally transfer from WS2 to the neighboring MoS2 and form an "electron-rich" configuration, which is beneficial to increase the HER rate and promote the overall performance. This thorough research will not only provide new thought to the analyses and elucidation of the inner mechanism of the HER process for this kind of two-dimensional composite but also guide further work on the basis of experimental and calculation results.
During the last decade, neoadjuvant chemotherapy (NACT) of early breast cancer (EBC) evolved from a therapy intended to enable operability to a standard treatment option aiming for increasing cure rates equivalent to adjuvant chemotherapy (ACT). In parallel, improvements in the quality control of breast cancer care have been established in specialized breast care units.
This study analyzed chemotherapy usage in patients with EBC treated at the Heidelberg University Breast Unit between January 2003 and December 2014.
Overall, 5703 patients were included in the analysis of whom 2222 (39 %) received chemotherapy, 817 (37 %) as NACT, and 1405 (63 %) as ACT. The chemotherapy usage declined from 48 % in 2003 to 34 % in 2014 of the cohort. Further, the proportion of NACT raised from 42 to 65 % irrespective of tumor subtype. In addition, frequency of pathologic complete response (pCR) defined as no tumor residues in breast and axilla (ypT0 ypN0) at surgery following NACT increased from 12 % in 2003 to 35 % in 2014. The greatest effect was observed in HER2+ breast cancer with an increase in patients achieving pCR from 24 to 68 %.
The results mirror the refined indication for chemotherapy in EBC and its preferred usage as NACT in Germany. The increase in pCR rate over time suggests improvement in outcome accomplished by a multidisciplinary decision-making process and stringent measures for quality control.
The major covalent adduct formed between a (13)C-labelled formaldehyde activated bis-amino mitoxantrone analogue (WEHI-150) and the hexanucleotide d(CG(5Me)CGCG)2 has been isolated by HPLC chromatography and the structure determined by NMR spectroscopy. The results indicate that WEHI-150 forms one covalent bond through a primary amine to the N-2 of the G2 residue, with the polycyclic ring structure intercalated at the (5Me)C3pG4/G10p(5Me)C9 site. Furthermore, the WEHI-150 aromatic ring system is oriented approximately parallel to the long axis of the base pairs, with one aliphatic side-chain in the major groove and the other side-chain in the minor groove. This study indicates that mitoxantrone derivatives like WEHI-150 should be capable of forming major-minor groove cross-linked adducts that will likely produce considerably different intracellular biological properties compared to known anthracycline and anthracenedione anticancer drugs.
This paper reports development and testing of a novel cross-flow wet electrostatic precipitator (WESP), recently patented at Ohio University, that utilizes vertical columns of permeable material in the form of polypropylene ropes placed in a cross-flow configuration within a flue gas stream. The cross-flow design has large surface area, which provides scrubbing action; therefore, it has the potential for removing multiple pollutants, including particulates, gases, vapors, and mists. In this new method, the ropes are kept wet by the liquid (water) introduced from the top of the cells running downward on the ropes by capillary action, making the permeable materials act as the ground electrode for capturing particles from the flue gas. Preliminary testing has shown an efficiency of well above 80% using two cells and three sets of discharge electrodes. Since the material of construction is primarily corrosion-resistant polymeric material, both weight and cost reductions are expected from this new design.
The newly invented cross-flow WESP exhibit particulate collection efficiency of well above 80% when introduced in particulate-laden exhaust flow. This value was obtained using a two cells and three discharge electrodes configuration. The electric field strength has a substantial effect on the collection efficiency. Also, the pressure drop test results indicate that there is a potential to increase the collection area, which, in turn, will increase the collection efficiency further.
A method to image taurine distributions within the central nervous system and other organs has long been sought. Since taurine is small and mobile, it cannot be chemically "tagged" and imaged using conventional immuno-histochemistry methods. Combining numerous indirect measurements, taurine is known to play critical roles in brain function during health and disease and is proposed to act as a neuro-osmolyte, neuro-modulator, and possibly a neuro-transmitter. Elucidation of taurine's neurochemical roles and importance would be substantially enhanced by a direct method to visualize alterations, due to physiological and pathological events in the brain, in the local concentration of taurine at or near cellular spatial resolution in vivo or in situ in tissue sections. We thus have developed chemically specific X-ray fluorescence imaging (XFI) at the sulfur K-edge to image the sulfonate group in taurine in situ in ex vivo tissue sections. To our knowledge, this represents the first undistorted imaging of taurine distribution in brain at 20 μm resolution. We report quantitative technique validation by imaging taurine in the cerebellum and hippocampus regions of the rat brain. Further, we apply the technique to image taurine loss from the vulnerable CA1 (cornus ammonis 1) sector of the rat hippocampus following global brain ischemia. The location-specific loss of taurine from CA1 but not CA3 neurons following ischemia reveals osmotic stress may be a key factor in delayed neurodegeneration after a cerebral ischemic insult and highlights the significant potential of chemically specific XFI to study the role of taurine in brain disease.
Gold nanoparticles (GNP) act as a radiosensitizer in radiation therapy. However, recent studies have shown contradictory evidence in terms of radiosensitization in the presence of GNP combined with X-ray megavoltage energy (MV) on different cell types. In this study, the effect of GNP on radiosensitization enhancement of HT-29 human colorectal cancer cells at MV X-ray energy was evaluated.
The cytotoxicity and radiosensitization of GNP were evaluated in HT-29 human colorectal cancer cells by MTS-assay and multiple MTS-assay, respectively. Cellular uptake was assayed using graphite furnace atomic absorption spectrometry (GFAAS). Apoptosis and cell cycle progression were determined by an Annexin V-FITC/propidium iodide (PI) kit and PI/RNase solution with flow cytometry, respectively.
Results showed that the cell viability of the HT-29 cells was not influenced by exposure to different concentrations of GNP (10-100 μM). GNP alone did not affect the cell cycle progression and apoptosis. In contrast, GNP, in combination with radiation (9 MV), induced more apoptosis. The interaction of GNP with MV energy resulted in a significant radiosensitization enhancement compared with irradiation alone.
It was concluded that GNP may work as bio-inert material on HT-29 cancer cells and their enhancement of radiosensitization may be due to increase in the absorbed irradiation dose.
Circulating T and B lymphocytes contribute to the pathogenesis of the neuroinflammatory autoimmune disease, multiple sclerosis (MS). Further progress in the development of MS treatments is dependent upon a greater understanding of the immunological disturbances that underlie the disease. Analyses of circulating immune cells by flow cytometry have revealed MS-associated alterations in the composition and function of T and B cell subsets, including temporal changes associated with disease activity. Disturbances in circulating immune populations reflect those observed in the central nervous system and include skewing towards proinflammatory CD4(+) and CD8(+) T cells and B cells, greater proportions of follicular T helper cells and functional defects in the corresponding T and B regulatory subsets. Utilizing the analytical power of modern flow cytometers, researchers are now well positioned to monitor immunological changes associated with disease activity or intervention, describe immunological signatures with predictive value and identify targets for therapeutic drug development. This review discusses the contribution of various T and B lymphocyte subsets to MS pathogenesis, provides current and relevant phenotypical descriptions to assist in experimental design and highlights areas of future research.
Novel 2-substitutedbenzyl-4(7)-phenyl-1H-benzo[d]imidazole compounds were synthesized and characterized. Although 2a and 2b were reported previously in the literature, 11 compounds were synthesized (nine of them were newly synthesized) and the tyrosinase inhibitory effects and antioxidant activities of these compounds were studied for the first time. All of the synthesized compounds displayed certain inhibitory effects on tyrosinase, with IC50 values ranging from 37.86 ± 0.24 to 75.81 ± 2.49 μM. Among the compounds, 2j exhibited similar tyrosinase inhibitory effect (IC50  = 37.86 ± 0.24 µM) to the positive control, kojic acid (IC50  = 21.93 ± 0.11 µM). Kinetic studies revealed it to act as non-competitive tyrosinase inhibitor with a Ki value of 50.2 µM. The antioxidant activities of the compounds were investigated by using in vitro antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). All of these results indicated that the compounds might have potential application as tyrosinase inhibitors.
Rnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations. Plexin-B1 has been reported to act as a GAP for R-Ras and/or Rap1 proteins. We show that all three plexin-B proteins interact with R-Ras and Rap1, but Rnd proteins do not alter this interaction or R-Ras or Rap1 activity. We demonstrate that plexin-B2 promotes Rnd3-induced cell rounding and loss of stress fibres, and enhances the inhibition of HeLa cell invasion by Rnd3. We identify the amino acids in Rnd3 that are required for plexin-B2 interaction, and show that mutation of these amino acids prevents Rnd3-induced morphological changes. These results indicate that plexin-B2 is a downstream target for Rnd3, which contributes to its cellular function.
Human lung fibroblasts (HLFs) act as innate immune sentinel cells that amplify the inflammatory response to injurious stimuli. Here, we use targeted lipidomics to explore the hypothesis that HLFs also play an active role in the resolution of inflammation. We detected cyclooxygenase-2 (COX-2)-dependent production of both proinflammatory and proresolving prostaglandins (PGs) in conditioned culture medium from HLFs treated with a proinflammatory stimulus, IL-1β. Among the proresolving PGs in the HLF lipidome were several known ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor whose activation in the lung yields potent anti-inflammatory, antifibrotic, and proresolving effects. Next, we used a cell-based luciferase reporter to confirm the ability of HLF supernatants to activate PPARγ, demonstrating, for the first time, that primary HLFs activated with proinflammatory IL-1β or cigarette smoke extract produce functional PPARγ ligands; this phenomenon is temporally regulated, COX-2- and lipocalin-type PGD synthase-dependent, and enhanced by arachidonic acid supplementation. Finally, we used luciferase reporter assays to show that several of the PGs in the lipidome of activated HLFs independently activate PPARγ and/or inhibit NFκB. These results indicate that HLFs, as immune sentinels, regulate both proinflammatory and proresolving responses to injurious stimuli. This novel endogenous resolution pathway represents a new therapeutic target for globally important inflammatory diseases such as chronic obstructive pulmonary disease.
The purpose of this review is to summarize current studies detailing the impact of unilateral sensorineural hearing loss in children and the most current modalities of treatment used in its management.
Current studies corroborate historic views on the impact of unilateral sensorineural hearing loss on patient wellbeing and academic success and stress the use of additional surveillance and studies to diagnose those patients that may pass standard screening practices and suffer from lack of prompt and proper care. With respect to management, notable findings include the continuous development of improved conventional and contralateral routing of signal amplification devices that may act to provide alternatives to percutaneous bone-anchored hearing aid implantation. These include improvements in more conventional hearing aid technology, so as to bridge the performance gap with the classical bone-anchored hearing aid implant, and the development of partially implanted transcutaneous bone conduction hearing devices. Due to dissatisfaction with sound localization, a new and significant development is the burgeoning accumulation of research on cochlear implantation for the treatment of unilateral sensorineural hearing loss in children.
With advances in technology in historic modalities of treatment, and the advent of new modalities such as cochlear implantation, the clinician has a wide armamentarium by which to provide treatment to patients based on clinical circumstances and patient desires.
The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) established an abbreviated approval pathway for biosimilar and interchangeable biological products that was intended to balance innovation and consumer interests. The FDA has published several guidance documents to facilitate implementation of the BPCI Act. Here we discuss the role of comparative clinical studies in the assessment of clinically meaningful differences and illustrate the underlying scientific concepts with a hypothetical example of a clinical study comparing a product to US-licensed bevacizumab. Clin Cancer Res; 22(21); 5167-70. ©2016 AACR.
Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF.
Bile acids are synthesized from cholesterol and are known to be involved with the emulsification and digestion of dietary lipids and fat-soluble vitamins. Outside of this role, bile acids can act as cell signaling effectors through binding and activating receptors on both the cell membrane and nucleus. Numerous reports have investigated these signaling pathways in conditions where the liver is damaged. More recently, effort has been made to investigate the role of bile acids in diseases outside of those associated with liver damage. This review summarizes recent findings on the influences that bile acids can exert in normal neurological function and their contribution to diseases of the nervous system, with the intent of highlighting the role of these metabolites as potential players in neurological disorders.-McMillin, M., DeMorrow, S. Effects of bile acids on neurological function and disease.
Despite the lack of evidence for effectiveness of the Flexible Assertive Community Treatment (Flexible ACT), the model is considered feasible and is well received by mental health professionals. No current studies have adequately examined mental health professional experiences of working with Flexible ACT.
The aim of this study was to explore mental health professional experiences of working with the Flexible ACT model compared with standard care.
The study was guided by grounded theory and based on the interviews with 19 theoretically chosen mental health professionals in Swedish urban areas primarily working with consumers with psychosis, who had worked with the Flexible ACT model for at least 6 months.
The analysis resulted in the core category: "Flexible ACT and the shared caseload create a common action space" and three main categories: (1) "Flexible ACT fills the need for a systematic approach to crisis intervention"; (2) "Flexible ACT has advantages in the psychosocial working environment"; and (3) "Flexible ACT increases the quality of care".
Mental health professionals may benefit from working with the Flexible ACT model through decreased job-strain and stress, increased feeling of being in control over their work situation, and experiences of providing higher quality of care.
Attention bias modification treatment (ABMT) targets threat-related attention biases in anxiety disorders. Most clinical trials of ABMT have focused on adults or small samples of youth. The current randomized controlled trial (RCT) examines ABMT efficacy in youth with social anxiety disorder (SAD) and tests possible moderators of treatment outcomes.
Sixty-seven youth with SAD were randomly assigned to ABMT or attention control training (ACT) conditions. Anxiety severity was measured at baseline, posttreatment, and 3-month follow-up. ClinicalTrials.gov name and identifier: Attention bias modification treatment for children with social anxiety, NCT01397032; http://www.clinicaltrials.gov.
Both ABMT and ACT induced significant reductions in clinician and self-rated social anxiety (ps < .001). An additional reduction was observed at the 3-month follow-up in clinician-rated anxiety symptoms (p = .03). Moderation effects were nonsignificant for the clinician-rated anxiety outcome, but age moderated self-reported anxiety. Older but not younger children, showed significant reduction in anxiety following ABMT relative to ACT (p < .001). Individual differences in attention control also moderated ABMT's effect on self-reported anxiety (p = .05). Children rated by their parents as lower on attention control benefited more from ABMT than those rated higher on attention control. Baseline attention bias did not moderate anxiety (p = .17).
Despite significant reductions in social anxiety, no specific evidence for ABMT was found relative to a control condition. Age and attention control moderated ABMT effects on self-reported SAD symptoms, with clinical effects for older relative to younger children and for those with lower attention control. These results highlight the need to consider developmental influences in the implementation of ABMT protocols.
Cannabinoids apparently act on inflammation through mechanisms different from those of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs). As a class, the cannabinoids are generally free from the adverse effects associated with NSAIDs. Their clinical development thus provides a new approach to treatment of diseases characterized by acute and chronic inflammation and fibrosis. A concise survey of the anti-inflammatory actions of the phytocannabinoids Δ(9)-tetrahydrocannabinol (THC), cannabidiol, cannabichromene, and cannabinol is presented. Mention is also made of the noncannabinoid plant components and pyrolysis products, followed by a discussion of 3 synthetic preparations-Cesamet (nabilone; Meda Pharmaceuticals, Somerset, NJ, USA), Marinol (dronabinol; THC; AbbVie, Inc., North Chicago, IL, USA), and Sativex (Cannabis extract; GW Pharmaceuticals, Cambridge United Kingdom)-that have anti-inflammatory effects. A fourth synthetic cannabinoid, ajulemic acid (AJA; CT-3; Resunab; Corbus Pharmaceuticals, Norwood, MA, USA), is discussed in greater detail because it represents the most recent advance in this area and is currently undergoing 3 phase 2 clinical trials by Corbus Pharmaceuticals. The endogenous cannabinoids, including the closely related lipoamino acids, are then discussed. The review concludes with a presentation of a possible mechanism for the anti-inflammatory and antifibrotic actions of these substances. Thus, several cannabinoids may be considered candidates for development as anti-inflammatory and antifibrotic agents. Of special interest is their possible use for treatment of chronic inflammation, a major unmet medical need.-Zurier, R. B., Burstein, S. H. Cannabinoids, inflammation, and fibrosis.
Fishes endemic to the Qinghai-Tibetan Plateau are comparatively well adapted to aquatic environments with low oxygen partial pressures (hypoxia). Here, we cloned the complete cDNA of hemoglobin (Hb) α and β from the Tibetan schizothoracine fish Schizopygopsis pylzovi, and then investigated changes in Hb mRNA and protein levels in spleen, liver and kidney in response to hypoxia. We applied severe hypoxia (4 h at PO2 = 0.6 kPa) and moderate hypoxia (72 h at PO2 = 6.0 kPa) to adult S. pylzovi. Changes of Hb expression under hypoxia, together with the investigations of spleen somatic index, kidney somatic index and Hb concentration in circulation, suggest that the kidney may not only serve as the erythropoietic organ, but also act as the major blood reservoir in S. pylzovi. From this perspective, the transcriptional activity of Hb in S. pylzovi, as reflected in the kidney, was turned down quickly after the onset of severe hypoxia, while under moderate hypoxia the transcriptional activity of Hb showed upregulation for a short time, but then the transcriptional machinery was turned down slowly on prolonged exposure. Notably, the changes in Hb protein levels in spleen, liver and kidney in response to severe and moderate hypoxia were not in line with the changes in mRNA levels, which are related with the blood reservoir in the kidney. Tibetan schizothoracine fish, at least S. pylzovi, show a particular response of the transcription regulation of Hb to moderate hypoxia, which is different from that of other fish species.
Targeted diagnosis and therapy enable precise tumor detection and treatment. Successful examples for precise tumor targeting are diagnostic and therapeutic radioligands. However, patients with tumors expressing low levels of the relevant molecular targets are deemed ineligible for such targeted approaches.
We performed a screen for drugs that upregulate the somatostatin receptor subtype 2 (sstr2). Then, we characterized the effects of these drugs on transcriptional, translational, and functional levels in vitro and in vivo.
We identified 9 drugs that act as epigenetic modifiers, including the inhibitor of DNA methyltransferase decitabine as well as the inhibitors of histone deacetylase tacedinaline and romidepsin. In vitro, these drugs upregulated sstr2 on transcriptional, translational, and functional levels in a time- and dose-dependent manner. Thereby, their combinations revealed synergistic effects. In vivo, drug-based sstr2 upregulation improved the tumor-to-background and tumor-to-kidney ratios, which are the key determinants of successful sstr2-targeted imaging and radiopeptide therapy.
We present an approach that uses epigenetic modifiers to improve sstr2 targeting in vitro and in vivo. Translation of this method into the clinic may potentially convert patients ineligible for targeted imaging and therapy to eligible candidates.
Primary and secondary school ages have been considered key moments to address the decrease of moderate-tovigorous physical activity (MVPA). Individual (eg, age, gender, and weight status) and contextual factors (moments of the day) need to be considered for a better explanation of the phenomenon. The quantity and quality of physical activity in Physical Education (PE), school recess (SR), and after school (AS) time need to be taken into account to solve the low levels of MVPA in youth.
A sample of adolescents (N = 231, 14.6 ± 1.2 years old) was studied using accelerometry to determine the objective MVPA level in PE, SR, and AS.
Results indicated statistically significant differences on MVPA between contexts (AS > PE > SR, P < .001) as well as regarding the individual factors: age (older > younger in PE and younger > older in SR time; P < .001), gender (boys > girls in all contexts, P < .001), and weight status (overweight > nonoverweight in AS, P < .01).
Because students did not meet the daily MVPA recommendations, some strategies have been provided in each of the contexts analyzed.
Few studies have examined the relationship of social anxiety with drinking game participation. Drinking games represent a popular form of drinking in university settings. Due to their structure, games may appeal to socially anxious drinkers, particularly among those seeking to fit in or cope with the social setting.
To examine the relationship of social anxiety with frequency of drinking game participation among a university undergraduate sample and to investigate if drinking motives moderate this association.
A total of 227 undergraduate students aged 18-24 years (73% female) who had consumed alcohol in the prior year were included in the current investigation. Hierarchical regression examined the influences of social anxiety and drinking motives on frequency of drinking game participation, as well the interactions of social anxiety with drinking for coping motives and conformity motives.
Social anxiety failed to emerge as a significant predictor of frequency of drinking game participation. However, drinking to cope moderated the relationship of social anxiety with frequency of drinking game participation. Socially anxious students who drank to cope were more likely to participate in drinking games on occasions when they consumed alcohol than those who did not endorse this drinking motive.
Results demonstrated the influence of drinking to cope in the relationship of social anxiety with frequency of drinking game participation. Future work should examine the relationship with other indicators of drinking game activity. Intervention efforts addressing social anxiety and drinking should consider motives for drinking, as well as drinking patterns.
Two Cheviot ewes homozygous for the A136L141R154Q171 (AL141RQ) prion protein (PrP) genotype were exposed intracerebrally to brain pools prepared using four field cases of atypical scrapie from the United Kingdom. Animals were clinically normal until the end of the experiment, when they were culled 7 years post-inoculation. Limited accumulation of disease-associated PrP (PrP(Sc)) was observed in the cerebellar molecular layer by immunohistochemistry, but not by western blot or enzyme-linked immunosorbent assay. In addition, PrP(Sc) was partially localized in astrocytes and microglia, suggesting that these cells have a role in PrP(Sc) processing, degradation or both. Our results indicate that atypical scrapie is transmissible to AL141RQ sheep, but these animals act as clinically silent carriers with long incubation times.
There is evidence indicating that parent training programmes including interaction coaching of parents of children with autism spectrum disorders (ASD) can increase parental responsiveness, promote language development and social interaction skills in children with ASD. However, there is a lack of research exploring precisely how healthcare professionals use language in interaction coaching.
To identify the speech acts of healthcare professionals during individual video-recorded interaction coaching sessions of a Hanen-influenced parent training programme with parents of children with ASD.
This retrospective study used speech act analysis. Healthcare professional participants included two speech-language therapists and one occupational therapist. Sixteen videos were transcribed and a speech act analysis was conducted to identify the form and functions of the language used by the healthcare professionals. Descriptive statistics provided frequencies and percentages for the different speech acts used across the 16 videos.
Six types of speech acts used by the healthcare professionals during coaching sessions were identified. These speech acts were, in order of frequency: Instructing, Modelling, Suggesting, Commanding, Commending and Affirming. The healthcare professionals were found to tailor their interaction coaching to the learning needs of the parents. A pattern was observed in which more direct speech acts were used in instances where indirect speech acts did not achieve the intended response.
The study provides an insight into the nature of interaction coaching provided by healthcare professionals during a parent training programme. It identifies the types of language used during interaction coaching. It also highlights additional important aspects of interaction coaching such as the ability of healthcare professionals to adjust the directness of the coaching in order to achieve the intended parental response to the child's interaction. The findings may be used to increase the awareness of healthcare professionals about the types of speech acts used during interaction coaching as well as the manner in which coaching sessions are conducted.
For interventions to be implemented effectively, fidelity must be documented. We evaluated fidelity delivery, receipt, and enactment of the 48-week Women's Lifestyle Physical Activity Program conducted to increase physical activity and maintain weight in African American women.
Three study conditions all received 6 group meetings; 1 also received 11 motivational interviewing personal calls (PCs), 1 received11 automated motivational message calls (ACs), and 1 received no calls. Group meeting delivery was assessed for adherence and competence. PC delivery was assessed with the Motivational Interviewing Treatment Integrity Code. Receipt was defined as group meeting attendance, completion of PCs, and listening to ACs. Enactment was number of weeks an accelerometer was worn.
For group meeting delivery, mean adherence was 80.8% and mean competence 2.9 of 3.0. Delivery of PCs did not reach criterion for competence. Receipt of more than one-half the dose was achieved for 84.9% of women for group meetings, 85.5% for PCs, and 42.1% for ACs. Higher group meeting attendance was associated with higher accelerometer steps at 24 weeks and lower BMI at 24 and 48 weeks.
Fidelity measurement and examination of intervention delivery, receipt, and enactment are important to explicate conditions in which interventions are successful.
Breaking periods of sitting with standing may prevent chronic diseases and increase energy expenditure (EE). Sit-to-stand height adjustable desks may promote workplace standing, but workers have to be willing to stand for portions of the workday.
For studies 1 and 2, EE was measured during word processing while sitting in a chair and while standing. Subjects scored their liking of each posture and time they would be willing to work in each posture during an 8-hour workday. Study 2 included an intervention of replacing subjects' sitting desks with a height adjustable desk. Liking of and willingness to work in each posture were measured before and after the 12-month intervention.
EE was 7.5 kcal/h greater when standing than when sitting. Subjects liked sitting more than standing in study 1. In study 2, liking of postures did not differ or change across 12 months use of height adjustable desks. Perceived willingness to stand decreased from 4.5 h/d at baseline to 3.4 h/d after 12 months.
Standing rather than sitting increased EE by 7.5 kcal/h. Use of a height adjustable desk for 12 months did not alter the hedonic value of standing or sitting, which is promising for long-term increases in standing.
Obesity is a critical problem among Mexican youth, but few studies have investigated associations among physical activity (PA) modes and anthropometrics in this population. This study examined associations among active commuting to school (ACS), sports or other organized PA, outdoor play, and body mass index (BMI) percentile and waist circumference (WC) among Mexican youth.
Parents of school children (N = 1996, ages 6 to 14 years, 53.1% female) in 3 Mexican cities reported PA participation using the (modified) fourth grade School Physical Activity and Nutrition Survey. Trained assessors measured BMI percentile and WC in person.
Parents reported that 52.3% of children engaged in ACS, 57.3% participated in sports or organized PA, and a median of 2 days in the previous week with at least 30 minutes of outdoor play. In complete case analyses (n = 857), ACS was negatively associated with BMI percentile, and outdoor play was negatively associated with WC after adjusting for school, age, sex, and income. In analyses incorporating data from multiple imputation (N = 1996), outdoor play was negatively associated with WC (all Ps < . 05).
ACS and outdoor play are favorably associated with anthropometrics and may help prevent childhood obesity in Mexico. ACS and outdoor play should be priorities for increasing youth PA in Mexico.
Parent support for child physical activity is a consistent predictor of increased childhood activity. Little is known about factors that prevent or facilitate support. The purpose of this research was to identify barriers to parent support for child physical activity in Appalachian parents.
A cross-sectional study assessed parents whose children participated in Coronary Artery Risk Detection in Appalachian Communities (CARDIAC) screenings in a rural Appalachian state. Barriers to parental support for physical activity, demographics, geographic location, and parental support for activity were measured.
A total of 475 parents completed surveys. The majority were mothers (86.7%), parents of kindergarteners (49.5%), white (89.3%), and living in a nonrural area (70.5%). Community-level factors were most frequently cited as barriers, particularly those related to the built environment. Rural and low-income parents reported significantly higher barriers. Community, interpersonal, and intrapersonal barriers were negatively correlated with parent support for child physical activity. Parents of girls reported a higher percentage of barriers related to safety.
Reported barriers in this sample differed from those reported elsewhere (Davison, 2009). Specific groups such as low-income and rural parents should be targeted in intervention efforts. Future research should explore gender differences in reported barriers to determine the influence of cultural stereotypes.
Physical activity (PA) has beneficial effects on older age physical functioning, but longitudinal studies with follow-ups extending up to decades are few. We investigated the association between leisure-time PA (LTPA) and occupational PA (OPA) from early to late adulthood in relation to later life performance-based physical functioning.
The study involved 1260 people aged 60 to 79 years who took part in assessments of physical functioning (Short Physical Performance Battery [SPPB] test, 10-m maximal walking test, and grip strength test). Participants' data on earlier life LTPA/OPA (age range 25 to 74 years) were received from the previous studies (average follow-up 13.4 years). Logistic, linear, and censored regression models were used to assess the associations between LTPA/OPA earlier in life and subsequent physical functioning.
A high level of LTPA earlier in life was associated with a lower risk of having difficulties on the SPPB test (odds ratio [OR]: 0.37; 95% confidence interval [CI], 0.24-0.58) and especially on the chair rise test (OR: 0.42; 95% CI, 0.27-0.64) in old age. Heavy manual work predicted difficulties on SPPB (OR: 1.91; 95% CI, 1.22-2.98) and the chair rise test (OR: 1.75; 95% CI, 1.14-2.69) and poorer walking speed (β = .10, P = .005).
This study highlights the importance of LTPA on later life functioning, but also indicates the inverse effects that may be caused by heavy manual work.
The role of psychosocial stress in the development of obesity and metabolic syndrome is receiving increased attention and has led to examination of whether physical activity may moderate the stress-metabolic syndrome relationship. The current study examined relationships among physical activity, stress, and metabolic syndrome in adolescents.
Participants (N = 126; 57 girls, 69 boys) were assessed for anthropometry, psychosocial stress, physical activity, and metabolic syndrome variables; t tests were used to examine sex differences, and regression analysis was used to assess relationships among variables controlling for sex and maturity status.
Mean body mass index approached the 75th percentile for both sexes. Typical sex differences were observed for systolic blood pressure, time spent in moderate and vigorous physical activity, and perceived stress. Although stress was not associated with MetS (β = -.001, P = .82), a modest, positive relationship was observed with BMI (β = .20, P = .04).
Strong relationships between physical activity and stress with MetS or BMI were not found in this sample. Results may be partially explained by overall good physical health status of the participants. Additional research in groups exhibiting varying degrees of health is needed.
We examined the relationship between parents' perception of neighborhood safety and children's physical activity and use of recreation facilities in a US nationally representative sample of fifth grade children.
We used data from the Early Childhood Longitudinal Study Kindergarten cohort, fifth grade sample (N = 9827). Multivariate logistic and linear regression models were used to examine associations between parents' perception of neighborhood safety for outside play and number of days children engage in physical activity, as well as children's use of recreational facilities for physical activity.
Children who used recreational facilities engaged in physical activity on more days of the week compared with children who did not use a facility (3.3 days vs. 3.8 days, P < .0001). Children from neighborhoods perceived as unsafe by parents engaged in almost 1 less day per week in physical activity (β = -.89, P < .0001). Children from neighborhoods perceived as unsafe were less likely to use recreational facilities compared with children from neighborhoods perceived as safe (odds ratio = 0.72, P < .0001). Children from less affluent families across rural and urban areas had half the odds of using recreational facilities compared with children from the wealthiest families living in urban areas.
Parents' perception of neighborhood safety for outside play can deter or promote children's physical activity and use of recreational facilities. Children from less affluent families are less likely to use facilities than children from wealthy families, regardless of place of residence.
Obesity and lack of physical activity are major public health problems in the United States. Well-designed, active living communities (ALCs) can help support physically active lifestyles. This study assessed attitudes of Hawaii decision makers in 2007 and 2013 to determine if priorities toward ALCs changed.
Elected and appointed state and county officials were mailed surveys both years. Respondents rated the importance of 23 specified problems, which included 1 obesity variable and 5 ALC variables.
The survey was completed by 126 (70.4%) respondents in 2007 and 117 (60.9%) in 2013. Among the specific problems, only obesity increased in rank from 14th to ninth place. Three variables fell more than 2 places: increasing traffic (fifth to seventh place), poorly planned development and sprawl (seventh to 11th place) and pedestrian safety (12th to 17th place). The other 2 stayed relatively the same: lack of pedestrian walkways, sidewalks, and crosswalks (16th to 15th place) and lack of recreational activities (22nd to 23rd place).
Across years, obesity concerns have increased but do not appear to be tied to increases in concern for ALC variables. More education for policymakers on the link between obesity, physical activity, and the built environment is necessary.
The purpose of this study was to compare the physical activity parenting practices (PAPPs) parents report using with the PAPPs incorporated in the published literature.
PAPPs in the literature were identified by reviewing the content of 74 published PAPP measures obtained from current systematic reviews supplemented with a literature search. The types of PAPPs used by parents were identified by surveying a stratified sample of 134 Canadian and US parents of 5- to 12 year-old children. Items from the literature and parent responses were coded using the same coding scheme. Differences between the PAPPs emphasized by the parents and the literature were examined.
Parents significantly emphasized different issues than what is measured in the literature (P < .001). Parents emphasized more control (13.6% vs. 6.9%), modeling and teaching (13.2% vs. 9.2%), and structural strategies (32.2% vs. 28.6%) and less autonomy support (11.8% vs. 14.0%), logistical support (9.9% vs. 12.8%), and responsiveness strategies (19.3% vs. 28.5%).
Physical activity practices most often employed by parents are not the ones emphasized in current measures. The extent to which putting more emphasis on the areas identified by parents will increase the predictive validity of the measures warrants further examination.
This study investigates the relationships between physical activity (PA), sports participation and sensation seeking or aggression and injury risk in young men.
A representative cohort study was conducted with 4686 conscripts for the Swiss army. Risk factors assessed at baseline were PA, the frequency of sports participation, sensation seeking, and aggression. The number of injuries during the past 12 months was reported 16 months after baseline. Exposure to moderate-tovigorous physical activity (MVPA) was estimated based on baseline PA.
Among conscripts, 48.5% reported at least 1 injury for the past 12 months. After accounting for exposure to MVPA, the most inactive individuals (reference group) had the highest injury risk and those with high levels of PA and weekly sports participation the lowest (Poisson regression analysis: incidence rate ratio = 0.14 [0.12-0.16]). Independent of activity level, sensation seeking increased cumulative injury incidence significantly (Logistic regression analysis [injured vs. not injured]: odds ratio = 1.29 [1.02-1.63]) and incidence rates marginally. Aggression was marginally associated only with cumulative injury incidence and only in those participating in daily sports.
When accounting for exposure to PA, being inactive is a strong injury risk factor in young men, whereas the roles of the personality variables are less clear.
Health-related fitness (HRF) and motor coordination (MC) can be influenced by children's environment and lifestyle behavior. This study evaluates the association between living environment and HRF, MC, and physical and sedentary activities of children in Suriname.
Tests were performed for HRF (morphological, muscular, and cardiorespiratory component), gross MC (Körperkoordinations Test für Kinder), fine MC (Movement Assessment Battery for Children), and self-reported activities in 79 urban and 77 rural 7-year-old Maroon children. Urban-rural differences were calculated by an independent sample t test (Mann-Whitney U test if not normally distributed) and χ(2) test.
No difference was found in body mass index, muscle strength, and the overall score of gross and fine MC. However, urban children scored lower in HRF on the cardiorespiratory component (P ≤ .001), in gross MC on walking backward (P = .014), and jumping sideways (P = 0.011). They scored higher in the gross MC component moving sideways (P ≤ .001) and lower in fine MC on the trail test (P = .036) and reported significantly more sedentary and fewer physical activities than rural children.
Living environment was associated with certain components of HRF, MC, and physical and sedentary activities of 7-year-old children in Suriname. Further research is needed to evaluate the development of urban children to provide information for possible intervention and prevention strategies.
A large proportion of children do not reach the recommended levels of physical activity for health. A quasiexperimental study with nonrandom assignment was performed to evaluate the effectiveness and feasibility of a school-based physical education intervention aimed at increasing the levels of moderate-to-vigorous physical activity (MVPA).
Ten classes from 4 primary schools, including 241 children aged 8 to 10 years, were recruited. The experimental group (n = 97) received 4 additional sessions/week of 60 minutes of MVPA for 8 months. The control group (n = 135) continued their standard program (2 sessions of 50 minutes/week). Motor abilities (standing long jump, handgrip strength, Harre circuit, sit and reach), physical fitness (Yo-Yo Intermittent Recovery Level-1), anthropometric measures (body mass index, waist to height ratio), and self-efficacy (Perceived Physical Ability Scale for Children) were evaluated at baseline and after the intervention.
The experimental group significantly improved in the Harre circuit both in males (P < .001) and females (P < .01), whereas physical fitness test improved only in males (P < .001). Males in the experimental group improved the perception of self-efficacy in coordinative abilities (P = .017).
The proposed school-based MVPA program showed effectiveness and feasibility. The differences observed by gender highlight the need to use different strategies to increase the involvement of all the participants.
The Newman's Every Child a Sports Person (NECaSP) intervention aspires to increase sport and physical activity (PA) participation among young people in the United Kingdom. The aims of this article are to report on a summative process evaluation of the NECaSP and make recommendations for future interventions.
Seventeen schools provided data from students aged 11 to 13 years (n = 1226), parents (n = 192), and teachers (n = 14) via direct observation and questionnaires. Means, SDs, and percentages were calculated for sociodemographic data. Qualitative data were analyzed via directed content analysis and main themes identified.
Findings indicate further administrative, educational, and financial support will help facilitate the success of the program in improving PA outcomes for young people and of other similar intervention programs globally. Data highlighted the need to engage parents to increase the likelihood of intervention success.
One main strength of this study is the mixed-methods nature of the process evaluation. It is recommended that future school-based interventions that bridge sports clubs and formal curriculum provision should consider a broader approach to the delivery of programs throughout the academic year, school week, and school day. Finally, changes in the school curriculum can be successful once all parties are involved (community, school, families).
Few studies have used ecological models to study multiple levels of association with objectively measured moderate-to-vigorous physical activity (MVPA) in young children from middle-income countries. The purpose of this study was to examine potential correlates of objectively measured MVPA in Brazilian children.
The sample consisted of 328 children. An Actigraph GT3X+ accelerometer was used to monitor MVPA over 7 days. Body mass index and body fat percentage were measured using a bioelectrical impedance scale. Questionnaires completed by the children, their parents, and school personnel queried individual, family and home, and school-level environmental correlates.
Children averaged 59.3 min/d in MVPA (44.5% met MVPA guidelines), and 51.8% were overweight/obese. For boys and girls combined, significant correlates (P < .05) of MVPA were waist circumference (β = -.007), travel mode to school (β = .140), maternal employment status (β = -.119) and TV in bedroom (β -.107). In boys, significant correlates of MVPA were waist circumference (β = -.011), travel mode to school (β = .133), and maternal employment status (β = -.195). In girls, the only significant correlate of MVPA was travel mode to school (β = .143).
Several factors were identified as correlates of MVPA in Brazilian children; however, only travel mode to school was common for both boys and girls.
One of the main reasons for cancer treatment resistance is the existence of cancer stem-like cells (CSCs). Here, we elucidated the relationship between low proteasome activity cells (LPACs) and CSCs.
The human colorectal cancer cell lines HCT116, SW480, DLD1, and KM12SM were engineered to stably express a green fluorescent molecule fused to the degron of ornithine decarboxylase, resulting in an accumulation of the fluorescence in LPACs. LPACs were isolated by flow cytometry. Treatment resistance (radio- and chemotherapy) and the capacity of LPACs to act as CSCs were analyzed. Microarray analysis was performed to reveal genes related to treatment resistance. The prognostic impact of potent genes was examined in 190 patients with colorectal cancer.
LPACs had a significantly increased capacity for radioresistance and chemoresistance (5-fluorouracil and oxaliplatin), significantly lower reactive oxygen species activity, and significantly increased sphere formation capacity compared with non-LPACs. The number of cells in the G0-G1 phase was significantly higher among LPACs. Subcutaneous injection of as few as 20 LPACs led to tumor formation in immunologically incompetent mice. Microarray analysis revealed that the expression of EP300-interacting inhibitor of differentiation 3 (EID3) was significantly increased in LPACs. In vitro assay revealed that EID3 positively controlled cell proliferation and treatment resistance. The high expression of EID3 was an adverse prognostic indicator in patients with colorectal cancer (P = 0.0400).
LPACs have characteristic treatment resistance and act as CSCs in colorectal cancer. In addition, EID3 is one of the potential regulators of treatment resistance in colorectal cancer and may be a potential therapeutic target. Clin Cancer Res; 22(21); 5277-86. ©2016 AACR.
Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.
There is limited Australian information on the prevalence and mental health consequences of bullying and ill-treatment at work. The aims of this study were to use data from an ongoing Australian longitudinal cohort study to (1) compare different measures of workplace bullying, (2) estimate the prevalence of bullying and ill-treatment at work, (3) evaluate whether workplace bullying is distinct from other adverse work characteristics and (4) examine the unique contribution of workplace bullying to common mental disorders in mid-life.
The sample comprised 1466 participants (52% women) aged 52-58 from wave four of the Personality and Total Health (PATH) through Life study. Workplace bullying was assessed by a single item of self-labelling measure of bullying and a 15-item scale of bullying-related behaviours experienced in the past 6 months. Factor analysis the identified underlying factor structure of the behavioural bullying scale.
Current bullying was reported by 7.0% of respondents, while 46.4% of respondents reported that they had been bullied at some point in their working life. Person-related and work-related bullying behaviours were more common than violence and intimidation. The multi-dimensional scale of bullying behaviours had greater concordance with a single item of self-labelled bullying (Area Under the Curve = 0.88) than other adverse work characteristics (all Area Under the Curves < 0.67). Self-labelled bullying and scales reflecting person-related and work-related bullying were independent predictors of depression and/or anxiety.
This study provides unique information on the prevalence and mental health impacts of workplace bullying and ill-treatment in Australia. Workplace bullying is a relatively common experience, and is associated with increased risk of depression and anxiety. Greater attention to identifying and preventing bullying and ill-treatment in the workplace is warranted.
In England and Wales, the Approved Mental Health Professional (AMHP) has final responsibility for applying under the Mental Health Act 1983 to admit an individual compulsorily and convey them to psychiatric hospital. The AMHP role is challenging and legally accountable and unique to the UK context.
To analyse the motivation of individuals to become AMHPs, and identify factors which may affect motivation.
Semi-structured interviews were conducted with 12 AMHPs from local authorities across Southern England. Ten participants were social workers, one was qualified as both a nurse and social worker, and one was a mental health nurse.
Participants identify career progression and professional development as significant as well as the status and independence of the role and enhanced job security. Social work participants value the Mental Health Act assessment as a contained piece of work, with a high degree of professional discretion.
AMHPs are motivated by an increase in professional status and job security, but also exercising independent judgment and authority in a time-limited intervention is emotionally and professionally rewarding.
There is a paucity of research into the experience of compulsory admissions under the Mental Health Act (MHA) 1983/2007, particularly for adults with psychosis, yet this diagnostic group reportedly account for the largest proportion of compulsory admissions.
This UK-based study's objective was to explore the compulsory admission experiences (under the MHA 1983/2007) of service users with psychosis, and to identify key characteristics of these experiences.
The qualitative method "Grounded Theory" (Glaser & Strauss, 1967) was employed as it worked inductively from the data. Seventeen participants (eight service users with psychosis, nine psychiatrists) were interviewed.
Five higher-order categories and 47 categories were identified and are displayed in a model entitled "A disturbing journey to and from detention".
This small-scale qualitative study achieved its objectives, exploring compulsory admission experiences (under the MHA 1983/2007) of service users with psychosis in England, and identified key characteristics of these experiences. Service and clinical implications are highlighted, with scope for further research.
Conflicting results on the role of secreted protein acidic and rich in cysteins (SPARC) expression have been reported in resected pancreatic ductal adenocarcinoma (PDAC), and its prognostic and/or predictive role in advanced PDAC (aPDAC) has not been extensively investigated yet. This study was designed to evaluate SPARC expression as a biomarker in aPDAC patients (pts) not receiving nab-paclitaxel.
Using immunohistochemistry, we examined the stromal as well as the tumoral (i.e., cytoplasmic) SPARC expression in tumour tissue (primary tumours and metastases) of 134 aPDAC pts participating in completed prospective clinical and biomarker trials. The SPARC expression levels were correlated to the pts' clinicopathological parameters and survival times.
Sixty-seven per cent of the analysed tumours showed high stromal SPARC expression, which was not associated with overall survival (OS, median 9.1 vs 7.6 months, P=0.316). A positive cytoplasmic SPARC expression was detected in 55% of the tumours and correlated significantly with inferior progression-free survival (PFS, 6.2 vs 8.6 months, P=0.004) and OS (7.8 vs 8.4 months, P=0.032). This association was strongest for pts, where primary tumour tissue was examined (PFS: 6.7 vs 10.8 months, P=0.004; OS: 7.9 vs 11.9 months, P=0.030), whereas no significant correlation was detected for pts, where only metastatic tissue was available (PFS: 5.8 vs 6.6 months, P=0.502; OS: 7.0 vs 7.8 months, P=0.452). In pts receiving gemcitabine-based chemotherapy cytoplasmic SPARC expression was significantly associated with an inferior PFS and OS (PFS: 6.2 vs 9.2 months, P=0.002; OS 7.3 vs 9.9 months, P=0.012), whereas no such association was detected for stromal SPARC expression or for pts receiving fluoropyrimidine-based chemotherapy.
We identified cytoplasmic SPARC expression in the primary tumour as a biomarker associated with inferior PFS and OS in aPDAC. Cytoplasmic SPARC expression may furthermore act as a negative predictive biomarker in pts treated with gemcitabine-based chemotherapy.
Near-infrared spectroscopy estimates cerebral regional tissue oxygen saturation (rSO2), which may decrease under hyperventilation. Propofol and sevoflurane act differently on cerebral blood vessels. Consequently, cerebral blood flow during hyperventilation with propofol and sevoflurane anaesthesia may differ.
The first aim of this study was to compare the changes in rSO2 between propofol and sevoflurane anaesthesia during hyperventilation. The second aim was to assess changes in rSO2 with ventilation changes.
A randomised, open-label study.
University of Yamanashi Hospital, Yamanashi, Japan from January 2014 to September 2014.
Fifty American Society of Anesthesiologists physical status 1 or 2 adult patients who were scheduled for elective abdominal surgery were assigned randomly to receive either propofol or sevoflurane anaesthesia. Exclusion criterion was a known history of cerebral disease such as cerebral infarction, cerebral haemorrhage, transient ischaemic attack and subarachnoid haemorrhage.
After induction of anaesthesia but before the start of surgery, rSO2, arterial carbon dioxide partial pressure (PaCO2) and arterial oxygen saturation were measured. Measurements were repeated at 5-min intervals during 15 min of hyperventilation with a PaCO2 around 30 mmHg (4 kPa), and again after ventilation was normalised.
The primary outcome was the difference of changes in rSO2 between propofol anaesthesia and sevoflurane anaesthesia during and after hyperventilation. The second outcome was change in rSO2 after the initiation of hyperventilation and after the normalisation of ventilation.
Changes of rSO2 during hyperventilation were -10 ± 7% (left) and -11 ± 8% (right) in the propofol group, and -10 ± 8% (left) and -9 ± 7% (right) in the sevoflurane group. After normalisation of PaCO2, rSO2 returned to baseline values. Arterial oxygen saturation remained stable throughout the measurement period. The rSO2 values were similar in the propofol and the sevoflurane groups at each time point.
The effects of hyperventilation on estimated rSO2 were similar with propofol and sevoflurane anaesthesia. Changes in rSO2 correlated well with ventilation changes.
Japan Primary Registries Network (JPRN); UMIN-CTR ID; UMIN000010640.
Resectable non-small cell lung cancer (NSCLC) treatment options most often consist of surgical resection along with adjuvant chemotherapy (ACT). The benefit of ACT however is modest and is accompanied by important side effects.
One central quest in the field is therefore the identification of a predictive marker of the response to ACT.
We applied an unbiased approach based on high content analysis of expression data generated from a discovery patient cohort.
We identified MMS19, a component of the cytoplasmic Iron-Sulfur Assembly (CIA) machinery important for the Nucleotide Excision Repair (NER) pathway as a pivotal gene for cisplatin toxicity. We then confirmed the association between MMS19 expression and the response to Cisplatin treatment in a panel of NSCLC cell lines. Finally we validated these pre-clinical data in a subgroup of JBR.10 trial patients through a hypothesis-driven analysis, and showed that MMS19 levels associated with ACT benefit.
We therefore propose the expression level of MMS19 as a candidate predictive marker of ACT benefit in resected NSCLC patients.
The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.
The microtubule cytoskeleton regulates cell polarity by spatially organizing membrane trafficking and signaling processes. In epithelial cells, microtubules form parallel arrays aligned along the apico-basal axis, and recent work has demonstrated that the members of CAMSAP/Patronin family control apical tethering of microtubule minus ends. Here, we show that in mammalian intestinal epithelial cells, the spectraplakin ACF7 (also known as MACF1) specifically binds to CAMSAP3 and is required for the apical localization of CAMSAP3-decorated microtubule minus ends. Loss of ACF7 but not of CAMSAP3 or its homolog CAMSAP2 affected the formation of polarized epithelial cysts in three-dimensional cultures. In short-term epithelial polarization assays, knockout of CAMSAP3, but not of CAMSAP2, caused microtubule re-organization into a more radial centrosomal array, redistribution of Rab11-positive (also known as Rab11A) endosomes from the apical cell surface to the pericentrosomal region and inhibition of actin brush border formation at the apical side of the cell. We conclude that ACF7 is an important regulator of apico-basal polarity in mammalian intestinal cells and that a radial centrosome-centered microtubule organization can act as an inhibitor of epithelial polarity.
The intestinal epithelium is the fastest renewing tissue in mammals and has a large flexibility to adapt to different types of damage. Lgr5(+) crypt base columnar (CBC) cells act as stem cells during homeostasis and are essential during regeneration. Upon perturbation, the activity of CBCs is dynamically regulated to maintain homeostasis and multiple dedicated progenitor cell populations can reverse to the stem cell state upon damage, adding another layer of compensatory mechanisms to facilitate regeneration. Here, we review our current understanding of how intestinal stem and progenitor cells contribute to homeostasis and regeneration, and the different signaling pathways that regulate their behavior. Nutritional state and inflammation have been recently identified as upstream regulators of stem cell activity in the mammalian intestine, and we explore how these systemic signals can influence homeostasis and regeneration.
Allergic rhinitis and asthma are common and closely related diseases. Recently, a Portugese questionnaire has been developed 'The Control of Allergic Rhinitis and Asthma Test' (CARATkids) that measures disease control of both diseases in children. This study aims to validate the CARATkids in Dutch children and for the first time in adolescents. In addition, to calculate the minimal clinically important difference (MCID).
A prospective observational study was conducted in an outpatient clinic. After translation of the CARATkids from Portuguese to Dutch, patients (6-18 years) with asthma or asthma and allergic rhinitis completed the CARATkids, Asthma Control Test (ACT) and visual analog scale (VAS) questionnaire three times. Baseline characteristics, mean scores, internal consistency, test-retest reliability, cross-sectional and longitudinal validity, discriminative properties, responsiveness and MCID of the CARATkids were assessed.
111 patients were included. 86% and 79% respectively completed the questionnaires at the second and third visit. All children had asthma and 85% had concomitant allergic rhinitis. The internal consistency was good with all expected a priori correlations met. CARATkids scores were higher in patients with uncontrolled asthma and patients with moderate-severe rhinitis compared to better controlled subjects. Patients with a variable asthma control had significantly higher scores during periods of uncontrolled asthma. Also the Guyatt's responsiveness index was good. The MCID was 2.8.
The CARATkids questionnaire is a reliable and valid tool to assess allergic rhinitis and asthma control among Dutch children. The tool can be used in adolescents. This article is protected by copyright. All rights reserved.
Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.
Although co-payments and deductibles are means of keeping health expenditures low, they have also been cited as barriers that inhibit patients from accessing necessary healthcare. We aimed to evaluate Rhode Island residents' experiences with cost-related access challenges within the state's healthcare system. We conducted a cross-sectional survey of resident experiences with healthcare in Rhode Island. Our survey instrument was composed of the RAND Corporation "Short-Form Patient Satisfaction Questionnaire (PSQ-18)", questions developed by the Rhode Island Office of the Health Insurance Commissioner, and ranking of health priorities based on prior community assessments conducted by the Rhode Island Department of Health. Data were collected at venues across the state as part of the Rhode Island Department of Health 2015 Statewide Health Inventory. From July to August 2015, 404 surveys were completed. We found that 40% of respondents had a co-pay of $20-$50, while 35.7% of respondents had a deductible of greater than $500. Further, one-third of respondents delayed receiving care due to financial barriers. This decision resulted in a worsening condition or hospital visit for nearly half of those respondents. Co-pays and deductibles pose challenges to Rhode Islanders accessing health care. Cost-related barriers to healthcare access should continue to be addressed, especially in the context of preventive care services, which are now being built into health insurance premiums through the Patient Protection and Affordable Care Act. [Full article available at http://rimed.org/rimedicaljournal-2016-11.asp].
There is currently no doubt about the serious threat that oxidative stress (OS) poses to human health. Therefore, a crucial strategy to maintain a good health status is to identify molecules capable of offering protection against OS through chemical routes. Based on the known efficiency of the phenolic and melatonin (MLT) families of compounds as antioxidants, it is logical to assume that phenolic MLT-related compounds should be (at least) equally efficient. Unfortunately, they have been less investigated than phenols, MLT and its non-phenolic metabolites in this context. The evidence reviewed here strongly suggests that MLT phenolic derivatives can act as both primary and secondary antioxidants, exerting their protection through diverse chemical routes. They all seem to be better free radical scavengers than MLT and Trolox, while some of them also surpass ascorbic acid and resveratrol. However, there are still many aspects that deserve further investigations for this kind of compounds.
In this work, a novel electrochemical aptasensor was developed for sensitive and selective detection of myoglobin based on meso-tetra (4-carboxyphenyl) porphyrin-functionalized graphene-conjugated gold nanoparticles (TCPP-Gr/AuNPs). Due to its good electric conductivity, large specific surface area, and excellent mechanical properties, TCPP-Gr/AuNPs can act as an enhanced material for the electrochemical detection of myoglobin. Meanwhile, it provides an effective matrix for immobilizing myoglobin-binding aptamer (MbBA). The electrochemical aptasensor has a sensitive response to myoglobin in a linear range from 2.0 × 10(-11) M to 7.7 × 10(-7) M with a detection limit of 6.7 × 10(-12) M (S/N = 3). Furthermore, the method has the merits of high sensitivity, low price, and high specificity. Our work will supply new horizons for the diagnostic applications of graphene-based materials in biomedicine and biosensors.
ZnO-carbon composite spheres were synthesized via starch hydrothermal carbonization (HTC) in the presence of a soluble zinc salt (acetate), followed by thermal processing under an argon atmosphere. Besides sustainability, the one-pot procedure represents a scalable synthesis of tailored carbon-metal oxide spheres with a structurally-ordered carbon matrix obtained at a relatively low temperature (700 °C). The ability of zinc cations to develop different linkages with starch's hydrophilic functional groups and to act as external nucleators determines an increase in HTC yield; the effect is obvious even in the presence of small concentrations of zinc in the reaction medium (0.005 M), thus providing a way to improve the carbonization process efficiency. It is also shown that zinc content is the control vector of the spherical composite's properties: a variation from 0.3 to 4.8 at% not only induces a variation in their size (200 nm-10 μm), interconnectivity (from disperse spheres to necklace-like aggregations), surface area and connected porosity (from micro- to mesoporosity), but also of their electrochemical and white light adsorption and emission features. Since the variation in zinc content is made by a simple adjustment of the raw material concentrations, the functionality of these carbon-based materials can be modulated in a straightforward manner.
Two enediyne based protein-capture compounds 1 and 2 were synthesized. Both these molecules have an aryl sulfonamide for reversible binding with Human Carbonic Anhydrase II (HCA II) and a pyrene moiety for the visualization of a capture event. While compound 1 has an aryl azide as a photo cross-linking agent, compound 2 lacks the azide moiety. Capture experiments with HCA II however show that both 1 and 2 can photo cross-link with the protein as indicated in gel electrophoresis as well as MALDI analysis after tryptic digestion of HCA II. This observation demonstrates the ability of the enediyne moiety to act as a photo-affinity label possibly via the addition of nucleophilic amino acids to the partially zwitterionic singlet form of the diradical generated by photo Bergman cyclization.
A literature review of 16 papers on occupational injury research in Malaysia published during a 13-year period from 2000-2013 was carried out. The objective of this review and article selection was based on relevance to the research theme and mention of areas for future research. Most of the publications have focused on descriptive epidemiology, management practices, worker's knowledge, attitude, training, and rehabilitation services. The transportation, agriculture and construction sectors were found to be the most hazardous sectors and would benefit the most from Occupational Safety & Health (OSH) research and interventions. There is a strong need to develop a national injury surveillance system and also a mechanism to ensure adherence to the Occupational Safety & Health Act(OSHA) 1994. Detailed description and identification of risk factors for occupational injury in the environment, including machinery and equipment used was generally lacking. Future research on occupational injury should focus on surveillance to determine the magnitude of occupational injuries, determination of risk factors, identifying costeffective interventions (such as enforcement of OSHA regulations), and assessment of rehabilitation services. Relevant government agencies, universities, corporate sector and occupational safety organizations need to play a proactive role in identifying priority areas and research capacity building. Funding for occupational injury should be commensurate with the magnitude of the problem.
Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA) from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system.
A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity.
This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.
In photosynthesis, the light-driven oxidation of water is a sustainable process, which converts solar to chemical energy and produces protons and oxygen. To enable biomimetic strategies, the mechanism of photosynthetic oxygen evolution must be elucidated. Here, we provide information concerning a critical step in the oxygen-evolving, or S-state, cycle. During this S3-to-S0 transition, oxygen is produced, and substrate water binds to the manganese-calcium catalytic site. Our spectroscopic and H2(18)O labeling experiments show that this S3-to-S0 step is associated with the protonation of an internal water cluster in a hydrogen-bonding network, which contains calcium. When compared to the protonated water cluster, formed during a preceding step, the S1-to-S2 transition, the S3-to-S0 hydronium ion is likely to be coordinated by additional water molecules. This evidence shows that internal water and the hydrogen bonding network act as a transient proton acceptor at multiple points in the oxygen-evolving cycle.
Strong professional priorities, evolving Affordable Care Act requirements, and a significantly limited public health nursing workforce prompted the University of Colorado College of Nursing to collaborate with the School of Public Health to implement one of the first Doctor of Nursing Practice/Master of Public Health dual degree programs in the nation. Federal grant funding supported the development, implementation, and evaluation of this unique post-baccalaureate dual degree program, for which there were no roadmaps, models, or best practices to follow. Several key issues emerged that serve as lessons learned in creating a new, novel higher education pathway for Advanced Public Health Nursing. This paper highlights two of those: (1) marketing, admission, and matriculation across two programs, and (2) enhancing curricula through distance coursework and interprofessional education. When collaboration with a school of public health is possible, the Doctor of Nursing Practice/Master of Public Health dual degree is an efficient way to prepare public health nurses with the highest level of public health knowledge, practice, and leadership expertise.
Current drugs against the influenza A virus (IAV) act by inhibiting viral neuraminidase (NA) enzymes responsible for the release of budding virions from sialoglycans on infected cells. Here, we describe an approach focused on a search for inhibitors that reinforce the protective functions of mucosal barriers that trap viruses en route to the target cells. We have generated mimetics of sialo-glycoproteins that insert into the viral envelope to provide a well-defined mucus-like environment encapsulating the virus. By introducing this barrier, which the virus must breach using its NA enzymes to infect a host cell, into a screening platform, we have been able to identify compounds that provide significant protection against IAV infection. This approach may facilitate the discovery of potent new IAV prophylactics among compounds with NA activities too weak to emerge from traditional drug screens.
The development of new antimalarial compounds remains a pivotal part of the strategy for malaria elimination. Recent large-scale phenotypic screens have provided a wealth of potential starting points for hit-to-lead campaigns. One such public set is explored, employing an open source research mechanism in which all data and ideas were shared in real time, anyone was able to participate, and patents were not sought. One chemical subseries was found to exhibit oral activity but contained a labile ester that could not be replaced without loss of activity, and the original hit exhibited remarkable sensitivity to minor structural change. A second subseries displayed high potency, including activity within gametocyte and liver stage assays, but at the cost of low solubility. As an open source research project, unexplored avenues are clearly identified and may be explored further by the community; new findings may be cumulatively added to the present work.
Mesangiogenic Progenitor Cells (MPCs) are human bone marrow-derived multipotent cells, isolated in vitro under selective culture conditions and shown to retain both mesengenic and angiogenic potential. MPCs also co-isolated with multipotent stromal cells (MSCs) when bone marrow primary cultures were set up for clinical applications, using human serum (HS) in place of fetal bovine serum (FBS). MPC culture purity (over 95%) is strictly dependent on HS supplementation with significant batch-to-batch variability. In the present paper we screened different sources of commercially available pooled human AB type serum (PhABS) for their ability to promote MPC production under selective culture conditions. As the majority of "contaminating" cells in MPC cultures were represented by MSC-like cells, we hypothesized a role by differentiating agents present in the sera. Therefore, we tested a number of growth factors (hGF) and found that higher concentrations of FGF-2, EGF, PDGF-AB, and VEGF-A as well as lower concentration of IGF-1 give sub-optimal MPC recovery. Gene expression analysis of hGF receptors was also carried out both in MSCs and MPCs, suggesting that FGF-2, EGF, and PDGF-AB could act promoting MSC proliferation, while VEGF-A contribute to MSC-like cell contamination, triggering MPC differentiation. Here we demonstrated that managing hGF contents, together with applying specific receptors inhibitors (Erlotinib-HCl and Nintedanib), could significantly mitigate the batch-to-batch variability related to serum supplementation. These data represent a fundamental milestone in view of manufacturing MPC-based medicinal products.
Transmuscular migration of the encircling band through rectus muscles and straddling of the cornea has only been reported in a few cases previously in the literature. This rare condition has never been associated with glaucoma. In this report, we aimed to describe a unique case with transmuscular migration of encircling buckle as a probable cause of glaucoma. A 17-year-old female presented with transmuscular migration of buckle and high intraocular pressure (IOP). Limbal/corneal migration of the silicone band was thought to be the main reason for the IOP rise; therefore, scleral band removal was performed. One month after removal, the patient was free of glaucoma medications and IOP was within normal limits. The retina remained attached during all postoperative visits. Transmuscular migration of the encircling band through rectus muscles and straddling of the cornea may act as a trigger for glaucoma.
Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds.
In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin.
In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay.
The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica.
Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture.
Aseptic loosening is the main long-term complication and indicates total hip replacement failure. There are many and often intricate causes for aseptic loosening. Incorrect placement of the implants is the most incriminated factor. Other patient and implant related factors may also predispose to loosening but to various degrees.
We conducted a retrospective study of 64 cases with aseptic loosening of total hip prosthesis to individuate patient, implant type and surgical technique related factors leading to aseptic loosening and to provide recommendations to minimize this risk.
This was an analytic retrospective study of 64 cases with aseptic loosening. The classification is that used by the French Society of Orthopedic Surgery and Traumatology. The average age of the patients at the time of the first arthroplasty was 40 years, 62 years at the time of loosening. Charnley stem was implanted in 55 cases, Muller stem in 9 cases. Acetabular component was well positioned in 69% of cases with an average inclination of 47.8 °. The stems were canal-filling in 86% of cases with Grade A cementation in 60% of cases. The mean time for loosening onset was 12 years. 72% of the prostheses had a survival longer than 10 years. Statistical analysis of the results identified risk factors leading to loosening, such as: age, body mass index, level of activity, cup inclination, femoral offset and cementation quality.
A significant reduction in aseptic loosening of total hip prostheses may only be achieved by a more rigorous selection of patients, a greater security in the technical act and a better choice of the implant to be placed.
The national incidence of adverse events (AEs) in Swedish orthopedic care has never been described. A new national database has made it possible to describe incidence, nature, preventability and consequences of AEs in Swedish orthopedic care.
We used national data from a structured two-stage record review with a Swedish modification of the Global Trigger Tool. The sample was 4,994 randomly selected orthopedic admissions in 56 hospitals during 2013 and 2014. The AEs were classified according to the Swedish Patient Safety Act into preventable or non-preventable.
At least one AE occurred in 733 (15 %, 95 % CI 13.7-15.7) admissions. Of 950 identified AEs, 697 (73 %) were judged preventable. More than half of the AEs (54 %) were of temporary nature. The most common types of AE were healthcare-associated infections and distended urinary bladder. Patients ≥65 years had more AEs (p < 0.001), and were more often affected by pressure ulcer (p < 0.001) and urinary tract infections (p < 0.01). Distended urinary bladder was seen more frequently in patients aged 18-64 years (p = 0.01). Length of stay was twice as long for patients with AEs (p < 0.001). We estimate 232,000 extra hospital days due to AEs during these 2 years. The pattern of AEs in orthopedic care was different compared to other hospital specialties.
Using a national database, we found AEs in 15 % of orthopedic admissions. The majority of the AEs was of temporary nature and judged preventable. Our results can be used to guide focused patient safety work.
The esthetic appreciation of music is strongly influenced by cultural background and personal taste. One would expect that this would complicate the utilizability of musical feedback in paradigms, such that music would only be perceived as a reward if it complies to personal esthetic appreciation. Here we report data where we assessed esthetic appreciation of music after 1. a physically strenuous music improvisation and 2. after passive music listening (where participants esthetically assessed similar music). Data are reported from two experiments with different patient groups: 1. Drug abuse patients, and 2. Chronic pain patients. Participants in both experiments performed Jymmin, a music feedback method where exercise equipment is modified in such a way that it can be played like musical instruments by modulating musical parameters in a composition software. This combines physical exertion with musical performance in a fashion that has previously been shown to have a number of positive psychological effects such as enhanced mood and reduced perceived exertion. In both experiments esthetic appreciation of musical presentations during Jymmin and a control condition without musical agency were compared. Data show that both patient groups perceived the musical outcome of their own performance as more esthetically pleasing than similar music they listened to passively. This suggests that the act of making music (when combined with physical exertion) is associated with a positivity bias about the perceived esthetical quality of the musical outcome. The outcome of personal musical agency thus tends to be perceived as rewarding even if it does not comply with personal esthetic appreciation. This suggests that musical feedback interventions may not always have to be highly individualized because individual taste may not always be crucial. The results also suggest that the method applied here may be efficient at encouraging music listeners to actively explore new musical styles that they might otherwise be reluctant to listen to (e.g., avant-garde music). The results also hint toward a deeper understanding of why musicians, who exert themselves physically during musical performances to generate music and regardless of the type of music they are playing, typically find the physically demanding experience esthetically satisfying.
It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.
Malaria-endemic countries have implemented community health worker (CHW) programs to provide malaria diagnosis and treatment to populations living beyond the reach of health systems. However, there is limited evidence describing the referral practices of CHWs. We examined the impact of malaria rapid diagnostic tests (mRDTs) on CHW referral in two cluster-randomized trials, one conducted in a moderate-to-high malaria transmission setting and one in a low-transmission setting in Uganda, between January 2010 and July 2012. All CHWs were trained to prescribe artemisinin-based combination therapy (ACT) for malaria and recognize signs and symptoms for referral to health centers. CHWs in the control arm used a presumptive diagnosis for malaria based on clinical symptoms, whereas intervention arm CHWs used mRDTs. CHWs recorded ACT prescriptions, mRDT results, and referral in patient registers. An intention-to-treat analysis was undertaken using multivariable logistic regression. Referral was more frequent in the intervention arm versus the control arm (moderate-to-high transmission, P < 0.001; low transmission, P < 0.001). Despite this increase, referral advice was not always given when ACTs or prereferral rectal artesunate were prescribed: 14% prescribed rectal artesunate in the moderate-to-high setting were not referred. In addition, CHWs considered factors alongside mRDTs when referring. Child visits during the weekends or the rainy season were less likely to be referred, whereas visits to CHWs more distant from health centers were more likely to be referred (low transmission only). CHWs using mRDTs and ACTs increased referral compared with CHWs using a presumptive diagnosis. To address these concerns, referral training should be emphasized in CHW programs as they are scaled-up.
Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.
Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisome protein docking complex PEX13-PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin-proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.
Conventional calculations of the global carbon budget infer the land sink as a residual between emissions, atmospheric accumulation, and the ocean sink. Thus, the land sink accumulates the errors from the other flux terms and bears the largest uncertainty. Here, we present a Bayesian fusion approach that combines multiple observations in different carbon reservoirs to optimize the land (B) and ocean (O) carbon sinks, land use change emissions (L), and indirectly fossil fuel emissions (F) from 1980 to 2014. Compared with the conventional approach, Bayesian optimization decreases the uncertainties in B by 41% and in O by 46%. The L uncertainty decreases by 47%, whereas F uncertainty is marginally improved through the knowledge of natural fluxes. Both ocean and net land uptake (B + L) rates have positive trends of 29 ± 8 and 37 ± 17 Tg C⋅y(-2) since 1980, respectively. Our Bayesian fusion of multiple observations reduces uncertainties, thereby allowing us to isolate important variability in global carbon cycle processes.
The American Society for Cell Biology Women in Cell Biology Sandra Masur Senior Award recognizes leadership in scientific accomplishments and in mentoring, which are intertwined. My development as a scientist reflects important mentors in my life, including my father and Joe Gall, who is my "Doktor Vater." In turn, as an established investigator, my scientific successes in researching 1) chromosomes, their replication and genomics, and 2) ribosomes, their structure, evolution, and biogenesis, reflects the hard work of my students and postdocs, for whom I act as a mentor, guiding them in their research and along their career paths.
The androgen receptor (AR) signaling axis drives all stages of prostate cancer, including the lethal, drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC), which arises after failure of androgen deprivation therapy (ADT). Persistent AR activity in spite of ADT and the second-generation AR-targeting agents enzalutamide and abiraterone is achieved in many cases by direct alterations to the AR signaling axis. Herein, we provide a detailed description of how such alterations contribute to the development and progression of CRPC. Aspects of this broad and ever-evolving field specifically addressed in this review include: the etiology and significance of increased AR expression; the frequency and role of gain-of-function mutations in the AR gene; the function of constitutively active, truncated forms of the AR termed AR variants and the clinical relevance of alterations to the activity and expression of AR coregulators. Additionally, we examine the novel therapeutic strategies to inhibit these classes of therapy resistance mechanisms, with an emphasis on emerging agents that act in a manner distinct from the current ligand-centric approaches. Throughout, we discuss how the central role of AR in prostate cancer and the constant evolution of the AR signaling axis during disease progression represent archetypes of two key concepts in oncology, oncogene addiction and therapy-mediated selection pressure.
Nanoemulsion has the potential to overcome several disadvantages in drug formulation. Loading poor water-soluble drugs in the appropriate nanoemulsions enhances their wettability and/or solubility. Consequently, this improves their pharmacokinetics and pharmacodynamics by different routes of administration. Associated with the optimum nanodroplets size or even combined with key components, the droplets act as a reservoir of drugs, enabling nanoemulsion to be multifunctional platform to treat diverse diseases. A number of important advantages, which comprise nanoemulsion attributes, such as efficient drug release with appropriate rate, prolonged efficacy, drug uptake control, low side effects and drug protection properties from enzymatic or oxidative processes, have been reported in last decade. The high flexibility of nanoemulsion includes also a variety of manufacturing process options and a combination of widely assorted components such as surfactants, liquid lipids or even drug-conjugates. These features provide alternatives for designing innovative nanoemulsions aiming at high-value applications. This review presents the challenges and prospects of different nanoemulsion types and its application. The drug interaction with the components of the formulation, as well as the drug mechanistic interaction with the biological environment of different routes of administration are also presented.
The role of photoperiod sensitivity (PS) of flowering genes have become well recognized in rice, whereas little attention has been drawn to the non-PS component of these genes, especially to their influence on gene-by-gene interactions. Rice populations in which the photoperiod-sensitive allele at Hd1 has become insensitive to photoperiod but continued to affect heading date (HD) were used in this study to fine-map a quantitative trait locus (QTL) for HD and analyze its genetic relationship to Hd1 The QTL was delimitated to a 96.3-kb region on the distal end of the long arm of chromosome 7. Sequence comparison revealed that this QTL is identical to Hd2 In the near-isogenic line (NIL) populations analyzed, Hd1 and Hd2 were shown to be photoperiod insensitive and have pleiotropic effects for HD, plant height and yield traits. The two genes were found to largely act additively in regulating HD and yield traits. The results indicate that non-PS components of flowering genes involved in photoperiod response play an important role in controlling flowering time and grain yield in rice, which should allow breeders to better manipulate pleiotropic genes for balancing adaptability and high-yielding accumulation.
Strigolactones are a recently identified class of hormone that regulate multiple aspects of plant development. The DWARF14 (D14) α/β fold protein has been identified as a strigolactone receptor, which can act through the SCF(MAX2) ubiquitin ligase, but the universality of this mechanism is not clear. Multiple proteins have been suggested as targets for strigolactone signalling, including both direct proteolytic targets of SCF(MAX2), and downstream targets. However, the relevance and importance of these proteins to strigolactone signalling in many cases has not been fully established. Here we assess the contribution of these targets to strigolactone signalling in adult shoot developmental responses. We find that all examined strigolactone responses are regulated by SCF(MAX2) and D14, and not by other D14-like proteins. We further show that all examined strigolactone responses likely depend on degradation of SMXL proteins in the SMXL6 clade, and not on the other proposed proteolytic targets BES1 or DELLAs. Taken together, our results suggest that in the adult shoot, the dominant mode of strigolactone signalling is D14-initiated, MAX2-mediated degradation of SMXL6-related proteins. We confirm that the BRANCHED1 transcription factor and the PIN-FORMED1 auxin efflux carrier are plausible downstream targets of this pathway in the regulation of shoot branching, and show that BRC1 likely acts in parallel to PIN1.
Due to its unique hierarchical structure, natural spider silk features exceptional mechanical properties such as high tensile strength and great extensibility, making it one of the toughest materials. Herein, we design bioinspired spider silk single-walled carbon nanotubes (BISS-SWCNTs) that combine the hierarchical structure of spider silk and the high strength and conductivity of SWCNTs. To imitate the hierarchical structure, Fe nanoparticles are embedded on the surface of directly synthesized SWCNTs skeleton followed by coating an amorphous carbon layer. The carbon layer forms the spider silk-featured skin-core structure with SWCNTs, thus making the tube junction tougher. The embedded Fe nanoparticles act as glue spots for preventing interfacial slippages between the BISS-SWCNTs and the reinforced matrix. With only 2.1 wt % BISS-SWCNTs added, the tensile strength and Young's modulus of the BISS-SWCNTs/PMMA composites can be improved by 300%. More importantly, the BISS-SWCNTs also retain the high conductivity and transmittance of the pristine SWCNTs film. This unique bioinspired material will be of great importance in applications of multifunctional composite materials and has important implications for the future of biomimetic materials.
Toroidal structures based on self-assembly of predesigned building blocks are well-established in the literature, but spontaneous self-organization to prepare such structures has not been reported to date. Here, organic-inorganic hybrid microtoroids synthesized by simultaneous coordination-driven assembly of amphiphilic molecules and hydrophilic polymers are reported. Mixing amphiphilic molecules with iron(III) chloride and hydrophilic polymers in water leads, within minutes, to the formation of starlike nanostructures. A spontaneous self-organization of these nanostructures is then triggered to form stable hybrid microtoroids. Interestingly, the toroids exhibit anisotropic hierarchical growth, giving rise to a layered toroidal framework. These microstructures are mechanically robust and can act as templates to host metallic nanoparticles such as gold and silver. Understanding the nature of spontaneous assembly driven by coordination multiple non-covalent interactions can help explain the well-ordered complexity of many biological organisms in addition to expanding the available tools to mimic such structures at a molecular level.
ZnO is a prime candidate for future use in transparent electronics; however, development of practical materials requires attention to factors including control of its unusual surface band bending and surface reactivity. In this work, we have modified the O-polar (0001̅), Zn-polar (0001), and m-plane (101̅0) surfaces of ZnO with phosphonic acid (PA) derivatives and measured the effect on the surface band bending and surface sensitivity to atmospheric oxygen. Core level and valence band synchrotron X-ray photoemission spectroscopy was used to measure the surface band bending introduced by PA modifiers with substituents of opposite polarity dipole moment: octadecylphosphonic acid (ODPA) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonic acid (F13OPA). Both PAs act as surface electron donors, increasing the downward band bending and the strength of the two-dimensional surface electron accumulation layer on all of the ZnO surfaces investigated. On the O-polar (0001̅) and m-plane (101̅0) surfaces, the ODPA modifier produced the largest increase in downward band bending relative to the hydroxyl-terminated unmodified surface of 0.55 and 0.35 eV, respectively. On the Zn-polar (0001) face, the F13OPA modifier gave the largest increase (by 0.50 eV) producing a total downward band bending of 1.00 eV, representing ∼30% of the ZnO band gap. Ultraviolet (UV) photoinduced surface wettability and photoconductivity measurements demonstrated that the PA modifiers are effective at decreasing the sensitivity of the surface toward atmospheric oxygen. Modification with PA derivatives produced a large increase in the persistence of UV-induced photoconductivity and a large reduction in UV-induced changes in surface wettability.
Check valves are often essential components in microfluidic devices, enabling automated sample processing for diagnostics at the point of care. However, there is an unmet need for a check valve design that is compatible with rigid thermoplastic devices during all stages of development-from initial prototyping with a laser cutter to final production with injection molding. Here, we present simple designs for a passive, normally closed check valve that is manufactured from commonly available materials with a CO2 laser and readily integrated into prototype and production thermoplastic devices. The check valve consists of a thermoplastic planar spring and a soft elastomeric pad that act together to seal against fluid backflow. The valve's cracking pressure can be tuned by modifying the spring's planar geometry and thickness. Seal integrity is improved with the addition of a raised annular boss beneath the elastomeric pad. To demonstrate the valve's usefulness, we employ these valves to create a finger-operated on-chip reagent reservoir and a finger-actuated pneumatic pump. We also apply this check valve to passively seal a device to enable portable detection of RNA from West Nile virus in a laser-cut device.
Registry-based clinical research in nephrolithiasis is critical to advancing quality in urinary stone disease management and ultimately reducing stone recurrence. A need exists to develop Health Insurance Portability and Accountability Act (HIPAA)-compliant registries that comprise integrated electronic health record (EHR) data using prospectively defined variables. An EHR-based standardized patient database-the Registry for Stones of the Kidney and Ureter (ReSKU™)-was developed, and herein we describe our implementation outcomes.
Interviews with academic and community endourologists in the United States, Canada, China, and Japan identified demographic, intraoperative, and perioperative variables to populate our registry. Variables were incorporated into a HIPAA-compliant Research Electronic Data Capture database linked to text prompts and registration data within the Epic EHR platform. Specific data collection instruments supporting New patient, Surgery, Postoperative, and Follow-up clinical encounters were created within Epic to facilitate automated data extraction into ReSKU.
The number of variables within each instrument includes the following: New patient-60, Surgery-80, Postoperative-64, and Follow-up-64. With manual data entry, the mean times to complete each of the clinic-based instruments were (minutes) as follows: New patient-12.06 ± 2.30, Postoperative-7.18 ± 1.02, and Follow-up-8.10 ± 0.58. These times were significantly reduced with the use of ReSKU structured clinic note templates to the following: New patient-4.09 ± 1.73, Postoperative-1.41 ± 0.41, and Follow-up-0.79 ± 0.38. With automated data extraction from Epic, manual entry is obviated.
ReSKU is a longitudinal prospective nephrolithiasis registry that integrates EHR data, lowering the barriers to performing high quality clinical research and quality outcome assessments in urinary stone disease.
Porous silicon (pSi) substrates are a promising platform for cell expansion, since pore size and chemistry can be tuned to control cell behavior. In addition, a variety of bioactives can be loaded into the pores and subsequently released to act on cells adherent to the substrate. Here, we construct a cell microarray on a plasma polymer coated pSi substrate that enables the simultaneous culture of human endothelial cells on printed immobilized protein factors, while a second soluble growth factor is released from the same substrate. This allows three elements of candidate pSi scaffold materials-topography, surface functionalization, and controlled factor release-to be assessed simultaneously in high throughput. We show that protein conjugation within printed microarray spots is more uniform on the pSi substrate than on flat glass or silicon surfaces. Active growth factors are released from the pSi surface over a period of several days. Using an endothelial progenitor cell line, we investigate changes in cell behavior in response to the microenvironment. This platform facilitates the design of advanced functional biomaterials, including scaffolds, and carriers for regenerative medicine and cell therapy.
Cyclotrimerization of alkynes catalyzed by transition metal complexes is a straightforward synthetic method for constructing a benzene skeleton in organic synthesis. Not only mononuclear complexes, but also multinuclear complexes act as catalysts for alkyne cyclotrimerization, and their reaction mechanisms have been intensively investigated toward developing highly efficient and regio- and chemo-selective catalysts. In this review, we summarize stoichiometric and catalytic alkyne coupling reactions on mononuclear and dinuclear scaffolds in relation to the reaction mechanism of alkyne cyclotrimerization, including our recent mechanistic approaches using dinuclear tantalum motifs.
Micro unmanned underwater vehicles (UUVs) need to house propulsion mechanisms that are small in size but sufficiently powerful to deliver on-demand acceleration for tight radius turns, burst-driven docking maneuvers, and low-speed course corrections. Recently, small-scale hydrogen peroxide (H2O2) propulsion mechanisms have shown great promise in delivering pulsatile thrust for such acceleration needs. However, the need for robust, high surface area nanocatalysts that can be manufactured on a large scale for integration into micro UUV reaction chambers is still needed. In this report, a thermal/electrical insulator, silicon oxide (SiO2) microfibers, is used as a support for platinum nanoparticle (PtNP) catalysts. The mercapto-silanization of the SiO2 microfibers enables strong covalent attachment with PtNPs, and the resultant PtNP-SiO2 fibers act as a robust, high surface area catalyst for H2O2 decomposition. The PtNP-SiO2 catalysts are fitted inside a micro UUV reaction chamber for vehicular propulsion; the catalysts can propel a micro UUV for 5.9 m at a velocity of 1.18 m/s with 50 mL of 50% (w/w) H2O2. The concomitance of facile fabrication, economic and scalable processing, and high performance-including a reduction in H2O2 decomposition activation energy of 40-50% over conventional material catalysts-paves the way for using these nanostructured microfibers in modern, small-scale underwater vehicle propulsion systems.
Novel magnetic biochars (MBC) were prepared by one-step pyrolysis of FeCl3-laden biomass and employed for Hg(0) removal in simulated combustion flue gas. The sample characterization indicated that highly dispersed Fe3O4 particles could be deposited on the MBC surface. Both enhanced surface area and excellent magnetization property were obtained. With the activation of FeCl3, more oxygen-rich functional groups were formed on the MBC, especially the C═O group. The MBC exhibited far greater Hg(0) removal performance compared to the nonmagnetic biochar (NMBC) under N2 + 4% O2 atmosphere in a wide reaction temperature window (120-250 °C). The optimal pyrolysis temperature for the preparation of MBC is 600 °C, and the best FeCl3/biomass impregnation mass ratio is 1.5 g/g. At the optimal temperature (120 °C), the Fe1.5MBC600 was superior in both Hg(0) adsorption capacity and adsorption rate to a commercial brominated activated carbon (Br-AC) used for mercury removal in power plants. The mechanism of Hg(0) removal was proposed, and there are two types of active adsorption/oxidation sites for Hg(0): Fe3O4 and oxygen-rich functional groups. The role of Fe3O4 in Hg(0) removal was attributed to the Fe(3+)(t) coordination and lattice oxygen. The C═O group could act as act as electron acceptors, facilitating the electron transfer for Hg(0) oxidation.
Trichinellosis is one of the most widespread parasitic zoonoses. Trichinella Owen, 1835 nematodes are found in pigs, horses, and humans in the domestic cycle, and in many carnivores and omnivores in the sylvatic cycle, such as wild boars, red foxes, raccoon dogs, and wolves. Carnivores are known to be involved in the circulation of Trichinella nematodes and they act as a reservoir in the sylvatic environment. The aim of this study was to determine the occurrence of Trichinella spp. infection in red foxes in Poland. Samples were collected from 2010 to 2015 in different regions of the country and then tested for Trichinella nematodes using HCl-pepsin digestion. Trichinella larvae were found in 10.02% of examined samples (145/1447). The larvae were identified as T. spiralis (11.03%), T. britovi (71.72%), and T. pseudospiralis (0.69%). No mixed infection was observed. The prevalence of infection varied between years and different voivodeships of the country. Our findings confirm that red foxes are involved in the maintenance of Trichinella spp. in the sylvatic cycle in Poland.
Nuclear receptors (NRs) are master regulators of broad genetic programs in metazoans. These programs are regulated in part by the small-molecule ligands that bind NRs and modulate their interactions with transcriptional coregulatory factors. X-ray crystallography is now delivering more complete pictures of how the multidomain architectures of NR homo- and heterodimers are physically arranged on their DNA elements and how ligands and coactivator peptides act through these complexes. Complementary studies are also pointing to a variety of novel mechanisms by which NRs access their DNA-response elements within chromatin. Here, we review the new structural advances together with proteomic discoveries that shape our understanding of how NRs form a variety of functional interactions with collaborating factors in chromatin.
Prolonged mechanical ventilation is associated with a longer intensive care unit (ICU) length of stay and higher mortality. Consequently, methods to improve ventilator weaning processes have been sought. Two recent Cochrane systematic reviews in ICU adult and paediatric populations concluded that protocols can be effective in reducing the duration of mechanical ventilation, but there was significant heterogeneity in study findings. Growing awareness of the benefits of understanding the contextual factors impacting on effectiveness has encouraged the integration of qualitative evidence syntheses with effectiveness reviews, which has delivered important insights into the reasons underpinning (differential) effectiveness of healthcare interventions.
1. To locate, appraise and synthesize qualitative evidence concerning the barriers and facilitators of the use of protocols for weaning critically-ill adults and children from mechanical ventilation;2. To integrate this synthesis with two Cochrane effectiveness reviews of protocolized weaning to help explain observed heterogeneity by identifying contextual factors that impact on the use of protocols for weaning critically-ill adults and children from mechanical ventilation;3. To use the integrated body of evidence to suggest the circumstances in which weaning protocols are most likely to be used.
We used a range of search terms identified with the help of the SPICE (Setting, Perspective, Intervention, Comparison, Evaluation) mnemonic. Where available, we used appropriate methodological filters for specific databases. We searched the following databases: Ovid MEDLINE, Embase, OVID, PsycINFO, CINAHL Plus, EBSCOHost, Web of Science Core Collection, ASSIA, IBSS, Sociological Abstracts, ProQuest and LILACS on the 26th February 2015. In addition, we searched: the grey literature; the websites of professional associations for relevant publications; and the reference lists of all publications reviewed. We also contacted authors of the trials included in the effectiveness reviews as well as of studies (potentially) included in the qualitative synthesis, conducted citation searches of the publications reporting these studies, and contacted content experts.We reran the search on 3rd July 2016 and found three studies, which are awaiting classification.
We included qualitative studies that described: the circumstances in which protocols are designed, implemented or used, or both, and the views and experiences of healthcare professionals either involved in the design, implementation or use of weaning protocols or involved in the weaning of critically-ill adults and children from mechanical ventilation not using protocols. We included studies that: reflected on any aspect of the use of protocols, explored contextual factors relevant to the development, implementation or use of weaning protocols, and reported contextual phenomena and outcomes identified as relevant to the effectiveness of protocolized weaning from mechanical ventilation.
At each stage, two review authors undertook designated tasks, with the results shared amongst the wider team for discussion and final development. We independently reviewed all retrieved titles, abstracts and full papers for inclusion, and independently extracted selected data from included studies. We used the findings of the included studies to develop a new set of analytic themes focused on the barriers and facilitators to the use of protocols, and further refined them to produce a set of summary statements. We used the Confidence in the Evidence from Reviews of Qualitative Research (CERQual) framework to arrive at a final assessment of the overall confidence of the evidence used in the synthesis. We included all studies but undertook two sensitivity analyses to determine how the removal of certain bodies of evidence impacted on the content and confidence of the synthesis. We deployed a logic model to integrate the findings of the qualitative evidence synthesis with those of the Cochrane effectiveness reviews.
We included 11 studies in our synthesis, involving 267 participants (one study did not report the number of participants). Five more studies are awaiting classification and will be dealt with when we update the review.The quality of the evidence was mixed; of the 35 summary statements, we assessed 17 as 'low', 13 as 'moderate' and five as 'high' confidence. Our synthesis produced nine analytical themes, which report potential barriers and facilitators to the use of protocols. The themes are: the need for continual staff training and development; clinical experience as this promotes felt and perceived competence and confidence to wean; the vulnerability of weaning to disparate interprofessional working; an understanding of protocols as militating against a necessary proactivity in clinical practice; perceived nursing scope of practice and professional risk; ICU structure and processes of care; the ability of protocols to act as a prompt for shared care and consistency in weaning practice; maximizing the use of protocols through visibility and ease of implementation; and the ability of protocols to act as a framework for communication with parents.
There is a clear need for weaning protocols to take account of the social and cultural environment in which they are to be implemented. Irrespective of its inherent strengths, a protocol will not be used if it does not accommodate these complexities. In terms of protocol development, comprehensive interprofessional input will help to ensure broad-based understanding and a sense of 'ownership'. In terms of implementation, all relevant ICU staff will benefit from general weaning as well as protocol-specific training; not only will this help secure a relevant clinical knowledge base and operational understanding, but will also demonstrate to others that this knowledge and understanding is in place. In order to maximize relevance and acceptability, protocols should be designed with the patient profile and requirements of the target ICU in mind. Predictably, an under-resourced ICU will impact adversely on protocol implementation, as staff will prioritize management of acutely deteriorating and critically-ill patients.
Surface nanobubbles, which are nanoscopic or microscopic gaseous domains forming at the solid/liquid interface, have a strong impact on the interface by changing the two-phase contact to a three-phase contact. Therefore, they are believed to affect the boundary condition and liquid flow. However, there are still disputes in the theoretical studies as to whether the nanobubbles can increase the slip length effectively. Furthermore, there are still no direct experimental studies to support either side. Therefore, an intensive study on the effective slip length for flows over bare surfaces with nanobubbles is essential for establishing the relation between nanobubbles and slip length. Here, we study the effect of nanobubbles on the slippage experimentally and theoretically. Our experimental results reveal an increase from 8 to 512 nm in slip length by increasing the surface coverage of nanobubbles from 1.7 to 50.8% and by decreasing the contact angle of nanobubbles from 42.8 to 16.6°. This is in good agreement with theoretical results. Our results indicate that nanobubbles could always act as a lubricant and significantly increase the slip length. The surface coverage, height, and contact angle are key factors for nanobubbles to reduce wall friction.
Androgens are essential for male development and reproductive function. They are transported to their site of action as blood-borne endocrine hormones but can also be produced within tissues to act in intracrine and paracrine fashions. Because of this, circulating concentrations may not accurately reflect the androgenic influence within specific tissue microenvironments. Mass spectrometry imaging permits regional analysis of small molecular species directly from tissue surfaces. However, due to poor ionization and localized ion suppression, steroid hormones are difficult to detect. Here, derivatization with Girard T reagent was used to charge-tag testosterone and 5α-dihydrotestosterone allowing direct detection of these steroids in mouse testes, in both basal and maximally stimulated states, and in rat prostate. Limits of detection were ∼0.1 pg for testosterone. Exemplary detection of endogenous steroids was achieved by matrix-assisted laser desorption ionization and either Fourier transform ion cyclotron resonance detection (at 150 μm spatial resolution) or quadrupole-time-of-flight detection (at 50 μm spatial resolution). Structural confirmation was achieved by collision induced fragmentation following liquid extraction surface analysis and electrospray ionization. This application broadens the scope for derivatization strategies on tissue surfaces to elucidate local endocrine signaling in health and disease.
What is the central question of this study? Do group III and IV muscle afferents act at the spinal or cortical level to affect the ability of the central nervous system to drive quadriceps muscles during fatiguing exercise? What is the main finding and its importance? The excitability of the motoneurone pool of vastus lateralis was unchanged by feedback from group III and IV muscle afferents. In contrast, feedback from these afferents may contribute to inhibition at the cortex. However, the excitability of the corticospinal pathway was not directly affected by feedback from these afferents. These findings are important for understanding neural processes during fatiguing exercise. In upper limb muscles, changes in afferent feedback, motoneurone excitability, and motor cortical output can contribute to failure of the central nervous system to recruit muscles fully during fatigue. It is not known whether similar changes occur with fatigue of muscles in the lower limb. We assessed the corticospinal pathway to vastus lateralis during fatiguing sustained maximal voluntary contractions (MVCs) of the knee extensors and during firing of fatigue-sensitive group III/IV muscle afferents maintained by postexercise ischaemia after fatiguing MVCs of the knee extensors and, separately, the flexors. In two experiments, subjects (n = 9) performed brief knee extensor MVCs before and after 2-min sustained MVCs of the knee extensors (experiment 1) or knee flexors (experiment 2). During MVCs, motor evoked potentials (MEPs) were elicited by transcranial magnetic stimulation over the motor cortex and thoracic motor evoked potentials (TMEPs) by electrical stimulation over the thoracic spine. During the 2-min extensor contraction, the size of vastus lateralis MEPs normalized to the maximal M-wave increased (P < 0.05), but normalized TMEPs were unchanged (P = 0.16). After the 2-min MVC, maintained firing of group III/IV muscle afferents had no effect on vastus lateralis MEPs or TMEPs (P = 0.18 and P = 0.50, respectively). Likewise, after the 2-min knee flexor MVC, maintained firing of these afferents showed no effect on vastus lateralis MEPs or TMEPs (P = 0.69 and P = 0.34, respectively). Motoneurones of vastus lateralis do not become less excitable during fatiguing isometric MVCs. Moreover, fatigue-sensitive group III/IV muscle afferents fail to affect the overall excitability of vastus lateralis motoneurones during MVCs.
Yeasts have sophisticated signaling pathways for sensing glucose, their preferred carbon source, to regulate its uptake and metabolism. One of these is the sensor/receptor-repressor (SRR) pathway, which detects extracellular glucose and transmits an intracellular signal that induces expression of HXT genes. The yeast casein kinases (Ycks) are key players in this pathway. Our model of the SRR pathway had the Ycks functioning downstream of the glucose sensors, transmitting the signal from the sensors to the Mth1 and Std1 corepressors that are required for repression of HXT gene expression. However, we found that overexpression of Yck1 fails to restore glucose signaling in a glucose sensor mutant. Conversely, overexpression of a glucose sensor suppresses the signaling defect of a yck mutant. These results suggest that the Ycks act upstream or at the level of the glucose sensors. Indeed, we found that the glucose sensor Rgt2 is phosphorylated on Yck consensus sites in its C-terminal tail in a Yck-dependent manner and that this phosphorylation is required for corepressor binding and ultimately HXT expression. This leads to a revised model of the SRR pathway in which the Ycks prime a site on the cytoplasmic tails of the glucose sensors to promote binding of the corepressors.
The molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in immobilised limbs. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFβ promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development.
Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.
In 2002, the Belgian Act on Euthanasia came into effect, regulating the intentional ending of life by a physician at the patient's explicit request. We undertook this study to describe trends in officially reported euthanasia cases in Belgium with regard to patients' sociodemographic and clinical profiles, as well as decision-making and performance characteristics.
We used the database of all euthanasia cases reported to the Federal Control and Evaluation Committee on Euthanasia in Belgium between Jan. 1, 2003, and Dec. 31, 2013 (n = 8752). The committee collected these data with a standardized registration form. We analyzed trends in patient, decision-making and performance characteristics using a χ(2) technique. We also compared and analyzed trends for cases reported in Dutch and in French.
The number of reported euthanasia cases increased every year, from 235 (0.2% of all deaths) in 2003 to 1807 (1.7% of all deaths) in 2013. The rate of euthanasia increased significantly among those aged 80 years or older, those who died in a nursing home, those with a disease other than cancer and those not expected to die in the near future (p < 0.001 for all increases). Reported cases in 2013 most often concerned those with cancer (68.7%) and those under 80 years (65.0%). Palliative care teams were increasingly often consulted about euthanasia requests, beyond the legal requirements to do so (p < 0.001). Among cases reported in Dutch, the proportion in which the person was expected to die in the foreseeable future decreased from 93.9% in 2003 to 84.1% in 2013, and palliative care teams were increasingly consulted about the euthanasia request (from 34.0% in 2003 to 42.6% in 2013). These trends were not significant for cases reported in French.
Since legalization of euthanasia in Belgium, the number of reported cases has increased each year. Most of those receiving euthanasia were younger than 80 years and were dying of cancer. Given the increases observed among non-terminally ill and older patients, this analysis shows the importance of detailed monitoring of developments in euthanasia practice.
Studies about the psychosocial issues concerning organ donation and transplantation tend to focus on the experiences of donor or recipient families. Little is known about the part played by correspondence exchanged between these two groups; in particular how they perceive the agency of organ donation. This is the first analysis to address the representation of the act of donation from the viewpoint of both donor and recipient families through interrogation of archived correspondence data, using linguistic techniques. The data was drawn from a collection of letters, from four USA organ procurement organisations, exchanged between donor and transplant recipient families. Donor families consistently linguistically ascribed agency and accountability for donation to the person who died, the donor. For the recipient families, on the other hand, the 'giver' was mainly implied, ambiguous or ascribed to the donor family.
We follow the history of nanobubbles from the earliest experiments pointing to their existence to recent years. We cover the effect of Laplace pressure on the thermodynamic stability of nanobubbles and why this implies that nanobubbles are thermodynamically never stable. Therefore, understanding bubble stability becomes a consideration of the rate of bubble dissolution, so the dominant approach to understanding this is discussed. Bulk nanobubbles (or fine bubbles) are treated separately from surface nanobubbles as this reflects their separate histories. For each class of nanobubbles, we look at the early evidence for their existence, methods for the production and characterization of nanobubbles, evidence that they are indeed gaseous, or otherwise, and theories for their stability. We also look at applications of both surface and bulk nanobubbles.
Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. We recently identified Sequoia (Seq), a C2H2 zinc-finger transcription factor with homology to Drosophila Tramtrack (Ttk) and the positive regulatory domain (PRDM) family, as important for lineage progression. Here, we find that seq mutants fail to execute the type I>0 daughter proliferation switch and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.
The provision of gifts to new mothers in Uganda is laden with significance that varies by the social location of the giver and receiver and the context and conditions under which the gift is made available. Here, we examine the act of gift giving and receiving within a Ugandan maternity care setting, describing the connections between these material objects and social relations. A study investigating the social organisation of maternity care in post-conflict northern Uganda found that gift-giving to new mothers functioned to create a material and discursive context wherein women's desire to access these goods was leveraged to create an incentive to attend formal maternity care during pregnancy and for delivery. In this article we describe the material and discursive processes surrounding gift-giving to new mothers in this global South health care setting. This article contributes critical analyses of the function of gifts in healthcare settings as constructing shared identities, social differences and normative values about health citizenship, and an incentive politic that affects equitable access to maternity care. Drawing on intersectional theory and analysis of how specific practices function ideologically to reward or incentivise pregnant women, we integrate material culture studies into the sociology of women's reproductive health.
Graphene oxide (GO) is one of the most appealing bidimensional materials able to interact non-covalently with achiral molecules and to act as chiral inducers. Vortexes can tune chirality and, consequently transfer a specific handedness to non-covalent host molecules, either when dispersed in water or when deposited on a solid surface.
Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.
Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD(+)) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3'-cordycepin, 5'- and 3'-phosphate and also to 5'-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5'-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2'-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2',1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1' of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.
Sexual signalling is predicted to shape the evolution of sex-specific ornamentation, and establishing the costs and benefits of ornamentation and the information that ornamentation provides to receivers is necessary to evaluating this adaptive function. Here, we assessed the adaptive function of a common colour ornament in insects, melanin wing ornamentation, using the dragonfly Pachydiplax longipennis. We hypothesized that greater ornamentation would improve territory-holding success by decreasing aggression that males receive from territorial rivals, but that more ornamented males may have shorter lifespans. Using mark-recapture field observations, we found that more ornamented males had greater territory-holding success and that viability selection did not act on wing melanization. We then compared the aggression of territorial rivals to decoy males before and after experimentally augmenting wing melanization, finding that males significantly reduced aggression following the manipulation. We next hypothesized that wing melanization would signal fighting ability to territorial rivals by reflecting condition via investment in the costly melanin synthesis pathway. We observed a positive relationship between ornamentation and the likelihood of winning territorial disputes, suggesting that wing melanization provides information about fighting ability to rivals. We also found a positive relationship between melanin-based immune defence and ornamentation, supporting a link between the signal and condition. We conclude that wing melanization is a condition-related signal of fighting ability and suggest that this may be a common mechanism promoting the evolution of melanin ornamentation.
Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels.
Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time-lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly-elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non-contractile, migratory phenotype with phagocytic capacity that may act as a macrophage-like cell.
Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a 'synthetic' migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1 μm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling.
Ion-stabilized nanobubbles in bulk aqueous solutions of various electrolytes were investigated. To understand the ion-specific mechanism of nanobubble stabilization, an approach based on the Poisson--Boltzmann equation at the nanobubble interface and in the near-surface layer was developed. It has been shown that the stabilization of nanobubbles is realized by the adsorption of chaotropic anions at the interface, whereas the influence of cosmotropic cations is weak. With increasing temperature, it should be accounted for by blurring the interface due to thermal fluctuations. As a result, the adsorbed state of ions becomes unstable: the nanobubble loses its stability and vanishes. This prediction was proven in our experiments. It turned out that in the case of liquid samples being kept in hermetically sealed ampules, where the phase equilibrium at the liquid-gas interface is fulfilled for any temperature, the volume number density of nanobubbles decreases with increasing temperature and this decrease is irreversible.
Over 15 million babies are born prematurely each year with approximately 1 million of these babies dying as a direct result of preterm delivery. β2 -Adrenoreceptor agonists that act via cAMP can reduce uterine contractions to delay preterm labour, but their ability to repress uterine contractions lasts ≤ 48 h and their use does not improve neonatal outcomes. Previous research has suggested that cAMP inhibits myometrial contractions via protein kinase A (PKA) activation, but this has yet to be demonstrated with PKA-specific agonists. We investigated the role of PKA in mediating cAMP-induced human myometrial relaxation, and the impact of prolonged cAMP elevation on myometrial contractility. Our findings suggest that PKA is not the sole mediator of cAMP-induced myometrial relaxation and that prolonged prophylactic elevation of cAMP alone is unlikely to prevent preterm labour (PTL).
Acute cAMP elevation inhibits myometrial contractility, but the mechanisms responsible are not fully elucidated and the long-term effects are uncertain. Both need to be defined in pregnant human myometrium before the therapeutic potential of cAMP-elevating agents in the prevention of preterm labour can be realised. In the present study, we tested the hypotheses that PKA activity is necessary for cAMP-induced myometrial relaxation, and that prolonged cAMP elevation can prevent myometrial contractions. Myometrial tissues obtained from term, pre-labour elective Caesarean sections were exposed to receptor-independent cAMP agonists to determine the relationship between myometrial contractility (spontaneous and oxytocin-induced), PKA activity, HSP20 phosphorylation and expression of contraction-associated and cAMP signalling proteins. Acute (1 h) application of cAMP agonists promoted myometrial relaxation, but this was weakly related to PKA activation. A PKA-specific activator, 6-Bnz-cAMP, increased PKA activity (6.8 ± 2.0 mean fold versus vehicle; P = 0.0313) without inducing myometrial relaxation. Spontaneous myometrial contractility declined after 24 h but was less marked when tissues were constantly exposed to cAMP agonists, especially for 8-bromo-cAMP (4.3 ± 1.2 mean fold versus vehicle; P = 0.0043); this was associated with changes to calponin, cofilin and HSP20 phosphorylated/total protein levels. Oxytocin-induced contractions were unaffected by pre-incubation with cAMP agonists despite treatments being able to enhance PKA activity and HSP20 phosphorylation. These data suggest that cAMP-induced myometrial relaxation is not solely dependent on PKA activity and the ability of cAMP agonists to repress myometrial contractility is lost with prolonged exposure. We conclude that cAMP agonist treatment alone may not prevent preterm labour.
The clinical diagnosis and recovery of acute kidney injury (AKI) are mainly based on the rapid decline of glomerular filtration rate (GFR) and its subsequent recovery. The factors that determine kidney recovery and reduce the risk of subsequent progression to chronic kidney disease (CKD), however, are poorly understood. Thus, there is a need to better define the magnitude and time pattern of changes in kidney function during AKI and its recovery that go beyond GFR. Tubular transport regulates body homeostasis and the associated transport work is a primary determinant of the kidneys' energy needs. The tubular system is at the center of the pathophysiology of AKI and its recovery. In particular, proximal tubules and thick ascending limbs have been proposed to act as sensors, effectors and injury recipients of AKI stimuli. Surprisingly little attention has been given to aspects of tubular transport function in AKI and the relevance for kidney recovery. This review aims to outline changes in tubular transport function in AKI, discusses their potential consequences and relevance for the diagnosis and prognosis of AKI and its recovery, including changes in GFR, and poses the question whether tubular transport provides an opportunity for intervention to rest the tubular system, which may have consequences for the progression to CKD. © 2016 S. Karger AG, Basel.
The electrolysis of aqueous solutions produces solutions that are supersaturated in oxygen and hydrogen gas. This results in the formation of gas bubbles, including nanobubbles ∼100 nm in size that are stable for ∼24 h. These aqueous solutions containing bubbles have been evaluated for cleaning efficacy in the removal of model contaminants bovine serum albumin and lysozyme from surfaces and in the prevention of the fouling of surfaces by these same proteins. Hydrophilic and hydrophobic surfaces were investigated. It is shown that nanobubbles can prevent the fouling of surfaces and that they can also clean already fouled surfaces. It is also argued that in practical applications where cleaning is carried out rapidly using a high degree of mechanical agitation the role of cleaning agents is not primarily in assisting the removal of soil but in suspending the soil that is removed by mechanical action and preventing it from redepositing onto surfaces. This may also be the primary mode of action of nanobubbles during cleaning.
What is the topic of this review? The review discusses how in atherosclerotic plaques, a combination of inflammatory mediators together with loss of anti inflammatory factors is most likely to be responsible for the excess of MMP over TIMP expression that causes plaque rupture and myocardial infarction. What advances does it highlight? Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) is divergent between human and mouse macrophages. There is prostaglandin E2 -dependent and -independent regulation. Inflammatory cytokines act through distinct (albeit overlapping) signalling pathways to elicit different patterns of MMP and TIMP expression. Transcriptional and epigenetic regulation occurs. Matrix metalloproteinases (MMPs) produced from macrophages contribute to plaque rupture, atherothrombosis and myocardial infarction. New treatments could emerge from defining the mediators and underlying mechanisms. In human monocytes, prostaglandin E2 (PGE2 ) stimulates MMP production, and inflammatory mediators such as tumour necrosis factor α, interleukin-1 and Toll-like receptor ligands can act either through or independently of PGE2 . Differentiation of human monocytes to non-foamy macrophages increases constitutive expression of MMP-7, -8, -9, -14 and -19 and tissue inhibitor of MMP (TIMP)-1 to -3 through unknown, PGE2 -independent mechanisms. Human macrophages express more MMP-1, -7 and -9 and TIMP-3 and less MMP-12 and -13 than mouse macrophages. Inflammatory mediators working through activator protein-1 and nuclear factor-κB transcription factor pathways upregulate MMP-1, -3, -10, -12 and -14 in human macrophages (MMP-9, -12 and -13 in mice), and studies with plaque tissue sections and isolated foam cells confirm this conclusion in vivo. Classical activation with granulocyte-macrophage colony-stimulating factor upregulates MMP-12, whereas interferon-γ upregulates MMP-12, -14 and -25 and downregulates TIMP-3 in human but not mouse macrophages. Alternative activation with interleukin-4 markedly stimulates the expression of only MMP-12 in humans and MMP-19 in mice. The anti-inflammatory cytokines interleukin-10 and transforming growth factor-β decrease production of several MMPs. Epigenetic upregulation of MMP-14 during foam cell formation or by granulocyte-macrophage colony-stimulating factor occurs by decreasing miRNA-24. A 'perfect storm' caused by a combination of these mechanisms is most likely to promote MMP-mediated macrophage invasion, tissue destruction and atherosclerotic plaque rupture.
Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration.
Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen-chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum.
MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples.
These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.
Since passage of the Affordable Care Act (ACA) in 2010, US stakeholders are increasingly being held accountable for the value of healthcare services and drugs administered to patients. Pharmacoeconomic analyses offer one method of demonstrating a product's value, yet there is a lack of resources specific to US drug costs relevant to each stakeholder. The aim of this study was to review current US drug costs (post-ACA). A literature review aimed at finding evidence on outpatient prescription drug costs was performed using the following sources: PubMed, governmental agencies, news websites, the Academy of Managed Care Pharmacy (AMCP) website, and Google Scholar. Articles were limited to those published in the years "2010-2016" and the "English" language, and those that described drug acquisition costs, reimbursement costs, and rebates or discounting for Medicare, Medicaid, and commercial payors. The Drug Cost Focus Group (DCFG) was convened to supplement the literature review; the DCFG provided their expertise on US drug costs and emerging issues affecting drug costs. ACA legislation increased drug rebates for manufacturers participating in the Medicaid Drug Rebate Program. Acquisition costs commonly referred to in the literature include the wholesale acquisition cost and average manufacture price. Drugs reimbursed by Medicaid are currently based on the actual acquisition cost and ACA-Federal Upper Limit. Evidence suggests that reimbursement methods in the public market are varied. Current gaps in the literature regarding commercial insurers' drug costs (post-ACA) present barriers to the application of relevant drug costs to pharmacoeconomic analyses.
The shortage of primary care providers and the provisions of the Affordable Care Act (ACA) have spurred discussion about expanding the number, scope of practice (SOP), and independence of primary care nurse practitioners (NPs). Such discussions in the media and among professional organizations may insinuate that changes to the laws governing NP practice will engender acrimony between practicing physicians and NPs. However, we lack empirical, descriptive data on how practicing professionals view NP independence in primary care.
The aim of the present study was to explore and describe the attitudes about NP independence among physicians and NPs working in primary care.
A qualitative study based on the principles of grounded theory.
Thirty primary care professionals in Missouri, USA, including 15 primary care physicians and 15 primary care NPs.
Semi-structured, in-depth interviews, with data analysis guided by grounded theory.
Participants had perspectives that were not well represented by professional organizations or the media. Physicians were supportive of a wide variety of NP roles and comfortable with high levels of NP independence and autonomy. Physicians and NPs described prerequisites to NP independence that were complementary. Physicians generally believed that NPs needed some association with physicians for patient safety, and NPs preferred having a physician readily accessible as needed. The theme "knowing your limits" was important to both NPs and physicians regarding NP independence, and has not been described previously in the literature.
NP and physician views about NP practice in primary care are not as divergent as their representative professional organizations and the news media would suggest. The significant agreement among NPs and physicians, and some of the nuances of their perspectives, supports recommendations that may reduce the perceived acrimony surrounding discussions of NP independent practice in primary care.
Despite vast improvements that have been made in the treatment of children with acute lymphoblastic leukemia (ALL), the majority of infant ALL patients (~80 %, < 1 year of age) that carry a chromosomal translocation involving the mixed lineage leukemia (MLL) gene shows a poor response to chemotherapeutic drugs, especially glucocorticoids (GCs), which are essential components of all current treatment regimens. Although addressed in several studies, the mechanism(s) underlying this phenomenon have remained largely unknown. A major drawback of most previous studies is their primary focus on individual genes, thereby neglecting the putative significance of inter-gene correlations. Here, we aimed at studying GC resistance in MLL-rearranged infant ALL patients by inferring an associated module of genes using co-expression network analysis. The implications of newly identified candidate genes with associations to other well-known relevant genes from the same module, or with associations to known transcription factor or microRNA interactions, were substantiated using literature data.
A weighted gene co-expression network was constructed to identify gene modules associated with GC resistance in MLL-rearranged infant ALL patients. Significant gene ontology (GO) terms and signaling pathways enriched in relevant modules were used to provide guidance towards which module(s) consisted of promising candidates suitable for further analysis.
Through gene co-expression network analysis a novel set of genes (module) related to GC-resistance was identified. The presence in this module of the S100 and ANXA genes, both well-known biomarkers for GC resistance in MLL-rearranged infant ALL, supports its validity. Subsequent gene set net correlation analyses of the novel module provided further support for its validity by showing that the S100 and ANXA genes act as 'hub' genes with potentially major regulatory roles in GC sensitivity, but having lost this role in the GC resistant phenotype. The detected module implicates new genes as being candidates for further analysis through associations with known GC resistance-related genes.
From our data we conclude that available systems biology approaches can be employed to detect new candidate genes that may provide further insights into drug resistance of MLL-rearranged infant ALL cases. Such approaches complement conventional gene-wise approaches by taking putative functional interactions between genes into account.
Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm(-1) leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.
Fibroblastic reticular cells (FRCs) of secondary lymphoid organs form distinct niches for interaction with hematopoietic cells. We found here that production of the cytokine IL-15 by FRCs was essential for the maintenance of group 1 innate lymphoid cells (ILCs) in Peyer's patches and mesenteric lymph nodes. Moreover, FRC-specific ablation of the innate immunological sensing adaptor MyD88 unleashed IL-15 production by FRCs during infection with an enteropathogenic virus, which led to hyperactivation of group 1 ILCs and substantially altered the differentiation of helper T cells. Accelerated clearance of virus by group 1 ILCs precipitated severe intestinal inflammatory disease with commensal dysbiosis, loss of intestinal barrier function and diminished resistance to colonization. In sum, FRCs act as an 'on-demand' immunological 'rheostat' by restraining activation of group 1 ILCs and thereby preventing immunopathological damage in the intestine.
The Enterobacteriaceae are a family of Gram-negative bacteria that include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine's microbiota, some of these organisms can bloom in the inflamed gut; expansion of enterobacteria is a hallmark of microbial imbalance known as dysbiosis. Microcins are small secreted proteins that possess antimicrobial activity in vitro, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit the expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limits the growth of competitors in the inflamed intestine, including commensal E. coli, adherent-invasive E. coli and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild-type, microcin-producing EcN to mice previously infected with S. enterica substantially reduced intestinal colonization by the pathogen. Our work provides the first evidence that microcins mediate inter- and intraspecies competition among the Enterobacteriaceae in the inflamed gut. Moreover, we show that microcins can act as narrow-spectrum therapeutics to inhibit enteric pathogens and reduce enterobacterial blooms.
We compared results of a modified version of the Asthma Control Test using parent proxy report (PP-ACT) with results reported by children and parents using the validated Childhood-Asthma Control Test (C-ACT). 104 parent-child dyads with a child aged 6 to 12 years with asthma were randomized to complete PP-ACT followed by C-ACT or C-ACT followed by PP-ACT. Scores ≤19 indicated uncontrolled asthma. We calculated sensitivity, specificity, positive predictive value, and negative predictive value for the PP-ACT in comparison with the C-ACT, and calculated concordance between the 2 scales. The PP-ACT had sensitivity of 86% and negative predictive value of 88% for detecting uncontrolled asthma. More than 75% of surveys were concordant (κ = 0.54, moderate agreement). Our results suggest that while the PP-ACT missed few children with uncontrolled asthma and may simplify reporting of asthma control in circumstances when child report is not feasible or creates barriers to survey receipt, limitations of proxy reporting should be considered.
The exercise paradox infers that, despite the well-established cardioprotective effects of repeated episodic exercise (training), the risk of acute atherothrombotic events may be transiently increased during and soon after an exercise bout. However, the acute impact of different exercise modalities on platelet function has not previously been addressed. We hypothesized that distinct modalities of exercise would have differing effects on in vivo platelet activation and reactivity to agonists which induce monocyte-platelet aggregate (MPA) formation. Eight middle-aged (53.5 ± 1.6 years) male participants took part in four 30 min experimental interventions (aerobic AE, resistance RE, combined aerobic/resistance exercise CARE, or no-exercise NE), in random order. Blood samples were collected before, immediately after, and 1 h after each intervention, and incubated with one of three agonists of physiologically/clinically relevant pathways of platelet activation (thrombin receptor activating peptide-6 TRAP, arachidonic acid AA, and cross-linked collagen-related peptide xCRP). In the presence of AA, TRAP, and xCRP, both RE and CARE evoked increases in MPAs immediately post-exercise (P < 0.01), whereas only AA significantly increased MPAs immediately after AE (P < 0.01). These increases in platelet activation post-exercise were transient, as responses approached pre-exercise levels by 1 h. These are the first data to suggest that exercise involving a resistance component in humans may transiently increase platelet-mediated thrombotic risk more than aerobic modalities.
Interactions among traits that build a complex structure may be represented as genetic covariation and correlation. Genetic correlations may act as constraints, deflecting the evolutionary response from the direction of natural selection. We investigated the relative importance of drift, selection, and constraints in driving skull divergence in a group of related toad species. The distributional range of these species encompasses very distinct habitats with important climatic differences and the species are primarily distinguished by differences in their skulls. Some parts of the toad skull, such as the snout, may have functional relevance in reproductive ecology, detecting water cues. Thus, we hypothesized that the species skull divergence was driven by natural selection associated with climatic variation. However, given that all species present high correlations among skull traits, our second prediction was of high constraints deflecting the response to selection. We first extracted the main morphological direction that is expected to be subjected to selection by using within- and between-species covariance matrices. We then used evolutionary regressions to investigate whether divergence along this direction is explained by climatic variation between species. We also used quantitative genetics models to test for a role of random drift versus natural selection in skull divergence and to reconstruct selection gradients along species phylogeny. Climatic variables explained high proportions of between-species variation in the most selected axis. However, most evolutionary responses were not in the direction of selection, but aligned with the direction of allometric size, the dimension of highest phenotypic variance in the ancestral population. We conclude that toad species have responded to selection related to climate in their skulls, yet high evolutionary constraints dominated species divergence and may limit species responses to future climate change.
Hippocampal granule cells generated in the weeks before and after an epileptogenic brain injury can integrate abnormally into the dentate gyrus, potentially mediating temporal lobe epileptogenesis. Previous studies have demonstrated that inhibiting granule cell production before an epileptogenic brain insult can mitigate epileptogenesis. Here, we extend upon these findings by ablating newly generated cells after the epileptogenic insult using a conditional, inducible diphtheria-toxin receptor expression strategy in mice. Diphtheria-toxin receptor expression was induced among granule cells born up to 5 weeks before pilocarpine-induced status epilepticus and these cells were then eliminated beginning 3 d after the epileptogenic injury. This treatment produced a 50% reduction in seizure frequency, but also a 20% increase in seizure duration, when the animals were examined 2 months later. These findings provide the first proof-of-concept data demonstrating that granule cell ablation therapy applied at a clinically relevant time point after injury can have disease-modifying effects in epilepsy.
These findings support the long-standing hypothesis that newly generated dentate granule cells are pro-epileptogenic and contribute to the occurrence of seizures. This work also provides the first evidence that ablation of newly generated granule cells can be an effective therapy when begun at a clinically relevant time point after an epileptogenic insult. The present study also demonstrates that granule cell ablation, while reducing seizure frequency, paradoxically increases seizure duration. This paradoxical effect may reflect a disruption of homeostatic mechanisms that normally act to reduce seizure duration, but only when seizures occur frequently.
Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.
Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins Impαβ or Impβ/7. Interestingly, in addition to its role in nuclear import, binding of the Impβ/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in Bowen-Conradi syndrome, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.
This report details the blood concentration of drugs found in motorists suspected of driving under the influence of drugs from 2010 to 2012 in England and Wales. This study was carried out as new legislation has come into place, setting fixed blood concentration limits for drugs in motorists. These include a cannabis (Δ9-THC) blood concentration of 2 µg/L, amphetamine 250 µg/L, benzoylecgonine (BZE) 50 µg/L, cocaine 10 µg/L, 6-monoacetylmorphine 5 µg/L, morphine 80 µg/L, diazepam 550 µg/L and methadone 500 µg/L. Samples were screened for opiates, methadone, benzodiazepines, cannabinoids, cocaine, amphetamines and methamphetamine. Cannabinoids were the most prevalent drug group (29.7%) followed by benzodiazepines (22.7%), opiates (18.8%), cocaine (16.3%), amphetamine (7%) and methadone (5.6%). The analytical results are compared with the new per se limits to give a reference of drug concentrations prior to this legislation coming into effect. Our studies show that 64.9% of the cannabis samples, 59.1% of the cocaine samples and 94.6% of the BZE samples would be above the new per se limits set under Section 5a of the Road Traffic Act. In contrast, the medicinal drugs such as benzodiazepines and opiates (morphine) were predominantly detected at concentrations below the new per se limit. Given its medical applications, amphetamines appear to have been grouped with the medicinal type drugs, with our data showing that 25.2% of the amphetamine positive samples would exceed the new specified limit. These data show that samples containing medicinal and prescription drugs are likely to be detected below the new legal limits, while illicit drugs were typically found at concentrations above the new specified limits.
Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.
Cerebral palsy (CP) impacts on the entire family in a manner that is long-term, complex and multifactorial. In addition, the quality of the parent-child relationship impacts on many and varied child outcomes, making the provision of easily accessible and evidence-based support to parents of children with CP a priority. This paper reports the protocol of a randomised controlled trial of an innovative and translatable online intervention, parenting acceptance and commitment therapy (PACT), for families of children with CP. We predict that participating in the PACT programme will be associated with improvements in the parent-child relationship, in child functioning and in adjustment and quality of life for both parent and child.
We aim to recruit 66 parents of children (2-10 years old) diagnosed with CP to this study. Families will be randomly assigned to two groups: wait-list control and PACT. PACT is a parenting intervention grounded in acceptance and commitment therapy (ACT) and developed into an online course 'PARENT101 Parenting with Purpose' using the edX platform. All participants will be offered PACT before completion of the study. Assessments will take place at baseline, following completion of PACT and at 6-month follow-up (retention) and will focus on the parent-child relationship, parent and child adjustment and parent and child quality of life. Analysis will follow standard methods for randomised controlled trials using general linear models, specifically analysis of variance or analysis of covariance.
Ethics approvals have been obtained through the Children's Health Queensland Hospital and Health Service Human Research Ethics Committee (HREC/15/QRCH/115) and The University of Queensland (2015001743). If efficacy is demonstrated, then the PARENT101 course has the potential to be disseminated widely in an accessible manner and at minimal cost. Further, the PACT framework may provide a blueprint for similar online courses with parents in a full range of contexts.
ACTRN12616000351415; Pre-results.
The abundance and diversity of the LINE-1 (L1) retrotransposon differ greatly among vertebrates. Mammalian genomes contain hundred of thousands L1s that have accumulated since the origin of mammals. A single group of very similar elements is active at a time in mammals, thus a single lineage of active families has evolved in this group. In contrast, non-mammalian genomes (fish, amphibians, reptiles) harbor a large diversity of concurrently transposing families, which are all represented by very small number of recently inserted copies. Why the pattern of diversity and abundance of L1 is so different among vertebrates remains unknown. To address this issue, we performed a detailed analysis of the evolution of active L1 in 14 mammals and in three non-mammalian vertebrate model species. We examined the evolution of base composition and codon bias, the general structure, and the evolution of the different domains of L1 (5'UTR, ORF1, ORF2, 3'UTR). L1s differ substantially in length, base composition and structure among vertebrates. The most variation is found in the 5'UTR, which is longer in amniotes, and in the ORF1, which tend to evolve faster in mammals. The highly divergent L1 families of lizard, frog and fish share species-specific features suggesting they are subjected to the same functional constraints imposed by their host. The relative conservation of the 5'UTR and ORF1 in non-mammalian vertebrates suggests that the repression of transposition by the host does not act in a sequence specific manner and did not result in an arms race, as is observed in mammals.
RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin-resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or 'LRKO' mice). Adipose tissue RBP4 expression and secretion remain intact in LRKO mice, and increase as expected in the setting of diet-induced insulin-resistance. However, circulating RBP4 is undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin-resistance. Adipocyte RBP4 may therefore play a more important autocrine or paracrine function confined within the adipose tissue compartment.
While supported by the Affordable Care Act, in the United States, interprofessional training often takes place after healthcare providers graduate and are practicing in the field. This article describes the implementation and evaluation of an interprofessional training for graduate-level healthcare trainees. A group of interprofessional healthcare faculty provided a weeklong interprofessional immersion for doctoral-level healthcare trainees (n = 24) in Pharmacy, Counselling Psychology, Nursing, and Family Medicine residents. Healthcare faculty and staff from each profession worked side-by-side to provide integrated training utilising the Interprofessional Education Collaborative core competency domains. Trainees were placed into small teams with representatives from each profession; each team observed, learned, and practiced working within teams to provide quality patient care. Qualitative and quantitative data were collected to identify the effect of the training on trainees' self-reported team skills, as well as the extent to which the trainees learned and utilised the competencies. The results suggest that after completing the training, trainees felt more confident in their ability to work within an interprofessional team and more likely to utilise a team-based approach in the future.
Objective: The unique characteristics of sexual assault (SA)-a toxic mix of an interpersonal harm, a violent exploitation of one's body, and a transformation of an act of connectedness into an act of submission-are postulated to negatively affect the self-concept. We sought to deepen the understanding of self-concept impairments among sexually assaulted women with varying levels of posttraumatic distress. To this end, we compared women with a main trauma of SA to women with a main trauma of motor-vehicle accident (MVA) and to nontraumatized (NT) women on several self-concept aspects. Our main hypotheses were (a) sexually assaulted women without PTSD exhibit impaired self-concept as compared with NT women and (b) SA is related to greater self-concept impairments as compared with MVA, even when posttraumatic distress is statistically controlled. Method: Women (N = 235: NT = 69, MVA = 87, SA = 79) completed a web-based survey including measures designed to assess the global and domain-specific contents and structure of the self-concept as well as background and clinical questionnaires. Results: Sexually assaulted women without PTSD reported impaired self-concept as compared with NT women. Furthermore, SA was related to greater self-concept impairments as compared with MVA, even when considering participants' levels of posttraumatic distress. Conclusions: SA is related to unique self-concept impairments that extend beyond symptoms, emphasizing the need to assess and address self-concept impairments in sexually assaulted women. The importance of adopting a multifaceted conceptualization of the self to gain a deeper understanding of the aftermath of trauma is highlighted. (PsycINFO Database Record
Graphene membranes act as highly sensitive transducers in nanoelectromechanical devices due to their ultimate thinness. Previously, the piezoresistive effect has been experimentally verified in graphene using uniaxial strain in graphene. Here, we report experimental and theoretical data on the uni- and biaxial piezoresistive properties of suspended graphene membranes applied to piezoresistive pressure sensors. A detailed model that utilizes a linearized Boltzman transport equation describes accurately the charge-carrier density and mobility in strained graphene and, hence, the gauge factor. The gauge factor is found to be practically independent of the doping concentration and crystallographic orientation of the graphene films. These investigations provide deeper insight into the piezoresistive behavior of graphene membranes.
Apoptotic cell death is critical for the early development of the nervous system, but once the nervous system is established, the apoptotic pathway becomes highly restricted in mature neurons. However, the mechanisms underlying this increased resistance to apoptosis in these mature neurons are not completely understood. We have previously found that members of the miR-29 family of microRNAs (miRNAs) are induced with neuronal maturation and that overexpression of miR-29 was sufficient to restrict apoptosis in neurons. To determine whether endogenous miR-29 alone was responsible for the inhibition of cytochrome c release in mature neurons, we examined the status of the apoptotic pathway in sympathetic neurons deficient for all three miR-29 family members. Unexpectedly, we found that the apoptotic pathway remained largely restricted in miR-29-deficient mature neurons. We therefore probed for additional mechanisms by which mature neurons resist apoptosis. We identify miR-24 as another miRNA that is upregulated in the maturing cerebellum and sympathetic neurons that can act redundantly with miR-29 by targeting a similar repertoire of prodeath BH3-only genes. Overall, our results reveal that mature neurons engage multiple redundant brakes to restrict the apoptotic pathway and ensure their long-term survival.
Testosterone (T) is an important mediator of reproductive behaviours and potential target for selection. However, there are few data relating natural variation in T to fitness estimates. Here, we used the GnRH challenge (an injection of gonadotropin-releasing hormone which stimulates maximal T release), to examine how individual differences in T relate to reproductive success and how T changes across date and breeding stage. We measured pre- and post-challenge T, in captive male Gouldian finches (Erythrura gouldiae), before and after introducing females, and across breeding stage. Post-challenge T before introducing females positively predicted breeding success. Post-challenge T levels were unrelated to date, but strongly related to stage; T production ability was strongly attenuated in incubating males. Prechallenge T levels related only to date. Our results suggest that T production ability is an important target for selection and that when males invest heavily in parental care they reduce their sensitivity to GnRH.
Colorectal malignancies with high microsatellite instability (MSI-H), either hereditary (Lynch syndrome) or sporadic, demonstrate better prognosis and altered response to 5FU chemotherapy. It is now recommended to perform MSI testing for all new cases of colorectal cancer regardless of being categorized as hereditary or sporadic. For MSI detection, immunohistochemistry or PCR-based protocols using a cohort of various sets of STR markers are recommended. Here we aimed to evaluate a simplified protocol using just a single STR marker, MT1XT20 mononucleotide repeat, for detection of MSI in Lynch syndrome patients. A Promega five-marker MSI testing panel and immunohistochemistry (IHC) were used as the gold standard in conjunction with MT1XT20.
Colorectal patients with a positive history of familial cancers were selected by evaluating medical records. Based on Amsterdam II criteria for Lynch syndrome 20 families were short listed. DNA was extracted from formalin fixed paraffin embedded tumour and adjacent normal tissues resected from the index case in each family. Extracted DNA was subjected to MT1XT20 mononucleotide marker analysis and assessment with a commercially available five marker MSI testing kit (Promega, USA). IHC also was performed on tissue sections and the results were compared with PCR based data.
Eight (40%), seven (35%) and five (25%) cases were MSI positive using with the Promega kit, IHC and MT1XT20, respectively. Among the markers included in Promega kit, BAT26 marker showed instability in all 8 samples. NR24 and NR21 markers showed instability in 7 (87.5%), and BAT25 and MONO 27 in 6 (75%) and 5 (62.5%).
Although MT1XT20 was earlier reported as a valid standalone marker for MSI testing in CRC patients, we could not verify this in our Iranian patients. Instead BAT26 among the markers included in Promega MSI testing kit showed instability in all 8 MSI-H CRC samples. Therefore, it seems BAT26 could act well as a single marker for MSI testing in Iranian CRC patients.
Prosocial behavior is arguably influenced by an interaction between intrinsic dispositions (e.g., group bias) and extrinsic factors (e.g., institutional regulations). The current study investigated this interaction developmentally. Preschoolers (3- to 4-year-olds) and kindergarteners (5- to 6-year-olds; N = 111) participated in a resource distribution task in which they had to consider both the recipients' group membership (minimal color-based groups), and their own teachers' preferences regarding how to distribute (give "all" or "none"). The results revealed that only kindergarteners were influenced by the experimental factors and differently across genders. Specifically, when the recommendation was to give "none," girls followed it indiscriminately toward in- and out-group recipients, but boys did so only toward out-group recipients. Thus, boys exploited an authority's legitimization to act antisocially, according to a parochial bias.
The ability of the human brain to escape the here and now (mind wandering) can take functional (problem solving) and dysfunctional (perseverative cognition) routes. Although it has been proposed that only the latter may act as a mediator of the relationship between stress and cardiovascular disease, both functional and dysfunctional forms of repetitive thinking have been associated with blood pressure (BP) reactivity of the same magnitude. However, a similar BP reactivity may be caused by different physiological determinants, which may differ in their risk for cardiovascular pathology.
To examine the way (hemodynamic profile) and the extent (compensation deficit) to which total peripheral resistance and cardiac output compensate for each other in determining BP reactivity during functional and dysfunctional types of repetitive thinking.
Fifty-six healthy participants randomly underwent a perseverative cognition, a mind wandering, and a problem solving induction, each followed by a 5-min recovery period while their cardiovascular parameters were continuously monitored.
Perseverative cognition and problem solving (but not mind wandering) elicited BP increases of similar magnitude. However, perseverative cognition was characterized by a more vascular (versus myocardial) profile compared to mind wandering and problem solving. As a consequence, BP recovery was impaired after perseverative cognition compared to the other two conditions.
Given that high vascular resistance and delayed recovery are the hallmarks of hypertension the results suggest a potential mechanism through which perseverative cognition may act as a mediator in the relationship between stress and risk for developing precursors to cardiovascular disease.
Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of cell-drug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.
Recent research progress has revealed that a novel type of interstitial cells termed cardiac telocytes (CTs) is found in the interstitium of the heart. We demonstrated that CTs are distributed both longitudinally and within the cross network in the myocardium and that the density of CTs in the atrium-atria and base of the myocardium is higher than that in the middle of the myocardium, while the density of CTs in the epicardium is higher than that in the endocardium. In addition, we documented, for the first time, that the network of CTs in the infarct zone of the myocardium is destroyed during myocardial infarction (MI). This fact shows that, in addition to the death of cardiac myocytes, the previously unrecognized death of CTs is an important mechanism that contributes to the structural damage and poor healing and regeneration observed in the infarcted myocardium. Furthermore, we demonstrated, for the first time, that transplantation of CTs in cases of MI decreases the infarct size and improves myocardial function. The mechanisms behind the beneficial effects of CT transplantation are increased angiogenesis at the infarct site and the border zone, decreased fibrosis in the infarct and non-infarct zones, improved pathological reconstruction of the left ventricle, and increased regeneration of CTs in the infarct zone. Our findings reveal that CTs can be specifically identified by the following characteristics: very small cell bodies, extreme prolongation with some dilation, predisposition to cell death under ischemia, and expression of molecular markers such as c-Kit, CD34, vimentin, and PDGFR-β. CTs act as a structural and functional niche microenvironment in the myocardium and play an essential role in maintaining the integrity of the myocardium and in the regeneration of damaged myocardium.
The telocytes have recently been described in the prostate gland. In mature gland, they exist in close association with the acini and their telopodes form networks whose functions remain unclear. In this chapter, our group gives a brief introduction to telocytes and explores the history that led to such a concept and then discusses hypotheses and presents new evidences about the roles exerted by telocytes in the prostate. First is given emphasis on the role that these cells possibly play in paracrine signaling employed in the differentiation of smooth muscle periacinar are then discussed other roles potentially performed by telocytes in the prostate, such as the organizational, where these cells would act in order to delimit stromal microenvironments, thereby assisting the differentiation of the prostatic anatomical components. In addition, the pacemaker function of smooth muscle cells contraction, as evidenced by the presence of caveolae and gap-type junction and, finally, the role of telocytes in prostate remodeling and the possible action as adult progenitor cells. Generally speaking, the chapter reaffirms the existence of telocytes as distinct cells of other stromal cells and the importance of this new cell type for normal metabolism and prostate development.
Over the last few years, in vitro models, based on patient-derived induced pluripotent stem cells (iPSCs), have received considerable attention for modeling different neurodegenerative disorders. Using this model, we analyzed transcription of 15 tripartite motif (trim) genes in iPSCs, derived from the different groups: Parkinson's disease (PD) patients bearing mutations in different genes, patient with the sporadic form of PD, and the healthy individuals. The transcription was observed during neuronal differentiation of the cells in vitro into neuronal stem cells and terminally differentiated neurons. The transcription of over 50 % of these genes, belonging to different sub-groups of the TRIM family, varied between PD patients and healthy individuals during the reprogramming of fibroblasts into iPSCs and the following neuronal differentiation. Moreover, the transcription of the trim6 and trim24 genes was different between cells, derived from PD patients, and control cells at all stages. The transcription of the four trim genes (trim5α, 26, 27, 31) remained unchanged during almost all investigated stages, compared with the controls. We suppose that the revealed changes in the transcription of several trim genes reflect their possible role in neurodegenerative processes at the early stages of PD. These genes may act as a gear unit between the PD progression and the deregulation of the immune system.
A large number of asthmatic patients, particularly females, present inadequate disease control. Depressive symptoms are reportedly common in asthma and have been related to poor disease control, but the mechanism of this association is still unclear. Poor quality sleep, frequently observed in asthmatics, is also a manifestation of depression and has been related to uncontrolled asthma. This study aimed to investigate the relationship between depressive symptoms, sleep quality, and asthma control.
This was a cross-sectional study of 123 women with previous diagnosis of asthma from a reference center in Fortaleza, Brazil. Depressive symptoms were assessed by the Beck Depression Inventory (BDI); quality of sleep was evaluated by the Pittsburgh Sleep Quality Index (PSQI), daytime sleepiness by the Epworth Sleepiness Scale (ESS), and asthma control by the Asthma Control Test (ACT).
Inadequate asthma control (ACT <20) was found in 94 (76.4 %) subjects, depressive symptoms in 92 (74.8 %), poor quality sleep (PSQI >5) in 99 (80.49 %), and excessive daytime sleepiness (ESS ≥10) in 34 (27.64 %). Depressive symptoms were associated with both poor quality sleep (R = 0.326) and inadequate asthma control (R = -0.299). Regression analysis showed that depressive symptoms and sleep quality were independent predictors of the level of asthma control.
Asthma control in women is independently associated with depressive symptoms and quality of sleep, suggesting that these patients might benefit from simple measures to promote healthy sleep behavior and sleep hygiene and also that routine screening for depression can be relevant, particularly, in poorly controlled cases.
In this report, a novel one-step chemical reduction method was reported for synthesis of water-soluble and stable fluorescent glutathione-templated silver nanocluster (GSH-Ag NCs) with ascorbic acid as an environmental-friendly reducing agent. On the basis of an oxidoreduction-induced fluorescence quenching mechanism, the prepared GSH-Ag NCs found to act as a cheap, non-toxic and highly sensitive "turn-off" fluorophore for ascorbic acid (AA). Furthermore, the fluorescence of the fluorophore/AA system could be recovered through addition of arginine (Arg), which made the system function as a highly selective "turn-on" sensor for arginine. Therefore, a "turn-off-on" switch sensor was proposed for detection of AA and Arg. Under optimized conditions, the probe gives a fluorescent response that is linear in the 2-300 μM concentration range of AA, with a detection limit of 0.1 μM. The probe for Arg, in turn, has a linear range in the 10-180 μM concentration range, and the limit of detection is 0.5 μM. In addition, the developed method showed great accuracy when employed to detect AA and Arg in human urine and serum, which shows its great potential in biological molecular recognition applications.
The cytokine interleukin-22 (IL-22) is a potent regulator of tissue responses during inflammation. Depending on the context of inflammation, IL-22 can have protective or inflammatory effects on epithelial cells. This dual nature of IL-22 leads us to hypothesize that its activity must be exquisitely regulated to prevent host tissue damage. Environmental factors may act as a cellular cue as to how cells respond to IL-22. Inflammatory environments are characterized by low oxygen and thus we examined whether cells respond differently to IL-22 hypoxia compared with normoxia. In this study, we show that hepatocyte responses to IL-22 stimulation are reduced in hypoxic environments. IL-22 stimulation of hepatocytes incubated in low oxygen led to reduced levels of activated signal transducer and activator of transcription 3 and further downstream effects such as reduced induction of the anti-microbial protein, lipocalin-2. This modulation appears to be independent of the hypoxia-inducible factor-1α signaling pathway. Thus, hypoxia that accompanies chronic inflammation may be a mechanism to regulate the bioactivity of the dual-natured IL-22 cytokine.Immunology and Cell Biology advance online publication, 22 November 2016; doi:10.1038/icb.2016.107.
A smart magnetic-targeting drug carrier γ-Fe2O3@p-silica comprising a γ-Fe2O3 core and porous shell has been prepared and characterized. The particles have a uniform size of about 60 nm, and a porous shell of thickness 3 nm. Abundant hydroxyl groups and a large surface area enabled the γ-Fe2O3@p-silica to be readily loaded with a large payload of the basic model drug rhodamine B (RB) (up to 73 mg g(-1)). Cytotoxicity assays of the γ-Fe2O3@p-silica particles indicated that the particles were biocompatible and suitable for carrying drugs. It was found that the RB was released rapidly at pH 5.5 but at pH 7.4 the rate and extent of release was greatly attenuated. The particles therefore demonstrate an excellent pH-triggered drug release. In addition, the γ-Fe2O3@p-silica particles could be tracked by magnetic resonance imaging (MRI). A clear dose-dependent contrast enhancement in both T 1-weighted and T 2-weighted MR images indicated the potential of the γ-Fe2O3@p-silica particles to act as dual-mode T 1 and T 2 MRI contrast agents.
To examine how the right to participation according to Article 19 of the United Nations' Convention on the Rights of Persons with Disabilities (UNCRPD) is promoted by personal assistance use in Sweden across age, gender and eligible person categories.
Register data and data from a questionnaire were used (N = 15,289). Principal component analysis was performed and the internal consistency was tested. Descriptive statistics (χ(2) test) were used across age, gender and eligible person categories and components.
An uneven distribution of personal assistance across the components Health and Care; Home, Leisure and Social Interaction; and Daily Occupation was found. Significant differences in personal assistance reported were found between children and adults, men and women and between the three eligible person categories.
The discrepancy between reported and expected outcome of personal assistance indicates that Article 19 of the UNCRPD has not been met. The unequal access to participation across age, gender and eligible person categories would seem to further signify that the Act concerning Support and Service for Persons with Certain Functional Impairments is promoting activities of a caring nature rather than fulfilling Article 19 of the UNCRPD, i.e. ensuring full participation in society. IMPLICATIONS FOR REHABILITATION Government assistance allowance were granted for predominantly health and care, i.e. basic needs presenting risk of undermining the intention of participation in society. Men reported more personal assistance use for activities promoting participation than women. The discrepancy found between reported and expected outcome of personal assistance underlines the importance of service providers and administrative officials being sensitive to policy intentions. There is a need of guidelines for service providers and administrative officials to promote disability rights of participation for persons eligible for personal assistance.
Despite the available research findings, recommendations and the South African Occupational Health and Safety Act (OHSA) (Act 85 of 1993), there are still challenges with regard to the implementation of selected sections and regulations of the OHSA. This is evidenced by the occupational injuries and illness claims registered with the compensation fund (South Africa, Department of Labour 1993).
To determine the extent to which the OHSA was implemented at an academic hospital in Johannesburg, from the senior professional nurses and nursing managers' perspective, and to describe recommendations in order to facilitate the implementation of the Act.
A contextual, quantitative, exploratory and descriptive survey was conducted. A purposive sampling method was used to select the participants that met the inclusion criteria. A structured Likert-scale questionnaire was used to collect data (Brink 2011). Stata version 12 was used to analyse the data. Cronbach's alpha, with a cut-off point of 0.7 was used to test for internal consistency. Ethical considerations were strictly adhered to. Results are presented in the form of graphs, frequency distributions and tables.
The study revealed that overall there is 93.3% non-implementation of the selected sections and regulations of the OHSA. These results have serious implications on the health and safety of employees in the workplace.
The study recommends that the replication of the study should be conducted in order to determine the extent of implementation of the selected sections and regulations of the OHSA in other government institutions.
The implementation of the South African Mental Health Care Act, which regulates care for clients with intellectual disabilities, impacted on the healthcare services provided to this population. Changes in the Act necessitated planning of new care packages, which resulted in the investigation of the current hospital client profile, as well as assessment data on patient abilities according to the occupational therapist and nursing staff as primary caregivers.
A retrospective, descriptive correlation study design was used as information was analysed from an existing database. Descriptive analysis of clients' demographic data, occupational performance and adaptive functioning were done, as well as a Spearman's rank correlation test and cluster analysis to describe the association between the levels of functioning as measured by the different professions.
The results indicated low levels of abilities, as well as a good to excellent correlation between results of the Fairview self-help scale and Creative Participation Assessment.
This study provided preliminary evidence that these two tools are valuable instruments for measuring occupational performance and adaptive functioning in institutions that provide care for this vulnerable and under-researched population.
Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, using organotypic cerebral cortex slices from mice expressing eGFP in Sox10-positive oligodendrocytes, that endogenously released GABA, acting on GABAA receptors, greatly reduces the number of oligodendrocyte lineage cells. The decrease in oligodendrocyte number correlates with a reduction in the amount of myelination but also an increase in internode length, a parameter previously thought to be set by the axon diameter or to be a property intrinsic to oligodendrocytes. Importantly, while TTX block of neuronal activity had no effect on oligodendrocyte lineage cell number when applied alone, it was able to completely abolish the effect of blocking GABAA receptors, suggesting that control of myelination by endogenous GABA may require a permissive factor to be released from axons. In contrast, block of AMPA/KA receptors had no effect on oligodendrocyte lineage cell number or myelination. These results imply that, during development, GABA can act as a local environmental cue to control myelination and thus influence the conduction velocity of action potentials within the CNS. GLIA 2016.
Responsive medicine is an appropriate training method which trains the graduates who can act effectively in initial and secondary aspects of health issues in the society.
This was a cross-sectional descriptive-analytic study which was done using quantitative method. The target population of this study was all the students of the Nutrition and Health School of Shiraz University of Medical Sciences. The sample was randomly selected in this study and 75 students were selected based on the methodologist's comments and similar studies and random-number table from a list obtained from the school's department of education. This questionnaire was a researcher-made one which consisted of 23 questions in 2 sections with 21 closed-ended questions and 2 open-ended questions; 70 questionnaires were completed correctly. The closed-ended questions had 4 aspects (completely agree to completely disagree) answered in 5-point Likert scale type. Its face validity was confirmed by 4 faculty members. The construct validity of the questionnaire was analyzed by factor analysis test and its reliability was assessed by a pilot on 20 students with a Cronbach's alpha of 0.85. The data were analyzed using descriptive statistical tests (mean, standard deviation, …) and the Pearson coefficient (p<0.001).
The results of this study showed that the maximum mean score was 3.58±0.65 which was related to the context of these courses and the minimum mean was 2.66±1.14 which was related to the logbook implementation. The 2 open-ended questions indicated that the most important strengths were the use of logbooks as a guide and determining the minimum training; of the weaknesses was the mismatch between the theoretical education and the practical activities. Also, developing the minimum training that an expert should know and using the common topics related to theoretical education were the most important points mentioned by the respondents.
The educational planning of the authorities for keeping the balance of the theoretical training with the practical activities and giving opportunities to the trainee or intern to face diseases and the common problems of the community seems to be necessary.
Increasing evidence suggests that cancer stem cells (CSCs) are a key occurrence in the process of many human cancers. Lung cancer is the most common aggressive malignancy and cause of cancer death worldwide. The research on lung cancer stem cells has been highlighted for many years. Lung CSCs seem to play a major role in lung cancer metastasis, drug resistance and tumour-self-renewal. MicroRNAs (miRNAs), a class of newly emerging small noncoding RNAs that act as post-transcriptional regulators of gene expression, have been demonstrated to serve as a vital player in fine-tuning a number of biological activities ranging from embryogenesis to programmed cell death as well as tumourigenesis. In recent years, several miRNAs have been highlighted to be specifically expressed in CSCs. The miRNA profile of CSCs is remarkably different from non-stem cancer cells. As such, many miRNAs have been shown to regulate self-renewal and differentiation properties of CSCs. In this review, we present the latest findings on miRNAs that regulate the tumour microenvironment of lung CSCs with the goal to prompt the development of novel therapeutic strategies for patients with lung cancer.
Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains-the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and βPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.
Supramolecular brush copolymers have attracted continuing interest due to their unusual architectures, fascinating properties, and potential applications in many fields involving smart stimuli-responsive drug delivery systems. Herein, the first pillararene-based amphiphilic supramolecular brush copolymer (P5-PEG-Biotin⊃PTPE) was constructed on the basis of the host-guest molecular recognition between a water-soluble pillar[5]arene (P5) and a viologen salt (M). P5-PEG-Biotin⊃PTPE self-assembled into supramolecular nanoparticles (SNPs), which were utilized as a self-imaging drug delivery vehicle by taking advantage of the aggregation-induced emission (AIE) effect. Encapsulation of anticancer drug doxorubicin (DOX) caused deactivation of the fluorescences of both the tetraphenylethene (TPE) and DOX chromophores due to the energy transfer relay (ETR) effect, mediated by Förster resonance energy transfer (FRET) and aggregation-caused quenching (ACQ). The release of loaded DOX molecules can be triggered by low pH and reductase, recovering the "silenced" fluorescence caused by the interruption of the ETR effect, achieving in situ visualization of the drug release process by observing the location and magnitude of the energy transfer-dependent fluorescence variation. The biotin ligands on the surfaces of the DOX-loaded SNPs act as targeting agents to deliver DOX preferentially to cancer cells over-expressing biotin receptor. In vitro studies demonstrated that the loading of DOX by this supramolecular nanomaterial exhibited selective cytotoxicity towards cancer cells over normal cells. The potency of this sophisticated supramolecular drug delivery system in cancer therapy was further evaluated in HeLa tumor-bearing mice. In vivo experiments confirmed that the DOX-loaded SNPs possess excellent antitumor efficacy with negligible systemic toxicity.
Feruloyl esterases (FAEs) represent a diverse group of carboxyl esterases that specifically catalyze the hydrolysis of ester bonds between ferulic (hydroxycinnamic) acid and plant cell wall polysaccharides. Therefore, FAEs act as accessory enzymes to assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass conversion. Their ability to release ferulic acid and other hydroxycinnamic acids from plant biomass makes FAEs potential biocatalysts in a wide variety of applications such as in biofuel, food and feed, pulp and paper, cosmetics, and pharmaceutical industries. This review provides an updated overview of the knowledge on fungal FAEs, in particular describing their role in plant biomass degradation, diversity of their biochemical properties and substrate specificities, their regulation and conditions needed for their induction. Furthermore, the discovery of new FAEs using genome mining and phylogenetic analysis of current publicly accessible fungal genomes will also be presented. This has led to a new subfamily classification of fungal FAEs that takes into account both phylogeny and substrate specificity.
Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo.
With the development of urban setting worldwide, the major issue of concern is the increase in the mortality rate in the population due to road traffic accidents. The face, being the most exposed region is susceptible to injuries and maybe associated with injuries to the adjacent neuro-cranium. The literature has conflicting views on the relationship between facial fractures and head injuries with some authors opining that the facial skeleton cushions the brain while some other authors claim that the facial fractures act as indicators for head injuries.
To analyze the correlation between the facial fractures and head injuries and to assess if the facial skeleton acts to protect the brain from injury.
A prospective study that included patients who reported to the emergency department of Basaveswar Teaching and General Hospital, Gulbarga, during 2 years, between August 2013 and July 2015 was conducted. A total of 100 patients with facial fractures were enrolled in the study.
Head injuries were sustained by 51 patients in the study. Maximum number of patients was in the age group of 20-29 with a male to female ratio of 10.1:1. The mandible was the most frequently fractured bone in the facial skeleton followed by the zygomatico-maxillary complex. A majority (96%) of patients with head injuries had fractures of either the upper third or the middle third of the face. Contusions and pneumocephalus were the most common head injury encountered. The Glasgow Coma Scale score was significantly lower in patients with associated head injuries as compared to those patients with facial trauma alone. The mortality rate in the study was 2% with both the victims having sustained middle third and upper third fractures respectively with associated head injuries.
The facial skeleton does not act to cushion the brain from injury but, in fact, the facial trauma victims should be considered potential head injury patients.
The objective of this study was to examine a novel profile: thiol-disulphide homeostasis in acute brucellosis. The study included 90 patients with acute brucellosis, and 27 healthy controls. Thiol-disulphide profile tests were analysed by a recently developed method, and ceruloplasmin levels were determined. Native thiol levels were 256.72 ± 48.20 µmol/L in the acute brucellosis group and 461.13 ± 45.37 µmol/L in the healthy group, and total thiol levels were 298.58 ± 51.78 µmol/L in the acute brucellosis group and 504.83 ± 51.05 µmol/L in the healthy group (p < 0.001, for both). The disulphide/native thiol ratios and disulphide/total thiol ratios were significantly higher, and native thiol/total thiol ratios were significantly lower, in patients with acute brucellosis than in the healthy controls (p < 0.001, for all ratios). There were positive or negative relationships between ceruloplasmin levels and thiol-disulphide parameters. The thiol-disulphide homeostasis was impaired in acute brucellosis. The strong associations between thiol-disulphide parameters and a positive acute-phase reactant reflected the disruption of the balance between the antioxidant and oxidant systems. Because thiol groups act as anti-inflammatory mediators, the alteration in the thiol-disulphide homeostasis may be involved in brucellosis.
We recently reported that the segment-specific noncoding regions (NCRs) of the HA and NA segments are subtype-specific, varying significantly in sequence and length at both the 3' and 5' ends. Interestingly, we found that nucleotides "CC" at positions 13-14 at the 3' end and "GUG" at positions 14' -16' at the 5' end are absolutely conserved among all HA subtype-specific NCRs. These HA segment-specific NCR nucleotides are located in the extended duplex region of the viral RNA promoter. In order to understand the significance of these highly conserved HA segment-specific NCR nucleotides in the virus life cycle, we performed extensive mutagenesis on the HA segment-specific NCR nucleotides and studied their functional significance in regulating influenza A virus replication in the context of the HA segment with both RNP reconstitution and virus infection systems. We found that the base-pairing at (3')13-(5')14' positions is critical for RNA promoter activity while the identity of the base pair is critical in determining HA segment packaging. Moreover, the identity of the residue at (3')14 is more functionally important in regulating virus genome packaging than in regulating viral RNA synthesis. Taken together, these results demonstrated that the HA segment-specific NCR nucleotides in the extended duplex region of the promoter not only form part of the promoter, but also play a key role in controlling virus selective genome packaging.
The segment-specific complementary nucleotides (13-15 in the 3' end and 14' -16' in the 5' end) in the extended duplex region of the influenza virus RNA promoter vary significantly among different segments and have rarely been studied. We here performed mutagenesis analysis of the highly conserved HA segment-specific nucleotides in the extended duplex region and examined their effects on virus replication in the context of the WSN virus infection. We found that these HA segment-specific nucleotides, not only act as a part of the RNA promoter, but also play a critical role in HA segment packaging. Therefore, we showed experimentally, for the first time, the requirement of the nucleotides in the extended duplex region for the RNA promoter, but also identified specific noncoding residues in regulating HA segment packaging. This work has implications for the development of attenuated vaccine strains and for elucidation the mechanisms of the virus genome packaging.
Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpA(NTD)), the Ebp pilus's adhesin. In a murine model, vaccination with EbpA(NTD) confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpA(NTD)-fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis, E. faecium, and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpA(NTD) antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpA(NTD)-fibrinogen interactions have considerable promise for treatment of CAUTI.
Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.
The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN), consistent with the high-affinity "collagen hug" mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation.
Cna is a collagen (Cn)-binding protein from Staphylococcus aureus that is involved in pathogenesis. Currently, we know little about the functional role of the repetitive B region of the protein. Here, we unravel the mechanical strength of Cna in living bacteria. We show that single Cna-Cn bonds are very strong, reflecting high-affinity binding by the collagen hug mechanism. We discovered that the B region behaves as a nanospring capable of sustaining high forces. This unanticipated mechanical response, not previously described for any staphylococcal adhesin, favors a model in which the B region has a mechanical function that is essential for strong ligand binding. Finally, we assess the antiadhesion activity of monoclonal antibodies against Cna, suggesting that they could be used to inhibit S. aureus adhesion.
The efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle in controlling Plasmodium falciparum malaria, thus the study of the origin and spread of associated mutations may provide some insights in the prevention of its emergence.This study reports the analysis of P. falciparum genetic diversity focusing antimalarial resistance associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. Present study took place eight years after a first one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), artesunate+amodiaquine.Genetic diversity was assessed by analysis of Pfmsp2 gene and neutral microsatellite loci Pfdhps and Pfdhfr alleles associated with sulphadoxine-pyrymethamine (SP) resistance and flanking microsatellite loci were investigated and prevalence of drug resistance associated point mutations of Pfcrt, Pfmdr1, Pfdhfr and Pfdhps genes was estimated. Further, to monitor the use of an ACT, we provide a baseline prevalence of K13-propeller mutations and Pfmdr1 copy numbers.After eight years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP and the spread of mutated genotypes differed according the setting. Regarding artemisinin resistance, although mutations reported as linked to artemisinin resistance were not present at the time, several SNPs in the K13 gene were observed, suggesting that a closer monitoring should be maintained to prevent a possible spread of artemisinin-resistance in Africa.
The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents which act directly on steps of the HCV lifecycle. Here we report the preclinical characteristics of ITMN-8187, a non-macrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other direct-acting antiviral agents in vitro displayed additive antiviral efficacy. A 30 mg/kg dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through Day 5 in a chimeric mouse model of HCV. A 3 mg/kg oral dose administered to rats, dogs, or monkeys yielded plasma concentrations 16 hours after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low inter-subject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.
Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affects the architecture of bacterial cells. Here we report a cell-biological study in model organisms of the genus Streptomyces, which are Actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, that contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed and polarisomes were redistributed to new sites, leading to emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act in different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth.
Polar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension of Streptomyces hyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influences Streptomyces tip growth. We report here that severe hyper-osmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip.
Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.
The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.
TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBR(act)-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis.
Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.
The family Lepilemuridae includes 26 species of sportive lemurs, most of which were recently described. The cryptic morphological differences confounded taxonomy until recent molecular studies; however, some species' boundaries remain uncertain. To better understand the genus Lepilemur, we analyzed 35 complete mitochondrial genomes representing all recognized 26 sportive lemur taxa and estimated divergence dates. With our dataset we recovered 25 reciprocally monophyletic lineages, as well as an admixed clade containing Lepilemur mittermeieri and Lepilemur dorsalis Using modern distribution data, an ancestral area reconstruction and an ecological vicariance analysis were performed to trace the history of diversification and to test biogeographic hypotheses. We estimated the initial split between the eastern and western Lepilemur clades to have occurred in the Miocene. Divergence of most species occurred from the Pliocene to the Pleistocene. The biogeographic patterns recovered in this study were better addressed with a combinatorial approach including climate, watersheds, and rivers. Generally, current climate and watershed hypotheses performed better for western and eastern clades, while speciation of northern clades was not adequately supported using the ecological factors incorporated in this study. Thus, multiple mechanisms likely contributed to the speciation and distribution patterns in Lepilemur.
ACT is an essential virulence factor of Bordetella pertussis and antibodies to ACT protect against B. pertussis infection in mice. The toxin is, therefore, a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we have developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG ELISA neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by polymerase chain reaction, anti-ACT IgG ELISA was positive in 72% and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity, and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.
Regulation of amino acid metabolism (RAM) domains are widely distributed among prokaryotes. In most cases, a RAM domain fuses with a DNA-binding domain to act as a transcriptional regulator. The extremely thermophilic bacterium, Thermus thermophilus, only carries a single gene encoding a RAM domain-containing protein on its genome. This protein is a stand-alone RAM domain protein (SraA) lacking a DNA-binding domain. Therefore, we hypothesized that SraA, which senses amino acids through its RAM domain, may interact with other proteins to modify its functions. In the present study, we identified anthranilate phosphoribosyltransferase (AnPRT), the second enzyme in the tryptophan biosynthetic pathway, as a partner protein that interacted with SraA in T. thermophilus. In the presence of tryptophan, SraA was assembled to a decamer and exhibited the ability to form a stable hetero-complex with AnPRT. An enzyme assay revealed that AnPRT was only inhibited by tryptophan in the presence of SraA. This result suggests a novel feedback control mechanism for tryptophan biosynthesis through an inter-RAM domain interaction in bacteria.
Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.
Fc-gamma receptors (FCGRs) are expressed on immune cells, bind to antibodies, and trigger antibody-induced cell-mediated anti-tumor responses when tumor-reactive antibodies are present. The affinity of the FCGR/antibody interaction is variable and dependent upon FCGR polymorphisms. Prior studies of cancer patients treated with immunotherapy indicate that FCGR polymorphisms can influence antitumor response for certain immunotherapies that act via therapeutically administered mAbs or via endogenous tumor-reactive antibodies induced from tumor antigen vaccines. The previously published "SELECT" trial of high-dose aldesleukin (HD-IL2) for metastatic renal cell carcinoma (mRCC) resulted in an objective response rate (ORR) of 25%. We evaluated the patients in this SELECT trial to determine whether higher affinity FCGR polymorphisms are associated with outcome.
Single nucleotide polymorphisms (SNPs) in FCGR2A, FCGR3A, and FCGR2C were analyzed, individually and in combination, for associations between genotype and clinical outcome.
When higher affinity genotypes for FCGR2A, FCGR3A and FCGR2C were considered together, they were associated with significantly increased tumor shrinkage and prolonged survival in response to HD-IL2.
While associations of higher affinity FCGR genotype with clinical outcome have been demonstrated with mAb therapy and with idiotype vaccines, to our knowledge, this is the first study to show associations of FCGR genotypes with outcome following HD-IL2 treatment. We hypothesize that endogenous anti-tumor antibodies may engage immune cells through their FCGRs, and HD-IL2 may enhance antibody-induced tumor destruction, or antibody-enhanced tumor antigen presentation, via augmented activation of innate or adaptive immune responses; this FCGR-mediated immune activity would be augmented through immunologically favorable FCGRs.
Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK.
Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.
Campylobacter is the leading cause of human gastroenteritis worldwide. Wild birds, including American crows, are abundant in urban, suburban, and agricultural settings and are likely zoonotic vectors of Campylobacter Their proximity to humans and livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. However, no studies have definitively demonstrated that crows are a vector for pathogenic Campylobacter We used genomics to evaluate the zoonotic and pathogenic potential of Campylobacter from crows to other animals with 184 isolates obtained from crows, chickens, cows, sheep, goats, humans, and nonhuman primates. Whole-genome analysis uncovered two distinct clades of Campylobacter jejuni genotypes; the first contained genotypes found only in crows, while a second genotype contained "generalist" genomes that were isolated from multiple host species, including isolates implicated in human disease, primate gastroenteritis, and livestock abortion. Two major β-lactamase genes were observed frequently in these genomes (oxa-184, 55%, and oxa-61, 29%), where oxa-184 was associated only with crows and oxa-61 was associated with generalists. Mutations in gyrA, indicative of fluoroquinolone resistance, were observed in 14% of the isolates. Tetracycline resistance (tetO) was present in 22% of the isolates, yet it occurred in 91% of the abortion isolates. Virulence genes were distributed throughout the genomes; however, cdtC alleles recapitulated the crow-only and generalist clades. A specific cdtC allele was associated with abortion in livestock and was concomitant with tetO These findings indicate that crows harboring a generalist C. jejuni genotype may act as a vector for the zoonotic transmission of Campylobacter IMPORTANCE: This study examined the link between public health and the genomic variation of Campylobacter in relation to disease in humans, primates, and livestock. Use of large-scale whole-genome sequencing enabled population-level assessment to find new genes that are linked to livestock disease. With 184 Campylobacter genomes, we assessed virulence traits, antibiotic resistance susceptibility, and the potential for zoonotic transfer to observe that there is a "generalist" genotype that may move between host species.
Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.
Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.
A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3.
The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.
Community health workers (CHW) may be effective in the delivery of tobacco dependence treatment with underserved groups. This study evaluated two evidence-based CHW models of treatment. It was hypothesized that smokers assigned to a CHW face-to-face condition would have higher abstinence at 12-month posttreatment than smokers enrolled in CHW referral to a state-sponsored quitline condition. Intrapersonal and treatment-related factors associated with abstinence at 12 months were determined.
A group-randomized trial was conducted with residents of 12 Ohio Appalachian counties with counties (n = 6) randomized to either a CHW face-to-face (F2F) or CHW quitline (QL) condition. Both conditions included behavioral counseling and free nicotine replacement therapy for 8 weeks. Follow-up data were collected at 3-, 6-, and 12-month posttreatment. Biochemically validated abstinence at 12 months served as the primary outcome.
Seven hundred and seven participants were enrolled (n = 353 CHWF2F; n = 354 CHWQL). Baseline sample characteristics did not differ by condition. Using an intent-to-treat analysis (85.4% retention at 12 months), 13.3% of CHWF2F participants were abstinent at 12 months, compared to 10.7% of CHWQL members (OR = 1.28; 95% confidence interval [CI] = 0.810, 2.014; p = .292). No differences in abstinence were noted at 3 or 6 months by condition. Age, marital status, and baseline levels of cigarette consumption, depressive symptoms, and self-efficacy for quitting in positive settings were associated with abstinence, as was counseling dose during treatment.
This research adds to the body of science evaluating the effectiveness of CHW models of tobacco dependence treatment. Both approaches may offer promise in low-resource settings and underserved regions.
This 12-county community-based group-randomized trial in Ohio Appalachia adds to the body of science evaluating the effectiveness of CHW models of tobacco dependence treatment. Both CHW approaches may offer promise in low-resource settings and underserved regions. These findings are useful to national, state, and local tobacco control agencies, as they expand delivery of preventive health care services postadoption of the Affordable Care Act in the United States.
Inflammation is essential for successful embryo implantation, pregnancy maintenance and delivery. In the last decade, important advances have been made in regard to endogenous, and therefore non-infectious, initiators of inflammation, which can act through the same receptors as pathogens. These molecules are referred to as damage-associated molecular patterns (DAMPs), and their involvement in reproduction has only recently been unraveled. Even though inflammation is necessary for successful reproduction, untimely activation of inflammatory processes can have devastating effect on pregnancy outcomes. Many DAMPs, such as uric acid, high-mobility group box 1 (HMGB1), interleukin (IL)-1 and cell-free fetal DNA, have been associated with pregnancy complications, such as miscarriages, preeclampsia and preterm birth in preclinical models and in humans. However, the specific contribution of alarmins to these conditions is still under debate, as currently there is lack of information on their mechanism of action. In this review, we discuss the role of sterile inflammation in reproduction, including early implantation and pregnancy complications. Particularly, we focus on major alarmins vastly implicated in numerous sterile inflammatory processes, such as uric acid, HMGB1, IL-1α and cell-free DNA (especially that of fetal origin) while giving an overview of the potential role of other candidate alarmins.
Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10(-5), OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10(-54) and 4.5x10(-4) respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10(-54); 1.0 × 10(-34)). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10(-8)) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4 Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.
Activin A is one of the members of transforming growth factor-β superfamily that is expressed in human large luteal cells, and may act in an autocrine/paracrine manner to regulate luteal function. Prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme and its derivative, prostaglandin E2 (PGE2), play significant roles in the regulation of corpus luteum formation and maintenance. To date, whether activin A can induce the expression of PTGS2 and the production of PGE2 in human granulosa-lutein cells is largely unknown. The aim of this study was to examine the effects of activin A on the regulation of PTGS2 expression and PGE2 production in human granulosa-lutein cells, and to investigate the underlying signal transduction mechanisms. In this study, the immortalized (SVOG cells) and primary human granulosa-lutein cells were used as the cell models. A TGF-β/activin type I receptor inhibitor, SB431542 and small interfering RNAs were used to investigate the activin A-induced downstream signaling pathway. We have demonstrated that activin A upregulated the expression of PTGS2 and increased the production of PGE2 via an ACVR1B-mediated SMAD2/3-SMAD4 signaling pathway. Our results suggest that activin A may be involved in the modulation of human corpus luteum formation via the induction of PTGS2 expression and PGE2 production.
Novel benzothiazole Schiff bases L(1) [1-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl) naphthalen-2-ol], L(2) [3-((4,6-difluorobenzo[d]thiazol-2-ylimino) methyl)benzene-1,2-diol], L(3) [2-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl)-5-methoxyphenol], L(4) [2-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl)-4-chlorophenol] and their binary Cu(II) complexes were synthesized. The structures of all the compounds have been discussed on the basis of elemental analysis, FT-IR, NMR, UV-Visible, ESI-Mass, TGA, ESR, SEM, powder XRD and magnetic moments. Based on the analytical and spectral data a square planar geometry has been assigned to all complexes in which the Schiff bases act as monobasic bidentate ligands, coordinating through the azomethine nitrogen and phenolic oxygen atom. DNA binding ability of these complexes was studied on CT-DNA by using UV-Vis absorption, fluorescence and viscometry. DNA cleavage ability of the complexes was examined on pBR322 DNA by using gel electrophoresis method. All the DNA binding studies reveal that they are good intercalators. The bioefficacy of the ligands and their complexes was examined against the growth of bacteria and fungi in vitro to evaluate their antimicrobial potential. The screening data revealed that the complexes showed more antimicrobial activity than the corresponding free ligands.
The potential importance of cytoplasmic incompatibility (CI)-inducing bacterial symbionts in speciation of their arthropod hosts has been debated. Theoretical advances have led to a consensus that a role is plausible when CI is combined with other isolating barriers. However, the insect model systems Nasonia and Drosophila are the only two experimental examples documented. Here, we analyzed the components of reproductive isolation between the parasitoid wasp Encarsia suzannae, which is infected by the CI-inducing symbiont Cardinium, and its uninfected sibling species Encarsia gennaroi. Laboratory crosses demonstrated that: (1) sexual isolation is incomplete; (2) hybrid offspring production is greatly reduced in the interspecific CI cross; (3) viable hybrids may be produced by curing E. suzannae males of Cardinium with antibiotics; (4) hybrid offspring production in the reciprocal cross is greatly reduced by hybrid inviability due to genetic incompatibilities; (5) hybrid sterility is nearly complete in both directions at the F1 stage. Thus, asymmetrical hybrid incompatibilities and CI act as complementary isolating mechanisms. We propose a new model for contributions of CI symbionts to speciation, with CI reducing gene flow between species in one direction, and in the other, a symbiont sweep resulting in accelerated mtDNA evolution, negative cytonuclear interactions, and hybrid incompatibilities.
Copper(I) complexes of the formula [Cu(L)(PPh3)2]X (1-4) (X = Cl(1), ClO4(2), BF4(3) and PF6(4)) [where L = N-(2-{[(2E)-2-(4-nitrobenzylidenyl)hydrazinyl]carbonyl}phenyl)benzamide; PPh3 = triphenylphosphine] have been prepared by the condensation of N-[2-(hydrazinocarbonyl)phenyl]benzamide with 4-nitrobenzaldehyde followed by the reaction with CuCl, [Cu(MeCN)4]ClO4, [Cu(MeCN)4]BF4 and [Cu(MeCN)4]PF6 in presence of triphenylphosphine as a coligand. Complexes 1-4 were then characterized by elemental analyses, FTIR, UV-visible and (1)H NMR spectroscopy. Mononuclear copper(I) complexes 1-4 were formed with L in its keto form by involvement of azomethine nitrogen and the carbonyl oxygen along with two PPh3 groups. A single crystal X-ray diffraction study of the representative complex [(Cu(L)(PPh3)2]CIO4 (2) reveals a distorted tetrahedral geometry around Cu(I). Crystal data of (2): space group = C2/c, a = 42.8596 (9) Å, b = 14.6207 (3) Å, c = 36.4643 (7) Å, V = 20,653.7 (7) Å(3), Z = 16. Complexes 1-4 exhibit quasireversible redox behaviour corresponding to a Cu(I)/Cu(II) couple. All complexes show blue-green emission as a result of fluorescence from an intra-ligand charge transition (ILCT), ligand to ligand charge transfer transition (LLCT) or mixture of both. Significant increase in size of the counter anion shows marked effect on quantum efficiency and lifetime of the complexes in solution.
DNA can be programmed to assemble into a variety of shapes and patterns on the nanoscale and can act as a template for hybrid nanostructures such as conducting wires, protein arrays and field-effect transistors. Current DNA nanostructures are typically in the sub-micrometre range, limited by the sequence space and length of the assembled strands. Here we show that on a patterned biochip, DNA chains collapse into one-dimensional (1D) fibres that are 20 nm wide and around 70 µm long, each comprising approximately 35 co-aligned chains at its cross-section. Electron beam writing on a photocleavable monolayer was used to immobilize and pattern the DNA molecules, which condense into 1D bundles in the presence of spermidine. DNA condensation can propagate and split at junctions, cross gaps and create domain walls between counterpropagating fronts. This system is inherently adept at solving probabilistic problems and was used to find the possible paths through a maze and to evaluate stochastic switching circuits. This technique could be used to propagate biological or ionic signals in combination with sequence-specific DNA nanotechnology or for gene expression in cell-free DNA compartments.
Polychlorinated biphenyls (PCBs) are a group of chemicals that persist in the environment, indoors, and humans. Lung exposure to airborne and food contaminants, such as PCBs, may cause possible lung disorders, such as cancer. In the present study, we investigated the effects of structurally different lower chlorinated (≤4Cl), noncoplanar PCB40, and coplanar PCB77 on human lung fibroblast cell line (HELF) cell proliferation, cell cycle progression, and possible molecular mechanisms. Noncoplanar PCB40 and coplanar PCB77 exhibited concentration- and time-dependent biphasic dose-response effects on HELF cell proliferation. Noncoplanar PCB40 and coplanar PCB77 induced 23 and 45% cytotoxicity at higher concentrations than the control. The flow cytometry analysis showed that exposure to PCB40 caused a significant increase in time spent in the G1 phase but decreased length of the S phase in a concentration- and time-dependent manner, whereas PCB77 exposure decreased time spent in the G1 and S phases but increased time spent in the G2 phase. Western blot analysis indicated that PCB77 increased the expression of cyclin E, CDK2, p21, and caspase-9, while PCB40 decreased the expression of these proteins (except CDK2 and p21). An increase in CDK expression after exposure to PCB77 suggests that it may cause carcinogenic effects on HELF cells at higher doses. Our results also demonstrate that the different cytotoxic effects induced by coplanar and nonplanar PCBs were correlated with their structural characteristics; the coplanar congener was more cytotoxic than the nonplanar congener. The study elaborates threshold levels for these chemicals and suggests that the cytotoxicity mechanisms by which PCB congeners act on HELF cells depend on their planarity and chemical structures. Furthermore, the study will be important for developing antidotes to the adverse effects and risk assessment practices for PCBs. © 2016 Wiley Periodicals, Inc. Environ Toxicol, 2016.
Influenza A virus (IAV) is an important pathogen that has a wide range of hosts and represents a threat to the health of humans and several animal species. IAV infection can induce the transcription of many genes in the host. In the present study, we demonstrated for the first time that three different strains of H1N1 IAV induce the expression of an IFN-stimulated gene, ISG20. We determined the antiviral activity of ISG20 against IAV because ISG20 inhibited viral protein expression and reduced the progeny viral titer dependent upon its exonuclease activity. To elucidate the detailed mechanism of ISG20, we further demonstrated that ISG20 impairs the polymerase activity and inhibits both the replication and transcription levels of the M1 and NP genes. Notably, we identified that ISG20 colocalizes and interacts with NP during IAV infection, while exonuclease-inactive mutant ISG20 lacked association with NP, indicating that ISG20 inhibits IAV replication by interacting with NP. Together, these data provide a detailed explanation for the specific antiviral action of ISG20 and suggest that ISG20 may act as a promising antiviral drug candidate against IAV.
Type 2 innate lymphoid cells (ILC2s) have been shown to produce large amounts of type 2 cytokines in a non-antigen-specific manner. These cytokines act upstream and downstream of ILC2 and are increasingly common in asthma drug development, thus warranting a closer investigation of the mechanism-related clinical manifestations of ILC2 in the selection of patients with asthma. We hypothesized that IL-13(+)ILC2s in the circulation might correlate with asthma control status as a result of persistent T-helper cell type 2 (Th2) inflammation in the lung. Furthermore, we aimed to explore ILC2s' responsiveness to glucocorticoid. The percentages of ILC2s and IL-13(+)ILC2s in different asthma subgroups were checked, and correlation analyses between ILC2s and asthma-related clinical parameters were performed. Dexamethasone treatments in ILC2s and Th2 cells were performed to clarify their response properties. ILC2s were identified as a Lin(-)CD45(hi)IL-7Rα(+)CRTH2(+) cell population distinct from human peripheral blood mononuclear cells. Frequencies of ILC2s were increased dramatically in those with asthma (0.04 ± 0.02%) compared with healthy donors (0.025 ± 0.011%). The percentages of IL-13(+)ILC2s were significantly higher in patients in the uncontrolled group (49.7 ± 16.9%) and partly controlled groups (30.8 ± 13.1%) than in those in the well-controlled group (16.7 ± 5.9%) and healthy control subjects (18.7 ± 8.7%). Effective treatment of uncontrolled IL-13(+)ILC2-positive patients with asthma resulted in dynamic modulation of IL-13(+)ILC2 levels back to baseline. ILC2s were more resistant to glucocorticoid than Th2 cells in vitro. ILC2s are strong responders to IL-25/IL-33 stimulation. IL-13(+)ILC2s show a positive correlation with patient asthma control status and are more resistant to glucocorticoid than Th2 cells in humans.
The fiduciary nature of the patient-physician relationship requires clinicians to act in the best interest of their patients. Patients are vulnerable due to their health status and lack of medical knowledge, which makes them dependent on the clinicians' expertise. Competent patients, however, may reject the recommendations of their physician, either refusing beneficial medical interventions or procedures based on their personal views that do not match the perceived medical indication. In some instances, the patients' refusal may jeopardize their health or life but also compromise the clinician's moral responsibility to promote the patient's best interests. In other words, health professionals have to deal with patients whose behavior and healthcare decisions seem counterproductive for their health, or even deteriorate it, because of lack of knowledge, bad habits or bias without being the patients' free voluntary choice. The moral dilemma centers on issues surrounding the limits of the patient's autonomy (rights) and the clinician's role to promote the well-being of the patient (duties). In this paper we argue that (1) the use of manipulative strategies, albeit considered beneficent, defeats the purpose of patient education and therefore should be rejected; and (2) the appropriate strategy is to empower patients through patient education which enhances their autonomy and encourages them to become full healthcare partners as opposed to objects of clinical intervention or entities whose values or attitudes need to be shaped and changed through education. First, we provide a working definition of the concept of patient education and a brief historical overview of its origin. Second, we examine the nature of the patient-physician relationship in order to delineate its boundaries, essential for understanding the role of education in the clinical context. Third, we argue that patient education should promote self-rebiasing, enhance autonomy, and empower patients to determine their therapeutic goals. Finally, we develop a moral framework for patient education.
Mitogen-activated protein kinases (MAPKs) are expressed in postmitotic neurons and act as important regulators in intracellular signaling. In addition to their nuclear distribution and roles in regulating gene expression, MAPKs, especially the extracellular signal-regulated kinase (ERK) subclass, reside in peripheral dendritic spines and synapses, including the postsynaptic density (PSD) microdomain. This peripheral pool of MAPKs/ERKs is either constitutively active or sensitive to changing synaptic input. Active MAPKs directly interact with and phosphorylate local substrates to alter their trafficking and subcellular/subsynaptic distributions, through which MAPKs regulate function of substrates and contribute to long-lasting synaptic plasticity. A number of physiologically relevant substrates of MAPKs have been identified at synaptic sites. Central among them are key synaptic scaffold proteins (PSD-95 and PSD-93), cadherin-associated proteins (δ-catenin), Kv4.2 K(+) channels, and metabotropic glutamate receptors. Through a reversible phosphorylation event, MAPKs rapidly and efficiently modulate the function of these substrates and thus determine the strength of synaptic transmission. This review summarizes the recent progress in cell biology of synaptic MAPKs and analyzes roles of this specific pool of MAPKs in regulating local substrates and synaptic plasticity.
Oxidative stress causes cellular damage by (i) altering protein stability, (ii) impairing organelle function, or (iii) triggering the formation of 4-HNE protein aggregates. The catabolic process known as autophagy is an antioxidant cellular response aimed to counteract these stressful conditions. Therefore, autophagy might act as a cytoprotective response by removing impaired organelles and aggregated proteins. In the present study, we sought to understand the role of autophagy in the clearance of 4-HNE protein aggregates in ARPE-19 cells under rotenone exposure. Rotenone induced an overproduction of reactive oxygen species (ROS), which led to an accumulation of 4-HNE inclusions, and an increase in the number of autophagosomes. The latter resulted from a disturbed autophagic flux rather than an activation of the autophagic synthesis pathway. In compliance with this, rotenone treatment induced an increase in LC3-II while upstream autophagy markers such as Beclin- 1, Vsp34 or Atg5-Atg12, were decreased. Rotenone reduced the autophagosome-to-lysosome fusion step by increasing tubulin acetylation levels through a ROS-mediated pathway. Proof of this is the finding that the free radical scavenger, N-acetylcysteine, restored autophagy flux and reduced rotenone-induced tubulin hyperacetylation. Indeed, this dysfunctional autophagic response exacerbates cell death triggered by rotenone, since 3-methyladenine, an autophagy inhibitor, reduced cell mortality, while rapamycin, an inductor of autophagy, caused opposite effects. In summary, we shed new light on the mechanisms involved in the autophagic responses disrupted by oxidative stress, which take place in neurodegenerative diseases such as Huntington or Parkinson diseases, and age-related macular degeneration.
c-Jun N-terminal kinases (JNKs), which belong to a mitogen-activated protein kinase (MAPK) family, are involved in the regulation of several physiological functions in mammals and act as mediators of apoptosis, obesity, and memory storage in the brain, including the processes of neuronal de- and regeneration. JNK subfamily is encoded by three separate but related genes: jnk1, jnk2, and jnk3, giving rise to at least ten distinct splice variants of the JNK proteins. JNK3 is thought to be a major contributor to neurodegeneration in mammalian brain. The role of JNK1 in the pathological processes affecting cognitive function, especially in diseases such as Alzheimer's disease (AD), is less clear. In order to evaluate the effects of JNK1 deficiency in an experimental model of familial Alzheimer's disease, double transgenic APPswe/PS1dE9 mice were crossed with the JNK1 heterozygous deficient animals (jnk1+/-). As expected, a ∼50 % reduction in JNK1 protein levels was observed in the hippocampi of 9-month-old APPswe/PS1dE9/jnk1+/- mice, compared with the APPswe/PS1dE9 group. JNK1 deficiency resulted in reduced BACE1 expression, suggesting alterations in amyloidogenic pathway. However, no significant inter-group differences in the total number of β-amyloid plaques were observed in the hippocampal region. In addition, protein levels of PPAR gamma coactivator-1α (PGC-1α), a molecule involved in mitochondrial biogenesis and energy homeostasis, were decreased in 9-month-old APPswe/PS1dE9 mice but not in APPswe/PS1dE9/jnk1+/- animals. Furthermore, JNK1 deficiency did not have an effect on pro-inflammatory marker expression in the hippocampus. Heterozygous deficiency of JNK1 results in the decrease of BACE1 protein levels, which is not accompanied by the reduction in the total number of β-amyloid plaques in the hippocampi of APPswe/PS1dE9 mice. Moreover, PGC-1α expression is restored in APPswe/PS1dE9/jnk1+/- animals, which indicates a possible role of JNK1 in brain mitochondrial regulation. Nevertheless, our results suggest that partial inhibition of JNK1 is not sufficient to prevent the neuropathological processes in this model. It may be necessary to inhibit both the JNK1 and JNK3 simultaneously, especially as previous studies suggest that JNK3 contributes to AD neuropathology.
Although previous studies have discussed the risk factors of unplanned suicide behavior in several countries, the unplanned suicide attempt in China was not explored in a large sample. We aim to look into the characteristics of unplanned suicide attempters in China and compare them with those suicide attempters with plans. Subjects were 791 medically serious suicide attempters aged 15-54 years in rural China. The sixth item of Beck's Suicide Intent Scale was used to estimate the planned and unplanned suicide attempt. Logistic regression analysis was performed to examine the factors related to planned or unplanned suicide attempt. The results showed that the planned suicide attempts were associated with higher education, hopelessness, and previous suicide act. The unplanned suicide attempt tends to be suicide by pesticide and store pesticide at home. A ban of lethal pesticides may be a method for suicide prevention in rural China.
Unsupported or "against-gravity" reaching and hand opening movements are greatly impaired in individuals with hemiparetic stroke. The reduction in reaching excursion and hand opening is thought to be primarily limited by abnormal muscle co-activation of shoulder abductors with distal limb flexors, known as flexion synergy, that results in a loss of independent joint control or joint individuation. Our laboratory employs several methods for quantifying this movement impairment, however the most documented techniques are sophisticated and laboratory-based. Here a series of robotic methods that vary in complexity from comprehensive (laboratory-based) to focused (clinically relevant) are outlined in detail in order to facilitate translation and make recommendations for utilization across the translational spectrum as part of Journal of NeuroEngineering and Rehabilitation thematic series, "Technically-advanced assessments in sensory motor rehabilitation." While these methods focus on our published work utilizing the device, ACT(3D), these methods can be duplicated using any mechatronic device with the appropriate characteristics. The common thread and most important aspect of the methods described is addressing the deleterious effects of abduction loading. Distal upper extremity joint performance is directly and monotonically modulated by proximal (shoulder abduction) joint demands. The employment of robotic metrics is the best tool for selectively manipulating shoulder abduction task requirements spanning the individual's full range of shoulder abduction strength. From the series of methods and the concluding recommendations, scientists and clinicians can determine the ideal robotic quantification method for the measurement of the impact of loss of independent joint control on reaching and hand function.
An evening chorus centered at near 2.2 kHz was detected across the years 2000 to 2014 from seabed receivers in 430-490 m depth overlooking the Perth Canyon, Western Australia. The chorus reached a maximum level typically 2.1 h post-sunset and normally ran for 2.1 h (between 3 dB down points). It was present at lower levels across most of the hours of darkness. Maximum chorus spectrum levels were 74-76 dB re 1 μPa(2)/Hz in the 2 kHz 1/3 octave band, averaging 6-12 dB and up to 30 dB greater than pre-sunset levels. The chorus displayed highest levels over April to August each year with up to 10 dB differences between seasons. The spatial extent of the chorus was not determined but exceeded the sampling range of 13-15 km offshore from the 300 m depth contour and 33 km along the 300 m depth contour. The chorus comprised short damped pulses. The most likely chorus source is considered to be fishes of the family Myctophidae foraging in the water column. The large chorus spatial extent and its apparent correlation with regions of high productivity suggest it may act as an acoustic beacon to marine fauna indicating regions of high biomass.
Glycoconjugates, molecules that contain sugar components, are major components of the cell envelopes of bacteria and cover much of their exposed surfaces. These molecules are involved in interactions with the surrounding environment and, in pathogens, play critical roles in the interplay with the host immune system. Despite the remarkable diversity in glycoconjugate structures, most are assembled by glycosyltransferases that act on lipid acceptors at the cytosolic membrane. The resulting glycolipids are then transported to the cell surface in processes that frequently begin with ATP-binding cassette transporters. This review summarizes current understanding of the structure and biosynthesis of glycolipid substrates and the structure and functions of their transporters. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.
Sulfur dioxide (SO2) is one of the main atmospheric pollutants worldwide, and is reported to be responsible for the formation of severe haze in China. Some studies have demonstrated a potential harmful effect of SO2 on the male reproductive system; however the underlying mechanism is still unknown. The purpose of this study is to investigate the roles of cytochrome P450 (P450), cAMP-responsive element modulator (CREM), and activator of CREM (ACT) in SO2-induced toxicity. Forty-eight male Wistar rats were randomly divided into an experimental and control group. The experiment group was exposed to SO2 in ambient air (10ppm, 4h/day), and the control group was treated with filtered air in the same conditions. After 2 weeks, the results showed a significant decrease in body weight and sperm motility, and an increase in the testis weight-to-body weight ratio as compared to the control group. Histological investigation suggested that SO2 exposure led to loose arrangement of the spermatogenic cells and local structural damage in the seminiferous tubules. Moreover, the expressions of P450, CREM and ACT proteins increased in the testes by 0.22%, 47.26% and 23.38%, respectively. Taken together, SO2 inhalation lowered sperm quality, altered testicular histology, and increased expressions of CREM and ACT proteins in the testes of rats. Overall, these results could contribute to a better understanding of SO2-induced male reproductive toxicity.
Lipoproteins are a set of natural nanoparticles whose main role is the transport of fats within the body. While much work has been done to develop synthetic nanocarriers to deliver drugs or contrast media, natural nanoparticles such as lipoproteins represent appealing alternatives. Lipoproteins are biocompatible, biodegradable, non-immunogenic and are naturally targeted to some disease sites. Lipoproteins can be modified to act as contrast agents in many ways, such as by insertion of gold cores to provide contrast for computed tomography. They can be loaded with drugs, nucleic acids, photosensitizers or boron to act as therapeutics. Attachment of ligands can re-route lipoproteins to new targets. These attributes render lipoproteins attractive and versatile delivery vehicles. In this review we will provide background on lipoproteins, then survey their roles as contrast agents, in drug and nucleic acid delivery, as well as in photodynamic therapy and boron neutron capture therapy.
The main goal of drug delivery systems is to target therapeutic cargoes to desired cells and to ensure their efficient uptake. Recently a number of studies have focused on designing bio-inspired nanocarriers, such as bacteriophages, and synthetic carriers based on the bacteriophage structure. Bacteriophages are viruses that specifically recognize their bacterial hosts. They can replicate only inside their host cell and can act as natural gene carriers. Each type of phage has a particular shape, a different capacity for loading cargo, a specific production time, and their own mechanisms of supramolecular assembly, that have enabled them to act as tunable carriers. New phage-based technologies have led to the construction of different peptide libraries, and recognition abilities provided by novel targeting ligands. Phage hybridization with non-organic compounds introduces new properties to phages and could be a suitable strategy for construction of bio-inorganic carriers. In this review we try to cover the major phage species that have been used in drug and gene delivery systems, and the biological application of phages as novel targeting ligands and targeted therapeutics.
Antiangiogenesis therapy has been served as a potent cancer treatment strategy for decades, yet disrupting neovasculature would provoke tumor cells into invasive growth and result in distal metastasis. The basic cause of cancer metastasis can be traced down to the presence of circulating tumor cells (CTCs) which detach from primary tumor site and act as 'seeds'. Epithelial cell adhesion molecule (EpCAM) is a potential biomarker for selective capture of epithelium-derived CTCs. Here, we integrated tumor neovessles-targetable ligands K237 peptide with Ep23 aptamer against EpCAM into a single drug-loaded nanoplatform using paclitaxel (PTX) as the model drug, aiming at damaging the primary tumor and neutralizing CTCs simultaneously to achieve a synergistic anti-tumor therapeutic effect. Enhanced cellular uptake, cell apoptosis-induction and cell-viability inhibition efficiency of the peptide and aptamer dual-functionalized nanoparticles (dTNP) were observed in both human umbilical vein endothelial cells (HUVEC) and 4T1 cells in vitro. Using cone-and-plate viscometer to create venous flow velocity, dTNP was also found to be able to capture CTCs under shear stress. The CTC-targeting and neutralization effect of dTNP in bloodstream and 4T1-GFP cell-derived lung metastasis mice model was confirmed via in vivo flow cytometry (IVFC), intravital imaging and confocal microscopy analysis. As a result, the orthotropic breast tumor-bearing mice administrated with PTX-loaded dTNP exhibited the optimal therapeutic effect. Taken together, the findings here provided direct evidence that the tumor neovasculature and CTCs dual-targeting drug delivery system could provide a novel modality for the treatment of highly-invasive breast cancer.
Gibberellins (GAs) are plant hormones that regulate most plant life cycle aspects, including flowering and fruit development. Here, we demonstrate the implication of GAs in ovule development. DELLA proteins, negative GA response regulators, act as positive factors for ovule integument development in a mechanism that involves transcription factor ABERRANT TESTA SHAPE (ATS). The seeds of the della global mutant, a complete loss-of-function of DELLA, and the ats-1 mutant are remarkably similar, with a round shape, a disorganized testa, and viviparism. These defects are the result of an alteration in integuments that fail to fully develop and are shorter than in wild-type plants. ats-1 also shows some GA-related phenotypes, for example, higher germination rates and early flowering. In fact, ats-1 has elevated GA levels due to the activation of GA biosynthesis genes, which indicates that ATS inhibits GA biosynthesis. Moreover, DELLAs and ATS proteins interact, which suggests the formation of a transcriptional complex that regulates the expression of genes involved in integument growth. Therefore, the repression of GA biosynthesis by ATS would result in the stabilization of DELLAs to ensure correct ATS-DELLA complex formation. The requirement of both activities to coordinate proper ovule development strongly argues that the ATS-DELLA complex acts as a key molecular factor. This work provides the first evidence for a role of GAs in ovule and seed development.
Administration of home parenteral support (HPS) has proven to be cost-effective over hospital care. Avoiding hospital readmissions became more of a focus for healthcare institutions in 2012 with the implementation of the Affordable Care Act. In 2010, our service developed a protocol to treat dehydration at home for HPS patients by ordering additional intravenous fluids to be kept on hand and to focus patient education on the symptoms of dehydration.
A retrospective analysis was completed through a clinical management database to identify HPS patients with dehydration. The hospital finance department and homecare pharmacy were utilized to determine potential cost avoidance.
In 2009, 64 episodes (77%) of dehydration were successfully treated at home versus 6 emergency department (ED) visits (7.5%) and 13 readmissions (15.5%). In 2010, we successfully treated 170 episodes (84.5%) at home, with 9 episodes (4.5%) requiring ED visits and 22 hospital readmissions (11%). The number of dehydration episodes per patient was significantly higher in 2010 (P < .001) and may be attributed to a shift in the patient population, with more patients having malabsorption as the indication for therapy in 2010 (P = .003).
There were more than twice as many episodes of dehydration identified and treated at home in 2010 versus 2009. Our protocol helped educate and provide the resources required to resolve dehydration at home when early signs were recognized. By reducing ED visits and hospital readmissions, healthcare costs were avoided by a factor of 29 when home treatment was successful.
Treatment with pulsed electromagnetic fields (PEMFs) is emerging as an interesting therapeutic option for patients with cancer. The literature has demonstrated that low-frequency/low-energy electromagnetic fields do not cause predictable effects on DNA; however, they can epigenetically act on gene expression. The aim of the present work was to study a possible epigenetic effect of a PEMF, mediated by miRNAs, on a human glioblastoma cell line (T98G). We tested a PEMF (maximum magnetic induction, 2 mT; frequency, 75 Hz) that has been demonstrated to induce autophagy in glioblastoma cells. In particular, we studied the effect of PEMF on the expression of genes involved in cancer progression and a promising synergistic effect with temozolomide, a frequently used drug to treat glioblastoma multiforme. We found that electromagnetic stimulation in combination with temozolomide can elicit an epigenetic pro-apoptotic effect in the chemo- and radioresistant T98G glioblastoma cell line.
Regulated mRNA translation plays an important role in normal cellular functions and cytoplasmic polyadenylation element binding proteins (CPEBs) are the key factors that control the elongation of poly(A) tail during translation. The expression of various CPEBs has been noted to be linked to tumorigenesis, tumor growth, invasiveness and angiogenesis; however, different CPEBs appear to play diverse roles in cancer. The evidence from the literature suggests that CPEB1 and CPEB3 act more likely as tumor suppressors; in contrast, CPEB2 and CPEB4 mainly exert oncogenic effects. In addition, different CPEB subtypes may interact with each other to regulate tumorigenesis. All four CPEB mRNAs contain multiple microRNA (miRNA) binding sites, while the functions of CPEBs are regulated by various miRNAs. These results indicate that CPEBs play a significant role in tumorigenesis; therefore, manipulation of the expression of different subtypes of CPEBs might modulate the behavior of cancer cells and provide new therapeutic concepts for cancer therapy. However, more studies are required to clarify their definite role in tumor development.
The paraventricular nucleus of the thalamus (PVT) has been implicated in behavioral responses to reward-associated cues. However, the precise role of the PVT in these behaviors has been difficult to ascertain since Pavlovian-conditioned cues can act as both predictive and incentive stimuli. The "sign-tracker/goal-tracker" rat model has allowed us to further elucidate the role of the PVT in cue-motivated behaviors, identifying this structure as a critical component of the neural circuitry underlying individual variation in the propensity to attribute incentive salience to reward cues. The current study assessed differences in the engagement of specific PVT afferents and efferents in response to presentation of a food-cue that had been attributed with only predictive value or with both predictive and incentive value. The retrograde tracer fluorogold (FG) was injected into the PVT or the nucleus accumbens (NAc) of rats, and cue-induced c-Fos in FG-labeled cells was quantified. Presentation of a predictive stimulus that had been attributed with incentive value elicited c-Fos in PVT afferents from the lateral hypothalamus, medial amygdala (MeA), and the prelimbic cortex (PrL), as well as posterior PVT efferents to the NAc. PVT afferents from the PrL also showed elevated c-Fos levels following presentation of a predictive stimulus alone. Thus, presentation of an incentive stimulus results in engagement of subcortical brain regions; supporting a role for the hypothalamic-thalamic-striatal axis, as well as the MeA, in mediating responses to incentive stimuli; whereas activity in the PrL to PVT pathway appears to play a role in processing the predictive qualities of reward-paired stimuli.
The mechanism of lithium's therapeutic action remains obscure, hindering the discovery of safer treatments for bipolar disorder. Lithium can act as an inhibitor of the kinase GSK3α/β, which in turn negatively regulates β-catenin, a co-activator of LEF1/TCF transcription factors. However, unclear is whether therapeutic levels of lithium activate β-catenin in the brain, and whether this activation could have a therapeutic significance. To address this issue we chronically treated mice with lithium. Although the level of non-phospho-β-catenin increased in all of the brain areas examined, β-catenin translocated into cellular nuclei only in the thalamus. Similar results were obtained when thalamic and cortical neurons were treated with a therapeutically relevant concentration of lithium in vitro. We tested if TCF7L2, a member of LEF1/TCF family that is highly expressed in the thalamus, facilitated the activation of β-catenin. Silencing of Tcf7l2 in thalamic neurons prevented β-catenin from entering the nucleus, even when the cells were treated with lithium. Conversely, when Tcf7l2 was ectopically expressed in cortical neurons, β-catenin shifted to the nucleus, and lithium augmented this process. Lastly, we silenced tcf7l2 in zebrafish and exposed them to lithium for 3 days, to evaluate whether TCF7L2 is involved in the behavioral response. Lithium decreased the dark-induced activity of control zebrafish, whereas the activity of zebrafish with tcf7l2 knockdown was unaltered. We conclude that therapeutic levels of lithium activate β-catenin selectively in thalamic neurons. This effect is determined by the presence of TCF7L2, and potentially contributes to the therapeutic response.
In this paper we present a comprehensive study for the ability of thermoresponsive nanogels (tNG) to act as cutaneous penetration enhancers. Given the unique properties of such molecular architectures with regard to their chemical composition and thermoresponsive properties, we propose a particular mode of penetration enhancement mechanism, i.e. hydration of the stratum corneum. Different tNG were fabricated using dendritic polyglycerol as a multifunctional crosslinker and three different kinds of thermoresponsive polymers as linear counterpart: poly(N-isopropylacrylamide) (pNIPAM), p(di(ethylene glycol) methyl ether methacrylate - co - oligo ethylene glycol methacrylate) (DEGMA-co-OEGMA475), and poly(glycidyl methyl ether - co - ethyl glycidyl ether) (tPG). Excised human skin was investigated by means of fluorescence microscopy, which enabled the detection of significant increment in the penetration of tNG as well as the encapsulated fluorescein. The morphology of the treated skin samples was thoroughly investigated by transmission electron microscopy and stimulated Raman spectromicroscopy. We found that tNG can perturbate the organization of both proteins and lipids in the skin barrier, which was attributed to tNG hydration effects. Interestingly, different drug delivery properties were detected and the ability of each investigated tNG to enhance skin penetration correlated well with the degree of induced stratum corneum hydration. The differences in the penetration enhancements could be attributed to the chemical structures of the nanogels used in this study. The most effective stratum corneum hydration was detected for nanogels having additional or more exposed polyether structure in their chemical composition.
Our previous study showed that IPPA08, a cis-configuration neonicotinoid compound with unique oxabridged substructure, acted as a specific synergist to neonicotinoid insecticides targeting nicotinic acetylcholine receptors (nAChRs). Heteropentamer nAChRs have diverse characteristics and can form canonical and noncanonical subunit interfaces. While canonical interfaces have been exploited as targets of many drugs, noncanonical interfaces have received less attention. In this study, the mechanism of IPPA08 synergism was evaluated on hybrid nAChRs consisting of three α1 subunits from the brown planthopper and two rat β1 subunits (Nlα1/rβ2) expressed in Xenopus oocytes. IPPA08 alone evoked inward currents, but only at very high concentrations, greater than 1 mM. However, at concentrations below 200 μM, IPPA08 slowed the decay of inward currents evoked by imidacloprid, but not by acetylcholine, and also increased the sensitivity of Nlα1/rβ2 to imidacloprid. Both modulations by IPPA08 were concentration-dependent in the same concentration range of 10-150 μM. Experimentally induced mutations in canonical (α+/β-) and noncanonical (β+/α-) interfaces of Nlα1/rβ2 receptors were also examined to evaluate the presence of possible binding sites for IPPA08 on the receptors. Our results showed that mutations in the canonical interfaces affected only the potency of IPPA08 as an agonist, while mutations in the noncanonical interfaces affected only the synergistic action of IPPA08. Based on these results, we propose that at low concentrations IPPA08 can act as a positive allosteric modulator of noncanonical interfaces, and likely slow the decay of currents through stabilizing the open-channel state caused by the action of imidacloprid on canonical interfaces.
Antibiotics inhibiting protein translation have long been used to treat and prevent infections by apicomplexan parasites. These compounds kill parasites by inhibiting organellar translation, and most act specifically against the apicoplast, a relict plastid in apicomplexans. Drug resistance in Plasmodium and other apicomplexans dictates a need for development of novel targets. Some apicoplast inhibitors have a delayed onset of action, so they cannot replace fast-acting drugs, although they still fulfil important roles in treating and preventing infections. The plethora of bacterial-like actors in the translation machinery of parasite mitochondria and plastids presents validated targets with strong potential for selectivity. Here we discuss existing drugs that inhibit organellar translation, and explore targets that may be further exploited in antiparasitic drug design.
Although the available pool of qualified underrepresented minority and women medical school graduates has expanded in recent decades, their representation in the radiological professions has improved only marginally. Recognizing this deficit in diversity, many professional medical societies, including the ACR, have incorporated these values as core elements of their missions and instituted programs that address previously identified barriers to a more diverse workforce. These barriers include insufficient exposure of underrepresented minorities and women to radiology and radiation oncology; misperception of these specialties as non-patient care and not community service; unconscious bias; and delayed preparation of candidates to compete successfully for residency positions. Critical success factors in expanding diversity and inclusion are well identified both outside and within the radiological professions; these are reviewed in the current communication. Radiology leaders are positioned to lead the profession in expanding the diversity and improving the inclusiveness of our professional workforce in service to an increasingly diverse society and patient population.
Scientists and policy-makers globally are calling for alternative approaches to conventional intensification of agriculture that enhance ecosystem services provided by biodiversity. The evidence reviewed here suggests that alternative approaches can achieve high crop yields and profits, but the performance of other socioeconomic indicators (as well as long-term trends) is surprisingly poorly documented. Consequently, the implementation of conventional intensification and the discussion of alternative approaches are not based on quantitative evidence of their simultaneous ecological and socioeconomic impacts across the globe. To close this knowledge gap, we propose a participatory assessment framework. Given the impacts of conventional intensification on biodiversity loss and greenhouse gas emissions, such evidence is urgently needed to direct science-policy initiatives, such as the United Nations (UN) 2030 Agenda for Sustainable Development.
Jehovah's Witnesses patients refuse blood transfusions for religious reasons. Anesthesiologists must master specific legal knowledge to provide care to these patients. Understanding how the Law and the Federal Council of Medicine treat this issue is critical to know how to act in this context. The aim of this paper was to establish a treatment protocol for the Jehovah's Witness patient with emphasis on ethical and legal duty of the anesthesiologist.
The article analyzes the Constitution, Criminal Code, resolutions of the Federal Council of Medicine, opinions, and jurisprudence to understand the limits of the conflict between the autonomy of will of Jehovah's Witnesses to refuse transfusion and the physician's duty to provide the transfusion. Based on this evidence, a care protocol is suggested.
The Federal Council of Medicine resolution 1021/1980, the penal code Article 135, which classifies denial of care as a crime and the Supreme Court decision on the HC 268,459/SP process imposes on the physician the obligation of blood transfusion when life is threatened. The patient's or guardian's consent is not necessary, as the autonomy of will manifestation of the Jehovah's Witness patient refusing blood transfusion for himself and relatives, even in emergencies, is no not forbidden.
Humans have coevolved over time to not only tolerate but also rely on trillions of microbes that aid in the development of our immune system, provide nutrients, break down potentially noxious substances, and act as a barrier against potentially pathogenic organisms. These microbes, collectively known as the microbiota, live in relatively stable communities on mucosal surfaces such as the respiratory tract and gastrointestinal tract. Changes to the microbiota are often transient, due to changes in diet, antibiotic exposure, and psychological stressor exposure. This chapter will discuss how psychological stressors can shape the intestinal microbial community and how these perturbations can contribute to stressor-induced changes in immune function, neurodevelopment, and behavioral deficits.
TRIM62 (tripartite motif containing 62) has been found to act as a tumor suppressor of several cancers. However, its precise biological role and related mechanism remain unknown in cervical cancer (CC).
Quantitative Real-time PCR and western blot were adopted to detect the mRNA and protein expression level of TRIM62 in both human CC cell lines and tissues. Immunohistochemistry was used to measure the TRIM62 expression in 30 normal cervical and 189 CC tissues. Univariate and multivariate Cox regression analyses and Kaplan-Meier survival analyses performed to investigate the association between TRIM62 expression and CC patients' prognosis. The effect of TRIM62 on CC growth and metastasis was studied in vitro and in vivo. Multi-pathway reporter array were utilized to identify the potential signaling manipulated by TRIM62.
TRIM62 was frequently down-regulated in both human CC cells and tissues. Low expression of TRIM62 in CC tissues was associated with aggressive clinicopathological features of CC patients. In addition, TRIM62 was also an independent poor prognostic factor for overall and disease-free survival of CC patients after surgery. Moreover, enforced expression of TRIM62 in CC cells significantly inhibited their abilities of proliferation, migration and invasion in vitro. Besides, subcutaneous xenograft tumor model and xenograft mouse metastatic model respectively displayed that TRIM62 impeded the growth and metastasis of CC in vivo. Furthermore, mechanism study exhibited that TRIM62 could suppress epithelial-mesenchymal transition (EMT) by inhibiting c-Jun/Slug signaling. The inhibitory role of TRIM62 in tumor proliferation might be through regulating cell cycle related proteins CyclinD1 and P27 by targeting c-Jun.
TRIM62 is a potential prognostic biomarker in CC and suppresses metastasis of CC via inhibiting c-Jun/Slug signaling-mediated EMT.
The majority of content in an Internet Support Group (ISG) is contributed by 1 % of the users ('super users'). Computational methods, such as topic modelling, can provide a large-scale quantitative objective description of this content. Such methods may provide a new perspective on the nature of engagement on ISGs including the role of super users and their possible effect on other users.
A topic model was computed for all posts (N = 131,004) in the ISG BlueBoard using Latent Dirichlet Allocation. A model containing 25 topics was selected on the basis of intelligibility as determined by diagnostic metrics and qualitative investigation. This model yielded 21 substantive topics for further analysis. Two chi-square tests were conducted separately for each topic to ascertain: (i) if the odds of super users' and other users' posting differed for each topic; and (ii) if for super users the odds of posting differed depending on whether the response was to a super user or to another user.
The 21 substantive topics covered a range of issues related to mental health and peer-support. There were significantly higher odds that super users wrote content on 13 topics, with the greatest effects being for Parenting Role (OR [95%CI] = 7.97 [7.85-8.10]), Co-created Fiction (4.22 [4.17-4.27]), Mental Illness (3.13 [3.11-3.16]) and Positive Change (2.82 [2.79-2.84]). There were significantly lower odds for super users on 7 topics, with the greatest effects being for the topics Depression (OR = 0.27 [0.27-0.28]), Medication (0.36 [0.36-0.37]), Therapy (0.55 [0.54-0.55]) and Anxiety (0.55 [0.55-0.55]). However, super users were significantly more likely to write content on 5 out of these 7 topics when responding to other users than when responding to fellow super users.
The findings suggest that super users serve the role of emotionally supportive companions with a focus on topics broadly resembling the consumer/carer model of recovery. Other users engage in topics with a greater focus on experiential knowledge, disclosure and informational support, a pattern resembling the clinical symptom-focussed approach to recovery. However, super users modify their content in response to other users in a manner consistent with being 'active help providers'.
The repertoire of free-living protozoa in contact lens solutions is poorly known despite the fact that such protozoa may act as direct pathogens and may harbor intra-cellular pathogens.
Between 2009 and 2014, the contact lens solutions collected from patients presenting at our Ophthalmology Department for clinically suspected keratitis, were cultured on non-nutrient agar examined by microscope for the presence of free-living protozoa. All protozoa were identified by 18S rRNA gene sequencing.
A total of 20 of 233 (8.6 %) contact lens solution specimens collected from 16 patients were cultured. Acanthamoeba amoeba in 16 solutions (80 %) collected from 12 patients and Colpoda steini, Cercozoa sp., Protostelium sp. and a eukaryotic more closely related to Vermamoeba sp., were each isolated in one solution. Cercozoa sp., Colpoda sp., Protostelium sp. and Vermamoeba sp. are reported for the first time as contaminating contact lens solutions.
The repertoire of protozoa in contact lens solutions is larger than previously known.
The extent to which sex reversal is associated with transitions in sex determining systems (XX-XY, ZZ-ZW, etc.) or abnormal sexual differentiation is predominantly unexplored in amphibians. This is in large part because most amphibian taxa have homomorphic sex chromosomes, which has traditionally made it challenging to identify discordance between phenotypic and genetic sex in amphibians, despite all amphibians having a genetic component to sex determination. Recent advances in molecular techniques such as genome complexity reduction and high throughput sequencing present a valuable avenue for furthering our understanding of sex determination in amphibians and other taxa with homomorphic sex chromosomes like many fish and reptiles.
We use DArTseq as a novel approach to identify sex-linked markers in the North American green frog (Rana clamitans melanota) using lab-reared tadpoles as well as wild-caught adults from seven ponds either in undeveloped, forested habitats or suburban ponds known to be subject to contamination by anthropogenic chemicals. The DArTseq methodology identified 13 sex-linked SNP loci and eight presence-absence loci associated with males, indicating an XX-XY system. Both alleles from a single locus show partial high sequence homology to Dmrt1, a gene linked to sex determination and differentiation throughout Metazoa. Two other loci have sequence similarities to regions of the chimpanzee and human X-chromosome as well as the chicken Z-chromosome. Several loci also show geographic variation in sex-linkage, possibly indicating sex chromosome recombination. While all loci are statistically sex-linked, they show varying degrees of female heterozygosity and male homozygosity, providing further evidence that some markers are on regions of the sex chromosomes undergoing higher rates of recombination and therefore further apart from the putative sex determining locus.
The ease of the DArTseq platform provides a useful avenue for future research on sex reversal and sex chromosome evolution in vertebrates, particularly for non-model species with homomorphic or cryptic or nascent sex chromosomes.
Lin28A and Lin28B are highly conserved RNA binding proteins with similar structure and functions. Recent studies demonstrated that both of them act as oncogenes and promote cancer progression. However, few researches compared the expression and functions of both oncogenes in human malignant tumors at same time. Additionally, although the expression and role of Lin28B in colon cancer is frequently reported, the expression and functions of Lin28A in colon cancer are largely unknown. In this study, we have systematically evaluated the expressional pattern, mutation status and correlation of both Lin28A and Lin28B in colon cancer tissues for the first time, and compared the roles of Lin28A and Lin28B in the proliferation, migration, invasion and apoptosis of colon cancer cells in vitro. We have showed that they are co-expressed and have functional similarities, however, the molecular mechanisms underlying their similar functions may not be identical. This study contributes to clarify the similarities and differences of Lin28A and Lin28B in colon cancer progression.
Flavonols with varied hydroxyl substitution can act as strong antioxidants. Thanks to their ability to chelate metals as well as to donate hydrogen atoms they have capacity to scavenge free radicals. Their metal complexes are often more active in comparison with free ligands. They exhibit interesting biological properties, e.g. anticancer, antiphlogistic and antibacterial. The relationship between molecular structure and their biological properties was intensively studied using spectroscopic methods (UV-Vis, IR, Raman, NMR, ESI-MS). The aim of this paper is review on spectroscopic analyses of molecular structure and biological activity of hydroxyflavonol metal complexes.
Most natural assets, including native biodiversity (our focus), are under increasing threat from direct (loss of habitat, hunting) and indirect (climate change) human actions. Most human impacts arise from increasing human populations coupled with rises in per capita resource use. The rates of change of human actions generally outpace those to which the biota can respond or adapt. If we are to maintain native biodiversity, then we must develop ways to envisage how the biota may be affected over the next several decades to guide management and policy responses. We consider the future for Australia's native biodiversity in the context of two assumptions. First, the human population in Australia will be 40million by 2050, which has been mooted by federal government agencies. Second, greenhouse gas emissions will track the highest rates considered by the Intergovernmental Panel on Climate Change. The scenarios are based on major drivers of change, which were constructed from seven key drivers of change pertinent to native biodiversity. Five scenarios deal with differing distributions of the human population driven by uncertainties in climate change and in the human responses to climate change. Other scenarios are governed largely by global change and explore different rates of resource use, unprecedented rates of technological change, capabilities and societal values. A narrative for each scenario is provided. The set of scenarios spans a wide range of possible future paths for Australia, with different implications for the future of native biodiversity.
On December 2, 1967, when Denise Darvall was hit by a car, a surgery that made medical history was unfold: Hamilton Naki, a black man, expertly removed her heart and gave it to Christian Barnard, who was preparing the receptor, Louis Washkansky, in an adjacent operating room. Naki's contribution was an outlaw act, a criminal offense under the laws of apartheid due to the difference of races; the law forbade him to cut white meat or touch white blood. Naki was perhaps the second most important man in the team that day. There were few photographs where he and Barnard appeared together, but because of the nature of society was Barnard who won the world's attention.
Reactive oxygen species act as important second messengers in cell signaling and homeostasis through the oxidation of protein thiols. However, the dynamic nature of protein oxidation and the lack of sensitivity of existing molecular probes have hindered our understanding of such reactions; therefore, new tools are required to address these challenges. We designed a bifunctional variant of the strained bicyclo[6.1.0]nonyne (BCN-E-BCN) that enables the tagging of intracellular protein sulfenic acids for biorthogonal copper-free click chemistry. In validation studies, BCN-E-BCN binds the sulfenylated form of the actin-severing protein cofilin, while mutation of the cognate cysteine residues abrogates its binding. BCN-E-BCN is cell permeable and reacts rapidly with cysteine sulfenic acids in cultured cells. Using different azide-tagged conjugates, we demonstrate that BCN-E-BCN can be used in various applications for the detection of sulfenylated proteins. Remarkably, cycloaddition of an azide-tagged fluorophore to BCN-E-BCN labelled proteins produced in vivo can be visualized by fluorescence microscopy to reveal their subcellular localization. These findings demonstrate a novel and multifaceted approach to the detection and trapping of sulfenic acids.
To demonstrate that it is possible to pursue teeth whitening treatment protocols during orthodontic treatment with no esthetic loss.
Many patients undergoing orthodontic treatment desire to have a straight and well aligned dentition, but also whiter teeth. For many years, it was believed that carrying out a whitening treatment with positioned orthodontic brackets in place would result in localized spots on the enamel labial surfaces of teeth. However, a deeper understanding of the bleaching process suggests that the oxidation caused by products, which results from hydrogen peroxide decomposition, are able to diffuse peripherally into the tooth structure and reach even that under the cemented brackets. Two in-office-bleaching treatments were performed in patients using orthodontic fixed braces in two or three 40-minute sessions using a 35% hydrogen peroxide.
In-office bleaching is possible and effective, even with orthodontic brackets in position. The teeth were successfully bleached despite the presence of brackets. All biological criteria have been fulfilled satisfying patients' expectations of aligned and whitened teeth in less time than if treatments had been performed separately, with satisfactory results and no esthetic loss.
The whitening of teeth is possible during orthodontic treatment with fixed braces without any esthetic loss. The in-office bleaching treatment with brackets in position also may act as a motivation factor, preventing patient withdrawal or treatment interruption. Therefore, at the end of the orthodontic treatment, the patient is able to display an aligned, functional and whitened smile.
The primitive genome's and the individual phenotype's modifications by epygenesys surveyes are crucial to evaluate the methabolic syndrome risk and its prevention. Through the most recent surveys, this analysis is aimed to several processes by which the various epigenetic types who are different from one to another subject may act on the methabolism key-genes, like that they even indirectly arouse the straight onset of one or more methabolic syndromes: the risk of passing this syndrome to next offsprings is more or less high. Through the new later surveyes who are originated from analysis undertaken on animals and experiments on men by in vitro and in vivo test on has proved how much the epigenetic modifications are affected either by the environmental compounds, often going back to maternal and prenatal nourishment, or by the more or less strong connection to the transcriptional genetic control : this occurs to the stochastic eventuality to pass the methabolic syndrome phenotype's to the following cohorts. Mainly in an individual observation these risks evaluation is until now indefined, buti t is going in progress and one has still waiting the issues of next new performed surveyes to can enlight on some interconnection that is following the histonic methilation and the micro-RNA not codifyng control role on some gene who is involved in these methabolic syndromes; this process is variable from one to another subject. All in conclusion is aiming to carry out the pharmacological treatment of the primary prevention to the methabolic diseases risk.
Accurately assessing the physiological status of firefighters during work in the heat is critical to ensuring their safety. Evaluating core temperatures (Tc) in the field is problematic due to cost and limitations in technology and accuracy. As such, fire services rely on individual perceptions of wellbeing. The present study aimed to establish whether perceptual responses measured using the perceptual strain index (PeSI), calculated from rate of perceived exertion (RPE) and thermal sensation (TS), could reliably predict the physiological strain (PSI) encountered by experienced firefighters working in a hot environment. We conducted two firefighting simulations (set-pace and self-paced) in a purpose built heat chamber (100 ± 5°C) comprised of two 20-minute periods separated by a 10-minute recovery outside the chamber. Physiological strain was measured via heart rate (HR) and gastrointestinal temperature (Tgi) and compared with PeSI at 5-min intervals. To evaluate the predictive ability of the PeSI for PSI, mean differences and the 95% limits of agreement (LOA) were established, along with correlation coefficients at each 5-min interval. Moderately significant correlations occurred in the second work bout of the self-paced trial only (10 min: r = 0.335, 15 min: r = 0.498, 20 min r = 0.439) with no other correlations observed at any other time during either trial or during the rest periods. Bland-Altman analysis revealed mean differences of -0.74 ± 2.70 (self-paced) and +0.04 ± 2.04 (set-paced) between PeSI and PSI with the 95% LOA being -4.77 to 3.28 (self-paced) and -4.01 to 2.01 (set-paced). The wide LOA and lack of correlations observed between perceptual and physiological strain in both self-paced and set-paced work trials indicate that PeSI is not sufficiently reliable as a sole measure of wellbeing for firefighters working in the heat. Hence, we recommend that fire services prioritise the development of reliable and effective monitoring tools for use in the field.
Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo and clinical studies (1990-2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area. A total of 29 studies were found by PubMed and www.webofknowledge.com searches: 20 were on skin (6 in vitro, 6 in vivo, 1 vitro/vivo, 7 clinical studies), while the other 9 regarded bone (1 in vitro, 2 in vivo, 1 clinical studies), cartilage (1 in vitro, 1 vitro/vivo, 2 clinical studies) or tendon (1 clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity and reduced pain and inflammation, through the activation of adenosine A2A receptor. PDRNs are currently used for bone, cartilage and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of 3 to a maximum of 5 times and they act trough the activation of A2A receptor. Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. This article is protected by copyright. All rights reserved.
The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.
Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.
Large-scale corporate projects, particularly those in extractive industries or hydropower development, have a history from early in the twentieth century of creating negative environmental, social, and health impacts on communities proximal to their operations. In many instances, especially for hydropower projects, the forced resettlement of entire communities was a feature in which local cultures and core human rights were severely impacted. These projects triggered an activist opposition that progressively expanded and became influential at both the host community level and with multilateral financial institutions. In parallel to, and spurred by, this activism, a shift occurred in 1969 with the passage of the National Environmental Policy Act in the United States, which required Environmental Impact Assessment (EIA) for certain types of industrial and infrastructure projects. Over the last four decades, there has been a global movement to develop a formal legal/regulatory EIA process for large industrial and infrastructure projects. In addition, social, health, and human rights impact assessments, with associated mitigation plans, were sequentially initiated and have increasingly influenced project design and relations among companies, host governments, and locally impacted communities. Often, beneficial community-level social, economic, and health programs have voluntarily been put in place by companies. These flagship programs can serve as benchmarks for community-corporate-government partnerships in the future. Here, we present examples of such positive phenomena and also focus attention on a myriad of challenges that still lie ahead.
Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis.
Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.
Globally, Arctic and Subarctic regions have experienced the greatest temperature increases during the last 30 years. These extreme changes have amplified threats to the freshwater ecosystems that dominate the landscape in many areas by altering water budgets. Several studies in temperate environments have examined the adaptive capacity of organisms to enhance our understanding of the potential repercussions of warming and associated accelerated drying for freshwater ecosystems. However, few experiments have examined these impacts in Arctic or Subarctic freshwater ecosystems, where the climate is changing most rapidly. To evaluate the capacity of a widespread ectotherm to anticipated environmental changes, we conducted a mesocosm experiment with wood frogs (Rana sylvatica) in the Canadian Subarctic. Three warming treatments were fully crossed with three drying treatments to simulate a range of predicted changes in wetland environments. We predicted wetland warming and drying would act synergistically, with water temperature partially compensating for some of the negative effects of accelerated drying. Across all drying regimes, a 1 °C increase in water temperature increased the odds of survival by 1.79, and tadpoles in 52-day and 64-day hydroperiod mesocosms were 4.1-4.3 times more likely to survive to metamorphosis than tadpoles in 45-day mesocosms. For individuals who survived to metamorphosis, there was only a weak negative effect of temperature on size. As expected, increased temperatures accelerated tadpole growth through day 30 of the experiment. Our results reveal that one of the dominant herbivores in Subarctic wetlands, wood frog tadpoles, are capable of increasing their developmental rates in response to increased temperature and accelerated drying, but only in an additive manner. The strong negative effects of drying on survival, combined with lack of compensation between these two environmental drivers, suggest changes in the aquatic environment that are expected in this ecosystem will reduce mean fitness of populations across the landscape.
Small GTPases in the Rho family act as major nodes with functions beyond cytoskeletal rearrangements shaping the Caenorhabditis elegans embryo during development. These small GTPases are key signal transducers that integrate diverse developmental signals to produce a coordinated response in the cell. In C. elegans, the best studied members of these highly conserved Rho family small GTPases, RHO-1/RhoA, CED-10/Rac, and CDC-42, are crucial in several cellular processes dealing with cytoskeletal reorganization. In this review, we update the functions described for the Rho family small GTPases in spindle orientation and cell division, engulfment, and cellular movements during C. elegans embryogenesis, focusing on the Rho subfamily Rac. Please also see the video abstract here.
The ability to fine-tune feature size in nanostructured thin films is critical, as many desirable properties of these materials are dictated by their nanostructure. Accordingly, there is a need for techniques that allow for modifying nanostructure while monitoring the morphological changes in situ. Here, we demonstrate a closed-loop electro-annealing system which enables in situ monitoring of morphology evolution in sub-micron nanoporous gold (np-Au) thin films. Np-Au is produced by a microfabrication-compatible self-assembly process that produces a network of interconnected ligaments with tunable diameter (10 s to 100 s of nanometers), making it a desirable material for numerous applications and fundamental studies alike. We specifically investigate the relationship between np-Au morphology (i.e., ligament diameter) and electrical resistance of the thin film. A strong correlation emerges between ligament size and electrical resistance, which puts forward resistance as an effective parameter for monitoring morphology evolution. Surprisingly, np-Au films with thicker ligaments lead to an increase in electrical resistance, which is unexpected since the extent of charge carrier scattering at the ligament surface should decrease with increasing ligament size. Further examination of np-Au morphology with high-resolution electron microscopy revealed grain growth on the ligaments in highly-annealed np-Au thin films. This suggests that grains act as scattering centers for charge carriers and this becomes the dominant mechanism in dictating electrical resistance in a percolated network of thin conductive ligaments.
Appropriate nutrition is an essential component of intensive care management of children with acute respiratory distress syndrome (ARDS) and is linked to patient outcomes. One out of every two children in the pediatric intensive care unit (PICU) will develop malnutrition or have worsening of baseline malnutrition and present with specific micronutrient deficiencies. Early and adequate enteral nutrition (EN) is associated with improved 60-day survival after pediatric critical illness, and, yet, despite early EN guidelines, critically ill children receive on average only 55% of goal calories by PICU day 10. Inadequate delivery of EN is due to perceived feeding intolerance, reluctance to enterally feed children with hemodynamic instability, and fluid restriction. Underlying each of these factors is large practice variation between providers and across institutions for initiation, advancement, and maintenance of EN. Strategies to improve early initiation and advancement and to maintain delivery of EN are needed to improve morbidity and mortality from pediatric ARDS. Both, over and underfeeding, prolong duration of mechanical ventilation in children and worsen other organ function such that precise calorie goals are needed. The gut is thought to act as a "motor" of organ dysfunction, and emerging data regarding the role of intestinal barrier functions and the intestinal microbiome on organ dysfunction and outcomes of critical illness present exciting opportunities to improve patient outcomes. Nutrition should be considered a primary rather than supportive therapy for pediatric ARDS. Precise nutritional therapies, which are titrated and targeted to preservation of intestinal barrier function, prevention of intestinal dysbiosis, preservation of lean body mass, and blunting of the systemic inflammatory response, offer great potential for improving outcomes of pediatric ARDS. In this review, we examine the current evidence regarding dose, route, and timing of nutrition, current recommendations for provision of nutrition to children with ARDS, and the current literature for immune-modulating diets for pediatric ARDS. We will examine emerging data regarding the role of the intestinal microbiome in modulating the response to critical illness.
Dental caries has been traditionally described as a multifactorial disease that involves the interaction of various factors like host, agent, substrate and time. Landmark studies have established the fact that Mutans Streptococci are the primary etiologic agents of dental caries. The prevention of dental caries by fluoride supplements in various vehicles, such as water and toothpaste, constitutes one of the most successful prevention measures.
The aim of the present study was to compare the clinical efficacy of four fluoride mouth rinses on Streptococcus mutans in high caries risk children and also to check the efficacy of the ingredient Triclosan which is present in two of the four mouth rinses.
The study is double blinded, consisting of 1000 children in age group 6-14yrs who were screened from residential schools. Of the total, 200 children were categorized as high caries risk group based on caries risk assessment tool form given by American Association of Pediatric Dentistry (AAPD) guidelines 2011. Prior to the study, salivary samples were collected and sent for microbial analysis to estimate Streptococcus mutans counts. Out of 200 salivary samples, 132 showed 10(6)CFU of Streptococcus mutans and these children were included in the study. The 132 children from each group received the assigned mouth wash for 14 consecutive days. On 15(th) day the salivary samples were collected and sent for microbial analysis and the obtained results were subjected to statistical analysis.
All the mouth washes showed a significant reduction in Colony Forming Units (CFU) counts of Streptococcus mutans. Among the four groups Group D (S flo) showed greater percentage reduction of Streptococcus mutans followed by Group A (Act), B (Kidodent) and C (Zerocary). There was no stastically significance reduction of Streptococcus mutans among the Triclosan containing and non containing groups.
The mean pre rinse CFU was significantly higher than post rinse CFU for all the study groups, suggesting that all the four mouth rinses were effective in decreasing the levels of Streptococcus mutans in the saliva. Both the Triclosan containing and non Triclosan groups showed the same amount of CFU count reduction.
Psychiatric diseases like anxiety, depression, schizophrenia and bipolar disorders are increasing at an alarming rate. These diseases can affect the quantity and quality of saliva leading to multiple oral diseases. Although many researchers have evaluated xerostomia in general population, its prevalence is not been assessed in patients suffering from different psychological disorders.
To investigate the prevalence of xerostomia and to assess the correlation between xerostomia and dryness of lip and mucosa in different psychological disorders.
A cross-sectional observational study was conducted over a period of six months in Department of Psychiatry and Department of Oral Medicine. Patients with anxiety, depression, schizophrenia and bipolar disorder, as diagnosed by an experienced psychiatrist, were given a questionnaire to evaluate the xerostomia. Patients with symptoms of xerostomia were subjected to oral examination by a skilled oral diagnostician to check for dryness of lips and mucosa. One hundred patients from each group of psychiatric diseases were included in the study using a consecutive sampling technique. An equal number of healthy individuals reporting to oral medicine department for routine oral screening were included as control group after initial psychiatric evaluation.
In this study statistically significant increase in the xerostomia in psychiatric patients was recorded when compared to the control group (p<0.01). Xerostomia was significantly higher in anxiety patients (51%) followed by depression (47%), bipolar disorder (41%), schizophrenia (39%) and control group (27%). The majority of the psychiatric patients had 'moderate' to 'severe' xerostomia whereas the control group had 'mild' xerostomia. Xerostomia was significantly higher in younger age group (18-49 years) than in older age group and females patients had higher xerostomia than male patients. Psychiatric patients had significantly more dryness of lip and mucosa than healthy controls. A moderate to strong spearman correlation (r=0.72) was observed between xerostomia and psychological alterations.
A positive association was established between psychological alterations and xerostomia and dryness of lip and mucosa. Emotional alterations may act as a precipitating factor that could influence the salivary secretion resulting in multiple oral diseases. Psychiatrists can screen for xerostomia and collaborate with dentists for comprehensive management of xerostomia in psychiatric patients.
Live Donor Liver Transplantation (LDLT) is an act of selflessness on the part of the donor who is subjected to a major hepatectomy. Ensuring safety and long-term well being of the donor is of utmost priority. We describe a 21-year-old otherwise healthy donor with perimembranous Ventricular Septal Defect (VSD) who successfully underwent donor hepatectomy after closure of the VSD. There is no literature available to guide regarding course of action in such a condition neither any study to substantiate the risk involved. Optimum anticoagulation, endocarditis prophylaxis and optimum interval between the two procedures are areas to be defined as our experience with similar cases increases. Our case emphasizes the importance of multidisciplinary approach and management of such patient at high volume centers.
Hymenolepis diminuta (H.diminuta) is prevalent worldwide, and a few hundred human cases have been reported till date. It is primarily a rodent parasite and humans (usually children) can act as accidental hosts. Infections are usually asymptomatic but abdominal pain, irritability, pruritis, mild diarrhoea and eosinophilia are among the existing symptoms in a few of the reported cases. Here, we report a case of an 11-year-old female child from Bijnor, who presented to us with complaints of abdominal pain, fatigue and irregular episodes of fever. Routine stool examination showed characteristic eggs of H.diminuta. Patient was given a single oral dose of praziquantel (25 mg/kg) and she improved. This case is presented to emphasize that till date there are very few reports on H.diminuta and there is limited data regarding its treatment protocols (dose and duration). Furthermore, albendazole which is commonly used drug for deworming helminthic infections is less effective in these infections.
Carbapenem-resistant Enterobacteriaceae (CRE) are drug-resistant Gram-negative bacteria that are present in the community as well as in hospitals. Their infection and colonisation puts critically ill patients at high risk due to the drug-resistant nature of the strains and possible spreading of these organisms, even in a hospital environment.
To examine the presence and types of Enterobacteriaceae species in patients admitted directly from the community.
The present study was a one-month pilot conducted in the ICU of a tertiary care hospital in Mumbai, India in 2015. Faecal samples of patients admitted from the community directly to the ICU were analysed using tests like MHT (Modified Hodge) and EDTA for the presence of IMP (action on Imipenem) and KPC (Klebsiella Test Pneumoniae Carbapenemase) producing strains of Enterobacteriaceae. Polymerase Chain Reaction (PCR) was performed to look for VIM, IMP, NDM1, OXA, and KPC genes. Antibiotic Sensitivity Test was carried out as per CLSI guidelines.
The results showed an alarming level of faecal carriage rates in adult ICU patients. Klebsiella pneumonia was the most common carbapenem-resistant isolate, closely followed by Escherichia coli. PCR results revealed nine strains were positive for bla(KPC) gene, from which 7 were Klebsiella pneumoniae and one each of Escherichia coli and Klebsiella oxytoca was observed. Antibiotic Sensitivity Test results showed that the isolates had maximum sensitivity to Colistin (100%) and Tigecycline (95%).
These levels indicate that in the absence of CRE screenings, proper isolation of carrier patients is not possible, leading to possible spreading of these resistant bacteria strains in ICUs. A longer period of study is required to obtain more substantial data to validate the results of this pilot.
Learning anatomy by dissection of cadavers is the best way to learn anatomy. Voluntary body donation is one of the sources of procuring cadavers. In the case of donations after hospital or non-institutional deaths, the family members of the deceased approach the hospital authorities regarding body donation of the deceased. There are situations, where there is no available accompanying near relatives of the deceased, which pose a challenge for personnel involved in the process of body donation. In two of the reported cases, the body donation was done by the live-in partner and a friend of the deceased. In another reported case, the son of the deceased was nominated by the donor at the time of registration. As the son of the deceased was not available at the time of death of the donor, donation was executed by another near relative. Anatomy Acts of individual States in the Union of India and Acts of other countries are examined about the consent for body donation by persons other than near relatives. None of the Anatomy Acts of various States in India provide an alternative in the absence of near relatives for claiming the body or for donating the body, except for Acts of Delhi and Kerala. There is a need to bring in a Unified Anatomy Act, common for all the States and Union territories in India and include: friend, live-in partner and a nominated person in the provisions of the Act to enable them to give consent for body donation.
Philosophical arguments stemming from the public health ethics arena suggest that public health interventions ought to be subject to normative inquiry that considers relational values, including concepts such as solidarity, reciprocity and health equity. As yet, however, the extent to which 'public' values influence the 'autonomous' decisions of the public remains largely unexplored. Drawing on interviews with 50 men in Vancouver, Canada, this study employs a critical discourse analysis to examine participants' decisions and motivations to voluntarily access HIV testing and/or to accept a routine HIV test offer. Within a sub-set of interviews, a transactional discourse emerged in which the decision to test features an arrangement of 'giving and receiving'. Discourses related to notions of solidarity emphasize considerations of justice and positions testing as a 'public' act. Lastly, 'individualistic' discourses focused on individual-level considerations, with less concern for the broader public 'good'. These findings underscore how normative dimensions pertaining to men's decisions to test are dialectically interrelated with the broader social and structural influences on individual and collective health-related behaviour, thereby suggesting a need to advance an explicit empirical-normative research agenda related to population and public health intervention research.
Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33-35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call "truncTALEs," for "truncated TAL effectors." We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it or a neighbor disengages. The presence of the 28 aa repeat, however, was not required for resistance suppression. TruncTALEs expand the paradigm for TAL effector-mediated effects on plants. We propose that Tal2h and other truncTALEs act as dominant negative ligands for an immune receptor encoded by the Xo1 locus, likely a nucleotide binding, leucine-rich repeat protein. Understanding truncTALE function and distribution will inform strategies for disease control.
Although several efforts have been made in the search for genetic and epigenetic patterns linked to diseases, a comprehensive explanation of the mechanisms underlying pathological phenotypic plasticity is still far from being clarified. Oxidative stress and inflammation are two of the major triggers of the epigenetic alterations occurring in chronic pathologies, such as neurodegenerative diseases. In fact, over the last decade, remarkable progress has been made to realize that chronic, low-grade inflammation is one of the major risk factor underlying brain aging. Accumulated data strongly suggest that phytochemicals from fruits, vegetables, herbs, and spices may exert relevant immunomodulatory and/or anti-inflammatory activities in the context of brain aging. Starting by the evidence that a common denominator of aging and chronic degenerative diseases is represented by inflammation, and that several dietary phytochemicals are able to potentially interfere with and regulate the normal function of cells, in particular neuronal components, aim of this review is to summarize recent studies on neuroinflammaging processes and proofs indicating that specific phytochemicals may act as positive modulators of neuroinflammatory events. In addition, critical pathways involved in mediating phytochemicals effects on neuroinflammaging were discussed, exploring the real impact of these compounds in preserving brain health before the onset of symptoms leading to inflammatory neurodegeneration and cognitive decline.
Growth of postmitotic neurons occurs during different stages of development, including metamorphosis, and may also be part of neuronal plasticity and regeneration. Recently we showed that growth of post-mitotic neuroendocrine cells expressing the basic helix loop helix (bHLH) transcription factor Dimmed (Dimm) in Drosophila could be regulated by insulin/IGF signaling and the insulin receptor (dInR). Dimm is also known to confer a secretory phenotype to neuroendocrine cells and can be part of a combinatorial code specifying terminal differentiation in peptidergic neurons. To further understand the mechanisms of Dimm function we ectopically expressed Dimm or Dimm together with dInR in a wide range of Dimm positive and Dimm negative peptidergic neurons, sensory neurons, interneurons, motor neurons, and gut endocrine cells. We provide further evidence that dInR mediated cell growth occurs in a Dimm dependent manner and that one source of insulin-like peptide (DILP) for dInR mediated cell growth in the CNS is DILP6 from glial cells. Expressing both Dimm and dInR in Dimm negative neurons induced growth of cell bodies, whereas dInR alone did not. We also found that Dimm alone can regulate cell growth depending on specific cell type. This may be explained by the finding that the dInR is a direct target of Dimm. Conditional gene targeting experiments showed that Dimm alone could affect cell growth in certain neuron types during metamorphosis or in the adult stage. Another important finding was that ectopic Dimm inhibits apoptosis of several types of neurons normally destined for programmed cell death (PCD). Taken together our results suggest that Dimm plays multiple transcriptional roles at different developmental stages in a cell type-specific manner. In some cell types ectopic Dimm may act together with resident combinatorial code transcription factors and affect terminal differentiation, as well as act in transcriptional networks that participate in long term maintenance of neurons which might lead to blocked apoptosis.
Understanding how new Medicaid enrollees are approaching their own health and health care in the shifting health care landscape of the Affordable Care Act has implications for future outreach and enrollment efforts, as well as service planning for this population. The objective of this study was to explore the health care experiences and expectations of new Medicaid expansion beneficiaries in the immediate post-enrollment period. We conducted semistructured, qualitative interviews with a random sample of 40 adults in Philadelphia who had completed an application for Medicaid through a comprehensive benefits organization after January 1, 2015, when the Medicaid expansion in Pennsylvania took effect. We conducted an inductive, applied thematic analysis of interview transcripts. The new Medicaid beneficiaries described especially high levels of pent-up demand for care. Dental care was a far more pressing and motivating concern than medical care. Preventive services were also frequently mentioned. Participants anticipated that insurance would reduce both stress and financial strain and improve their experience in the health care system by raising their social standing. Participants highly valued the support of telephone application counselors in the Medicaid enrollment process to overcome bureaucratic obstacles they had encountered in the past. Dental care and preventive services appear to be high priorities for new Medicaid enrollees. Telephone outreach and enrollment support services can be an effective way to overcome past experiences with administrative barriers.
The chemokine receptor CXCR4 and its chemokine ligand CXCL12 mediate directed cell migration during organogenesis, immune responses and also metastatic disease. Yet the mechanisms governing CXCL12/CXCR4-dependent chemotaxis remain poorly understood. Here, we show that the β-arrestin1/STAM1 complex, initially identified to govern lysosomal trafficking of CXCR4, also mediates CXCR4-dependent chemotaxis. Expression of minigene fragments from β-arrestin1 or STAM1, known to disrupt the β-arrestin1/STAM1 complex, and RNAi against β-arrestin1 or STAM1, attenuates CXCL12-induced chemotaxis. The β-arrestin1/STAM1 complex is necessary for promoting autophosphorylation of focal adhesion kinase (FAK). FAK is necessary for CXCL12-induced chemotaxis and associates with and localizes with β-arrestin1 and STAM1 in a CXCL12-dependent manner. Our data reveal previously unknown roles for β-arrestin1 and STAM1 in CXCR4-dependent chemotaxis, which we propose act in concert to regulate FAK signaling. The β-arrestin1/STAM1 complex is a promising target for blocking CXCR4-promoted FAK autophosphorylation and chemotaxis.
PGC-1-related coactivator (PRC) has a dual function in growth-regulated mitochondrial biogenesis and as a sensor of metabolic stress. PRC induction by mitochondrial inhibitors, intracellular ROS, or topoisomerase I inhibition orchestrates an inflammatory program associated with the adaptation to cellular stress. Activation of this program is accompanied by the coordinate expression of c-MYC, which is linked kinetically to that of PRC in response to multiple stress inducers. Here, we show that the c-MYC inhibitor 10058-F4 blocks the induction of c-MYC, PRC, and representative PRC-dependent stress genes by the respiratory chain uncoupler, carbonyl cyanide m-chlorophenyl hydrazine (CCCP). This result, confirmed by the suppression of PRC induction by c-MYC siRNA silencing, demonstrates a requirement for c-MYC in orchestrating the stress program. PRC steady-state expression was markedly increased upon mutation of two GSK-3 serine phosphorylation sites within the carboxyl-terminal domain. The negative control of PRC expression by GSK-3 was consistent with the phosphor-inactivation of GSK-3β by CCCP and by the induction of PRC by the GSK-3 inhibitor AZD2858. Unlike PRC, which was induced post-translationally through increased protein half-life, c-MYC was induced predominantly at the mRNA level. Moreover, suppression of Akt activation by the Akt inhibitor MK-2206 blocked the CCCP induction of PRC, c-MYC, and representative PRC stress genes, demonstrating a requirement for Akt signaling. MK-2206 also inhibited the phosphor-inactivation of GSK-3β by CCCP, a result consistent with the ability of Akt to phosphorylate, and thereby suppress GSK-3 activity. Thus, PRC and c-MYC can act in concert through Akt-GSK-3 signaling to reprogram gene expression in response to mitochondrial stress.
Recent evidence suggests involvement of coagulation factor XIa (FXIa) in thrombotic event development. This study was conducted to explore possible synergies between tissue factor (TF) and exogenous FXIa (E-FXIa) in thrombin generation.
In thrombin generation assays, for increasing concentrations of E-FXIa with low, but not with high TF concentrations, peak thrombin significantly increased whereas lag time and time to peak significantly decreased. Similar dependencies of lag times and rates of thrombin generation were found in mathematical model simulations. In both in vitro and in silico experiments that included E-FXIa, thrombin bursts were seen for TF levels much lower than those required without E-FXIa. For in silico thrombin bursts initiated by the synergistic action of TF and E-FXIa, the mechanisms leading to the burst differed substantially from those for bursts initiated by high TF alone. For the synergistic case, sustained activation of platelet-bound FIX by E-FXIa, along with the feedback-enhanced activation of platelet-bound FVIIIa and FXa, was needed to elicit a thrombin burst. Furthermore, the initiation of thrombin bursts by high TF levels relied on different platelet FIX/FIXa binding sites than those involved in bursts initiated by low TF levels with E-FXIa.
Low concentrations of TF and exogenous FXIa, each too low to elicit a burst in thrombin production alone, act synergistically when in combination to cause substantial thrombin production. The observation about FIX/FIXa binding sites may have therapeutic implications.
The present study aimed to investigate the effects of mood stabilizers, specifically lithium (Li) and valproate (VPA), on PI3K/Akt signaling pathway in the brains of rats subjected to the ouabain (OUA)-induced animal model of mania. In addition, it was evaluated the effects of AR-A0144818, a GSK-3β inhibitor, on manic-like behaviour induced by OUA. In the first experimental protocol Wistar rats received a single ICV injection of OUA or artificial cerebrospinal fluid (aCSF). From the day following ICV injection, the rats were treated for 6 days with intraperitoneal injections of saline, Li or VPA twice a day. In the second experimental protocol, rats received OUA, aCSF, OUA plus AR-A0144818, or aCSF plus AR-A0144818. In the 7th day after OUA injection, locomotor activity was measured using the open-field test. In addition, we analyzed levels of p-PI3K, p-MAPK, p-Akt, p-GSK-3β in the brain of rats by immunoblot. Li and VPA reversed OUA-related hyperactivity. OUA decreased pPI3K, pAkt and pGSK-3β levels. Li and VPA improved these OUA-induced cellular dysfunctions; however, the effects of the mood stabilizers were dependent on the protein and brain region analyzed. In addition, AR-A0144818 reversed the manic-like behavior induced by OUA. These findings suggest that the manic-like effects of ouabain are associated with the activation of GSK-3β, and that Li and VPA exert protective effects against OUA-induced inhibition on GSK-3β pathway.
Oxidative stress is involved in various and numerous pathological states including several age-related neurodegenerative diseases. Peroxidation of the membrane lipid bilayer is one of the major sources of free radical-mediated injury that directly damages neurons causing increased membrane rigidity, decreased activity of membrane-bound enzymes, impairment of membrane receptors and altered membrane permeability and eventual cell death. Moreover, the peroxidation of polyunsaturated fatty acids leads to the formation of aldehydes, which can act as toxic by-products. One of the most abundant and cytotoxic lipid -derived aldehydes is 4-hydroxy 2-nonenal (HNE). HNE toxicity is mainly due to the alterations of cell functions by the formation of covalent adducts of HNE with proteins. A key marker of lipid peroxidation, HNE-protein adducts, were found to be elevated in brain tissues and body fluids of Alzheimer disease, Parkinson disease, Huntington disease and amyotrophic lateral sclerosis subjects and/or models of the respective age-related neurodegenerative diseases. Although only a few proteins were identified as common targets of HNE modification across all these listed disorders, a high overlap of these proteins occurs concerning the alteration of common pathways, such as glucose metabolism or mitochondrial function that are known to contribute to cognitive decline. Within this context, despite the different etiological and pathological mechanisms that lead to the onset of different neurodegenerative diseases, the formation of HNE-protein adducts might represent the shared leit-motif, which aggravates brain damage contributing to disease specific clinical presentation and decline in cognitive performance observed in each case.
Biglycan, a small leucine-rich proteoglycan, has been shown to play an important role in stabilizing fibrotic scars after experimental myocardial infarction. However, the role of biglycan in the development and regression of cardiomyocyte hypertrophy and fibrosis during cardiac pressure overload and unloading remains elusive. Thus, the aim of the present study was to assess the effect of biglycan on cardiac remodeling in a mouse model of left ventricular pressure overload and unloading.
Left ventricular pressure overload induced by transverse aortic constriction (TAC) in mice resulted in left ventricular dysfunction, fibrosis and increased biglycan expression. Fluorescence- and magnetic-assisted sorting of cardiac cell types revealed upregulation of biglycan in the fibroblast population, but not in cardiomyocytes, endothelial cells or leukocytes after TAC. Removal of the aortic constriction (rTAC) after short-term pressure overload (3weeks) improved cardiac contractility and reversed ventricular hypertrophy but not fibrosis in wild-type (WT) mice. Biglycan ablation (KO) enhanced functional recovery but did not resolve cardiac fibrosis. After long-term TAC for 9weeks, ablation of biglycan attenuated the development of cardiac hypertrophy and fibrosis. In vitro, biglycan induced hypertrophy of neonatal rat cardiomyocytes and led to activation of a hypertrophic gene program. Putative downstream mediators of biglycan signaling include Rcan1, Abra and Tnfrsf12a. These genes were concordantly induced by TAC in WT but not in biglycan KO mice.
Left ventricular pressure overload induces biglycan expression in cardiac fibroblasts. Ablation of biglycan improves cardiac function and attenuates left ventricular hypertrophy and fibrosis after long-term pressure overload. In vitro biglycan induces hypertrophy of cardiomyocytes, suggesting that biglycan may act as a signaling molecule between cell types to modulate cardiac remodeling.
Staphylococcal infection and neutrophilic inflammation can act in concert to establish a profoundly hypoxic environment. In this review we summarise how neutrophils and Staphylococcus aureus are adapted to function under hypoxic conditions, with a particular focus on the impaired ability of hypoxic neutrophils to effect Staphylococcus aureus killing.
A new series of beta amino acids, which act as CXCR3 antagonists, has been identified. The formerly optimized N,N-disubstituted benzylamine derivatives with carboxylic acid function on the N-atom was used as starting point and compounds with carboxyl function not attached to the N-atom were investigated. Affinity, metabolic stability in human and mouse liver microsomes and Caco-2 permeability were optimized. Compounds with double-digit nanomolar CXCR3 affinity, favourable microsomal stability and Caco-2 permeability have been identified.
Social media tools, including blogs, social networks, and media-sharing sites, help nutrition and dietetics practitioners reach broader audiences and connect directly with the public. In many ways, social media has transformed the practice of dietetics and has opened up new avenues for communicating food and nutrition information. Social media has been an effective tool for virtual nutrition counseling, patient education, peer-to-peer support, and public health campaigns. Increasingly, nutrition and dietetics practitioners are using social media to network and collaborate with colleagues, conduct a job search, stay current with new research, champion a cause, promote products or services, and build a business. The potential role of social media in the profession is far reaching, yet there are important guidelines to follow related to ethics and professionalism. When using social media, nutrition and dietetics practitioners must remember that they are governed by the same Code of Ethics that guides all other aspects of practice. In addition, it is critical to have a thorough understanding of all the factors related to social media professionalism, including disclosure rules from the Federal Trade Commission, patient/client privacy and confidentiality as covered by the Health Insurance Portability and Accountability Act, and copyright laws that protect intellectual property. In today's digital age, it is essential for nutrition and dietetics practitioners to recognize the professional opportunities and challenges of social media. Failing to effectively and ethically use social media can reflect poorly on the individual practitioner and the profession. Certain violations may have legal implications. The purpose of this Academy of Nutrition and Dietetics practice paper is to provide guidance on social media's relevance, potential applications, best practices, benefits, and risks.
While subepidermal skin inking as a fashion trend has rapidly gained popularity in Western societies, systemic anaphylaxis as a complication of tattooing has only been described once in refereed literature. Furthermore the previously reported case was from a patient who already suffered from severe allergies and no attempt to pinpoint the actual causes was made.
We present the case of a 59-year-old man, who developed a progressive swelling and redness five hours after receiving a tattoo. Another hour later he appeared in the emergency room with a grade 3 systemic anaphylaxis. He presented with rapidly progressing swelling and redness of the tattooed left arm, left cheek and lips as well as tongue. Allergies were not previously known in this patient. He responded well to treatment with prednisolone and antihistamines. Further workup identified formaldehyde, nickel, and manganese in the inks as potential chemical triggers of the patient's symptoms. The patient refused further allergological work-up, such as prick testing.
Clinicians should be alert to the potential capacity of tattoo inks to act as triggers of systemic anaphylaxis. Policymakers should attempt to better restrict the use of known allergenic compounds in commercial tattoo inks.
Cardiac troponins are specific for the heart, but not for the acute coronary syndrome. We wanted to assess how common elevated cardiac troponin concentrations were, in a population with significant non-cardiac disease.
We measured both hs-cTnT and hs-cTnI on all samples submitted to the laboratory during one 24h period, and assessed the magnitude of the cTn concentration with the location and severity of disease of the patient.
Community patients and patients from the maternity ward had the lowest cTn concentrations with results above the 99th percentile being only 0-2% of the total. As expected, the highest proportion of results >99th percentile came from Coronary Care and Intensive Care. However, substantial numbers of persons on Medical and Surgical wards, without a primary diagnosis of cardiac disease, also had cTn >99th percentile. Particularly for cTnT, there was a highly significant odds ratio predicting mortality when results above and below the 99th percentile were compared.
Significant illnesses apart from the acute coronary syndrome are important causes of a rise in cTn to above the 99th percentile, and appear to reflect the total body burden of disease. Even when the high hs-cTn concentration is not due to the acute coronary syndrome, there is a significant association with all-cause mortality.
In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building.
Entirely differing from the common templating-based multistep strategy for fabricating multifunctional hollow mesoporous silica nanoparticles (HMSN), a facile and template-free synthetic strategy has been established to construct a unique hollow/mesoporous organosilica nanocapsule (OSNC) concurrently encapsulating both isopentyl acetate (PeA) liquid and superparamagnetic iron oxides inside (denoted as PeA@OSNC). This novel material exhibits ultrasmall and uniform particle size (∼82 nm), high surface area (∼534 m(2)·g(-1)), and excellent colloidal stability in aqueous solution. The oil-phase PeA with relatively low boiling point (142 °C) and high volatility not only plays a crucial role in formation of a large hollow cavity from the viewpoint of structural design but also enables the PeA@OSNC to act as an efficient enhancement agent in high-intensity focused ultrasound (HIFU) therapy. Moreover, the unique satellite-like distribution of Fe3O4 nanoparticles (NP) on the organosilica shell offered excellent magnetic resonance imaging (MRI) contrast capability of PeA@OSNC in vitro and in vivo. More importantly, such a novel theranostic agent has favorable biosafety, which is very promising for future clinical application in MRI-guided HIFU therapy.
Diabetes induces a decrease in the number and function of different pro-angiogenic cell types generically designated as putative endothelial progenitor cells (EPC), which encompasses cells from myeloid origin that act in a paracrine fashion to promote angiogenesis and putative "true" EPC that contribute to endothelial replacement. This not only compromises neovasculogenesis in ischemic tissues but also impairs, at an early stage, the reendotheliziation process at sites of injury, contributing to the development of endothelial dysfunction and cardiovascular complications. Hyperglycemia, insulin resistance and dyslipidemia promote putative EPC dysregulation by affecting the SDF-1/CXCR-4 and NO pathways and the p53/SIRT1/p66Shc axis that contribute to their mobilization, migration, homing and vasculogenic properties. To optimize the clinical management of patients with hypoglycemic agents, statins and renin-angiotensin system inhibitors, which display pleiotropic effects on putative EPC, is a first step to improve their number and angiogenic potential but specific strategies are needed. Among them, mobilizing therapies based on G-CSF, erythropoietin or CXCR-4 antagonism have been developed to increase putative EPC number to treat ischemic diseases with or without prior cell isolation and transplantation. Growth factors, genetic and pharmacological strategies are also evaluated to improve ex vivo cultured EPC function before transplantation. Moreover, pharmacological agents increasing in vivo the bioavailability of NO and other endothelial factors demonstrated beneficial effects on neovascularization in diabetic ischemic models but their effects on endothelial dysfunction remain poorly evaluated. More experiments are warranted to develop orally available drugs and specific agents targeting p66Shc to reverse putative EPC dysfunction in the expected goal of preventing endothelial dysfunction and diabetic cardiovascular complications.
The standard of 5-Hydroxymethylfurfural (5-HMF) existed in dextrose injection as an inevitable by-product during high-temperature setrilization has been included in pharmacopoeias considering its hazardous effects on human health. We found that the concentrations of 5-HMF in some traditional Chinese medicine injections (TCMIs) far exceeded its limit in dextrose injection. Besides, we detected 5, 5'-Oxydimethylenebis (2-furfural) (OMBF) in those TCMIs containing high concentrations of 5-HMF. We investigated the in vivo immunomodulatory effects of 5-HMF and OMBF at three dose levels using the reporter antigen popliteal lymph node assay (RA-PLNA), which allows the straightforward examination and mechanistic study of immunotoxicity of low molecular weight compounds. We found that 5-HMF increased the production of IgG2a and IFN-γ when co-injected with TNP-OVA, indicating its capability of providing a co-stimulatory signal to evoke a typical type-1 immune response. Compared with the 5-HMF, OMBF elevated the production of IgG1, IgG2, IL-4 and IFN-γ in response to both reporter antigens, suggesting that OMBF can act as a neo-antigen or neo-epitope to elicit a mixed type-1 and type-2 immune response. It indicates that both 5-HMF and OMBF have immunosensitizing potential with different mechanisms, and exposure to 5-HMF and OMBF may represent a safety concern for humans.
Why do people act altruistically? One theory is that empathy is a driver of morality. Experimental studies of this are often confined to laboratory settings, which often lack ecological validity. In the present study we investigated whether empathy traits predict if people will act altruistically in a real-world setting, "in the wild". We staged a situation in public that was designed to elicit helping, and subsequently measured empathic traits in those who either stopped to help or walked past and did not help. Results show that a higher number of empathic traits are a significant and positive predictor for altruistic behavior in a real-life situation. This supports the theory that the act of doing good is correlated with empathy.
There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (∼30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a "sweet spot" for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.
Sensitivity to the rewarding properties of appetitive substances has long been implicated in excessive consumption of palatable foods and drugs of abuse. Previous research focusing on individual differences in reward responsiveness has found heightened trait reward sensitivity to be associated with binge-eating, hazardous drinking, and illicit substance use. Food addiction has been proposed as an extreme form of compulsive-overeating and has been associated with genetic markers of heightened reward responsiveness. However, little research has explicitly examined the association between reward sensitivity and food addiction. Further, the processes by which individual differences in this trait are associated with excessive over-consumption has not been determined. A total of 374 women from the community completed an online questionnaire assessing reward sensitivity, food addiction, emotional, externally-driven, and hedonic eating. High reward sensitivity was significantly associated with greater food addiction symptoms (r = 0.31). Bootstrapped tests of indirect effects found the relationship between reward sensitivity and food addiction symptom count to be uniquely mediated by binge-eating, emotional eating, and hedonic eating (notably, food availability). These indirect effects held even when controlling for BMI, anxiety, depression, and trait impulsivity. This study further supports the argument that high levels of reward sensitivity may offer a trait marker of vulnerability to excessive over-eating, beyond negative affect and impulse-control deficits. That the hedonic properties of food (especially food availability), emotional, and binge-eating behavior act as unique mediators suggest that interventions for reward-sensitive women presenting with food addiction may benefit from targeting food availability in addition to management of negative affect.
Steroids are the most efficacious anti-inflammatory agents. However, their toxicities and side-effects compromise their clinical application. Various strategies and major efforts were dedicated for formulating viable liposomal glucocorticosteroids (GCs), so far none of these were approved.
To evaluate these approaches for formulating GC-delivery systems, especially liposomes, and with focus on the Barenholz Lab experience.
We developed PEGylated nano-liposomes (NSSL) remotely loaded with water-soluble amphipathic weak acid GC-prodrugs. Their remote loading results in high, efficient and stable loading to the level that enables human clinical use. We characterized them for their physical chemistry and stability. We demonstrated their therapeutic efficacy in relevant animal models and studied their pharmacokinetics (PK), biodistribution (BD) and pharmacodynamics advantages over the free pro-drugs.
Our steroidal nano-drugs demonstrate much superior PK, BD, tolerability and therapeutic efficacies compared to the free pro-drugs and to most drugs currently used to treat these diseases. These nano-drugs act as robust immune-suppressors, affecting cytokines secretion and diminishing hemorrhage and edema.
The combination of improved physical-chemistry, PK, BD, tolerability and therapeutic efficacy of these steroidal nano-drugs over the pro-drugs "as-is" support their further clinical development as potential therapeutic agents for treating inflammatory diseases.
Human platelets contain conventional (α and β) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives.
Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe(2+) recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.
Strong social capital is increasingly recognized as an organizational advantage. Better knowledge sharing and reduced transaction costs increase work efficiency. To mimic the formation of the associated communication network, we propose the Expert Game, where each individual must find a specific expert and receive her help. Participants act in an impersonal environment and under time constraints that provide short-term incentives for noncooperative behavior. Despite these constraints, we observe cooperation between individuals and the self-organization of a sustained trust network, which facilitates efficient communication channels with increased information flow. We build a behavioral model that explains the experimental dynamics. Analysis of the model reveals an exploitation protection mechanism and measurable social capital, which quantitatively describe the economic utility of trust.
This review categorizes functionally validated actions of defined scorpion toxin (SCTX) neuropeptides across ion channel subclasses, highlighting key trends in this rapidly evolving field. Scorpion envenomation is a common event in many tropical and subtropical countries, with neuropharmacological actions, particularly autonomic nervous system modulation, causing significant mortality. The primary active agents within scorpion venoms are a diverse group of small neuropeptides that elicit specific potent actions across a wide range of ion channel classes. The identification and functional characterisation of these SCTX peptides has tremendous potential for development of novel pharmaceuticals that advance knowledge of ion channels and establish lead compounds for treatment of excitable tissue disorders. This review delineates the unique specificities of 320 individual SCTX peptides that collectively act on 41 ion channel subclasses. Thus the SCTX research field has significant translational implications for pathophysiology spanning neurotransmission, neurohumoral signalling, sensori-motor systems and excitation-contraction coupling.
Antagonistic coevolution between hosts and parasites is a key process in the genesis and maintenance of biological diversity. Whereas coevolutionary dynamics show distinct patterns under favourable environmental conditions, the effects of more realistic, variable conditions are largely unknown. We investigated the impact of a fluctuating environment on antagonistic coevolution in experimental microcosms of Pseudomonas fluorescens SBW25 and lytic phage SBWΦ2. High-frequency temperature fluctuations caused no deviations from typical coevolutionary arms race dynamics. However, coevolution was stalled during periods of high temperature under intermediate- and low-frequency fluctuations, generating temporary coevolutionary cold spots. Temperature variation affected population density, providing evidence that eco-evolutionary feedbacks act through variable bacteria-phage encounter rates. Our study shows that environmental fluctuations can drive antagonistic species interactions into and out of coevolutionary cold and hot spots. Whether coevolution persists or stalls depends on the frequency of change and the environmental optima of both interacting players.
A combination of nickel and photoredox catalysts promoted novel cross-coupling reactions of aryl halides with 4-alkyl-1,4-dihydropyridines. 4-Alkyl-1,4-dihydropyridines act as formal nucleophilic alkylation reagents through a photoredox-catalyzed carbon-carbon (C-C) bond-cleavage process. The present strategy provides an alternative to classical carbon-centered nucleophiles, such as organometallic reagents.
A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets.
Aromatase inhibitors (AIs) are a class of drugs that act by blocking the production of estrogens from androgens. The current review concentrates on the prenatal developmental toxicity of AIs in experimental models. Available data indicate that AIs may affect pregnancy at human therapeutic or lower doses. The window of vulnerability to AIs is not limited to organogenesis, but also includes the preimplantation stage and fetal periods. Decreased embryo/fetal survival was the prominent treatment-related effect. Morphological anomalies noted in fetuses exposed to AIs included skeletal anomalies, abnormal head morphology, increased ano-genital distance in female fetuses, and minor urinary tract system anomalies. Placental enlargement was consistently reported in rats and non-human primates after maternal treatment with several AIs. In conclusion, data from basic scientific research suggest that low intensity exposure to AIs applied during a wide gestational window can profoundly affect prenatal development.
Current research supports the notion that the apparently innate trait Sensory Processing Sensitivity (SPS) may act as a modulator of development as function of the environment. Interestingly, the common serotonin transporter linked polymorphic region (5-HTTLPR) does the same. While neural mechanisms underlying SPS are largely unknown, those associated with the 5-HTTLPR have been extensively investigated. We perform a comparative analysis of research findings on sensory processing facets associated with the trait and polymorphism to: 1. detect shared phenotypes and frame a hypothesis towards neural mechanisms underlying SPS; 2. increase the understanding of 5-HTTLPR-associated behavioral patterns. Trait and polymorphism are both associated with differential susceptibility to environmental stimuli; additionally, both involve 1. having stronger emotional reactions, 2. processing of sensory information more deeply, 3. being more aware of environmental subtleties, and 4. being easily overstimulated. We discuss neural mechanisms and environmental conditions that may underlie these four facets. Besides urging the actual assessment of the link between the two, the conclusions of our analyses may guide and focus future research strategies.
A new steroidal saponin, furotrilliumoside (FT) was isolated from the roots and rhizomes of Trillium tschonoskii Maxim. Its structure was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. FT showed superior activity of inhibiting NO production of RAW264.7 cells induced by lipopolysaccharide (LPS) in the preliminary biological screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of FT were investigated in LPS-induced RAW264.7 cells. The results showed that FT could reduce LPS-induced expression of inducible nitric oxide synthase (iNOS) and then resulted in the decrement of NO production. More meaningful, FT could down-regulate the expression of cyclooxygenase-2 (COX-2) and decrease the expressions of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), in both gene and protein levels. In mechanism study, FT blocked the LPS-induced upregulation of phosphorylated phosphoinositide-3-kinase and Akt (PI3K/Akt). Furthermore, FT inhibited the translocation of nuclear factor-kappa B (NF-κB) through the prevention of inhibitory factor kappa B alpha (IκBα) phosphorylation and degradation and also suppressed the mitogen-activated protein kinases (MAPK) signaling pathway in LPS-stimulated RAW264.7 macrophages. In addition, FT upregulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). Taken together, FT might act as a natural agent to treat some inflammatory diseases by targeting PI3K/Akt, MARK and Nrf2/HO-1 pathways.
Endometrioid endometrial carcinoma (EEC) is the most common gynecologic malignancy around the world. Epithelial-to-mesenchymal transition (EMT) is a core process during EEC cell invasion. The abnormal expression of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) or miR-200 family members were shown to facilitate EMT in multiple human cancers, but the regulatory mechanism by which MALAT1 and miR-200 act remains unknown. Previous studies have shown that miR-200 family members are enriched in EEC as well as melanoma and some ovarian carcinomas. In the present study, we first showed that miR-200c levels were higher in most EEC specimens than in non-tumor tissues, while MALAT1 levels were lower. Moreover, we found that miR-200c bound directly to MALAT1 using luciferase reporter and qRT-PCR assays. MALAT1 and miR-200c are reciprocally repressed, and TGF-β increased MALAT1 expression by inhibiting miR-200c. When the interaction between miR-200c/MALAT1 was interrupted, the invasive capacity of EEC cells was decreased and EMT markers expression were altered in vitro. A xenograft tumor model was used to show that targeting the miR-200c/MALAT1 axis inhibited EEC growth and EMT-associated protein expression in vivo. In summary, miR-200c/MALAT1 axis is a target with therapeutic potential in EEC. However, different expression model of miR-200c and MALAT1 in EEC with that in other organ carcinomas needs further mechanism researches.
Circumcision is defined as the partial or total excision of the prepuce. It is one of the most frequently performed surgical procedures around the world. This practice primarily involves minors. The aim of this study was to review the legal aspects of ritual circumcision in France. We report the cases of an 11-year-old and a 15-year-old boy examined in the forensic unit at the Hôtel-Dieu hospital in Paris, after their father complained to the Minors Protection Brigade of Paris following the discovery of their circumcision. The examination conducted in the forensic unit found that the two boys had undergone recent circumcision. The total incapacity of work (ITT) was assessed as 5 days for the 15-year-old boy and 1 day for the 11-year-old boy. Ritual "medicalized" circumcision is legal in France, and does not fall under article 222-1 of the Criminal Code (torture and acts of barbarism) or article 16-1 of the Civil Code (inviolability of the human body). Circumcision is treated as a "serious act" by several areas of jurisprudence, which means it is imperative to obtain the consent of both parents. Moreover, it must be performed in a hospital by knowledgeable staff.
Few studies have attempted to characterize how co-occurring risk factors for substance use disorders intersect. A recent study examined this question regarding cigarette smoking and demonstrated that co-occurring risk factors generally act independently. The present study examines whether that same pattern of independent intersection of risk factors extends to illicit drug abuse/dependence using a U.S. nationally representative sample (National Survey on Drug Use and Health, 2011-2013). Logistic regression and classification and regression tree (CART) modeling were used to examine risk of past-year drug abuse/dependence associated with a well-established set of risk factors for substance use (age, gender, race/ethnicity, education, poverty, smoking status, alcohol abuse/dependence, mental illness). Each of these risk factors was associated with significant increases in the odds of drug abuse/dependence in univariate logistic regressions. Each remained significant in a multivariate model examining all eight risk factors simultaneously. CART modeling of these 8 risk factors identified subpopulation risk profiles wherein drug abuse/dependence prevalence varied from <1% to >80% corresponding to differing combinations of risk factors present. Alcohol abuse/dependence and cigarette smoking had the strongest associations with drug abuse/dependence risk. These results demonstrate that co-occurring risk factors for illicit drug/abuse dependence generally intersect in the same independent manner as risk factors for cigarette smoking, underscoring further fundamental commonalities across these different types of substance use disorders. These results also underscore the fundamental importance of differences in the presence of co-occurring risk factors when considering the often strikingly different prevalence rates of illicit drug abuse/dependence in U.S. population subgroups.
The glycosylated and halogenated dialkylresorcinol (DAR) compounds bartolosides A-D (1-4) were recently discovered from marine cyanobacteria and represent a novel family of glycolipids, encoded by the brt biosynthetic gene cluster. Here, we report the isolation and NMR- and MS-based structure elucidation of monoglycosylated bartolosides E-K (5-11), obtained from Synechocystis salina LEGE 06099, a strain closely related to the cyanobacterium that produces the diglycosylated 2-4. In addition, a genome region containing orthologues of brt genes was identified in this cyanobacterium. Interestingly, the major bartoloside in S. salina LEGE 06099 was 1 (above 0.5% dry wt), originally isolated from the phylogenetically distant filamentous cyanobacterium Nodosilinea sp. LEGE 06102. Compounds 5-11 are analogues of 1, with different alkyl chain lengths or halogenation patterns. Their structures and the organization of the brt genes suggest that the DAR-forming ketosynthase BrtD can generate structural diversity by accepting fatty acyl-derived substrates of varying length. Compound 9 features a rare midchain gem-dichloro moiety, indicating that the putative halogenase BrtJ is able to act twice on the same midchain carbon.
We evaluated the effects of ACEA (selective cannabinoid (CB)1 receptor agonist), WIN 55,212-2 mesylate (WIN; non-selective CB1 and CB2 receptor agonist) and N-palmitoylethanolamine (PEA; an endogenous fatty acid of ethanolamide) in DBA/2 mice, a genetic model of reflex audiogenic epilepsy. PEA, ACEA or WIN intraperitoneal (i.p.) administration decreased the severity of tonic-clonic seizures. We also studied the effects of PEA, WIN or ACEA after co-administration with NIDA-41020 (CB1 receptor antagonist) or GW6471 (PPAR-α antagonist) and compared the effects of WIN, ACEA and PEA in order to clarify their mechanisms of action. PEA has anticonvulsant features in DBA/2 mice mainly through PPAR-α and likely indirectly on CB1 receptors, whereas ACEA and WIN act through CB1 receptors. The co-administration of ineffective doses of ACEA, PEA and WIN with some antiepileptic drugs (AEDs) was examined in order to identify potential pharmacological interactions in DBA/2 mice. We found that PEA, ACEA and WIN co-administration potentiated the efficacy of carbamazepine, diazepam, felbamate, gabapentin, phenobarbital, topiramate and valproate and PEA only also that of oxcarbazepine and lamotrigine whereas, their co-administration with levetiracetam and phenytoin did not have effects. PEA, ACEA or WIN administration did not significantly influence the total plasma and brain levels of AEDs; therefore, it can be concluded that the observed potentiation was only of pharmacodynamic nature. In conclusion, PEA, ACEA and WIN show anticonvulsant effects in DBA/2 mice and potentiate the effects several AEDs suggesting a possible therapeutic relevance of these drugs and their mechanisms of action.
Despite therapeutic advances, lung cancer remains one of the most common causes of cancer-related deaths worldwide. The ZNF703 gene has been identified as the driver of the 8p11-12 region and its amplification or overexpression has been associated with several types of cancers. It has also been shown that ZNF703 overexpression can activate the Akt/mTOR signaling pathway. The aim of our study was to investigate the role of the ZNF703 gene in association with Akt/mTOR activation in non-small cell lung cancer (NSCLC). Expression levels in tumors and matched noncancerous tissue samples from 47 patients were analyzed by qRT-PCR and the Akt phosphorylation levels were investigated by Western blotting. Our results show that ZNF703 is up-regulated in 63.4% of NSCLC tumor samples. Althogh the correlation did not reach a statistically significant level Akt phosphorylation was increased in tumor tissues expressing high levels of ZNF703. The role of the ZNF703 gene has not been investigated in NSCLC. Our data show that ZNF703 may contribute to tumor development in NSCLC by activating the Akt/mTOR pathway.
Carotenoids and retinoids have several similar biological activities such as antioxidant properties, the inhibition of malignant tumour growth and the induction of apoptosis. Supplementation with carotenoids can affect cell growth and modulate gene expression and immune responses. Epidemiological studies have shown a correlation between a high carotenoid intake in the diet with a reduced risk of breast, cervical, ovarian, colorectal cancers, and cardiovascular and eye diseases. Cancer chemoprevention by dietary carotenoids involves several mechanisms, including effects on gap junctional intercellular communication, growth factor signalling, cell cycle progression, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/β-catenin pathway and inflammatory cytokines. Moreover, carotenoids can stimulate the proliferation of B- and T-lymphocytes, the activity of macrophages and cytotoxic T-cells, effector T-cell function and the production of cytokines. Recently, the beneficial effects of carotenoid-rich vegetables and fruits in health and in decreasing the risk of certain diseases has been attributed to the major carotenoids, β-carotene, lycopene, lutein, zeaxanthin, crocin (/crocetin) and curcumin, due to their antioxidant effects. It is thought that carotenoids act in a time- and dose-dependent manner. In this review, we briefly describe the biological and immunological activities of the main carotenoids used for the treatment of various diseases and their possible mechanisms of action.
Ulinastatin, a urinary trypsin inhibitor (UTI), is widely used to clinically treat lipopolysaccharide (LPS)-related inflammatory disorders recently. Adherent pathogen-associated molecular patterns (PAMPs), of which LPS is the best-studied and classical endotoxin produced by Gram-negative bacteria, act to increase the biological activity of osteopedic wear particles such as polymethyl-methacrylate (PMMA) and titanium particles in cell culture and animal models of implant loosening. The present study was designed to explore the inhibitory effect of UTI on osteoclastogenesis and inflammatory osteolysis in LPS/PMMA-mediated Raw264.7 cells and murine osteolysis models, and investigate the potential mechanism. The in vitro study was divided into the control group, LPS-induced group, PMMA-stimulated group and UTI-pretreated group. UTI (500 or 5000 units/ml) pretreatment was followed by PMMA (0.5 mg/ml) with adherent LPS. The levels of inflammatory mediators including tumour necrosis factor-α (TNF-α), matrixmetallo-proteinases-9 (MMP-9) and interleukin-6 (IL-6), receptor activation of nuclear factor NF-κB (RANK), and cathepsin K were examined and the amounts of phosphorylated I-κB, MEK, JNK and p38 were measured. In vivo study, murine osteolysis models were divided into the control group, PMMA-induced group and UTI-treated group. UTI (500 or 5000 units/kg per day) was injected intraperitoneally followed by PMMA suspension with adherent LPS (2×10(8) particles/25 μl) in the UTI-treated group. The thickness of interfacial membrane and the number of infiltrated inflammatory cells around the implants were assessed, and bone mineral density (BMD), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), relative bone volume over total volume (BV/TV) of distal femur around the implants were calculated. Our results showed that UTI pretreatment suppressed the secretion of proinflammatory cytokines including MMP-9, IL-6, TNF-α, RANK and cathepsin K through down-regulating the activity of nuclear factor kappa B (NF-κB) and MAPKs partly in LPS/PMMA-mediated Raw264.7 cells. Finally, UTI treatment decreased the inflammatory osteolysis reaction in PMMA-induced murine osteolysis models. In conclusion, these results confirm the anti-inflammatory potential of UTI in the prevention of particle disease.
MicroRNAs (miRNAs) are small non-coding RNAs which can act as master regulators of gene expression, modulate almost all biological process and are essential for maintaining cellular homeostasis. Dysregulation of miRNA expression has been associated with aberrant gene expression and may lead to pathological conditions. Evidence suggests that miRNA expression profiles are altered between health and disease and as such may be considered as biomarkers of disease. Evidence is increasing that miRNAs are particularly important in lung homeostasis and development and have been demonstrated to be the involved in many pulmonary diseases such as asthma, COPD, sarcoidosis, lung cancer and other smoking related diseases. Better understanding of the function of miRNA and the mechanisms underlying their action in the lung, would help to improve current diagnosis and therapeutics strategies in pulmonary diseases. Recently, some miRNA-based drugs have been introduced as possible therapeutic agents. In this review we aim to summarize the recent findings regarding the role of miRNAs in the airways and lung and emphasise their potential therapeutic roles in pulmonary diseases.
Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.
The Controlled Substances Staff of the Center for Drug Evaluation and Research at the US Food and Drug Administration and the Pharmaceutical Research Manufacturers Association (PhRMA) conducted a series of open forum dialog sessions between 2006 and 2016. A Cross Company Abuse Liability Council (CCALC) was formed during the process of this unique collaborative effort between Industry and Federal Regulators whose goals were to establish the development of standards for the preclinical screening of new molecular entities for schedule control actions required as part of every New Drug Application process. The draft guidance document was published and disseminated in 2010, which allowed for alternative approaches to each study protocol requirement needed for NDA review, if the approach satisfied the requirements of the applicable statutes and regulations (i.e., the controlled substance act). In a series of recent pre-study protocol reviews requested by confidential Pharmaceutical Sponsors of MPI Research, the CSS staff appeared to change its policy and set forth to require all drug discrimination study data to be generated under "extinction" test sessions. MPI Research is a Contract Research Organization acting on behalf of pharmaceutical companies and bound under separate confidentiality agreements.
The purpose of this review is to highlight the data appearing in peer-reviewed scientific journals that do not support the regulatory administrative constraints on one specific testing methodology (extinction) to the exclusion of another (reinforced test sessions).
This mind shift represents a restrictive administrative policy by the FDA that is not supported by the published data.
Symptomatic chronic Chagas disease affects up to 40% of patients infected with Trypanosoma cruzi. The lack of reliable early markers of cure after therapy hinders disease management and clinical trials with new drugs. We performed a study with 18 months of follow-up to compare changes in immune parameters and T. cruzi-specific immune responses as surrogate markers of response to therapy between patients treated with benznidazole and untreated patients. This was a pilot, open-label, randomised clinical trial of treatment with benznidazole versus no treatment in patients with indeterminate chronic T. cruzi infection. In both groups we investigated changes in T-cell activation, T-cell subpopulations, regulatory T-cell counts, IL6, and sCD14 levels, and T. cruzi-specific immune responses (Th1, Th2, and Th17 responses). Fourteen patients were included in the study (seven in each group). Median age was 35 years (P25-75 31-43), 57% were female, and 93% were Bolivian. Benznidazole was administered at 5mg/kg/day for 60days. Three patients discontinued benznidazole owing to adverse reactions and were not evaluated. At the end of the follow-up period, treated patients showed significantly less immune activation and lower regulatory T-cell counts, with an increased Th17 and Th1 response. This randomised pilot clinical trial administering benznidazole to patients with indeterminate chronic Chagas disease brings about changes in the adaptive immunity, leading to a general decrease in inflammatory status. This apparently beneficial response could act as the basis for monitoring new antiparasitic drugs.
Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical α/β fold hydrolase, was previously reported to have both γ-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any γ-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities.
Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of α/β fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.
The potential efficacy of cannabinoid receptor ligands for the treatment of epilepsy remains controversial; cannabis components that act via cannabinoid type 1 (CB1) receptors produce anticonvulsant effects in animal models despite treatment with the CB receptor agonist reliably inducing convulsions in various species. Moreover, the potential role of cannabinoid receptor type 2 (CB2) to modulate seizures remains under-investigated. This study assessed the effects of the selective CB2 receptor agonist, AM1241, on pentylenetetrazole (PTZ)-induced seizures in rats. A stereotactically placed guide cannula was surgically implanted into the right lateral ventricle in adult Wistar rats which, 5-6days later, received an acute intracerebroventricular (i.c.v.) microinfusion of AM1241 (0.01, 1 or 10μg/2μl or vehicle) 5min before intraperitoneal (i.p.) injection of PTZ (70mg/kg). Rats were observed for 30min and the seizure severity behavior measured using a modified Racine's scale. Additional groups of rats were pretreated with a single low dose of the selective CB2 receptor antagonist, AM630 (dose 1mg/kg; i.p.), or vehicle, 30min prior to i.c.v. microinfusion of AM1241 (1μg/2μl). AM1241 administration significantly increased tonic-clonic seizure incidence and severity while also decreasing the onset of generalized seizures (AM1241 1 and 10μg/2μl). Pretreatment with AM630 prevented the proconvulsant effects of AM1241. This study shows, for the first time, that selective activation of CB2 receptors can increase generalized seizure susceptibility and suggests that pathological hyperexcitability phenomena can be differentially regulated by targeting CB1 and CB2 receptors.
Antigen-binding fragments (Fab) and F(ab')2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab')2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab')2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab')2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.
 Visceral leishmaniasis (VL) is a potentially fatal parasitic disease associated with fever, cachexia and impaired protective T-cell responses against the parasite.
 Peripheral blood leukocytes from 105 subjects with VL and healthy control subjects from the endemic region of Muzaffarpur, Bihar, India, were compared using flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. Findings were correlated with clinical data.
 An expanded population of low-density neutrophils that expressed HLA-DR, CD80 and CD86 was observed in subjects with VL. This neutrophil population contracted after successful treatment of disease. Plasma from patients with acute VL was able to induce similar high-level HLA-DR expression in neutrophils from healthy subjects. HLA-DR(+) neutrophils from subjects with VL did not stimulate T-cell proliferation, but they did express higher programmed cell death ligand-1 (PDL1) than other neutrophils, and lymphocytes of the same subjects expressed high programmed cell death 1 (PD1).
 Patients with acute VL have expanded circulating low-density neutrophils expressing markers of antigen presentation, which diminish after treatment. Development of HLA-DR(+) neutrophils is stimulated, at least in part, by components of plasma from patients with acute disease. Although we found no evidence that they act as antigen-presenting cells, these neutrophils expressed markers implicating a role in T-cell exhaustion.
Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and autism. FXS results from the absence of FMRP, an RNA binding protein associated to ribosomes that influences the translation of specific mRNAs in post-synaptic compartments of neurons. The main molecular consequence of the absence of FMRP is an excessive translation of neuronal protein in several areas of the brain. This local protein synthesis deregulation is proposed to underlie the defect in synaptic plasticity responsible for FXS. Recent findings in neurons of the fragile X mouse model (Fmr1-KO) uncovered another consequence of the lack of FMRP: a deregulation of the diacylglycerol (DAG)/phosphatidic acid (PA) homeostasis. DAG and PA are two interconvertible lipids that influence membrane architecture and that act as essential signaling molecules that activate various downstream effectors, including master regulators of local protein synthesis and actin polymerization. As a consequence, DAG and PA govern a variety of cellular processes, including cell proliferation, vesicle/membrane trafficking and cytoskeletal organization. At the synapse, the level of these lipids is proposed to influence the synaptic activation status. FMRP appears as a master regulator of this neuronal process by controlling the translation of a diacylglycerol kinase enzyme that converts DAG into PA. The deregulated levels of DAG and PA caused by the absence of FMRP could represent a novel therapeutic target for the treatment of FXS.
RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by X-ray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain.
Fusarium langsethiae is a fungal pathogen of cereal crops that is an increasing problem in northern Europe, but much of its epidemiology is poorly understood. The species produces the mycotoxins T-2 and HT-2, which are highly toxic. It was hypothesized that grain aphids, Sitobion avenae, may transmit F. langsethiae inoculum between wheat plants, and a series of transmission experiments and volatile chemical analyses was performed to test this. Manual translocation of aphids from inoculated to uninfected hosts resulted in pathogen DNA accumulation in hosts. However, the free movement of wingless aphids from infected to healthy plants did not. The addition of winged aphids reared on F. langsethiae-inoculated wheat seedlings to wheat plants also did not achieve successful pathogen transfer. While our data suggested that aphid transmission of the pathogen was not very efficient, we observed an increase in disease when aphids were present. After seedling inoculation, an increase in pathogen DNA accumulation in seedling leaves was observed upon treatment with aphids. Furthermore, the presence of aphids on wheat plants with F. langsethiae-inoculated ears not only led to a rise in the amount of F. langsethiae DNA in infected grain but also to an increase in the concentrations of T-2 and HT-2 toxins, with more than 3-fold higher toxin levels than diseased plants without aphids. This work highlights that aphids increase the susceptibility of wheat host plants to F. langsethiae and that aphid infestation is a risk factor for accumulating increased levels of T-2 and HT-2 in wheat products.
Fusarium langsethiae is shown here to cause increased contamination levels of grain with toxins produced by fungus when aphids share the host plant. This effect has also recently been demonstrated with Fusarium graminearum, yet the two fungal species show stark differences in their effect on aphid populations. In both cases, aphids improve the ability of the pathogens to cause and initiate Fusarium head blight (FHB) disease in wheat, but F. langsethiae may be able to act as a dispersal agent. F. langsethiae contributes harmful toxins to wheat grain that need to be controlled, but as yet, its epidemiology is unresolved. This work reveals insights into the role aphids play in promoting the successful colonization of this species in wheat and the benefit of controlling aphid populations on crops that are at high risk of FHB.
Organisms often balance among reproduction, growth and survival. When these processes are in competition, selection may act to drive functional dimorphism. Unlike seed plants, ferns use their foliar surfaces for reproduction and carbon fixation. Across species, ferns exhibit a gradient of fertile-sterile dimorphy: from the production of highly reduced fertile fronds (holodimorphic) to no reduction (monomorphic) in laminar area between fronds. Here the physiological impacts of fertile-sterile dimorphy were investigated through a series of observational and experimental field manipulations.
Temporal shifts in photosynthesis, respiration and percent nitrogen (%N) were examined to evaluate changes in physiological behaviour over the growing season of two species of fern of similar ecological niche, yet of different degrees of fertile-sterile frond dimorphism: Osmundastrum cinnamomeum (holodimorphic) and Osmunda regalis (hemidimorphic). These data are combined with experimental fertile and sterile frond removal to evaluate relative costs of reproduction in both species. Finally, labelled δ(13)C gas was used to follow carbon allocation across the growing season.
The data demonstrate that reproductive structures in the holodimorphic O. cinnamomeum come at more significant carbon and nitrogen costs relative to those in the hemidimorphic O. regalis Excision experiments demonstrate that investment in fertile fronds strongly impacted future allocation to reproduction in the holodimorphic species but had a lesser effect on the hemidimorphic species. The labelling experiments showed that fixed carbon is translocated to the rhizomes only, but at different times in the two species. Investment in underground resources probably allows these plants to manage the costs of reproduction associated with increased dimorphy.
Fertile-sterile dimorphy has evolved multiple times in ferns in spite of the apparent physiological costs associated with a reduction in photosynthetically active tissues. These apparent costs may be offset by an increase in potential spore dispersal distance and/or increased spore production. The phenomenon may further influence species ecology as dimorphic taxa often occupy resource-rich environments.
All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.
The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.
Buccal micronucleus cytome assay was carried out in 47 exposed (sprayers and leaf harvesters), 47 non-exposed (controls) to determine the extent of damage working in the tea plantations of Terai region of West Bengal, India. As the pesticide exposed male workers were found to consume alcohol and smoked cigarettes/bidis, 35 smokers and 30 alcoholics were also included for comparison. Results showed a significant difference in micronuclei (9.91 ± 2.74, p ≤ .001), nuclear bud (4.98 ± 1.31, p ≤ .001), binucleate (6.26 ± 2.84, p ≤ .001), karyorrhectic (8.36 ± 2.28, p ≤ .001), pyknotic (5.62 ± 1.78, p ≤ .05) as well as karyolytic (6.81 ± 3.00, p ≤ .001) nuclei compared with control. Comparison also revealed a higher frequency of micronuclei (6.11 ± 2.55, p ≤ .01), nuclear bud (4.06 ± 1.97, p ≤ .05), binucleate (4.34 ± 1.85, p ≤ .001), karyorrhectic (6.83 ± 2.12, p ≤ .001), and karyolytic (6.20 ± 2.54, p ≤ .001) nuclei except pyknotic cell in the smoker than control. Frequency of binucleate (3.80 ± 1.73, p ≤ .05), karyorrhectic (5.57 ± 2.34, p ≤ .05), pyknotic (5.50 ± 1.36, p ≤ .05), and karyolytic (6.30 ± 2.71, p ≤ .001) nuclei was higher in the alcoholics than control (non-alcoholics), whereas the micronuclei and nuclear bud were found to be non-significant compared with the control. Our analyses also revealed a higher proportion of the micronucleus and the cell death parameters in the pesticide exposed males than females, which indicated that pesticide, smoking, and alcohol may act synergistically to cause more damage to the buccal epithelial cells. However, age and the exposure duration have no influence on the micronucleus and other cell death parameters.
Current evidence suggests that many GWAS and IL1 SNPs are associated with periodontal diseases but their functional role remains ambiguous. Therefore, it is imperative to elucidate the molecular pathways through which these SNPs might act on the development of the disease. The purpose of this review was to highlight the regulatory elements of noncoding regions of the genome and provide insights on the functional role of periodontitis-associated GWAS and IL1 SNPs.
A search was performed using ENCODE data available on different browsers.
GWAS and IL1 SNPs overlap DNase I hypersensitivity sites, histone modifications and transcription binding sites. Some of these noncoding variants influenced the transcription activity of inflammatory genes.
SNPs associated with periodontal diseases may contribute to the development of the disorder through their functional roles. Unraveling the character of genetic components might explain the diversity of clinical phenotypes among population groups as well as disease susceptibility.
Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.
Communication has been suggested as a mechanism to stabilize cooperation. In bacteria, chemical communication, termed quorum sensing (QS), has been hypothesized to fill this role, and extracellular public goods are often induced by QS at high cell densities. Here we show, with the bacterium Vibrio harveyi, that QS provides strong resistance against invasion of a QS defector strain by maximizing the cellular growth rate at low cell densities while achieving maximum productivity through protease upregulation at high cell densities. In contrast, QS mutants that act as defectors or unconditional cooperators maximize either the growth rate or the growth yield, respectively, and thus are less fit than the wild-type QS strain. Our findings provide experimental evidence that regulation mediated by microbial communication can optimize growth strategies and stabilize cooperative phenotypes by preventing defector invasion, even under well-mixed conditions. This effect is due to a combination of responsiveness to environmental conditions provided by QS, lowering of competitive costs when QS is not induced, and pleiotropic constraints imposed on defectors that do not perform QS.
Cooperation is a fundamental problem for evolutionary biology to explain. Conditional participation through phenotypic plasticity driven by communication is a potential solution to this dilemma. Thus, among bacteria, QS has been proposed to be a proximate stabilizing mechanism for cooperative behaviors. Here, we empirically demonstrate that QS in V. harveyi prevents cheating and subsequent invasion by nonproducing defectors by maximizing the growth rate at low cell densities and the growth yield at high cell densities, whereas an unconditional cooperator is rapidly driven to extinction by defectors. Our findings provide experimental evidence that QS regulation prevents the invasion of cooperative populations by QS defectors even under unstructured conditions, and they strongly support the role of communication in bacteria as a mechanism that stabilizes cooperative traits.
Aspirin has been used in the treatment and chemoprevention of many malignant cancers. The mechanism of its anti-cancer activity mainly involves the inhibition of cyclooxygenase-2 (COX-2). However, the application of aspirin is limited by the serious gastric mucosal damage that accompanies its usage. We have previously reported the preparation of a novel aspirin derivative that we named Ca-Asp, and showed that it causes less damage to gastric mucosa of rat and inhibits the expression of COX-2 to higher degree than Asp. However, the anti-cancer effect and mechanism of Ca-Asp was not demonstrated. In this study, the anti-cancer effect of Ca-Asp was investigated and compared with those of Asp and Hydroxyapatite (Hap) at the cell level. The results showed that treatment of SGC-7901 cells (human gastric cancer cell line) with 200-400μg/ml Ca-Asp resulted in significant reduction in cell viability, compared to treatment with either Asp or Hap, and at a higher concentration (500μg/ml). Subsequent investigation into the possible underlying mechanism showed that Ca-Asp induced apoptosis and caused cell cycle arrest at the G1 phase. Ca-Asp also up-regulated the levels of caspase-3 and p53, but down regulated the level of cyclin D1, NF-κB, COX-2 and PGE2. Furthermore, simultaneous treatment of SGC-7901 cells with Ca-Asp and exogenous PGE2 reduced the anti-proliferative effect of Ca-Asp on the cells. Taken together, the results suggested that Ca-Asp might act as a potential anti-cancer drug, and that its suppression of PGE2 production might constitute an important part of its anti-cancer activity.
Contamination of aquatic habitats with anthropogenic nutrients has been associated with an increase in mosquito larval populations but the underlying mechanisms remain poorly understood. We examined the individual and combined effects of two synthetic fertilizers (ammonium sulfate and potassium chloride) on Aedes albopictus survival, development time, and sex ratio. The bacterial and fungal communities of water samples from different fertilizer treatments were also characterized by MiSeq sequencing of the 16S rRNA gene (bacteria) and internal transcribed spacer 1 (fungi) and their relationship with mosquito survival and development determined. Mosquitoes from ammonium sulfate treatment had significantly lower survival rates and longer development times compared to those from control, potassium chloride or a mixture of the two fertilizers. Fertilizer treatment had no significant effects on Ae. albopictus sex ratio although ammonium sulfate treatment tended to be more biased towards males relative to the other treatments. There were no significant effects of fertilizer treatment on fungal communities. However, potassium chloride treatments had lower bacterial diversity compared to the other treatments and the bacterial community structure of control and potassium chloride treatments differed significantly from that of ammonium sulfate and a mixture of the two fertilizers. Microbial composition but not diversity was significantly associated with mosquito survival and development. These findings suggest that anthropogenic nutrients can have a profound impact on mosquito survival and development. In addition to any potential direct effects on mosquito physiology, our results suggest that fertilizers can act indirectly by disrupting the microbial communities that provide a critical food resource for mosquito larvae.
Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine. Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance. In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.
To evaluate the relationship between ampicillin dosing, exposure, and seizures.
This was a retrospective observational cohort study of electronic health record (EHR) data combined with pharmacokinetic model derived drug exposure predictions. We used the EHR from 348 Pediatrix Medical Group neonatal intensive care units from 1997 to 2012. We included all infants 24-41 weeks gestational age, 500-5400 g birth weight, first exposed to ampicillin prior to 25 days postnatal age. Using a 1-compartment pharmacokinetic model and EHR data, we simulated maximum ampicillin concentration at steady state (Cmaxss, µg/mL) and area under the concentration time curve from 0 to 24 hours (AUC24, µg*h/dL). Using multivariable logistic regression, we evaluated association between ampicillin dosing, exposure, and seizures as documented in the EHR.
We identified 131 723 infants receiving 134 041 courses of ampicillin for 653 506 infant-days of exposure. The median daily dose was 200 mg/kg/d (25th, 75th percentile; 100, 200). Median Cmaxss and AUC24 were 256.6 µg/mL (164.3, 291.5) and 2593 µg*h/dL (1917, 3334). On multivariable analysis, dosing was not associated with seizures. However increasing Cmaxss (OR = 1.10, 95% CI 1.03, 1.17) and AUC24 (OR 1.11, 95% CI 1.05, 1.18) were associated with increased odds of seizures.
In this cohort of hospitalized infants, higher ampicillin exposure was associated with seizures as documented in the EHR.
Arsenic and polycyclic aromatic hydrocarbon (PAH) exposures affect many people worldwide leading to cancer and other diseases. Arsenite (As(+3)) and certain PAHs are known to cause genotoxicity. However, there is limited information on the interactions between As(+3) and PAHs at environmentally relevant concentrations. The thymus is the primary immune organ for T cell development in mammals. Our previous studies showed that environmentally relevant concentrations of As(+3) induce genotoxicity in mouse thymus cells through Poly(ADP-ribose) polymerase (PARP) inhibition. Certain PAHs, such as the metabolites of benzo(a)pyrene (BaP), are known to cause DNA damage by forming DNA adducts. In the present study, primary mouse thymus cells were examined for DNA damage following 18 hr in vitro treatments with 5 or 50 nM As(+3) and 100 nM BaP, benzo[a]pyrene-7,8-dihydrodiol (BP-Diol), or benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). An interactive increase in genotoxicity and apoptosis were observed following treatments with 5 nM As (+)  (3 )+( )100 nM BP-diol and 50 nM As (+)  (3 )+( )100 nM BPDE. We attribute the increase in DNA damage to inhibition of PARP inhibition leading to decreased DNA repair. To further support this hypothesis, we found that a PARP inhibitor, 3,4-dihydro-5[4-(1-piperindinyl) butoxyl]-1(2H)-isoquinoline (DPQ), also interacted with BP-diol to produce an increase in DNA damage. Interestingly, we also found that As(+3) and BP-diol increased CYP1A1 and CYP1B1 expression, suggesting that increased PAH metabolism may also contribute to genotoxicity. In summary, these results show that the suppression of PARP activity and induction of CYP1A1/CYP1B1 may act together to increase DNA damage produced by As(+3) and PAHs.
INTRODUCTION Hemicolectomies are not tailored in right-sided colon cancer resections, despite significant variation in the incidence and origin of the right colic artery (RCA). Early evidence suggests that removal of the relevant lymphovascular package and associated cancer as part of complete mesocolic excision (CME), rather than the entire right colon, may produce better outcomes. Advancing laparoscopic techniques are making this possible, and so it is increasingly important to more precisely define the anatomy of the RCA. METHODS To demonstrate the incidence and variation of the RCA, 25 formalin embalmed cadavers were dissected. Consent to dissection and photography was obtained under Human Tissue Act regulations. RESULTS Eleven female and 14 male cadavers (mean age 79.7 years, range 41-95 years) were included. The RCA originated from the right branch of the middle colic artery in nine cadavers (36%), while it arose from the superior mesenteric artery in eight cases (32%) and from the ileocolic or root of the middle colic artery in a smaller number of specimens. The RCA was absent in two individuals. CONCLUSIONS The RCA arises from the right branch of the middle colic artery in a considerable number of cases. The literature to date does not reflect the precision of anatomical understanding required for CME; hence, a new definition for the right colic vessel is proposed.
Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T-Vec (talimogene laherparepvec), a second-generation oncolytic herpes simplex virus type 1 (HSV-1) armed with GM-CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T-Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX-594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM-CSF-expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild-type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47∆, a third-generation oncolytic HSV-1, is ongoing in glioblastoma patients. G47∆ was recently designated as a "Sakigake" breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47∆ with priority reviews and a fast-track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor-specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients.
Omalizumab (Xolair) dosing in severe allergic asthma is based on serum IgE and bodyweight. In Australia, patients eligible for omalizumab but exceeding recommended ranges for IgE (30-1500 IU/mL) and bodyweight (30-150 kg) may still receive a ceiling dose of 750 mg/4 weeks. About 62% of patients receiving government-subsidized omalizumab are enrolled in the Australian Xolair Registry (AXR).
To determine whether AXR participants above the recommended dosing ranges benefit from omalizumab and to compare their response to within-range participants.
Data were stratified according to dose range status (above-range or within-range). Further sub-analyses were conducted according to the reason for being above the dosing range (IgE only vs. IgE and weight).
Data for 179 participants were analysed. About 55 (31%) were above recommended dosing criteria; other characteristics were similar to within-range participants. Above-range participants had higher baseline IgE [812 (IQR 632, 1747) IU/mL vs. 209 (IQR 134, 306) IU/mL] and received higher doses of omalizumab [750 (IQR 650, 750) mg] compared to within-range participants [450 (IQR, 300, 600) mg]. At 6 months, improvements in Juniper 5-item Asthma Control Questionnaire (ACQ-5, 3.61 down to 2.01 for above-range, 3.47 down to 1.93 for within-range, P < 0.0001 for both) and Asthma Quality of Life Questionnaire (AQLQ mean score (3.22 up to 4.41 for above-range, 3.71 up to 4.88 for within-range, P < 0.0001) were observed in both groups. Forced expiratory volume in one second (FEV1 ) improved among above-range participants. There was no difference in response between above-range and within-range participants. Above-range participants due to either IgE alone or IgE and weight had similar improvements in ACQ-5, AQLQ and FEV1 .
Patients with severe allergic asthma above recommended dosing criteria for omalizumab have significantly improved symptom control, quality of life and lung function to a similar degree to within-range participants, achieved without dose escalation above 750 mg.
Programmed cell death (PCD) is a fundamental plant process in growth and development and in response to both biotic and abiotic stresses. Nitric oxide (NO) is a central component in determining whether a cell undergoes PCD, either as a direct elicitor of the response or as a factor in signal transduction from various hormones. Both NO and hormones that use NO as a signal transducer are mobile in the plant. Why do one set of cells die while adjacent cells remain alive, if this is the case? There is evidence to suggest that phytoglobins (Pgbs; previously termed non-symbiotic hemoglobins) may act as binary switches to determine plant cellular responses to perturbations. There are anywhere from one to five Pgb genes in plants that are expressed in response to growth and development and to stress. One of their main functions is to scavenge NO. This review will discuss how Pgb modulates cellular responses to auxin, cytokinin, and jasmonic acid during growth and development and in response to stress. The moderation in the production of reactive oxygen species (ROS) by Pgbs and the effects on PCD will also be discussed. An overall mechanism for Pgb involvement will be presented.
Physicians and hospital systems often have relationships with biomedical manufacturers to develop new ideas, products, and further education. Because this relationship can influence medical research and practice, reporting disclosures are necessary to reveal any potential bias and inform consumers. The Sunshine Act was created to develop a new reporting system of these financial relationships called the Open Payments database. Currently all disclosures submitted with research to scientific meetings are at the discretion of the physician. We hypothesized that financial relationships between authors and the medical industry are underreported.
We aimed to describe concordance between physicians' financial disclosures listed in the abstract book from the 41st annual scientific meeting of the Society of Gynecologic Surgeons to physician payments reported to the Center for Medicaid and Medicare Services Open Payments database for the same year.
Authors and scientific committee members responsible for the content of the 41st annual scientific meeting of the Society of Gynecologic Surgeons were identified from the published abstract book; each abstract listed disclosures for each author. Abstract disclosures were compared with the transactions recorded on the Center for Medicaid and Medicare Services Open Payments database for concordance. Two authors reviewed each nondisclosed Center for Medicaid and Medicare Services listing to determine the relatedness between the company listed on the Center for Medicaid and Medicare Services and abstract content.
Abstracts and disclosures of 335 physicians meeting inclusion criteria were reviewed. A total of 209 of 335 physicians (62%) had transactions reported in the Center for Medicaid and Medicare Services, which totaled $1.99 million. Twenty-four of 335 physicians (7%) listed companies with their abstracts; 5 of those 24 physicians were concordant with the Center for Medicaid and Medicare Services. The total amount of all nondisclosed transactions was $1.3 million. Transactions reported in the Center for Medicaid and Medicare Services associated with a single physician ranged from $11.72 to $405,903.36. Of the 209 physicians with Center for Medicaid and Medicare Services transactions that were not disclosed, the majority (68%) had at least 1 company listed in the Center for Medicaid and Medicare Services that was determined after review to be related to the subject of their abstract.
Voluntary disclosure of financial relationships was poor, and the majority of unlisted disclosures in the abstract book were companies related to the scientific content of the abstract. Better transparency is needed by physicians responsible for the content presented at gynecological scientific meetings.
Few studies have assessed the dietary quality of children who eat meals from home compared with school meals according to the 2010 Dietary Guidelines for Americans.
The objective of this study was to examine diet quality for elementary school students in relation to source of breakfast and lunch (whether school meal or from an outside source).
An observational study was conducted of students in 43 schools in San Diego, CA, during the 2011-2012 school year.
Fourth- and fifth-grade students (N=3,944) completed a diary-assisted 24-hour food recall.
The Healthy Eating Index-2010 (HEI-2010) scores of children who ate breakfast and lunch at school were compared with the HEI-2010 scores of children who obtained their meals from home and a combination of both school and home.
Analysis of variance, χ(2) test, and generalized estimating equation models adjusted for age, sex, race/ethnicity, grade, language, and school level clustering were performed.
School lunch eaters had a higher mean±standard deviation overall diet quality score (HEI-2010=49.0±11.3) compared with students who ate a lunch obtained from home (46.1±12.2; P=0.02). There was no difference in overall diet quality score by breakfast groups. Students who ate school breakfast had higher total fruit (P=0.01) and whole fruit (P=0.0008) scores compared with students who only ate breakfast obtained from home. Students who ate school foods had higher scores for dairy (P=0.007 for breakfast and P<0.0001 for lunch) and for empty calories from solid fats and added sugars (P=0.01 for breakfast and P=0.007 for lunch).
Eating school lunch was associated with higher overall diet quality compared with obtaining lunch from home. Future studies are needed that assess the influence of the Healthy Hunger-Free Kids Act on children's diet quality.
In a study by Gelstein et al., we found that human emotional tears act as a social chemosignal. In the first of three different experiments in that study we observed that sniffing women's emotional tears reduced the sexual attractiveness attributed by men to pictures of women's faces. In a study partly titled "Chemosignaling effects of human tears revisited", Gračanin et al. claim failed replication of this effect in a series of experiments, one they described as "exactly the same procedure" as Gelstein. Given that Gračanin et al. refused our extended offer to jointly replicate the experiment at our expense, we can merely comment on their effort. We find that Gračanin, who are not a chemosignaling laboratory, used methodology that falls short of standards typically applied in chemosignaling research. Thus, their experiments were profoundly different from Gelstein. Finally, we found that in reanalysing their raw data we could in fact replicate the effect from Gelstein. Thus, we conclude that the failed replication in Gračanin is neither a replication nor failed.
Self-report is the most common means of obtaining mammography screening data. The purpose of this study was to assess the accuracy of minority women's self-reported mammography by comparing their self-reported dates of mammograms with those in their medical records from a community-based randomized control trial. We found that out of 192 women, 116 signed the Health Information Portability and Accountability Act form and, among these, 97 had medical records that could be verified (97 / 116 = 83.6%). Ninety-two records matched where both sources confirmed a mammogram; 48 of 92 (52.2%) matched perfectly on self-reported date of mammogram. Complexities in the verification process warrant caution when verifying self-reported mammography screening in minority populations. In spite of some limitations, our findings support the usage of self-reported data on mammography as a validated tool for other researchers investigating mammography screening among minority women who continue to have low screening rates.
Wide and discriminate use of antibiotics has resulted in serious biological and ecological concerns, especially the emergence of antibiotic resistance. Probiotics, known as beneficial microbes, are being proposed as an effective and eco-friendly alternative to antibiotics. They were first applied in aquaculture species more than three decades ago, but considerable attention had been given only in the early 2000s. Probiotics are defined as live or dead, or even a component of the microorganisms that act under different modes of action in conferring beneficial effects to the host or to its environment. Several probiotics have been characterized and applied in fish and a number of them are of host origin. Unlike some disease control alternatives being adapted and proposed in aquaculture where actions are unilateral, the immense potential of probiotics lies on their multiple mechanisms in conferring benefits to the host fish and the rearing environment. The staggering number of probiotics papers in aquaculture highlights the multitude of advantages from these microorganisms and conspicuously position them in the dynamic search for health-promoting alternatives for cultured fish. This paper provides an update on the use of probiotics in finfish aquaculture, particularly focusing on their modes of action. It explores the contemporary understanding of their spatial and nutritional competitiveness, inhibitory metabolites, environmental modification capability, immunomodulatory potential and stress-alleviating mechanism. This timely update affirms the importance of probiotics in fostering sustainable approaches in aquaculture and provides avenues in furthering its research and development.
Jehovah's Witnesses patients refuse blood transfusions for religious reasons. Anesthesiologists must master specific legal knowledge to provide care to these patients. Understanding how the Law and the Federal Council of Medicine treat this issue is critical to know how to act in this context. The aim of this paper was to establish a treatment protocol for the Jehovah's Witness patient with emphasis on ethical and legal duty of the anesthesiologist.
The article analyzes the Constitution, Criminal Code, resolutions of the Federal Council of Medicine (FCM), opinions, and jurisprudence to understand the limits of the conflict between the autonomy of will of Jehovah's Witnesses to refuse transfusion and the physician's duty to provide the transfusion. Based on this evidence, a care protocol is suggested.
The FCM resolution 1021/1980, the penal code Article 135, which classifies denial of care as a crime and the Supreme Court decision on the HC 268,459/SP process imposes on the physician the obligation of blood transfusion when life is threatened. The patient's or guardian's consent is not necessary, as the autonomy of will manifestation of the Jehovah's Witness patient refusing blood transfusion for himself and relatives, even in emergencies, is no not forbidden.
In the last decades, a number of new antimuscarinic drugs have been introduced for treatment of the overactive bladder (OAB), defined symptomatically (OAB syndrome) or urodynamically (detrusor overactivity). Recently, three new drug principles have been approved for clinical use, the β3 -adrenoceptor agonist, mirabegron, the phosphodiesterase-5 inhibitor, tadalafil and the blocker of afferent and efferent nerves, botulinum toxin. However, new alternatives are continuously being explored. OAB is a filling disorder, and ATP is involved in the generation of afferent impulses. One way of blocking the ATP afferent pathway is through the use of P2X3 receptor antagonists. In animal models, this strategy appears to work very well, but whether it translates effectively to man remains to be established. Evidence suggests that components of the endocannabinoid system are involved in regulation of bladder function. Clinical studies of cannabinoid extracts on LUTS are scarce and essentially restricted to patients with MS, and the results have so far not been convincing. Amplification of endocannabinoid activity by inhibiting their degradation via fatty acid amide hydrolase inhibitors may be an attractive approach, but no clinical experiences in OAB have been reported. Studies of the lower urinary tract have indicated that several transient receptor potential (TRP) channels, including TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1, are expressed in the bladder and may act as sensors of stretch and/or chemical irritation. Animal studies have shown that inhibition of these pathways can be effective for the reduction in bladder activity. However, the roles of these channels for normal function and in pathological states have not been established, and so far adverse effects (hyperthermia) have hampered development of antagonists.
Chronic Kidney Disease (CKD) is a common health problem affecting 1 in 12 Americans. It is associated with elevated risks of mortality, cardiovascular disease, and high costs for the treatment of renal failure with dialysis or transplantation. Advances in CKD care are impeded by the lack of biomarkers for early diagnosis, assessment of the extent of tissue injury, estimation of disease progression, and evaluation of response to therapy. Such biomarkers should improve the performance of existing measures of renal functional impairment (estimated glomerular filtration rate, eGFR) or kidney damage (proteinuria). MicroRNAs (miRNAs) a class of small, non-coding RNAs that act as post-transcriptional repressors are gaining momentum as biomarkers in a number of disease areas. In this review, we examine the potential utility of miRNAs as promising biomarkers for renal disease. We explore the performance of miRNAs as biomarkers in two clinically important forms of CKD, diabetes and the nephropathy developing in kidney transplant recipients. Finally, we highlight the pitfalls and opportunities of miRNAs and provide a broad perspective for the future clinical development of miRNAs as biomarkers in CKD beyond the current gold standards of eGFR and albuminuria.
Following the Health and Social Care Act in England, public health teams were formally transferred from the NHS to local authorities in April 2013.
Online survey of Directors of Public Health (DsPH) in local authorities in England (n = 152) to investigate their experience within local government 1 year on. Tests of association were used to explore relationships between the perceived integration and influence of public health, and changes in how the public health budget was being spent.
The organization of and managerial arrangements for public health within councils varied. Most DsPH felt that good relationships had been established within the council, and the move had made them more able to influence priorities for health improvement, even though most felt their influence was limited. Changes in commissioning using the public health budget were already widespread and included the de-commissioning of services.
There was a widespread feeling amongst DsPH that they had greater influence since the reforms, and that this went across the local authority and beyond. Public health's influence was most apparent when the transfer of staff to local government had gone well, when collaborative working relationships had developed, and when local partnership groups were seen as being effective.
Traditionally, the cost object in health care has been either a service line (e.g. orthopaedics) or a clinical intervention (e.g. hip replacement). In the mid-2000s, the Department of Health recommended that in the future the patient should be the cost object, to enable a better analysis of cost drivers in health care, resulting in patient-level information and costing systems (PLICSs). Monitor (the economic regulator for health care) proposes that PLICS data will now form the basis for mandatory prices for health-care services across all care settings.
Our main aim was to investigate the use of PLICSs.
We surveyed all English foundation trusts and NHS trusts, and undertook four case studies of foundation trusts. Three trusts were generalist and one was specialist. We also surveyed commissioning support units to explore the potential for PLICSs in commissioning.
The most significant use of PLICSs was cost improvement within the trusts. There was only modest utilisation of PLICSs to allocate resources across services and settings. We found that trusts had separate reporting systems for costs and clinical outcomes, engendering little use for PLICSs to link cost with quality. Although there was significant potential for PLICSs in commissioning, 74% of survey respondents at trusts considered their PLICS data to be commercially sensitive and only 5% shared the data with commissioners. The use of PLICSs in community services was, generally, embryonic because of the absence of units of health care for which payment can be made, service definitions and robust data collection systems. The lack of PLICS data for community services, allied with the commercial sensitivity issue, resulted in little PLICS presence in collaborative cross-organisational initiatives, whether between trusts or across acute and community services. PLICS data relate to activities along the patient pathway. Such costs make sense to clinicians. We found that PLICSs had created greater clinical engagement in resource management despite the fact that the trust finance function had actively communicated PLICSs as a new costing tool and often required its use in, for example, business cases for clinical investment. Operational financial management at the trusts was undertaken through service line reporting (SLR) and traditional directorate budgets. PLICSs were considered more of a strategic tool.
Both PLICSs and SLR identify and interrogate service line profitability. Although trusts currently cross-subsidise to support loss-making areas under the tariff, they are actively considering disinvesting in unprofitable service lines. Financial pressure within the NHS, along with its current competitive, business-oriented ethos, induces trusts to act in their own interests rather than those of the whole health economy. However, many policy commentators suggest that care integration is needed to improve patient care and reduce costs. Although the Health and Social Care Act 2012 (Great Britain. Health and Social Care Act 2012. London: The Stationery Office; 2012) requires both competition and the collaboration needed to achieve care integration, the two are not always compatible. We conclude that competitive forces are dominant in driving the current uses of PLICSs. Future research should interrogate the use of PLICSs in New Care Models – Vanguard Sites (NHS England. New Care Models – Vanguard Sites. NHS England; 2015) and initiatives to deliver the ‘Five Year Forward View’ (Monitor and NHS England. Reforming the Payment System for NHS Services: Supporting the Five Year Forward View. London: Monitor; 2015).
The National Institute for Health Research Health Services and Delivery Research programme.
The purpose of this study was to identify current practices of school-based speech-language pathologists (SLPs) in the United States for bilingual language assessment and compare them to American Speech-Language-Hearing Association (ASHA) best practice guidelines and mandates of the Individuals with Disabilities Education Act (IDEA, 2004).
The study was modeled to replicate portions of Caesar and Kohler's (2007) study and expanded to include a nationally representative sample. A total of 166 respondents completed an electronic survey.
Results indicated that the majority of respondents have performed bilingual language assessments. Furthermore, the most frequently used informal and standardized assessments were identified. SLPs identified supports, and barriers to assessment, as well as their perceptions of graduate preparation.
The findings of this study demonstrated that although SLPs have become more compliant to ASHA and IDEA guidelines, there is room for improvement in terms of adequate training in bilingual language assessment.
P-Rex proteins are guanine nucleotide exchange factors (GEFs) that act on the Rho/Rac family of GTP binding proteins. The activity of P-Rex proteins is regulated by several extracellular stimuli. In fact, activation of growth factor receptors has been reported to activate a phosphorylation/dephosphorylation cycle of P-Rex1. Such cycle includes dephosphorylation of serines 313 and 319 which negatively regulate the GEF activity of P-Rex1, together with phosphorylation of serines 605 and 1169 which favour P-Rex1 GEF activity. However, the kinases that regulate phosphorylation at these different regulatory sites are largely unknown. Here we have investigated the potential regulatory action of several kinases on the phosphorylation of P-Rex1 at S313, S319, S605 and S1169. We show that activation of protein kinase C (PKC) caused phosphorylation of S313, S319 and S1169. Activation of growth factor receptors induced phosphorylation of S1169 through a mechanism that was independent of PKC, indicating that distinct kinases and mechanisms control the phosphorylation of P-Rex1 at different regulatory serines. Genetic and biochemical studies confirmed that the PKC isoform PKCδ was able to directly phosphorylate P-Rex1 at S313. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S313 was substituted by alanine, indicated that phosphorylation at that residue negatively regulated P-Rex1 exchange activity. We suggest that control of P-Rex1 activity depends on a highly dynamic interplay among distinct signalling routes and its multisite phosphorylation is controlled by the action of different kinases.
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor. Acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) are both precursor lesions that lead to the development of PDAC. Reg family proteins (Reg1A, 1B, 3A/G, 4) are a group of calcium-dependent lectins that promote islet growth in response to inflammation and/or injuries. The aim of this study was to establish a role for Reg proteins in the development of PDAC and their clinical value as biomarkers. We found that Reg1A and Reg3A/G were highly expressed in the ADM tissues by immunohistochemistry. In the 3-dimensional culture of mouse acinar cells, Reg3A promoted ADM formation with concurrent activation of mitogen-acitvated protein kinase. Upregulation of Reg1A and Reg1B levels was observed as benign ductal epithelium progresses from PanIN to invasive PDAC. Patients with PDAC showed significantly higher serum levels of Reg1A and Reg1B than matching healthy subjects. These results were further validated by the quantification of Reg 1A and 1B mRNA levels in the microdissected tissues (22- and 6-fold increases vs. non-tumor tissues). Interestingly, patients with higher levels of Reg1A and 1B exhibited improved survival rate than those with lower levels. Furthermore, tissue expressions of Reg1A, Reg1B, and Reg4 could differentiate metastatic PDAC in the liver from intrahepatic cholangiocarcinoma with 92% sensitivity and 95% specificity. Overall, our results demonstrate the upregulation of Reg proteins during PDAC development. If validated in larger scale, Reg1A and Reg1B could become clinical markers for detecting early stages of PDAC, monitoring therapeutic response, and/or predicting patient's prognosis.
Irregular metallic growth at the anode during recharging of batteries can seriously influence the safety of batteries. To address this problem, we have attempted to design active anode materials with anion charge carriers and recently observed the formation and dissolution of an electrochemical film by triflate anions (CF3SO3(-)) at the surface of magnesium in an ionic liquid (IL) electrolyte of Mg(CF3SO3)2, which represents a rare anode material. The effect of heterogeneous cations on film formation was examined in this work. In an IL that dissolves NaCF3SO3, sodium ions with a lower reduction potential than Mg(2+)/Mg would not be expected to assist film formation. However, to our surprise, we discovered that some sodium ions are involved in film formation. The sodium ions are believed to act as a cross-linking point for the formation of a film network, which resulted in fairly good reversibility for film formation. In a Ce(CF3SO3)3-IL electrolyte, an electrochemically formed film free of Ce(3+) was obtained. The trivalent cerium cations were deactivated and transformed to an oxide on Mg metal. However, the reversibility of film formation in the Ce(CF3SO3)3 system did not meet the expected level. By coupling the film formation and dissolution behavior with a V2O5 cathode, a rechargeable battery was fabricated with dual ion transport species of Na(+) or Ce(3+) for the cathode and CF3SO3(-) for the anode. The unique battery with NaCF3SO3 is demonstrated to exhibit good discharge/charge performance with long-term cyclability.
Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.
Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control.
The mitogen-activated protein kinase (MAPK) signaling pathway is a cascade of protein kinases that act in a sequential and predominantly linear fashion, albeit displaying some cross talk with other signaling cascades. Mutations in proteins integral to the MAPK signaling pathway are present in more than 50% of cutaneous melanomas. The most frequently mutated protein is v-raf murine sarcoma viral oncogene homolog B (BRAF), followed by neuroblastoma Ras viral oncogene homolog (NRAS). Recently, the development of targeted drugs for the treatment of BRAF-mutant melanoma has led to the widespread implementation of molecular assays for the detection of specific BRAF mutations. There have been some attempts to standardize testing of BRAF mutations, but this has not been achieved so far. Here we provide an updated review on the role of the MAPK signaling pathway in the pathogenesis of cutaneous melanoma, focusing on several different BRAF mutations and their diagnostic and therapeutic implications.
The advent of click chemistry has had a profound influence on almost all branches of chemical science. This is particularly true of radiochemistry and the synthesis of agents for positron emission tomography (PET), single photon emission computed tomography (SPECT), and targeted radiotherapy. The selectivity, ease, rapidity, and modularity of click ligations make them nearly ideally suited for the construction of radiotracers, a process that often involves working with biomolecules in aqueous conditions with inexorably decaying radioisotopes. In the following pages, our goal is to provide a broad overview of the first ten years of research at the intersection of click chemistry and radiochemistry. The discussion will focus on four areas that we believe underscore the critical advantages provided by click chemistry: (i) the use of prosthetic groups for radiolabeling reactions, (ii) the creation of coordination scaffolds for radiometals, (iii) the site-specific radiolabeling of proteins and peptides, and (iv) the development of strategies for in vivo pretargeting. Particular emphasis will be placed on the four most prevalent click reactions - the Cu-catalyzed azide-alkyne cycloaddition (CuAAC), the strain-promoted azide-alkyne cycloaddition (SPAAC), the inverse electron demand Diels-Alder reaction (IEDDA), and the Staudinger ligation - though less well-known click ligations will be discussed as well. Ultimately, it is our hope that this review will not only serve to educate readers but will also act as a springboard, inspiring synthetic chemists and radiochemists alike to harness click chemistry in even more innovative and ambitious ways as we embark upon the second decade of this fruitful collaboration.
Cytohesin family proteins act as guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor family of small GTP-binding proteins. Aplysia Sec7 (ApSec7), a member of the cytohesin family in Aplysia, plays key roles in neurite outgrowth in Aplysia neurons. Although ApSec7 has a conserved coiled-coil (CC) domain, its role was not clear. In this study, we found that the CC domain of ApSec7 and ARNO/cytohesin 2 are involved in homodimer formation, leading to efficient plasma membrane targeting of ApSec7 and ARNO/cytohesin 2 in HEK293T cells. Therefore, deletion of the CC domain of ApSec7 and ARNO/cytohesin 2 may result in a loss of dimerization and reduce plasma membrane localization. In addition, the CC domains of ApSec7 and ARNO/cytohesin 2 have partially or fully CRM1-dependent nuclear export signals, respectively. Taken together, our results suggest that the CC domain of cytohesin family proteins, including ApSec7 and ARNO/cytohesin 2, has dual roles in intracellular targeting: increased plasma membrane targeting through homodimer formation and nuclear exclusion through either a CRM1-dependent or a CRM1-independent pathway.
Protein micropatterning techniques, including microfluidic devices and protein micro-contact printing, enable the generation of highly controllable substrates for spatial manipulation of intracellular and extracellular signaling determinants to examine the development of cultured dissociated neurons in vitro. In particular, culture substrates coated with proteins of interest in defined stripes, including cell adhesion molecules and secreted proteins, have been successfully used to study neuronal polarization, a process in which the neuron establishes axon and dendrite identities, a critical architecture for the input/output functions of the neuron. We have recently used this methodology to pattern the extracellular protein Semaphorin 3A (Sema3A), a secreted factor known to control neuronal development in the mammalian embryonic cortex. We showed that stripe-patterned Sema3A regulates axon and dendrite formation during the early phase of neuronal polarization in cultured rat hippocampal neurons. Here, we describe microfabrication and substrate stripe micropatterning of Sema3A. We note that same methodologies can be applied to pattern other extracellular proteins that regulate neuronal development in the embryonic brain, as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and Netrin-1. We describe modifications of these methodologies for stripe micropatterning of membrane-permeable analog of the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP), intracellular regulators of neuronal polarization that might act downstream of Sema3A.
Semaphorin guidance molecules act through different receptor complexes to activate multiple signaling cascades leading to changes in axonal growth cone behavior and morphology. We describe here approaches for studying the effect of individual Semaphorins on isolated forebrain neurons from mouse embryos and dissecting downstream signaling pathways. These approaches include the production of recombinant Semaphorin ligands, the culture of dissociated primary neurons, the manipulation of gene expression by electroporation in primary neurons, and functional assays to assess axon outgrowth and growth cone collapse.
Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.
A new system of slits called `spiderweb slits' have been developed for depth-resolved powder or polycrystalline X-ray diffraction measurements. The slits act on diffracted X-rays to select a particular gauge volume of sample, while absorbing diffracted X-rays from outside of this volume. Although the slit geometry is to some extent similar to that of previously developed conical slits or spiral slits, this new design has advantages over the previous ones in use for complex heterogeneous materials and in situ and operando diffraction measurements. For example, the slits can measure a majority of any diffraction cone for any polycrystalline material, over a continuous range of diffraction angles, and work for X-ray energies of tens to hundreds of kiloelectronvolts. The design is generated and optimized using ray-tracing simulations, and fabricated through laser micromachining. The first prototype was successfully tested at the X17A beamline at the National Synchrotron Light Source, and shows similar performance to simulations, demonstrating gauge volume selection for standard powders, for all diffraction peaks over angles of 2-10°. A similar, but improved, design will be implemented at the X-ray Powder Diffraction beamline at the National Synchrotron Light Source II.
This study examined the contributions of individual muscles to changes in energetic cost of transport (COT) over seven walking speeds, and compared results between healthy young and elderly subjects. Twenty six participants (13 young aged 18-30; 13 old aged 70-80) were recruited. COT (O2/kg body mass/km) was calculated by standardizing the mean oxygen consumption recorded during steady state walking. Electromyography signals from 10 leg muscles were used to calculate the cumulative activity required to traverse a unit of distance (CMAPD) for each muscle at each speed. In the old group CMAPD was correlated with COT, presented higher and more variable values, and showed greater increases around optimal speed for all studied muscles. Soleus CMAPD was independent of speed in the young group, but this was not evident with aging. Greater energy cost of walking in older individuals seems to be attributable to increased energy cost of all lower limb muscles.
There are significant inequalities in physical health and life expectancy between people with and without a mental illness. Understanding perspectives of people with mental illness on personal meanings of physical health is essential to ensuring health services are aligned with consumer understandings, needs, and values. A qualitative exploratory study was undertaken involving focus groups with 31 consumers in The Australian Capital Territory, Australia. Participants were asked: "What does physical health mean to you?" Thematic analysis was applied to interview transcripts. Five themes are discussed, representing different emphases in the meaning of physical health: (1) physical and mental are interconnected, (2) absence of disease, (3) moving the body, (4) struggling for healthy diet, and (5) functioning and participation. Physical pain was a difficulty that arose across these themes. Mental health consumers see physical health as always connected with well-being. Nurses would benefit from been informed by consumer understandings of physical health. In addition, there should be more attention to quality of life measures of people with mental illness as these are more congruent with consumer perspectives on physical health than biomedical measures.
Issue: Although predictions that the Affordable Care Act (ACA) would lead to reductions in employer-sponsored health coverage have not been realized, some of the law’s critics maintain the ACA is nevertheless driving higher premium and deductible costs for businesses and their workers. Goal: To compare cost growth in employer-sponsored health insurance before and after 2010, when the ACA was enacted, and to compare changes in these costs relative to changes in workers’ incomes. Methods: The authors analyzed federal Medical Expenditure Panel Survey data to compare cost trends over the 10-year period from 2006 to 2015. Key findings and conclusions: Compared to the five years leading up to the ACA, premium growth for single health insurance policies offered by employers slowed both in the nation overall and in 33 states and the District of Columbia. There has been a similar slowdown in growth in the amounts employees contribute to health plan costs. Yet many families feel pinched by their health care costs: despite a recent surge, income growth has not kept pace in many areas of the U.S. Employee contributions to premiums and deductibles amounted to 10.1 percent of U.S. median income in 2015, compared to 6.5 percent in 2006. These costs are higher relative to income in many southeastern and southern states, where incomes are below the national average.
There is increasing interest in identifying natural bioactive compounds that can improve mitochondrial functionality and regulate apoptosis. The brewery industry generates wastewater that could yield a natural extract containing bioactive phenolic compounds. Polyphenols act as antioxidants and have been documented to protect the human body from degenerative diseases such as cardiovascular diseases or cancer. The main aims of our research were to determine the phenolic profile of a crude extract obtained (at pilot scale) from a brewery waste stream and to evaluate the biochemical activity of this extract on the mitochondrial function of a cancer cell line (SH-SY5Y). This work is a basic translational pilot study. The total phenolic content was determined by the Folin-Ciocalteu assay, which revealed that 2.30% of the extract consisted of phenolic compounds. The polyphenols, identified and quantified by reverse-phase-high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS), were mainly flavonoids. After cell culture, the tumoral cells treated with the polyphenolic extract showed enhanced mitochondrial oxidative function, which is likely related to a decrease in oxidative stress and an increase in mitochondrial biogenesis. This type of brewery waste stream, properly treated, may be a promising source of natural antioxidants to replace the synthetic antioxidants currently used in the food industry.
In our previous study, NKB/NK3R system has been shown to act at the pituitary level to up-regulate SLα synthesis and secretion in grass carp. However, whether NK3R expression can serve as a regulatory target at the pituitary level and contribute to NKB interactions with other SLα regulators is still unclear. In current study, using grass carp pituitary cells as a model, we have a novel finding that co-treatment of SLα/SLβ with carp TAC3 gene products, could induce a noticeable enhancement in SLα mRNA expression and these potentiating effects occurred with a parallel rise in NK3R transcript level after SLα/SLβ treatment. Interestingly, the stimulatory effects of SLα/SLβ on NK3R gene expression could be further potentiated by co-treatment with IGF-I/-II and simultaneous exposure of carp pituitary cells to SLα/SLβ and IGF-I/-II in the presence of TAC3 gene products was found to markedly elevated SLα mRNA expression (20 fold increase) and this synergistic stimulation was mediated by cAMP/PKA-, PLC/PKC- and Ca(2+) -dependent cascades functionally coupled with NK3R activation. These findings suggest that local release of SLα via functional interactions with IGF-I/-II and TAC3/NK3R system may constitute a potent stimulatory signal for SLα gene expression in the carp pituitary via up-regulation of NK3R expression.
Spin-Hall oscillators (SHO) are promising sources of spin-wave signals for magnonics applications, and can serve as building blocks for magnonic logic in ultralow power computation devices. Thin magnetic layers used as "free" layers in SHO are in contact with heavy metals having large spin-orbital interaction, and, therefore, could be subject to the spin-Hall effect (SHE) and the interfacial Dzyaloshinskii-Moriya interaction (i-DMI), which may lead to the nonreciprocity of the excited spin waves and other unusual effects. Here, we analytically and micromagnetically study magnetization dynamics excited in an SHO with oblique magnetization when the SHE and i-DMI act simultaneously. Our key results are: (i) excitation of nonreciprocal spin-waves propagating perpendicularly to the in-plane projection of the static magnetization; (ii) skyrmions generation by pure spin-current; (iii) excitation of a new spin-wave mode with a spiral spatial profile originating from a gyrotropic rotation of a dynamical skyrmion. These results demonstrate that SHOs can be used as generators of magnetic skyrmions and different types of propagating spin-waves for magnetic data storage and signal processing applications.
There is international interest in, and continued concern about, the potential long-term impact of involuntary admission to psychiatric institutions, and the effect this coercive action has on a person's well-being and human rights. Involuntary detention in hospital remains a controversial process that involves stakeholders with competing concerns and who often describe negative experiences of the process, which can have long-lasting effects on the therapeutic relationship with service users. The aim of the present study was to explore the perspectives of key stakeholders involved in the involuntary admission and detention of people under the Mental Health Act 2001 in Ireland. Focus groups were used to collect data. Stakeholders interviewed were service users, relatives, general practitioners, psychiatrists, mental health nurses, solicitors, tribunal members, and police. Data were analysed using a general inductive approach. Three key categories emerged: (i) getting help; (ii) detention under the Act; and (iii) experiences of the tribunal process. This research highlights gaps in information and uncertainty about the involuntary admission process for stakeholders, but particularly for service users who are most affected by inadequate processes and supports. Mental health law has traditionally focussed on narrower areas of detention and treatment, but human rights law requires a greater refocussing on supporting service users to ensure a truly voluntary approach to care. The recent human rights treaty, the UN Convention on the Rights of Persons with Disabilities, is to guarantee a broad range of fundamental rights, such as liberty and integrity, which can be affected by coercive processes of involuntary admission and treatment.
Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. We report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa, and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species. This article is protected by copyright. All rights reserved.
Polycystic ovary syndrome (PCOS) was associated with a number of polymorphisms of genes involved in insulin signaling. So far, they have been studied separately. The aim of this study was to verify the impact of the coexistence of two polymorphisms of insulin signaling.
One hundred consecutive PCOS women (diagnosed by Rotterdam criteria) and 45 age-matched healthy women were genotyped for two polymorphisms: Gly972Arg of IRS-1 and Lys121Gln of PC-1. Also, they underwent clinical evaluation, blood sampling for measurement of metabolic and hormonal indices, and a 75-g oral glucose tolerance test (OGTT).
Comparing PCOS women with controls, the rate of homo-/heterozygosity was significantly greater (50 vs. 24.5%, P = 0.004) for IRS-1 polymorphism, but insignificantly greater (20 vs. 13.3%, P = 0.33) for PC-1 polymorphism. In PCOS women, compared with controls, the genotypes IRS-1 hetero/PC-1 wild type (WT) (36 vs. 17.8%, P = 0.03) and IRS-1 hetero/PC-1 hetero (14 vs. 6.7%, P = 0.20) were overrepresented at the expense of IRS-1 WT/PC-1 WT (44 vs. 68.8%, P = 0.005), while IRS-1 WT/PC-1 hetero was similarly represented (6 vs. 6.7%). Based on genotype, metabolic and hormonal indices changed significantly. For instance, six indices (HOMA-IR, fasting insulin, insulin area under the curve at OGTT, triglycerides, total and calculated free testosterone) were the highest in IRS-1 hetero/PC-1 WT women.
Genetic variations in insulin signaling contribute to the extent and the variability of metabolic and hormonal derangement.
Healthcare provider burnout is considered a factor in quality of care, yet little is known about the consistency and magnitude of this relationship. This meta-analysis examined relationships between provider burnout (emotional exhaustion, depersonalization, and reduced personal accomplishment) and the quality (perceived quality, patient satisfaction) and safety of healthcare.
Publications were identified through targeted literature searches in Ovid MEDLINE, PsycINFO, Web of Science, CINAHL, and ProQuest Dissertations & Theses through March of 2015. Two coders extracted data to calculate effect sizes and potential moderators. We calculated Pearson's r for all independent relationships between burnout and quality measures, using a random effects model. Data were assessed for potential impact of study rigor, outliers, and publication bias.
Eighty-two studies including 210,669 healthcare providers were included. Statistically significant negative relationships emerged between burnout and quality (r = -0.26, 95 % CI [-0.29, -0.23]) and safety (r = -0.23, 95 % CI [-0.28, -0.17]). In both cases, the negative relationship implied that greater burnout among healthcare providers was associated with poorer-quality healthcare and reduced safety for patients. Moderators for the quality relationship included dimension of burnout, unit of analysis, and quality data source. Moderators for the relationship between burnout and safety were safety indicator type, population, and country. Rigor of the study was not a significant moderator.
This is the first study to systematically, quantitatively analyze the links between healthcare provider burnout and healthcare quality and safety across disciplines. Provider burnout shows consistent negative relationships with perceived quality (including patient satisfaction), quality indicators, and perceptions of safety. Though the effects are small to medium, the findings highlight the importance of effective burnout interventions for healthcare providers. Moderator analyses suggest contextual factors to consider for future study.
Effective immune responses require the precise regulation of dynamic interactions between hematopoietic and non-hematopoietic cells. The Rho subfamily of GTPases, which includes RhoA, is rapidly activated downstream of a diverse array of biochemical and biomechanical signals, and is emerging as an important mediator of this cross-talk. Key downstream effectors of RhoA are the Rho kinases, or ROCKs. The ROCKs are two serine-threonine kinases that can act as global coordinators of a tissue's response to stress and injury because of their ability to regulate a wide range of biological processes. Although the RhoA-ROCK pathway has been extensively investigated in the non-hematopoietic compartment, its role in the immune system is just now becoming appreciated. In this commentary, we provide a brief overview of recent findings that highlight the contribution of this pathway to lymphocyte development and activation, and the impact that dysregulation in the activation of RhoA and/or the ROCKs may exert on a growing list of autoimmune and lymphoproliferative disorders.
Several studies have reported age-associated changes in DNA methylation in the first few years of life and in adult populations, but the extent of such changes during childhood is less well studied. The goals of this study were to investigate to what degree intra-individual changes in DNA methylation are associated with aging during childhood and dissect the methylation changes directly associated with aging from the effect mediated through variation in cell-type composition (CTC).
We performed reduced representation bisulfite sequencing (RRBS) in peripheral whole-blood samples collected at 2, 10, and 16 years of age. We identified age-associated longitudinal changes in DNA methylation at 346 CpGs in 178 genes. Analyses separating the effect mediated by CTC variability across age identified 26 CpGs located in 12 genes that associated directly with age. Hence, the CTC changes across age appear to act as a mediator of the observed DNA methylation associated with age. The results were replicated using EpiTYPER in a second sample set selected from the same cohort. Gene ontology analyses revealed enrichment of transcriptional regulation and developmental processes. Further, comparisons of the mean DNA methylation differences between the time points reveal greater differences between 2 to 10 years and 10 to 16 years, suggesting that the identified age-associated DNA methylation patterns manifests in early childhood.
This study reveals insights into the epigenetic dynamics associated with aging early in life. Such information could ultimately provide clues and point towards molecular pathways that are susceptible to aging-related disease-associated epigenetic dysregulation.
To examine the expression of ALDOB in gastric cancer (GC) tissue and to reveal its potential clinicopathological and prognostic significance.
We screened for genes that were differentially expressed between GC and nontumor tissues using a microarray, specifically the Affymetrix U133 Plus 2.0 Array platform. We then verified the transcriptional and translational levels of ALDOB by performing quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). In addition, a merged data set based on the Gene Expression Omnibus was generated and a survival analysis performed.
The microarray analysis revealed that ALDOB was downregulated (more than sevenfold) in GC compared with nontumor tissue. Both qRT-PCR and IHC validated the decrease of ALDOB in GC tissue. Moreover, we found that the expression of ALDOB was significantly related to tumor-invasion depth, lymph-node metastasis, distant metastasis, and TNM stage. The survival analysis, based on the IHC and merged data set, indicated that the overall survival was better in patients with high ALDOB expression. The Cox regression analysis showed that ALDOB expression was an independent prognostic factor for GC.
The expression of ALDOB in GC tissue was significantly related to the clinicopathological features and prognosis of the disease, thus suggesting that ALDOB could act as a novel molecular marker for GC.
The dengue virus (DENV) nonstructural protein 5 (NS5) contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds) to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol) were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo.
In order to advance the assisted reproductive technologies used in animals and human beings, it is important to accumulate basic informations about underlying molecular mechanisms that shape the biological processes of reproduction. From within seminal plasma, proteins perform a wide variety of distinct functions that regulate major reproductive events such as fertilization. The ability of such proteins to bind and interact with different antagonistic ions and biomolecules such as polysaccharides, lipids, and other proteins present in the male and female reproductive tract define these capabilities. Over the last two decades, extensive work has been undertaken in an attempt to define the role of seminal plasma proteins, of which, Gelatin binding proteins (GBPs) represent a large family. GBPs comprise of known group of Bovine seminal plasma (BSP) protein family, matrix metallo proteinases (MMP 2 and MMP 9) and fibronectin, which have been widely studied. The presence of a type II repeat is a characteristic feature of GBPs, which is similar in structure to the fibronectin type II domain (fn2), which has ability to bind multiple ligands including gelatin, glycosaminoglycans, choline phospholipids, and lipoproteins. Two fn2 domains are present within the BSP protein family, while, three fn2 domains are found in gelatinases (MMP-2 and MMP9), and ELSPBP1 (Epididymosomes Transfer Epididymal Sperm Binding Protein 1) contains four long fn2 domains. For the most part BSP proteins are exclusively expressed in seminal vesicles although mBSPH1, mBSPH2 and hBSPH1 are all expressed in the epididymis. The expression of gelatinases has been demonstrated in several organs and tissues such as the prostate, testis, epididymis, ovary, human placenta, cervix and endometrial wall. This review intends to bring current updates on the role of GBPs in reproductive physiology to light, which may act as basis for future studies on GBPs.
To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes.
A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 3' untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA.
Let7i was decreased in patients with acute ischemic stroke (fold change -1.70, p < 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r = -0.32, p = 0.02), infarct volume (r = -0.21, p = 0.04), and plasma MMP9 (r = -0.46, p = 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling.
Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke. Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1.
Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signalling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signalling molecule is sphingosine-1-phosphate (S1P) whose breakdown is catalysed by sphingosine-1-phosphate lyase (S1PL), a pyridoxal 5 '-phosphate (PLP) dependent enzyme that catalyses the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine (PE). Here we show the pathogenic bacterium Burkholderia pseudomallei K96243 encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established mass spectrometry-based methodology we show that recombinant S1PL2021 is catalytically active. Using recombinant human fatty aldehyde dehydrogenase (FALDH) we developed a spectrophotometric, enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the x-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.
Affordable Care Act provisions implemented in 2014 could have influenced employers' decisions to offer health insurance. Using data for 2014 from the Medical Expenditure Panel Survey-Insurance Component, we found little change in employer-sponsored health insurance offerings: More than 95 percent of employers either continued offering coverage or continued not offering it between 2013 and 2014. Fewer than 3.5 percent of employers dropped coverage, and 1.1 percent added coverage.
To describe a dynamic three-dimensional (3D) computed tomography (CT) technique for the upper airway and compare the required radiation dose to that used for common clinical studies of a similar anatomical area, such as for subjects undergoing routine clinical facial CT.
Dynamic upper-airway CT was performed on eight subjects with persistent obstructive sleep apnea, four of whom were undergoing magnetic resonance imaging and an additional four subjects who had a contraindication to magnetic resonance imaging. This Health Insurance Portability and Accountability Act-compliant study was approved by our institutional review board, and informed consent was obtained. The control subjects (N = 41) for comparison of radiation dose were obtained from a retrospective review of the clinical picture-archiving computer system to identify 10 age-matched patients per age-based control group undergoing facial CT.
Dynamic 3D CT can be performed with an effective radiation dose of less than 0.38 mSv, a dose that is less than or comparable to that used for clinical facial CT. The resulting data- set is a uniquely complete, dynamic 3D volume of the upper airway through a full respiratory cycle that can be processed for clinical and modeling analyses.
A dynamic 3D CT technique of the upper airway is described that can be performed with a clinically reasonable radiation dose and sets a benchmark for future use.
Lung cancer is a leading cause of cancer-related deaths worldwide. Lung cancer risk factors, including smoking and exposure to environmental carcinogens, have been linked to chronic inflammation. An integral feature of inflammation is the activation, expansion and infiltration of diverse immune cell types, including CD4(+) T cells. Within this T cell subset are immunosuppressive regulatory T (Treg) cells and pro-inflammatory T helper 17 (Th17) cells that act in a fine balance to regulate appropriate adaptive immune responses.In the context of lung cancer, evidence suggests that Tregs promote metastasis and metastatic tumor foci development. Additionally, Th17 cells have been shown to be an integral component of the inflammatory milieu in the tumor microenvironment, and potentially involved in promoting distinct lung tumor phenotypes. Studies have shown that the composition of Tregs and Th17 cells are altered in the tumor microenvironment, and that these two CD4(+) T cell subsets play active roles in promoting lung cancer progression and metastasis.We review current knowledge on the influence of Treg and Th17 cells on lung cancer tumorigenesis, progression, metastasis and prognosis. Furthermore, we discuss the potential biological and clinical implications of the balance among Treg/Th17 cells in the context of the lung tumor microenvironment and highlight the potential prognostic function and relationship to metastasis in lung cancer.
In drug development, in vitro human model systems are absolutely essential prior to the clinical trials, considering the increasing number of chemical compounds in need of testing, and, keeping in mind that animals cannot predict all the adverse human health effects and reactions, due to the species-specific differences in metabolic pathways. The liver plays a central role in the clearance and biotransformation of chemicals and xenobiotics. In vitro liver model systems by using highly differentiated human cells could have a great impact in preclinical trials. Membrane biohybrid systems constituted of human hepatocytes and micro- and nano-structured membranes, represent valuable tools for studying drug metabolism and toxicity. Membranes act as an extracellular matrix for the adhesion of hepatocytes and compartmentalise them in a well-defined physical and chemical microenvironment with high selectivity. Advanced 3-D tissue cultures are furthermore achieved by using membrane bioreactors (MBR), which ensure the continuous perfusion of cells protecting them from shear stress. MBRs with different configurations allow the culturing of cells at high density and under closely monitored high perfusion, similarly to the natural liver. These devices that promote the long-term maintenance and differentiation of primary human hepatocytes with preserved liver specific functions can be employed in drug testing for prolonged exposure to chemical compounds and for assessing repeated-dose toxicity. The use of primary human hepatocytes in membrane bioreactors is the only system providing a faster and more cost-effective method of analysis for the prediction of in vitro human drug metabolism and enzyme induction alternative and/or complementary to animal experimentation. In this paper, in vitro models for studying drug metabolism and toxicity as advanced biohybrid membrane systems and membrane bioreactors will be reviewed.
G protein-coupled receptors (GPCRs) comprise the largest membrane protein family. These receptors sense a variety of signaling molecules, activate multiple intracellular signal pathways, and act as the targets of over 40% of marketed drugs. Recent progress on GPCR structural studies provides invaluable insights into the structure-function relationship of the GPCR superfamily, deepening our understanding about the molecular mechanisms of GPCR signal transduction. Here, we review recent breakthroughs on GPCR structure determination and the structural features of GPCRs, and take the structures of chemokine receptor CCR5 and purinergic receptors P2Y1 R and P2Y12 R as examples to discuss the importance of GPCR structures on functional studies and drug discovery. In addition, we discuss the prospect of GPCR structure-based drug discovery. © 2016 IUBMB Life, 68(11):894-903, 2016.
The World Health Organization (WHO) classification from 2014 differentiates between different subtypes of mucinous adenocarcinoma of the uterine cervix. A gastric subtype was recently described that showed no association with high-risk human papillomavirus (HPV) infections, has a poor prognosis, is mainly diagnosed in women of Asian origin and can occur in patients with Peutz-Jeghers syndrome. Although no clear grading system has been recommended in the WHO classification, it is likely that grading of adenocarcinomas of the uterine cervix will partly be based on the different patterns of invasion. Deep stromal infiltration of macroinvasive carcinomas is defined as an infiltration of >66 % of the cervical stroma. In the near future a maximum tumor size of 2 cm could act as a discriminator for planning of less radical surgery. Parameters of the histopathological report that are relevant for the prognostic assessment as well as the choice of adjuvant treatment and function as quality indicators during certification are described. The histological type of an adenocarcinoma alone is of no predictive or prognostic relevance for patients undergoing primary surgical treatment, neoadjuvant chemotherapy, combined chemo-radiotherapy or treatment with angiogenesis inhibitors. Currently, molecular parameters and biomarkers are of no relevance.
The Affordable Care Act encourages integration of behavioral health into primary care. We aim to estimate the level of under-reporting of drug use in federally qualified health centers (FQHCs) among self-reported risky drug users.
Adult patients in the waiting rooms of 4 FQHCs who self-reported risky drug use on the screening instrument World Health Organization's Alcohol, Smoking and Substance Involvement Screening Test (score 4-26), who participated in the "Quit Using Drugs Intervention Trial," submitted urine samples for drug testing. Under-reporters were defined as patients who denied use of a specific drug via questionnaire, but whose urine drug test was positive for that drug. Descriptive statistics, Pearson chi-square test, and logistic regression were used for analysis.
Of the 192 eligible participants, 189 (96%) provided urine samples. Fifty-four samples were negative or indeterminate, yielding 135 participants with positive urine drug tests for this analysis: 6 tested positive for amphetamines, 18 opiates, 21 cocaine, 97 marijuana. Thirty patients (22%) under-reported drug use and 105 (78%) reported drug use accurately. Under-reporting by specific substances was: amphetamines 66%, opiates 45%, cocaine 14%, and marijuana 7%. Logistic regression revealed that under-reporting of any drug was associated with history of incarceration and older age (odds ratios 2.6 and 3.3, respectively; P < 0.05).
Under-reporting of drug use is prevalent even among self-reported drug users in primary care patients of FQHCs (22%), but varied considerably based on the substance used. Further research is indicated to assess the extent of under-reporting among all primary care patients, regardless of their self-reported drug use status.
If good intentions pave the road to hell, what paves the road to heaven? We propose that moral judgments are based, in part, on the degree of effort exerted in performing the immoral or moral act. Because effort can serve as an index of goal importance, greater effort in performing immoral acts would lead to more negative judgments, whereas greater effort in performing moral acts would lead to more positive judgments. In support of these ideas, we found that perceived effort intensified judgments of both immoral (Studies 1-2) and moral (Studies 2-7) agents. The effect of effort on judgment was independent of the outcome (Study 3) and of perceptions of the outcome extremity (Study 6). Furthermore, the effect of effort on judgment was mediated by perceived goal importance (Studies 4-6), even when controlling for perceived intentions (Studies 5-6). Finally, we demonstrate that perceived effort can influence actual behavior, such as the assignment of monetary rewards (Study 7). We discuss the possible implications of effort as a causal motivational factor in moral judgment and social retribution. (PsycINFO Database Record
Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the use of chemical acaricides; however, their uncontrolled use has increased tSresistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. The only anti-tick vaccine commercially available at present in the world is based on intestinal Bm86 protein, and shows a variable effectiveness depending on tick strains or geographic isolates. Therefore, there is a need to characterize new antigens in order to improve immunological protection. The aim of this work was to identify immunogenic proteins from ovarian tissue extracts of R. microplus, after cattle immunization. Results showed that ovarian proteins complexed with the adjuvant Montanide ISA 50 V generated a strong humoral response on vaccinated cattle. IgG levels peaked at fourth post-immunization week and remained high until the end of the experiment. 1D and 2D SDS-PAGE-Western blot assays with sera from immunized cattle recognized several ovarian proteins. Reactive bands were cut and analyzed by LC-MS/MS. They were identified as Vitellogenin, Vitellogenin-2 precursor and Yolk Cathepsin. Our findings along with bioinformatic analysis indicate that R. microplus has several Vitellogenin members, which are proteolytically processed to generate multiple polypeptide fragments. This apparent complexity of vitellogenic tick molecular targets gives the opportunity to explore their potential usefulness as vaccine candidates but, at the same time, imposes a challenge on the selection of the appropriate set of antigens.
Pluripotent mouse embryonic stem cells maintain their identity throughout virtually infinite cell divisions. This phenomenon, referred to as self-renewal, depends on a network of sequence-specific transcription factors (TFs) and requires daughter cells to accurately reproduce the gene expression pattern of the mother. However, dramatic chromosomal changes take place in mitosis, generally leading to the eviction of TFs from chromatin. Here, we report that Esrrb, a major pluripotency TF, remains bound to key regulatory regions during mitosis. We show that mitotic Esrrb binding is highly dynamic, driven by specific recognition of its DNA-binding motif and is associated with early transcriptional activation of target genes after completion of mitosis. These results indicate that Esrrb may act as a mitotic bookmarking factor, opening another perspective to molecularly understand the role of sequence-specific TFs in the epigenetic control of self-renewal, pluripotency and genome reprogramming.
In many birds, males are presumed to protect their paternity by closely guarding their mate or copulating frequently with her. Both these costly behaviors are assumed to reduce the risk and/or intensity of sperm competition. However, despite many studies on avian extra-pair paternity, it remains unclear how strongly these behaviors are related to fitness and other key life-history traits. Here, we conduct meta-analyses to address two questions. First, are mate guarding and/or frequent copulation positively correlated with a male's share of paternity at his nest? We find a significant positive correlation between both presumed paternity protection behaviors and paternity share. The relationship is, however, weak (r = 0.08-0.23). This is perhaps unsurprising if the risk of partner infidelity, hence the need to protect paternity, varies among males. For example, more attractive males might have less need to protect their paternity. Second, do males with higher indices of so-called male "quality" (phenotypic measures, usually subjectively defined by researchers as predictors of male attractiveness) exhibit lower levels of paternity protection behavior? We find a negative correlation between male quality and paternity protection. This finding might partly explain the weak relationship between paternity protection and paternity, although we discuss other, nonmutually exclusive possibilities.
Biologists have taken the concept of organism largely for granted. However, advances in the study of chimerism, symbiosis, bacterial-eukaryote associations, and microbial behavior have prompted a redefinition of organisms as biological entities exhibiting low conflict and high cooperation among their parts. This expanded view identifies organisms in evolutionary time. However, the ecological processes, mechanisms, and traits that drive the formation of organisms remain poorly understood. Recognizing that organismality can be context dependent, we advocate elucidating the ecological contexts under which entities do or do not act as organisms. Here we develop a "contextual organismality" framework and provide examples of entities, such as honey bee colonies, tumors, and bacterial swarms, that can act as organisms under specific life history, resource, or other ecological circumstances. We suggest that context dependence may be a stepping stone to the development of increased organismal unification, as the most integrated biological entities generally show little context dependence. Recognizing that organismality is contextual can identify common patterns and testable hypotheses across different entities. The contextual organismality framework can illuminate timeless as well as pressing issues in biology, including topics as disparate as cancer emergence, genomic conflict, evolution of symbiosis, and the role of the microbiota in impacting host phenotype.
Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.
Helicoverpa armigera is a major agricultural pest that is distributed across Europe, Asia, Africa and Australasia. This species is hypothesized to have spread to the Americas 1.5 million years ago, founding a population that is at present, a distinct species, Helicoverpa zea. In 2013, H. armigera was confirmed to have re-entered South America via Brazil and subsequently spread. The source of the recent incursion is unknown and population structure in H. armigera is poorly resolved, but a basic understanding would highlight potential biosecurity failures and determine the recent evolutionary history of region-specific lineages. Here, we integrate several end points derived from high-throughput sequencing to assess gene flow in H. armigera and H. zea from populations across six continents. We first assemble mitochondrial genomes to demonstrate the phylogenetic relationship of H. armigera with other Heliothine species and the lack of distinction between populations. We subsequently use de novo genotyping-by-sequencing and whole-genome sequences aligned to bacterial artificial chromosomes, to assess levels of admixture. Primarily, we find that Brazilian H. armigera are derived from diverse source populations, with strong signals of gene flow from European populations, as well as prevalent signals of Asian and African ancestry. We also demonstrate a potential field-caught hybrid between H. armigera and H. zea, and are able to provide genomic support for the presence of the H. armigera conferta subspecies in Australasia. While structure among the bulk of populations remains unresolved, we present distinctions that are pertinent to future investigations as well as to the biosecurity threat posed by H. armigera.
Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
Plasminogen is a major plasma protein and the zymogen of the broad spectrum protease plasmin. Plasmin activity leads to tissue degradation, direct and through activation of metalloproteinases. Infected tooth root canals, as a consequence of the inflammatory response and eventual necrosis, contain tissue fluid and blood components. These will coat the root canal walls and act as conditioning films that allow bacterial biofilms to grow and be a potential source of hematogenously spreading bacteria. We investigated the effect of in vitro surface conditioning with human plasminogen on the initial adhesion of bacteria. Four bacterial species, L. salivarius, E. faecalis, A. naeslundii, and S. gordonii, isolated from dental root canals, and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown in albumin- or plasminogen-coated flow chambers and studied by confocal laser scanning microscopy using the cell viability staining LIVE/DEAD and 16S rRNA fluorescence in situ hybridization (FISH). A. naeslundii, L. salivarius and in particular S. gordonii showed a higher initial adhesion to the plasminogen-coated surfaces. E. faecalis did not show any preference for plasminogen. Four-species biofilms cultured for 96 h showed that streptococci increased their proportion with time. Further experiments aimed at studying different streptococcal strains. All these adhered more to plasminogen-coated surfaces than to albumin-coated control surfaces. The specificity of the binding to plasminogen was verified by blocking lysine-binding sites with epsilon-aminocaproic acid. Plasminogen is thus an important plasma component for the initial adhesion of oral bacteria, in particular streptococci. This binding may contribute to their spread locally as well as to distant organs or tissues.
To assess the sensitivity of non-localized, whole-head (1)H-MRS to an individual's serial changes in total-brain NAA, Glx, Cr and Cho concentrations - metabolite metrics often used as surrogate markers in neurological pathologies.
In this prospective study, four back-to-back (single imaging session) and three serial (successive sessions) non-localizing, ~3min (1)H-MRS (TE/TR/TI=5/10(4)/940ms) scans were performed on 18 healthy young volunteers: 9 women, 9 men: 29.9±7.6 [mean±standard deviation (SD)] years old. These were analyzed by calculating a within-subject coefficient of variation (CV=SD/mean) to assess intra- and inter-scan repeatability and prediction intervals. This study was Health Insurance Portability and Accountability Act compliant. All subjects gave institutional review board-approved written, informed consent.
The intra-scan CVs for the NAA, Glx, Cr and Cho were: 3.9±1.8%, 7.3±4.6%, 4.0±3.4% and 2.5±1.6%, and the corresponding inter-scan (longitudinal) values were: 7.0±3.1%, 10.6±5.6%, 7.6±3.5% and 7.0±3.9%. This method is shown to have 80% power to detect changes of 14%, 27%, 26% and 19% between two serial measurements in a given individual.
Subject to the assumption that in neurological disorders NAA, Glx, Cr and Cho changes represent brain-only pathology and not muscles, bone marrow, adipose tissue or epithelial cells, this approach enables us to quantify them, thereby adding specificity to the assessment of the total disease load. This will facilitate monitoring diffuse pathologies with faster measurement, more extensive (~90% of the brain) spatial coverage and sensitivity than localized (1)H-MRS.
The number of trained otolaryngologists available is insufficient to supply current and projected US health care needs. The goal of this study was to assess available databases and present accurate data on the current otolaryngology workforce, examine methods for prediction of future health care needs, and explore potential issues with forecasting methods and policy implementation based on these predictions.
Retrospective analysis of research databases, public use files, and claims data.
The total number of otolaryngologists and current practices in the United States was tabulated using the databases of the American Academy of Otolaryngology-Head and Neck Surgery, American Medical Association, American Board of Otolaryngology, American College of Surgeons, Association of American Medical Colleges, National Center for Health Statistics, and Department of Health and Human Services. Otolaryngologists were identified as surgeons and classified into surgical groups using a combination of AMA primary and secondary self-reported specialties and American Board of Medical Specialties certifications. Data gathered were cross-referenced to rule out duplications to assess total practicing otolaryngologists. Data analyzed included type of practice: 1) academic versus private and 2) general versus specialty; and demographics: 1) urban versus rural, 2) patient age, 3) reason for visit (referral, new, established, surgical follow-up), 4) reason for visit (diagnosis), and 5) payer type.
Analysis from the above resources estimates the total number of otolaryngologists practicing in the United States in 2011 to be 12,609, with approximately 10,522 fully trained practicing physicians (9,232-10,654) and 2,087 in training (1,318 residents and 769 fellows/others). Based on 2011 data, workforce projections would place the fully trained and practicing otolaryngology workforce at 11,088 in 2015 and 12,084 in 2025 unless changes in training occur. The AAO-HNS Physicians Resource Committee performed an extensive analysis of collated data from multiple sources in 2014 and identified 10,800 practicing otolaryngologists and 2,087 in training. It is estimated that the current attrition rate is approximately 306 otolaryngologists per year. Percentage distribution of office visits by patient age was found to be 20% <15 years old, 7% 15 to 24 years old, 21% 25 to 44 years old, 32% 45 to 64 years old, 11% 65 to 74 years old, and 10% ≥75 years old. Reason for visit was 34% new, 29% chronic, 17% chronic with exacerbation, and 15% pre- or postsurgical follow-up. The top diagnoses consisted of otitis media, chronic sinusitis, and impacted cerumen. Payer mix consisted of 59% private insurance, 19% Medicare, and 12% Medicaid/Children's Health Insurance Program.
Despite past findings and predictions of 8,000 to 8,500 otolaryngologists practicing in the United States, collated data from above resources places the total at 12,887, with 10,800 fully trained and practicing in 2014. This 30% to 50% underestimation of the otolaryngology workforce has an impact on future predictions and resource utilization analysis. Even when this correction is considered, the available trained otolaryngologists required to serve the otolaryngologic health care needs of the US population are still insufficient and understaffed. The impact of an aging population and the estimated 30 to 47 million newly insured citizens under the 2010 Patient Protection and Affordable Care Act are also unprecedented variables that must be considered. Further analysis of differences in physician productivity and geographic population density, and model formation of current otolaryngology workforce utilization, are needed to predict future public health needs.
NA Laryngoscope, 126:5-11, 2016.
The aim of this study was to provide evidence that actions performed by an individual influence the sensorimotor memory processing and, in particular, the integration process. We conducted 3 experiments that highlighted the multimodal aspect of memory traces. The 1st experiment consisted of a short-term priming paradigm based on 2 phases: a learning phase, consisting of the association between a shape and a sound, and a test phase, examining the priming effect of the shape seen in the learning phase on the processing of target tones. The participants' motor response became a factor in Experiments 2 and 3, allowing us to observe its influence on the integration between the shape and the sound. In Experiment 1, we showed that (a) the prime associated with the sound in the learning phase had an effect on target processing and (b) the component reactivated by the prime was perceptual in nature (i.e., auditory). Experiment 2 showed that the participants' responses were faster when the association of a shape and a sound had been learned with a motor response rather than without. Experiment 3 showed that the integration process required the individual to act while learning the association between the shape and the sound; otherwise no integration effect was observed. Our results highlight the role of motor responses as a necessary criterion for the integration process to take place. (PsycINFO Database Record
Essentials Patients with α-1-antitrypsin (α1-AT) Pittsburgh exhibit a mild bleeding tendency. A new case of α1-AT Pittsburgh with suspected high antifibrinolytic potential was studied. We showed that α1-AT Pittsburgh inhibits tissue plasminogen activator and plasmin. The antifibrinolytic potential of the variant contributes to explaining the mild bleeding phenotype.
α1 -Antitrypsin (α1 -AT) Pittsburgh has a Met358 to Arg substitution at the reactive Met-Ser site of α1 -AT, which enables the protein to act as a potent thrombin inhibitor. Four patients with α1 -AT Pittsburgh have been described to date. An additional young girl was recently diagnosed with α1 -AT Pittsburgh in our center after presenting with a large hematoma in the forearm. Interestingly, all of these patients showed a potent thrombin inhibitor in the plasma and a mild bleeding phenotype. This observation suggests that the in vivo consequences of the mutation may contribute to the maintenance of normal hemostatic balance. We assessed inhibition of the fibrinolytic system by the variant protein by evaluating the fibrinolysis inhibitory potential of the patient's plasma, purified wild-type α1 -AT and purified Pittsburgh α1 -AT with an electrophoretic zymography system, western blotting, and clot fibrinolysis. Our results indicate that the patient's plasma and purified α1 -AT Pittsburgh have strong potential to inhibit tissue-type plasminogen activator and plasmin.
Scaffolds provide a physical support for osteoblasts and act as the medium to transfer mechanical stimuli to cells. To verify our hypothesis that the surface chemistry of scaffolds regulates the perception of cells to mechanical stimuli, the sensitivity and tolerability of osteoblasts to fluid shear stress (FSS) of various magnitudes (5, 12, 20 dynes/cm(2) ) were investigated on various surface chemistries (-OH, -CH3 , -NH2 ), and their follow-up effects on cell proliferation and differentiation were examined as well. The sensitivity was characterized by the release of adenosine triphosphate (ATP), nitric oxide (NO) and prostaglandin E2 (PGE2 ) while the tolerability was by cellular membrane integrity. The cell proliferation was characterized by S-phase cell fraction and the differentiation by ALP activity and ECM expression (fibronectin and type I collagen). As revealed, osteoblasts demonstrated higher sensitivity and lower tolerability on OH and CH3 surfaces, yet lower sensitivity and higher tolerability on NH2 surfaces. Observations on the focal adhesion formation, F-actin organization and cellular orientation before and after FSS exposure suggest that the potential mechanism lies in the differential control of F-actin organization and focal adhesion formation by surface chemistry, which further divergently mediates the sensitivity and tolerability of ROBs to FSS and the follow-up cell proliferation and differentiation. These findings are essentially valuable for design/selection of desirable surface chemistry to orchestrate with FSS stimuli, inducing appropriate cell responses and promoting bone formation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2978-2991, 2016.
Joint pain is common in haemophilia and may be acute or chronic. Effective pain management in haemophilia is essential to reduce the burden that pain imposes on patients. However, the choice of appropriate pain-relieving measures is challenging, as there is a complex interplay of factors affecting pain perception. This can manifest as differences in patients' experiences and response to pain, which require an individualized approach to pain management. Prophylaxis with factor replacement reduces the likelihood of bleeds and bleed-related pain, whereas on-demand therapy ensures rapid bleed resolution and pain relief. Although use of replacement or bypassing therapy is often the first intervention for pain, additional pain relief strategies may be required. There is an array of analgesic options, but consideration should be paid to the adverse effects of each class. Nevertheless, a combination of medications that act at different points in the pain pathway may be beneficial. Nonpharmacological measures may also help patients and include active coping strategies; rest, ice, compression, and elevation; complementary therapies; and physiotherapy. Joint aspiration may also reduce acute joint pain, and joint steroid injections may alleviate chronic pain. In the longer term, increasing use of prophylaxis or performing surgery may be necessary to reduce the burden of pain caused by the degenerative effects of repeated bleeds. Whichever treatment option is chosen, it is important to monitor pain and adjust patient management accordingly. Beyond specific pain management approaches, ongoing collaboration between multidisciplinary teams, which should include physiotherapists and pain specialists, may improve outcomes for patients.
Responding to changes in the road ahead is essential for successful driving. Steering control can be modeled using 2 complementary mechanisms: guidance control (to anticipate future steering requirements) and compensatory control (to stabilize position-in-lane). Drivers seem to rapidly sample the visual information needed for steering using active gaze patterns, but the way in which this perceptual information is combined remains unclear. Influential models of steering capture many steering behaviors using just 'far' and 'near' road regions to inform guidance and compensatory control respectively (Salvucci & Gray, 2004). However, optic flow can influence steering even when road-edges are visible (Kountouriotis, Mole, Merat, & Wilkie, 2016). Two experiments assessed whether flow selectively interacted with compensatory and/or guidance levels of steering control, under either unconstrained gaze or constrained gaze conditions. Optic flow speed was manipulated independent of the veridical road-edges so that use of flow would lead to predictable understeering or oversteering. Steering was found to systematically vary according to flow speed, but crucially the Flow-Induced Steering Bias (FISB) magnitude depended on which road-edge components were visible. The presence of a guidance signal increased the influence of flow, with the largest FISB in 'Far' and 'Complete' road conditions, whereas the smallest FISB was observed when only 'Near' road-edges were visible. Gaze behaviors influenced steering to some degree, but did not fully explain the interaction between flow and road-edges. Overall the experiments demonstrate that optic flow can act indirectly upon steering control by modulating the guidance signal provided by a demarcated path. (PsycINFO Database Record
The efficacy, safety, and ease of use of rivaroxaban may reduce anticoagulation-treatment burden and improve nonvalvular atrial fibrillation (NVAF) patient satisfaction compared with vitamin K antagonists (VKAs).
Transitioning from a VKA to rivaroxaban improves treatment satisfaction in routine practice.
Xarelto for Prevention of Stroke in Patients With Atrial Fibrillation (XANTUS) is a prospective, noninterventional study in patients with NVAF prescribed rivaroxaban for prevention of stroke in routine practice. Patients receiving a VKA 4 weeks prior to the initial XANTUS study visit and switched to rivaroxaban were asked to complete the Anti-Clot Treatment Scale (ACTS). Changes from the initial visit to the first follow-up visit at ∼ 3 months (corresponding to a comparison of rivaroxaban vs prior VKA) for ACTS burden and benefit scores were calculated using and reported as least squared mean differences (LSMDs) with 95% confidence intervals (CIs).
The study included 1291 NVAF patients with prior VKA treatment. The mean baseline ACTS burden and benefit scores were 50.51 ± 8.42 and 10.30 ± 2.70, respectively. After ∼ 3 months of rivaroxaban treatment, LSMDs were 4.38 points (95% CI: 2.53-6.22, P < 0.0001) for the burden and 1.01 points (95% CI: 0.27-1.75, P = 0.0075) for the benefit score. Fifty-four percent and 48% of patients reported experiencing at least a minimally important clinical difference in burden and benefit scores, respectively.
Within this XANTUS cohort, switching from a VKA to rivaroxaban yielded statistically and clinically significant improvements in ACT burden and benefit scores.
Caspase-8 is a key initiator of apoptotic cell death where it functions as the apical protease in death receptor-mediated apoptosis triggered via the death-inducing signalling complex (DISC). However, the observation that caspase-8 is upregulated in many common tumour types led to the discovery of alternative non-apoptotic, pro-survival functions, many of which are contingent on phosphorylation of a tyrosine residue (Y380) found in the linker region between the two catalytic domains of the enzyme. Furthermore, Src-mediated Y380 phosphorylation leads to increased resistance to CD95-induced apoptosis; however, the mechanism underlying this impaired response to extrinsic apoptotic stimuli has not been identified. Consequently, we have employed a number of model systems to further dissect this protective mechanism. First, using an in vitro DISC model together with recombinant procaspase-8 variants, we show that Y380 phosphorylation inhibits procaspase-8 activation at the CD95 DISC, thereby preventing downstream activation of the caspase cascade. Second, we validated this finding in a cellular context using transfected neuroblastoma cell lines deficient in caspase-8. Reconstitution of these lines with phosphomimetic-caspase-8 results in increased resistance to CD95-mediated apoptosis and enhanced cell migration. When the in vitro DISC is assembled in the presence of cell lysate, caspase-8 Y380 phosphorylation attenuates DISC activity by inhibiting procaspase-8 autoproteolytic activity but not recruitment or homodimerization of caspase-8 within the complex. Once incorporated into the DISC, phosphorylated caspase-8 is unable to be released from the complex; this inhibits further cycling and release of active catalytic subunits into the cytoplasm, thus resulting in increased apoptotic resistance. Taken together, our novel findings expand our understanding of the key mechanisms underlying the anti-apoptotic functions of caspase-8 which may act as a critical block to existing antitumour therapies. Importantly, reversal or inhibition of caspase-8 phosphorylation may prove a valuable avenue to explore for sensitization of resistant tumours to extrinsic apoptotic stimuli.
Succinate dehydrogenase inhibitor (SDHI) fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk.
Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 times less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than the least sensitive of the baseline isolates. These isolates were also insensitive to most commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates.
This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined. © 2016 Society of Chemical Industry.
Intentional deception is a common act that often has detrimental social, legal, and clinical implications. In the last decade, brain activation patterns associated with deception have been mapped with functional magnetic resonance imaging (fMRI), significantly expanding our theoretical understanding of the phenomenon. However, despite substantial criticism, polygraphy remains the only biological method of lie detection in practical use today. We conducted a blind, prospective, and controlled within-subjects study to compare the accuracy of fMRI and polygraphy in the detection of concealed information. Data were collected between July 2008 and August 2009.
Participants (N = 28) secretly wrote down a number between 3 and 8 on a slip of paper and were questioned about what number they wrote during consecutive and counterbalanced fMRI and polygraphy sessions. The Concealed Information Test (CIT) paradigm was used to evoke deceptive responses about the concealed number. Each participant's preprocessed fMRI images and 5-channel polygraph data were independently evaluated by 3 fMRI and 3 polygraph experts, who made an independent determination of the number the participant wrote down and concealed.
Using a logistic regression, we found that fMRI experts were 24% more likely (relative risk = 1.24, P < .001) to detect the concealed number than the polygraphy experts. Incidentally, when 2 out of 3 raters in each modality agreed on a number (N = 17), the combined accuracy was 100%.
These data justify further evaluation of fMRI as a potential alternative to polygraphy. The sequential or concurrent use of psychophysiology and neuroimaging in lie detection also deserves new consideration.
Impulsivity, the tendency to act quickly without adequate planning or concern for consequences, is a commonly cited risk factor for suicidal thoughts and behaviour. There are many definitions of impulsivity and how it relates to suicidality is not well understood. Mood instability, which describes frequent fluctuations of mood over time, is a concept related to impulsivity that may help explain this relationship. The purpose of this study was to determine whether impulsivity could predict suicidal thoughts after controlling for mood instability.
This study utilized longitudinal data from the 2000 Adult Psychiatric Morbidity Survey (N = 2,406). There was a time interval of 18 months between the two waves of the study. Trait impulsivity and mood instability were measured with the Structured Clinical Interview for DSM-IV Axis II Personality Disorders. Logistic regression analyses were used to evaluate baseline impulsivity and mood instability as predictors of future suicidal thoughts.
Impulsivity significantly predicted the presence of suicidal thoughts, but this effect became non-significant with mood instability included in the same model.
Impulsivity may be a redundant concept when predicting future suicidal thoughts if mood instability is considered. The significance is that research and therapy focusing on mood instability along with impulsivity may be useful in treating the suicidal patient.
Mood instability and impulsivity both predict future suicidal thoughts. Impulsivity does not predict suicidal thoughts after controlling for mood instability. Assessing and treating mood instability could be important aspects of suicide prevention and risk management.
Physical inactivity has been recognized, by the World Health Organization as the fourth cause of death (5.5 % worldwide). On the contrary, physical activity (PA) has been associated with improved quality of life and decreased risk of several diseases (i.e., stroke, hypertension, myocardial infarction, obesity, malignancies). Bone turnover is profoundly affected from PA both directly (load degree is the key determinant for BMD) and indirectly through the activation of several endocrine axes. Several molecules, secreted by muscle (myokines) and adipose tissues (adipokines) in response to exercise, are involved in the fine regulation of bone metabolism in response to the energy availability. Furthermore, bone regulates energy metabolism by communicating its energetic needs thanks to osteocalcin which acts on pancreatic β-cells and adipocytes. The beneficial effects of exercise on bone metabolism depends on the intermittent exposure to myokines (i.e., irisin, IL-6, LIF, IGF-I) which, instead, act as inflammatory/pro-resorptive mediators when chronically elevated; on the other hand, the reduction in the circulating levels of adipokines (i.e., leptin, visfatin, adiponectin, resistin) sustains these effects as well as improves the whole-body metabolic status. The aim of this review is to highlight the newest findings about the exercise-dependent regulation of these molecules and their role in the fine regulation of bone metabolism.
Multilayer cellular stacks of crosslinked, electrospun 25 wt % hydroxyapatite (HA)-gelatin and pure gelatin fiber scaffolds, seeded with human fetal osteoblasts (hFOBs), were studied for up to 18 days in static and dynamic cell culture. Two types of stack models were investigated: a four-layer stack with cells seeded at the bottom surface of the first/top layer and the top surface of the fourth/bottom layer, so that the two middle layers were not seeded with cells with the aim to act as continuing conduits of culture medium and nutrients supply to the adjacent cell-populated zones; a three-layer stack with cells seeded at the bottom surface of each layer. hFOBs exhibited lower migration rate through the stack thickness for the 25 wt % HA-gelatin scaffolds as compared to the pure gelatin scaffolds, due to the small pores of the former. Hence, the regularly seeded three-layer stack maintained cell-free porous zones in all layers through which the culture medium could continuously perfuse, while good fusion was achieved at the interface of all layers via the cross-migrating cells with a preference to downwards vertical migration attributed to gravity. Dynamic cell culture conditions enhanced overall cell growth by about 6% for the regularly seeded three-layer stack. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2016.
Although modes of action (MOAs) play a key role in the understanding of the toxic mechanism of chemicals, the MOAs have not been investigated for antibiotics to green algae. This paper is to discriminate excess toxicity from baseline level and investigate the MOAs of 13 different antibiotics to algae by using the determined toxicity values. Comparison of the toxicities shows that the inhibitors of protein synthesis to bacteria, such as azithromycin, doxycycline, florfenicol and oxytetracycline, exhibit significantly toxic effects to algae. On the other hand, the cell wall synthesis inhibitors, such as cefotaxime and amoxicillin, show relatively low toxic effects to the algae. The concentrations determined by HPLC indicate that quinocetone and amoxicillin can be easily photodegraded or hydrolyzed during the toxic tests. The toxic effects of quinocetone and amoxicillin to the algae are attributed to not only their parent compounds, but also their metabolites. Investigation on the mode of action shows that, except rifampicin, all the tested antibiotics exhibit excess toxicity to Pseudokirchneriella subcapitata (P. subcapitata). These antibiotics can be identified as reactive modes of action to the algae. They act as electrophilic mechanism of action to P. subcapitata. These results are valuable for the understanding of the toxic mechanism to algae.
Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two CoA molecules bound in both the canonical AcCoA/CoA-binding site and the acceptor-substrate-binding site. Our work also provides initial clues regarding the substrate specificity of these two enzymes; however, their native function(s) remain unknown. We found both proteins act as N- rather than O-acetyltransferases and preferentially acetylate l-threonine. The combination of structural and kinetic analyses of these two previously uncharacterized GNATs provides fundamental knowledge and a framework on which future studies can be built to elucidate their native functions.
This is a cross-sectional study with a quantitative approach, establishing the epidemiological profile of people with physical disabilities resident in the municipality of Florianópolis, in the Southern Brazilian state of Santa Catarina, and analyzing it in relation to the public policies related to that population. The minimum sample was determined by a statistical calculation, considering the population of the municipality with disabilities, according to data from the 2010 Population Census. The data were collected using an electronic form installed in mobile devices and stored at an online provider. The data were analyzed and handled with the software Statistical Package for the Social Sciences. A total of 139 questionnaires were processed. A separation of the results was made using these categories: Individual attributes; Social attributes; and Characteristics related to disability. As well as the descriptive profile of the disabled people, the study analyses and discusses the distance, worldwide and in Brazil, between the proposals for public policies and the actual actions of care directed towards the rights of these people. The conclusion is that there is a fundamental need to act in relation to the real needs of this population and consolidate proposals for health promotion, health protection and health rehabilitation for them.
This article discusses the main advances and challenges for understanding and evaluating disability as a restriction for social participation. This new understanding has its origins in the 2006 WHO International Classification of Functioning, Disability, and Health - ICF, the 2001 UN Convention on the Rights of Persons with Disabilities, and more recently, the July 2015 Brazilian Inclusion of People with Disabilities Act (IPDA), also known as the Statute on Persons with Disabilities. The change in the understanding of disability from a merely biomedical perspective, to an understanding that is based on oppression and social inequality reinforces the idea that disability is not an individual attribute, but the result of a society that is not prepared for human diversity. Based on a legislative analysis of the many documents on policies regarding persons with disabilities, notably the IPDA and the evaluations of disability that the ICF already uses in Brazil, the main contention proposed is that classifying and valuing disability is challenging for professional evaluators as well as for Brazilian public policy. This is mainly due to the challenges of recognizing the barriers and environmental factors that hamper the full participation in society of people with disabilities.
Do individuals intuitively favor certain moral actions over others? This study explores the role of intuitive thinking-induced by time pressure and cognitive load-in moral judgment and behavior. We conduct experiments in three different countries (Sweden, Austria, and the United States) involving over 1,400 subjects. All subjects responded to four trolley type dilemmas and four dictator games involving different charitable causes. Decisions were made under time pressure/time delay or while experiencing cognitive load or control. Overall we find converging evidence that intuitive states do not influence moral decisions. Neither time-pressure nor cognitive load had any effect on moral judgments or altruistic behavior. Thus we find no supporting evidence for the claim that intuitive moral judgments and dictator game giving differ from more reflectively taken decisions. Across all samples and decision tasks men were more likely to make utilitarian moral judgments and act selfishly compared to women, providing further evidence that there are robust gender differences in moral decision-making. However, there were no significant interactions between gender and the treatment manipulations of intuitive versus reflective decision-making.
Susceptibility to phosphine (PH3 ) and sulfuryl fluoride (SF) and cross resistance to SF were evaluated in two life stages (eggs and adults) of key grain insect pests, Rhyzopertha dominca (F.), Sitophilus oryzae (L.), Cryptolestes ferrugineus (Stephens), and Tribolium castaneum (Herbst). This study was performed with an aim to integrate SF into phosphine resistance management program in Australia.
Characterisation of susceptibility and resistance to phosphine in eggs and adults showed that C. ferrugineus was the most tolerant as well as resistant species. Mortality responses of eggs and adults to SF at 25 °C revealed T. castaneum to be the most tolerant species followed by S. oryzae, C. ferrugineus and R. dominica. A high dose range of SF, 50.8-62.2 mg L(-1) over 48 h, representing a c (concentration) x t (time) products of 2438-2985 gh m(-3) was required for complete control of eggs of T. castaneum, whereas eggs of the least tolerant R. dominca required only 630 gh m(-3) for 48 h (13.13 mg L(-1) ). Mortality response of eggs and adults of phosphine-resistant strain to SF in all four species confirmed the lack of cross resistance to SF.
Our research concludes that phosphine resistance does not confer cross resistance to SF in grain insect pests irrespective of the variation in levels of tolerance to SF itself or resistance to phosphine in their egg and adult stages. While our study confirms that SF has potentials as a "phosphine resistance breaker", the observed higher tolerance in eggs stresses the importance of developing SF fumigation protocols with longer exposure periods.
In 1990, the US Congress amended the Clean Air Act (CAA) to reduce regional-scale ecosystem degradation from SO x and NO x emissions which have been responsible for acid deposition in regions such as the Adirondack Mountains of New York State. An ecosystem assessment project was conducted from 1994 to 2012 by the Darrin Fresh Water Institute to determine the effect of these emission reduction policies on aquatic systems. The project investigated water chemistry and biota in 30 Adirondack lakes and ponded waters. Although regulatory changes made in response to the 1990 CAA amendments resulted in a reduction of acid deposition within the Adirondacks, the ecosystem response to these reductions is complicated. A statistical analysis of SO4, pH, Al, and DOC data collected during this project demonstrates positive change in response to decreased deposition. The changes in water chemistry also have lowered the risk of Al toxicity to brook trout (Salvelinus fontinalis [Mitchill]), which allowed the re-introduction of this species to Brooktrout Lake from which it had been extirpated. However, pH and labile aluminum (Alim) fluctuate and are not strongly correlated to changes in acid deposition. As such, toxicity to S. fontinalis also is cyclic and provides rationale for the difficulties inherent in re-establishing resident populations in impacted aquatic environments. Overall, aquatic ecosystems of the Adirondacks show a positive response to reduced deposition driven by changes in environmental policy, but the response is more complex and indicates an ecosystem-wide interaction between aquatic and watershed components of the ecosystem.
A primary pathogeny of epilepsy is excessive activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs). To find potential molecules to inhibit AMPARs, high-throughput screening was performed in a library of tetrapeptides in silico. Computational results suggest that some tetrapeptides bind stably to the AMPAR. We aligned these sequences of tetrapeptide candidates with those from in vitro digestion of the trout skin protein. Among salmon-derived products, Glu-Gly-Ala-Arg (EGAR) showed a high biological affinity toward AMPAR when tested in silico. Accordingly, natural EGAR was hypothesized to have anticonvulsant activity, and in vitro experiments showed that EGAR selectively inhibited AMPAR-mediated synaptic transmission without affecting the electrophysiological properties of hippocampal pyramidal neurons. In addition, EGAR reduced neuronal spiking in an in vitro seizure model. Moreover, the ability of EGAR to reduce seizures was evaluated in a rodent epilepsy model. Briefer and less severe seizures versus controls were shown after mice were treated with EGAR. In conclusion, the promising experimental results suggest that EGAR inhibitor against AMPARs may be a target for antiepilepsy pharmaceuticals. Epilepsy is a common brain disorder characterized by the occurrence of recurring, unprovoked seizures. Twenty to 30 % of persons with epilepsy do not achieve adequate seizure control with any drug. Here we provide a possibility in which a natural and edible tetrapeptide, EGAR, can act as an antiepileptic agent. We have combined computation with in vitro experiments to show how EGAR modulates epilepsy. We also used an animal model of epilepsy to prove that EGAR can inhibit seizures in vivo. This study suggests EGAR as a potential pharmaceutical for the treatment of epilepsy.
It is now widely established that health care organizations are well advised not only to identify and act upon the concerns of all patient groups but also to encourage and enable them to voice their concerns in the first place. That said, research has begun to reveal that patients differ substantially in their readiness to complain, with many deciding to remain silent even after experiencing severe adverse events. Little research has explored whether patients at the margins (e.g., elderly, disabled, or mentally ill patients) are more likely to remain silent.
We examined the extent to which patients' social (being elderly or poorly educated), physical (having a permanent impairment such as deafness, blindness, or a chronic physical condition), and mental marginality (having a mental illness or learning disability) is associated with their intention and perceived ability to complain.
We matched survey and patient record data for hospital inpatients treated in the English National Health Service in 2007. We then computed two-stage probit selection models to estimate the cross-sectional association between patients' social, physical, and mental marginality and their intention (Stage 1, N1 = 58,062) and perceived ability to complain (Stage 2, N2 = 3,765).
Only 6.47% of all patients intended to complain. Of these, only 10.41% indicated that hospital staff provided them with all the information they needed to complain. An additional 14.70% reported to have received at least some of the information needed for this purpose. Patients above 80 not only exhibited significantly lower intentions to complain than their mid-aged counterparts (-1.16%) but also felt considerably less well informed to file a complaint (-5.45%). Similarly, patients suffering from blindness or a severe vision impairment showed a significantly lower perceived ability to complain (-5.20%).
Patients at the margins, especially elderly patients and those with a severe vision impairment, will often remain silent and require special attention, if health care organizations are to listen to-and learn from-the voices of all patients. Our results indicate the need for inclusive complaint procedures designed to fuel organizational learning. Dedicated roles such as case managers and complaint officers might help to make such feedback channels accessible to all patients.
Mechanical force and Wnt signaling activate β-catenin-mediated transcription to promote proliferation and tissue expansion. However, it is unknown whether mechanical force and Wnt signaling act independently or synergize to activate β-catenin signaling and cell division. We show that mechanical strain induced Src-dependent phosphorylation of Y654 β-catenin and increased β-catenin-mediated transcription in mammalian MDCK epithelial cells. Under these conditions, cells accumulated in S/G2 (independent of DNA damage) but did not divide. Activating β-catenin through Casein Kinase I inhibition or Wnt3A addition increased β-catenin-mediated transcription and strain-induced accumulation of cells in S/G2. Significantly, only the combination of mechanical strain and Wnt/β-catenin activation triggered cells in S/G2 to divide. These results indicate that strain-induced Src phosphorylation of β-catenin and Wnt-dependent β-catenin stabilization synergize to increase β-catenin-mediated transcription to levels required for mitosis. Thus, local Wnt signaling may fine-tune the effects of global mechanical strain to restrict cell divisions during tissue development and homeostasis.
Interventions to increase cooking skills (CS) and food skills (FS) as a route to improving overall diet are popular within public health. This study tested a comprehensive model of diet quality by assessing the influence of socio-demographic, knowledge- and psychological-related variables alongside perceived CS and FS abilities. The correspondence of two measures of diet quality further validated the Eating Choices Index (ECI) for use in quantitative research.
A cross-sectional survey was conducted in a quota-controlled nationally representative sample of 1049 adults aged 20-60 years drawn from the Island of Ireland. Surveys were administered in participants' homes via computer-assisted personal interviewing (CAPI) assessing a range of socio-demographic, knowledge- and psychological-related variables alongside perceived CS and FS abilities. Regression models were used to model factors influencing diet quality. Correspondence between 2 measures of diet quality was assessed using chi-square and Pearson correlations.
ECI score was significantly negatively correlated with DINE Fat intake (r = -0.24, p < 0.001), and ECI score was significantly positively correlated with DINE Fibre intake (r = 0.38, p < 0.001), demonstrating a high agreement. Findings indicated that males, younger respondents and those with no/few educational qualifications scored significantly lower on both CS and FS abilities. The relative influence of socio-demographic, knowledge, psychological variables and CS and FS abilities on dietary outcomes varied, with regression models explaining 10-20 % of diet quality variance. CS ability exerted the strongest relationship with saturated fat intake (β = -0.296, p < 0.001) and was a significant predictor of fibre intake (β = -0.113, p < 0.05), although not for healthy food choices (ECI) (β = 0.04, p > 0.05).
Greater CS and FS abilities may not lead directly to healthier dietary choices given the myriad of other factors implicated; however, CS appear to have differential influences on aspects of the diet, most notably in relation to lowering saturated fat intake. Findings suggest that CS and FS should not be singular targets of interventions designed to improve diet; but targeting specific sub-groups of the population e.g. males, younger adults, those with limited education might be more fruitful. A greater understanding of the interaction of factors influencing cooking and food practices within the home is needed.
Based on the assumption of parental influence on adolescent behavior, multicomponent school-based dietary interventions often include a parental component. The effect of this intervention component is seldom reported and the evidence is inconsistent. We conducted a systematic process evaluation of the parental component and examined whether the leveal of parental involvement in a large multi-component intervention: the Boost study was associated with adolescents' fruit and vegetable (FV) intake at follow-up.
The Boost study was targeting FV intake among 1,175 Danish 7(th) graders (≈13- year-olds) in the school year 2010/11. The study included a school component: free FV in class and curricular activities; a local community component: fact sheets for sports- and youth clubs; and a parental component: presentation of Boost at a parent-school meeting, 6 newsletters to parents, 3 guided student-parent curricular activities, and a student-parent Boost event.
Students whose parent replied to the follow-up survey (n = 347).
Questionnaire data from students, parents and teachers at 20 intervention schools. Process evaluation measures: dose delivered, dose received, appreciation and level of parental involvement. Parental involvement was trichotomized into: low/no (0-2 points), medium (3 points) and high (4-6 points). The association between level of parental involvement and self-reported FV intake (24-h recall), was analyzed using multilevel regression analyses.
The Boost study was presented at a parent-school meeting at all intervention schools. The dose delivered was low to moderate for the three other parental elements. Most parents appreciated the intervention and talked with their child about Boost (83.5 %). High, medium and low parental involvement was found among 30.5 %, 29.6 % and 39.4 % of the students respectively. Parental involvement was highest among women. More men agreed that the parental newsletters provided new information. Students with a medium and high level of parental involvement ate 47.5 and 95.2 g more FV per day compared to students with low level/no parental involvement (p = 0.02).
Students with a high level of parental involvement ate significantly more FV at follow-up compared to students with a low level/no parental involvement. Parental involvement in interventions may improve adolescents' FV intake if challenges of implementation can be overcome.
ISRCTN11666034 . Registered 06/01/2012. Retrospectively registered.
Brazil created Health Councils to bring together civil society groups, heath professionals, and government officials in the discussion of health policies and health system resource allocation. However, several studies have concluded that Health Councils are not very influential on healthcare policy. This study probes this issue further by providing a descriptive account of some of the challenges civil society face within Brazil's Health Councils.
Forty semi-structured interviews with Health Council Members at the municipal, state and national levels were conducted in June and July of 2013 and May of 2014. The geographical location of the interviewees covered all five regions of Brazil (North, Northeast, Midwest, Southeast, South) for a total of 5 different municipal Health Councils, 8 different state Health Councils, and the national Health Council in Brasilia. Interview data was analyzed using a thematic approach.
Health Councils are limited by a lack of legal authority, which limits their ability to hold the government accountable for its health service performance, and thus hinders their ability to fulfill their mandate. Equally important, their membership guidelines create a limited level of inclusivity that seems to benefit only well-organized civil society groups. There is a reported lack of support and recognition from the relevant government that negatively affects the degree to which Health Council deliberations are implemented. Other deficiencies include an insufficient amount of resources for Health Council operations, and a lack of training for Health Council members. Lastly, strong individual interests among Health Council members tend to influence how members participate in Health Council discussions.
Brazil's Health Councils fall short in providing an effective forum through which civil society can actively participate in health policy and resource allocation decision-making processes. Restrictive membership guidelines, a lack of autonomy from the government, vulnerability to government manipulation, a lack of support and recognition from the government and insufficient training and operational budgets have made Health Council largely a forum for consultation. Our conclusions highlight, that among other issues, Health Councils need to have the legal authority to act independently to promote government accountability, membership guidelines need to be revised in order include members of marginalized groups, and better training of civil society representatives is required to help them make more informed decisions.
Swamp deer (Rucervus duvaucelii) is an endemic, Scheduled I species under the Wildlife (Protection) Act 1972, India. According to variations in antler size, it has been classified into three subspecies, namely Western (R. duvaucelii duvaucelii), Central (R. duvaucelii branderi), and Eastern (R. duvaucelii ranjitsinhii). For planning effective ex situ and in situ conservation of a wide-ranging species in different bioclimatic regions and in wildlife forensic, the use of genetic characterization in defining morpho/ecotypes has been suggested because of the geographic clines and reproductive isolation. In spite of these morphotypes, very little is known about the genetic characteristics of the three subspecies, hence no strict subspecies-based breeding plan for retaining the evolutionary characteristics in captive populations for subsequent re-introduction is available except for a few studies. We describe the genetic characteristics of these three subspecies using cytochrome b of the mtDNA genome (400 bp). The DNA sequence data indicated 11 variable sites within the three subspecies. Two paraphyletic clades, namely the Central India and Western-Eastern populations were found, whereas the Western and Eastern populations are monophyletic with a bootstrap value of 69% within the clade. We suggest the need of sorting these three subspecies using different molecular mtDNA markers in zoos for captive breeding purposes so as to retain the genetic diversity of the separate geographic clines and to use a subspecies-specific fixed-state nucleotide to assess the extent of poaching to avoid any population demography stochastically in India.
The activation of the complement system by canonical and non-canonical mechanisms results in the generation of multiple C3 and C5 cleavage fragments including anaphylatoxins C3a and C5a as well as opsonizing C3b/iC3b. It is now well appreciated that anaphylatoxins not only act as pro-inflammatory mediators but as immunoregulatory molecules that control the activation status of cells and tissue at several levels. Likewise, C3b/iC3b is more than the opsonizing fragment that facilitates engulfment and destruction of targets by phagocytes. In the circulation, it also facilitates the transport and delivery of bacteria and immune complexes to phagocytes, through a process known as immune adherence, with consequences for adaptive immunity. Here, we will discuss non-classical immunoregulatory properties of C3 and C5 cleavage fragments. We highlight the influence of anaphylatoxins on Th2 and Th17 cell development during allergic asthma with a particular emphasis on their role in the modulation of CD11b(+) conventional dendritic cells and monocyte-derived dendritic cells. Furthermore, we discuss the control of anaphylatoxin-mediated activation of dendritic cells and allergic effector cells by adaptive immune mechanisms that involve allergen-specific IgG1 antibodies and plasma or regulatory T cell-derived IL-10 production. Finally, we take a fresh look at immune adherence with a particular focus on the development of antibacterial cytotoxic T-cell responses.
Mannose-binding lectin (MBL), collectin-10, collectin-11, and the ficolins (ficolin-1, ficolin-2, and ficolin-3) are soluble pattern recognition molecules in the lectin complement pathway. These proteins act as mediators of host defense and participate in maintenance of tissue homeostasis. They bind to conserved pathogen-specific structures and altered self-antigens and form complexes with the pentraxins to modulate innate immune functions. All molecules exhibit distinct expression in different tissue compartments, but all are found to a varying degree in the circulation. A common feature of these molecules is their ability to interact with a set of serine proteases named MASPs (MASP-1, MASP-2, and MASP-3). MASP-1 and -2 trigger the activation of the lectin pathway and MASP-3 may be involved in the activation of the alternative pathway of complement. Furthermore, MASPs mediate processes related to coagulation, bradykinin release, and endothelial and platelet activation. Variant alleles affecting expression and structure of the proteins have been associated with a variety of infectious and non-infectious diseases, most commonly as disease modifiers. Notably, the severe 3MC (Malpuech, Michels, Mingarelli, and Carnevale) embryonic development syndrome originates from rare mutations affecting either collectin-11 or MASP-3, indicating a broader functionality of the complement system than previously anticipated. This review summarizes the characteristics of the molecules in the lectin pathway.
Innate immunity is fundamental to our defense against microorganisms. Physiologically, the intravascular innate immune system acts as a purging system that identifies and removes foreign substances leading to thromboinflammatory responses, tissue remodeling, and repair. It is also a key contributor to the adverse effects observed in many diseases and therapies involving biomaterials and therapeutic cells/organs. The intravascular innate immune system consists of the cascade systems of the blood (the complement, contact, coagulation, and fibrinolytic systems), the blood cells (polymorphonuclear cells, monocytes, platelets), and the endothelial cell lining of the vessels. Activation of the intravascular innate immune system in vivo leads to thromboinflammation that can be activated by several of the system's pathways and that initiates repair after tissue damage and leads to adverse reactions in several disorders and treatment modalities. In this review, we summarize the current knowledge in the field and discuss the obstacles that exist in order to study the cross-talk between the components of the intravascular innate immune system. These include the use of purified in vitro systems, animal models and various types of anticoagulants. In order to avoid some of these obstacles we have developed specialized human whole blood models that allow investigation of the cross-talk between the various cascade systems and the blood cells. We in particular stress that platelets are involved in these interactions and that the lectin pathway of the complement system is an emerging part of innate immunity that interacts with the contact/coagulation system. Understanding the resulting thromboinflammation will allow development of new therapeutic modalities.
As a representative photocatalyst for photoelectrochemical solar water splitting, TiO2 has been intensively studied but most researches have focused on the rutile and anatsase phases because brookite, another important crystalline polymorph of TiO2, rarely exists in nature and is difficult to synthesize. In this work, hydrogen doped brookite (H:brookite) nanobullet arrays were synthesized via a well-designed solution reaction for the first time. H:brookite shows highly improved PEC properties with excellent stability, enhanced photocurrent, and significantly high Faradaic efficiency for overall solar water splitting. To support the experimental data, ab initio density functional theory calculations were also conducted. At the interstitial doping site that has minimum formation energy, the hydrogen atoms act as shallow donors and exist as H(+). which has the minimum formation energy among three states of hydrogen (H(+). H(0), and H(-)). The calculated density of states of H:brookite shows a narrowed bandgap and an increased electron density compared to the pristine brookite. The combined experimental and theoretical results provide frameworks for the exploration of the PEC properties of doped brookite and extend our knowledge regarding the undiscovered properties of brookite of TiO2.
The no-cloning theorem states that an unknown quantum state cannot be cloned exactly and deterministically due to the linearity of quantum mechanics. Associated with this theorem is the quantitative no-cloning limit that sets an upper bound to the quality of the generated clones. However, this limit can be circumvented by abandoning determinism and using probabilistic methods. Here, we report an experimental demonstration of probabilistic cloning of arbitrary coherent states that clearly surpasses the no-cloning limit. Our scheme is based on a hybrid linear amplifier that combines an ideal deterministic linear amplifier with a heralded measurement-based noiseless amplifier. We demonstrate the production of up to five clones with the fidelity of each clone clearly exceeding the corresponding no-cloning limit. Moreover, since successful cloning events are heralded, our scheme has the potential to be adopted in quantum repeater, teleportation and computing applications.
Polyphenols are widespread constituents of different food commodities. These are regarded as essential micronutrients because of their well-documented health benefits. These health benefits depend on the amount of polyphenols consumed and their bioavailability in the gut. The microbial transformation of polyphenols in gut is poorly characterized, where, these polyphenols may act as promoting factors for proliferation of beneficial gut inhabitants and inhibiting the pathogenic species. The aim of this review is to present a holistic view on occurrence of polyphenols, their health benefits and influence of dietary polyphenols on gut microbiota.
Workplace learning (WPL) placements are a mandatory part of occupational therapy courses. There is some evidence that suggests WPL placements in international settings are beneficial for students' learning, and personal and professional development. The aim of this study was to explore the impact an international WPL placement in Vietnam had on the perceived personal and professional development of a group of Australian occupational therapy graduates.
Interpretative phenomenological analysis was used to explore the perceptions of how participation in the Charles Sturt University School of Community Health's Vietnam placement influenced the personal and professional development of occupational therapy graduates. Individual semi-structured interviews were conducted with nine graduates who participated in the Vietnam placement when they were final year occupational therapy students. Interviews were audio-recorded, transcribed verbatim and individually analysed to identify key themes.
Two major themes emerged from the analysis: becoming resourceful, resilient and confident, and becoming respectful of difference. The participants indicated that participation in the Vietnam placement had a positive impact on their personal and professional development.
Participants indicated that the Vietnam placement enabled them to develop their resourcefulness, resilience, reasoning skills, cultural competence, confidence and independence, beyond what they felt would have achieved on a domestic placement. For these reason these participants found the placement a beneficial and worthwhile experience.
There is little evidence to direct health systems toward providing efficient interventions to address medical errors, defined as an unintended act of omission or commission or one not executed as intended that may or may not cause harm to the patient but does not achieve its intended outcome. We believe that lack of guidance on what is the most efficient way to reduce medical errors and improve the quality of health-care limits the scale-up of health system improvement interventions. Challenges to economic evaluation of these interventions include defining and implementing improvement interventions in different settings with high fidelity, capturing all of the positive and negative effects of the intervention, using process measures of effectiveness rather than health outcomes, and determining the full cost of the intervention and all economic consequences of its effects. However, health system improvement interventions should be treated similarly to individual medical interventions and undergo rigorous economic evaluation to provide actionable evidence to guide policy-makers in decisions of resource allocation for improvement activities among other competing demands for health-care resources.
The aim of this study was to evaluate the effects of low-volume high-intensity interval training and continuous low to moderate intensity training on quality of life, functional capacity and cardiovascular disease risk factors in cancer survivors.
Cancer survivors within 24 months post-diagnosis were randomly assigned into the low-volume high-intensity interval training group (n = 8) or the continuous low to moderate intensity training group (n = 8) group for 36 sessions (12 weeks) of supervised exercise. The low-volume high-intensity interval training (LVHIIT) group performed 7 × 30 s intervals (≥85% maximal heart rate) and the continuous low to moderate intensity training (CLMIT) group performed continuous aerobic training for 20 min (≤55% maximal heart rate) on a stationary bike or treadmill.
Significant improvements (time) were observed for 13 of the 23 dependent variables (ES 0.05-0.61, p ≤ 0.05). An interaction effect was observed for six minute walk test (18.53% [32.43-4.63] ES 0.50, p ≤ 0.01) with the LVHIIT group demonstrating greater improvements.
These preliminary findings suggest that both interventions can induce improvements in quality of life, functional capacity and selected cardiovascular disease risk factors. The LVHIIT program was well tolerated by the participants and our results suggest that LVHIIT is the preferred modality to improve fitness (6MWT); it remains to be seen which intervention elicits the most clinically relevant outcomes for patients. A larger sample size with a control group is required to confirm the significance of these findings.
Established under Section 25 of the HIV Prevention and Control Act of 2006, the HIV and AIDS Tribunal of Kenya is the only HIV-specific statutory body in the world with the mandate to adjudicate cases relating to violations of HIV-related human rights. Yet, very limited research has been done on this tribunal. Based on findings from a desk research and semi-structured interviews of key informants conducted in Kenya, this article analyzes the composition, mandate, procedures, practice, and cases of the tribunal with the aim to appreciate its contribution to the advancement of human rights in the context of HIV. It concludes that, after a sluggish start, the HIV and AIDS Tribunal of Kenya is now keeping its promise to advance the human rights of people living with and affected by HIV in Kenya, notably through addressing barriers to access to justice, swift ruling, and purposeful application of the law. The article, however, highlights various challenges still affecting the tribunal and its effectiveness, and cautions about the replication of this model in other jurisdictions without a full appraisal.
One thousand people die every day in India as a result of TB, a preventable and treatable disease, even though the Constitution of India, government schemes, and international law guarantee available, accessible, acceptable, quality health care. Failure to address the spread of TB and to provide quality treatment to all affected populations constitutes a public health and human rights emergency that demands action and accountability. As part of a broader strategy, health activists in India employ Public Interest Litigation (PIL) to hold the state accountable for rights violations and to demand new legislation, standards for patient care, accountability for under-spending, improvements in services at individual facilities, and access to government entitlements in marginalized communities. Taking inspiration from right to health PIL cases (PILs), lawyers in a New Delhi-based rights organization used desk research, fact-findings, and the Right To Information Act to build a TB PIL for the Delhi High Court, Sanjai Sharma v. NCT of Delhi and Others (2015). The case argues that inadequate implementation of government TB schemes violates the Constitutional rights to life, health, food, and equality. Although PILs face substantial challenges, this paper concludes that litigation can be a crucial advocacy and accountability tool for people living with TB and their allies.
Backround: Abnormal expression of CXC chemokine receptor 1 (CXCR1) has shown the ability to promote tumor angiogensis, invasion and metastasis in several cancers. The purpose of our curret study is to discover the clinical prognostic significance of CXCR1 in resectable gastric cancer.
330 gastric cancer patients who underwent R0 gastrectomy with standard D2 lymphadenectomy at Zhongshan Hospital, Fudan University between 2007 and 2008 were enrolled. CXCR1 expression was evaluated with use of immunohistochemical staining. The relation between CXCR1 expression and clinicopathological features and postoperative prognosis was respectively inspected.
In both discovery and validation data sets, CXCR1 high expression indicated poorer overall survival (OS) in TNM II and III patients. Furthermore, multivariate analysis identified CXCR1 expression and TNM stage as two independent prognostic factors for OS. Incorporating CXCR1 expression into current TNM staging system could generate a novel clinical predictive model for gastric cancer, showing better prognostic accuracy with respect to patients' OS. More importantly, TNM II patients with higher CXCR1 expression were shown to significantly benefit from postoperative 5-fluorouracil (5-FU) based adjuvant chemotherapy (ACT).
CXCR1 in gastric cancer was identified as an independent adverse prognostic factor. Combining CXCR1 expression with current TNM staging system could lead to better risk stratification and more accurate prognosis for gastric cancer patients. High expression of CXCR1 identified a subgroup of TNM stage II gastric cancer patients who appeared to benefit from 5-FU based ACT.
Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by (51)Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.
Accumulation of anthropogenic litter (AL) on marine beaches and its ecological effects have been a major focus of research. Recent studies suggest AL is also abundant in freshwater environments, but much less research has been conducted in freshwaters relative to oceans. The Adopt-a-BeachTM (AAB) program, administered by the Alliance for the Great Lakes, organizes volunteers to act as citizen scientists by collecting and maintaining data on AL abundance on Great Lakes beaches. Initial assessments of the AAB records quantified sources and abundance of AL on Lake Michigan beaches, and showed that plastic AL was >75% of AL on beaches across all five Great Lakes. However, AAB records have not yet been used to examine patterns of AL density and composition among beaches of all different substrate types (e.g., parks, rocky, sandy), across land-use categories (e.g., rural, suburban, urban), or among seasons (i.e., spring, summer, and fall). We found that most AL on beaches are consumer goods that most likely originate from beach visitors and nearby urban environments, rather than activities such as shipping, fishing, or illegal dumping. We also demonstrated that urban beaches and those with sand rather than rocks had higher AL density relative to other sites. Finally, we found that AL abundance is lowest during the summer, between the US holidays of Memorial Day (last Monday in May) and Labor Day (first Monday in September) at the urban beaches, while other beaches showed no seasonality. This research is a model for utilizing datasets collected by volunteers involved in citizen science programs, and will contribute to AL management by offering priorities for AL types and locations to maximize AL reduction.
Nucleotides are key subunits for nucleic acids and provide energy for intracellular metabolism. They can also be released from cells to act physiologically as extracellular messengers or pathologically as danger signals. Extracellular nucleotides stimulate membrane receptors in the P2 and P1 family. P2X are ATP-activated cation channels; P2Y and P1 are G-protein coupled receptors activated by ATP, ADP, UTP, and UDP in the case of P2 or adenosine for P1. Renal P2 receptors influence both vascular contractility and tubular function. Renal cells also express ectonucleotidases that rapidly hydrolyze extracellular nucleotides. These enzymes integrate this multireceptor purinergic-signaling complex by determining the nucleotide milieu to titrate receptor activation. Purinergic signaling also regulates immune cell function by modulating the synthesis and release of various cytokines such as IL1-β and IL-18 as part of inflammasome activation. Abnormal or excessive stimulation of this intricate paracrine system can be pro- or anti-inflammatory, and is also linked to necrosis and apoptosis. Kidney tissue injury causes a localized increase in ATP concentration, and sustained activation of P2 receptors can lead to renal glomerular, tubular, and vascular cell damage. Purinergic receptors also regulate the activity and proliferation of fibroblasts, promoting both inflammation and fibrosis in chronic disease. In this short review we summarize some of the recent findings related to purinergic signaling in the kidney. We focus predominantly on the P2X7 receptor, discussing why antagonists have so far disappointed in clinical trials and how advances in our understanding of purinergic signaling might help to reposition these compounds as potential treatments for renal disease.
Cell senescence is a process of central importance to the understanding of aging as well as to the development of new drugs. It is related with genomic instability, which has been shown to occur in the presence of autophagy deficiency. Yet, the mechanism that triggers genomic instability and senescence from a condition of autophagy deficiency remains unknown. By analyzing the consequences of treating human keratinocytes (HaCaT) with the pentacyclic triterpenoid Betulinic Acid (BA) we were able to propose that cell senescence can develop as a response to parallel damage in the membranes of mitochondria and lysosome.
We performed biochemical, immunocytochemical and cytometric assays after challenging HaCaT cells with BA. We also evaluated membrane leakage induced by BA in liposomes and giant unilamellar vesicles.
By destabilizing lipid bilayers of mitochondria and lysosomes, BA triggers the misbalance in the mitochondrial-lysosomal axis leading to perceived autophagy impairment, lipofuscinogenesis, genomic instability and cell senescence. The progressive accumulation of mitochondria and lipofuscin, which comes from imperfect mitophagy triggered by BA, provides a continuous source of reactive species further damaging lysosomes and leading to cell aging.
This work reveals that the initial trigger of cell senescence can be the physical damage in the membranes of lysosomes and mitochondria.
This concept will help in the search of new drugs that act as senescence-inductors. BA is under evaluation as chemotherapeutic agent against several types of tumors and induction of cell senescence should be considered as one of its main mechanisms of action.
Activin A (Act A), a member of the transforming growth factor-beta (TGF-β), reduces neuronal apoptosis during cerebral ischemia through Act A/Smads signaling pathway. However, little is known about the effect of Act A/Smads pathway on autophagy in neurons. Here, we found that oxygen-glucose deprivation (OGD)-induced autophagy was suppressed by exogenous Act A in a concentration-dependent manner and enhanced by Act A/Smads pathway inhibitor (ActRIIA-Ab) in neuronal PC12 cells. These results indicate that Act A/Smads pathway negatively regulates autophagy in OGD-treated PC12 cells. In addition, we found that c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways are involved in the OGD-induced autophagy. The activation of JNK and p38 MAPK pathways in OGD-treated PC12 cells was suppressed by exogenous Act A and enhanced by ActRIIA-Ab. Together, our results suggest that Act A/Smads signaling pathway negatively regulates OGD-induced autophagy via suppression of JNK and p38 MAPK pathways in neuronal PC12 cells.
The biochemical model of C3 photosynthesis by Farquhar, von Caemmerer and Berry (FvCB) assumes that photosynthetic CO2 assimilation is limited by one of three biochemical processes that are not always easily discerned. This leads to improper assessments of biochemical limitations that limit the accuracy of the model predictions. We use the sensitivity of rates of CO2 assimilation and photosynthetic electron transport to changes in O2 and CO2 concentration in the chloroplast to evaluate photosynthetic limitations. Assessing the sensitivities to O2 and CO2 concentrations reduces the impact of uncertainties in the fixed parameters to a minimum and simultaneously entirely eliminates the need to determine the variable parameters of the model, such as Vcmax , J, or TP . Our analyses demonstrate that Rubisco limits carbon assimilation at high temperatures, while it is limited by triose phosphate utilization at lower temperatures and at higher CO2 concentrations. Measurements can be assigned a priori to one of the three functions of the FvCB model, allowing testing for the suitability of the selected fixed parameters of the model. This approach can improve the reliability of photosynthesis models on scales from the leaf level to estimating the global carbon budget.
Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.
Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non-coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non-vesicular circulating miRNAs exhibit a potential role as mediators of the cross-talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. J. Cell. Physiol. 9999: 1-9, 2016. © 2016 Wiley Periodicals, Inc.
Many fish that are exposed to a threat release disturbance cues, which are chemicals that alert conspecifics to the presence of the threat. The release of disturbance cues has been well demonstrated in various species of laboratory-reared fish. Migratory fish species often exhibit increased cortisol levels and are exposed to numerous stressors during their migrations, which could trigger the release of disturbance cues. We tested the responses of wild migrating sockeye salmon (Oncorhynchus nerka) and pink salmon (O. gorbuscha) to the odours of disturbed and undisturbed conspecifics to determine whether these fish release disturbance cues following exposure to a simulated stressor. Furthermore, we tested the responses of sockeye salmon to water-borne cortisol, following evidence from past studies that this chemical is excreted through the gills of stressed fish, and speculation that endogenous correlates of stress might function as disturbance cues. We found that sockeye salmon avoid the odour of disturbed conspecifics, whereas pink salmon do not. Avoidance occurred in both female and male sockeye salmon, and was associated with an increase in plasma cortisol levels in females, but not in males. We also found no behavioural response to water-borne cortisol, which suggests this chemical does not act as an exogenous disturbance cue in sockeye salmon. Avoidance of disturbed conspecifics could limit exposure to risks during the sockeye salmon spawning migration, but could also delay the rate of migration and thereby accrue reproductive costs.
Studies have been mounting in support of the finding that plants release aerobic methane (CH4 ), and that these emissions are increased by both short-term and long-term environmental stress. It remains unknown whether or not they are affected by variation in light quantity and quality, whether emissions change over time, and whether they are influenced by physiological parameters. Light is the primary energy source of plants, and therefore an important regulator of plant growth and development. Both shade-intolerant sunflower and shade-tolerant chrysanthemum were investigated for the release of aerobic CH4 emissions, using either low or high light intensity, and varying light quality, including control, low or normal red:far-red ratio (R:FR), and low or high levels of blue, to discern the relationship between light and CH4 emissions. It was found that low levels of light act as an environmental stress, facilitating CH4 release from both species. R:FR and blue lights increased emissions under low light, but the results varied with species, providing evidence that both light quantity and quality regulate CH4 emissions. Emission rates of 6.79-41.13 ng g(-1) DW h(-1) and 18.53-180.25 ng g(-1) DW h(-1) were observed for sunflower and chrysanthemum, respectively. Moreover, emissions decreased with age as plants acclimated to environmental conditions. Since effects were similar in both species, there may be a common trend among a number of shade-tolerant and shade-intolerant species. Light quantity and quality are influenced by factors including cloud covering, so it is important to know how plants will be affected in the context of aerobic CH4 emissions.
Gemini supra-amphiphiles with different spacer lengths, [M-n-M](2+)@2[DBS](-) (n = 2, 6, 10), were easily constructed. The conformational flexibility and hydrophobicity of the spacer group can be effectively tailored through regulating the spacer length, leading to the fine control of the topologies and subsequent hierarchical self-assemblies of [M-n-M](2+)@2[DBS](-). Vesicles are primarily fabricated by [M-n-M](2+)@2[DBS](-), and then successively fused into vesicle clusters, nanotubes, and planar bilayers, whose bilayer curvatures are gradually decreased, with increasing spacer length. Coating [M-10-M](2+) with β-CD can reduce the flexibility and hydrophobicity of the decyl spacer, resulting in the reversion from planar bilayers to vesicles. Furthermore, stable aqueous two-phase systems (ATPSs) that spontaneously formed via vesicle fusion in the solutions of [M-n-M](2+)@2[DBS](-) (n = 6, 10) can act as functional supramolecular systems in the isolation and purification of oil-soluble biomaterials.
A comparative chromatographic evaluation of chlorinated decarboxylated betanins and betanidins generated under activity of hypochlorous acid exerted upon these highly antioxidative potent decarboxylated pigments derived from natural sources was performed by LC-DAD-ESI-MS/MS. Comparison of the chromatographic profiles of the chlorinated pigments revealed two different directions of retention changes in relation to the corresponding substrates. Chlorination of all betacyanins that are decarboxylated at carbon C-17 results in an increase of their retention times. In contrast, all other pigments (the non-decarboxylated betacyanins as well as 2-decarboxy- and 15-decarboxy-derivatives) exhibit lower retention after chlorination. During further chromatographic experiments based upon chemical transformation of the related pigments (decarboxylation and deglucosylation), the compounds' structures were confirmed. The elaborated method for determination of chlorinated pigments enabled analysis of a chlorinated red beet root extract that was submitted to the MPO/H2O2/Cl(-) system acting under inflammation-like conditions (pH 5). This indicates a promising possibility for measurement of these chlorinated pigments as indicators of specific inflammatory states wherein betacyanins and decarboxylated betacyanins act as hypochlorite scavengers.
Monosomal karyotype (MK) is known as a far end of the unfavorable cytogenetics in adult acute myeloid leukemia (AML), while available data in childhood AML is scarce. In this study, we investigated the prevalence and prognostic value of MK with retrospectively analyzed 119 patients newly diagnosed with childhood de novo AML. Ten patients (8.4%) revealed to have MK. All MK-positive (MK(+)) AML were associated with complex cytogenetic abnormalities and belonged to the cytogenetic adverse-risk group. Nine of MK(+) patients (90%) achieved complete remission. The event-free survival (EFS) and overall survival (OS) of MK(+) adverse group were comparable to the ESF and OS of MK-negative non-adverse group (EFS 60.0±15.5% vs 59.0±5.1%, P=0.925; OS 70.0±14.5% vs 58.1±5.3%, P=0.696). In multivariate analysis, MK was not an independent adverse prognostic factor for EFS (hazard ratio 0.45, 95% C.I. 0.13-1.50, P=0.194). In addition, 7 of 9 MK(+) patients who received allogeneic hematopoietic stem cell transplantation (HSCT) survived event-free, with a median follow-up of 64 months. In conclusion, MK did not act as an adverse prognostic factor in childhood de novo AML. Allogeneic HSCT might have contributed to the excellent outcome of MK(+) childhood de novo AML.
Significant breakthroughs in the field of injury prevention and childhood safety have occurred during the past half-century. For example, the Poison Prevention Packaging Act of 1970 and the institution of child passenger safety laws are responsible for a significant reduction in injuries among children and adolescents. This review will focus on the following three topics because of their significant contribution to pediatric injury morbidity, especially among adolescents, and their promise for further effective prevention research. Opioid overdoses by adolescents and young adults are increasing; however, the use of naloxone by bystanders represents a life-saving development in opioid overdose prevention that deserves further investigation. Youth firearm injury remains a major cause of death and disability in adolescents. Despite a lack of robust injury prevention research on the topic, the development of novel approaches to access and examine firearm injury data is leading to exploration of public health approaches to reduce these injuries. Finally, despite legislative and educational efforts surrounding child passenger safety and graduated driver license laws, motor vehicle crashes are still a leading cause of injury for both children and adolescents; however, research on these laws holds the opportunity for significant reduction in injuries. Focused efforts to reduce unintentional injuries from opiate overdoses, firearms and motor vehicle crashes may produce a breakthrough in the field of injury prevention similar to that of the Poison Prevention Packaging Act.Pediatric Research (2016); doi:10.1038/pr.2016.193.
Adrenocortical tumors (ACTs) represent less than 0.2% of all childhood neoplasms. Frequent clinical manifestations are virilization, hypercortisolism, and peripheral precocious puberty (PPP).
We describe two cases in which ACTs were responsible for virilization (case 1) and PPP (case 2) in prepubertal girls. In both cases an ACT diagnosis was made after 5-6 months from the first appearance of clinical signs. Surgery was performed within 1 month of diagnosis, and the benign nature of tumors was histologically confirmed. Despite complete tumor resection, virilizing features persisted.
Adrenocortical tumors should be considered early in the assessment of PPP. There is often a significant delay between the onset of symptoms and accurate diagnosis but early treatment is essential to limit the clinical manifestations of androgen overproduction.
Gemini analogs of calcitriol, characterized by the extension of the C21-methyl group of calcitriol with a second chain, act as agonists of the vitamin D receptor (VDR). This second side chain of gemini is accommodated in a new cavity inside the VDR created by the structural rearrangement of the protein core. The resulting conformational change preserves the active state of the receptor and bestows gemini compounds with biological activities that exceed those of calcitriol. Of particular interest are gemini's anti-cancer properties, and in this study we demonstrate anti-proliferative and tumor-reducing abilities of BXL0124 and BXL0097, differing only by the presence or absence, respectively, of the methylene group on the A ring. BXL0124 acts as a more potent VDR agonist than its 19-nor counterpart by activating VDR-mediated transcription at lower concentrations. In a similar manner, BXL0124 is more active than BXL0097 in growth inhibition of breast cancer cells and reduction of tumor volume. Structural comparisons of BXL0097 and BXL0124, as their VDR complexes, explain the elevated activity of the latter.
Despite increased leptin concentrations during pregnancy, fat mass and food intake are increased. The satiety response to central leptin is suppressed, indicating a state of leptin insensitivity in the hypothalamus. Although the regulation of food intake is a major function of leptin, this hormone also influences a wide range of functions within the body. These actions include the regulation of glucose homeostasis, which undergoes major adaptation in the maternal body to generate optimal conditions for foetal development and growth. The present study aimed to investigate the effects of central leptin treatment on glucose homeostasis in pregnant rats to determine whether pregnancy-induced leptin insensitivity is functionally specific, and to further investigate changes in glucose homeostasis during pregnancy. After an overnight fast, nonpregnant and day 14 pregnant rats received an i.c.v. injection of leptin (100 ng or 4 μg) or vehicle then underwent a glucose tolerance test (GTT). Further groups of nonpregnant and day 14 pregnant rats were killed 30 min after leptin (doses ranging from 40 ng-4 μg) or vehicle i.c.v. injections for western blot analysis of phospho-signal transducer and activator of transcription 3 (STAT3) and phospho-Akt in various hypothalamic nuclei. Central leptin injection prior to a GTT lead to lowered basal insulin concentrations and impaired glucose tolerance in nonpregnant female rats, whereas the same doses of leptin had no significant effect on glucose tolerance in day 14 pregnant rats, indicating that, similar to the satiety actions of leptin, the effects of leptin on glucose homeostasis are suppressed during pregnancy. Furthermore, in the arcuate nucleus and ventromedial and dorsomedial nuclei of the hypothalamus, comprising three leptin-sensitive areas, there was no evidence that leptin induced Akt phosphorylation despite significant increases in phospho-STAT3, suggesting that leptin does not act through phospho-Akt in these areas in female rats.
Across culture, healthy infants show a high interest in infant-directed (ID) talking and singing. Despite ID talking and ID singing being very similar in physical properties, infants differentially respond to each of them. The mechanisms underpinning these different responses are still under discussion.
This study explored the behavioral (n = 26) and brain (n = 14) responses from 6- to 8-month-old infants to ID talking and ID singing during a face-to-face mother-infant interaction with their own mother. Behavioral response was analyzed from offline video coding, and brain response was estimated from the analysis of electrophysiological recordings.
We found that during ID talking, infants displayed a significantly higher number of visual contacts, vocalizations, and body movements than during ID singing. Moreover, only during ID talking were the number of visual contacts and vocalizations positively correlated with the number of questions and pauses in the mother's speech.
Our results suggest that ID talking provides infants with specific cues that allow them not only to react to mother stimulation, but also to act toward them, displaying a rudimentary version of turn-taking behavior. Brain activity partially supported that interpretation. The relevance of our results for bonding is discussed.
Endophytic bacteria may act individually or in consortia in controlling certain plant diseases. In this study, pepper plants (Capsicum annuum L. cv. Nokkwang) were cultivated in glasshouse conditions using field soils collected from two different geographic locations, Deokso (DS) and Gwangyang (GY) in Korea. Community structure and antifungal activity of pepper endophytic bacteria were analyzed using culture-independent (PCR-DGGE) and culture-dependent (plating) methods, respectively. Dissimilarities were observed between DGGE profiles of DS and GY samples at all plant tissues. However, sequencing of the major DGGE bands revealed an enrichment of Firmicutes in the leaves of plants propagated in either soil. Similar results were observed with the culturable assays. Firmicutes dominated the isolates from both leaf samples, DS leaf (100 %) and GY leaf (83.3 %), although the genus compositions of DS leaf and GY leaf isolates were different. We assessed the antifungal activity of each isolate recovered to better understand the potential role that these endophytic bacteria may play. Of the 27 representative isolates from DS plant samples, 17 isolates (63.0 %) had antagonistic activity against at least one of the fungi tested. Seventeen isolates from GY plant samples (58.6 %) displayed antagonistic properties. The results show that the endophytic communities differ in the same plant species when propagated in different soils. Exploring the internal tissues of plants growing in diverse soil environments could be a way to find potential candidates for biocontrol agents.
Animal and human studies have consistently demonstrated that cortical regions are important for pain perception and pain-related emotional changes. Studies of the anterior cingulate cortex (ACC) have shown that adult cortical synapses can be modified after peripheral injuries, and long-term changes at synaptic level may contribute to long-lasting suffering in patients. It also explains why chronic pain is resistant to conventional analgesics that act by inhibiting synaptic transmission. Insular cortex (IC), another critical cortical area, is found to be highly plastic and can undergo long-term potentiation (LTP) after injury. Inhibiting IC LTP reduces behavioral sensitization caused by injury. LTP of glutamatergic transmission in pain related cortical areas serves as a key mechanism for chronic pain.
Effective Anatomical Acts transformed medical education and curtailed grave-robbing. William S. Forbes, Demonstrator of Anatomy at Jefferson Medical College in Philadelphia, authored the Pennsylvania Anatomy Act of 1867, but it was ineffective. In December of 1882, Forbes and accomplices were charged with grave-robbing. Forbes was acquitted in early 1883, but his accomplices were all convicted; nevertheless, these events precipitated a strengthened Anatomy Act in 1883. Forbes was crowned the Father of the Pennsylvania Anatomy Act and was revered by the Philadelphia medical community for his personal sacrifices for medical education; they even paid his legal fees. Over the remainder of his life, Forbes received many honors. However, there was a second major player, rural doctor William J. McKnight, a convicted grave-robber and State Senator. The evidence shows that Forbes precipitated the crisis, which was a racial powder keg, and then primarily focused on his trial, while McKnight, creatively working behind the scenes in collaboration with Jefferson, Anatomy Professor William H. Pancoast, used the crisis to draft and pass transformative legislation enabling anatomical dissection at Pennsylvania medical schools. While not minimizing Forbes suffering throughout these events, McKnight should be appropriately recognized for his initiative and contributions, which far exceeded those of Forbes.
LncRNA-ROR has been reported to be involved in many kinds of human cancers. However, whether LncRNA-ROR is involved in gallbladder cancer progression remains largely unknown. The objective of this study is to investigate the role of LncRNA-ROR in gallbladder cancer. We found that LncRNA-ROR expression level was upregulated in gallbladder cancer tissues (P < 0.05) and was significantly associated with tumor sizes (P < 0.05) and lymph node metastasis (P < 0.05). High expression of LncRNA-ROR was significantly associated with poor prognosis in gallbladder cancer patients (P < 0.05). Moreover, knockdown of LncRNA-ROR inhibited cell proliferation, migration, and invasion. The epithelial-mesenchymal transition (EMT) phenotype induced by TGF-β1 was reversed after LncRNA-ROR knocking down in SGC-996 and Noz cells. LncRNA-ROR plays an important role in the development of gallbladder cancer and mediates the EMT in gallbladder cancer. LncRNA-ROR might act as a marker of prognosis and therapeutic target for gallbladder cancer.
Lead compounds that target tubulin are being developed as agents against the human malaria parasite, Plasmodium falciparum. It is important to define the binding sites of these molecules on the tubulin dimer: Taxol, Vinca domains or novel binding pockets; however, extraction of native parasite tubulin is difficult.
This report aims to develop assays that allow the rapid assessment of binding sites of compounds on the tubulin dimer.
We have developed a simple growth assay, using a combination of two anti-microtubule drugs that have overlapping binding sites, to study whether the two drugs act in synergistic, antagonistic or neutral manner. Additionally, Molecular docking was used to predict the binding sites of the drugs.
The combination assay shows antagonistic interactions between drugs having overlapping binding sites. In contrast, drugs that do not bind to overlapping sites show no interactions or synergism in this combination assay. Molecular docking predictions show that indeed, drugs with antagonistic interactions in the growth assay do bind to overlapping sites.
These two assays can be a simple preliminary screen for the binding sites of novel anti-tubulin compounds being developed for malaria therapeutics.
Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory system. It is generally chronic and challenging to treat. The recommended pharmacotherapy for neuropathic pain includes the use of some antidepressants, such as tricyclic antidepressants (TCAs) (amitriptyline…) or serotonin and noradrenaline re-uptake inhibitors (duloxetine…), and/or anticonvulsants such as the gabapentinoids gabapentin or pregabalin. Antidepressant drugs are not acute analgesics but require a chronic treatment to relieve neuropathic pain, which suggests the recruitment of secondary downstream mechanisms as well as long-term molecular and neuronal plasticity. Noradrenaline is a major actor for the action of antidepressant drugs in a neuropathic pain context. Mechanistic hypotheses have implied the recruitment of noradrenergic descending pathways as well as the peripheral recruitment of noradrenaline from sympathetic fibers sprouting into dorsal root ganglia; and importance of both α2 and β2 adrenoceptors have been reported. These monoamine re-uptake inhibitors may also indirectly act as anti-proinflammatory cytokine drugs; and their therapeutic action requires the opioid system, particularly the mu (MOP) and/or delta (DOP) opioid receptors. Gabapentinoids, which target the voltage-dependent calcium channels α2δ-1 subunit, inhibit calcium currents, thus decreasing the excitatory transmitter release and spinal sensitization. Gabapentinoids also activate the descending noradrenergic pain inhibitory system coupled to spinal α2 adrenoceptors. Gabapentinoid treatment may also indirectly impact on neuroimmune actors, like proinflammatory cytokines. These drugs are effective against neuropathic pain both with acute administration at high dose and with repeated administration. This review focuses on mechanistic knowledge concerning chronic antidepressant treatment and gabapentinoid treatment in a neuropathic pain context.
To study plasma levels of matrix metalloproteinases (MMPs) as potential markers of recovery during intensive rehabilitation therapy (IRT) after stroke.
Prospective and descriptive 3-month follow-up study.
Rehabilitation unit and research center.
Patients with first-ever ischemic stroke (n=15) enrolled to IRT (≥3h/d and 5d/wk) and healthy volunteers (n=15) (N=30).
Not applicable.
The primary outcome was to measure plasma MMP3, MMP12, and MMP13 levels and evaluate potential associations with motor/functional scales using a battery of tests (National Institutes of Health Stroke Scale, modified Rankin scale, Barthel Index, Fugl-Meyer Assessment, Functional Ambulation Categories, Medical Research Council scale, Chedoke Arm and Hand Activity Inventory, and the 10-m walk test) before IRT and at 1- and 3-month follow-ups. The secondary outcome was to evaluate the use of these MMPs as biomarkers as predictors of patient's outcome.
MMP levels remained stable during the study period and were similar to those in the healthy volunteer group. However, baseline MMP12 and MMP13 levels were strongly associated with stroke severity and were found to be elevated in those patients with the poorest outcomes. Interestingly, plasma MMP3 was independent of baseline stroke characteristics but was found to be increased in patients with better motor/functional recovery and in patients with larger improvements during rehabilitation.
MMPs might act as biologic markers of recovery during rehabilitation therapy related to their roles in both injury and tissue remodeling. Future confirmatory investigations in multicenter studies are warranted by our data.
In order to discover plant-derived signaling pathway inhibitors with antifungal properties, a two-component screening system utilizing the calcineurin and Hog1 mitogen-activated protein kinase pathways responsible for the virulence networks of Cryptococcus neoformans was employed, owing to the counter-regulatory actions of these pathways. Of the 1,000 plant extracts tested, two bioactive compounds from Miliusa sinensis were found to act specifically on the calcineurin pathway of C. neoformans. These compounds, identified as pashanone and 5-hydroxy-6,7-dimethoxyflavanone, exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentration values ranging from 4.0 to >128 μg/ml.
During the screening of endophytes obtained from Glycyrrhiza glabra Linn., the extract from a fungal culture designated as GG1F1 showed significant antimicrobial activity. The fungus was identified as a species of the genus Phoma and was most closely related to Phoma cucurbitacearum. The chemical investigation of the GG1F1 extract led to the isolation and characterization of two thiodiketopiperazine derivatives. Both the compounds inhibited the growth of several bacterial pathogens especially that of Staphylococcus aureus and Streptococcus pyogenes, with IC50 values of less than 10 μM. The compounds strongly inhibited biofilm formation in both the pathogens. In vitro time kill kinetics showed efficient bactericidal activity of these compounds. The compounds were found to act synergistically with streptomycin while producing varying effects in combination with ciprofloxacin and ampicillin. The compounds inhibited bacterial transcription/translation in vitro, and also inhibited staphyloxanthin production in S. aureus. Although similar in structure, they differed significantly in some of their properties, particularly the effect on the expression of pathogenecity related genes in S. aureus at sub-lethal concentrations. Keeping in view the antimicrobial potential of these compounds, it would be needful to scale up the production of these compounds through fermentation technology and further explore their potential as antibiotics using in vivo models.
Acidobacteria is one of the most abundant phyla in soils and has been detected in rhizosphere mainly based on cultivation-independent approaches such as 16S rRNA gene survey. Although putative interaction of Acidobacteria with plants was suggested, so far no plant-bacterial interactions were shown. Therefore, we performed several in vitro tests to evaluate Acidobacteria-plant interactions and the possible mechanisms involved in such interaction. We observed that Arabidopsis thaliana inoculated with three strains belonging to Acidobacteria subdivision 1 showed increase in biomass of roots and shoots as well as morphological changes in root system. Our results indicate that the plant hormone indole-3-acetic acid production and iron acquisition are plausibly involved in the plant and Acidobacteria interactions. Here, we confirm for the first time that Acidobacteria can actively interact with plants and act as plant growth-promoting bacteria. In addition, we show that Acidobacteria strains produce exopolysaccharide which supports the adhesion of bacteria to the root surfaces.
Migraine is the third most common disease worldwide, the most common neurological disorder, and one of the most common pain conditions. Despite its prevalence, the basic physiology and underlying mechanisms contributing to the development of migraine are still poorly understood and development of new therapeutic targets is long overdue. Until recently, the major contributing pathophysiological event thought to initiate migraine was cerebral and meningeal arterial vasodilation. However, the role of vasodilation in migraine is unclear and recent findings challenge its necessity. While vasodilation itself may not contribute to migraine, it remains possible that vessels play a role in migraine pathophysiology in the absence of vasodilation. Blood vessels consist of a variety of cell types that both release and respond to numerous mediators including growth factors, cytokines, adenosine triphosphate (ATP), and nitric oxide (NO). Many of these mediators have actions on neurons that can contribute to migraine. Conversely, neurons release factors such as norepinephrine and calcitonin gene-related peptide (CGRP) that act on cells native to blood vessels. Both normal and pathological events occurring within and between vascular cells could thus mediate bi-directional communication between vessels and the nervous system, without the need for changes in vascular tone. This review will discuss the potential contribution of the vasculature, specifically endothelial cells, to current neuronal mechanisms hypothesized to play a role in migraine. Hypothalamic activity, cortical spreading depression (CSD), and dural afferent input from the cranial meninges will be reviewed with a focus on how these mechanisms can influence or be impacted by blood vessels. Together, the data discussed will provide a framework by which vessels can be viewed as important potential contributors to migraine pathophysiology, even in light of the current uncertainty over the role of vasodilation in this disorder.
The distribution and levels of TNIP1 in malignant and normal gastric mucosa are different, but it is not known whether TNIP1 polymorphisms are related to gastric carcinogenesis. To assess the association between four TNIP1 SNPs (rs3792792, rs4958881, rs7708392, rs10036748) and carcinogenesis, we used Sequenom Mass-ARRAY technology to determine the genotypes of 302 gastric carcinoma patients and 300 healthy controls in a Northwest Chinese Han population. These data were then compared using the Chi-square test/Fisher's exact test, genetic model analysis, and haplotype analysis. Odds ratios (OR) and 95% confidence intervals (CI) were used to evaluate the correlation. We observed that patients with the "G" allele of rs7708392 and the "C" allele of rs10036748 showed an increased risk of gastric carcinoma (OR= 1.335, 95%CI: 1.021-1.745, P= 0.035; OR= 1.358, 95%CI: 1.039-1.774, P= 0.025, respectively). Conversely, the haplotype "CT" of TNIP1 (rs7708392-rs10036748) may act as a genetic protective factor for gastric carcinoma (adjusted OR= 0.731, 95%CI: 0.552-0.970, P= 0.030). Our results are the first to suggest that genetic variation in TNIP1 gene is associated with gastric carcinoma, though, this finding must be confirmed in other populations with larger sample size.
Environmentally transmitted opportunistic pathogens shuttle between two substantially different environments: outside-host and within-host habitats. These environments differ from each other especially with respect to nutrient availability. Consequently, the pathogens are required to regulate their behavior in response to environmental cues in order to survive, but how nutrients control the virulence in opportunistic pathogens is still poorly understood. In this study, we examined how nutrient level in the outside-host environment affects the gene expression of putative virulence factors of the opportunistic fish pathogen Flavobacterium columnare. The impact of environmental nutrient concentration on bacterial virulence was explored by cultivating the bacteria in various nutrient conditions, measuring the gene expression of putative virulence factors with RT-qPCR and, finally, experimentally challenging rainbow trout (Oncorhynchus mykiss) fry with these bacteria. Our results show that increased environmental nutrient concentration can increase the expression of putative virulence genes, chondroitinase (cslA) and collagenase, in the outside-host environment and may lead to more rapid fish mortality. These findings address that the environmental nutrients may act as significant triggers of virulence gene expression and therefore contribute to the interaction between an environmentally transmitted opportunistic pathogen and its host.
Ubiquitin-conjugating enzyme E2T (UBE2T) is a member of the E2 family that mediates the ubiquitin-proteasome system and regulates gene expression. It is a major oncogene in several cancers such as lung cancer and breast cancer, while the potential functions of UBE2T in gastric cancer (GC) remains largely unknown. Here, we identified the roles of UBE2T in GC progression and its potential to act as a prognostic marker of GC. Our data demonstrated that UBE2T was significantly upregulated in gastric cancer tissues, and the high expression of UBE2T was significantly correlated with poor differentiation, high T classification, and poor prognosis. In vitro experiments indicated that UBE2T promoted cell proliferation and inhibited cell cycle arrest. In addition, we observed that UBE2T modulated cell mobility by inducing epithelial-mesenchymal transition. Collectively, these findings suggest that UBE2T plays an important role in the tumorigenesis of gastric cancer and could act as a potential independent prognostic factor for cancer therapy.
The main focus for the development of adenosine targets as analgesics to date has been A1Rs due to its antinociceptive profile in various preclinical pain models. The usefulness of systemic A1R agonists may be limited by other effects (cardiovascular, motor), but enhanced selectivity for pain might occur with partial agonists, potent and highly selective agonists, or allosteric modulators. A2AR agonists exhibit some peripheral pronociceptive effects, but also act on immune cells to suppress inflammation and on spinal glia to suppress pain signaling and may be useful for inflammatory and neuropathic pain. A2BR agonists exhibit peripheral proinflammatory effects on immune cells, but also spinal antinociceptive effects similar to A2AR agonists. A3Rs are now demonstrated to produce antinociception in several preclinical neuropathic pain models, with mechanistic actions on glial cells, and may be useful for neuropathic pain. Endogenous adenosine levels can be augmented by inhibition of metabolism (via adenosine kinase) or increased generation (via nucleotidases), and these approaches have implications for pain. Endogenous adenosine contributes to antinociception by several pharmacological agents, herbal remedies, acupuncture, transcutaneous electrical nerve stimulation, exercise, joint mobilization, and water immersion via spinal and/or peripheral effects, such that this system appears to constitute a major pain regulatory system. Finally, caffeine inhibits A1-, A2A- and A3Rs with similar potency, and dietary caffeine intake will need attention in trials of: (a) agonists and/or modulators acting at these receptors, (b) some pharmacological and herbal analgesics, and (c) manipulations that enhance endogenous adenosine levels, all of which are inhibited by caffeine and/or A1R antagonists in preclinical studies. All adenosine receptors have effects on spinal glial cells in regulating nociception, and gender differences in the involvement of such cells in chronic neuropathic pain indicate gender may also need attention in preclinical and human trials evaluating the efficacy of adenosine-based analgesics.
A fullerene-based photosensitizer is incorporated postsynthetically into a Zr6 -based MOF, NU-1000, for enhanced singlet oxygen production. The structural organic linkers in the MOF platform also act as photosensitizers which contribute to the overall generation of singlet oxygen from the material under UV irradiation. The singlet oxygen generated by the MOF/fullerene material is shown to oxidize sulfur mustard selectively to the less toxic bis(2-chloroethyl)sulfoxide with a half-life of only 11 min.
Individuals show significant variations in performing a motor act. Previous studies in the action observation literature have largely ignored this ubiquitous, if often unwanted, characteristic of motor performance, assuming movement patterns to be highly similar across repetitions and individuals. In the present study, we examined the possibility that individual variations in motor style directly influence the ability to understand and predict others' actions. To this end, we first recorded grasping movements performed with different intents and used a two-step cluster analysis to identify quantitatively 'clusters' of movements performed with similar movement styles (Experiment 1). Next, using videos of the same movements, we proceeded to examine the influence of these styles on the ability to judge intention from action observation (Experiments 2 and 3). We found that motor styles directly influenced observers' ability to 'read' others' intention, with some styles always being less 'readable' than others. These results provide experimental support for the significance of motor variability for action prediction, suggesting that the ability to predict what another person is likely to do next directly depends on her individual movement style.
Based on complex network theory, we propose a computational methodology which addresses the spatial distribution of fuel breaks for the inhibition of the spread of wildland fires on heterogeneous landscapes. This is a two-level approach where the dynamics of fire spread are modeled as a random Markov field process on a directed network whose edge weights are determined by a Cellular Automata model that integrates detailed GIS, landscape and meteorological data. Within this framework, the spatial distribution of fuel breaks is reduced to the problem of finding network nodes (small land patches) which favour fire propagation. Here, this is accomplished by exploiting network centrality statistics. We illustrate the proposed approach through (a) an artificial forest of randomly distributed density of vegetation, and (b) a real-world case concerning the island of Rhodes in Greece whose major part of its forest was burned in 2008. Simulation results show that the proposed methodology outperforms the benchmark/conventional policy of fuel reduction as this can be realized by selective harvesting and/or prescribed burning based on the density and flammability of vegetation. Interestingly, our approach reveals that patches with sparse density of vegetation may act as hubs for the spread of the fire.
The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.
After endocytic uptake, influenza viruses transit early endosomal compartments and eventually reach late endosomes. There, the viral glycoprotein hemagglutinin (HA) triggers fusion between endosomal and viral membrane, a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus. Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules. While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA, the effect of intracellular transport dynamics on the progress of infection remains largely unclear. In this study, we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection. We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA. We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection. Further, we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus.
In the brain, the striatum and prefrontal cortex interact to gauge the value of actions and the self-regulatory demands in a given environment. New research, involving manipulation of the neural circuitry, has revealed multiple routes by which 'imbalances' in circuit function cause regulation deficits.
Mitochondria in the protist Brevimastigomonas motovehiculus are in the process of dismantling their mitochondrial electron transport chain complexes as they adapt to anaerobic environments. Novel protein interactions suggest a highly complicated process rather than the simple removal of unnecessary genes.
The lamprey belongs to the phylogenetically oldest group of vertebrates that diverged from the mammalian evolutionary line 560 million years ago. A comparison between the lamprey and mammalian basal ganglia establishes a detailed similarity regarding its input from cortex/pallium and thalamus, as well as its intrinsic organisation and projections of the output nuclei. This means that the basal ganglia circuits now present in rodents and primates most likely had evolved already at the dawn of vertebrate evolution. This includes the 'direct pathway' with striatal projection neurons (SPNs) expressing dopamine D1 receptors, which act to inhibit the tonically active GABAergic output neurons in globus pallidus interna and substantia nigra pars reticulata that at rest keep the brainstem motor centres under tonic inhibition. The 'indirect pathway' with dopamine D2 receptor-expressing SPNs and intrinsic basal ganglia nuclei is also conserved. The net effect of the direct pathway is to disinhibit brainstem motor centres and release motor programs, while the indirect pathway instead will suppress motor activity. Transmitters, connectivity and membrane properties are virtually identical in lamprey and rodent basal ganglia. We predict that the basal ganglia contains a series of modules each controlling a given pattern of behaviour including locomotion, eye-movements, posture, and chewing that contain both the direct pathway to release a motor program and the indirect pathway to inhibit competing behaviours. The phasic dopamine input serves value-based decisions and motor learning. During vertebrate evolution with a progressively more diverse motor behaviour, the number of modules will have increased progressively. These new modules with a similar design will be used to control newly developed patterns of behaviour - a process referred to as exaptation.
The Americans with Disabilities Act prohibits discrimination on the basis of disability and requires schools to provide reasonable accommodations for persons with disabilities. The profession of nursing is striving for diversity and inclusion, but barriers still exist to realizing accommodations for people with disabilities. Promoting disclosure, a supportive and enabling environment, resilience, and realistic expectations are important considerations if we are to include among our ranks health professionals who can understand, based on similar life experiences of disability, a fuller range of perspectives of the patients we care for.
Monocytes are short-lived mononuclear phagocytes that circulate in the bloodstream and comprise two main subpopulations that in the mouse are best defined by the Ly6C marker. Intravascular functions of "classical" Ly6C+ monocytes and their interactions with other lymphoid and myeloid leukocytes in the circulation remain poorly understood. Rather, these cells are known to efficiently extravasate into tissues. Indeed, Ly6C+ monocytes and their descendants have emerged as a third, highly plastic and dynamic cellular system that complements the two classical, tissue-resident mononuclear phagocyte compartments, i.e., macrophages and dendritic cells, on demand. Following recruitment to injured tissue, Ly6C+ monocytes respond to local cues and can critically contribute to the initiation and resolution of inflammatory reactions. The second main murine monocyte subset, Ly6C- cells, derive in steady state from Ly6C+ monocytes and remain in the vasculature, where the cells act as scavengers. Moreover, a major fraction of Ly6C- monocytes adheres to the capillary endothelium and patrols the vessel wall for surveillance. Given the central role of monocytes in homeostasis and pathology, in-depth study of this cellular compartment can be highly informative on the health state of the organism and provides an attractive target for therapeutic intervention.
Triglycerides (TGs) are among the most efficacious stimulators of incretin secretion; however, the relative importance of FFA1 (G Protein-coupled Receptor [GPR] 40), FFA4 (GPR120), and GPR119, which all recognize TG metabolites, ie, long-chain fatty acid and 2-monoacylglycerol, respectively, is still unclear. Here, we find all 3 receptors to be highly expressed and highly enriched in fluorescence-activated cell sorting-purified GLP-1 and GIP cells isolated from transgenic reporter mice. In vivo, the TG-induced increase in plasma GIP was significantly reduced in FFA1-deficient mice (to 34%, mean of 4 experiments each with 8-10 animals), in GPR119-deficient mice (to 24%) and in FFA1/FFA4 double deficient mice (to 15%) but not in FFA4-deficient mice. The TG-induced increase in plasma GLP-1 was only significantly reduced in the GPR119-deficient and the FFA1/FFA4 double deficient mice, but not in the FFA1, and FFA4-deficient mice. In mouse colonic crypt cultures the synthetic FFA1 agonists, TAK-875 stimulated GLP-1 secretion to a similar extent as the prototype GLP-1 secretagogue neuromedin C; this, however, only corresponded to approximately half the maximal efficiency of the GPR119 agonist AR231453, whereas the GPR120 agonist Metabolex-209 had no effect. Importantly, when the FFA1 agonist was administered on top of appropriately low doses of the GPR119 agonist, a clear synergistic, ie, more than additive, effect was observed. It is concluded that the 2-monoacylglycerol receptor GPR119 is at least as important as the long-chain fatty acid receptor FFA1 in mediating the TG-induced secretion of incretins and that the 2 receptors act in synergy, whereas FFA4 plays a minor if any role.
Diffusion-weighted imaging (DWI) and (18) F-fluorodeoxyglucose-positron emission tomography ((18) F-FDG-PET) independently correlate with malignancy in breast cancer, but the relationship between their structural and metabolic metrics is not completely understood. This study spatially correlates diffusion, perfusion, and glucose avidity in breast cancer with simultaneous PET/MR imaging and compares correlations with clinical prognostics.
In this Health Insurance Portability and Accountability Act-compliant prospective study, with written informed consent and approval of the institutional review board and using simultaneously acquired FDG-PET and DWI, tissue diffusion (Dt ), and perfusion fraction (fp ) from intravoxel incoherent motion (IVIM) analysis were registered to FDG-PET within 14 locally advanced breast cancers. Lesions were analyzed using 2D histograms and correlation coefficients between Dt , fp , and standardized uptake value (SUV). Correlations were compared with prognostics from biopsy, metastatic burden from whole-body PET, and treatment history.
SUV||Dt correlation coefficient significantly distinguished treated (0.11 ± 0.24) from nontreated (-0.33 ± 0.26) patients (P = 0.005). SUV||fp correlations were on average negative for the whole cohort (-0.17 ± 0.13).
Simultaneously acquired and registered FDG-PET/DWI allowed quantifiable descriptions of breast cancer microenvironments that may provide a framework for monitoring and predicting response to treatment. Magn Reson Med, 2016. © 2016 International Society for Magnetic Resonance in Medicine.
Cardiomyopathy and associated heart failure continues to be one of the most severe complications that threaten a large population. Curcumin, one of the three curcuminoids of the spice turmeric, is very well known for a multitude of health benefits and functions. Norepinephrine (NE), a catecholamine and also a stress hormone may cause the cardiomyocytes to develop increased sensitivity to death with its increasing concentrations. In this study, we investigated the cardioprotective effect of curcumin in NE-induced cardiac apoptosis using several fluorescent and nonfluorescent microscopic techniques like DAPI, PI, Giemsa, PicroSirius and TUNEL. The aim of the study was to assess the effect of curcumin in preventing the occurrence of features underlying apoptosis such as nuclear disruption, chromatin condensation, DNA fragmentation and alterations in mitochondrial membrane permeability. Our results show that curcumin protects the cardiomyocytes against apoptosis significantly and also helps them to revert to their normal physiological state. Hence, we propose that curcumin has the potential to act as a therapeutic agent for the attenuation of NE-induced cardiac cell death and modulation of apoptosis in H9c2 cardiomyocytes.
Simvastatin (SIM), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been reported to inhibit the activity of hepatitis B virus (HBV), however, the mechanism underlying its antiviral function remains unknown. Minichromosome maintenance (MCM) 7, a component of the MCM complex, has been reported to act as an important host factor aiding virus genome replication in host cells. The present study demonstrated that downregulation of MCM7 inhibited the expression of proteins transferred by adenoviral vectors. This suggests an association between MCM7 and viral DNA expression. Thus, the current study aimed to investigate whether SIM affected MCM7 expression. Notably, the results of the present study indicated that following exposure to SIM the protein expression levels of MCM7 in HepG2.2.15, a human HBV‑transfected liver cell line, was decreased. In addition, the HBV DNA replication in the cell line was suppressed. As quantitative polymerase chain reaction experiments demonstrated that SIM did not downregulate the mRNA expression level of MCM7, the current study further investigated whether SIM affects the translation of MCM7. Western blot experiments indicated that SIM improved the activation of eukaryotic initiation factor‑2α (eIF2α), a protein synthesis initiation factor, and upregulated the upstream factors of eIF2α, protein kinase RNA‑like endoplasmic reticulum kinase, which is regulated by the liver kinase B1 (LKB1)‑AMP‑activated protein kinase (AMPK) signaling pathway. These results indicated that SIM induced HBV downregulation via an MCM‑dependent mechanism, and SIM may inhibit MCM7 expression by increasing the phosphorylation of eIF2α, which is mediated by the LKB1-AMPK signaling pathway.
The Forkhead box O (FOXO) protein family is predominantly involved in apoptosis, oxidative stress, DNA damage/repair, tumor angiogenesis, glycometabolism, regulating life span and other important biological processes. Its activity is affected by a variety of posttranslational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, methylation and glycosylation. When cells are subjected to different environments, the corresponding PTMs act on the FOXO protein family, to change transcriptional activity or subcellular localization, and the expression of downstream target genes, will ultimately affect the biological behavior of the cells. In this review, we will discuss the biological characteristics of FOXO protein PTMs.
MicroRNAs (miRNAs) were reported to be involved in the development of clear cell renal cell carcinoma (ccRCC). However, the study on miRNAs in ccRCC is far from complete. The present study identified miRNAs which could act as potential novel prognostic markers for ccRCC, and analyzed its possible mechanism. We found that miR-19a correlated with poor prognosis of ccRCC patients via promoting cell proliferation and suppressing PTEN/SMAD4 expression. Both the microarray screening result and TCGA KIRC dataset analysis showed that miR-19a was significantly upregulated in ccRCC tissues, and further analysis of TCGA data revealed that the upregulated level of miR-19a was strongly associated with advanced T stage and poor prognosis of ccRCC patients. Consistent with clinical observations, miR-19a overexpression significantly promoted ccRCC cell proliferation in vitro. To further explore the mechanism by which miR-19a correlated with cell proliferation and poor prognosis of ccRCC, we performed gene set enrichment analysis (GSEA) for target genes of miR-19a in ccRCC patients. Result indicated that the key target genes of miR-19a included SMAD4 and PTEN. In ccRCC tissues, expression levels of SMAD4 and PTEN were negatively correlated with expression level of miR-19a, revealing that miR-19a suppressed the expression of SMAD4 and PTEN in ccRCC patients. miR-19a overexpression significantly suppressed the expression of SMAD4 and PTEN in vitro, further verifying that SMAD4 and PTEN were the target genes of miR-19a in ccRCC cells. Our results elucidated the tumor promoting role of miR-19a and established miR-19a as a potential novel prognostic marker for ccRCC.
Veno-venous extra-corporeal membrane oxygenation (vv-ECMO) is an established salvage therapy for severe respiratory failure, and may provide an alternative form of treatment for trauma-induced acute respiratory distress syndrome (ARDS) when conventional treatments have failed. The need for systemic anticoagulation is a relative contraindication in patients with bleeding risks, especially in multi traumatic injury.
We describe a case series of four trauma patients with ARDS who were managed with ECMO admitted to the Neuro Critical Care Unit (NCCU) at Addenbrooke's Hospital, Cambridge (UK), from January 2000 to January 2016. We performed a systematic review of the available literature in order to investigate the safety and efficacy of vv-ECMO in post-traumatic ARDS focusing on the use of different anticoagulation strategies and risk of bleeding in polytrauma patients.
Thirty-one patients were included. A heparin bolus was given in 16 cases. Eleven patients developed complications during treatment with ECMO with 3 cases of major bleeding. In all documented cases of bleeding a bolus and infusion of heparin was administered, aiming for an ACT target > 150 s. Two patients treated with heparin-free ECMO developed thromboembolic complications. Four patients died and death was never directly or indirectly related to use of ECMO.
Vv-ECMO can be lifesaving in respiratory failure. Our experience and our literature review suggests that vv-ECMO should be considered as rescue treatment for the management of severe hypoxemic respiratory failure secondary to ARDS in trauma patients.In patients with a high risk of bleeding, the use of ECMO with no initial anticoagulation could be considered a valid option. In patients with a moderate risk of bleeding, use of a heparin infusion keeping an ACT target < 150s can be appropriate.
Level III - Systematic Review.
Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the μ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the μ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the μ-opioid receptor. Our recent data indicate that a radiolabelled [(3)H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no μ-opioid receptors, such as rodent cerebellum, or brain of μ-opioid receptor deficient (MOPr(-/-)) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.
Dioxiranes are powerful oxidants that can act via two different mechanisms: 1) homolytic (H abstraction and oxygen rebound) and 2) heterolytic (electrophilic oxidation). So far, it has been reported that the nature of the substrate dictates the reaction mode independently from the dioxirane employed. Herein, we report an unprecedented case in which the nature of the dioxirane rules the oxidation chemoselectivity. In particular, a switch from C-H to N-H oxidation is observed in the oxidation of lactams moving from dimethyl dioxirane (DDO) to methyl(trifluoromethyl)dioxirane (TFDO). A physical organic chemistry study, which combines the oxidation with two other dioxiranes methyl(fluoromethyl)dioxirane, MFDO, and methyl(difluoromethyl)dioxirane, DFDO, with computational studies, points to a diverse ability of the dioxiranes to either stabilize the homo or the heterolytic pathway.
Toinvestigate the effects of psychosocial work factors (PWF) and psychological distress (PD) on self-assessed work ability.
This follow-up study included 7,810 individuals (55%women) with good work ability at baseline. PWFandPD (measured by GHQ-12) were assessed at baseline and work ability at 7-year follow-up. Effects of PWF and PD on work ability were analyzed by logistic regression, odds ratios (OR) with 95% CI, and by mediation analysisusing 4-way decomposition.
Low support was associated with poor work ability for both women and men (OR 1.78 and 1.89). For men, also low skill discretion was associated with poor work ability (OR 2.07). For both women and men, PD was associated with poor work ability (OR 3.41 and 1.84). PD did not act as an intermediate variablein the association between PWF and work ability.
Strategies for sustainable work ability should focus on both working conditions and health factors. Am. J. Ind. Med. © 2016 Wiley Periodicals, Inc.
Roughly focused extracorporeal shock waves therapy (ESWT) is characterized by a wide focal area, a large therapy zone, easy positioning, and less pain during treatment. The purpose of this study was to investigate the effects of roughly focused ESWT on the expression of osteoprotegerin (OPG) and bone morphogenetic protein-2 (BMP-2) in osteoporotic fractures in rats.
Seventy-two female Sprague-Dawley (SD) rats, 3 months old, were divided into sham-operated group (n = 6) and an ovariectomized (OVX) group (n = 66). Sixty OVX SD rats were used as a model of double proximal tibial osteotomy and inner fixation. The osteotomy site in the left tibia was treated with roughly focused ESWT once at an energy density of 0.26 mJ/mm2, 60 doses/min, and 2000 pact quantities. The contralateral right tibia was left untreated and served as a control. Expression of OPG and BMP-2 in the callus of the osteoporotic fracture area was assessed using immunohistochemistry, real-time polymerase chain reaction (PCR), and Western blotting analysis.
Bone mineral density (BMD) at the proximal tibia, femur, and L5 spine was significantly reduced after ovariectomy. BMD of proximal tibia was 12.9% less in the OVX group than that in the sham-operated group. Meanwhile, bilateral oophorectomy resulted in a lower trabecular bone volume fraction (BV/TV) in the proximal tibia of the sham-OVX animals. Three months after bilateral oophorectomy, BV/TV was 14.29% of baseline BV/TV in OVX legs versus 45.91% in the sham-OVX legs (P < 0.001). These data showed that the SD rats became a suitable model of osteoporosis, 3 months after they were OVX. Immunohistochemical analysis showed higher levels of BMP-2 and OPG expression in the treatment group than those in the control group. Compared with the contralateral controls, decreased expression of OPG and BMP-2 at 3 days after roughly focused ESWT, followed by a later increase at 7 days, was indicated by real-time PCR and Western blotting analysis. The OPG messenger RNA (mRNA) expression levels peaked at 6 weeks after the shock wave treatment, paired with a much earlier (at 4 weeks) increase of BMP-2, and declined close to normal at 8 weeks.
Roughly focused ESWT may promote the expression of OPG and BMP-2 in the osteoporotic fracture area in rats. BMP-2 and OPG may act synergistically and may lead to a significant enhancement of bone formation and remodeling.
Huang-Lian-Jie-Du-Decoction (HLJDD, Oren-gedoku-to in Japanese) is commonly used in traditional Chinese medicine (TCM) to treat ischemic stroke. This study investigated the efficacy of various combinations of the major components of HLJDD, berberine (A), baicalin (B), and jasminoidin (C), on the treatment of ischemic stroke modeled by middle cerebral artery occlusion (MCAO) in rats. The effects of A, B and C individually and their combinations were investigated using proton nuclear magnetic resonance (1H NMR)-based metabolomics complemented with neurologic deficit scoring, infarct volume measurement, biochemistry, histopathology and immunohistochemistry, as well as quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Ischemic stroke produces severe oxidative stress, which induces further damage. Our results show that the ABC combination treatment increased levels of cellular antioxidants that scavenged reactive oxygen species during ischemia-reperfusion via the nuclear erythroid 2-related factor 2 (Nrf2) signaling cascade. These protective effects were not observed with the other treatments. These results suggest that a combination of component herbs in HLJDD exhibit stronger effects than the individual herbs alone. Our integrated metabolomics approach also provides a tractable, powerful tool for understanding the science behind TCM formulations.
To set up an operational basis of the Realizing the European Network of Biodosimetry (RENEB) network within which the application of seven established biodosimetric tools (the dicentric assay, the FISH assay, the micronucleus assay, the PCC assay, the gamma-H2AX assay, electron paramagnetic resonance and optically stimulated luminescence) will be compared and standardized among the participating laboratories.
Two intercomparisons were organized where blood samples and smartphone components were irradiated, coded and sent out to participating laboratories for dosimetric analysis. Moreover, an accident exercise was organized during which each RENEB partner had the chance to practice the procedure of activating the network and to handle large amounts of dosimetric results.
All activities were carried out as planned. Overall, the precision of dose estimates improved between intercomparisons 1 and 2, clearly showing the value of running such regular activities.
The RENEB network is fully operational and ready to act in case of a major radiation emergency. Moreover, the high capacity for analyzing radiation-induced damage in cells and personal electronic devices makes the network suitable for large-scale analyses of low doses effects, where high numbers of samples must be scored in order to detect weak effects.
Prisoner populations are characterized by high rates of hepatitis C (HCV), up to thirty times that of the general population in Australia. Within Australian prisons, less than 1% of eligible inmates access treatment. Public health strategies informed by social capital could be important in addressing this inequality in access to HCV treatment. Twenty-eight male inmates participated in qualitative interviews across three correctional centres in New South Wales, Australia. All participants had recently tested as HCV RNA positive or were receiving HCV treatment. Analysis was conducted with participants including men with experiences of HCV treatment (n=10) (including those currently accessing treatment and those with a history of treatment) and those who were treatment naïve (n=18). Social capital was a resourceful commodity for inmates considering and undergoing treatment while in custody. Inmates were a valuable resource for information regarding HCV treatment, including personal accounts and reassurance (bonding social capital), while nurses a resource for the provision of information and care (linking social capital). Although linking social capital between inmates and nurses appeared influential in HCV treatment access, there remained opportunities for increasing linking social capital within the prison setting (such as nurse-led engagement within the prisons). Bonding and linking social capital can be valuable resources in promoting HCV treatment awareness, uptake and adherence. Peer-based programmes are likely to be influential in promoting HCV outcomes in the prison setting. Engagement in prisons, outside of the clinics, would enhance opportunities for linking social capital to influence HCV treatment outcomes.
Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.
This study assessed the width of the trophic niche of four characid species (Bryconops giacopinii, Bryconops inpai, Hyphessobrycon aff. melazonatus and Iguanodectes geisleri) found under different co-occurrence circumstances in Amazonian upland streams. The study was conducted during the rainy season of 2011 at eight sites of two micro-basins of the Ducke Reserve, Manaus, Amazonas, Brazil. The four species were studied in the following circumstances: only one of the species occurring in the stream; two species co-occurring; three species co-occurring. The relative volume of the food items in the fish stomachs was used to calculate Hurlbert's trophic-niche breadth for the individuals of each species in the different co-occurrence circumstances. Hyphessobrycon aff. melazonatus changed their diet when occurring in syntopy with other characid species of similar feeding habits, as shown by a significant narrowing of its trophic niche. The opportunistic habits and great feeding flexibility of these characid species make the partitioning of food resources possible and act as an important ecological mechanism that facilitates the coexistence of different species, possibly by attenuating the effects of direct competition for food. In addition, the low carrying capacity of these upland forest streams may be an important environmental factor influencing the results of this study.
On 4 August 2011 a modification of the infection protection act became law, meaning that antibiotic consumption surveillance and evaluation of data on the local resistance situation became obligatory for hospitals. Four years after the modification of the infection protection act became law, the Munich public health department aimed to evaluate the state of implementation of antibiotic consumption surveillance in Munich hospitals and to ascertain which antibiotic stewardship (ABS) structures have already been established.
A questionnaire was sent to Munich hospitals about their antibiotic management structural data and state of implementation of legal requirements.
Only 32 % of the hospitals have qualified ABS-experts available. In 76 % of the hospitals persons responsible for antibiotic consumption surveillance are appointed, while in 12 % persons responsible for antibiotic consumption surveillance are at least partly released from their normal work for this activity. Twenty-one hospitals (21 %) conduct antibiotic consumption surveillance taking into account all antiinfective agents mentioned in the Robert Koch-Institute (RKI) guidelines. Of these, 19 (76 %) did this on basis of World Health Organizations ATC/DDD-system (anatomical therapeutic chemical [ATC] classification system which uses defined daily doses [DDD]). The data on antibiotic consumption is evaluated in 72 % of the hospitals, 68 % take account of the local resistance situation. In 96 %, in-house lists of antiinfectives are available; in 80 %, in-house guidelines for antibiotic treatment are available. Fourty-four percent of the hospitals take part in a national surveillance.
For the first time data were collected on implementation of antibiotic consumption surveillance in hospitals, which has been required by law since 2011. An incomplete implementation of legal requirements was demonstrated. It was found that structural and personnel prerequisites are often missing, that release from normal work for this additional duty is rarely granted and that the evaluation of data is often either missing or does not correspond to the legal requirements by omitting to take account of the local resistance situation. The hygienic control of hospitals by the public health service was extended by an additional subject. To carry out this very specialized task in a competent way, an ABS-qualification for public health staff is essential from the perspective of the authors.
Optimized dosage regimens of antibiotics have remained obscure since their introduction. During the last two decades pharmacokinetic(PK)-pharmacodynamic(PD) relationships, originally established in animal experiments, have been increasingly used in patients. The action of betalactams is believed to be governed by the time the plasma concentration is above the minimum inhibitory concentration (MIC). Aminoglycosides act as planned when the peak concentration is a multiple of the MIC and vancomycin seems to work best when the area under the plasma vs. time curve (AUC) to MIC has a certain ratio. Clinicians should be aware that these relationships can only be an indication in which direction dosing should go. Larger studies with sufficiently high numbers of patients and particularly severely sick patients are needed to prove the concepts. In times where all antibiotics can be measured with new technologies, the introduction of therapeutic drug monitoring (TDM) is suggested for ICUs (Intensive Care Unit). The idea of a central lab for TDM of antibiotics such as PEAK (Paul Ehrlich Antibiotika Konzentrationsmessung) is supported.
This study aimed to identify the characteristics of claimed mental disorders. Because the workers believed the cause of the mental disorders was work-related stress or a specific event, we could identify the major work-related stressor for claimed cases.
We included claimed cases of occupational mental disorder or suicide reported during 2010-2014 to the Korea Workers Compensation and Welfare Service (KCOMWEL), established by Industrial Accidents Insurance (IACI) Act. We conducted qualitative analysis using a form specifically developed for this study as well as a quantitative analysis.
Of the 569 claimed cases, 142 cases were recognized as occupational mental disorder or suicide. The approval rate was 24.9 %. Suicide was the most commonly approved mental disorder (23.0 %), followed by major depressive disorder (14.9 %). Regarding profession, 109 workers were managers, and 95 workers were office clerks. The main work-related stressors of the approved cases were acute stressful events (76 cases), long working hours (12 cases), and changes in workload (6 cases). The primary stressful events were work-related legal problems, workplace violence, and employment status-related issues.
Claims due to mental disorders or suicide increased during the 5-year study period, and the approval rate was approximately 33 %, and the main stressor of the claimed cases was an acute stressful event such as physiologic trauma, employment-related issues, fear of legal or financial responsibility, abrupt change in organizational responsibility, or workplace violence.
Provision of suitable habitat for waterbirds is a major challenge for environmental managers in arid and semiarid regions with high spatial and temporal variability in rainfall. It is understood in broad terms that to survive waterbirds must move according to phases of wet-dry cycles, with coastal habitats providing drought refugia and inland wetlands used during the wet phase. However, both inland and coastal wetlands are subject to major anthropogenic pressures, and the various species of waterbird may have particular habitat requirements and respond individualistically to spatiotemporal variations in resource distribution. A better understanding of the relationships between occurrence of waterbirds and habitat condition under changing climatic conditions and anthropogenic pressures will help clarify patterns of habitat use and the targeting of investments in conservation. We provide the first predictive models of habitat availability between wet and dry phases for six widely distributed waterbird species at a large spatial scale. We first test the broad hypothesis that waterbirds are largely confined to coastal regions during a dry phase. We then examine the contrasting results among the six species, which support other hypotheses erected on the basis of their ecological characteristics. There were large increases in area of suitable habitat in inland regions in the wet year compared with the dry year for all species, ranging from 4.14% for Australian White Ibis to 31.73% for Eurasian Coot. With over half of the suitable habitat for three of the six species was located in coastal zones during drought, our study highlights the need to identify and conserve coastal drought refuges. Monitoring of changes in extent and condition of wetlands, combined with distribution modeling of waterbirds, will help support improvements in the conservation and management of waterbirds into the future.
Recent empirical and conceptual papers have highlighted the potential for metabolism to act as a proximate mechanism for behavior that could explain animal personality (consistency over time). Under this hypothesis, individuals with consistently high levels of behavioral activity should also have high resting metabolic rate (RMR) as it can reflect capacity to process food and generate energy. We tested for the predicted positive covariance between RMR and three behaviors that differ in energy demands in 30 male guppies, using multivariate mixed models; we repeatedly measured their activity (10 times each), courtship displays (nine times), voracity (10 times), and metabolism (four-times). Resting metabolic rate (measured overnight in respirometry trials) did not consistently differ among males, whereas initial peak metabolism measured during those same trials (R = 0.42), and all behaviors were repeatable (R = 0.33-0.51). RMR declined over time suggesting habituation to the protocol, whereas peak metabolism did not. Initial peak metabolism was negatively correlated with courtship display intensity, and voracity was positively correlated with activity, but all other among-individual correlations were not significant. We conclude that RMR does not provide a proximate explanation for consistent individual differences in behavior in male guppies, and therefore the potential for independent evolution of these physiological and behavioral traits seems possible. Finally, we identify peak metabolism as a potential measure of the stress response to confinement, which highlights the value of considering various aspects of metabolic rates recording during respirometry trials.
Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX).
The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants.
This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.
Major depressive disorder (MDD) affects around 350 million people worldwide; however, the underlying genetic basis remains largely unknown. In this study, we took into account that MDD is a gene-environment disorder, in which stress is a critical component, and used whole-genome screening of functional variants to investigate the 'missing heritability' in MDD. Genome-wide association studies (GWAS) using single- and multi-locus linear mixed-effect models were performed in a Los Angeles Mexican-American cohort (196 controls, 203 MDD) and in a replication European-ancestry cohort (499 controls, 473 MDD). Our analyses took into consideration the stress levels in the control populations. The Mexican-American controls, comprised primarily of recent immigrants, had high levels of stress due to acculturation issues and the European-ancestry controls with high stress levels were given higher weights in our analysis. We identified 44 common and rare functional variants associated with mild to moderate MDD in the Mexican-American cohort (genome-wide false discovery rate, FDR, <0.05), and their pathway analysis revealed that the three top overrepresented Gene Ontology (GO) processes were innate immune response, glutamate receptor signaling and detection of chemical stimulus in smell sensory perception. Rare variant analysis replicated the association of the PHF21B gene in the ethnically unrelated European-ancestry cohort. The TRPM2 gene, previously implicated in mood disorders, may also be considered replicated by our analyses. Whole-genome sequencing analyses of a subset of the cohorts revealed that European-ancestry individuals have a significantly reduced (50%) number of single nucleotide variants compared with Mexican-American individuals, and for this reason the role of rare variants may vary across populations. PHF21b variants contribute significantly to differences in the levels of expression of this gene in several brain areas, including the hippocampus. Furthermore, using an animal model of stress, we found that Phf21b hippocampal gene expression is significantly decreased in animals resilient to chronic restraint stress when compared with non-chronically stressed animals. Together, our results reveal that including stress level data enables the identification of novel rare functional variants associated with MDD.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.174.
Hippocampal neurogenesis has been proposed to participate in a myriad of behavioral responses, both in basal states and in the context of neuropsychiatric disorders. Here, we identify activating protein 2γ (AP2γ, also known as Tcfap2c), originally described to regulate the generation of neurons in the developing cortex, as a modulator of adult hippocampal glutamatergic neurogenesis in mice. Specifically, AP2γ is present in a sub-population of hippocampal transient amplifying progenitors. There, it is found to act as a positive regulator of the cell fate determinants Tbr2 and NeuroD, promoting proliferation and differentiation of new glutamatergic granular neurons. Conditional ablation of AP2γ in the adult brain significantly reduced hippocampal neurogenesis and disrupted neural coherence between the ventral hippocampus and the medial prefrontal cortex. Furthermore, it resulted in the precipitation of multimodal cognitive deficits. This indicates that the sub-population of AP2γ-positive hippocampal progenitors may constitute an important cellular substrate for hippocampal-dependent cognitive functions. Concurrently, AP2γ deletion produced significant impairments in contextual memory and reversal learning. More so, in a water maze reference memory task a delay in the transition to cognitive strategies relying on hippocampal function integrity was observed. Interestingly, anxiety- and depressive-like behaviors were not significantly affected. Altogether, findings open new perspectives in understanding the role of specific sub-populations of newborn neurons in the (patho)physiology of neuropsychiatric disorders affecting hippocampal neuroplasticity and cognitive function in the adult brain.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.169.
Understanding the rates, spectra, and fitness effects of spontaneous mutations is fundamental to answering key questions in evolution, molecular biology, disease genetics and conservation biology. To estimate mutation rates and evaluate the effect of selection on new mutations, we propagated mutation accumulation (MA) lines of Daphnia pulex for more than 82 generations and maintained a non-MA population under conditions where selection could act. Both experiments were started with the same obligate asexual progenitor clone. By sequencing 30 genomes and implementing a series of validation steps that informed the bioinformatic analyses, we identified a total of 477 single nucleotide mutations (SNMs) in the MA lines, corresponding to a mutation rate of 2.30 × 10(-9) (95% CI 1.90 - 2.70 × 10(-9)) per nucleotide per generation. The high overall rate of loss of heterozygosity (LOH) of 4.82 × 10(-5) per site per generation was due to a large ameiotic recombination event spanning an entire arm of a chromosome (~6 Mb) and several hemizygous deletion events spanning ~2 kb each. In the non-MA population, we found significantly fewer mutations than expected based on the rate derived from the MA experiment, indicating purifying selection was likely acting to remove new deleterious mutations. We observed a surprisingly high level of genetic variability in the non-MA population, which we propose to be driven by balancing selection. Our findings suggest that both positive and negative selection on new mutations is powerful and effective in a strictly clonal population.
A novel class of mesoionic pyrido[1,2-a]pyrimidinones has been discovered with exceptional insecticidal activity controlling a number of insect species, particularly hemiptera and lepidoptera. Mode-of-action studies showed that they act on nicotinic acetylcholine receptors (nAChRs) primarily as inhibitors. Here we report the discovery, evolution, and preparation of this class of chemistry. Our efforts in structure-activity relationship elucidation and biological activity evaluation are also presented.
With increasing evidence showing the involvement of oxidative stress in the pathogenesis of various diseases, the effects of clinical drugs possessing antioxidant functions have received much attention. The unregulated oxidative modification of biological molecules leading to diseases is mediated by multiple oxidants including free radicals, peroxynitrite, hypochlorite, lipoxygenase, and singlet oxygen. The capacity of antioxidants to scavenge or quench oxidants depends on the nature of oxidants. In the present study, the antioxidant effects of several clinical drugs against plasma lipid oxidation induced by the aforementioned five kinds of oxidants were investigated from the production of lipid hydroperoxides, which have been implicated in the pathogenesis of various diseases. Troglitazone acted as a potent peroxyl radical scavenger, whereas probucol and edaravone showed only moderate reactivity and carvedilol, pentoxifylline, and ebselen did not act as radical scavenger. Probucol and edaravone suppressed plasma oxidation mediated by peroxynitrite and hypochlorite. Troglitazone and edaravone inhibited 15-lipoxygenase mediated plasma lipid oxidation, the IC50 being 20 and 34μM respectively. None of the drugs used in this study suppressed plasma lipid oxidation by singlet oxygen. This study shows that the antioxidant effects of drugs depend on the nature of oxidants and that antioxidants against multiple oxidants are required to cope with oxidative stress in vivo.
TP53 is a critical tumor suppressor gene that regulates cell cycle progression, apoptosis, cellular senescence and many other properties critical for control of normal cellular growth and death. Due to the pleiotropic effects that TP53 has on gene expression and cellular physiology, mutations at this tumor suppressor gene result in diverse physiological effects. T53 mutations are frequently detected in numerous cancers. The expression of TP53 can be induced by various agents used to treat cancer patients such as chemotherapeutic drugs and ionizing radiation. Radiation will induce Ataxia telangiectasia mutated (ATM) and other kinases that results in the phosphorylation and activation of TP53. TP53 is also negatively regulated by other mechanisms, such as ubiquitination by ligases such as MDM2. While TP53 has been documented to control the expression of many "classical" genes (e.g., p21(Cip-1), PUMA, Bax) by transcriptional mechanisms for quite some time, more recently TP53 has been shown to regulate microRNA (miR) gene expression. Different miRs can promote oncogenesis (oncomiR) whereas others act to inhibit tumor progression (tumor suppressor miRs). Targeted therapies to stabilize TP53 have been developed by various approaches, MDM2/MDM4 inhibitors have been developed to stabilize TP53 in TP53-wild type (WT) tumors. In addition, small molecules have been isolated that will reactivate certain mutant TP53s. Both of these types of inhibitors are in clinical trials. Understanding the actions of TP53 may yield novel approaches to suppress cancer, aging and other health problems.
Plants are sessile organisms exposed constantly to potential virulent microbes seeking for full pathogenesis in hosts. Different from animals employing both adaptive and innate immune systems, plants only rely on innate immunity to detect and fight against pathogen invasions. Plant innate immunity is proposed to be a two-tiered immune system including pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In PTI, PAMPs, the elicitors derived from microbial pathogens, are perceived by cell surface-localized proteins, known as pattern recognition receptors (PRRs), including receptor-like kinases (RLKs) and receptor-like proteins (RLPs). As single-pass transmembrane proteins, RLKs and RLPs contain an extracellular domain (ECD) responsible for ligand binding. Recognitions of signal molecules by PRR-ECDs induce homo- or heterooligomerization of RLKs and RLPs to trigger corresponding intracellular immune responses. RLKs possess a cytoplasmic Ser/Thr kinase domain that is absent in RLPs, implying that protein phosphorylations underlie key mechanism in transducing immunity signalings and that RLPs unlikely mediate signal transduction independently, and recruitment of other patterns, such as RLKs, is required for the function of RLPs in plant immunity. Receptor-like cytoplasmic kinases, resembling RLK structures but lacking the ECD, act as immediate substrates of PRRs, modulating PRR activities and linking PRRs with downstream signaling mediators. In this chapter, we summarize recent discoveries illustrating the molecular machines of major components of PRR complexes in mediating pathogen perception and immunity activation in plants.
The positively charged amino acids are commonly used excipients in biopharmaceutical formulations for stabilization of therapeutic proteins, yet the mechanisms for their modulation of protein stability are poorly understood. In this study, both lysine and histidine are shown to affect the thermal stability of myoglobin, bovine serum albumin, and lysozyme through a combination of mechanisms governed by their respective functional side chains and glycine, similar to arginine. This study provides evidence that at low concentrations, lysine and histidine interact with proteins by a combination of (1) direct electrostatic interactions with negatively charged side chains, (2) possible binding to high-affinity hydrophobic binding sites, and (3) glycine-mediated weak interactions with peptide backbone and polar side chains. At high concentrations, lysine and histidine act via (4) glycine-mediated competition for water between the unfolding protein and the excipient and (5) sidechain-mediated interaction with apolar regions exposed during unfolding (histidine). Lysine and histidine are useful for biopharmaceutical formulations as they were less destabilizing of the protein structures tested than arginine at concentrations above 100 mM.
Caring for someone with Alzheimer's disease (AD) is very challenging. Social support may play a crucial role in helping caregivers to adapt better to their caregiving role. The aim of this study is to explore the role of social support as a moderator variable of the relationship between depression and life satisfaction in caregivers for patients with AD in Saudi Arabia.
In this cross-sectional study, 122 caregivers for patients with AD completed questionnaires assessing informal social support, depressive symptoms, and general life satisfaction.
The demographic characteristics showed that 79% (n = 96) of caregivers were females and between the ages of 20 and 50. Higher levels of social support positively correlated with reported higher levels of life satisfaction (r = 0.483, p < 0.001). Depression was negatively correlated with social support (r = -0.418, p < 0.001) and life satisfaction scores (r = -0.553, p = < 0.001). Social support was found to be a partial mediator variable, mediating approximately 23.05% of the total effect of depression on life satisfaction (Sobel = -3.065, p = 0.002).
Informal social support can act as a mediator variable in the relationship of depression and life satisfaction in caregivers of AD patients. Improving the informal social support networks may help in coping with caregiving burden and better quality of life.
This study used qualitative methods to investigate the relationship between geographic access and gendered intra-household hierarchies and how these influence treatment-seeking decision-making for childhood fever within the Chikwawa district of Malawi. Previous cross-sectional survey findings in the district indicated that distance from facility and associated costs are important determinants of health facility attendance in the district. This paper uses qualitative data to add depth of understanding to these findings by exploring the relationship between distance from services, anticipated costs and cultural norms of intra-household decision-making, and to identify potential intervention opportunities to reduce challenges experienced by those in remote locations. Qualitative data collection included 12 focus group discussions and 22 critical incident interviews conducted in the local language, with primary caregivers of children who had recently experienced a febrile episode.
Low geographic accessibility to facilities inhibited care-seeking, sometimes by extending the 'assessment period' for a child's illness episode, and led to delays in seeking formal treatment, particularly when the illness occurred at night. Although carers attempted to avoid incurring costs, cash was often needed for transport and food. Whilst in all communities fathers were normatively responsible for treatment costs, mothers generally had greater access to and control over resources and autonomy in decision-making in the matrilineal and matrilocal communities in the central part of the district, which were also closer to formal facilities.
This study illustrates the complex interplay between geographic access and gender dynamics in shaping decisions on whether and when formal treatment is sought for febrile children in Chikwawa District. Geographic marginality and cultural norms intersect in remote areas both to increase the logistical and anticipated financial barriers to utilising services and to reduce caretakers' autonomy to act quickly once they recognize the need for formal care. Health education campaigns should be based within communities, engaging all involved in treatment-seeking decision-making, including men and grandmothers, and should aim to promote the ability of junior women to influence the treatment-seeking process. Both mothers' financial autonomy and fathers financial contributions are important to enable timely access to effective healthcare for children with malaria.
Antagonistic coevolution between bacteria and their viral parasites, phage, drives continual evolution of resistance and infectivity traits through recurrent cycles of adaptation and counter-adaptation. Both partners are vulnerable to extinction through failure of adaptation. Environmental conditions may impose unequal abiotic selection pressures on each partner, destabilising the coevolutionary relationship and increasing the extinction risk of one partner. In this study we explore how the degree of population mixing and resource supply affect coevolution-induced extinction risk by coevolving replicate populations of Pseudomonas fluorescens SBW25 with its associated lytic phage SBW25Ф2 under four treatment regimens incorporating low and high resource availability with mixed or static growth conditions.
We observed an increased risk of phage extinction under population mixing, and in low resource conditions. High levels of evolved bacterial resistance promoted phage extinction at low resources under both mixed and static conditions, whereas phage populations could survive when phage susceptible bacterial genotypes rose to high frequency.
These findings demonstrate that phage extinction risk is influenced by multiple abiotic conditions, which together act to destabilise the bacteria-phage coevolutionary relationship. The risk of coevolution-induced extinction is therefore dependent on the ecological context.
Although a number of natural materials have been used as hemostatic agents, many substances do not act quickly enough. Here, we created a novel dressings using collagen and chitosan with recombinant batroxobin (r-Bat) to promote faster and more effective hemostasis. We hypothesized that r-Bat would promote synergetic blood coagulation because it contains a blood coagulation active site different than those of collagen and chitosan. Our results suggest that each substances can maintain hemostatic properties while in the mixed dressings and that our novel hemostatic dressings promotes potent control of bleeding, as demonstrated by a whole blood assay and rat hemorrhage model. In a rat femoral artery model, the scaffold with a high r-Bat concentration more rapidly controlled excessive bleeding. This novel dressings has enormous possible for rapidly controlling bleeding and it improves upon the effect of collagen and chitosan used alone. Our novel r-Bat dressings is a possible candidate for improving preoperative care and displays promising properties as an absorbable agent in hemostasis.
Despite the excellent hemostatic properties of collagen and chitosan pads, they reported to brittle behavior and lack sufficient hemostatic effect within relevant time. Therefore, we created a novel pad using collagen and chitosan with recombinant batroxobin (r-Bat). r-Bat acts as a thrombin-like enzyme in the coagulation cascade. Specifically, r-Bat, in contrast to thrombin, only splits fibrinopeptide A off and does not influence other hemostatic factors or cells, which makes it clinically useful as a stable hemostatic agent. Also the materials in the pad have synergetic effect because they have different hemostatic mechanisms in the coagulation cascade. This report propose the novel hemostatic pad isreasonable that a great potential for excessive bleeding injury and improve effects of natural substance hemostatic pad.
Parasporins are novel protein toxins preferentially cytotoxic against human cancer cells. They are obtained from parasporal inclusions of Bacillus thuringiensis and, accordingly, are considered congeners of the insecticidal Cry toxins. Two types of parasporins have been identified: the three-domain Cry toxin type and the β-pore-forming-toxin (β-PFT) type. Crystal structures of representative members of the two types, PS1Aa1 and PS2Aa1, have been determined and compared with those of well-studied toxins. PS1Aa1 has a typical architecture characteristic of the three-domain insecticidal Cry toxins, though it is cleaved into two polypeptides. It has an extra N-terminal segment found only in the inactive form of the Cry toxins and, hence, it is presumed to act through another mechanism as an activator in the apoptotic signaling pathway rather than a pore-forming toxin. PS2Aa1 shows a remarkable structural similarity to the aerolysin-type β-PFTs, which is much greater than expected from its limited sequence identity to those toxins. This strongly suggests that a pore-forming mechanism similar to that of β-PFTs is involved in the action of this type of parasporin. The structural comparison of PS2Aa1 to other aerolysin-type β-PFTs indicates conserved oligomerization and pore-forming structures in domains 2 and 3, and highly diverse putative receptor binding region structures in domain 1, likely accounting for enhanced cancer cell cytotoxicity as compared to normal control cells. The structural implications for the mechanism of action and cellular specificity of both Cry and β-PFT type parasporins will be enhanced by further experimental validation.
The polyphenol resveratrol (RSV) is found in the skin of red grapes and has been reported to exhibit anticancer properties. The antitumor effects of RSV in the gastrointestinal tract have gained considerable interest due to the high exposure of this tissue to this dietary compound. One of the hallmarks of cancer cells is their particular metabolism mainly relying on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. Although RSV has been described to act as a calorie-restriction mimetic, modulating energy metabolism in normal tissues, little efforts have been done to study the effects of this polyphenol in the metabolism of cancer cells. Taking this into account, the aim of this study was to explore metabolic effects of this polyphenol in colon cancer.
Oxygen consumption, ATP levels, Western blotting and other molecular biology techniques were carried out to characterize the metabolic signature of RSV in SW620 colon cancer cells.
Paradoxically, the cytotoxic effects of RSV were associated with an increase in oxygen consumption supported by mitochondrial biogenesis and increased fatty acid oxidation. This partial reversion of the Warburg effect was followed by hyperpolarization of mitochondrial membrane and ROS production, leading to an increased apoptosis.
Our results propose that the anticancer mechanisms of RSV could reside in targeting cancer cell metabolism, promoting mitochondrial electron transport chain overload and, ultimately, increasing ROS production.
These results shed new light into the anticancer mechanism of RSV supporting the ability of this compound in potentiating the effects of chemotherapy.
Venoms from various predatory species, such as fish hunting molluscs scorpions, snakes and arachnids contain a large spectrum of toxins that include blockers of voltage-gated calcium channels. These peptide blockers act by two principal manners - physical occlusion of the pore and prevention of activation gating. Many of the calcium channel-blocking peptides have evolved to tightly occupy their binding pocket on the principal pore forming subunit of the channel, often rendering block poorly reversible. Moreover, several of the best characterized blocking peptides have developed a high degree of channel subtype selectivity. Here we give an overview of different types of calcium channel-blocking toxins, their mechanism of action, channel subtype specificity, and potential use as therapeutic agents.
Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous myeloid cells that can suppress antitumor immunity. MDSCs are divided into granulocytic (G‑MDSCs) and monocytic subsets. In the present study, the microRNA profiles of the G‑MDSCs were determined and the differential expression of microRNAs between G‑MDSCs from tumor‑bearing mice and tumor‑free mice was examined. The number of G‑MDSCs in spleens of Lewis lung carcinoma (LLC)‑bearing mice was ~6‑fold higher than in spleens of normal mice (13.54±1.74% vs. 2.14±1.44%; P<0.01) and G‑MDSCs account for about 72.9% of all MDSCs. The microRNA (miRNA) profiles of the G‑MDSCs from spleen of LLC‑bearing mice were obtained using a microRNA microarray and compared with their counterparts from spleens of tumor‑free mice. A total of 43 miRNAs with >1.3‑fold increased or decreased change were differentially expressed between the experimental and control group mice. The levels of nine of these differentially expressed miRNAs, miRNA‑468 (miR‑486), miR‑192, miR‑128, miR‑125a, miR‑149, miR‑27a, miR‑125b, miR‑350 and miR‑328, were also analyzed by RT‑qPCR to validate the microarray data. The concordance rate between the results tested by the two methods was 88.9%. Bioinformatics analyses revealed that these miRNAs may act on various target genes, including Adar, Pik3r1, Rybp and Rabgap1, to regulate the survival, differentiation and the function of tumor‑induced granulocytic MDSCs. The results revealed microRNAs and potential targets that may be vital for regulating survival, differentiation and function of G‑MDSCs induced by LLC. Further investigation should be performed to clarify the roles of these microRNAs in regulating LLC‑induced granulocytic MDSCs and the target genes that mediate their functions.
Protocadherin genes (PCDHs) have been suggested to act as tumor suppressor genes in various tumor types. Previous studies have demonstrated the upregulation of certain PCDH‑γ subfamily genes in nodal and duodenal follicular lymphoma (FL) using gene expression analyses. However, the mechanisms and associated molecular function of PCDH‑γ subfamily gene upregulation in FL remain to be elucidated. The present study examined the expression of PCDHGA3, an upregulated PCDH‑γ gene subfamily member, in B‑cell lymphoma 2 (BCL2)‑positive and ‑negative FL, and evaluated its association with tumor cell proliferation in an FL‑derived cell line. Immunohistochemical analysis demonstrated that the majority of FL grade 1‑2 samples (19/20; 95%) and over half of grade 3A FL samples (5/9; 56%) were PCDHGA3‑positive, whereas only 1/17 reactive lymphoid hyperplasia samples was positive. Notably, this positivity was widely observed in samples of BCL2‑negative FL (13/15; 87%) and FL with diffuse area (10/10; 100%). The FL‑derived cell line FL18 exhibited strong PCDHGA3 expression, similar to the patient samples, and its proliferation was suppressed by PCDHGA3 gene knockdown. Genes expressed concomitantly with PCDHGA3 were selected from gene expression data, and TNFRSF6B, a member of the tumor necrosis factor receptor superfamily, was among the top five most strongly correlated genes. Coexpression of TNFRSF6B and PCDHGA3 was observed immunohistochemically in FL18 cells, suggesting potential cooperation in tumor cell maintenance. In conclusion, the results of the present study indicated that PCDHGA3 was expressed in FL irrespective of BCL2 status and grading and was associated with cell proliferation. Further studies involving molecular genetic analyses are required to elucidate the mechanisms underlying the activity of PCDHGA3 in FL.
Genome sequencing of large cohorts of tumors has revealed that mutations in genes encoding chromatin regulators are frequent in cancer. However, the precise contribution of these mutations to tumor development often remains elusive. Here, we review the current knowledge concerning the alterations of the Polycomb machinery in cancer, with a particular focus on the Polycomb repressive complex 2 (PRC2), a key chromatin modifier involved in the maintenance of transcriptional silencing. A broad variety of alterations can impair PRC2 activity; yet, overall, only one type of alteration is found in a given class of tumor. We discuss the potential impact of the various types of PRC2 alterations on gene expression. We propose that the distinct set of genes regulated by PRC2, depending on tumor etiology, constrain the type of alteration of PRC2 that can fuel tumor development. Beyond this specificity, we propose that PRC2 and, more generally, chromatin regulators act as gatekeepers of transcriptional integrity, a role that often confers a tumor-suppressive function.
We previously reported that tetrodotoxin (TTX) perfusion into the median raphe nucleus (MRN), which contains the cell bodies of serotonin (5-HT) neurons, induced a considerable body temperature reduction under normal and low ambient temperatures (23 and 5°C, respectively) in freely moving rats but showed no such effect under high ambient temperature (35°C). In the present study, we aimed to determine the mechanism(s) of body temperature reduction after TTX perfusion into the MRN by measuring tail skin temperature (an index of heat loss), heart rate (an index of heat production), and locomotor activity (Act) under normal ambient temperature (23°C). We performed similar experiments in the dorsal raphe nucleus (DRN), another area containing cell bodies of 5-HT neurons, to compare any functional differences with the MRN. TTX perfusion into the MRN or DRN induced significant hypothermia (from 37.4±0.2 to 33.7±0.4°C or from 37.4±0.1 to 34.5±0.4°C, respectively; P<0.001) with increased tail skin temperature (from 26.1±0.8 to 31.1±1.3°C or from 26.3±0.9 to 31.7±0.4°C, respectively; P<0.001), but no change in heart rate. However, TTX perfusion into the MRN or DRN differentially affected Act. TTX perfusion into the MRN induced hyperactivity (from 10.7±4.6 to 67.6±25.1 counts/min; P<0.01), whereas perfusion into the DRN induced immobility. Thus, the 5-HT projections from the MRN and DRN may play similar roles in thermoregulation, both in the heat production system and in the heat loss system, but their roles in the regulation of Act might be distinct and opposite.
Magnetic Resonance Imaging (MRI) is a noninvasive radiology technique used to examine the internal organs of human body. It is useful for the diagnosis of structural abnormalities in the body. Contrast agents are used to increase the sensitivity of this technique. 1,4,7,10-Tetraazacyclododecane (cyclen) is a macrocyclic tetraamine. Its derivatives act as useful ligands to produce stable complexes with Gd(3+) ion. Such chelates are investigated as MRI contrast agents. Free Gd(3+) ion is extremely toxic for in vivo use. Upon complexation with a cyclen-based ligand, it is trapped in the preformed central cavity of the ligand resulting in the formation of a highly stable Gd(3+)-chelate. Better kinetic and thermodynamic stability of cyclen-based MRI contrast agents decrease their potential toxicity for in vivo use. Consequently, such agents have proved to be safest for clinical applications. Relaxivity is the most important parameter used to measure the effectiveness of a contrast agent. A number of factors influence this parameter. This article elucidates detailed strategies to increase relaxivity of cyclen-based MRI contrast agents. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) are two key ligands derived from cyclen. They also act as building blocks for the synthesis of novel ligands. A few important methodologies for the synthesis of DOTA and DO3A derivatives are described. Moreover, the coordination geometry of chelates formed by these ligands and their derivatives is discussed as well. Novel ligands can be developed by the appropriate derivatization of DOTA and DO3A. Gd(3+)-chelates of such ligands prove to be useful MRI contrast agents of enhanced relaxivity, greater stability, better clearance, lesser toxicity and higher water solubility.
Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. Here, we show by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 act together in the lung mesenchyme to maintain branching morphogenesis. Expression of both genes depends on epithelially derived Shh signaling, with additional modulation by Bmp, Wnt, and Tgfβ signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with our previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b, we conclude that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.
Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various clinical applications, molecular mechanisms involved are still poorly understood. The aim of the study was to investigate the ability of laser stimulation to increase cell proliferation through an early activation of three redox sensitive pathways, namely Nrf-2, NF-κB and ERK in a rat odontoblast-like cell line (MDPC-23 cells). MDPC-23 cells were irradiated with different energy settings (0-50J, corresponding to 0-32.47J/cm(2)) and cell proliferation was evaluated by cell counting. Nrf-2, NF-κB and ERK signaling pathways activation was investigated through Western blot analysis. Our results show that a single 25J laser stimulation is able to increase cell proliferation and that this effect could be increased by repeating the stimulation twice with a time lapse of 24h. Western blot experiments demonstrated that laser stimulation is able to induce an early activation response in intracellular signaling, with an overlapping time pattern between the three considered pathways. Results discussed in this paper reveal a complex mechanism underlying near-infrared induced increase in pre-odontoblasts proliferation, involving three survival pathways that can act both separately or through reciprocal crosstalk. In particular, data presented suggest an important role for ERK pathway that could act directly by stimulating cell proliferation but can also induce both Nrf-2 and NF-κB activation, acting as a critical cellular checkpoint in response to imbalanced redox state generated by a laser induced increase in ROS production.
In the present study, the interaction of model transport proteins Human serum albumin (HSA) and Bovine serum albumin (BSA) with a photoactive dye, Azure B (AZB) were studied by spectroscopic and in silico methods. The absorption spectral behavior of AZB in the presence of varying concentrations of serum albumins (HSA and BSA) revealed the formation of dye aggregates within the protein cavity. The binding parameters computed from the emission quenching data showed that AZB bind to HSA and BSA with significant affinity and it was revealed that both the serum proteins (HSA and BSA) can bind AZB at more than one binding sites having at least one high-affinity binding site with different affinities (non-independent). The existence of static quenching mechanism was further evidenced from the time-resolved fluorescence spectroscopic analysis. Site-competitive replacement experiments with specific site markers showed that AZB binds to site I of HSA and BSA. AutoDock based blind docking approach and molecular dynamics simulation studies were used to analyze the most probable binding location of AZB in HSA and BSA. The AZB induced unfolding of HSA and BSA was established by using absorption, circular dichroism and FT-IR spectral studies. The influence of AZB complexation on the biological function of HSA and BSA was evaluated by probing the hydrolysis of p-nitrophenyl acetate.
The estrogen receptor beta (ERβ) selective agonist is considered a promising candidate for the treatment of estrogen deficiency symptoms in ERβ-expressing tissues, without the risk of breast cancer, and multiple classes of compounds have been reported as ERβ selective agonists. Among them, 6-6 bicyclic ring-containing structures (e.g., isoflavone phytoestrogens) are regarded as one of the cyclized analogues of isobutestrol 5b, and suggest that other cyclized scaffolds comprising 5-6 bicyclic rings could also act as selective ERβ ligands. In this study, we evaluated the selective ERβ agonistic activity of 1-(4-hydroxybenzyl)indan-5-ol 7a and studied structure-activity relationship (SAR) of its derivatives. Some functional groups improved the properties of 7a; introduction of a nitrile group on the indane-1-position resulted in higher selectivity for ERβ (12a), and further substitution with a fluoro or a methyl group to the pendant phenyl ring was also preferable (12b, d, and e). Subsequent chiral resolution of 12a identified that R-12a has a superior profile over S-12a. This is comparable to diarylpropionitrile (DPN) 5c, one of the promising selective ERβ agonists and indicates that this indane-based scaffold has the potential to provide better ERβ agonistic probes.
Understanding language requires more than the use of fixed conventions and more than decoding combinatorial structure. Instead, comprehenders make exquisitely sensitive inferences about what utterances mean given their knowledge of the speaker, language, and context. Building on developments in game theory and probabilistic modeling, we describe the rational speech act (RSA) framework for pragmatic reasoning. RSA models provide a principled way to formalize inferences about meaning in context; they have been used to make successful quantitative predictions about human behavior in a variety of different tasks and situations, and they explain why complex phenomena, such as hyperbole and vagueness, occur. More generally, they provide a computational framework for integrating linguistic structure, world knowledge, and context in pragmatic language understanding.
The heterometallic complexes (NH4 )2 [Co(H2 O)6 ]2 [V10 O28 ]⋅4 H2 O (1) and (NH4 )2 [Co(H2 O)5 (β-HAla)]2 [V10 O28 ]⋅4 H2 O (2) have been synthesized and used for the preparation of mixed oxides as catalysts for water oxidation. Thermal decomposition of 1 and 2 at relatively low temperatures (<500 °C) leads to the formation of the solid mixed oxides CoV2 O6 /V2 O5 (3) and Co2 V2 O7 /V2 O5 (4). The complexes (1, 2) and heterogeneous materials (3, 4) act as catalysts for photoinduced water oxidation. A modification of the thermal decomposition procedure allowed the deposition of mixed metal oxides (MMO) on a mesoporous TiO2 film. The electrodes containing Co/V MMOs in TiO2 films were used for electrocatalytic water oxidation and showed good stability and sustained anodic currents of about 5 mA cm(-2) at 1.72 V versus relative hydrogen electrode (RHE). This method of functionalizing TiO2 films with MMOs at relatively low temperatures (<500 °C) can be used to produce other oxides with different functionality for applications in, for example, artificial photosynthesis.
Structural maintenance of chromosome 1 alpha (SMC1A) gene has been reported to be related to tumor development in some types of human cancers. However, the misregulation of SMC1A and its functions in castration-resistant prostate cancer (CRPC) have not been well understood. In the present study, we found that SMC1A was elevated in androgen-independent PCa cell lines PC-3 and DU-145 compared to androgen sensitive LNCap and 22RV1 cells by qPCR and western blot assay. Knockdown of SMC1A inhibited cell growth, colony formation and cell migration abilities of PC-3 and DU145 cells by MTT, colony formation and transwell assays, and affected cell cycle progression in PC-3 and DU145 cells by flow cytometry. Moreover, SMC1A knockdown significantly reduced tumor growth in vivo in a nude mouse model. Additionally, we also found that the expression of SMC1A gene was higher in prostate cancer tissues than in the adjacent normal tissues by immunohistochemical staining, and was positively correlated to tumor metastasis and recurrence by Oncomine database mining. Taken together, the present study indicates that SMC1A may play an important role in malignant transformation of PCa under conditions of androgen deprivation and act as a new target for PCa diagnosis and treatment.
Melatonin is synthesized by the pineal gland and is released into the blood. In the last several years, some studies have shown that melatonin has anticancer properties; however, the mechanisms behind the antitumour traits are unclear, especially in pancreatic cancer. Therefore, in the present study, we investigated the antitumour effects of melatonin on the human pancreatic carcinoma cell line MIA PaCa‑2 and explored its biological mechanisms. MIA PaCa‑2 cells were treated with melatonin, and we used a CCK‑8 assay to evaluate the cell viability. We also used flow cytometry to observe cell apoptosis and western blot analysis to assess the protein expression. Our study found that melatonin inhibited cell viability, suppressed colony formation and reduced cell migration and invasion and induced cell apoptosis in MIA PaCa‑2 cells. Our results showed that melatonin treatment inhibited NF‑κB p65 activation. Moreover, melatonin treatment activated the mitogen‑activated protein kinase pathways (c‑jun N‑terminal kinase and extracellular‑regulated kinase 1/2), which increased Bax protein expression and caspase‑3 cleavage and decreased Bcl‑2 protein expression. These new developments demonstrate that melatonin plays a potential role in anticancer treatment and may act as an effective therapeutic agent in the future.
Previously, we reported that salicylate-based analogs of bryostatin protect cells from chikungunya virus (CHIKV)-induced cell death. Interestingly, 'capping' the hydroxyl group at C26 of a lead bryostatin analog, a position known to be crucial for binding to and modulation of protein kinase C (PKC), did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a pathway independent of PKC. The work detailed in this study demonstrates that salicylate-derived analog 1 and two capped analogs (2 and 3) are not merely cytoprotective compounds, but act as selective and specific inhibitors of CHIKV replication. Further, a detailed comparative analysis of the effect of the non-capped versus the two capped analogs revealed that compound 1 acts both at early and late stages in the chikungunya virus replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with the PKC inhibitors sotrastaurin and Gö6976 counteracts the antiviral activity of compound 1 without affecting that of capped analogs 2 and 3, providing further evidence that the latter elicit their anti-CHIKV activity independently of PKC. Remarkably, treatment of CHIKV-infected cells with a combination of compound 1 and a capped analog resulted in a pronounced synergistic antiviral effect. Thus, these salicylate-based bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway.
To investigate whether microRNA (miRNA) miR-21 regulates dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression through binding 3'-UTR region directly in human umbilical venous endothelial cells (HUVECs) and to explore whether DDAH1-V2/V3 transcripts can function as microRNA sponge, thereby modulating DDAH1-V1 expression.
The DDAH1 3'-UTR containing miR-21 recognizing sequence was cloned into PmirGLO dual-luciferase miRNA target expression plasmid to construct PmirGLO-miR-21. The plasmid and miR-21 (at concentrations of 25, 50, 100 nM, respectively) or negative control (100 nM) were co-transfected into HUVECs, luciferase activity was detected at 24 h. HUVECs were incubated with 2 μg/ml Actinomycin D for the indicated time after miR-21 (25 nM) transfection, half-lives of DDAH1 mRNA were determined. HUVECs were transfected with PmirGLO-miR-21 alone or co-transfected with miR-21 for 24 h, DDAH1 transcripts mRNA, eNOS activity and DDAH1 protein expression were determined.
MiR-21 decreased luciferase activity of PmirGLO-miR-21 in a dose-dependent manner (P < 0.05 for 25 nM miR-21, P < 0.01 for 50 nM and 100 nM miR-21), and miR-21 inhibitor increased reporter activity of PmirGLO-miR-21 and mRNA expression of all three DDAH1 transcript variants significantly (P < 0.05, respectively). The degree of increase in endogenous DDAH1 mRNA expression by miR-21 inhibitor was more obvious for DDAH1-V3. Overexpression of miR-21 decreased mRNA expression and mRNA half-life time of all DDAH1 transcripts significantly (P < 0.05), and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1 and -V3 with or without miR-21 transfection (P < 0.05, respectively). MiR-21 (100 nM) decreased DDAH1 protein expression and eNOS activity significantly (P < 0.05), which was reversed by PmirGLO-miR-21 transfection (P < 0.05). Transfection of PmirGLO-miR-21 alone increased intracellular miR-21 expression by approximately 5.6-fold, but only showed a trend of increase in DDAH1 protein expression.
Our results confirmed DDAH1 3'-UTR as a target for miR-21, and endogenous miR-21 showed increased inhibitory effect on DDAH1-V3 transcript. DDAH1 3'-UTR, especially for DDAH1-V3, may function as miR-21 sponge to regulate DDAH1 protein expression. Modulation of miR-21-DDAH1 interaction may provide a new approach for tackling cardiovascular diseases.
In an approaching scenario of soil nutrient depletion, root association with soil microorganisms can be key for plant health and sustainability [1-3]. Symbiotic arbuscular mycorrhizal (AM) fungi are major players in helping plants growing under nutrient starvation conditions. They provide plants with minerals like phosphate and, furthermore, act as modulators of plant growth altering the root developmental program [4, 5]. However, the precise mechanisms involved in this latter process are not well understood. Here, we show that AM fungi are able to modulate root cortex development in Medicago truncatula by activating a novel GRAS-domain transcription factor, MIG1, that determines the size of cortical root cells. MIG1 expression peaks in arbuscule-containing cells, suggesting a role in cell remodeling during fungal accommodation. Roots ectopically expressing MIG1 become thicker due to an increase in the number and width of cortical cells. This phenotype is fully counteracted by gibberellin (GA) and phenocopied with a GA biosynthesis inhibitor or by expression of a dominant DELLA (Δ18DELLA1) protein. MIG1 downregulation leads to malformed arbuscules, a phenotype rescued by Δ18DELLA1, suggesting that MIG1 intersects with the GA signaling to control cell morphogenesis through DELLA1. DELLA1 was shown to be a central node controlling arbuscule branching [6-8]. Now we provide evidence that, together with MIG1, DELLA1 is responsible for radial cortical cell expansion during arbuscule development. Our data point toward DELLA proteins being not only longitudinal root growth repressors [9] but also positive regulators of cortical radial cell expansion, extending the knowledge of how DELLAs control root growth.
Plexins (Plexs) are a large family of phylogenetically conserved guidance receptors that bind specifically to semaphorins (Semas), another large family of guidance molecules. In the Drosophila embryonic central nervous system (CNS), the secreted semaphorins Sema-2a and Sema-2b both act as ligands for PlexB, but mediate mutually independent and opposite functions (repulsive and attractive guidance, respectively). PlexB is also known to regulate motor axon guidance in the embryonic peripheral nervous system (PNS). However, it is unclear whether the mechanisms of ligand regulation of PlexB seen in the CNS are similar or the same as those that exist in PNS motor axon guidance. Here, we find that two distinct modes of ligand regulation underlie differential roles of PlexB in PNS motor axon pathfinding during embryonic development. Epistasis analyses in the intersegmental nerve b (ISNb) pathway suggest that PlexB serves as a receptor for both Sema-2a and Sema-2b and integrates their mutually dependent but opposite guidance functions. Furthermore, we present evidence that PlexB mediates not only Sema-2a/2b-dependent guidance functions, but also Sema-2a/2b-independent target recognition in establishing the segmental nerve a (SNa) motor axon pathway. These results demonstrate that a single guidance receptor can elicit diverse effects on the establishment of neuronal connectivity via regulation of its ligands themselves.
Colorectal cancer (CRC), one of the most prevalent malignant cancers, has high rates pf incidence and is the fourth leading cause of cancer-related deaths for both men and women worldwide. MicroRNAs (miRNAs) play critical roles in the development of various types of cancers. miRNA‑330-5p has been implicated in the progression of prostate, neuronal and pancreatic cancers by regulating proliferation, migration, invasion and epithelial-mesenchymal transition of cells. The purpose of the present study was to investigate the expression of miR-330-5p in CRC and identify its target gene(s) that may act in CRC tumorigenesis. We found that miR-330-5p expression was significantly lower in CRC tissues than that in adjacent non-tumorous tissues. Furthermore, we identified integrin α5 (ITGA5) as a new target of miR-330-5p and found that it inhibits ITGA5 expression by directly binding to the 3' untranslated region of ITGA5 mRNA. These results suggest that downregulation of miR-330-5p expression may affect CRC development via modulation of ITGA5 expression.
Curcumin, a natural polyphenolic compound extracted from rhizomes of Curcuma longa (turmeric), a plant in the ginger family (Zingiberaceae) has been used worldwide and extensively in Southeast Asia. Curcumin exhibited numerous biological and pharmacological activities including potent antioxidant, cardiovascular disease, anticancer, anti-inflammatory effects and neurodegenerative disorders in cell cultures and animal models. Hence, the present study was designed in order to explore the possible neuroprotective role of curcumin against rotenone induced cognitive impairment, oxidative and mitochondrial dysfunction in mice. Chronic administration of rotenone (1mg/kg i.p.) for a period of three weeks significantly impaired cognitive function (actophotometer, rotarod and open field test), oxidative defense (increased lipid peroxidation, nitrite concentration and decreased activity of superoxide dismutase, catalase and reduced glutathione level) and mitochondrial complex (II and III) enzymes activities as compared to normal control group. Three weeks of curcumin (50, 100 and 200mg/kg, p.o.) treatment significantly improved behavioral alterations, oxidative damage and mitochondrial enzyme complex activities as compared to negative control (rotenone treated) group. Curcumin treated mice also mitigated enhanced acetylcholine esterase enzyme level as compared to negative control group. We found that curcumin restored motor deficits and enhanced the activities of antioxidant enzymes suggesting its antioxidant potential in vivo. The findings of the present study conclude neuroprotective role of curcumin against rotenone induced Parkinson's in mice and offer strong justification for the therapeutic prospective of this compound in the management of PD.
Endotoxic shock is the primary cause of morbidity and mortality in hospital patients, creating an urgent need to explore the mechanisms involved in sepsis. Our previous studies showed that T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) attenuated the inflammatory response through regulating the functions of macrophages. However, the mechanism by which Tim-4 does this has not been fully elucidated. In this study, we found that Tim-4 expression was increased in lipopolysaccharide (LPS)-induced endotoxic shock. Interestingly, the survival rate of mice in the Tim-4 overexpression group was higher than that of the control group after LPS administration. To investigate the function of Tim-4 in LPS-induced inflammation, we further demonstrated that Tim-4 attenuated LPS-induced endotoxic shock by inhibiting cytokine production by macrophages. Blocking expression of Tim-4 and nuclear factor-kappa B (NF-κB) signal inhibition showed that Tim-4 inhibited cytokine production via NF-κB signaling pathway. This study indicates that Tim-4 may exert its immune modulation by regulating inflammatory factor secretion and might act as a novel potential target for inflammatory diseases, especially endotoxic shock.
Engineered nanoparticles (NPs) in the environment can act both as contaminants, when they are unintentionally released, and as remediation agents when injected on purpose at contaminated sites. In this work two carbon-based NPs are considered, namely CARBO-IRON®, a new material developed for contaminated site remediation, and single layer graphene oxide (SLGO), a potential contaminant of the next future. Understanding and modeling the transport and deposition of such NPs in aquifer systems is a key aspect in both cases, and numerical models capable to simulate NP transport in groundwater in complex 3D scenarios are necessary. To this aim, this work proposes a modeling approach based on modified advection-dispersion-deposition equations accounting for the coupled influence of flow velocity and ionic strength on particle transport. A new modeling tool (MNM3D - Micro and Nanoparticle transport Model in 3D geometries) is presented for the simulation of NPs injection and transport in 3D scenarios. MNM3D is the result of the integration of the numerical code MNMs (Micro and Nanoparticle transport, filtration and clogging Model - Suite) in the well-known transport model RT3D (Clement et al., 1998). The injection in field-like conditions of CARBO-IRON® (20g/l) amended by CMC (4g/l) in a 2D vertical tank (0.7×1.0×0.12m) was simulated using MNM3D, and compared to experimental results under the same conditions. Column transport tests of SLGO at a concentration (10mg/l) representative of a possible spill of SLGO-containing waste water were performed at different values of ionic strength (0.1 to 35mM), evidencing a strong dependence of SLGO transport on IS, and a reversible blocking deposition. The experimental data were fitted using the numerical code MNMs and the ionic strength-dependent transport was up-scaled for a full scale 3D simulation of SLGO release and long-term transport in a heterogeneous aquifer. MNM3D showed to potentially represent a valid tool for the prediction of the long-term behavior of engineered nanoparticles released in the environment (e.g. from landfills), and the preliminary design of in situ aquifer remediation through injection of suspensions of reactive NPs.
Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.
Patients with Jervell and Lange-Nielsen syndrome (JLNS) exhibit severe phenotypes that are characterized by congenital deafness, very long QT intervals, and high risk of life-threatening arrhythmias. Current treatment strategies include high doses of beta-blocker medication, left cardiac sympathetic denervation, and ICD placement, which is challenging in young children.
The purpose of this study was to evaluate the safety and effect of pacing in addition to beta-blocker treatment in children with JLNS.
All genetically confirmed patients with JLNS born since 1999 in Norway were included in the study. Data on history of long QT syndrome-related symptoms, QT interval, and beta-blocker and pacemaker treatment were recorded.
A total of 9 patients with QT intervals ranging from 510 to 660 ms were identified. Eight patients developed long QT syndrome-related symptoms, and 1 patient died before diagnosis. The survivors received beta-blocker medication. Seven patients also received a pacemaker; 1 had a ventricular lead and 6 had atrial leads. The patient with the ventricular lead died during follow-up. The 6 patients with atrial leads survived without events at a mean follow-up of 6.9 years after pacemaker implantation. Two patients received prophylactic upgrade to a 2-chamber ICD.
No arrhythmic events occurred in 6 very young JLNS patients who received atrial pacing in combination with increased doses of beta-blockers during 7-year follow-up. If confirmed in additional patients, this treatment strategy may prevent life-threatening arrhythmias in this high-risk patient group and may act as a bridge to insertion of a 2-chamber ICD when left cardiac sympathetic denervation is not available.
Pathogen emergence can drive major changes in host population demography, with implications for population dynamics and sensitivity to environmental fluctuations. The amphibian disease chytridiomycosis, caused by infection with the fungal pathogen Batrachochytrium dendrobatidis (Bd), is implicated in the severe decline of over 200 amphibian species. In species that have declined but not become extinct, Bd persists and can cause substantial ongoing mortality. High rates of mortality associated with Bd may drive major changes in host demography, but this process is poorly understood. Here, we compared population age structure of Bd-infected populations, Bd-free populations and museum specimens collected prior to Bd emergence for the endangered Australian frog, Litoria verreauxii alpina (alpine tree frog). We then used population simulations to investigate how pathogen-associated demographic shifts affect the ability of populations to persist in stochastic environments. We found that Bd-infected populations have a severely truncated age structure associated with very high rates of annual adult mortality. Near-complete annual adult turnover in Bd-infected populations means that individuals breed once, compared with Bd-free populations where adults may breed across multiple years. Our simulations showed that truncated age structure erodes the capacity of populations to withstand periodic recruitment failure; a common challenge for species reproducing in uncertain environments. We document previously undescribed demographic shifts associated with a globally emerging pathogen and demonstrate how these shifts alter host ecology. Truncation of age structure associated with Bd effectively reduces host niche width and can help explain the contraction of L. v. alpina to perennial waterbodies where the risk of drought-induced recruitment failure is low. Reduced capacity to tolerate other sources of mortality may explain variation in decline severity among other chytridiomycosis-challenged species and highlights the potential to mitigate disease impacts through minimizing other sources of mortality.
The microglia, once thought only to be supporting cells of the central nervous system (CNS), are now recognized to play essential roles in many pathologies. Many studies within the last decades indicated that the neuro-immune interaction underlies the generation and maintenance of neuropathic pain. Through a large number of receptors and signaling pathways, the microglial cells communicate with neurons, astrocytes and other cells, including those of the immune system. A disturbance or loss of CNS homeostasis causes rapid responses of the microglia, which undergo a multistage activation process. The activated microglia change their cell shapes and gene expression profiles, which induce proliferation, migration, and the production of pro- or antinociceptive factors. The cells release a large number of mediators that can act in a manner detrimental or beneficial to the surrounding cells and can indirectly alter the nociceptive signals. This review discusses the most important microglial intracellular signaling cascades (MAPKs, NF-kB, JAK/STAT, PI3K/Akt) that are essential for neuropathic pain development and maintenance. Our objective was to identify new molecular targets that may result in the development of powerful tools to control the signaling associated with neuropathic pain.
The FK506-binding protein 14 (FKBP14) is a subfamily of immunophilins, has been implicated in various biochemical processes. However, its effects on the primary malignant bone tumor, osteosarcoma, are unclear. Here, we reported that FKBP14 may be an oncogene as it overexpressed in osteosarcoma tissues and cell lines, and FKBP14 expression was correlated with metastases, recurrence, tumor maximum diameter and poor survival time. FKBP14 was associated with the biological pathways including cell cycle, apoptosis and metastasis. Furthermore, we detected FKBP14 knockdown induced cell cycle arrest, apoptosis, invasion and adhesion in vitro. FKBP14 knockdown decreased the protein levels of PCNA, CDK1 and CCNB1 that promotes cell cycle, increased Bax, caspase-3 and caspase-7 protein involved in promoting cell apoptosis, and increased KIF4A expression as well as decreased SMC4 and TMEM33 proteins that contribute to cell invasion and adhesion. In addition, FKBP14 knockdown also caused a significant inhibition in tumor growth in vivo. Then, we found that the protein RhoA was identified as a binding partner of FKBP14. Taken together, FKBP14 may act as an oncogene in osteosarcoma via suppressing apoptosis and promoting invasion and adhesion in osteosarcoma carcinogenesis. FKBP14 may be a prognostic factor and potential target for osteosarcoma treatment.
Concurrent chemoradiation therapy (CCRT) is the predominant treatment in esophageal cancer, however resistance to therapy and tumor recurrence are exceedingly common. Elevated ERBB2/Her2 may be at least partially responsible for both the high rates of recurrence and resistance to CCRT. This receptor tyrosine kinase is upregulated in 10-20% of esophageal squamous cell carcinoma (ESCC) tissues, and amplification of ERBB2 has been correlated with poor prognosis in esophageal cancer. Tissues from 131 ESCC patients, along with cell and animal models of the disease were used to probe the underlying mechanisms by which ERBB2 upregulation occurs and causes negative outcomes in ESCC. We found that overexpression of ERBB2 inhibited radiosensitivity in vitro. Furthermore, miR-193a-5p reduced ERBB2 expression by directly targeting the 3'UTR. Increased miR-193a-5p enhanced radiosensitivity and inhibited tumorigenesis in vitro and in vivo. Additionally, low miR-193a-5p expression correlated with poor prognosis in ESCC patients, and ESCC patients with good CCRT response exhibited higher miR-193a-5p expression. Our data suggest that patients with high miR-193a-5p will likely benefit from CCRT treatment alone, however a combination of CCRT with Herceptin may be beneficial for patients with low miR-193a-5p expression.
Vascular mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novo vascular networks, which promoted tumor metastasis. The aim of this study was evaluate the impact of VM on recurrence-free survival (RFS) in urothelial carcinoma of the bladder (UCB). Records from 202 patients treated with radical cystectomy (RC) for UCB at Zhongshan Hospital between 2002 and 2014 were reviewed. The presence of VM was identified by CD31-PAS double staining. Positive VM staining occurred in 19.3% (39 of 202) UCB cases, and it was associated with increased risks of recurrence (Log-Rank p<0.001). VM was identified as an independent prognostic factor (p=0.002). In the cohort with MIBC, patients with VM negative got CSS benefit from the use of ACT (p = 0.048). As for lung metastasis, the combination of VM and TNM stage (AUC 0.792) showed a better prognostic value than TNM stage alone (AUC 0.748, p = 0.008) or VM alone (AUC 0.714, p = 0.023). Vascular mimicry could be a potential prognosticator for recurrence-free survival in patients with UCB after RC. Vascular mimicry seems to predict risk of developing lung metastases after RC. The presence of VM identified a subgroup of patients with MIBC who appeared to benefit from adjuvant chemotherapy.
Ginseng is one of the most widely used natural medicines in the world. Recent studies have suggested Panax ginseng has a wide range of beneficial effects on aging, central nervous system disorders, and neurodegenerative diseases. However, knowledge about the specific bioactive components of ginseng is still limited. This work aimed to screen for the bioactive components in Panax ginseng that act against neurodegenerative diseases, using the target cell-based bioactivity screening method. Firstly, component analysis of Panax ginseng extracts was performed by UPLC-QTOF-MS, and a total of 54 compounds in white ginseng were characterized and identified according to the retention behaviors, accurate MW, MS characteristics, parent nucleus, aglycones, side chains, and literature data. Then target cell-based bioactivity screening method was developed to predict the candidate compounds in ginseng with SH-SY5Y cells. Four ginsenosides, Rg2, Rh1, Ro, and Rd, were observed to be active. The target cell-based bioactivity screening method coupled with UPLC-QTOF-MS technique has suitable sensitivity and it can be used as a screening tool for low content bioactive constituents in natural products.
The expression of estrogen receptor alpha (ERα) varies across brain regions and changes with age and according to the previous history of estradiol exposure. ERα is regulated by a number of mechanisms including the level of mRNA (Esr1) expression. For this study, we took advantage of regional differences in hippocampal ERα expression to investigate DNA ERα promoter methylation at CpG dinucleotide sites as a potential epigenetic mechanism for regulating gene expression. Young and aged female Fischer 344 rats were ovariectomized, and Esr1 expression and ERα promoter methylation were examined in hippocampal regions CA1 and CA3, either 3 or 14 weeks following surgery. The results indicate that reduced Esr1 expression in region CA1 relative to CA3 was associated with an increase in DNA methylation in region CA1, particularly for the first CpG site. Additionally, differential methylation of distal CpG sites, 11-17, was associated with altered Esr1 expression during aging or following long-term hormone deprivation. The results support the idea that methylation of site 1 may be the primary regulatory region for cross-regional patterns in ERα expression, while distal sites are modifiable across the life span and may act as a feedback mechanism for ERα activity.
While some evidence supports the beneficial effects of integrating neglected tropical disease (NTD) programs to optimize coverage and reduce costs, there is minimal information regarding when or how to effectively operationalize program integration. The lack of systematic analyses of integration experiences and of integration processes may act as an impediment to achieving more effective NTD programming. We aimed to learn about the experiences of NTD stakeholders and their perceptions of integration.
We evaluated differences in the definitions, roles, perceived effectiveness, and implementation experiences of integrated NTD programs among a variety of NTD stakeholder groups, including multilateral organizations, funding partners, implementation partners, national Ministry of Health (MOH) teams, district MOH teams, volunteer rural health workers, and community members participating in NTD campaigns. Semi-structured key informant interviews were conducted. Coding of themes involved a mix of applying in-vivo open coding and a priori thematic coding from a start list.
In total, 41 interviews were conducted. Salient themes varied by stakeholder, however dominant themes on integration included: significant variations in definitions, differential effectiveness of specific integrated NTD activities, community member perceptions of NTD programs, the influence of funders, perceived facilitators, perceived barriers, and the effects of integration on health system strength. In general, stakeholder groups provided unique perspectives, rather than contrarian points of view, on the same topics. The stakeholders identified more advantages to integration than disadvantages, however there are a number of both unique facilitators and challenges to integration from the perspective of each stakeholder group.
Qualitative data suggest several structural, process, and technical opportunities that could be addressed to promote more effective and efficient integrated NTD elimination programs. We highlight a set of ten recommendations that may address stakeholder concerns and perceptions regarding these key opportunities. For example, public health stakeholders should embrace a broader perspective of community-based health needs, including and beyond NTDs, and available platforms for addressing those needs.
The Affordable Care Act intended to "extend affordable coverage" and "ensure access" for vulnerable patient populations. This investigation examined whether the type of insurance (Medicaid, Medicare, Blue Cross, cash pay) carried by trauma patients influences access to pain management specialty care.
Investigators phoned 443 board-certified pain specialists, securing office visits with 235 pain physicians from 8 different states. Appointments for pain management were for a patient who sustained an ankle fracture requiring surgery and experiencing difficulty weaning off opioids. Offices were phoned 4 times assessing responses to the 4 different payment methodologies.
Fifty-three percent of pain specialists contacted (235 of 443) were willing to see new patients to manage pain medication. Within the 53% of positive responses, 7.2% of physicians scheduled appointments for Medicaid patients, compared with 26.8% for cash-paying patients, 39.6% for those with Medicare, and 41.3% with Blue Cross (P < 0.0001). There were no differences in appointment access between states that had expanded Medicaid eligibility for low-income adults versus states that had not expanded Medicaid eligibility. Neither Medicaid nor Medicare reimbursement levels for new patient visits correlated with ability to schedule an appointment or influenced wait times.
Access to pain specialists for management of pain medication in the postoperative trauma patient proved challenging. Despite the Affordable Care Act, Medicaid patients still experienced curtailed access to pain specialists and confronted the highest incidence of barriers to receiving appointments.
The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models.
Cotinine is the major metabolite of nicotine and has displayed some capacity for improving cognition in mouse models following chronic administration. We tested if acute cotinine treatment is capable of improving cognition in the mouse model of Fragile X syndrome, Fmr1(-/-) knockout mice, and if this is related to inhibition by cotinine treatment of glycogen synthase kinase-3β (GSK3β), which is abnormally active in Fmr1(-/-) mice. Acute cotinine treatment increased the inhibitory serine-phosphorylation of GSK3β and the activating phosphorylation of AKT, which can mediate serine-phosphorylation of GSK3β, in both wild-type and Fmr1(-/-) mouse hippocampus. Acute cotinine treatment improved cognitive functions of Fmr1(-/-) mice in coordinate and categorical spatial processing, novel object recognition, and temporal ordering. However, cotinine failed to restore impaired cognition in GSK3β knockin mice, in which a serine9-to-alanine9 mutation blocks the inhibitory serine phosphorylation of GSK3β, causing GSK3β to be hyperactive. These results indicate that acute cotinine treatment effectively repairs impairments of these four cognitive tasks in Fmr1(-/-) mice, and suggest that this cognition-enhancing effect of cotinine is linked to its induction of inhibitory serine-phosphorylation of GSK3. Taken together, these results show that nicotinic receptor agonists can act as cognitive enhancers in a mouse model of Fragile X syndrome and highlight the potential role of inhibiting GSK3β in mediating the beneficial effects of cotinine on memory.
As the medical community awaits the release of final rules to implement the new Medicare payment system under the Medicare Access and CHIP Reauthorization Act (MACRA), physicians can take steps now to prepare for the transition.
One of the goals of the Affordable Care Act aims to provide affordable health insurance through the health insurance exchange marketplace (the Marketplace). This study explores enrollments in the Marketplace in Texas and in rural vs urban areas in the East, South, and West regions of the state. Data are derived from the US Census Bureau and the Department of Health and Human Services. A total of 92.7% of eligible non-elderly adult Texans (NEATs) had enrolled in Marketplace insurance as of February 2015. Rural residents were less likely than urban residents to use the Marketplace. Most enrollees (85%) had received tax credits, and 58.6% had received cost-sharing reductions. The number of uninsured NEATs was reduced by 710,000 by 2014, which is equivalent to two-thirds of the enrollees in the Marketplace. One-third of the enrollees previously had private or employer-based insurance before enrollment into the Marketplace.
Food intake depends on homeostatic and non-homeostatic factors. In order to use grape seed proanthocyanidins (GSPE) as food intake limiting agents, it is important to define the key characteristics of their bioactivity within this complex function. We treated rats with acute and chronic treatments of GSPE at different doses to identify the importance of eating patterns and GSPE dose and the mechanistic aspects of GSPE. GSPE-induced food intake inhibition must be reproduced under non-stressful conditions and with a stable and synchronized feeding pattern. A minimum dose of around 350 mg GSPE/kg body weight (BW) is needed. GSPE components act by activating the Glucagon-like peptide-1 (GLP-1) receptor because their effect is blocked by Exendin 9-39. GSPE in turn acts on the hypothalamic center of food intake control probably because of increased GLP-1 production in the intestine. To conclude, GSPE inhibits food intake through GLP-1 signaling, but it needs to be dosed under optimal conditions to exert this effect.
In the infected human hepatocyte, expression of the hepatitis B virus (HBV) accessory protein X (HBx) is essential to maintain viral replication in vivo. HBx critically interacts with the host damaged DNA binding protein 1 (DDB1) and the associated ubiquitin ligase machinery, suggesting that HBx functions by inducing the degradation of host proteins. To identify such host proteins, we systematically analyzed the HBx interactome. One HBx interacting protein, talin-1 (TLN1), was proteasomally degraded upon HBx expression. Further analysis showed that TLN1 levels indeed modulate HBV transcriptional activity in an HBx-dependent manner. This indicates that HBx-mediated TLN1 degradation is essential and sufficient to stimulate HBV replication. Our data show that TLN1 can act as a viral restriction factor that suppresses HBV replication, and suggest that the HBx relieves this restriction by inducing TLN1 degradation.
This work first reports the preparation of super-amphiphilic silica-nanogel composites to reduce the contact angle of water to increase the diffusion of pollutant into adsorbents. In this respect, the silica nanoparticles were encapsulated into nanogels based on ionic and nonionic polyacrylamides by dispersion polymerization technique. The morphologies and the dispersion stability of nanogel composites were investigated to clarify the ability of silica-nanogel composites to adsorb at different interfaces. The feasibility of silica polyacrylamide nanogel composites to act as a high-performance adsorbent for removal of methylene blue (MB) dye and heavy metals (Co(2+) and Ni(2+)) from aqueous solution was investigated. The surface tension, contact angle, average pore size, and zeta potential of the silica-nanogel composites have been evaluated. The MB dye and heavy metal adsorption capacity achieved Qmax = 438-387 mg/g which is considerably high. The adsorption capacity results are explained from the changes in the morphology of the silica surfaces as recorded from scanning electron microscopy (SEM).
Levels of physical activity (PA) affect health already at four years of age. The aims of this study were to describe levels and patterns of PA and sedentary time (ST) in a sample of four-year-old Swedish children and to assess the number of children achieving PA guidelines throughout the week.
Data from 540 four-year old children enrolled in the population-based PRIMROSE trial was used. PA was measured for a period of one week by the Actigraph GT3X+ accelerometer. Average PA, time spent in light PA, moderate-to-vigorous PA (MVPA) and ST were assessed.
On average children spent 6.7% of the day in MVPA and 45% of the day being sedentary and 33% (n=178) of the children met the PA guidelines of 60 minutes of MVPA per day. Boys spent 56.8 (SD 21.8) minutes/day in MVPA, while girls spent 43.0 (SD 18.1) minutes/day in MVPA (P<0.001).
Four-year-old children spent almost half of the day being sedentary and only one-third meet the recommended PA guidelines. This finding is alarming as higher levels of PA, already at four years of age, seem to reduce the risk of childhood obesity and provides long-term health benefits.
In Latin America, more than 80% of adolescent girls are physically inactive. Inactivity may be reinforced by female stereotypes and objectification in the Latin American sociocultural context.
We examined the influence of objectification on the adoption of an active lifestyle among 192 adolescents (14 -17 y/old) from urban and rural areas in Costa Rica. Analyses of 48 focus-groups sessions were grounded in Objectification Theory.
Vigorous exercises were gender-typed as masculine while girls had to maintain an aesthetic appearance at all times. Adolescents described how girls were anxious around the prospect of being shamed and sexually-objectified during exercises. This contributed to a decrease in girls' desire to engage in physical activities. Among males, there is also a budding tolerance of female participation in vigorous sports, as long as girls maintained a feminine stereotype outside their participation.
Self-objectification influenced Costa Rican adolescent girls' decisions to participate in physical activities. Interventions may include: procuring safe environments for physical activity where girls are protected from fear of ridicule and objectification; sensitizing boys about girl objectification and fostering the adoption of a modern positive masculine and female identities to encourage girls participation in sports.
Limited research has examined park use and physical activity among economically and ethnically diverse families. This study fills that gap.
Parents (n=326) completed a questionnaire about their park use (yes/no, parks visited, reasons for non-use) and physical activity (Godin Leisure Time questionnaire). Geographical Information Systems was used to calculate the distance from respondents' home address to their nearest park. The number of activity features (e.g., playgrounds) in parks was determined objectively using the Community Park Audit Tool.
Half of respondents were sufficiently active; 87.6% reported park use in the prior 6 months. Among sufficiently active respondents, 92.4% reported park use (p=.011). We found no difference in park proximity between respondents who did and did not report distance as a park use barrier. An objective assessment confirmed fewer activity features in parks near the homes of respondents reporting few activity features as a barrier to park use. The most often visited parks were significantly larger than the less-often visited parks.
Parks might best support physial activity for families when activity features are carefully planned and equitably distributed across parks. Efforts to promote families' awareness of park locations might be warranted to reduce perceived proximity barriers.
Many young children are not meeting the Canadian physical activity guidelines. In an effort to change this, the term "active play" has been used to promote increased physical activity levels. Within this young cohort, physical activity is typically achieved in the form of active play behaviour. The current study aimed to review and synthesize the literature to identify key concepts used to define and describe active play among young children. A secondary objective was to explore the various methods adopted for measuring active play.
A systematic review was conducted by searching seven online databases for English-language, original research or reports, and were eligible for inclusion if they defined or measured active play among young children (i.e., 2-6 years).
Nine studies provided a definition or description of active play, six measured active play, and 13 included both outcomes. While variability in active play definitions did exist, common themes included: increased energy exerted, rough and tumble, gross motor movement, unstructured, freely chosen, and fun. Alternatively, many researchers described active play as physical activity (n = 13) and the majority of studies used a questionnaire (n = 16) to assess active play among young children.
Much variability in the types of active play, methods of assessing active play, and locations where active play can transpire were noted in this review. As such, an accepted and consistent definition is necessary, which we provide herein.
The goal of this study was to assess the physical activity (PA) and its determinants of older people living in the three different environments.
Three equal (n=693 each) groups of individuals aged ≥65 years living in urban, rural and institutional environments took part in this study. PA was measured by the Seven-Day Recall PA Questionnaire (energy expenditure - PA-EE) and the Stanford Usual Activity Questionnaire (health-related behaviors - PA-HRB).
PA-EE was highest in the rural environment and lowest in nursing homes. PA-HRB were most common in urban area. Older age, lower education level, several concomitant diseases and the number of systematically used medications were consistently related to lower PA-EE and PA-HRB. Smoking habit, presence of hypertension, musculoskeletal and gastrointestinal disorders had different association to PA-EE and PA-HRB in the three environments.
Sub-populations of older people differ from the general population with regard to their level of PA and its association with sociodemographic data and concomitant diseases. Concomitant serious diseases significantly decrease the level of PA of older subjects. The relationship between PA and non-debilitating disorders may vary depending on the living environment or PA assessment methodology.
Public parks hold promise for promoting population-level PA, but studies show a significant portion of park use is sedentary. Past research has documented the effectiveness of message-based strategies for influencing diverse behaviors in park settings and for increasing PA in non-park contexts. Therefore, to inform message-based interventions (e.g., point-of-decision prompts) to increase park-based PA, the purpose of this study was to elicit insights about key attitudes, perceived norms, and personal agency that affect park use and park-based PA in low-income urban neighborhoods.
This study used six focus groups with youth and adults (n=41) from low-income urban areas in Kansas City, MO, to examine perceptions of key attitudinal outcomes and motivations, perceived norms, key referents, and personal agency facilitators and constraints that affect park use and park-based PA.
Participant attitudes reflected the importance of parks for mental and physical health, with social interaction and solitude cited as key motivations. Of 10 themes regarding perceived norms, influential others reflected participants' ethnic makeup but little consensus emerged among groups. Social and safety themes were cited as both facilitators and constraints, along with park offerings and setting.
Information about attitudes, perceived norms, and personal agency can increase understanding of theoretically-derived factors that influence park-based PA and help park and health professionals create communication strategies to promote PA.
The current study evaluated the feasibility and effectiveness of a diet and physical activity intervention for homeless adults.
Shelter residents (N = 32) were randomly assigned to a 4-week diet and physical activity intervention (n = 17) or an assessment-only control group (n = 15). Intervention participants received tailored educational newsletters, pedometers with step goals, and twice daily fruit/vegetable snacks. Key measures included 24-hour dietary recall interviews and accelerometer-measured moderate to vigorous intensity physical activity (MVPA).
At baseline, 68.8% of participants were overweight or obese, 93.8% reported food insecurity, and 43.8% reported activity levels below physical activity guidelines. Baseline dietary recall interviews indicated low fruit/vegetable consumption, and elevated intake of added sugar, saturated fat, and sodium relative to current dietary recommendations. During the 4-week study period, intervention participants engaged in significantly greater accelerometer-measured daily MVPA (p<.001) than controls (median=60 daily minutes p vs. 41 daily minutes). Between groups differences in fruit/vegetable consumption at the end of treatment did not reach statistical significance. Most participants reported that the intervention was helpful for increasing fruit/vegetable intake and physical activity.
Findings highlight the potential to improve dietary quality and increase physical activity among sheltered homeless adults.
Examine the independent association of sedentary behavior and cognitive function in older adults, as well as whether or not physical activity attenuates this potential association.
Data from the 1999-2002 National Health and Nutrition Examination Survey were used (N=2,472 adults 60-85 yrs). Sedentary behavior was subjectively assessed and the Digit Symbol Substitution Test (DSST) was employed to assess cognitive function.
Among an unadjusted and an adjusted model not accounting for physical activity, only 5+ hrs/day (vs. < 1 hr) of sedentary time was independently associated with lower DSST scores (β = -3.1; 95% CI: -5.8 to -0.4; P=0.02). However, a fully adjusted model (adding in moderate-to-vigorous physical activity as a covariate) did not yield a statistically significant association between 5+ hrs/day of sedentary time and DSST scores (β = -2.5; 95% CI: -5.1-0.2; P=0.07).
Accumulated daily sedentary behavior of 5+ hrs is associated with lower cognitive function in an older adult population when physical activity is not taken into account. However, physical activity may account for 19% of the total association between sedentary behavior and cognitive function, thus attenuating the sedentary-cognitive function association. Efforts should be made to promote physical activity in the aging population.
Evidence has shown that physical activity (PA) is associated with low mortality risk. However, data about reduced mortality due to PA are scarce in developing countries and the dose-response relationship between PA from different domains and all-cause mortality remains unclear. Thus, the aim of this study is to investigate the association of PA from different domains on all-cause mortality among Brazilian adults.
679 males and females composed the study sample. Participants were divided into quartile groups according to PA from different domains (occupational, sports, and leisure-time). Medical records were used to identify the cause of the death. Cox regression analysis was performed to determine the independent associations of PA from different domains and all-cause mortality.
During the follow-up period, 59 participants died. The most prevalent cause of death was circulatory system diseases (n=20; 33.9% [21.8% to 45.9%]). Higher scores of occupational (HR= 0.45 [95% CI: 0.20 to 0.97]), sports (HR= 0.44 [95% CI: 0.20 to 0.95]) and overall PA (HR= 0.40 [95% CI: 0.17 to 0.90]) were associated with lower mortality, even after adjustment for confounders.
The findings in this study showed the importance of being active in different domains to reduce mortality risk.
Proximity to urban green spaces (UGS) and open sports spaces (OSS) benefits health, promotes physical activity (PA) and sports practice (SP).
Analyse the association between PA or SP according to distances between UGS or OSS and teenagers' residences or schools.
We evaluated 1333 (53.9% girls) teenagers (13year-old) living and studying in Porto, Portugal (EPITeen cohort). PA was classified as light or moderate/vigorous. Distances were the shortest routes from residences or schools to UGS/OSS, and classified in: ≤250m; >250m to≤500m; >500m to≤750m; >750m. Chi-square test and chi-square for trends were used to compare proportions; associations were measured using logistic regression, through odds ratio and 95% confidence intervals, adjusting to BMI and parental education.
Regarding vicinity' of schools, the prevalence of moderate/vigorous PA among boys, decreases as distances to OSS increases. For girls, the prevalence of sports decreases as distances to UGS increase. For boys, we found an association between moderate/vigorous PA and proximity to OSS in the vicinity of schools: considering ≤250m as reference, the odds of moderate/vigorous PA is 0.20(0.06-0.63) for >250m to≤500m; 0.21(0.07-0.61) for >500m to≤750m and 0.19(0.06-0.58) for >750m.
Vicinities of schools seem to influence teenagers to be more physical active and increase sports participation.
The aim was to investigate the association between distant green space and physical activity modified by local green space.
Information about physical activity, demographic and socio-economic background at the individual level was extracted from New South Wales Population Health Survey. The proportion of a postcode that was parkland was used as a proxy measure for access to parklands and was calculated for each individual.
There was a significant relationship between distant green space and engaging in moderate to vigorous physical activity (MVPA) at least once a week. No significant relationship was found between adequate physical activity and distant green space. No significant relationships were found between adequate physical activity, engaging in MVPA, and local green space. However, if respondents lived in greater local green space (≥25%), there was a significant relationship between engaging in MVPA at least once a week and distance green space of ≥20%.
This study highlights the important effect of distant green space on physical activity. Our findings also suggest that moderate size of local green space together with moderate size of distant green space are important lever for participation of physical activity.
This quasi-experimental study assessed the effects of new workplace showers on physical activity behaviors in a sample of downtown employees in Austin Texas USA.
The study design was quasi-experimental with two comparison groups. Data were collected via internet-based surveys prior to and four months after shower installation at one worksite. Differences across study groups in the ranks of change in past-week minutes of physical activity from baseline to follow-up were assessed. Adjusted odds ratios and 95% confidence intervals for reporting an increase of ≥10 min past-week physical activity and workday physical activity among those with new showers and existing showers relative to those with no showers were also assessed.
No significant differences in changes in physical activity from baseline to follow-up across study groups were found. One-quarter of participants with new workplace showers and 46.9% of those with existing workplace showers at baseline reported ever using the showers.
This prospective study did not find significant changes in employee physical activity four months after installation of worksite showers. Worksite shower users were highly active at baseline, suggesting a possible early adopter effect, with potential for diffusion. Future studies may benefit from longer exposure times and larger samples.
Background Aim of the study was to test the 12-month effects of a multicomponent physical activity (PA) intervention at schoolyards on recess PA levels of 6th and 7th grade children in primary schools, combining Global Positioning System (GPS) and accelerometry data. Methods A quasi-experimental study design was used with 20 paired intervention and control schools. GPS confirmatory analyses were applied to validate attendance at schoolyards during recess. Data from 376 children from 7 pairs of schools were included in the final analyses. Pooled intervention effectiveness was tested by multilevel linear regression analyses, whereas effectiveness of intervention components was tested by multivariate linear regression analyses. Results Children exposed to the multicomponent intervention increased their time spent in light PA (+5.9%) during recess. No pooled effects on Moderate-to-Vigorous PA were found. In depth-analyses on intervention components showed that physical schoolyard interventions particularly predicted a decrease in time spent in sedentary behavior during recess at follow-up. Intervention intensity and school's commitment to the project strengthened this effect. Conclusions The multicomponent schoolyard PA intervention was effective in making children spend a larger proportion of recess time in light PA, which was most likely the result of a shift from sedentary behavior to light PA.
The association between walking and environmental attributes depends on walking purpose. This study, based on a large survey of U.S. adults, examined the association between perceived neighborhood safety and built environment attributes, and walking for transportation and leisure.
Data were obtained on transportation and leisure-time walking, perceived neighborhood safety and built environment attributes, and demographic characteristics from the summer wave of the 2012 ConsumerStyles survey of 3,951 U.S. adults. Associations were examined by demographic characteristics.
Seventy-five percent of respondents reported walking for either transportation (54%) or leisure (56%) in the past week, 59% reported no safety concern, and 36% reported absence of any built environment attribute of walkability nearby. Respondents with more education, and those who lived in metropolitan areas were more likely to report built environment attributes supportive of walking. All built environment attributes examined, as well as safety concern due to speeding vehicles, were associated with walking after adjustment for demographic characteristics.
Walking, particularly for transportation, is associated with many built environment attributes among U.S. adults. These attributes may be important to consider when designing and modifying the built environment of communities, especially those which are less walkable.
In order to overcome one of the greatest challenges in malaria treatment, drug resistance, new drug candidates are urgently needed, which should preferably act via novel mechanisms. Successful optimization of a phenotypic screening hit based on a quinoline-4-carboxamide derivative resulted in the highly promising lead structure 4, which according to the Medicines for Malaria Venture (MMV) met the efficacy and drug metabolism and pharmacokinetics (DMPK) requirements for a malaria drug target candidate and consequently was selected for preclinical development.
Janus and patchy particles are emerging as models for studying complex directed assembly patterns and as precursors of new structured materials and composites. Here we show how lipid-induced capillary bridging could serve as a new and nonconventional method of assembling patchy particles into ordered structures. Iron oxide surface patches on latex microspheres were selectively wetted with liquid lipid, driving the particle assembly into two- and three-dimensional clusters via interparticle capillary bridge formation. The liquid phase of the bridges allows local reorganization of the particles within the clusters and assists in forming true equilibrium configurations. The temperature-driven fluid-to-gel and gel-to-fluid phase transitions of the fatty acids within the bridge act as a thermal switch for cluster assembly and disassembly. By complementing the experiments with Monte Carlo simulations, we show that the equilibrium cluster morphology is determined by the patch characteristics, namely, their size, number, and shape. This study demonstrates the ability of capillary bridging as a versatile tool to assemble thermoresponsive clusters and aggregates. This method of binding particles is simple, robust, and generic and can be extended further to assemble particles with nonspherical shapes and complex surface chemistries enabling the formation of sophisticated colloidal molecules.
Little is known with certainty about the mechanisms of idiosyncratic drug reactions (IDRs); however, there is substantive evidence that reactive metabolites are involved in most, but not all, IDRs. In addition, evidence also suggests that most IDRs are immune mediated. That raises the question of how reactive metabolites induce an immune response that can lead to an IDR. The dominant hypotheses are the hapten and danger hypotheses. These are complementary hypotheses: a reactive metabolite can act as a hapten to produce neoantigens, and it can also cause cell damage leading to the release of danger-associated molecular pattern molecules that activate antigen presenting cells. Both are required for an immune response. In addition, drugs may induce an immune response through inflammasome activation. We have found examples in which the ability to activate inflammasomes differentiated drugs that cause IDRs from similar drugs that do not. There are other hypotheses that do not involve an immune mechanism such as mitochondrial injury and bile salt export pump (BSEP) inhibition. With some possible exception, these hypotheses are unlikely to be able to completely explain IDRs. However, some types of mitochondrial injury or BSEP inhibition could produce danger signals. The major mechanism that protects us from IDRs appears to be immune tolerance. Consistent with this hypothesis, we used checkpoint inhibition to develop the first animal model of idiosyncratic drug-induced liver injury that has the same characteristics as the idiosyncratic injury in humans. This was accomplished by treating Pd-1-/- mice with anti-CTLA-4 antibodies and amodiaquine. The combination of the Pd-1-/- mouse and anti-CTLA-4 also unmasks the ability of other drugs such as isoniazid to cause delayed type liver injury. This model should allow rigorous testing of mechanistic hypotheses that was impossible in the past.
Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 =12 μm.
Climate shifts at decadal scales can have environmental consequences, and therefore, identifying areas that act as environmental refugia is valuable in understanding future climate variability. Here we illustrate how, given appropriate geohydrology, a rift basin and its catchment can buffer vegetation response to climate signals on decadal time-scales, therefore exerting strong local environmental control. We use time-series data derived from Normalised Difference Vegetation Index (NDVI) residuals that record vegetation vigour, extracted from a decadal span of MODIS images, to demonstrate hydrogeological buffering. While this has been described previously it has never been demonstrated via remote sensing and results in relative stability in vegetation vigour inside the delta, compared to that outside. As such the Delta acts as a regional hydro-refugium. This provides insight, not only to the potential impact of future climate in the region, but also demonstrates why similar basins are attractive to fauna, including our ancestors, in regions like eastern Africa. Although vertebrate evolution operates on time scales longer than decades, the sensitivity of rift wetlands to climate change has been stressed by some authors, and this work demonstrates another example of the unique properties that such basins can afford, given the right hydrological conditions.
Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or 'podocytes', the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes.
In locusts, two lobula giant movement detector neurons (LGMDs) act as looming object detectors. Their reproducible responses to looming and their ethological significance makes them models for single neuron computation. But there is no comprehensive picture of the neurons that connect directly to each LGMD. We used high-through-put serial block-face scanning-electron-microscopy to reconstruct the network of input-synapses onto the LGMDs over spatial scales ranging from single synapses and small circuits, up to dendritic branches and total excitatory input. Reconstructions reveal that many trans-medullary-afferents (TmAs) connect the eye with each LGMD, one TmA per facet per LGMD. But when a TmA synapses with an LGMD it also connects laterally with another TmA. These inter-TmA synapses are always reciprocal. Total excitatory input to the LGMD 1 and 2 comes from 131,000 and 186,000 synapses reaching densities of 3.1 and 2.6 synapses per μm(2) respectively. We explored the computational consequences of reciprocal synapses between each TmA and 6 others from neighbouring columns. Since any lateral interactions between LGMD inputs have always been inhibitory we may assume these reciprocal lateral connections are most likely inhibitory. Such reciprocal inhibitory synapses increased the LGMD's selectivity for looming over passing objects, particularly at the beginning of object approach.
In this paper, a two-layer network is built to simulate the mechanism of visual selection and shifting based on the mapping dynamic model for instantaneous frequency. Unlike the differential equation model using limit cycle to simulate neuron oscillation, we build an instantaneous frequency mapping dynamic model to describe the change of the neuron frequency to avoid the difficulty of generating limit cycle. The activity of the neuron is rebuilt based on the instantaneous frequency and in this work, we use the first layer of neurons to implement image segmentation and the second layer of neurons to act as visual selector. The frequency of the second neuron (central neuron) is always changing, while central neuron resonates with the neurons corresponding to an object, the object is selected, then with the central neuron frequency changing, the selected object loses attention, the process goes on.
MicroRNAs (miRNAs) may act as either tumor suppressors or oncogenes in various types of cancers. Previous studies have indicated that miR-17-5p is involved in the initiation and development of human tumors. However, its mechanism and function in nasopharyngeal carcinoma (NPC) remain largely unclear. In this study, we evaluated the expression profiles of miR-17-5p and p21 in NPC cell lines and tissues by quantitative real-time PCR (qRT-PCR). For the analysis, we have established a stable overexpression or depletion of miR-17-5p NPC cell lines for analyzing the effects of cell proliferation by MTT, colony formation, and cell cycle assay. A nude mice xenograft model was used to verify the tumor growth in vivo. MiR-17-5p was overexpressed, whereas the expression of p21 was downregulated in NPC cell lines and tissues. The miR-17-5p expression level was inversely correlated with the p21 mRNA level in NPC samples. Furthermore, analysis of 2(-ΔΔCt) value in 81 NPC patients suggested that the elevated expression level of miR-17-5p or the downregulated expression level of p21 was significantly correlated with tumor size (T classification) and tumor stage, and Kaplan-Meier survival analysis revealed a correlation between miR-17-5p or p21 expression level and overall survival times in 81 NPC patients. MiR-17-5p promoted cell growth in vivo and in vitro by directly targeting p21. Our results indicate that miR-17-5p can promote the occurrence of NPC and it may serve as a potential novel diagnostic maker or therapeutic target for NPC in the future.
Malnutrition is a significant issue in the hospital setting. This cross-sectional, observational study determined the prevalence of malnutrition amongst 189 adult inpatients in a teaching hospital using the Patient-Generated Subjective Global Assessment tool and compared data to control groups for coding of malnutrition to determine the estimated unclaimed financial reimbursement associated with this comorbidity. Fifty-three percent of inpatients were classified as malnourished. Significant associations were found between malnutrition and increasing age, decreasing body mass index, and increased length of stay. Ninety-eight percent of malnourished patients were coded as malnourished in medical records. The results of the medical history audit of patients in control groups showed that between 0.9 and 5.4% of patients were coded as malnourished which is remarkably lower than the 52% of patients who were coded as malnourished from the point prevalence study data. This is most likely to be primarily due to lack of identification. The estimated unclaimed annual financial reimbursement due to undiagnosed or undocumented malnutrition based on the point prevalence study was AU$8,536,200. The study found that half the patients were malnourished, with older adults being particularly vulnerable. It is imperative that malnutrition is diagnosed and accurately documented and coded, so appropriate coding, funding reimbursement, and treatment can occur.
Various factors determine the rate at which mutations are generated and fixed in viral genomes. Viral evolutionary rates may vary over the course of a single persistent infection and can reflect changes in replication rates and selective dynamics. Dedicated statistical inference approaches are required to understand how the complex interplay of these processes shapes the genetic diversity and divergence in viral populations. Although evolutionary models accommodating a high degree of complexity can now be formalized, adequately informing these models by potentially sparse data, and assessing the association of the resulting estimates with external predictors, remains a major challenge. In this article, we present a novel Bayesian evolutionary inference method, which integrates multiple potential predictors and tests their association with variation in the absolute rates of synonymous and non-synonymous substitutions along the evolutionary history. We consider clinical and virological measures as predictors, but also changes in population size trajectories that are simultaneously inferred using coalescent modelling. We demonstrate the potential of our method in an application to within-host HIV-1 sequence data sampled throughout the infection of multiple patients. While analyses of individual patient populations lack statistical power, we detect significant evidence for an abrupt drop in non-synonymous rates in late stage infection and a more gradual increase in synonymous rates over the course of infection in a joint analysis across all patients. The former is predicted by the immune relaxation hypothesis while the latter may be in line with increasing replicative fitness during the asymptomatic stage.
A phylogeny has been calculated by maximum likelihood comparisons of the concatenated consensus protein sequences of 29 tobamoviruses shown to be non-recombinant. This phylogeny has statistically significant support throughout, including its basal branches. The viruses form eight lineages that are congruent with the taxonomy of the hosts from which each was first isolated and, with the exception of three of the twenty-nine species, all fall into three clusters that have either asterid or rosid or caryophyllid hosts (i.e. the major subdivisions of eudicotyledonous plants). A modified Mantel permutation test showed that the patristic distances of virus and host phylogenies are significantly correlated, especially when the three anomalously placed viruses are removed. When the internal branches of the virus phylogeny were collapsed the congruence decreased. The simplest explanation of this congruence of the virus and host phylogenies is that most tobamovirus lineages have co-diverged with their primary plant hosts for more than 110 million years, and only the brassica-infecting lineage originated from a major host switch from asterids to rosids. Their co-divergence seems to have been 'fuzzy' rather than 'strict', permitting viruses to switch hosts within major host clades. Our conclusions support those of a coalesence analysis of tobamovirus sequences, that used proxy node dating, but not a similar analysis of nucleotide sequences from dated samples, which concluded that the tobamoviruses originated only 100 thousand years ago.
Certain genes and neurobiology ('neurogenetics') may predispose some people to violent behavior. Increasingly, defendants introduce neurogenetic evidence as a mitigating factor during criminal sentencing. Identifying the cause of a criminal act, biological or otherwise, does not necessarily preclude moral or legal liability. However, valid scientific evidence of an inherited proclivity sometimes should be considered when evaluating whether a defendant is less morally culpable for a crime and perhaps less deserving of punishment. This Note proposes a two-pronged test to understand whether and when neurogenetic evidence should be considered to potentially mitigate an individual's culpability for criminal behavior. The first prong normatively assesses whether a defendant meets a threshold of having meaningfully managed his risk of harming others based on what he knew, or should have known, about his own proclivities to violence. The second prong considers the admissibility of the evidence based on whether the specific neurogenetic proclivity claimed by the defendant is relevant and adequately supported by science so as to be reliable. This proposed two-pronged test, beginning with an ethical threshold and followed by a scientific hurdle, can help judges and juries establish when to accept arguments for neurogenetic mitigation at sentencing, and when to reject them.
A defendant is criminally responsible for his action only if he is shown to have engaged in a guilty act-actus reus (eg for larceny, voluntarily taking someone else's property without permission)-while possessing a guilty mind-mens rea (eg knowing that he had taken someone else's property without permission, intending not to return it)-and lacking affirmative defenses (eg the insanity defense or self-defense). We therefore first review neuroscientific studies that bear on the nature of voluntary action, and so could, potentially, tell us something of importance about the actus reus of crimes. Then we look at studies of intention, perception of risk, and other mental states that matter to the mens rea of crimes. And, last, we discuss studies of self-control, which might be relevant to some formulations of the insanity defense. As we show, to date, very little is known about the brain that is of significance for understanding criminal responsibility. But there is no reason to think that neuroscience cannot provide evidence that will challenge our understanding of criminal responsibility.
This commentary focuses on the tenuous line between health and disease and the conflicting characterizations of genetic predisposition that sometimes place it on one side of that line, and sometimes on the other. For example, GINA uses the line between health and disease to distinguish between, respectively, the healthy (including, those with genetic predispositions), who are shielded from discrimination, and those with 'manifested illness,' who are not. At the same time, some have argued that the Americans with Disabilities Act protects individuals with genetic predispositions, relying on a label akin to disability, as opposed to health, to characterize this group. Similarly, courts have described genetic predisposition as a disease of sorts to justify insurance payment for medical intervention. Attempts to fit genetic predisposition neatly into the binary world of health or illness can be problematic because this dichotomy doesn't capture the complex continuum between those states. Some individuals reside in yet another 'liminal' state when they develop mild symptoms or biomarkers, placing them somewhere between genetic predisposition and actual disease manifestation. As a result, they may be unprotected under existing frameworks. Liminal states are therefore problematic not only with respect to insurance reimbursement, but in other areas as well.
Several recent articles have called for the regulation of consumer transcranial direct current stimulation (tDCS) devices, which provide low levels of electrical current to the brain. However, most of the discussion to-date has focused on ethical or normative considerations; there has been a notable absence of scholarship regarding the actual legal framework in the United States. This article aims to fill that gap by providing a pragmatic analysis of the consumer tDCS market and relevant laws and regulations. In the five main sections of this manuscript, I take into account (a) the history of the do-it-yourself tDCS movement and the subsequent emergence of direct-to-consumer devices; (b) the statutory language of the Federal Food, Drug and Cosmetic Act and how the definition of a medical device-which focuses on the intended use of the device rather than its mechanism of action-is of paramount importance for discussions of consumer tDCS device regulation; (c) how both the Food and Drug Administration (FDA) and courts have understood the FDA's jurisdiction over medical devices in cases where the meaning of 'intended use' has been challenged; (d) an analysis of consumer tDCS regulatory enforcement action to-date; and (e) the multiple US authorities, other than the FDA, that can regulate consumer brain stimulation devices. Taken together, this paper demonstrates that rather than a 'regulatory gap,' there are multiple, distinct pathways by which consumer tDCS can be regulated in the United States.
Emotion comprehension (EC) is known to be a key correlate and predictor of prosociality from early childhood. In the present study, we examined this relationship within the broad theoretical construct of social understanding which includes a number of socio-emotional skills, as well as cognitive and linguistic abilities. Theory of mind, especially false-belief understanding, has been found to be positively correlated with both EC and prosocial orientation. Similarly, language ability is known to play a key role in children's socio-emotional development. The combined contribution of false-belief understanding and language to explaining the relationship between EC and prosociality has yet to be investigated. Thus, in the current study, we conducted an in-depth exploration of how preschoolers' false-belief understanding and language ability each contribute to modeling the relationship between children's comprehension of emotion and their disposition to act prosocially toward others, after controlling for age and gender. Participants were 101 4- to 6-year-old children (54% boys), who were administered measures of language ability, false-belief understanding, EC and prosocial orientation. Multiple mediation analysis of the data suggested that false-belief understanding and language ability jointly and fully mediated the effect of preschoolers' EC on their prosocial orientation. Analysis of covariates revealed that gender exerted no statistically significant effect, while age had a trivial positive effect. Theoretical and practical implications of the findings are discussed.
We studied multi-loci variants to identify the contribution of six candidate genes (ADIPOQ, CDH13, LYPLAL1, MC4R, PPARG and PGC1A) in the development of obesity and overweight.
We genotyped 404 chromosomes with eleven SNPs in Mexican female adolescents, who were subdivided into two groups (obesity-overweight and normal-weight) using the World Health Organization parameters. Genomic (800 chromosomes) and ancestral (208 chromosomes) controls were included to reduce the population bias. Anthropometric measurements, biochemical parameters, and caloric intake were obtained only in the groups of Mexican female adolescents.
A positive genotype-phenotype association was found that involves the multi-allelic combination of three risk alleles (one in PPARG and two in LYPLAL1) with obesity and overweight (OR=3.1, P=.010). This combination also exhibited a significant association with waist circumference (P=.030) and triglycerides levels (P=.030). These associations were supported by a logistic regression analysis adjusted for several confounding variables.
Our data suggest the joint participation of PPARG-LYPLAL1 genes in metabolic disorders development. Hence, these genes could act as potential biomarkers in obesity and overweight. Our findings underscore the complexity of metabolic disorders and provide evidence about the importance of multi-loci analysis to study complex diseases.
This article briefly explains the food and nutrition implications of the new standards, tax penalties and reporting requirements for non-profit hospitals and healthcare systems to maintain a tax-exempt or charitable status under section 501(c)(3) of the Federal Internal Revenue Code set forth in The Patient Protection and Affordable Care Act (P.L. 111-148, Sec. 9007). The newly created 501(r) of the Internal Revenue Code requires, beginning with the first tax year on or after March 23, 2012, that such hospitals demonstrate community benefit by conducting a community health needs assessment (CHNA) at least once every three years and annually file information by means of a Schedule H (Form 990) regarding progress towards addressing identified needs. As hospitals conduct their CHNA and work further and collaboratively with community stakeholders on developing and monitoring their proposed action plans, the breadth and depth of food and nutrition activities occurring as a result of the Affordable Care Act Hospital Community Benefit Program will likely increase. The CHNA requirement, along with other emerging initiatives focused on improving the food environments and nutrition-related activities of hospitals and healthcare systems offer fruitful opportunities for food and nutrition professionals to partner on innovative ways to leverage hospital infrastructure and capacity to influence those residing, working or visiting the hospital campus, as well as the surrounding community.
Ventral hernia repair (VHR) is a commonly performed surgical procedure. Unfortunately, long term prospective information about quality of life and outcomes after VHR has been challenging to obtain. Decoupling follow up from clinical visits via patient reported outcomes (PROs) has been proposed as a means of achieving better long term assessments after VHR. The Americas Hernia Society Quality Collaborative (AHSQC) is a national quality improvement (QI) effort in hernia repair which uses PROs to obtain long term follow up. However, the modality of PRO engagement to maximize participation has not been well established. A formal QI initiative was undertaken to determine if long term PRO follow up could be increased at a single AHSQC site by adding telephone communication to email communication for long term postoperative VHR assessment.
Between September 2015 and July 2016, the long term (greater than 1 year) AHSQC PRO completion rates after VHR at our institution were analyzed using Plan-Do-Study-Act cycles. Two interventions were implemented: 1). Contacting patients using telephone 2). Changing timing of telephone calls.
Two-hundred-thirty-two patients were identified of which 99 (42.7%) met eligibility criteria. Prior to this initiative, the long term PRO completion rate was 16.3% in postoperative VHR patients. The completion rate after introducing telephone calls (intervention 1) was 35.7% and after changing the timing of telephone calls (intervention 2) was 55.1%. The mean participation rate was 45.4% (±9.7%).
A telephone based approach markedly improved long term PRO participation rates in postoperative VHR patients. Ultimately, a combination of email and telephone communication may be necessary to achieve higher levels of PRO follow up in the VHR population.
Chromatin plays a central role in maintaining hematopoietic stem cells and during their stepwise differentiation. Although a large number of histone modifications and chromatin-modifying enzymes have been identified, how these act in concert to produce specific phenotypic outcomes remains to be established. MOZ (KAT6A) is a lysine acetyltransferase and enhances transcription at target gene loci. In contrast, the Polycomb group protein BMI1 (PCGF4) is part of the transcriptionally repressive PRC1 complex. Despite their opposing effects on transcription, MOZ and BMI1 regulate biological systems in a similar manner. MOZ and BMI1 are required for the development of transplantable HSCs, for restraining cellular senescence, for the proper patterning of the anterior-posterior axis during development and for the specification and maintenance of the B-cell lineage. Thus, we set out to explore the relationship between MOZ and BMI1. We recently established that MOZ and BMI1 have opposing effects on the initiation of Hox gene expression during embryonic development and that defects in body segment identity specification observed in single Moz and Bmi1 mutants were rescued in compound mutants. We report here the relationship between MOZ and BMI1 in hematopoiesis. Using Moz(+/-);Bmi1(+/-) compound mutant mice, we found that MOZ and BMI1, but not the BMI1-related protein MEL18 (PCGF2), play synergistic roles in maintaining adult HSCs. Although BMI1 restrains premature senescence, we established that MOZ acts to maintain the quiescent state of HSCs. Our work revealed that MOZ and BMI1 regulate HSCs in a synergistic manner by acting on distinct processes required to maintain HSCs.
Between 2006 and 2015, the Act on the State Emergency Medical System was the key act governing the organization, financing and provision of emergency care in Poland. From the moment it entered into force, it had been heavily criticized. The critique focused, among others, on the lack of provisions allowing for emergency medical services (EMS) to be performed outside the EMS units, the lack of a separate Act regulating the profession of a medical rescuer and the lack of a separate professional organization representing medical rescuers. As early as 2008 a team of specialists was set up to work on amending the Act and these works resulted in the draft Act on the State Emergency Medical System that was submitted to public consultations on 19 August, 2014. This draft was further reworked in 2015 and was signed by the President on 25 September of the same year. The Act addressed some of the shortcomings of the EMS legislation that was previously in place. However, the new Act did not meet the key demands of medical rescuers; namely, it did not introduce a separate legal act regulating this profession nor established a professional organisation representing their interests. An analysis of the vested interests of various groups of medical professionals indicates that these interests are likely to have influenced the final legislative outcome. The Act, as well as its implementing executive regulation from April 2016, may reduce support of certain medical professional groups during the Act's implementation as well as create tensions between these groups, especially between medical rescuers and nurses.
Techniques to improve solder joint reliability have been the recent research focus in the electronic packaging industry. In this study, Cu/SAC305/Cu solder joints were fabricated using a low-power high-frequency ultrasonic-assisted reflow soldering approach where non-ultrasonic-treated samples were served as control sample. The effect of ultrasonic vibration (USV) time (within 6s) on the solder joint properties was characterized systematically. Results showed that the solder matrix microstructure was refined at 1.5s of USV, but coarsen when the USV time reached 3s and above. The solder matrix hardness increased when the solder matrix was refined, but decreased when the solder matrix coarsened. The interfacial intermetallic compound (IMC) layer thickness was found to decrease with increasing USV time, except for the USV-treated sample with 1.5s. This is attributed to the insufficient USV time during the reflow stage and consequently accelerated the Cu dissolution at the joint interface during the post-ultrasonic reflow stage. All the USV-treated samples possessed higher shear strength than the control sample due to the USV-induced-degassing effect. The shear strength of the USV-treated sample with 6s was the lowest among the USV-treated samples due to the formation of plate-like Ag3Sn that may act as the crack initiation site.
To evaluate the therapeutic value, safety, and long-term clinical outcomes of percutaneous lumbar facet synovial cyst (LFSC) rupture.
Our study was institutional review board (IRB)-approved and Health Insurance Portability and Accountability Act (HIPAA)-compliant. The study group comprised 71 patients (44 women, mean age: 65 ± 17 years) who underwent CT- or fluoroscopy-guided percutaneous LFSC rupture. The technical success of LFSC rupture, the long-term clinical outcome, including repeat procedures or surgery, and imaging findings on MRI and CT were recorded.
Seventy-nine LFSC ruptures were performed in 71 patients. CT guidance was used in 57 cases and fluoroscopy guidance in 22 cases. LFSC rupture was technically successful in 58 out of 79 cases (73 %). Mean injection volume for cyst rupture was 3.6 ± 2.2 mL and a combination of steroid and anesthetic was injected in all cases. Over a mean follow-up time of 44 months, 12 % of patients underwent repeat cyst rupture, and 46 % eventually underwent surgery, whereas the majority of patients (55 %) experienced symptomatic relief and did not undergo surgery. There was no significant association between a successful outcome and age, sex, level, or size of LFSC (p > 0.1). LFSCs with T2 hypointensity were more likely to require surgery (p = 0.02). There was one complication, a bacterial skin infection that completely resolved following antibiotic therapy.
Percutaneous LFSC rupture is an effective and safe nonsurgical treatment option for LFSC. More than half of treated patients were able to avoid subsequent surgery. Therefore, percutaneous LFSC rupture should be considered before surgical intervention.
This pooled analysis study aimed to reveal the prognostic relevance of microRNAs (miRNAs) in patients with diffuse large B-cell lymphoma (DLBCL).
We examined the impact of miRNAs on clinical outcome. Eligible studies were identified and quality assessed using multiple search strategies. Data were extracted from included studies which correlated survival with expression of miRNAs (serum or tissue).
We pooled proper studies, and combined the hazard ratios with 95% confidence intervals to estimate strength of the correlations. There were 18 studies including 1950 patients with DLBCL eligible for pooled analysis. We found significant combined HRs for poor overall survival for high expression of miR-21 and low expression of miR-224 in tumor tissue, but for favorable relapse free survival for high expression of miR-21 in serum. Progression free survival was shortened in patients with low expression of miR-199a/b, miR-146b-5p, miR-224 and high expression of miR-222.
MiRNAs may act as independent prognostic factors in patients with DLBCL, and useful in risk stratification.
Examine the prevalence of alcohol and its contributory role in unintentional fatal river drowning in Australia to inform strategies for prevention.
Cases of unintentional fatal river drowning in Australia, 1-July-2002 to 30-June-2012, were extracted from the National Coronial Information System. Cases with positive alcohol readings found through autopsy or toxicology reports were retained for analysis. Discrete analysis was conducted on cases with a Blood Alcohol Content (BAC) of ≥0.05% (0.05grams of alcohol in every 100 millilitres of blood).
Alcohol was known to be involved in 314 cases (40.8%), 279 recorded a positive BAC, 196 (70.3%) recorded a BAC of ≥0.05%. 40.3% of adult victims had a BAC of ≥0.20%. Known alcohol involvement was found to be more likely for victims who drowned as a result of jumping in (χ(2)=7.8; p<0.01), identify as Aboriginal and Torres Strait Islander (χ(2)=8.9; p<0.01) and drowned in the evening (χ(2)=7.8; p<0.01) and early morning (χ(2)=16.1; p<0.01) hours.
The number of people who drown with alcohol in their bloodstream is concerning and challenging for prevention. To assist with the prevention of alcohol related river drowning improved data quality, as well as a greater understanding of alcohol's contribution and consumption patterns at rivers (especially those <18 years of age) is required.
Alcohol contributes to fatal unintentional drowning in Australian rivers. Although prevention is challenging, better data and exposure studies are the next step to enhance prevention efforts.
Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. Many animal studies have found ZEA to possess a disruptive effect on the hormonal balance, mainly due to its similarity to naturally-occurring estrogens. With increasing consciousness of the adverse effects of endocrine disruptors on human health, it is becoming more important to monitor ZEA concentrations in food and identify its potential effects on human health. Based on a review of recent studies on animal models and molecular pathways in which ZEA is reported to have an influence on humans, we postulate that ZEA might act as an endocrine disruptor in humans in a similar way to animals. Moreover, its endocrine-disrupting effect might be also a causative factor in carcinogenesis. This review article summarizes the latest knowledge about the influence of ZEA on the human hormonal balance.
Laterality of function in the orofacial musculature suggests there may be side-to-side asymmetry of proprioceptive acuity in lip movement compared to the temporomandibular joint (TMJ). In the present work, 14 young adults were tested for acuity of lip and TMJ closure movements onto plugs varying from 5 to 8mm without visual feedback. Testing was conducted on both left and right sides, using the same psychophysical task and stimuli. Results showed superior proprioceptive acuity at the lips, with no significant side effect. However, there was side-to-side asymmetry in the correlations between proprioceptive performance for the two anatomical structures, with performance on the right side strongly correlated but not on the left. This is consistent with the need for coordination between structures during chewing. When acuity at different points in the stimulus range was examined, the right side lips were better with small stimuli. Overall, results support enhanced use-specific proprioception.
The interaction of aminoacids (Glycine, Proline, Lysine) with brushite based bone cements has been investigated by several techniques (FTIR spectroscopy, Thermogravimetry-TG, Scanning Electron Microscopy-SEM, mechanical properties studies), with the aim to elucidate the properties of the resulting composite materials and the interaction occurring at molecular level between the inorganic matrix and the organic moieties. Brushite phase is predominantly obtained also in the presence of aminoacids added during preparation of the bone cement. Focusing on Glycine incorporation, the presence of a fraction of bulk Glycine, weakly interacting with the inorganic matrix, together with Glycine specifically interacting with adsorption sites can be envisaged, as pointed out by FT IR and thermogravimetric data. In detail, FT-IR data evidenced changes in shape and position of bands associated to stretching modes of the carboxylic groups in Glycine structure, which can be explained by the coordination of these functional groups with the Ca ions in the matrix. Heating this composite at controlled temperature results in the detection of a condensation products, either cyclic condensation product, either dipeptide. Diffuse and not specific H-bonding seems to be the main form of interaction of Proline and Lysine with brushite. Due to the coordination with Ca ions here described, Glycine can act as retardant during brushite preparation, allowing good workability of the resulting composite.
The growing interest in the use of recyclable and biodegradable natural materials has become a relevant topic in pharmaceutics. In this work, we suggest the use and valorization of natural horny skeleton of marine sponges (Porifera, Dictyoceratida) as bio-based dressing for topical drug delivery. Biomaterial characterization focusing on morpho-functional traits, swelling behavior, fluid uptake performances, glycosaminoglycans content and composition and microbiological quality assessment was carried out to investigate the collagenic skeleton properties. After grinding and sieving processes, l-cysteine hydrochloride-loaded formulations were designed in form of powder or polymeric film by testing various drug concentrations and different drying parameters. Drug content, SEM analyses and in vitro permeation studies were performed to test the suitability of skeleton-based formulations. To this respect, drying time and temperature are key parameters for skeleton-mediated drug crystallization. Consequently, this behavior seems to influence drug loading and permeation profiles of formulations. The high percentages of drug are found after absorption into sponge powder and in vitro permeation studies demonstrate that cysteine is released more slowly than the pure drug within 1h. Such a system is attractive because it combines the known healing properties of cysteine with the advantageous potentials of the collagen/proteoglycan network, which can act as biocompatible carrier able to absorb the excess of the wound exudate while releasing the drug. Furthermore, due to its glycosaminoglycans content, natural sponge skeletal scaffold might act as bioactive-biomimetic carrier regulating the wound healing processes.
New generation of barrier membranes has been developed, which not only act as barriers but also as delivery devices to release specific growth factors. This study observed biological behaviors of bone morrow mesenchymal stem cells (BMMSCs) pretreated by bFGF or BMP-2 in vitro and evaluated differential bone regeneration process induced by bFGF and BMP-2 loaded acellular dermal matrix (ADM) membrane using critical-size rat calvarial defect model in vivo. The results showed that the proliferation capability of BMMSCs pretreated by bFGF was stronger than that by BMP-2, while there was temporally differential effect of bFGF and BMP-2 pretreatment on MSC osteogenic differentiation potentials. During healing process of rat calvarial defects, 2-fold more CD34-/CD90+ MSCs in group of bFGF-ADM was observed than in any other treatment group at 2weeks. However, there were similar amount of new bone formation and expression of osteopotin in newly-formed bone tissue in groups of bFGF- and BMP-2-ADM at 8weeks, which were more than those in ADM alone and blank control. Taken together, bFGF-ADM guided similar bone regeneration to BMP-2 through more efficient recruitment of MSCs, and moreover, BMMSCs pretreated by bFGF showed stronger proliferation at 1-5days and osteogenic differentiation potentials at 14days compared with BMP-2 pretreatment.
During the Pleistocene, shifts of species distributions and their isolation in disjunct refugia led to varied outcomes in how taxa diversified. Some species diverged, others did not. Here, we begin to address another facet of the role of the Pleistocene in generating today's diversity. We ask which processes contributed to divergence in semi-arid southern Australian birds. We isolated 11 autosomal nuclear loci and one mitochondrial locus from a total of 29 specimens of the sister species pair, Chestnut Quail-thrush Cinclosoma castanotum and Copperback Quail-thrush C. clarum.
A population clustering analysis confirmed the location of the current species boundary as a well-known biogeographical barrier in southern Australia, the Eyrean Barrier. Coalescent-based analyses placed the time of species divergence to the Middle Pleistocene. Gene flow between the species since divergence has been low. The analyses suggest the effective population size of the ancestor was 54 to 178 times smaller than populations since divergence. This contrasts with recent multi-locus studies in some other Australian birds (butcherbirds, ducks) where a lack of phenotypic divergence was accompanied by larger historical population sizes. Post-divergence population size histories of C. clarum and C. castanotum were inferred using the extended Bayesian skyline model. The population size of C. clarum increased substantially during the late Pleistocene and continued to increase through the Last Glacial Maximum and Holocene. The timing of this expansion across its vast range is broadly concordant with that documented in several other Australian birds. In contrast, effective population size of C. castanotum was much more constrained and may reflect its smaller range and more restricted habitat east of the Eyrean Barrier compared with that available to C. clarum to the west.
Our results contribute to awareness of increased population sizes, following significant contractions, as having been important in shaping diversity in Australian arid and semi-arid zones. Further, we improve knowledge of the role of Pleistocene climatic shifts in areas of the planet that were not glaciated at that time but which still experienced that period's cyclical climatic fluctuations.
Antipsychotic drugs, all of which block the dopamine D2 receptor to a greater or lesser extent, are the mainstay for the pharmacological treatment of schizophrenia. Engaging in a deeper understanding of how antipsychotics act on the brain and body, at the cellular, molecular and physiological level is vital to comprehend both the beneficial and potentially harmful actions of these medications and stimulate development of novel therapeutics. To address this, we review recent advances in our understanding of neuroadaptations to antipsychotics, focusing on (1) treatment efficacy, (2) impact on brain volume and (3) evidence from human post-mortem studies that attempt to dissect neuropathological effects of antipsychotic drugs from organic schizophrenia neurobiology and (4) cardio-metabolic side effects. Our aim is to stimulate discussion on these highly clinically relevant topics and consider how we might use current and evolving knowledge and new methodologies in the fields of neuropharmacology and neuroscience, to advance our understanding of the long-term impact of antipsychotic treatment. Ultimately, this may inform the clinical use of these drugs.
The proneural gene, Atoh1, is necessary and in some contexts sufficient for early inner ear hair cell development. Its function is the subject of intensive research, not least because of the possibility that it could be used in therapeutic strategies to reverse hair cell loss in deafness. However, it is clear that Atoh1's function is highly context dependent. During inner ear development, Atoh1 is only able to promote hair cell differentiation at specific developmental stages. Outside the ear, Atoh1 is required for differentiation of a variety of other cell types, for example in the intestine and cerebellum. The reasons for this context dependence are poorly understood. So far, the pathways and key players that instruct Atoh1 to act as a mechanosensory cell fate determinant in the context of the inner ear are largely unknown. Here we review evidence that suggests that Atoh1 function in hair cell differentiation is modulated by interaction with other transcription factors. We particularly focus on the possible roles of Gfi1 and Pou4f3, drawing from studies in mouse, Drosophila and C. elegans.
Environmental gradients, and particularly climatic variables, exert a strong influence on plant distribution and, potentially, population genetic diversity and differentiation. Differences in water availability can cause among-population variation in ecological processes and can thus interrupt populations' connectivity and isolate them environmentally. The present study examines the effect of environmental heterogeneity on plant populations due to environmental isolation unrelated to geographic distance. Using AFLP markers, we analyzed genetic diversity and differentiation among 12 Salvia spinosa populations and 13 Salvia syriaca populations from three phytogeographical regions (Mediterranean, Irano-Turanian and Saharo-Arabian) representing the extent of the species' geographic range in Jordan. Differences in geographic location and climate were considered in the analyses. For both species, flowering phenology varied among populations and regions. Irano-Turanian and Saharo-Arabian populations had higher genetic diversity than Mediterranean populations, and genetic diversity increased significantly with increasing temperature. Genetic diversity in Salvia syriaca was affected by population size, while genetic diversity responded to drought in S. spinosa. For both species, high levels of genetic differentiation were found as well as two well-supported phytogeographical groups of populations, with Mediterranean populations clustering in one group and the Irano-Turanian and Saharo-Arabian populations in another. Genetic distance was significantly correlated to environmental distance, but not to geographic distance. Our data indicate that populations from moist vs. arid environments are environmentally isolated, where environmental gradients affect their flowering phenology, limit gene flow and shape their genetic structure. We conclude that environmental heterogeneity may act as driver for the observed variation in genetic diversity.
Emerging evidence suggests that small nucleolar RNAs (snoRNAs) and their host genes (SNHGs) have malfunctioning roles in the development of human cancers. We globally investigated the molecular mechanisms by which snoRNA host gene 6 (SNHG6) promotes hepatocellular carcinoma (HCC) progression using human tissues and cell lines. We found that SNHG6 is overexpressed in HCC tissues and in hepatoma cell lines and is closely associated with histologic grade, hepatitis B virus DNA, Barcelona Clinic Liver Cancer stage and portal vein tumor thrombus in patients with HCC. Knockdown of SNHG6 induced apoptosis and repressed cell cycle progression in hepatoma cell lines, whereas transgenic expression of SNHG6 in the immortalized human hepatic cell line L02 had opposite effects. Xenograft tumors grown from SNHG6-knockdown cells had smaller mean volumes than did tumors grown from control cells. SNHG6 may act as a competing endogenous RNA, effectively becoming a sink for miR-101-3p and thereby modulating the derepression of zinc finger E-box binding homeobox 1, imposing an additional level of post-transcriptional regulation. Functionally, SNHG6 promotes tumor growth and metastasis by inducing epithelial to mesenchymal transition. Further investigations showed that SNHG6 could affect HCC tumorigenesis by binding to up-frameshift protein 1 and regulating Smad7 expression.
Human milk contains growth factors that maintain intestinal mucosal homeostasis, but the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. In this study, IEC-6 (rat intestinal epithelial cells) cells were used as a model to study cell differentiation mediated by transforming growth factor-β2 (TGF-β2), the most abundant growth factor in human milk. We focused on the transcription factor early growth response-1 (EGR-1), as we found a robust and rapid response in our initial transcription factor screen. Immunoblotting and immunofluorescent assays confirmed the phenotype change upon TGF-β2 treatment and EGR-1 stimulation in the nucleus, with maximum expression occurring at 1 h. Chromatin immunoprecipitation sequencing was performed to map genome-wide EGR-1 binding sites on more than 1800 genes, widely involved in processes such as gene expression, transcription, membrane invagination and metabolism. In particular, more than 15 Wnt signaling pathway genes have EGR-1 binding sites; among them, Axin1 was the limiting factor, ensuring proper β-catenin accumulation in the cytoplasm. We further used chromatin immunoprecipitation quantitative PCR to validate that EGR-1 binds to the region of -636/-454 bp and -454/-200 bp of the Axin1 promoter and functionally activates gene expression. The effect of TGF-β2 on maintaining small intestinal cell homeostasis was partially explained by Axin1 activation through EGR-1.
Plastocyanin (petE) plays an essential role in photosynthesis as an electron carrier between cytochrome b6 f and photosystem I, and in some cyanobacteria it can be replaced by the haem-containing protein, cytochrome c6 (petJ). In Synechocystis sp. PCC 6803, transcription of petE and petJ is activated and repressed, respectively, by Cu. Here, we show that Ni can act similarly to Cu in inducing petE and repressing petJ, thus leading to a partial switch between cytochrome c6 and plastocyanin. Transcription of these genes is only altered by Ni in Cu-depleted medium, and none of the Ni-dependent transcription factors described in Synechocystis, NrsR and InrS seem to be involved in this regulation. Finally, we show that plastocyanin is essential for growth under conditions of excess Ni.
Electrospun mats containing cyclodextrin polymers (poly-αCD or poly-βCD) were developed to act as wound dressings showing tunable release rate of the antifungal agent fluconazole incorporated forming inclusion complexes. Poly-αCD and poly-βCD were prepared via cross-linking with epichlorohydrin (EPI) as water-soluble large molecular weight polymers. Then, polyCDs forming complexes with fluconazole were mixed with poly-(ε-caprolactone) (PCL) or poly(N-vinylpyrrolidone) (PVP) for electrospinning. Obtained bead-free fibers showed a random distribution, diameters in the 350-850nm range, and a variety of physical stability behaviors in aqueous environment. Mats were coated by hexamethyldisiloxane (HMDSO) plasma polymerization to create a hydrophobic layer that prevented rapid drug diffusion. HMDSO coating was evidenced by the Si content of mat surface (EDX analysis) and by the increase in the water contact angle (up to 130°). In physiological-mimicking medium, non-treated mats showed burst release of fluconazole, whereas HMDSO-coated mats sustained the release and delayed disintegration of PVP-based mats. Antifungal tests evidenced that both coated and non-coated mats efficiently inhibited the growth of Candida albicans.
Unexpected presentation of a startling auditory stimulus (SAS>120 decibels) in a reaction time (RT) paradigm results in the startle reflex and an early release (<100ms) of the preplanned motor response (StartReact effect). Mechanical perturbations applied to the upper limbs elicit short- (M1) and long-latency (M2) stretch reflexes and have also been shown to initiate intended motor responses early (<100ms). Ravichandran et al. (2013) recently proposed that unexpected delivery of a perturbation could also elicit a startle response and therefore the StartReact effect may be responsible for the early trigger of a preplanned response. To investigate this further, we examined startle incidence, RT, and stretch reflex modulation for both expected and unexpected perturbations. In Experiment 1, participants performed active (ACT) and passive (DNI) conditions to an expected large perturbation (similar to previous studies examining M2). The startle response was not observed; however, the perturbation still elicited the voluntary response at short latency (<100ms) and goal-dependent modulation of the M2 response was observed. In Experiment 2, participants performed ACT and DNI conditions to a weak auditory stimulus or a small wrist perturbation. On unexpected trials we probed startle circuitry with a large perturbation or SAS. The SAS consistently elicited a startle response in both ACT and DNI conditions, but startle-like activity was only observed on 17.4% of ACT perturbation probe trials. Our findings suggest that while unexpected upper limb perturbations can be startling, startle triggering of the preplanned voluntary response is not the primary mechanism responsible for goal-dependent modulation of the M2 response.
This opinion article explores how sustained neural firing in association areas allows high-order mental representations to be coactivated over multiple perception-action cycles, permitting sequential mental states to share overlapping content and thus be recursively interrelated. The term "state-spanning coactivity" (SSC) is introduced to refer to neural nodes that remain coactive as a group over a given period of time. SSC ensures that contextual groupings of goal or motor-relevant representations will demonstrate continuous activity over a delay period. It also allows potentially related representations to accumulate and coactivate despite delays between their initial appearances. The nodes that demonstrate SSC are a subset of the active representations from the previous state, and can act as referents to which newly introduced representations of succeeding states relate. Coactive nodes pool their spreading activity, converging on and activating new nodes, adding these to the remaining nodes from the previous state. Thus, the overall distribution of coactive nodes in cortical networks evolves gradually during contextual updating. The term "incremental change in state-spanning coactivity" (icSSC) is introduced to refer to this gradual evolution. Because a number of associated representations can be sustained continuously, each brain state is embedded recursively in the previous state, amounting to an iterative process that can implement learned algorithms to progress toward a complex result. The longer representations are sustained, the more successive mental states can share related content, exhibit progressive qualities, implement complex algorithms, and carry thematic or narrative continuity. Included is a discussion of the implications that SSC and icSSC may have for understanding working memory, defining consciousness, and constructing AI architectures.
Prospective memory (PM), the ability to remember to do something at the appropriate time in the future, is crucial in everyday life. One way to improve PM performance is to increase the salience of a cue announcing that it is time to act. Multiple sclerosis (MS) patients often report PM failures and there is growing evidence of PM deficits among this population. However, such deficits are poorly characterized and their relation to cognitive status remains unclear. To better understand PM deficits in MS patients, this study investigated the impact of cue salience on PM, and its relation to retrospective memory (RM) and executive deficits.
Thirty-nine (39) MS patients were compared to 18 healthy controls on a PM task modulating cue salience during an ongoing general knowledge test.
MS patients performed worse than controls on the PM task, regardless of cue salience. MS patients' executive functions contributed significantly to the variance in PM performance, whereas age, education and RM did not. Interestingly, low- and high-executive patients' performance differed when the cue was not salient, but not when it was, suggesting that low-executive MS patients benefited more from cue salience.
These findings add to the growing evidence of PM deficits in MS and highlight the contribution of executive functions to certain aspects of PM. In low-executive MS patients, high cue salience improves PM performance by reducing the detection threshold and need for environmental monitoring.
p,p'-Dichlordiphenyldichloroethylene (DDE) is a metabolite of the insecticide dichlorodiphenyltrichloroethane (DDT), an organochloride which was massively used from its discovery in 1939 until the early 1970's. Due to the tremendous half-life of DDT and DDE, both substances are to date environmentally relevant. Furthermore, DDT is still employed in many African countries in the context of the WHO's antimalaria campaign. In amphibians, DDE was found to act as antiandrogenic endocrine disrupting chemical (EDC), whereas in other species DDE was found to act as an estrogen. To determine the mode of action (MOA) of DDE in adult male Xenopus laevis, we exposed adult male frogs to different concentrations of DDE, as well as to the estrogenic EDC ethinylestradiol (EE2) and the antiandrogenic fungicide vinclozolin (VIN) for four consecutive nights. We then analyzed the mate calling behavior, which was previously shown to be affected by (anti)androgenic and (anti)estrogenic EDC in a MOA-specific manner, in order to assess whether DDE exposure results in estrogen-specific or antiandrogen-specific alterations of the mate calling behavior. Our results demonstrate that DDE alters the reproductive behavior of male X. laevis. Lowered sexual arousal of exposed males was indicated by a decreased production of advertisement calls and higher amounts of calls that suggest a sexually unaroused state of the males. Our results further indicate that DDE can display both, estrogenic and antiandrogenic MOA, either of which can have adverse effects on reproductive physiology and behavior in X. laevis. The disruption of the affected mating behavior, which is crucial for a successful reproduction, might result in a reduced reproductive success of DDE exposed animals.
Social marketing managers promote desired behaviors to the audience by tangibilizing them in the form of environmental opportunities in order to enhance benefits and reduce barriers. This study proposed "benchmarks" modified from those found in the past literature that would match important concepts of the social marketing framework and whose inclusion would ensure behavior change effectiveness as well as analyzed behavior change interventions on a "social marketing continuum" to assess whether the number of benchmarks and the role of specific benchmarks influence the effectiveness of physical activity promotion efforts.
A systematic review of social marketing interventions available in academic studies published between 1997 and 2013 revealed 173 conditions in 92 interventions.
Findings based on chi-square, Mallow's Cp, and Logical Analysis of Data tests revealed that the presence of more benchmarks in interventions increased the likelihood of success in promoting physical activity. The presence of more than 3 benchmarks improved the success of the interventions; specifically, all interventions were successful when more than 7.5 benchmarks were present. Further, primary formative research, core product, actual product, augmented product, promotion, and behavioral competition had a significant influence on the effectiveness of interventions.
Social marketing is an effective approach in promoting physical activity among adults when a substantial number of benchmarks are employed and when managers understand the audience, make the desired behavior tangible, and promote the desired behavior persuasively.
Obesity is associated with arterial stiffening and diminished quality of life. Bikram yoga may be a feasible alternative to traditional exercise among obese individuals. Accordingly, the purpose of this study was to investigate the impact of Bikram yoga, a heated style of hatha yoga, on arterial stiffness in normal and overweight/obese adults.
Forty three (23 normal body mass index or BMI; 20 overweight/obese) apparently heathy participants completed an 8-week Bikram yoga intervention. Body composition was estimated via dual energy x-ray absorptiometry (DXA), arterial stiffness was measured via brachial-ankle pulse wave velocity and health-related quality of life was assessed via RAND 36-Item Short Form survey at baseline and at the end of the 8-week intervention.
After the intervention, brachial-ankle pulse wave velocity decreased (p<0.05) in overweight/obese participants while no such changes were observed in normal BMI participants. In the quality of life measures, emotional well-being improved (p<0.05) in both groups, and general health improved (p<0.05) only in the normal weight BMI group.
Bikram yoga ameliorates arterial stiffness in overweight/obese adults and can positively impact quality of life regardless of BMI.
The aim of this study was to use a social-ecological approach to examine the influence of individual, social, and environmental factors on moderate-to-vigorous physical activity (MVPA) and screen-time in a sample of nine year old children in Ireland.
The sample was 1509 boys and girls from the Growing Up in Ireland (GUI) national study. MVPA, screen time, and individual, social, and environmental variables were assessed via questionnaires completed by children, their parents, and their teachers. Multiple regression was used to identify factors that correlated with children's MVPA and screen-time levels.
For boys, factors such as activity with friends (p<.0001) and popularity (p<.01) were associated with MVPA, while factors such as BMI (p<.01) and MVPA (p<.01) were associated with screen time. Similarly for girls, factors such as activity with friends (p<.0001) and sociability were associated with MVPA, however factors such as BMI (p<.05), and access to play space (p<.05) were more closely associated with screen time.
Social factors were more closely associated with MVPA, while individual factors were significantly correlated with screen time for both boys and girls. Correlates differed for boys and girls, suggesting that interventions should consider both the target population as well as the activity behavior.
This study evaluated a physical activity (PA)-related obesity model on mortality.
Data from the 1999-2006 NHANES were used (N=16,077), with follow-up through 2011. PA was subjectively assessed, with body mass index (BMI) and waist circumference (WC) objectively measured. From these, 12 mutually exclusive groups (G) were evaluated, including: G1: Normal BMI, Normal WC and Active; G2: Normal BMI, Normal WC and Inactive; G3: Normal BMI, High WC and Active; G4: Normal BMI, High WC and Inactive; G5: Overweight BMI, Normal WC and Active; G6: Overweight BMI, Normal WC and Inactive; G7: Overweight BMI, High WC and Active; G8: Overweight BMI, High WC and Inactive; G9: Obese BMI, Normal WC and Active; G10: Obese BMI, Normal WC and Inactive; G11: Obese BMI, High WC and Active; and G12: Obese BMI, High WC and Inactive.
Compared to G2, the following had a reduced mortality risk: G1, G3, G5, G6, G7, G8, G9, and G11. Compared to G12, the following had a reduced mortality risk: G1, G3, G5, G7, G9, and G11. In each respective group for BMI and WC, the active group had a reduced mortality risk.
Across all BMI and WC combinations, PA improved mortality risk identification.
Older adults with osteoarthritis (OA) often report that their disease symptoms are exacerbated by weather conditions. This study examines the association between outdoor physical activity (PA) and weather conditions in older adults from six European countries and assesses whether outdoor PA and weather conditions are more strongly associated in older persons with OA than in those without the condition.
The American College of Rheumatology classification criteria were used to diagnose OA. Outdoor PA was assessed using the LASA Physical Activity Questionnaire. Data on weather parameters were obtained from weather stations.
Of the 2439 participants (65-85 years), 29.6% had OA in knee, hand and/or hip. Participants with OA spent fewer minutes in PA than participants without OA (Median=42.9, IQR=20.0-83.1 versus Median=51.4, IQR=23.6-98.6; p<0.01). In the full sample, temperature (B=1.52; p<0.001) and relative humidity (B=-0.77; p<0.001) were associated with PA. Temperature was more strongly associated with PA in participants without OA (B=1.98; p<0.001) than in those with the condition (B=0.48; p=0.47).
Weather conditions are associated with outdoor PA in older adults in the general population. Outdoor PA and weather conditions were more strongly associated in older adults without OA than in their counterparts with OA.
Promoting physical activity (PA) in low- and middle-income countries is an important public health topic as well as a challenge for practice. This study aimed to assess the effect of a school-based intervention on different PA-related variables among students.
This cluster-randomized-controlled trial included 548 students in the intervention group and 537 in the control group (11-18 years-old) from six schools in neighborhoods with low Human Development Index (from 0.170 to 0.491) in Fortaleza, Brazil. The intervention included strategies focused on training teachers, opportunities for PA in the school environment and health education. Variables measured at baseline and again at the 4-months follow-up included the weekly time in different types of moderate-to-vigorous PA (MVPA), preference for PA during leisure-time, PA behavioral change stage and active commuting to school. Generalized linear models and binary logistic regressions were used.
An intervention effect was found by increasing the weekly time in MVPA (effect size=0.17), popular games (effect size=0.35), and the amount of PA per week (effect size=0.27) among students (all p<0.05).
The intervention was effective in promoting improvements in some PA outcomes, but the changes were not sufficient to increase the proportion of those meeting PA recommendations.
Exposure to green space has been associated with increased physical activity. However, it is not clear whether this association is because active people preferentially live in greener areas. Relationships between exposure to green space and physical activity during pregnancy are not well defined. Our objective was to determine whether exposure to green space was associated with physical activity in pregnant women.
The current study was completed within the Growing Up in New Zealand cohort study of 6772 pregnant women. The proportion of green space in each census area unit was determined and geocoded to residential address. The association between exposure to green space and physical activity was determined using logistic regression analyses after controlling for confounding variables.
Exposure to green space was not associated with participation in physical activity during first trimester and the remainder of pregnancy once preference for living in greener neighborhoods was taken into account.
The lack of association between green space and physical activity found in this study does not necessarily mean that living in green space will not translate into better pregnancy health. Preference for living in greener neighborhoods should be considered when investigating relationships between green space and physical activity.
Transit-related walking provides a potential opportunity to promote general walking behavior, yet few studies have examined this issue. Since people's decisions tend to vary as they walk between home and transit and between transit and destination, this study separated trips made in each direction.
This study identified the associations between socio-demographics and the two-step process of transit-related walking: 1) whether transit users walked for home-transit trip or transitdestination trip, and 2) the walking duration for home-transit trip or transit-destination trip among those who walked.
This cross-sectional study used the 2009 National Household Travel Survey and used the Heckman 2-step selection model by including 4,042 respondents (10,105 trips) who walked all portions for home-transit trip and 3,756 (8,075 trips) for transit-destination trip.
The mean walking duration for home-transit trips (7.60 minutes) was shorter than transit-destination trips (7.87 minutes). Hispanics were more likely to walk for both directions and had higher walking durations than did whites. Respondents living in low-income households were more likely to walk for home-transit trip, but not for transit-destination trips.
This study illustrated several implications regarding to transit-related walking, such as creating short home-transit distances and targeting whites in promoting transit-related walking.
The suitability of smartphone applications (Apps) currently used to track walking/running may differ depending on a person's weight condition. This study aimed to examine the validity and reliability of Apps for both normal-weight and overweight/obese young adults.
Thirty normal-weight (aged 21.7±1.0 years, BMI 21.3±1.9 kg/m(2)) and 30 overweight/obese young adults (aged 21.0±1.4 years, BMI 28.6±3.7 kg/m(2)) wore a smartphone and pedometer on their right hip while walking/running at 3 different intensities on treadmills. Apps was randomly assigned to each individual for measuring average velocity, step count, distance, and energy expenditure (EE), and these measurements were then analyzed.
The Apps were not accurate in counting most of the measured variables and data fell significantly lower in the parameters than those measured with standard-reference instruments in both light and moderate intensity activity among the normal-weight group. Among the overweight and obese group, the Apps were not accurate in detecting velocity, distance, or EE during either light or vigorous intensities. The percentages of mean difference were 30.1%-48.9%.
Apps may not have sufficient accuracy to monitor important physical parameters of human body movement. Apps need to be developed that can, in particular, respond differently based on a person's weight status.
Numerous studies have focused on the role of environments in promoting physical activity, but few studies have examined the specific locations where children are active and whether being active in these locations is associated with physical activity levels over time.
Self-reported locations of where physical activity occurred and physical activity measured via accelerometry were obtained for a cohort of 520 children in 5(th) and 6(th) grades. Latent class analysis was used to generate classes of children defined by the variety of locations where they were active (i.e., home, school grounds, gyms, recreational centers, parks or playgrounds, neighborhood, and church). Latent transition analyses were used to characterize how these latent classes change over time and to determine whether the latent transitions were associated with changes in physical activity levels.
Two latent classes were identified at baseline with the majority of children in the class labeled as 'limited variety'. Most children maintained their latent status over time. Physical activity levels declined for all groups, but significantly less so for children who maintained their membership in the 'greater variety' latent status.
Supporting and encouraging physical activity in a variety of locations may improve physical activity levels in children.
To examine whether accelerometer-measured PA-based reactivity was present in a nationally representative sample of U.S. children (6-11 yrs), adolescents (12-17 yrs) and adults (≥ 20 yrs).
Data from the 2003-2006 NHANES (N = 674, 6-85 years) were used. PA was assessed using the ActiGraph 7164 accelerometer, with PA assessed over 7 days of monitoring. Two PA metrics were assessed, including activity counts per day (CPD) and time spent in moderate-to-vigorous physical activity (MVPA). Evidence of reactivity was defined as a statistically significantly change in either of these two PA metrics from day 1 of monitoring to days 2 or 3, with day 1 of monitoring being a Monday.
Suggestion of reactivity was observed only for the adult population where CPD from days two and three (297140.6 ± 7920.3 and 295812.9 ± 8364.9), respectively, differed significantly from day one (309611.5 ± 9134.9) over the monitoring period (4.0 - 4.5% change). The analysis was conducted 2 additional times with differing start days (Tuesday and Wednesday), and this approach failed to demonstrated a reactive presence.
In this national sample of U.S. children, adolescents and adults, we did not observe sufficient evidence of accelerometer reactivity.
Physical inactivity in midlife women is associated with increased intra-abdominal adipose tissue development. We describe an innovative multimethod study 1) to better understand barriers to physical activity (PA) and 2) engage midlife women to product test physical activities and identify local community-based providers and sustainable and fun PA experiences.
Formative research on PA barriers from the Chicago site SWAN ancillary study of midlife women was utilized to develop a pilot testing measure. Feasibility, acceptability and sustainability of the PA activities were determined using the measure.
Desirable locations and/or instructors were identified. The first 2 groups identified, pilot tested, and then ranked activities for their ability to promote sustained PA. The six topranked were: circuit training, total body fitness, kickboxing, Zumba, Pilates and pedometer. The final group pilot tested highly ranked PA in two-week blocks, and ranked pedometer and Zumba in their top three.
Consensus was reached regarding activities that could be valuable in promoting sustained PA in midlife women. Choosing convenient sites and popular instructors further facilitates sustainability. Building relationships with key community partners is essential for sustainability. Community-based participant involvement in study design is a critical element in developing a healthy living intervention.
Nowadays, the majority of adolescents exceed the AAP guidelines for screen use and this is likely to be a risk factor for obesity. The present study aims at investigating adolescent screen viewing in the context of home and neighborhood environment.
A sample of 1141 adolescents as well as their parents participated in this survey. Adolescents were asked to complete a questionnaire about time spent on screen viewing behaviors. Respectively, parents completed a questionnaire concerning environmental predictors.
Almost 2/3 of the adolescents surveyed spend more than 2 hours per day on screen entertainment, with boys dealing with PCs and electronic games more than girls. The likelihood for an adolescent to exceed 2 hours of screen time is 3.87 times more when he has his meals in front of a TV screen on a daily basis, 1.69 times more when the TV is on, often as not on his return from school and 1.74 times more when there is a PC in the adolescent's bedroom.
Certain environmental predictors influence adolescents' screen time, as a result, corrective intervention should aim at the family as a whole, as this whole shapes home environment.
As a first line of defense against insect herbivores many plants store high concentrations of toxic and deterrent secondary metabolites in glandular trichomes. Plant Pleiotropic Drug Resistance (PDR)-type ABC transporters are known secondary metabolite transporters, and several have been implicated in pathogen or herbivore defense. Here, we report on Petunia hybrida PhPDR2 as a major contributor to trichome-related chemical defense. PhPDR2 was found to localize to the plasma membrane and be predominantly expressed in multicellular glandular trichomes of leaves and stems. Down-regulation of PhPDR2 via RNA interference (pdr2) resulted in a markedly higher susceptibility of the transgenic plants to the generalist foliage feeder Spodoptera littoralis. Untargeted screening of pdr2 trichome metabolite contents showed a significant decrease in petuniasterone and petuniolide content, compounds, which had previously been shown to act as potent toxins against various insects. Our findings suggest that PhPDR2 plays a leading role in controlling petuniasterone levels in leaves and trichomes of petunia, thus contributing to herbivory resistance.
To examine the combined effects of body mass index (BMI), physical activity (PA) and sitting on incident type 2 diabetes mellitus (T2DM) among Australian adults.
A sample of 29,572 adults aged ≥45 years from New South Wales, Australia, completed baseline (2006-2008) and follow-up (2010) questionnaires. Incident T2DM was defined as self-reported, physician-diagnosed diabetes at follow-up. BMI was categorised as normal/overweight/obese. PA was tertiled into low/medium/high. Sitting was dichotomised as higher/lower sitting (≥ or <8 hours/day). Odds ratios (OR) were estimated for developing T2DM using logistics regression for individual and combined risk factors, and data stratified by BMI categories.
During a mean 2.7 (SD: 0.9) years of follow-up, 611 (2.1%) participants developed T2DM. In fully adjusted models, BMI was the only independent risk factor for incident T2DM. In stratified analyses, the association between BMI and T2DM did not differ significantly across sitting or PA categories. Overweight/obese individuals with high PA and lower sitting had higher odds of incident T2DM than normal counterparts with low PA and higher sitting.
High PA/low sitting did not attenuate the risk of T2DM associated with overweight/obesity. Maintaining a healthy weight, by adopting healthy lifestyle behaviours, is critical for T2DM prevention.
This study aims to identify patterns of use and preferences related to technology platforms that could support physical activity (PA) programs in an underserved population.
A 29-item questionnaire was administered at five health and wellness sites targeting low income communities in Chicago. Frequency tables were generated for Internet, cell phone, and social media use and preferences. Chi-squared analysis was used to evaluate differences across age and income groups.
A total of 291 individuals participated and were predominantly female (69.0%). Majority reported incomes less than $30,000 (72.9%) and identified as African American/Black/Caribbean (49.3%) or Mexican/Mexican American (34.3%). Most participants regularly used smartphones (63.2%) and the Internet (75.9%). Respondents frequently used Facebook (84.8%), and less commonly used Instagram (43.6%), and Twitter (20.0%). Free Internet-based exercise programs were the most preferred method to increase PA levels (31.6%), while some respondents (21.0%) thought none of the surveyed technology applications would help.
Cell phone, Internet, and social media use is common among the surveyed underserved population. Technology preferences to increase PA levels varied, with a considerable number of respondents not preferring the surveyed technology platforms. Creating educational opportunities to increase awareness may maximize the effectiveness of technology-based PA interventions.
It is currently not known how much walking should be advocated for good health in adolescent girls. The aim of this study was therefore to recommend health referenced standards for step defined physical activity relating to appropriate health criterion/indicators in a group of adolescent girls.
Two hundred and thirty adolescent girls aged between 12-15years volunteered to take part in the study. Each participant undertook measurements (BMI, waist circumference, % body fat and blood pressure) to define health status. Activity data were collected by pedometer and used to assess daily step counts and accumulated daily activity time over seven consecutive days.
Individuals classified as 'healthy' did not take significantly more steps·day(-1) nor spend more time in moderate intensity activity than individuals classified as at health risk or with poor health profiles.
'Healthy' adolescent girls do not walk significantly more in term of steps·day(-1) or time spent in activity than girls classified as 'unhealthy'. This could suggest that adolescent girls may not walk enough to stratify health and health related outcomes and as a result the data could not be used to inform an appropriate step guideline for this population.
This study examined associations of perceived and objectively-measured neighborhood environmental attributes with leisure-time sitting for transport among middle-to-older aged Japanese adults.
Data were collected using a postal survey of 998 adults aged 40-69 years. Generalized linear modelling with a gamma distribution and a log link was used to examine associations of perceived (International Physical Activity Questionnaire-Environmental module) and Geographic Information Systems (GIS)-derived built environment attributes with self-reported leisure-time sitting for transport.
Mean leisure-time sitting time for transport was 20.4 min/day. After adjusting for potential confounders, perceived higher residential density, GIS-measured higher population density, better access to destinations, better access to public transport, longer sidewalk length, and higher street connectivity, were associated significantly with lower sitting time for transport.
Residents living in neighborhoods with attributes previously found to be associated with more walking tended to spend less time sitting for transport during leisure-time. The health benefits of walkability-related attributes may accrue not only through increased physical activity, but also through less sedentary time.
The studyaim was to assess the physical activity levels as well as the intention to become physically active in patients with stable coronary heart disease (CHD) with a special focus on the association with their risk profile.
Analyses are based on the cross-sectional EUROASPIRE IV surveys. Information was available on 8966 patients in EUROASPIRE III and on 7998 coronary patients was available in EUROASPIRE IV. Physical activity level according to patients risk profile and medical management was assessed, the intention to become physically active was investigated and a time trend analysis was performed.
A better cardiovascular risk profile as well as receiving physical activity advice or weight loss advice was associated with better physical activity levels. The physical activity status improved significantly over time, the proportion of patients reporting vigorous physical activity for at least 20 minutes ≥ 3 times/week increased from 14.1% to 20.2% (p<0.001). Similarly, a significantly greater proportion of patients are in the maintenance stage (36.6% vs. 27.4%) and a smaller proportion in the precontemplation stage (43.2% vs. 52.3%.).
Although an increase was seen in the proportion of patients being adequately physical active, physical activity levels remain suboptimal in many CHD patients.
Promoting regular physical activity (PA) and lessening sedentary behaviors (SB) constitute a public health priority. Recent evidence suggests that PA and SB are not only related to reflective processes (e.g., behavioral intentions), but also to impulsive approach-avoidance tendencies (IAAT). This study aims to test the effect of a computerized IAAT intervention on an exercise task.
Participants (N = 115) were randomly assigned to one of three experimental conditions, in which they were either trained to approach PA and avoid SB (ApPA-AvSB condition), to approach SB and avoid PA (ApSB-AvPA condition), or to approach and avoid PA and SB equally often (active control condition). The main outcome variable was the time spent carrying out a moderate intensity exercise task.
IAAT towards PA decreased in the ApSB-AvPA condition, tended to increase in the ApPA-AvSB condition, and remained stable in the control condition. Most importantly, the ApPA-AvSB manipulation led to more time spent exercising than the ApSB-AvPA condition. Sensitivity analyses excluding individuals who were highly physically active further revealed that participants in the ApPA-AvSB condition spent more time exercising than participants in the control condition.
These findings provide preliminary evidence that a single intervention session can successfully change impulsive approach tendencies towards PA and can increase the time devoted to an exercise task, especially among individuals who need to be more physically active. Potential implications for health behavior theories and behavior change interventions are outlined.
The purpose of this study was to examine changes in physical activity and healthy eating knowledge and behaviors associated with the level of exposure to POWERPLAY, a men-centered workplace health promotion program.
This study is based on a quasi-experimental pre-post design. Using a computer assisted telephone interview survey, data regarding program exposure and physical activity and health eating knowledge and behaviors were collected from men (N=103) in 4 workplaces.
Exposure scores were calculated and participants were categorized as having low (n=54) or high exposure (n=49) to POWERPLAY. Compared to the low exposure group, those reporting high exposure scored significantly higher on physical activity knowledge (F (1, 99) =14.17, p < .001, eta(2) = .125) and health eating knowledge (F (1, 99) =14.37, p = .001, eta(2) = .111). The high exposure group also reported significantly more minutes walked place to place (F (2, 206) = 3.91, p = .022, eta(2) = .037) and on minutes walked for leisure (F (2, 230) = 3.08, p = .048, eta(2) = .026).
POWERPLAY shows significant promise as a workplace health promotion approach and may have an even greater impact when program exposure is augmented with environmental and policy changes.
School districts in the United States are turning towards new sources of revenue to maintain their interscholastic sports programs. One common revenue generating policy is the implementation of participation fees, also known as pay-to-play. One concern of the growing trend of participation fees is how it impacts student participation opportunities. This study looks at how pay-to-play fees are impacting participation opportunities and participation rates in the state of Michigan.
Through merging three school-level data sets, Civil Rights Data Collection, the Common Core of Data, and participation information from MHSAA (Michigan High School Athletic Association), bivariate analysis and ordinary least squares regression were utilized in our analysis.
Our findings indicate that certain types of schools are able to support pay-to-play fees: relatively large schools that are located in suburban, white communities, with relatively low poverty rates. We also found that participation fees are not decreasing the number of sport opportunities for students, participation opportunities are higher in schools with fees; but participation rates are similar between schools with and without participation fees.
Participation fee policy implications are discussed and we offer suggestions for future research.
Harmful health effects associated with sedentary behaviour may be attenuated by breaking up long periods of sitting by standing or walking. However, studies assess interruptions in sitting time differently, making comparisons between studies difficult. It has not previously been described how the definition of minimum break duration affects sitting outcomes. Therefore, the aim was to address how definitions of break length affect total sitting time, number of sit-to-stand transitions, prolonged sitting periods and time accumulated in prolonged sitting periods among office workers.
Data were collected from 317 office workers. Thigh position was assessed with an ActiGraph GT3x+ fixed on the right thigh. Data were exported with varying bout length of breaks. Afterwards, sitting outcomes were calculated for the respective break lengths.
Absolute numbers of sit-to-stand transitions decreased, and number of prolonged sitting periods and total time accumulated in prolonged sitting periods increased, with increasing minimum break length. Total sitting time was not influenced by varying break length.
The definition of minimum break length influenced the sitting outcomes with the exception of total sitting time. A standard definition of break length is needed for comparison and interpretation of studies in the evolving research field of sedentary behaviour.
The role of parents' perceptions of the neighborhood environment in determining children's active commuting to and from school (ACS) is understudied. This study examined the association between parents' perceptions of neighborhood social cohesion, perceived neighborhood safety, and their children's ACS.
This cross-sectional analysis (n=857 from 81 elementary schools in Texas) examined baseline data from the Texas Childhood Obesity Prevention Policy Evaluation project. Participants had a mean age of 9.6 (0.6) years, and 50% were girls. Mixed effects logistic regression models were used to assess gender-stratified associations between parent's perceived social cohesion and children's ACS and their perception of neighborhood safety.
A positive significant association was observed between levels of perceived social cohesion and children's ACS for boys (p-value=0.047); however, an inverse significant association was observed among girls (p-value=0.033). Parents of boys living in neighborhoods with medium to high social cohesion were more likely to perceive their neighborhood as safe compared to parents living in neighborhoods with low social cohesion, though non-significant. Perceived neighborhood safety for walking and biking was associated with greater ACS among boys (p-value=0.003).
Our study findings indicate that the both social and physical environments are important factors in determining ACS among boys.
There is little evidence on prevalence or risk factors for soil transmitted helminth infections in Timor-Leste. This study describes the epidemiology, water, sanitation and hygiene, and socioeconomic risk factors of STH and intestinal protozoa amongst communities in Manufahi District, Timor-Leste. As part of a cluster randomised controlled trial, a baseline cross-sectional survey was conducted across 18 villages, with data from six additional villages. Stool samples were assessed for soil transmitted helminth and protozoal infections using quantitative PCR (qPCR) and questionnaires administered to collect water, sanitation and hygiene and socioeconomic data. Risk factors for infection were assessed using multivariable mixed-effects logistic regression, stratified by age group (preschool, school-aged and adult). Overall, soil transmitted helminth prevalence was 69% (95% Confidence Interval 67-71%), with Necator americanus being most common (60%; 95% Confidence Interval 58-62%) followed by Ascaris spp. (24%; 95% Confidence Interval 23-26%). Ascaris-N. americanus co-infection was common (17%; 95% Confidence Interval 15%-18%). Giardia duodenalis was the main protozoan identified (13%; 95% Confidence Interval 11-14%). Baseline water, sanitation and hygiene infrastructure and behaviours were poor. Although risk factors varied by age of participants and parasite species, risk factors for N. americanus infection included, generally, age in years, male sex, and socioeconomic quintile. Risk factors for Ascaris included age in years for children, and piped water to the yard for adults. In this first known assessment of community-based prevalence and associated risk factors in Timor-Leste, soil transmitted helminth infections were highly prevalent, indicating a need for soil transmitted helminth control. Few associations with water, sanitation and hygiene were evident, despite water, sanitation and hygiene being generally poor. In our water, sanitation and hygiene we will investigate implications of improving WASH on soil transmitted helminth infection in impoverished communities.
PDRG1, is short for P53 and DNA damage-regulated gene, which have been found over 10 years. Although severe studies have described the roles of PDRG1 separately in many kinds of tumors, how to act as an oncogene are unclear. To better verify the function of PDRG1 in lung cancer, both loss-function and gain-function of PDRG1 studies based on two human lung cancer lines were performed. Following the transfection of PDRG1, both A549 and 95-D cells showed significant changes in cell viability, the expression of some protein and apoptosis, which were all implied the PDRG1 is an oncogene. Another interesting finding is PDRG1 could promote radioresistance involved the ATM-p53 signaling pathway in lung cancer. If we combine radiotherapy with gene-targeted therapy together effectively, predominant effect may be acquired, which is a huge milestone in clinical cure about lung cancer.
Regulatory T cells (Tregs) play pivotal roles in limiting the duration and magnitude of immune response against infectious agents and self-antigens. This is accomplished through contact-dependent and -independent mechanisms that involve crosstalk between Treg cells and other immune and tissue-specific cell types. The same machinery is employed by Tregs to regulate immune responses to cancer, limiting both pro-tumor inflammation and anti-tumor immunity. Factors produced by Treg cells also act directly on transformed epithelial cells and exert opposing effects during different stages of cancer development. Therefore, the immune regulatory cell population serves as a double-edged sword for the development, progression, and treatment of cancers. In this review, we summarize current knowledge on the roles of Treg lymphocytes during cancer development, as well as the underlying cellular and molecular mechanism.
Reduced mitochondrial function is an important cause of aging and age-related diseases. We previously revealed a relatively higher level of mitochondrial DNA (mtDNA) content in centenarians. However, it is still unknown whether such an mtDNA content pattern of centenarians could be passed on to their offspring and how it was regulated. To address these issues, we recruited 60 longevity families consisting of 206 family members (cohort 1) and explored their mtDNA copy number. The results showed that the first generation of the offspring (F1 offspring) had a higher level of mtDNA copy number than their spouses (p < 0.05) independent of a gender effect. In addition, we found a positive association of mtDNA copy number in centenarians with that in F1 offspring (r = 0.54, p = 0.0008) but not with that in F1 spouses. These results were replicated in another independent cohort consisting of 153 subjects (cohort 2). RNA sequencing analysis suggests that the single-stranded DNA-binding protein 4 was significantly associated with mtDNA copy number and was highly expressed in centenarians as well as F1 offspring versus the F1 spouses, thus likely regulates the mtDNA copy number in the long-lived family members. In conclusion, our results suggest that the pattern of high mtDNA copy number is likely inheritable, which may act as a favorable factor to familial longevity through assuring adequate energy supply.
Previous studies indicated that reduced androgen levels may contribute to both physical and cognitive disorders in men, including Alzheimer's disease. New drug candidates for Alzheimer's disease in patients with androgen deficiency should ideally be able to act not only on multiple brain targets but also to correct impaired endocrine functions in hypogonadal men with Alzheimer's disease. Ropren(®) is one such candidate for the treatment of Alzheimer's disease in men with an imbalance of androgens. Accordingly, the aim of the current study was to examine the effects of long-term Ropren(®) administration (8.6mg/kg, orally, once daily, for 28 days) on the anxiety-like behavior and monoamines levels in the rat hippocampus using a β-amyloid (25-35) rat model of Alzheimer's disease following gonadectomy. Ropren(®) was administered to the gonadectomized (GDX) rats and GDX rats treated with testosterone propionate (TP, 0.5mg/kg, subcutaneous, once daily, for 28 days). Anxiety-like behavior was assessed in the elevated plus maze (EPM) and the light-dark test (LDT), locomotor and grooming activities were assessed in the open field test (OFT). Ropren(®) alone or in combination with TP-induced anxiolytic effects as evidenced in the EPM and in the LDT and increased locomotor activity in the OFT. Additionally, it was observed that dopamine (DA) and serotonin (5-HT) levels increased while 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in the hippocampus decreased. Our results indicate that Ropren(®) has a marked anxiolytic-like action due to an increase in the monoamines levels in the experimental rat model of Alzheimer's disease with altered levels of androgens.
HBV infection has been reported to be closely associated with HCC development; however, the underlying mechanisms are unclear. Emerging evidence has indicated that long non-coding RNAs (lncRNAs) play important regulatory roles in the pathogenesis and progression of cancers. To investigate the important role and mechanism of lncRNAs in the progression of HBV-related HCC, we screened lncRNAs in HBV-positive and HBV-negative HCC tissues. We identified a novel lncRNA, lncRNA-Unigene56159, which is highly expressed in HBV-related HCC tissues, and further analysis showed that this lncRNA was induced by HBV in vitro. Functionally, Unigene56159 significantly promoted cell migration/invasion and epithelial-mesenchymal transition (EMT) in HCC. Mechanistically, Unigene56159 could directly bind to miR-140-5p and effectively act as a competing endogenous RNA (ceRNA) for miR-140-5p to de-repress the expression of the target gene Slug. Collectively, our findings indicate that the Unigene56159/miR-140-5p/Slug axis contributes to HCC cell migration and invasion, which may provide novel insights into the function of lncRNA-driven hepatocarcinogenesis.
Incidental and secondary findings have become an important by-product of diagnostic testing, and their ramifications affect clinical care, research, and policy. Given parallels in the reporting and management of such findings on diagnostic imaging, radiologists may draw from ongoing discussions in medical genetics to rethink more patient-centered approaches to analogous clinical, ethical, and medicolegal dilemmas. Low-risk incidental findings in particular may be drivers of unnecessary testing, invasive procedures, and overtreatment, with associated financial, psychological, and clinical consequences. As radiologists act in patients' best interests by strengthening standardized guidelines on how each finding merits further diagnostic testing or treatment, perhaps the greatest challenge for producing such guidelines is for low-risk incidental findings, for which adverse consequences are unlikely but associated with substantial uncertainty because of the lack of strong evidence on which to base the recommendations. More uniform recommendations for managing low-risk radiologic incidental findings should therefore aim to provide reasonable options that apply across a spectrum of patient preferences. These will require evaluation through research and will ultimately influence the quality of care. Specific areas for exploration may include (1) better gauging of patient attitudes and preferences regarding low-risk incidental findings, (2) using patient preferences to inform more uniform recommendations for low-risk findings that apply across a spectrum of preferences and help guide shared decision making, and (3) when patients endorse a strong preference not to discover low-risk incidental findings, how it might be possible for professional standards to curtail their generation in specific circumstances.
Because of the broad incidence, morbidity and mortality associated with prostate-derived cancer, the development of more effective new technologies continues to be an important goal for the accurate detection and treatment of localized prostate cancer, lymphatic involvement and metastases. Prostate-specific membrane antigen (PSMA; Glycoprotein II) is expressed in high levels on prostate-derived cells and is an important target for visualization and treatment of prostate cancer. Radiolabeled peptide targeting technologies have rapidly evolved over the last decade and have focused on the successful development of radiolabeled small molecules that act as inhibitors to the binding of the N-acetyl-l-aspartyl-l-glutamate (NAAG) substrate to the PSMA molecule. A number of radiolabeled PSMA inhibitors have been described in the literature and labeled with SPECT, PET and therapeutic radionuclides. Clinical studies with these agents have demonstrated the improved potential of PSMA-targeted PET imaging agents to detect metastatic prostate cancer in comparison with conventional imaging technologies. Although many of these agents have been evaluated in humans, by far the most extensive clinical literature has described use of the (68)Ga and (177)Lu agents. This review describes the design and development of these agents, with a focus on the broad clinical introduction of PSMA targeting motifs labeled with (68)Ga for PET-CT imaging and (177)Lu for therapy. In particular, because of availability from the long-lived (68)Ge (T1/2=270days)/(68)Ga (T1/2=68min) generator system and increasing availability of PET-CT, the (68)Ga-labeled PSMA targeted agent is receiving widespread interest and is one of the fastest growing radiopharmaceuticals for PET-CT imaging.
RECQ1, the most abundant one of the human RecQ helicases family, has been identified as a prometastasis gene in breast and cervical cancers. However, the effects of RECQ1 on non-small cell lung cancer (NSCLC) and the underlying molecular mechanisms are still unclear. In the present study, RECQ1 expression (in three NSCLC cell lines and one bronchial epithelial cell line) was detected by real-time quantitative PCR (RT-qPCR). Expression of RECQ1 in A549 cells was knocked down by lentivirus-mediated RNA interference technique (RNAi). The effects of RECQ1 knockdown on cell proliferation, migration and invasion were assessed by Cell Counting Kit-8 (CCK-8) assay and transwell assays. Epithelial-mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin as well as vimentin) were detected by RT-qPCR and western blotting analyses. We found that RECQ1 expression was significantly higher in three NSCLC cell lines than that in a normal human bronchial epithelial cell line. Knocking down RECQ1 significantly suppressed A549 cell proliferation, migration and invasion. The expressions of the epithelial marker, E-cadherin were elevated in both mRNA and protein levels, whereas the expressions of the mesenchymal markers, N-cadherin and vimentin were decreased. Taken together, our findings suggest that RECQ1 may act as an important mediator in promoting lung cancer progression via modulation of the EMT. RECQ1 might represent a potential therapeutic target in NSCLC.
To present the treatment patterns, disease activity, and visual outcomes of eyes in the maintenance phase of a treat-and-extend regimen for neovascular age-related macular degeneration (nAMD). To compare the maintenance phase behavior of eyes with a shorter induction phase (≤3 injections) with those requiring a longer induction phase (>3 injections).
Database observational study.
Eyes with nAMD receiving anti-vascular endothelial growth factor (VEGF) treatment using a treat-and-extend protocol. Persistently active eyes were excluded, as were eyes with <12 months follow-up during the maintenance phase.
Clinical information from a large prospective international voluntary registry of nAMD was analyzed. The maintenance phase was defined as starting at the first clinician-reported grading of lesion inactivity.
For analyses by eye: treatment interval at first reactivation; time to first reactivation; and visual acuity change during the study period. For analyses by visit: choroidal neovascular membrane activity graded by the treating physician; time since previous injection; and visual acuity loss since previous injection (>0 letters and ≥15 letters).
The mean change in visual acuity during the maintenance phase was +1.0 letters at 12 months -0.6 letters at 24 months and -1.5 at 36 months. Median treatment interval increased from 35 days at study entry to 63 days at 12 months and was 60 days at 36 months. 38.5% of eyes remained inactive at all observed visits during the maintenance phase (minimum 1 year follow-up, mean 945 days). The most common treatment interval at first reactivation was 8 weeks. Treatment intervals beyond 12 weeks seemed to be associated with increased risk of disease reactivation, with risk of reactivation reaching 37.4% at treatment intervals of ≥20 weeks. Eyes with a longer induction phase had worse visual outcomes in the maintenance phase, and earlier and more-frequent disease reactivation, although they received injections less frequently.
The detailed behavior of eyes in the maintenance phase of treat-and-extend management for nAMD is presented. Visual acuity was well maintained during the study period. The most common interval at which reactivation first occurred was 8 weeks. Longer duration of induction phase was associated with worse visual acuity outcomes and earlier disease reactivation, perhaps because of undertreatment.
Diaporine is a novel fungal metabolic product and has an unprecedented symmetric polyketide with a novel framework from diaporthe sp. In the current study, we researched the anti-cancer role of this natural compound. Firstly, we evaluate the sensitivities of diaporine in several cancer cells and the results show that diaporine has proliferation-inhibitory effect against cancer cells via MTT assay, especially on breast cancer cells. In addition, we find that diaporine can disrupt the morphology and viability of breast cancer cells and their migration and invasion capacities are also impaired tested by using wound healing method and transwell. Further investigation show that diaporine can efficiently induce an intensive breast cancer cell cycle arrest especially in G2 phase and finally leads to cancer cell apoptosis by flow cytometer analysis. For the mechanism by which mediated the breast cancer cell death, we find that diaporine can induce an increase of ROS level in breast cancer cells. When depressing ROS generation by using anti-oxidant agent N-acetyl-l-cysteine or Vitamin C, the apoptosis effects of breast cancer cells are disrupted. Collectively, our results suggest that diaporine can act as an anti-cancer agent and this function is dependent on promoting high level ROS release of cancer cells.
Liver resection has been associated with high morbidity and mortality, and the most serious complication is liver failure. Patient evaluation is limited to risk scales. The 50-50 criteria and bilirubin peak>7mg/dl have been used as mortality predictors.
The aim of this study was to determine the risk factors associated with morbidity and mortality for liver resection in our population.
A retrospective study was carried out on 51 patients that underwent liver resection. Sociodemographic variables, pathology, and the surgical act were analyzed, together with morbidity and mortality and their associated factors.
Fifty-one patients, 23 men and 28 women, were analyzed. They had a mean age of 51.4±19.13 years, 64.7% had concomitant disease, and their mean MELD score was 7.49±1.79. The mean size of the resected lesions was 7.34±3.47cm, 51% were malignant, and 34 minor resections were performed. The Pringle maneuver was used in 64.7% of the cases and the mean blood loss was 1,090±121.76ml. Morbidity of 25.5% was associated with viral hepatitis infection, greater blood loss, transfusion requirement, the Pringle maneuver, lower hemoglobin and PTT values, and higher MELD, INR, bilirubin, and glucose values. A total 3.9% mortality was associated with hyperbilirubinemia, hyperglycemia, and greater blood loss and transfusions.
The main risk factors associated with the morbidity and mortality of liver resection in our population were those related to the preoperative biochemical parameters of the patient and the factors that occurred during the surgical act.
Poor quality of sleep is frequent in euthymic bipolar patients and conveys worse clinical outcomes. We investigated the features of euthymic bipolar patients associated with poor sleep quality, with a focus on the effect of childhood trauma.
493 euthymic patients with DSM-IV-defined bipolar disorders were recruited in FondaMental Advanced Centers of Expertize for Bipolar Disorders (FACE-BD) between 2009 and 2014. Clinical variables were recorded. Subjective sleep quality and history of childhood trauma were respectively measured by the Pittsburgh Sleep Quality Index (PSQI) and the Childhood Trauma Questionnaire (CTQ).
Poor sleepers were older, less professionally active, had significantly higher anxiety levels, took more anxiolytic drugs and did endorse more suicide attempts and suicidal ideas than good sleepers after adjusting for anxiety levels and age. Emotional abuse was associated with poor sleep quality after adjustment for BMI, age, professional activity, and bipolar disorders (BD) type (OR=1.83; 95% CI [1.30; 3.10]; p=0.02). However, this association was lost after adjustment for anxiety levels, anxiolytic treatment and suicide ideation/attempts.
The main limitation was the type of sleep assessment, which only measured the subjective part of sleep complaints.
A history of emotional abuse might underlie sleep problems in many bipolar patients but anxiety seems to act as a confounding factor in this relationship. New studies are needed to elucidate the role of childhood maltreatment on poor sleep among bipolar patients.
MicroRNAs (miRNAs) play an important role in the development of various cancers, including cervical cancer (CC). The dysregulation of miRNA expression is associated with oncogenic transformation and miRNA often act as tumor suppressors. In this study, we aimed to analyze the effect on and mechanism of miR-139-3p in the progression of CC. The result of real-time PCR showed that miR-139-3p was down-regulated in CC tissues and cell lines. Overexpression of miR-139-3p significantly suppressed HeLa cell proliferation, migration and invasion and induced cell apoptosis. Bioinformatics analysis and luciferase reporter gene assay confirmed that NOB1 was targeted by miR-139-3p at the 3'-untranslated region (3'UTR) of its mRNA sequence. Furthermore, overexpression of NOB1 counteracted the effects of miR-139-3p suppression. Our results suggest that miR-139-3p may act as a tumor suppressor that can inhibit CC cell proliferation, migration and invasion and induce cell apoptosis through down-regulation of NOB1 expression. Taken together, this study provides a novel potential therapeutic strategy for the treatment of CC.
MicroRNA-622 has been proven down-regulated in many human malignancies and correlated with tumor progression. However, its role in esophageal squamous cell carcinoma (ESCC) is still unclear. The aim of this study was to explore the expression and function of miR-622 in ESCC.
Using quantitative RT-PCR, we detected miR-622 expression in ESCC cell lines and primary tumor tissues. The association of miR-622 expression with clinicopathological factors and prognosis was also analyzed. Then, the effects of miR-622 on the biological behavior of ESCC cells were investigated. At last, the potential regulatory function of miR-622 on E2F1 expression was confirmed.
miR-622 was found to be down-regulated in ESCC tissues and cell lines. Decreased miR-622 expression was closely correlated with aggressive clinicopathological features and poor overall survival. Multivariate regression analysis corroborated that low level of miR-622 expression was an independent unfavourable prognostic factor for patients with ESCC. Up-regulation of miR-622 could significantly reduce ESCC cell proliferation, enhance cell apoptosis, and impair cell invasion and migration in vitro, while down-regulation of miR-622 showed opposite effects. Further, E2F1 was confirmed as a direct target of miR-622 by using Luciferase Reporter Assay.
These findings indicate that miR-622 may act as a tumor suppressor in ESCC and would serve as a potential therapy target for this disease.
Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H2O2), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H2O2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H2O2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H2O2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg(-1) FW range. Commonly used additives for H2O2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue.
Kindler syndrome (KS) is an autosomal recessive blistering skin disease resulting from pathogenic mutations in FERMT1. This gene encodes kindlin-1, a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective cell adhesion and migration. Electric fields also act as intrinsic regulators of adhesion and migration in the skin, but the molecular mechanisms by which this occurs are poorly understood. Here we show that keratinocytes derived from KS patients are unable to undergo electrotaxis, and this defect is restored by overexpression of wild-type kindlin-1 but not a W612A mutation that prevents kindlin-integrin binding. Moreover, deletion of the pleckstrin homology domain of kindlin-1 also failed to rescue electrotaxis in KS cells, indicating that both integrin and lipid binding are required for this function. Kindlin-1 was also required for the maintenance of lamellipodial protrusions during electrotaxis via electric field-activated β1 integrin. Indeed, inhibition of β1 integrins also leads to loss of electrotaxis in keratinocytes. Our data suggest that loss of kindlin-1 function may therefore result in epithelial insensitivity to electric fields and contribute to KS disease pathology.
Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organisms.
The key role of RUNX3 in physiological T-cell differentiation has been extensively documented. However, information on its relevance for the development of human T-cell lymphomas or leukemias is scarce. Here, we show that alterations of RUNX3 by either heterozygous deletion or methylation of its distal promoter can be observed in the tumor cells of 15 of 21 (71%) patients suffering from Sézary syndrome, an aggressive variant of cutaneous T-cell lymphoma. As a consequence, mRNA levels of RUNX3/p46, the isoform controlled by the distal promoter, are significantly lower in Sézary syndrome tumor cells. Re-expression of RUNX3/p46 reduces cell viability and promotes apoptosis in a RUNX3/p46(low) cell line of cutaneous T-cell lymphoma. Based on this, we present evidence that RUNX3 can act as a tumor suppressor in a human T-cell malignancy and suggest that this effect is predominantly mediated through transcripts from its distal promoter, in particular RUNX3/p46.
Previous studies have examined participation in specific leisure-time physical activities (PA) among US adults. The purpose of this study was to identify specific activities that contribute substantially to total volume of leisure-time PA in US adults.
Proportion of total volume of leisure-time PA moderate-equivalent minutes attributable to 9 specific types of activities was estimated using self-reported data from 21,685 adult participants (≥ 18 years) in the National Health and Nutrition Examination Survey 1999-2006.
Overall, walking (28%), sports (22%), and dancing (9%) contributed most to PA volume. Attributable proportion was higher among men than women for sports (30% vs. 11%) and higher among women than men for walking (36% vs. 23%), dancing (16% vs. 4%), and conditioning exercises (10% vs. 5%). The proportion was lower for walking, but higher for sports, among active adults than those insufficiently active and increased with age for walking. Compared with other racial/ethnic groups, the proportion was lower for sports among non- Hispanic white men and for dancing among non- Hispanic white women.
Walking, sports, and dance account for the most activity time among US adults overall, yet some demographic variations exist. Strategies for PA promotion should be tailored to differences across population subgroups.
Although individual studies have reported on the number of steps/day taken by individuals with COPD, this evidence has not been systematically reviewed or synthesized.
MEDLINE and PsycINFO were searched for studies reporting objectively-measured steps/day and percent predicted forced expiratory volume in one second (FEV1%) in patients with COPD. Meta-analyses were used to estimate steps/day across studies, while meta-regression was used to estimate between-study variance based on clinical and demographic factors (year and location of study, activity monitor brand, number of days wearing the monitor, whether participants were about to enter pulmonary rehabilitation, 6 minute walk distance (6MWD), FEV1%, age, and sex).
38 studies including 2621 participants met inclusion criteria. The pooled mean estimate was 4579 steps/day (95% CI:4310-5208) for individuals with COPD. Only 6MWD, FEV1% and whether or not patients were about to undergo pulmonary rehabilitation explained a significant portion of the variance (p<0.1) in univariate meta-regression. In a multivariate model including the above risk factors, only FEV1% was associated with steps/day after adjustment for other covariates.
These results indicate that patients with COPD achieve extremely low levels of physical activity as assessed by steps/day, and that severity of airflow obstruction is associated with activity level.
Physical activity level is an important tool to identify individuals predisposed to developing chronic diseases, which represent a major concern worldwide.
To identify correlates of daily step counts measured using pedometers, as well as analyze the associations between health outcomes and three different amounts of daily physical activity.
The sample comprised 278 participants with a mean age of 46.51±9.02 years, of both sexes (126 men and 153 women). Physical activity was assessed using pedometers for seven consecutive days and three amounts of daily physical activity were considered: ≥10,000 steps/day, ≥7,500 steps/day and <5,000 steps/day. Sleep quality was assessed through a questionnaire and Dual Energy X-Ray Absorptiometry was used to measure body fatness. Sociodemographic and anthropometric data were also collected.
The percentages of adults achieving at least 10,000 and 7,500 steps/day on a minimum of five days of the evaluated week were 12.9% and 30.9%, respectively. Adults who reached ≥7,500 steps/day presented a lower likelihood of being obese (OR= 0.38 [0.17-0.85]) and reported worse sleep quality (OR= 0.58 [0.34-0.99]), while reaching <5,000 steps/day was associated with a higher likelihood of worse sleep quality (OR= 2.11 [1.17-3.82]).
Physical activity measured by pedometer in adulthood constituted a behavior related to lower adiposity and better sleep quality.
There are few studies on determinants of cardiorespiratory fitness (CRF) among girls and boys separately in population samples of children.
We therefore investigated the determinants of CRF, assessed by maximal workload per height using allometric scaling, in a population sample of 162 girls and 177 boys aged 6-8 years using automated bootstrap feature selection and linear regression models.
The strongest determinants of CRF among girls were maximal heart rate (HR) (standardized regression coefficient ß=0.31, p<0.001), unsupervised physical activity (ß=0.29, p <0.001), lean body mass (ß=0.23, p=0.001) and errors in static balance test (ß=-0.16, p=0.02) accounting altogether for 25.7% of variation in CRF. In boys, unsupervised physical activity (ß=0.24, p<0.001), resting HR (ß=-0.25, p<0.001), hand grip strength (ß=0.21, p=0.001), errors in static balance test (ß=-0.16, p=0.01), organized football (ß =0.16, p=0.01) and unsupervised trampoline jumping (ß=0.14, p=0.04) were the strongest determinants of CRF accounting altogether for 29.7% of variation in CRF.
These findings suggest that unsupervised physical activity is sufficient in improving CRF in both genders. Furthermore, larger muscle mass and better balance are associated with higher CRF that has to be taken into account when assessing CRF using maximal cycle ergometer exercise test among children.
Screen-time of adolescents has been associated with obesity and other adverse health outcomes; however, little is known about screen-based behaviours in Asian adolescents. The purpose of this study was to describe the prevalence, patterns and correlates of recreational screen-based behaviours among adolescents in Bangladesh.
A total of 758 students, aged 13-16 years and 52% girls, from eight secondary schools of Dhaka city, Bangladesh completed a survey where the Adolescent Sedentary Activity Questionnaire was used to collect information on screen-time. Total screen-time was categorised as ≤2 h/day (low) or >2 h/day (high).
About 79% of the adolescents had high recreational screen-time, with similar proportions between boys (78%) and girls (80%). Median reported recreational screen-time was 4.0 h/day with longer times among boys (4.3 h/day) than girls (3.6 h/day). Multivariable analyses showed that high screen-time was more common among boys than girls, and positively associated with commuting to school by car, consumption of fast food ≥3 times/week, having sleep disturbance, and high family income.
This study identified high rates of recreational screen-time among urban adolescents in Bangladesh and specific correlates of prolonged screen-time, and underscores the needs for developing pragmatic strategies to reduce sedentariness among adolescents in Bangladesh.
The present study compared the effects of a single yoga group exercise class to a resistance group exercise class on state body satisfaction and social physique anxiety in women.
A pre-test post-test design was used. Participants (N = 46) completed both a resistance and yoga group exercise class, in a counter-balanced order. Measures of body satisfaction and social physique anxiety were completed immediately before and after each class.
A 2 (time) x 2 (class type) repeated-measures multiple analysis of variance showed a significant overall time x class type interaction, F(2, 44) = 5.69, p < .01, ƞp(2) = .21. There was a significant increase in body satisfaction following the yoga class. Following both classes, there was a significant decrease in social physique anxiety but the magnitude of the changes was larger following the yoga class than the resistance class.
Both types of exercise class were associated with improvements in body image, with greater improvements following the yoga class. This study provided evidence of the positive effects of yoga for reducing state social physique anxiety and increasing state body satisfaction, adding to correlational evidence suggesting yoga session be particularly beneficial for improving body image-related outcomes in women.
Complete streets require evaluation to determine if they encourage active transportation.
Data were collected before and after a street intervention provided new light rail, bike lanes, and better sidewalks in Salt Lake City, Utah. Residents living near (<800 m) and far (≥801-2000 m) from the street were compared, with sensitivity tests for alternative definitions of near (<600 and <1000 m). Dependent variables were accelerometer/global positioning system (GPS) measures of transit trips, non-transit walking trips, and biking trips that included the complete street corridor.
Active travel trips for Near-Time 2 residents, the group hypothesized to be the most active, were compared to the other three groups (Near-Time 1, Far-Time 1, and Far-Time 2), net of control variables. Near-Time 2 residents were more likely to engage in complete street transit walking trips (35%, adjusted) and non-transit walking trips (50%) than the other three groups (24-25% and 13-36%, respectively). Bicycling was less prevalent, with only one of three contrasts significant (10% of Near-Time 2 residents had complete street bicycle trips compared to 5% of Far-Time 1 residents).
Living near the complete street intervention supported more pedestrian use and possibly bicycling, suggesting complete streets are also public health interventions.
It remains unclear whether self-regulated exercise is sufficient to maintain the benefits acquired during formal cardiac rehabilitation (CR). This retrospective observational study investigated the effects of a home-based exercise intervention after discharge from CR upon anthropometric and aerobic capacity markers in clinically stable patients.
Fifty patients with cardiovascular disease were discharged after 6 months of CR and encouraged to maintain aerobic exercise without supervision. Subsequent to 6 months of follow-up, patients were assigned to compliant (n=34) or non-compliant (n=16) groups according to their compliance to the home-based program. Maximal aerobic capacity (VO2peak) and anthropometric data were assessed before CR, at discharge, and after 6 months of follow-up.
No statistical differences between compliant and non-compliant groups were observed at baseline and at discharge from CR. At the end of the follow-up, statistical differences across groups were not found for body mass or body mass index, but increases in VO2peak (+3.6 vs. -0.6 ml/kg.min, P=0.004) and oxygen pulse (+1.5 vs. +0.2 ml/bpm, P=0.03) were greater in compliant than non-compliant group.
Self-regulated exercising following CR discharge seems to be effective to maintain gains in exercise capacity acquired during supervised center-based programs.
The aim of this study was to evaluate the association between physical activity from 11 to 15 years of age and pulmonary function (PF) gain from 15 to 18 years of age among adolescents in a birth cohort in Brazil.
Longitudinal analysis of the individuals participating in the 1993 Pelotas Birth Cohort Study. Physical activity was assessed by self-report at ages 11 and 15, spirometry was performed at ages 15 and 18 (n=3,571). Outcome variables assessed were gains in forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and peak expiratory flow (PEF). Crude and adjusted linear regressions, stratified by sex, and mediation analyses were performed.
Boys who were active (leisure-time and total physical activity) at ages 11 and 15 had higher gains in FEV1, FVC, and PEF than those who were inactive. Vigorous-intensity physical activity in boys was also associated with FEV1 and FVC gains. Mediation analyses showed that height at age 18 accounted for 5-75% of the association between physical activity and PF gains. No significant associations were found amongst girls.
Physical activity in early adolescence is associated with gains in PF by the end of adolescence in boys.
Effective interventions are needed to increase physical activity in the general population. To target interventions we need knowledge of insufficiently active groups in society. This study aims to identify demographic and health-related correlates of leisure time physical inactivity in a general Scandinavian population.
Study participants comprised 5734 controls, age 18-70 years, from two ongoing Swedish case-control studies. Participants self-reported their leisure time physical activity level. The odds of being physically inactive were calculated using logistic regression.
A total of 42% of participants were classified as physically inactive during leisure time. A lower prevalence of inactivity was associated with middle age, higher education, having previous experience of sports participation, following a GI/Mediterranean diet and having a light physical workload. A high prevalence of inactivity was associated with high age, high body mass index, smoking, being a never drinker of alcohol, having children, a weak social network or lower levels of emotional support, and a low vegetable intake.
Several factors were associated with leisure time physical inactivity. Directing interventions to target groups defined by specific factors associated with physical inactivity could be an efficient way to increase activity and improve health in the general population.
Erbin, Lano, Scribble, and Densin-180 belong to LAP (leucine-rich repeats and PDZ domain) adaptor proteins involved in cell signaling pathways. Previously, we identified Erbin, Lano, and Scribble, but not Densin-180, in muscle cells, where they are involved in regulating the aggregation of nicotinic acetylcholine receptors in vitro. Here, we analyzed their cellular localization at the neuromuscular junction (NMJ) in skeletal muscles of mice. Erbin, Lano, and Scribble were significantly accumulated at NMJs and localized in different synaptic cells. Moreover, we used mouse mutants to analyze the role of Erbin at the NMJ. We used two Erbin mutant mouse strains that either completely lack Erbin protein (Erbin(null/null) ) or express a truncated Erbin mutant where the carboxy-terminal PDZ domain is replaced by β-galactosidase (Erbin(ΔC/ΔC) ) thereby abolishing its interaction with ErbB receptor tyrosine kinases. Neither the lack of the PDZ domain of Erbin, nor its complete absence interfered with the general localization of LAP proteins at NMJs, but Lano and Scribble transcript levels were up-regulated in homozygous Erbin-null muscles. Furthermore, grip strength was reduced and neural transmission impaired in homozygous aged Erbin-null but not Erbin-ΔC mice. Erbin-null skeletal muscles did not reveal any conspicuous impairment of the muscle fiber. Localization of other NMJ marker proteins was not affected either. Quantitative 3D morphometry showed that NMJs of Erbin-null muscles were significantly smaller and fragmented in the soleus. We speculate that Erbin, Lano, and Scribble act at the post-synaptic membrane of NMJs in a concerted fashion to regulate nicotinic acetylcholine receptors cluster morphology and neural transmission. Cover Image for this issue: doi: 10.1111/jnc.13340.
At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).
Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes.
Data on physical inactivity, a known risk factor for non-communicable diseases and its correlates in sub-Saharan Africa are almost absent. We assessed physical activity patterns and associated factors among adults.
A population-based study of 1,208 adults was conducted in the Iganga-Mayuge Health and Demographic Surveillance Site, Uganda. Physical activity was assessed using a pedometer for seven days. Physical inactivity was defined as a daily average of < 7,500 steps while sedentary behaviour was defined as a daily average of < 5,000 steps. Logistic regression was conducted to identify factors associated with physical inactivity and sedentary behaviour.
Of the participants, 18.8% were sedentary (10.6% of men; 26.9% of women, p < 0.001), 37.6% were physically inactive (28.5% of men; 46.6% of women, p<0.001). Factors associated with sedentary behaviour were being female, ≥ 65 years, peri-urban residence, being a domestic worker, formal employment and lower primary education. Factors associated with physical inactivity were being female, 55-64 years, ≥ 65 years, peri-urban residence, overweight and obesity.
Sedentary behaviour and physical inactivity were prevalent among the adult population. Targeted physical activity promotion interventions are needed.
Electro-fermentation (EF) is a novel process that consists of electrochemically controlling microbial fermentative metabolism with electrodes. The electrodes can act as either electron sinks or sources that allow unbalanced fermentation. They can also modify the medium by changing the redox balance. Such electrochemical control exerts significant effects not only on microbial metabolism and cellular regulation but also on interspecies interactions and the selection of bacterial populations in mixed microbial cultures. In this paper we propose some basics and principles to better define the EF concept within the field of bioelectrochemistry. We also explore the up-to-date strategies to put EF into practice and propose hypothetical mechanisms that could explain the first EF results reported in the literature.
Emergency physicians frequently interact with law enforcement officers and patients in their custody. As always, the emergency physician's primary professional responsibility is to promote patient welfare, and his or her first duty is to the patient. Emergency physicians should treat criminals, suspects, and prisoners with the same respect and attention they afford other patients while ensuring the safety of staff, visitors, and other patients. Respect for patient privacy and protection of confidentiality are of paramount importance to the patient-physician relationship. Simultaneously, emergency physicians should attempt to accommodate law enforcement personnel in a professional manner, enlisting their aid when necessary. Often this relates to the emergency physician's socially imposed duties, governed by state laws, to report infectious diseases, suspicion of abuse or neglect, and threats of harm. It is the emergency physician's duty to maintain patient confidentiality while complying with Health Insurance Portability and Accountability Act regulations and state law.
There are lay midwives worldwide, interchangeably and universally called traditional birth attendants or traditional midwives by organisations such as the World Health Organization and the International Confederation of Midwives.
This study aimed to explore the history of lay midwives (village midwives) in Brunei, describe the evolution from their previous to current roles and determine if they are still needed by women today.
This qualitative, descriptive study included in-depth, semi-structured interviews with eight women who had received care from village midwives. Data analysis was based on the principles underpinning thematic analysis and used a constant comparative method.
Village midwives have been popular in Brunei since the 1900s, with their major role being to assist women with childbirth. However, since the 1960s, their roles and practices have changed to focus on pre-conception, antenatal, postnatal and women's general healthcare. Traditional practices were influenced by religion, culture and the social context of and within Brunei.
The major changes in village midwives' roles and practices resulted from the enforcement of the Brunei Midwives' Act in 1956. Village midwives' traditional practices became juxtaposed with modern complementary alternative medicine practices, and they began charging a fee for their services.
Brunei village midwives are trusted by women, and their practices may still be widely accepted in Brunei. Further research is necessary to confirm their existence, determine the detailed scope and appropriateness of their practices and verify the feasibility of them working together with healthcare professionals.
In, many high and middle-income countries, childbearing women have a variety of birthplaces available to them including home, birth centres and traditional labour wards. There is good evidence indicating that birthplace impacts on outcomes for women but less is known about the impact on midwives.
To explore the way that birthplace impacts on midwives in Australia and the United Kingdom.
A qualitative descriptive study was undertaken. Data were gathered through focus groups conducted with midwives in Australia and in the United Kingdom who worked in publicly-funded maternity services and who provided labour and birth care in at least two different settings.
Five themes surfaced relating to midwifery and place including: 1. practising with the same principles; 2. creating ambience: controlling the environment; 3. workplace culture: being watched 4. Workplace culture: "busy work" versus "being with"; and 5. midwives' response to place.
While midwives demonstrate a capacity to be versatile in relation to the physicality of birthplaces, workplace culture presents a challenge to their capacity to "be with" women.
Given the excellent outcomes of midwifery led care, we should focus on how we can facilitate the work of midwives in all settings. This study suggests that the culture of the birthplace rather than the physicality is the highest priority.
Adipocytokines play an important role in adipose tissue homeostasis, especially in obesity-associated disorders such as non-alcoholic fatty liver and their complications including hepatocellular carcinoma (HCC). Although visfatin is an adipocytokine highly expressed in visceral fat that has been demonstrated to play a critical role in the progression of human malignancies, little is known about the role of visfatin in HCC associated with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. In this study, we investigated whether plasma visfatin levels were altered in patients with HCC and the association between plasma visfatin levels and pretreatment hematologic profiles. Plasma visfatin levels were measured by enzyme-linked immunosorbent assays in 193 patients with different stages of HBV or HCV infection, and 92 healthy control subjects. The patients with HCC and chronic HCV or HBV infection had higher levels of visfatin than patients with HBV, HCV, and cirrhosis. In multivariate logistic regression analysis, levels of alpha-fetoprotein (AFP) (OR: 1.13, p=0.003), and plasma visfatin (OR: 1.17, p=0.046) were independently associated with HCC. Multiple stepwise regression analysis showed that plasma visfatin level was positively associated with age, aspartate aminotransferase to platelet ratio index (APRI), and AFP. Trend analyses confirmed that plasma visfatin concentration was associated with AFP>8ng/mL, cirrhosis, HCC, tumor size>5cm, and Barcelona Clinic Liver Cancer-C stage. These results suggested that the plasma visfatin level is associated with the presence of HCC, and that a higher plasma visfatin level may be important in the pathogenesis of HCC. Visfatin may act as both a protective and pro-inflammatory factor. Plasma visfatin concentration may serve as an additional tool to identify patients with more advanced necroinflammation.
Hendra virus (HeV) causes potentially fatal respiratory and/or neurological disease in both horses and humans. Although Australian flying-foxes of the genus Pteropus have been identified as reservoir hosts, the precise mechanism of HeV transmission has yet to be elucidated. To date, there has been limited investigation into the role of haematophagous insects as vectors of HeV. This mode of transmission is particularly relevant because Australian flying-foxes host the bat-specific blood-feeding ectoparasites of the genus Cyclopodia (Diptera: Nycteribiidae), also known as bat flies. Using molecular detection methods, we screened for HeV RNA in 183 bat flies collected from flying-foxes inhabiting a roost in Boonah, Queensland, Australia. It was subsequently demonstrated that during the study period, Pteropus alecto in this roost had a HeV RNA prevalence between 2 and 15% (95% CI [1, 6] to [8, 26], respectively). We found no evidence of HeV in any bat flies tested, including 10 bat flies collected from P. alecto in which we detected HeV RNA. Our negative findings are consistent with previous findings and provide additional evidence that bat flies do not play a primary role in HeV transmission.
Enzymatic electrodes are becoming increasingly common for energy production and sensing applications. Research over the past several decades has addressed a major issue that can occur when using these biocatalysts, i.e., slow heterogeneous electron transfer, by incorporation of a redox active species to act as an electron shuttle. There are several advantages to immobilizing both the enzyme and mediator at the enzyme surface, including increased electron transfer rates, decreased enzyme leaching, and minimized diffusion limitations. Redox polymers consisting of a redox active center attached to a polymer backbone are a particularly attractive option because they have high self-exchange rates for electron transfer and tunable redox potential. Osmium (Os) polymers are the most well studied of this type of polymer for bioelectrocatalysis. Here, we describe the methods to synthesize one of the most common Os redox polymers and how it can be used to fabricate glucose oxidase electrodes. Procedures are also outlined for evaluating the enzymatic electrodes.
As part of its single technology appraisal (STA) process, the National Institute for Health and Care Excellence (NICE) invited the company that manufactures cabazitaxel (Jevtana(®), Sanofi, UK) to submit evidence for the clinical and cost effectiveness of cabazitaxel for treatment of patients with metastatic hormone-relapsed prostate cancer (mHRPC) previously treated with a docetaxel-containing regimen. The School of Health and Related Research Technology Appraisal Group at the University of Sheffield was commissioned to act as the independent Evidence Review Group (ERG). The ERG produced a critical review of the evidence for the clinical and cost effectiveness of the technology based upon the company's submission to NICE. Clinical evidence for cabazitaxel was derived from a multinational randomised open-label phase III trial (TROPIC) of cabazitaxel plus prednisone or prednisolone compared with mitoxantrone plus prednisone or prednisolone, which was assumed to represent best supportive care. The NICE final scope identified a further three comparators: abiraterone in combination with prednisone or prednisolone; enzalutamide; and radium-223 dichloride for the subgroup of people with bone metastasis only (no visceral metastasis). The company did not consider radium-223 dichloride to be a relevant comparator. Neither abiraterone nor enzalutamide has been directly compared in a trial with cabazitaxel. Instead, clinical evidence was synthesised within a network meta-analysis (NMA). Results from TROPIC showed that cabazitaxel was associated with a statistically significant improvement in both overall survival and progression-free survival compared with mitoxantrone. Results from a random-effects NMA, as conducted by the company and updated by the ERG, indicated that there was no statistically significant difference between the three active treatments for both overall survival and progression-free survival. Utility data were not collected as part of the TROPIC trial, and were instead taken from the company's UK early access programme. Evidence on resource use came from the TROPIC trial, supplemented by both expert clinical opinion and a UK clinical audit. List prices were used for mitoxantrone, abiraterone and enzalutamide as directed by NICE, although commercial in-confidence patient-access schemes (PASs) are in place for abiraterone and enzalutamide. The confidential PAS was used for cabazitaxel. Sequential use of the advanced hormonal therapies (abiraterone and enzalutamide) does not usually occur in clinical practice in the UK. Hence, cabazitaxel could be used within two pathways of care: either when an advanced hormonal therapy was used pre-docetaxel, or when one was used post-docetaxel. The company believed that the former pathway was more likely to represent standard National Health Service (NHS) practice, and so their main comparison was between cabazitaxel and mitoxantrone, with effectiveness data from the TROPIC trial. Results of the company's updated cost-effectiveness analysis estimated a probabilistic incremental cost-effectiveness ratio (ICER) of £45,982 per quality-adjusted life-year (QALY) gained, which the committee considered to be the most plausible value for this comparison. Cabazitaxel was estimated to be both cheaper and more effective than abiraterone. Cabazitaxel was estimated to be cheaper but less effective than enzalutamide, resulting in an ICER of £212,038 per QALY gained for enzalutamide compared with cabazitaxel. The ERG noted that radium-223 is a valid comparator (for the indicated sub-group), and that it may be used in either of the two care pathways. Hence, its exclusion leads to uncertainty in the cost-effectiveness results. In addition, the company assumed that there would be no drug wastage when cabazitaxel was used, with cost-effectiveness results being sensitive to this assumption: modelling drug wastage increased the ICER comparing cabazitaxel with mitoxantrone to over £55,000 per QALY gained. The ERG updated the company's NMA and used a random effects model to perform a fully incremental analysis between cabazitaxel, abiraterone, enzalutamide and best supportive care using PASs for abiraterone and enzalutamide. Results showed that both cabazitaxel and abiraterone were extendedly dominated by the combination of best supportive care and enzalutamide. Preliminary guidance from the committee, which included wastage of cabazitaxel, did not recommend its use. In response, the company provided both a further discount to the confidential PAS for cabazitaxel and confirmation from NHS England that it is appropriate to supply and purchase cabazitaxel in pre-prepared intravenous-infusion bags, which would remove the cost of drug wastage. As a result, the committee recommended use of cabazitaxel as a treatment option in people with an Eastern Cooperative Oncology Group performance status of 0 or 1 whose disease had progressed during or after treatment with at least 225 mg/m(2) of docetaxel, as long as it was provided at the discount agreed in the PAS and purchased in either pre-prepared intravenous-infusion bags or in vials at a reduced price to reflect the average per-patient drug wastage.
Based on in-depth interviews with 16 heterosexual men, this study focuses on participants' meaning-making surrounding a common and controversial sexual act in pornography: ejaculation on a woman's face (EOWF). We analyze the ways that male consumers decoded EOWF and the ways that EOWF, as a sexual script, was included in the men's accounts of their sexual desires and practices. The majority of the men decoded EOWF through the preferred (encoded) meaning as an act of male dominance and sexual aggression and that they wanted to engage in it despite their general belief that women would not be interested in it.
To describe the amyotrophic lateral sclerosis (ALS) patients who sought medication under the Washington State Death with Dignity (DWD) Act since its inception in 2009.
Chart review at 3 tertiary medical centers in the Seattle/Puget Sound region and comparison to publicly available data of ALS and all-cause DWD cohorts from Washington and Oregon.
In Washington State, 39 patients with ALS requested DWD from the University of Washington, Virginia Mason, and Swedish Medical Centers beginning in 2009. The median age at death was 65 years (range 46-86). Seventy-seven percent of the patients used the prescriptions. All of the patients who used the medications passed away without complications. The major reasons for patients to request DWD as reported by participating physicians were loss of autonomy and dignity and decrease in enjoyable activities. Inadequate pain control, financial cost, and loss of bodily control were less commonly indicated. These findings were similar to those of the 92 patients who sought DWD in Oregon. In Washington and Oregon, the percentage of patients with ALS seeking DWD is higher compared to the cancer DWD cohort. Furthermore, compared to the all-cause DWD cohort, patients with ALS are more likely to be non-Hispanic white, married, educated, enrolled in hospice, and to have died at home.
Although a small number, ALS represents the disease with the highest proportion of patients seeking to participate in DWD. Patients with ALS who choose DWD are well-educated and have access to palliative or life-prolonging care. The use of the medications appears to be able to achieve the patients' goals without complications.
Research has shown that people who feel powerful are more likely to act than those who feel powerless, whereas people who feel ambivalent are less likely to act than those whose reactions are univalent (entirely positive or entirely negative). But what happens when powerful people also are ambivalent? On the basis of the self-validation theory of judgment, we hypothesized that power and ambivalence would interact to predict individuals' action. Because power can validate individuals' reactions, we reasoned that feeling powerful strengthens whatever reactions people have during a decision. It can strengthen univalent reactions and increase action orientation, as shown in past research. Among people who hold an ambivalent judgment, however, those who feel powerful would be less action oriented than those who feel powerless. Two experiments provide evidence for this hypothesized interactive effect of power and ambivalence on individuals' action tendencies during both positive decisions (promoting an employee; Experiment 1) and negative decisions (firing an employee; Experiment 2). In summary, when individuals' reactions are ambivalent, power increases the likelihood of inaction.
Cilia are plasma membrane protrusions that act as cellular propellers or antennae. To perform these functions, cilia must maintain a composition distinct from those of the contiguous cytosol and plasma membrane. The specialized composition of the cilium depends on the ciliary gate, the region at the ciliary base separating the cilium from the rest of the cell. The ciliary gate's main structural features are electron dense struts connecting microtubules to the adjacent membrane. These structures include the transition fibers, which connect the distal basal body to the base of the ciliary membrane, and the Y-links, which connect the proximal axoneme and ciliary membrane within the transition zone. Both transition fibers and Y-links form early during ciliogenesis and play key roles in ciliary assembly and trafficking. Accordingly, many human ciliopathies are caused by mutations that perturb ciliary gate function.
Troubleshooting of deep brain stimulators (Activa SC/PC/RC Medtronic PLC) sometimes results in a decision to replace a tunneled stretch-coil extension cable. We present a simple technique to accomplish this atraumatically without a tunneling tool.
In the treatment of patients with deep brain stimulators, complication avoidance and efficiency of operative time are paramount. We sought to find the most safe, effective, and rapid method for performing the conceptually-simple yet technically-nuanced act of replacing lead extension cables.
#6 (8.0 metric) surgical steel 18" (45cm) monofilament (Ethicon US, LLC), also known as #6 sternal wire, was connected in-line with DBS extension cables (Medtronic DBS Extension 37086-60) in novel fashion to overcome intra-procedural hurdles encountered during the past decade in a busy functional neurosurgery service.
Patients tolerate the procedure well and return home shortly after recovery with no complications.
A less expensive and faster technique for passing pulse generator extension cables may be the use of a sternal wire. Using the described technique, pulse generators may be quickly and safely adjusted from side-to-side and site-to-site as the clinical situation dictates.
To determine the effectiveness and cost savings of a real-world, continuous, pharmacist-delivered service with an employed patient population with diabetes over a 5-year period.
The Patients, Pharmacists Partnerships (P(3) Program) was offered as an "opt-in" benefit to employees of 6 public and private self-insured employers in Maryland and Virginia. Care was provided in ZIP code-matched locations and at 2 employers' worksites.
Six hundred two enrolled patients with type 1 and 2 diabetes were studied between July 2006 and May 2012 with an average follow-up of 2.5 years per patient. Of these patients, 162 had health plan cost and utilization data. A network of 50 trained pharmacists provided chronic disease management to patients with diabetes using a common process of care. Communications were provided to patients and physicians.
Employers provided incentives for patients who opted in, including waived medication copayments and free diabetes self-monitoring supplies. The service was provided at no cost to the patient. A Web-based, electronic medical record that complied with the Health Insurance Portability and Accountability Act helped to standardize care. Quality assurance was conducted to ensure the standard of care.
Glycosylated hemoglobin (A1c), blood pressure, and total health care costs (before and after enrollment).
Statistically significant improvements were shown by mean decreases in A1c (-0.41%, P <0.001), low-density lipoprotein levels (-4.7 mg/dL, P = 0.003), systolic blood pressure (-2.3 mm Hg, P = 0.001), and diastolic blood pressure (-2.4 mm Hg, P <0.001). Total annual health care costs to employers declined by $1031 per beneficiary after the cost of the program was deducted. This 66-month real-world study confirms earlier findings. Employers netted savings through improved clinical outcomes and reduced emergency and hospital utilization when comparing costs 12 months before and after enrollment.
The P(3) program had positive clinical outcomes and economic outcomes. Pharmacist-provided comprehensive medication therapy management services should be included as a required element of insurance offered by employers and health insurance exchanges.
To compare Hounsfield unit (HU) data obtained from true-unenhanced (TUE) and virtual-unenhanced (VUE) imaging obtained with a fast kv-switching dual-energy computed tomography (CT) scanner using multimaterial decomposition algorithm.
In this Institutional Review Board-approved, Health Insurance Portability and Accountability Act-compliant, retrospective cohort study, CT scans of 19 patients undergoing multiphasic renal protocol abdominal CT on a fast kv-switching dual-energy CT scanner were reviewed. CT numbers were measured on the matched TUE and VUE generated using a multimaterial decomposition algorithm with selective iodine suppression, and postcontrast images at predefined locations in seven organs. Six hundred sixty regions of interest were placed at 132 locations. Agreement was assessed with paired t test, Pearson's correlation, and Bland-Altman analysis.
Mean TUE and VUE measurements were not significantly different in the corticomedullary (P = 0.25) or nephrographic (P = 0.10) phases. There was a strong correlation between TUE and VUE CT numbers (corticomedullary: r = 0.90, nephrographic: r = 0.90, each P < 0.001). Discrepancies ≥5 HU occurred 46 times (35%, 46 of 132) in the corticomedullary phase and 44 times (33%, 44 of 132) in the nephrographic phase. Discrepancies ≥10 HU occurred in 7% (9 of 132 in both corticomedullary and nephrographic phases). Interphase, intrasubject VUE CT numbers were strongly correlated (r = 0.93, P < 0.001), but discrepancies ≥5 HU (22% [29 of 132]) and ≥10 HU (2% [3 of 132]) occurred. There was no significant correlation between the true postcontrast CT number and the magnitude of VUE-TUE discrepancy (r = -0.04, P = 0.6).
CT numbers on VUE images generated from fast kv-switching dual-energy CT scans strongly correlate with TUE CT numbers on a population basis, but commonly vary 5-9 HU on a per-patient basis.
Peptide ligand-receptor kinase interactions have emerged as a key component of plant growth and development. Now, highly related small signaling peptides have been shown to act antagonistically on the same receptor kinase, providing new insights into how plants optimize developmental processes using competitive peptides.
Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated I. scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of I. scapularis-associated virus-1, which was reported in a recent metagenomic study of I. scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks.
A simple Schiff base (BMSA) prepared from salicylaldehyde and 2-(1H-benzo[d]imidazol-2-yl)aniline was evaluated as an efficient fluorescent chemosensor for the selective recognition of Al(3+)and Cu(2+) over other common metal ions. This sensor could detect Al3(+) in CH3OH/PBS with distinct emission red-shift (the detection limit 0.31μM)and Cu(2+)in CH3OH/Tris-HCL (the detection limit 0.54μM) with obvious fluorescence quenching. The obtained BMSA-Al(3+) and BMSA-Cu(2+) complexes could act as cascade sensors for detecting F(-) and S(2-), respectively. The recognizing behavior of BMSA toward Al(3+)and Cu(2+) has been investigated in detail through Job's Plot, FT-IR NMR, and HRMS analysis. Moreover, this chemosensor was verified to be of low cytotoxicity and good imaging characteristics for the detection of Al(3)(+) and Cu(2+), and further for the recognition of F(-) and S(2-) in living cells, suggesting that BMSA was proved to be a useful tool for tracking Al3(+)/Cu(2+)and F(-)/S(2-) ions in vivo.
The emergence in 2009 of Plasmodium falciparum parasites resistant to the primary therapies currently in use (artemisinin-based combination therapy, ACT) in Southeast Asia threatens to set back decades of global progress in malaria control and elimination. Progress to date through multiple sets of initiatives and partners to contain or eliminate these parasites has been hampered due to a wide range of organizational, financial, and health systems-level challenges. In this commentary, a set of seven specific and concrete actions are proposed to directly address these issues and to accelerate P. falciparum elimination within the Greater Mekong Subregion to avert a wider public health crisis. These actions are specifically needed to elevate the situation and response mechanisms to those of a true emergency; to address systems-level challenges with personnel limitations and stock-outs of key commodities; and to restructure the response mechanisms to be well-aligned with the required outcomes. Consideration of these issues is especially pressing with planning meetings for renewal of the Regional Artemisinin-resistance Initiative (RAI) framework slated for late 2016 and into 2017, but these suggestions are also relevant for malaria programmes globally.
Whilst multi-parametric magnetic resonance imaging (mp-MRI) has been a significant advance in the diagnosis of prostate cancer, scanning all patients with elevated prostate specific antigen (PSA) levels is considered too costly for widespread National Health Service (NHS) use, as the predictive value of PSA levels for significant disease is poor. Despite the fact that novel blood and urine tests are available which may predict aggressive disease better than PSA, they are not routinely employed due to a lack of clinical validity studies. Furthermore approximately 40 % of mp-MRI studies are reported as indeterminate, which can lead to repeat examinations or unnecessary biopsy with associated patient anxiety, discomfort, risk and additional costs.
We aim to clinically validate a panel of minimally invasive promising blood and urine biomarkers, to better select patients that will benefit from a multiparametric prostate MRI. We will then test whether the performance of the mp-MRI can be improved by the addition of an advanced diffusion-weighted MRI technique, which uses a biophysical model to characterise tissue microstructure called VERDICT; Vascular and Extracellular Restricted Diffusion for Cytometry in Tumours. INNOVATE is a prospective single centre cohort study in 365 patients. mp-MRI will act as the reference standard for the biomarker panel. A clinical outcome based reference standard based on biopsy, mp-MRI and follow-up will be used for VERDICT MRI.
We expect the combined effect of biomarkers and VERDICT MRI will improve care by better detecting aggressive prostate cancer early and make mp-MRI before biopsy economically viable for universal NHS adoption.
INNOVATE is registered on ClinicalTrials.gov, with reference NCT02689271 .
Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection.
The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems.
Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.
Mechanical loading is the primary functional determinant of bone mass and architecture, and osteocytes play a key role in translating mechanical signals into (re)modelling responses. Although the precise mechanisms remain unclear, Wnt signalling pathway components, and the anti-osteogenic canonical Wnt inhibitor Sost/sclerostin in particular, play an important role in regulating bone's adaptive response to loading. Increases in loading-engendered strains down-regulate osteocyte sclerostin expression, whereas reduced strains, as in disuse, are associated with increased sclerostin production and bone loss. However, while sclerostin up-regulation appears to be necessary for the loss of bone with disuse, the role of sclerostin in the osteogenic response to loading is more complex. While mice unable to down-regulate sclerostin do not gain bone with loading, Sost knockout mice have an enhanced osteogenic response to loading. The molecular mechanisms by which osteocytes sense and transduce loading-related stimuli into changes in sclerostin expression remain unclear but include several, potentially interlinked, signalling cascades involving periostin/integrin, prostaglandin, estrogen receptor, calcium/NO and Igf signalling. Deciphering the mechanisms by which changes in the mechanical environment regulate sclerostin production may lead to the development of therapeutic strategies that can reverse the skeletal structural deterioration characteristic of disuse and age-related osteoporosis and enhance bones' functional adaptation to loading. By enhancing the osteogenic potential of the context in which individual therapies such as sclerostin antibodies act it may become possible to both prevent and reverse the age-related skeletal structural deterioration characteristic of osteoporosis.
A small but growing body of research documents associations between structural forms of stigma (e.g., same-sex marriage bans) and sexual minority health. These studies, however, have focused on a limited number of outcomes and have not examined whether sociodemographic characteristics, such as race/ethnicity and education, influence the relationship between policy change and health among sexual minorities.
To determine the effect of civil union legalization on sexual minority women's perceived discrimination, stigma consciousness, depressive symptoms, and four indicators of hazardous drinking (heavy episodic drinking, intoxication, alcohol dependence symptoms, adverse drinking consequences) and to evaluate whether such effects are moderated by race/ethnicity or education.
During the third wave of data collection in the Chicago Health and Life Experiences of Women study (N = 517), Illinois passed the Religious Freedom Protection and Civil Union Act, legalizing civil unions in Illinois and resulting in a quasi-natural experiment wherein some participants were interviewed before and some after the new legislation. Generalized linear models and interactions were used to test the effects of the new legislation on stigma consciousness, perceived discrimination, depression, and hazardous drinking indicators. Interactions were used to assess whether the effects of policy change were moderated by race/ethnicity or education.
Civil union legislation was associated with lower levels of stigma consciousness, perceived discrimination, depressive symptoms, and one indicator of hazardous drinking (adverse drinking consequences) for all sexual minority women. For several other outcomes, the benefits of this supportive social policy were largely concentrated among racial/ethnic minority women and women with lower levels of education.
Results suggest that policies supportive of the civil rights of sexual minorities improve the health of all sexual minority women, and may be most beneficial for women with multiply marginalized statuses.
Ketotifen has recently been reported to inhibit the growth of both asexual and sexual malaria parasites. A parasite transporter, PfgABCG2, has been implicated in its mechanism of action. Human dihydrofolate reductase (hDHFR) is the most commonly used selectable marker to create transgenic Plasmodium falciparum cell lines. Growth assays using transgenic P. falciparum parasites with different selectable markers revealed that the presence of hDHFR rather than the absence of PfgABCG2 is responsible for a shift in the parasite's sensitivity to ketotifen. Employing a range of in vitro assays and liquid chromatography-mass spectrometry we show that ketotifen influences hDHFR activity, but it is not metabolised by the enzyme. Our data also highlights potential pitfalls when functionally characterising transgenic parasites.
In Saccharomyces cerevisiae, nuclear exosome along with TRAMP and DRN selectively eliminates diverse aberrant messages. These decay apparatuses appear to operate as independent mechanisms in the nucleus. Here, using genetic and molecular approach we systematically investigate the functional relationship between exosome, TRAMP and DRN mechanisms by examining their relative contributions in the degradation of diverse classes of aberrant nuclear mRNAs generated at various phases of mRNP biogenesis. Our findings suggest that nuclear exosome in association with the TRAMP complex exclusively degrades the transcription assembly-defective mRNPs and splice-defective intron-containing pre-mRNAs, whereas nuclear exosome along with DRN solely degrades the export-defective messages. The degradation of aberrant read-through transcripts with 3(')-extensions, in contrast, requires the activity of TRAMP and DRN together along with nuclear exosome function. Thus, the profile of substrate specificity of these nuclear decay machines reflects dependency of the nuclear exosome for either TRAMP or DRN function to degrade distinct nuclear mRNAs. We propose that DRN apparatus may act as a novel ancillary factor required for the nuclear exosome function to degrade specific classes of aberrant messages.
This study investigated functional and transcriptional modulation of the Multixenobiotic resistance (MXR) system as a cytoprotective mechanism contributing to the physiological chemoresistance of haemocytes in the Mediterranean mussel. Basal transport activity was assessed using the model substrate rhodamine 123 and specific inhibitors for the MXR-related transporters P-glycoprotein (ABCB mRNA) and Multidrug resistance-related protein (ABCC mRNA). Results showed that MXR activity in mussel haemocytes was mainly supported by the Mrp-mediated efflux. In agreement, ABCC was expressed at higher levels than ABCB. Activation of the cyclic-AMP (cAMP) dependent protein kinase A (PKA) resulted in increased rhodamine efflux, which was counteracted by the selective PKA inhibitor H89. Although serotonin, a physiological modulator of cAMP/PKA signaling and ABCB transcription in haemocytes, did not affect basal MXR transport, the environmental pharmaceuticals fluoxetine, propranolol, and carbamazepine, which interact in different ways with the adrenergic and serotoninergic pathways, were showed to act as modulators and substrates of MXR-related transporters and to affect cell viability. While the increased MXR activity may have lowered the cytotoxic effects of propranolol and carbamazepine, the lack of MXR efflux induction by fluoxetine may play a role in the observed cytotoxicity of the compound.
Salmonella enterica serovar typhimurium (S. typhimurium) is globally distributed and causes massive morbidity and mortality in humans and animals. S. typhimurium carries Salmonella plasmid virulence (spv) locus, which is highly conserved and closely related to bacterial pathogenicity, while its exact role in host immune responses during infection remains to be elucidated. To counteract the invaders, the host has evolved numerous strategies, among which the innate immunity and autophagy act as the first defense. Recently, zebrafish has been universally accepted as a valuable and powerful vertebrate model in analyzing bacteria-host interactions. To investigate whether spv locus enhances the virulence of Salmonella by exerting an effect on the host early defense, zebrafish larvae were employed in this study. LD50 of S. typhimurium to zebrafish larvae and bacterial dissemination were analyzed. Sudan black B and neutral red staining were performed to detect the responses of neutrophils and macrophages to Salmonella infection. Autophagy agonist Torin1 and inhibitor Chloroquine were used to interfere in autophagic flux, and the protein level of Lc3 and p62 were measured by western blotting. Results indicated that spv locus could decrease the LD50 of S. typhimurium to zebrafish larvae, accelerate the reproduction and dissemination of bacteria by inhibiting the function of neutrophils and macrophages. Moreover, spv locus restrained the formation of autophagosomes in the earlier stage of autophagy. These findings suggested the virulence of spv locus involving in suppressing host innate immune responses for the first time, which shed new light on the role of spv operon in Salmonella pathogenicity.
Several gill claudin (Cldn) tight junction proteins in Japanese medaka are regulated by salinity (cldn10 paralogs and cldn28b), while others are constitutively expressed (cldn27a, cldn28a and cldn30c). The role of the endocrine system in this regulation has yet to be understood. The in vitro effects of cortisol and prolactin on cldn expression in gill explant cultures were investigated in medaka. ncc2b and cftr were used as markers of specific ionocytes associated with freshwater- and seawater-acclimation, respectively. Concentration-response experiments were performed by overnight incubation with 0, 0.1, 1 and 10μgmL(-1) cortisol or 0, 0.01, 0.1 and 1μgmL(-1) ovine prolactin. Cortisol significantly up-regulated cftr, ncc2b, cldn10 paralogs, cldn27a and cldn30c from 1.2- to 5-fold control levels at 10μgmL(-1). Cortisol had no effect on cldn28a and cldn28b. Prolactin had a concentration-dependent effect, decreasing expression of cftr (1μgmL(-1), 2.2-fold) while increasing ncc2b (from 0.1μgmL(-1), 6-7-fold). Prolactin up-regulated expression of 3 cldns: cldn28b (0.1 and 1μgmL(-1)), cldn10c and cldn10f (1μgmL(-1)), with up to 2-, 2.5- and 2-fold of control level, respectively. A combination experiment with both hormones showed that they act in synergy on cldn28b and have an additive effect on cftr, ncc2b, cldn10c and cldn10f. Our results showed that cortisol and prolactin are essential to maintain the expression of specific branchial claudins. This work also provides evidence that both hormones act directly on gill of medaka to modulate determinants of paracellular ion movement.
The interaction of water with cellulose stages many unresolved questions. Here (2)H MAS NMR and IR spectra recorded under carefully selected conditions in (1)H-(2)H exchanged, and re-exchanged, cellulose samples are presented. It is shown here, by a quantitative and robust approach, that only two of the three available hydroxyl groups on the surface of cellulose fibrils are exchanging their hydrogen with the surrounding water molecules. This finding is additionally verified and explained by MD simulations which demonstrate that the (1)HO(2) and (1)HO(6) hydroxyl groups of the constituting glucose units act as hydrogen-bond donors to water, while the (1)HO(3) groups behave exclusively as hydrogen-bond acceptors from water and donate hydrogen to their intra-chain neighbors O(5). We conclude that such a behavior makes the latter hydroxyl group unreactive to hydrogen exchange with water.
The goals of this article are to review the pertussis cocooning strategy, which has been recommended in France since 2004 to protect infants not yet vaccinated from becoming infected by vaccinating their immediate entourage, and to present room for improvement. The analysis of the literature between 2004 and 2015 shows that pertussis vaccine coverage in new parents is lower than 50% and that attempts that have already been implemented to increase it are effective. Pertussis vaccine coverage improvement requires all health actors to collaborate and be trained in informing and motivating parents to get vaccinated before, during and after pregnancy (the parents then will act as relays to their relatives); generalization in maternity wards of systematic checking of the vaccination card; extension to the midwives of the right to prescribe and administer pertussis vaccine to spouses; vaccination facilitation in maternity wards with the support of health organizations. Exchange and sharing of experiences between health care professionals are essential. Pregnancy is the ideal period to promote pertussis vaccination.
The laboratory of genetics and physiology 2 (LGP2) is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may participate in the immune regulation process. The role of LGP2 on modulating signaling was ambiguous, some researchers suggested that the regulation mechanism of LGP2 to melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) were different. In this study, the bioinformatics and functions of LGP2 from miiuy croaker (mmLGP2) were characterized. Comparative genomic analysis showed that the evolution of LGP2 in mammals was more conserved than it in fish. LGP2 contains three structural domains: ResIII, HelicaseC and RD, and ResIII structural domain of LGP2 was extremely conservative. The mmLGP2 was ubiquitously expressed in the tested miiuy croaker tissues and the expressions were significantly upregulated after stimulation with poly(I:C), indicating that LGP2 might participate in the immune response, especially antiviral immunity. Furthermore, immunofluorescence of miiuy croaker LGP2 presents in the cytoplasm in Hela cells. The overexpression of mmLGP2 can activate ISRE, but cannot activate NF-κB luciferase reporter, implying that mmLGP2 might act as a positive regulator in immune responses through activating ISRE to induce the expression of IFN. The research of mmLGP2 will enrich the information of fish LGP2, and the functional experiments will be helpful for the future research about fish immune systems.
Soon after the emergence of 2009 pandemic H1N1, the first outbreaks in breeder turkey operations were reported that implicated human-to-turkey transmission. In the spring of 2016, the reoccurrence of 2009 pandemic H1N1 lineage viruses infecting breeder turkey flocks in Ontario and Manitoba, Canada, also implicated human-to-turkey transmission. In addition to raising concerns over biosecurity and vaccine failures, these cases once again raise the issue of whether turkeys have the potential to act as a bridge species to generate novel influenza A virus reassortants with public health implications.
Human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1) is shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding GS-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-δ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.
Significant heterogeneity between different tumors prevents the discovery of cancer driver genes, especially in a patient-specific manner. We previously prioritized five personalized candidate mutation-driver genes in a hyper-mutated hepatocellular carcinoma patient using a multi-omics strategy. However, the roles of the prioritized driver genes and patient-specific mutations in hepatocarcinogenesis are unclear. We investigated the impact of the tumor-mutated allele on structure-function relationship of the encoded protein and assessed both loss- and gain-of-function of these genes and mutations on hepatoma cell behaviors in vitro. The prioritized mutation-driver genes act as tumor suppressor genes and inhibit cell proliferation and migration. In addition, the loss-of-function effect of the patient-specific mutations promoted cell proliferation and migration. Of note, the HNF1A S247T mutation significantly reduced the HNF1A transcriptional activity for hepatocyte nuclear factor 4 alpha (HNF4A) but did not disrupt nuclear localization of HNF1A. The results provide evidence for supporting the validity of our proposed multi-omics strategy, which supplies a new avenue for prioritizing mutation-drivers towards personalized cancer therapy.
Big Data is having an impact on many areas of research, not the least of which is biomedical science. In this review paper, big data and machine learning are defined in terms accessible to the clinical chemistry community. Seven myths associated with machine learning and big data are then presented, with the aim of managing expectation of machine learning amongst clinical chemists. The myths are illustrated with four examples investigating the relationship between biomarkers in liver function tests, enhanced laboratory prediction of hepatitis virus infection, the relationship between bilirubin and white cell count, and the relationship between red cell distribution width and laboratory prediction of anaemia.
Perinatal network the "Security birth" Pays de la Loire (RSN), in collaboration with the regional network "Sexual health" (RRSS) organized a satisfaction survey in 2014 among women who had an induced abortion in the centers in Pays de la Loire. The objective of the study is to evaluate the satisfaction of women who resort to abortion centers, study the factors and propose actions for improvement.
A questionnaire, developed with professionals, was offered to women after the end of the abortion act. The scales of satisfaction were based on Likert scales to 4 degrees. They were collected for the period of contact centers, for the abortion itself, and overall. Data were collected on the civil status, the care pathway to get to the abortion center, features centers and professionals met, acts anesthesia and end of abortion, wait times and overall satisfaction of women. The women with rather poor satisfaction were compared with other women by univariate analysis with odds ratios (OR) and multivariate with adjusted odds ratios (ORa).
Of the 18 centers, 13 participated and 319 responses were analyzed. The abortions were performed on average 8±SA 2. Among the methods, 40.1 % were drug, and 59.9 % with anesthesia (14.0 % with general anesthesia [AG] and 45.9 % under local anesthesia [AL]). The first contacts on average to 5.7 SA were mostly general practitioners (38 %), then the abortion centers directly (22 %), and planning centers (16 %). The information was considered clear (94 %), the appropriate orientation (97 %), with respect to the application (98 %). The first meeting at the center was done 7 days after the call (6.7 SA), and met women overall. The abortion was performed 16 days after the first call center (8.0 SA) with satisfaction by field 78 % (waiting time) to 98 % (confidentiality, privacy…). The overall satisfaction rate was 89.0 % and 8.2 % were not satisfied. Factors related to the non-satisfaction in multivariate analysis were the least easy access to the center (ORa=0.31 [.11 to 0.86; p=0.02]), pain perceived≥4 (ORa=3 50 [1.32 to 9.28], p=0.02), the lack of explanation (ORa=0.23 [0.05 to 0.97]; p=0.04), and the accompanying inability (ORa=0.27 [0.10 to 0.70], p=0.007). Finally, 12.6 % of women reported for improvement with clear remarks.
This is the first regional survey on the satisfaction of women resorting to abortion. Delays from the first contact are satisfactory and show no organizational problem. The various dimensions of satisfaction showed a high satisfaction rate in the 13 participating centers. The least satisfactory factors are related to organizational problems (waiting circuits especially trips to the operating room and maternity) and problems related to the care itself (not enough explanations, reduced ability to reassure and support Savory absence, lack of choice of the method of anesthesia, pain experienced). Five areas for improvement have been proposed to the care-givers of the centers.
This article briefly reviews 'aid-in-dying' options such as euthanasia and physician-assisted suicide in Europe and the US Physician-assisted suicide is now legal in four US States. Current practices, medications used and statistics relating to prescription frequency and death rates from the participating States are briefly discussed. This paper also examines the role of pharmacists in assisted suicides; legal, ethical and professional challenges that they face, and future implications on pharmacist education to enable them to make an educated decision about their involvement in aid-in-dying practices.
The highly conserved RAS-mitogen activated protein kinase (MAPK) signaling pathway is involved in a wide range of cellular processes including differentiation, proliferation, and survival. Somatic mutations in genes encoding RAS-MAPK components frequently occur in many tumors, making the RAS-MAPK a critical pathway in human cancer. Since the pioneering study reporting that let-7 miRNA acted as tumor suppressor by repressing the RAS oncogene, growing evidence has suggested the importance of miRNAs targeting the RAS-MAPK in oncogenesis. MiRNAs alterations in human cancers may act as a rheostat of the oncogenic RAS signal that is often amplified as cancers progress. However, specific mechanisms leading to miRNAs deregulation and their functional consequences in cancer are far from being fully elucidated. In this review, we provide an experimental-validated map of RAS-MAPK oncomiRs and tumor suppressor miRNAs from transmembrane receptor to downstream ERK proteins. MiRNAs could be further considered as potential genetic biomarkers for diagnosis, prognosis, or therapeutic purpose.
Providing an evidence base for wildlife population management is difficult, due to limited opportunities for experimentation and study replication at the population level. We utilized an opportunity to assess the outcome of a test and cull programme aimed at limiting the spread of Mycobacterium bovis in African buffalo. Buffalo act as reservoirs of M. bovis, the causative agent of bovine tuberculosis (BTB), which can have major economic, ecological and public health impacts through the risk of infection to other wildlife species, livestock and surrounding communities. BTB prevalence data were collected in conjunction with disease control operations in Hluhluwe-iMfolozi Park, South Africa, from 1999 to 2006. A total of 4733 buffalo (250-950 per year) were tested for BTB using the single comparative intradermal tuberculin (SCIT) test, with BTB-positive animals culled, and negative animals released. BTB prevalence was spatially and temporally variable, ranging from 2.3% to 54.7%. Geographic area was a strong predictor of BTB transmission in HiP, owing to relatively stable herds and home ranges. Herds experiencing more intensive and frequent captures showed reduced per capita disease transmission risk and less increase in herd prevalence over time. Disease hot spots did not expand spatially over time, and BTB prevalence in all but the hot spot areas was maintained between 10% and 15% throughout the study period. Our data suggest that HiP's test and cull programme was effective at reducing BTB transmission in buffalo, with capture effort and interval found to be the crucial components of the programme. The programme was thus successful with respect to the original goals; however, there are additional factors that should be considered in future cost/benefit analyses and decision-making. These findings may be utilized and expanded in future collaborative work between wildlife managers, veterinarians and scientists, to optimize wildlife disease control programmes and mitigate conflict at the interface of conservation, agricultural and urban areas.
Story listening in children relies on brain regions supporting speech perception, auditory word recognition, syntax, semantics, and discourse abilities, along with the ability to attend and process information (part of executive functions). Speed-of-processing is an early-developed executive function. We used functional and structural magnetic resonance imaging (MRI) to demonstrate the relationship between story listening and speed-of-processing in preschool-age children. Eighteen participants performed story-listening tasks during MRI scans. Functional and structural connectivity analysis was performed using the speed-of-processing scores as regressors. Activation in the superior frontal gyrus during story listening positively correlated with speed-of-processing scores. This region was functionally connected with the superior temporal gyrus, insula, and hippocampus. Fractional anisotropy in the inferior frontooccipital fasciculus, which connects the superior frontal and temporal gyri, was positively correlated with speed-of-processing scores. Our results suggest that speed-of-processing skills in preschool-age children are reflected in functional activation and connectivity during story listening and may act as a biomarker for future academic abilities.
Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities.
Systemic lupus erythematosus (SLE) is a severe autoimmune disease that causes multiple-organ dysfunction mainly affecting women in their childbearing years. Type I IFN synthesis is usually triggered by viruses, and its production is tightly regulated and limited in time in health individuals. However, many patients with systemic autoimmune diseases including SLE have signs of aberrant production of type I interferon (IFN) and display an increased expression of IFN-inducible genes. Continuous type I IFNs derived from activated plasmacytoid dendritic cells (pDCs) by interferogenic immune complexes (ICs) and migration of these cells to tissues both break immune tolerance and promote an on-going autoimmune reaction in human body. By the means of detecting type I IFNs and IFN-inducible genes, it can help with diagnosis and evaluation of SLE in early stage and more efficiently. Anti-IFN-α monoclonal antibodies in SLE patients were recently reported and is now being investigated in phase II clinical trails. In this review, we focus on recent research progress in type I IFN and IFN-inducible genes. Possible mechanisms behind the dysregulated type I IFN system in SLE and how they contribute to the development of an autoimmune process, and act as a biomarker and therapeutic target will be reviewed.
Because of increased law enforcement and subsequent media attention, methamphetamine users appear in the public's imagination as diseased, zombie-like White trash. We explore methamphetamine users' perceptions about whether the images, people, and situations in anti-methamphetamine campaigns reflect their own lives and experiences using meth.
To explore these perceptions, we used photo-elicitation interviews with 47 people who used methamphetamine (30 former and 17 active). Specifically, we presented participants with images from the Faces of Meth and the Montana Meth Project campaigns to stimulate discussion.
We found that participants believed these ads did not reflect their personal experiences and consequently were ineffective at curtailing their own methamphetamine use. They believed that the ads represented a certain type of methamphetamine user, particularly those they defined as dysfunctional users. They created symbolic boundaries between themselves and those portrayed in the ads to show how they differed, which allowed them to believe that the ads were not relevant to their experiences.
Findings suggest that there are unintended consequences to inauthentic/dramatic imagery. Participants did not believe they were like those in the ads-thus saw no reason to quit or seek help. Consequently, overly stigmatizing portraits of users may act as barriers to desistence. The findings have implications for designing anti-methamphetamine campaigns.
Social class is a robust predictor of health, with risk for disease and mortality increasing towards the lower end of the socioeconomic (SES) spectrum. While certain psychological characteristics, such as high sense of control, can protect low-SES individuals from adverse health outcomes, very few studies have investigated the biological mechanisms underlying these relationships. In this study, we tested whether sense of control mitigated the associations between SES and cortisol activity, and SES and physical health in daily life (i.e., number and severity of physical symptoms). Next, we tested whether individual differences in cortisol secretion would act as a mechanism by which SES and perceived control influenced physical health. In a large national sample from the Midlife in the United States (MIDUS) survey, we found that SES interacted with perceived control in predicting morning cortisol levels, cortisol slopes, number of physical symptoms, and severity of physical symptoms. Specifically, SES disparities in these health outcomes were more pronounced among individuals reporting low levels of perceived control than among individuals endorsing high levels of perceived control. Further, we found that a flatter cortisol slope mediated the link between lower SES and greater number and severity of physical symptoms for those individuals who reported lower levels of perceived control, but not for individuals reporting higher levels of perceived control. These findings suggest that perception of greater control may act as a buffer against the effect of low SES on health-related physiological processes.
Evidence suggests the putative human pheromone Δ4,16-androstadien-3-one (androstadienone), a natural component of human sweat, increases attention to emotional information when passively inhaled, even in minute amounts. However, the neural mechanisms underlying androstadienone's impact on the perception of emotional stimuli have not been clarified. To characterize how the compound modifies neural circuitry while attending to emotional information, 22 subjects (11 women) underwent two fMRI scanning sessions, one with an androstadienone solution and one with a carrier control solution alone on their upper lip. During each session, participants viewed blocks of emotionally positive, negative, or neutral images. The BOLD response to emotional images (relative to neutral images) was greater during exposure to androstadienone in right orbitofrontal and lateral prefrontal cortex, particularly during positive image blocks. Androstadienone did not impact the response to social images, compared to nonsocial images, and results were not related to participant sex or olfactory sensitivity. To examine how androstadienone influences effective connectivity of this network, a dynamic causal model was employed with primary visual cortex (V1), amygdala, prefrontal cortex, and orbitofrontal cortex on each side. These models indicated that emotional images increased the drive from V1 to the amygdala during the control session. With androstadienone present, this drive to amygdala was decreased specifically for positive images, which drove downstream increases in orbitofrontal and prefrontal activity. This evidence suggests that androstadienone may act as a chemical signal to increase attention to positively valenced information via modifications to amygdala connectivity.
Cancer radiotherapy (RT) is a clinically used tumor treatment strategy applicable for a wide range of solid tumors. However, during RT treatment of tumors, only a small portion of applied ionizing irradiation energy is absorbed by the tumor, in which the largely hypoxic microenvironment also limits the anti-tumor efficacy of RT. In this work, we rationally fabricate polyethylene glycol (PEG) stabilized perfluorocarbon (PFC) nano-droplets decorated with TaOx nanoparticles (TaOx@PFC-PEG) as a multifunctional RT sensitizer. The obtained TaOx@PFC-PEG nanoparticles on one hand can absorb X-ray by TaOx to concentrate radiation energy within tumor cells, on the other hand after saturating PFC with oxygen will act as an oxygen reservoir to gradually release oxygen and improve tumor oxygenation. As the result, remarkably enhanced in vivo RT treatment is achieved with TaOx@PFC-PEG nanoparticles in our mouse tumor model experiments. Our work thus presents a new nanotechnology strategy to enhance RT-induced tumor treatment by simultaneously concentrating radiation energy within tumors and improving tumor oxygenation, using one multifunctional agent.
Homeodomain proteins are evolutionary conserved proteins present in the entire eukaryote kingdom. They execute functions that are essential for life, both in developing and adult organisms. Most homeodomain proteins act as transcription factors and bind DNA to control the activity of other genes. In contrast to their similar DNA binding specificity, homeodomain proteins execute highly diverse and context-dependent functions. Several factors, including genome accessibility, DNA shape, combinatorial binding and the ability to interact with many transcriptional partners, diversify the activity of homeodomain proteins and culminate in the activation of highly dynamic, context-specific transcriptional programs. Clarifying how homeodomain transcription factors work is central to our understanding of development, disease and evolution.
There is extensive literature to show that nucleic acids can be taken up by cells under experimental conditions and that foetal DNA can be detected in maternal tissues. The uptaken DNA can integrate into host cell genomes and can be transcribed and translated into proteins. They can also cause chromosomal damage and karyotype alterations. Cell-free nucleic acids (cfNAs)-based non-invasive DNA diagnostic techniques are being extensively researched in the field of cancer with the potential to advance new prognostic parameters and direct treatment decisions. However, whether extracellular cfNAs that are released into circulation from dying cells as a consequence of normal physiology have any functional significance has not been explored. A recent study has demonstrated that circulating cfNAs have the ability to cause DNA damage and mutagenesis by illegitimately integrating into healthy cells of the body, thereby acting as mobile genetic elements. Fluorescently-labeled cfNAs isolated from sera of cancer patients and healthy volunteers were shown to be readily taken up by host cells followed by activation of a DNA-damage-repair-response which led their large scale integration into the host cell genomes. The latter caused dsDNA breaks and apoptosis in cells in vitro and in those of vital organs when injected intravenously into mice. Cell-free chromatin was consistently more active than cell-free DNA, while cfNAs derived from cancer patients were significantly more active than those from healthy volunteers. This study suggests that circulating extracellular cfNAs act as physiological continuously arising DNA mutagens with implications for ageing, cancer and a host of other degenerative human pathologies.
Calcium entry via voltage gated calcium channels mediates a wide range of physiological functions, whereas calcium channel dysregulation has been associated with numerous pathophysiological conditions. There are myriad cell signaling pathways that act on voltage gated calcium channels to fine tune their activities and to regulate their cell surface expression. These regulatory mechanisms include the activation of G protein-coupled receptors and downstream phosphorylation events, and their control over calcium channel trafficking through direct physical interactions. Calcium channels also undergo post-translational modifications that alter both function and density of the channels in the plasma membrane. Here we focus on select aspects of these regulatory mechanisms and highlight recent developments.
The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.
Why do forest productivity and biomass decline with elevation? To address this question, research to date generally has focused on correlative approaches describing changes in woody growth and biomass with elevation. We present a novel, mechanistic approach to this question by quantifying the autotrophic carbon budget in 16 forest plots along a 3300 m elevation transect in Peru. Low growth rates at high elevations appear primarily driven by low gross primary productivity (GPP), with little shift in either carbon use efficiency (CUE) or allocation of net primary productivity (NPP) between wood, fine roots and canopy. The lack of trend in CUE implies that the proportion of photosynthate allocated to autotrophic respiration is not sensitive to temperature. Rather than a gradual linear decline in productivity, there is some limited but nonconclusive evidence of a sharp transition in NPP between submontane and montane forests, which may be caused by cloud immersion effects within the cloud forest zone. Leaf-level photosynthetic parameters do not decline with elevation, implying that nutrient limitation does not restrict photosynthesis at high elevations. Our data demonstrate the potential of whole carbon budget perspectives to provide a deeper understanding of controls on ecosystem functioning and carbon cycling.
Worldwide, Salmonella spp. is a significant cause of disease for both humans and wildlife, with wild birds adapted to urban environments having different opportunities for pathogen exposure, infection, and transmission compared to their natural conspecifics. Food provisioning by people may influence these factors, especially when high-density mixed species flocks aggregate. White Ibises (Eudocimus albus), an iconic Everglades species in decline in Florida, are becoming increasingly common in urbanized areas of south Florida where most are hand-fed. We examined the prevalence of Salmonella shedding by ibises to determine the role of landscape characteristics where ibis forage and their behavior, on shedding rates. We also compared Salmonella isolated from ibises to human isolates to better understand non-foodborne human salmonellosis. From 2010-2013, 13% (n = 261) adult/subadult ibises and 35% (n = 72) nestlings sampled were shedding Salmonella. The prevalence of Salmonella shedding by ibises significantly decreased as the percent of Palustrine emergent wetlands and herbaceous grasslands increased, and increased as the proportion of open-developed land types (e.g. parks, lawns, golf courses) increased, suggesting that natural ecosystem land cover types supported birds with a lower prevalence of infection. A high diversity of Salmonella serotypes (n = 24) and strain types (43 PFGE types) were shed by ibises, of which 33% of the serotypes ranked in the top 20 of high significance for people in the years of the study. Importantly, 44% of the Salmonella Pulsed-Field Gel Electrophoresis patterns for ibis isolates (n = 43) matched profiles in the CDC PulseNet USA database. Of these, 20% came from Florida in the same three years we sampled ibis. Importantly, there was a negative relationship between the amount of Palustrine emergent wetland and the number of Salmonella isolates from ibises that matched human cases in the PulseNet database (p = 0.056). Together, our results indicate that ibises are good indicators of salmonellae strains circulating in their environment and they have both the potential and opportunity to transmit salmonellae to people. Finally, they may act as salmonellae carriers to natural environments where other more highly-susceptible groups (nestlings) may be detrimentally affected.
Respiratory infectious disease epidemics and pandemics are recurring events that levy a high cost on individuals and society. The health-protective behavioral response of the public plays an important role in limiting respiratory infectious disease spread. Health-protective behaviors take several forms. Behaviors can be categorized as pharmaceutical (e.g., vaccination uptake, antiviral use) or non-pharmaceutical (e.g., hand washing, face mask use, avoidance of public transport). Due to the limitations of pharmaceutical interventions during respiratory epidemics and pandemics, public health campaigns aimed at limiting disease spread often emphasize both non-pharmaceutical and pharmaceutical behavioral interventions. Understanding the determinants of the public's behavioral response is crucial for devising public health campaigns, providing information to parametrize mathematical models, and ultimately limiting disease spread. While other reviews have qualitatively analyzed the body of work on demographic determinants of health-protective behavior, this meta-analysis quantitatively combines the results from 85 publications to determine the global relationship between gender and health-protective behavioral response. The results show that women in the general population are about 50% more likely than men to adopt/practice non-pharmaceutical behaviors. Conversely, men in the general population are marginally (about 12%) more likely than women to adopt/practice pharmaceutical behaviors. It is possible that factors other than pharmaceutical/non-pharmaceutical status not included in this analysis act as moderators of this relationship. These results suggest an inherent difference in how men and women respond to epidemic and pandemic respiratory infectious diseases. This information can be used to target specific groups when developing non-pharmaceutical public health campaigns and to parameterize epidemic models incorporating demographic information.
An 11-year-old healthy girl was noted to have intermittent, spontaneous, and bilateral hemolacria of 14 months' duration. Examination and workup failed to identify an organic or psychogenic cause. The patient was expectantly managed with eventual spontaneous resolution. This report draws attention to this unusual and often concerning clinical entity. The case report is in compliance with the Health Insurance Portability and Accountability Act.
Global population aging has raised academic interest in successful aging to a public policy priority. Currently there is no consensus regarding the definition of successful aging. However, a synthesis of research shows successful aging can be defined as a late-life process of change characterized by high physical, psychological, cognitive, and social functioning. Masters athletes systematically train for, and compete in, organized forms of team and individual sport specifically designed for older adults. Masters athletes are often proposed as exemplars of successful aging. However, their aging status has never been examined using a comprehensive multidimensional successful aging definition. Here, we examine the successful aging literature, propose a successful aging definition based on this literature, present evidence which suggests masters athletes could be considered exemplars of successful aging according to the proposed definition, and list future experimental research directions.
Despite the exercise benefits, disparities among diverse older adults continue to exist where African American women have the lowest percentage of any population group in meeting national recommended activity guidelines. Drawing on the philosophical tradition of phronesis (practical reasoning) introduced by Aristotle, we studied perceptions of the exercise value among 14 older African American women. Three themes included: 1) exercise goals (e.g., effort exerted), 2) exercise reasons (e.g., health benefits, enjoyment and convenience, and activity recommendation), and 3) inactivity reasons (e.g., health issues, lack of motivation, and family responsibilities/priorities). Although most women reported being active, only three participants met the Healthy People 2020 guidelines for aerobic and muscle-strengthening activities, while two individuals were inactive. Exercise promoters should consider the values that motivate older African American women to exercise, such as the provision of accessible, non-discriminatory exercise facilities, and not to exercise, such as concerns about neighborhood safety, in designing programs.
This study aimed to examine whether muscle force and tendon stiffness in a muscle -tendon complex alter synchronously following 8-week whole-body vibration (WBV) training in older people. Forty older women aged 65 years and older, were randomly assigned into Control (CON, n=15) and WBV training groups (Exposure Time (ET, n=13) and Vibration Intensity (VI, n=12)). For the training groups, 4-week detraining period was subjected following the training period. Throughout the training/detraining period, force of the medial gastrocnemius (MG) muscle and stiffness of the Achilles tendon were assessed 4 times (0, 4, 8, and 12-week) using a combined system of dynamometer and ultrasonography. While muscle force gradually increased throughout the training period (p<0.05), significant increase in tendon stiffness was observed after 8 weeks (p<0.05). These findings indicated that, during the early phase of WBV training, muscle force and tendon stiffness changed asynchronously, which might be a factor to possible musculotendinous injuries.
The aim of this study was to compare postural sway during a series of static balancing tasks and during five chair rises between healthy young (mean (SEM) age 26(1) yrs), healthy old (age 67(1) yrs) and master athlete runners (age 67(1) yrs; competing and training for the previous 51(5) yrs) using the Microsoft Kinect One. The healthy old had more sway than young in all balance tasks. The master athletes had similar sway to young during two-leg balancing and one leg standing with eyes open. When balancing on one-leg with eyes closed, both the healthy old and the master athletes had around 17-fold more sway than young. The healthy old and master athletes also had less anterio-posterior movement during chair rising compared with young. These results suggest that masters runners are not spared from the age-associated decline in postural stability and may benefit from specific balance training.
This investigation examined differences in functional sit-to-stand power/velocity between cohorts of adults aged 18-97 years. This study included 264 healthy adults classified into 4 cohorts (18-40, C1; 60-69, C2; 70-79, C2; ≥80, C4). Participants completed the sit-to-stand task 5 times. Power and velocity were measured via the TENDO power analyzer. Absolute average power was maintained from C1-C3, but decreased (p<.01) in C4. Absolute peak power decreased between C1-C2 (p<.01), was similar between C2-C3, and decreased in C4 (p<.01). Relative (to body weight) average and peak power decreased between C1-C2 (p<.01), was similar between C2-C3, and decreased in C4 (p<.01). Average velocity was similar between C1 and C2, but decreased in C3 (p<.01) and C4 (p<.01), respectively. Peak velocity was significantly different between all cohorts (p<.01). Declines in functional power may plateau during the 7th and 8th decades, accelerating after 80 years.
To determine how care home managers negotiate the conflict between maintaining a safe environment while enabling the autonomy of residents with dementia. This is important because there is limited research with care home managers; yet, they are key agents in the implementation of national policies.
Semi-structured interviews were conducted with 18 managers from care homes offering dementia care in the Northwest of England. Data were analysed using a thematic analysis approach.
There were three areas in which care home staff reported balancing safety and risk against the individual needs of residents. First, the physical environment created a tension between safety and accessibility to the outside world, which meant that care homes provided highly structured or limited access to outdoor space. Second, care home managers reflected a balancing act between an individual's autonomy and the need to protect their residents' dignity. Finally, care home managers highlighted the ways in which an individual's needs were framed by the needs of other residents to the extent that on some occasions an individual's needs were subjugated to the needs of the general population of a home.
There was a strong, even dominant, ethos of risk management and keeping people safe. Managing individual needs while maintaining a safe care home environment clearly is a constant dynamic interpersonal process of negotiating and balancing competing interests for care home managers.
The energy-dependent cross section of the ^{7}Be(n,α)^{4}He reaction, of interest for the so-called cosmological lithium problem in big bang nucleosynthesis, has been measured for the first time from 10 meV to 10 keV neutron energy. The challenges posed by the short half-life of ^{7}Be and by the low reaction cross section have been overcome at n_TOF thanks to an unprecedented combination of the extremely high luminosity and good resolution of the neutron beam in the new experimental area (EAR2) of the n_TOF facility at CERN, the availability of a sufficient amount of chemically pure ^{7}Be, and a specifically designed experimental setup. Coincidences between the two alpha particles have been recorded in two Si-^{7}Be-Si arrays placed directly in the neutron beam. The present results are consistent, at thermal neutron energy, with the only previous measurement performed in the 1960s at a nuclear reactor. The energy dependence reported here clearly indicates the inadequacy of the cross section estimates currently used in BBN calculations. Although new measurements at higher neutron energy may still be needed, the n_TOF results hint at a minor role of this reaction in BBN, leaving the long-standing cosmological lithium problem unsolved.
Exploration of interfacial hydration networks of zwitterion and nonionized trialanine has been performed using DFT-M062X quantum chemical computations explicitly considering up to 41 water molecules. The step-by-step water molecules peptide surrounding, carried out for unfolded extended (β), polyproline II (PPII) conformations reveals the crucial importance of explicit solvent effects in stabilizing the zwitterion form and the left-handed PPII-helix ubiquitously found at room temperature for short polyalanines. Hydration effects are much greater for the ionized form of the peptide; thus, the zwitterion is about 10 kcal mol(-1) more stable than the nonionized form. For the β → PPII transformation, the two components of free Gibbs energy act in the opposite direction; thus, it is favored by enthalpy but not by entropy. These findings agree with experimental data that report an equilibrium between these conformers modulated by temperature. Thermodynamic functions of the four conformers (β-β, β-PPII, PPII-β, and PPII-PPII) for zwitterion trialanine are similar to those derived for the protonated one (Ala3H(+)); therefore, the peptidic conformation is independent of the pH of the solution. Rather, it reflects the high propensity of alanine toward PPII helix. The enthalpic preference of the PPII has electrostatic origin and it is owing to a more favorable interaction of dipole of each peptidic residue with water dipole of H-bonded molecules.
Owing to their ability to act as light-harvesting scaffolds, porphyrin-containing metal-organic frameworks (MOFs) are in the forefront of research on the application of highly ordered molecular materials to problems in solar-energy conversion. In this work, solvent-assisted linker exchange (SALE) is performed on a pillared paddlewheel porphyrin containing MOF thin film to collapse a 3D framework to a 2D framework. The change in dimensionality of the framework is confirmed by a decrease in the film thickness, the magnitude of which is in agreement with crystallographic parameters for related bulk materials. Furthermore, NMR spectroscopy performed on the digested sample suggests a similar change in geometry is achieved in bulk MOF samples. The decreased distance between the porphyrin chromophores in the 2D MOF film compared to the 3D film results in enhanced energy transfer through the film. The extent of energy transport was probed by assembling MOF thin film where the outermost layers are palladium porphyrin (P2) units, which act as energy traps and fluorescence quenchers. Steady-state emission spectroscopy together with time-resolved emission spectroscopy indicates that excitons can travel through about 9-11 layers (porphyrin layers) in 2D films, whereas in 3D films energy transfer occurs through no more than about 6-8 layers. The results are difficult to understand if only changes in MOF interlayer spacing are considered but become much more understandable if dipole-dipole coupling distances are considered.
Carbohydrates are important in signaling, energy storage and metabolism. Depending on their function, carbohydrates can be part of larger structures, such as glycoproteins, glycolipids or other functionalities (glycoside). To this end, polymers can act as carriers of carbohydrates in so-called glycopolymers, which mimic the multivalent carbohydrate functionalities. We chose a biocompatible poly(2-ethyl-2-oxazoline) (PEtOx) as the basis for making glycopolymers. Via the partial hydrolysis of PEtOx, a copolymer of PEtOx and polyethyleneimine (PEI) was obtained; the subsequent reductive amination with the linear forms of glucose and maltose yielded the glycopolymers. The ratios of PEtOx and carbohydrates were varied systematically, and the solution behaviors of the resulting glycoconjugates are discussed. Dynamic light scattering (DLS) revealed that depending on the carbohydrate ratio, the glycopolymers were either fully water-soluble or formed agglomerates in a temperature-dependent manner. Finally, these polymers were tested for their biological availability by studying their lectin binding ability with Concanavalin A.
This document provides the final text of regulations governing employee protection (retaliation or whistleblower) claims under section 1558 of the Affordable Care Act, which added section 18C to the Fair Labor Standards Act to provide protections to employees who may have been subject to retaliation for seeking assistance under certain affordability assistance provisions (for example, health insurance premium tax credits) or for reporting potential violations of the Affordable Care Act's consumer protections (for example, the prohibition on rescissions). An interim final rule (IFR) governing these provisions and request for comments was published in the Federal Register on February 27, 2013. Thirteen comments were received; eleven were responsive to the IFR. This rule responds to those comments and establishes the final procedures and time frames for the handling of retaliation complaints under section 18C, including procedures and time frames for employee complaints to the Occupational Safety and Health Administration (OSHA), investigations by OSHA, appeals of OSHA determinations to an administrative law judge (ALJ) for a hearing de novo, hearings by ALJs, review of ALJ decisions by the Administrative Review Board (ARB) (acting on behalf of the Secretary of Labor), and judicial review of the Secretary of Labor's (Secretary's) final decision. It also sets forth the Secretary's interpretations of the Affordable Care Act whistleblower provision on certain matters.
Taxon-specific DNA tests are applied to many ecological and management questions, increasingly using environmental DNA (eDNA). eDNA facilitates noninvasive ecological studies but introduces additional risks of bias and error. For effective application, PCR primers must be developed for each taxon and validated in each system. We outline a nine step framework for the development and validation of taxon-specific primers for eDNA analysis in ecological studies, involving reference database construction, phylogenetic evaluation of the target gene, primer design, primer evaluation in silico, and laboratory evaluation of primer specificity, sensitivity and utility. Our framework makes possible a rigorous evaluation of likely sources of error. The first five steps can be conducted relatively rapidly and (where reference DNA sequences are available) require minimal laboratory resources, enabling assessment of primer suitability before investing in further work. Steps six to eight require more costly laboratory analyses but are essential to evaluate risks of false-positive and false-negative results, while step 9 relates to field implementation. As an example, we have developed and evaluated primers to specifically amplify part of the mitochondrial ND2 gene from Australian bandicoots. If adopted during the early stages of primer development, our framework will facilitate large-scale implementation of well-designed DNA tests to detect specific wildlife from eDNA samples. This will provide researchers and managers with an understanding of the strengths and limitations of their data and the conclusions that can be drawn from them.
Laser trapping and manipulation of blood cells without mechanical contact have become feasible with implication of laser tweezers. They open up new horizons for the hemorheologic researches, offer new possibilities for studying live cells interactions on individual cell level under the influence of different endogenous and exogenous factors. The operation principle of laser tweezers is based on the property of strongly focused laser beam to act on a dielectric microparticle located in the vicinity of the beam waist with a force that drives the particle to the equilibrium location and holds it there. If the beam waist position is manipulated, so is the position of the particle. The displacement of the particle from the equilibrium position by external forces can be calibrated so that these forces can be precisely measured in the range ca. 0.1-100 pN. This is the range of forces of elastic deformation of blood cells and of their interaction with each other and with vessel walls. Being able to measure these forces without mechanical contact allows for studying on single cell level the mechanisms of interactions that was impossible earlier. Here we discuss the basic features of these techniques and give some examples of challenging hemorheologic studies.
Hemoglobinopathies, particularly β-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways.
Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production.
We show that SIN3A genomic variants are associated both with β-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in β-hemoglobinopathies patients.
Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.
NAD is a pyridine nucleotide that is involved in cell metabolism and signaling of plant growth and stress. Recently, we reported on the multifaceted nature of NAD-inducible immunity in Arabidopsis. We identified NAD as an integral regulator of multiple defense layers such as production of ROS, deposition of callose, stimulation of cell death and modulation of defense metabolism including the defense hormones SA, JA and ABA, and other defense-associated metabolites. Altogether, NAD-induced immune effects confer resistance to diverse pathogenic microbes. Our addendum to this work further demonstrates an impact of NAD on the cytosolic calcium pool, a well-known component of early plant defense response.
Managing invasive alien species in Canada requires reliable taxonomic identification as the basis of rapid-response management. This can be challenging, especially when organisms are small and lack morphological diagnostic features. DNA-based techniques, such as DNA barcoding, offer a reliable, rapid, and inexpensive toolkit for taxonomic identification of individual or bulk samples, forensic remains, and even environmental DNA. Well suited for this requirement, they could be more broadly deployed and incorporated into the operating policy and practices of Canadian federal departments and should be authorized under these agencies' articles of law. These include Fisheries and Oceans Canada, Canadian Food Inspection Agency, Transport Canada, Environment Canada, Parks Canada, and Health Canada. These efforts should be harmonized with the appropriate provisions of provincial jurisdictions, for example, the Ontario Invasive Species Act. This approach necessitates that a network of accredited, certified laboratories exists, and that updated DNA reference libraries are readily accessible. Harmonizing this approach is vital among Canadian federal agencies, and between the federal and provincial levels of government. Canadian policy and law must also be harmonized with that of the USA when detecting, and responding to, invasive species in contiguous lands and waters. Creating capacity in legislation for use of DNA-based identifications brings the authority to fund, train, deploy, and certify staff, and to refine further developments in this molecular technology.
Molecular characterization of neuron populations, particularly those controlling threat responses, is essential for understanding the cellular basis of behaviour and identifying pharmacological agents acting selectively on fear-controlling circuitry. Here we demonstrate a comprehensive workflow for identification of pharmacologically tractable markers of behaviourally characterized cell populations. Thy1-eNpHR-, Thy1-Cre- and Thy1-eYFP-labelled neurons of the BLA consistently act as fear inhibiting or 'Fear-Off' neurons during behaviour. We use cell-type-specific optogenetics and chemogenetics (DREADDs) to modulate activity in this population during behaviour to block or enhance fear extinction. Dissociated Thy1-eYFP neurons are isolated using FACS. RNA sequencing identifies genes strongly upregulated in RNA of this population, including Ntsr2, Dkk3, Rspo2 and Wnt7a. Pharmacological manipulation of neurotensin receptor 2 confirms behavioural effects observed in optogenetic and chemogenetic experiments. These experiments identify and validate Ntsr2-expressing neurons within the BLA, as a putative 'Fear-Off' population.
Renal cell carcinoma (RCC) is a seventh ranked malignancy with poor prognosis. RCC is lethal at metastatic stage as it does not respond to conventional systemic treatments, and there is an urgent need to find out promising novel biomarkers for effective treatment. The goal of this study was to evaluate the biomarkers that can be potential therapeutic target and predict effective inhibitors to treat the metastatic stage of RCC.
We conducted transcriptomic profiling to identify differentially expressed genes associated with RCC. Molecular pathway analysis was done to identify the canonical pathways and their role in RCC. Tissue microarrays (TMA) based immunohistochemical stains were used to validate the protein expression of cyclinD1 (CCND1) and were scored semi-quantitatively from 0 to 3+ on the basis of absence or presence of staining intensity in the tumor cell. Statistical analysis determined the association of CCND1 expression with RCC. Molecular docking analyses were performed to check the potential of two natural inhibitors, rutin and curcumin to bind CCND1.
We detected 1490 significantly expressed genes (1034, upregulated and 456, downregulated) in RCC using cutoff fold change 2 and p value < 0.05. Hes-related family bHLH transcription factor with YRPW motif 1 (HEY1), neuropilin 2 (NRP2), lymphoid enhancer-binding factor 1 (LEF1), and histone cluster 1 H3h (HIST1H3H) were most upregulated while aldolase B, fructose-bisphosphate (ALDOB), solute carrier family 12 (SLC12A1), calbindin 1 (CALB1) were the most down regulated genes in our dataset. Functional analysis revealed Wnt/β-catenin signaling as the significantly activated canonical pathway (z score = 2.53) involving cyclin D1 (CCND1). CCND1 was overexpressed in transcriptomic studies (FC = 2.26, p value = 0.0047) and TMA results also showed the positive expression of CCND1 in 53 % (73/139) of RCC cases. The ligands - rutin and curcumin bounded with CCND1 with good affinity.
CCND1 was one of the important upregulated gene identified in microarray and validated by TMA. Docking study showed that CCND1 may act as a potential therapeutic target and its inhibition could focus on the migratory, invasive, and metastatic potential of RCC. Further in vivo and in vitro molecular studies are needed to investigate the therapeutic target potential of CCND1 for RCC treatment.
Purple acid phosphatases (PAPs) are binuclear metallo-hydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals, PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. The aim of the present study was to investigate inhibitory effect of synthesized diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives as potential red kidney bean PAP (rkbPAP) inhibitors accompanied by experimental and molecular modeling assessments. Enzyme kinetic data showed that they are good rkbPAP inhibitors whose potencies improve with increasing alkyl chain length. Hexadecyl derivatives, as most potent compounds (Ki = 1.1 µM), inhibit rkbPAP in the mixed manner, while dodecyl derivatives act as efficient noncompetitive inhibitor. Also, analysis by molecular modeling of the structure of the rkbPAP-inhibitor complexes reveals factors, which may be important for the determination of inhibition specificity.
A novel series of thiophene derived Schiff bases and their transition metal- [Co(II), Cu(II), Zn(II), Ni(II)] based compounds are reported. The Schiff bases act as tridentate ligands toward metal ions via azomethine-N, deprotonated-N of ammine substituents and S-atom of thienyl moiety. The synthesized ligands along with their metal complexes were screened for their in vitro antibacterial activity against six bacterial pathogens (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and for antifungal activity against six fungal pathogens (Trichophytonlongifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata). The results of antimicrobial studies revealed the free ligands to possess potential activity which significantly increased upon chelation.
The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein-protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ∼10 residues on the solvent exposed surface of palm domain. Protein-peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.
Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers.
Vibrio cholerae serogroup O1 is the main causative agent of cholera diseases defined by life threatening rice watery diarrhea. Cholera routine vaccination has failed in controlling epidemics in developing countries because of their hard and expensive production. In this study, our aim was to investigate phage displayed mimotopes that could mimic V. cholerae lipopolysaccharide (LPS). Although LPS of Vibrio, as an endotoxin, can stimulate the immune system, thereby making it a suitable candidate for cholera vaccine, its toxicity remains as a main problem. Phage particles displaying 12 amino acid peptides were selected from phage library mimicking the antigenic epitopes of LPS from vibrio. The screening was carried out using single-domain antibody fragment VHH against LPS as target through three rounds of selection. Three clones with highest affinity to VHH were selected. To find out a new and efficient vaccine against cholera, these three phage particles containing high-affinity peptides were administered to mice to investigate the active and passive immunity. Out of 20 particles, three showed the highest affinity toward VHH. ELISA was carried out with immunized mice sera using LPS and three selected phages particles individually. ETEC, Shigella sonnei, and clinical isolates were used as bacterial targets. These three selected phages (individually or in combination) could stimulate mice immune system producing active and passive immunity. The mice immunized with phage particles could protect about 14 LD50 of V. cholerae. In conclusion, these peptides are mimicking LPS and can potentially act as vaccine candidates against V. cholerae. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Utilization of social media both in the private and professional arenas has grown rapidly in the last decade. The rise of social media use within health care can be viewed as the Internet-based corollary of the patient-centered care movement, in which patient perspectives and values are central to the delivery of quality care. For orthopedic surgeons and their practices, general-purpose online social networks, such as Facebook and Twitter, are convenient platforms for marketing, providing patient education and generating referrals. Virtual health communities are used less frequently by orthopedic surgeons but provide forums for patient engagement and active surgeon-to-patient communication via blogs and ask-the-doctor platforms. This commentary reviews the current state of social media use in orthopedic practice, with particular emphasis on managing the extension of the surgeon-patient relationship online, including the unique practice risks social media poses, such as privacy concerns, potential liability, and time consumption.
In the last 20 years it has been widely demonstrated that cell nucleus contains neutral and polar lipids localized in nuclear membranes, nucleoli, nuclear matrix and chromatin. Nuclear lipids may show specific organization forming nuclear lipid microdomains and have both structural and functional roles. Depending on their localization, nuclear lipids play different roles such as the regulation of nuclear membrane and nuclear matrix fluidity but they also can act as platforms for vitamin and hormone function, for active chromatin anchoring, and for the regulation of gene expression, DNA duplication and transcription. Crosstalk among different kinds of lipid signalling pathways influence the physiopathology of numerous cell types. In neural cells the nuclear lipids are involved in cell proliferation, differentiation, inflammation, migration and apoptosis. Abnormal metabolism of nuclear lipids might be closely associated with tumorigenesis and neurodegenerative diseases such as Alzheimer disease and Parkinson disease among others.
This manuscript reports the identification and characterization of five transcription factors binding to the promoter of OsNHX1 in a salt stress tolerant rice genotype (Hasawi). Although NHX1 encoding genes are known to be highly regulated at the transcription level by different abiotic stresses, namely salt and drought stress, until now only one transcription factor (TF) binding to its promoter has been reported. In order to unveil the TFs regulating NHX1 gene expression, which is known to be highly induced under salt stress, we have used a Y1H system to screen a salt induced rice cDNA expression library from Hasawi. This approach allowed us to identify five TFs belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) binding to the OsNHX1 gene promoter. We have also shown that these TFs act either as transcriptional activators (OsPCF2, OsNIN-like4) or repressors (OsCPP5, OsNIN-like2) and their encoding genes are differentially regulated by salt and PEG-induced drought stress in two rice genotypes, Nipponbare (salt-sensitive) and Hasawi (salt-tolerant). The transactivation activity of OsNIN-like3 was not possible to determine. Increased soil salinity has a direct impact on the reduction of plant growth and crop yield and it is therefore fundamental to understand the molecular mechanisms underlying gene expression regulation under adverse environmental conditions. OsNHX1 is the most abundant K(+)-Na(+)/H(+) antiporter localized in the tonoplast and its gene expression is induced by salt, drought and ABA. To investigate how OsNHX1 is transcriptionally regulated in response to salt stress in a salt-tolerant rice genotype (Hasawi), a salt-stress-induced cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsNHX1 promoter as bait. Five transcription factors (TFs) belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) were identified as binding to OsNHX1 promoter. Transactivation activity assays performed in Arabidopsis and rice protoplasts showed that OsPCF2 and OsNIN-like4 are activators of the OsNHX1 gene expression, while OsCPP5 and OsNIN-like2 act as repressors. The transactivation activity of OsNIN-like3 needs to be further investigated. Gene expression studies showed that OsNHX1 transcript level is highly induced by salt and PEG-induced drought stress in both shoots and roots in both Nipponbare and Hasawi rice genotypes. Nevertheless, OsNHX1 seems to play a particular role in shoots in response to drought. Most of the TFs binding to OsNHX1 promoter showed a modest transcriptional regulation under stress conditions, however, in response to most of the conditions studied, the OsPCF2 was induced earlier than OsNHX1, indicating that OsPCF2 may activate OsNHX1 gene expression. In addition, although the OsNHX1 response to salt and PEG-induced drought stress in either shoots or roots was quite similar in both rice genotypes, the expression of OsPCF2 in roots under salt stress and the OsNIN-like4 in roots subjected to PEG was mainly up-regulated in Hasawi, indicating that these TFs may be associated with the salt and drought stress tolerance observed for this genotype.
The public health risks associated with dairy farming intensification are an emerging concern. We examine the association between dairy cattle density and cryptosporidiosis risk in children <5 years old in New Zealand from 1997 to 2008, a period of rapid intensification of the dairy industry. Multi-level Poisson regression was used to model reported cryptosporidiosis (N = 3869 cases) incidence in relation to dairy cattle densities across urban and rural areas separately, after controlling for microbiological quality of public drinking water supplies and neighbourhood socio-economic factors using the Census Area Unit of residence. Within urban areas, the risk of cryptosporidiosis in children less than 5 years old was significantly, positively associated with medium and high dairy cattle density IRR 1.3 (95% CI 1.2, 1.5) and 1.5 (95% CI 1.2, 1.9) respectively, when compared to areas with no dairy cattle. Within rural areas, the incidence risk of cryptosporidiosis in children less than 5 years old were significantly, positively associated with medium and high dairy cattle density: IRR 1.7 (95% CI 1.3, 2.3) and 2.0 (95% CI 1.5, 2.8) respectively, when compared to areas with no dairy cattle. These results have public health implications for children living on and in proximity to intensively stocked dairy cattle farms.
Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.
The detection of pyrophosphatase (PPase) activity is of great significance in diagnosing diseases and understanding the function of PPase-related biological events. This study constructed a turn off-on-off fluorescent system for PPase activity assay based on PPase-regulated competitive coordination of Cu(2+) between a water-soluble fluorescent probe 6,7-dihydroxycoumarin (DHC) and pyrophosphate (PPi). The probe DHC can coordinate with Cu(2+) and consequently display on-off type fluorescence response. Furthermore, the in situ formed nonfluorescent Cu(2+)-DHC complex can act as an effective off-on type fluorescent probe for sensing PPi due to the higher coordination reactivity between Cu(2+) and PPi than that between Cu(2+) and DHC. The subsequent addition of PPase to the mixture containing Cu(2+), DHC, and PPi leads to the fluorescence requenching of the system again (an off state) because PPase catalyzes the hydrolysis of PPi into orthophosphate in the reaction system. Under the optimum conditions, the decrease of the fluorescence intensity of DHC-Cu(2+)-PPi system was linear with the increase of the PPase activity in the range from 0.1 to 0.3 U. The detection limit was down to 0.028 U PPase (S/N = 3). Moreover, the as-established system was also applied to evaluate PPase inhibitor. This study offers a simple yet effective method for the detection of PPase activity.
Stand up paddle boarding (SUP) is a rapidly growing sport and recreational activity where anecdotal evidence exists for its proposed health, fitness and injury rehabilitation benefits. While limited scientific evidence exists to substantiate these claims, previous studies have shown that high levels of fitness, strength and balance exists amongst participants of this sport. The purpose of this study was to conduct a training intervention on a group of previously untrained individuals to ascertain the potential of SUP on various health parameters.
An intervention study was conducted where after being tested initially, subjects were left for 6 weeks to act as their own control before the SUP intervention began. A total of 13 SUP participants completed the training study (nine males, four females) which was comprised of three 1 h sessions per week for 6 weeks.
No significant changes occurred during the initial control period. Significant (P < 0.05) improvements were made in aerobic (+23.57 %) and anaerobic fitness (+41.98 %), multidirectional core strength tests (prone +19.78 %, right side +26.19 %, left side +28.31 %, Biering Sorensen +21.33 %) and self-rated quality of life questionnaires in the physical (+19.99 %) and psychological (+17.49 %) domains. No significant changes were detected in static or dynamic balance over the duration of the training intervention.
These results demonstrate the cardiovascular, musculoskeletal and psychological improvements achievable for the novice when utilizing SUP as a training tool. The result from this study provides some evidence to substantiate the claims of health and fitness benefits SUP.
Wine lees, a major waste product of winemaking, is a rich source of polyphenolic compounds. LED-light irradiation at 400-nm elicited microbicidal activity of aqueous extract from wine lees (WLE) against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, in addition to reactive oxygen species (ROS) formation, including hydroxyl radical (·OH) and hydrogen peroxide (H2O2). Although treatment for 20 min of photoirradiation alone exerted bactericidal activity with a 2- to 3-log reduction, photoirradiated WLE for 20 min achieved a 5-log or greater reduction in viable S. aureus and P. aeruginosa cells. Regarding C. albicans, a 1-log reduction (90 % reduction) of viable cells was achieved by photoirradiated WLE for 40 min, whereas photoirradiation alone did not show any fungicidal effect. ROS analyses revealed that approximately 170 μM ·OH and 600 μM H2O2 were generated in photoirradiated WLE for 20 min. Because the bactericidal activity of photoirradiated WLE was abolished by ·OH scavengers, ROS, especially highly oxidative ·OH, may be responsible for the microbicidal activity of photoirradiated WLE. In addition to its microbicidal activity, WLE may act as an antioxidant as it exerted radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl, a stable free radical.
Global warming influences a series of ecological processes and ecosystems' stability. Although comprehensive studies have been done to investigate responses of various ecosystem processes to rising air temperatures, less is known about changes in soil temperatures and their impact on below-ground processes, particularly in deep layers. Herein, we used 50 y of temperature data (1962-2011) from 360 sites in China to assess spatio-temporal changes in soil temperatures from the surface to a depth of 3.20 m. We determined, apparently for the first time, that soil surface temperature increased 31% more than air temperature, potentially leading to more carbon release to the atmosphere than predicted. Annual mean surface temperature increased by 2.07-4.04 and 0.66-2.21 °C in northern and southern China, respectively, with the greatest in winter. Warming occurred as deep as 3.20 m. The soil temperature rise was predicted to have increased soil respiration by up to 28%, reinforcing climate warming and extending the potential growing season by up to 20 d across China. However, use of only air temperature to estimate soil temperature changes would underestimate those impacts. In conclusion, these results highlighted the importance of soil warming and of using soil temperature to assess and predict soil processes.
The mammalian Hedgehog (Hh) signaling pathway is required for development and for maintenance of adult stem cells, and overactivation of the pathway can cause tumorigenesis. All responses to Hh family ligands in mammals require the primary cilium, an ancient microtubule-based organelle that extends from the cell surface. Genetic studies in mice and humans have defined specific functions for cilium-associated microtubule motor proteins: they act in the construction and disassembly of the primary cilium, they control ciliary length and stability, and some have direct roles in mammalian Hh signal transduction. These studies highlight how integrated genetic and cell biological studies can define the molecular mechanisms that underlie cilium-associated health and disease.
High performance liquid chromatography (HPLC) is a kind of efficient separation technology and has been used widely in many fields. Micro-sized porous silica microspheres as the most popular matrix have been used for fast separation and analysis in HPLC. In this paper, the monodisperse large-porous silica microspheres with controllable size and structure were successfully synthesized with polymer microspheres as the templates and characterized. First, the poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) microspheres (PGMA-EDMA) were functionalized with tetraethylenepentamine (TEPA) to generate amino groups which act as a catalyst in hydrolysis of tetraethyl orthosilicate (TEOS) to form Si-containing low molecular weight species. Then the low molecular weight species diffused into the functionalized PGMA-EDMA microspheres by induction force of the amino groups to form polymer/silica hybrid microspheres. Finally, the organic polymer templates were removed by calcination, and the large-porous silica microspheres were obtained. The compositions, morphology, size distribution, specific surface area and pore size distribution of the porous silica microspheres were characterized by infrared analyzer, scanning-electron microscopy, dynamic laser scattering, the mercury intrusion method and thermal gravimetric analysis, respectively. The results show that the agglomeration of the hybrid microspheres can be overcome when the templates were functionalized with TEPA as amination reagent, and the yield of 95.7% of the monodisperse large-porous silica microspheres can be achieved with high concentration of polymer templates. The resulting large-porous silica microspheres were modified with octadecyltrichlorosilane (ODS) and the chromatographic evaluation was performed by separating the proteins and the digest of BSA. The baseline separation of seven kinds of protein standards was achieved, and the column delivered a better performance when separating BSA digests comparing with the commercial one currently available. The high column efficiency and good reproducibility present that the large-porous silica microspheres obtained can be used as a matrix for peptide and protein separation.
Many plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants.
Here we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine.
This work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA).
Of the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine.
The results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.
The genus Xylaria has been reported as a rich source of biologically active secondary metabolites. In the present study, an endophytic fungus Xylaria psidii has been isolated from the leaf sample of Aegle marmelos (L.) Corr., characterized on the basis of its morphological features and sequence data for the ITS region (KU291350) of the nuclear ribosomal DNA. Biological screening of ethyl acetate extract of Xylaria psidii displayed a potential therapeutic effect on pancreatic cancer cells.
This study was designed systematically to explore Xylaria psidii, an endophytic fungus for the identification of biologically active secondary metabolites against pancreatic cancer cells.
While exploring the bioactive secondary metabolites, a sensitive and reliable LC-MS based dereplication approach was applied to identify four compounds A-D from fungal extract. Further bioactivity guided isolation of fungal extract yielded two major metabolites 1 and 2. The structures of 1 and 2 have been determined by detailed spectroscopic analysis including MS, NMR, IR and UV data and similarity with published data. Xylarione A (1) is new whereas (-) 5-methylmellein (2) is reported for the first time from X. psidii. Both the isolated compounds were screened for their effect on the viability and proliferation against a panel of cancer cell lines (MCF-7, MIA-Pa-Ca-2, NCI-H226, HepG2 and DU145) of different tissue origin.
Compounds 1 and 2 exhibited cytotoxicity against pancreatic cancer (MIA-Pa-Ca-2) cells with IC50 values of 16.0 and 19.0 µm, respectively. The cell cycle distribution in MIA-Pa-Ca-2 cells, confirmed a cell cycle arrest at the sub-G1 phase. Cell death induced by 1 and 2 displayed features characteristic of apoptosis. Flow cytometry based analysis of 1 and 2 using Rhodamine-123 displayed substantial loss of mitochondrial membrane potential in a concentration dependent manner by both the compounds.
Results conclude that the isolated compounds 1 and 2 are responsible for the activity shown by crude ethyl acetate extract and may act as potential leads for medicinal chemists for designing more potent analogs.
Molecular clocks drive oscillations in leaf photosynthesis, stomatal conductance, and other cell and leaf-level processes over ~24 h under controlled laboratory conditions. The influence of such circadian regulation over whole-canopy fluxes remains uncertain; diurnal CO2 and H2O vapor flux dynamics in the field are currently interpreted as resulting almost exclusively from direct physiological responses to variations in light, temperature and other environmental factors. We tested whether circadian regulation would affect plant and canopy gas exchange at the Montpellier European Ecotron. Canopy and leaf-level fluxes were constantly monitored under field-like environmental conditions, and under constant environmental conditions (no variation in temperature, radiation, or other environmental cues).
We show direct experimental evidence at canopy scales of the circadian regulation of daytime gas exchange: 20-79 % of the daily variation range in CO2 and H2O fluxes occurred under circadian entrainment in canopies of an annual herb (bean) and of a perennial shrub (cotton). We also observed that considering circadian regulation improved performance by 8-17 % in commonly used stomatal conductance models.
Our results show that circadian controls affect diurnal CO2 and H2O flux patterns in entire canopies in field-like conditions, and its consideration significantly improves model performance. Circadian controls act as a 'memory' of the past conditions experienced by the plant, which synchronizes metabolism across entire plant canopies.
Parental self-efficacy (PSE) refers to beliefs of parents to effectively engage in behaviors that result in desired outcomes for their children. There are several instruments of PSE for promoting healthy dietary or physical activity (PA) behaviors in children. These measures typically assess PSE in relation to some quantity or frequency of behavior, for example, number of servings or times per week. However, measuring PSE in relation to contextual circumstances, for example, psychological states and situational demands, may be a more informative approach. The purpose of the present study was to develop and psychometrically evaluate a context-based PSE instrument.
Swedish mothers of five-year-old children (n = 698) responded to the Parental Self-Efficacy for Healthy Dietary and Physical Activity Behaviors in Preschoolers Scale (PDAP) and a questionnaire on dietary and PA behaviors in children. Interviews were conducted to explore participant perceptions of the quality of the PDAP items. Psychometric evaluation was conducted using exploratory and confirmatory factor analyses. Spearman correlations between PSE and child behaviors were examined.
Twenty-seven interviews were conducted with participants, who perceived the items as highly comprehensible, relevant and acceptable. A four-factor model of a revised 21-item version of the PDAP fitted the data, with different factors of PSE for promoting healthy dietary or PA behaviors in children depending on whether circumstances were facilitating or impeding successful performance. Internal consistency was excellent for total scale (Cronbach's α = .94), and good for factors (α = .84-.88). Correlations were in the expected direction: positive correlations between PSE and healthy behaviors, and negative correlations between PSE and unhealthy behaviors (all r ss ≤ .32).
Psychometric evaluation of the PDAP provided preliminary support of construct validity and internal consistency.
To explore the perspective of Turkish-Dutch general practitioner (GP) patients on informal interpreting from an integrated theory base, focusing on interpreters' roles, trust and power.
Semi-structured in depth interviews were conducted with 21 first-generation Turkish-Dutch migrant patients who made use of informal interpreters to communicate with their GPs. An interview guide was designed based on the theoretical framework of interpreter's roles, trust and power, covering questions about interpreters' role, trust in informal/professional interpreters and power division in the medical consultation. The interviews were transcribed verbatim and analyzed according to the constant comparative method.
Besides providing linguistic translation, informal interpreters were expected to perform the roles of advocates and caregivers of the patients. Informal interpreters were trusted more than professional interpreters, mainly for fidelity reasons, that is, because the patients assumed that informal interpreters would act in their best interests. Although informal interpreters were often perceived as the primary interlocutor, the patients did not feel dominated by them, but rather empowered by their presence.
Our findings indicate a connection between the role of the advocate, the fidelity dimension of trust and the perceived empowerment of the patients. By linking interpreters' role to trust and power, this study contributes to theory building in the field of informal interpreting, which is needed to design evidence-based interventions to improve health care delivery to patients with insufficient language ability and thus to advance health care delivery to migrant patients, which is currently lagging behind.
Immune thrombocytopenia purpura (ITP) is characterized by the production of pathological autoantibodies that cause reduction in platelet counts. The disease can have serious medical consequences, leading to uncontrolled bleeding that can be fatal. Current widely used therapies for the treatment of ITP are non-specific and can, at times, result in complications that are more burdensome than the disease itself. In the present study, the use of platelet membrane-coated nanoparticles (PNPs) as a platform for the specific clearance of anti-platelet antibodies is explored. The nanoparticles, whose outer layer displays the full complement of native platelet surface proteins, act as decoys that strongly bind pathological anti-platelet antibodies in order to minimize disease burden. Here, we study the antibody binding properties of PNPs and assess the ability of the nanoparticles to neutralize antibody activity both in vitro and in vivo. Ultimately, we leverage the neutralization capacity of PNPs to therapeutically treat a murine model of antibody-induced thrombocytopenia and demonstrate considerable efficacy as shown in a bleeding time assay. PNPs represent a promising platform for the specific treatment of antibody-mediated immune thrombocytopenia by acting as an alternative target for anti-platelet antibodies, thus preserving circulating platelets with the potential of leaving broader immune function intact.
Restricted and repetitive behaviors are a defining feature of autism, which can be expressed as a cognitive flexibility deficit or stereotyped, motor behaviors. There is limited knowledge about the underlying neuropathophysiology contributing to these behaviors. Previous findings suggest that central 5HT2A receptor activity is altered in autism, while recent work indicates that systemic 5HT2A receptor antagonist treatment reduces repetitive behaviors in an idiopathic model of autism. 5HT2A receptors are expressed in the orbitofrontal cortex and striatum. These two regions have been shown to be altered in autism. The present study investigated whether 5HT2A receptor blockade in the dorsomedial striatum or orbitofrontal cortex in the BTBR mouse strain, an idiopathic model of autism, affects the phenotype related to restricted and repetitive behaviors. Microinfusion of the 5HT2A receptor antagonist, M100907 into the dorsomedial striatum alleviated a reversal learning impairment and attenuated grooming behavior. M100907 infusion into the orbitofrontal cortex increased perseveration during reversal learning and potentiated grooming. These findings suggest that increased 5HT2A receptor activity in the dorsomedial striatum may contribute to behavioral inflexibility and stereotyped behaviors in the BTBR mouse. 5HT2A receptor signaling in the orbitofrontal cortex may be critical for inhibiting a previously learned response during reversal learning and expression of stereotyped behavior. The present results suggest which brain areas exhibit abnormalities underlying repetitive behaviors in an idiopathic mouse model of autism, as well as which brain areas systemic treatment with M100907 may principally act on in BTBR mice to attenuate repetitive behaviors.
The first quaternary ammonium-group-containing [FeFe]-hydrogenase models [(μ-PDT)Fe2 (CO)4 {κ(2) -(Ph2 P)2 N(CH2 )2 NMe2 BzBr}] (2; PDT=propanedithiolate) and [(μ-PDT)Fe2 (CO)4 {μ-(Ph2 P)2 N(CH2 )2 NMe2 BzBr}] (4) have been prepared by the quaternization of their precursors [(μ-PDT)Fe2 (CO)4 {κ(2) -(Ph2 P)2 N(CH2 )2 NMe2 }] (1) and [(μ-PDT)Fe2 (CO)4 {μ-(Ph2 P)2 N(CH2 )2 NMe2 }] (3) with benzyl bromide in high yields. Although new complexes 1-4 have been fully characterized by spectroscopic and X-ray crystallographic studies, the chelated complexes 1 and 2 converted into their bridged isomers 3 and 4 at higher temperatures, thus demonstrating that these bridged isomers are thermodynamically favorable. An electrochemical study on hydrophilic models 2 and 4 in MeCN and MeCN/H2 O as solvents indicates that the reduction potentials are shifted to less-negative potentials as the water content increases. This outcome implies that both 2 and 4 are more easily reduced in the mixed MeCN/H2 O solvent than in MeCN. In addition, hydrophilic models 2 and 4 act as electrocatalysts and achieve higher icat /ip values and turnover numbers (TONs) in MeCN/H2 O as a solvent than in MeCN for the production of hydrogen from the weak acid HOAc.
Depending on the amount of methanol present in solution, CO2 adducts of N-heterocyclic carbenes (NHCs) and N-heterocyclic olefins (NHOs) have been found to be in fully reversible equilibrium with the corresponding methyl carbonate salts [EMIm][OCO2 Me] and [EMMIm][OCO2 Me]. The reactivity pattern of representative 1-ethyl-3-methyl-NHO-CO2 adduct 4 has been investigated and compared with the corresponding NHC-CO2 zwitterion: The protonation of 4 with HX led to the imidazolium salts [NHO-CO2 H][X], which underwent decarboxylation to [EMMIm][X] in the presence of nucleophilic catalysts. NHO-CO2 zwitterion 4 can act as an efficient carboxylating agent towards CH acids such as acetonitrile. The [EMMIm] cyanoacetate and [EMMIm]2 cyanomalonate salts formed exemplify the first C-C bond-forming carboxylation reactions with NHO-activated CO2 . The reaction of the free NHO with dimethyl carbonate selectively led to methoxycarbonylated NHO, which is a perfect precursor for the synthesis of functionalized ILs [NHO-CO2 Me][X]. The first NHO-SO2 adduct was synthesized and structurally characterized; it showed a similar reactivity pattern, which allowed the synthesis of imidazolium methyl sulfites upon reaction with methanol.
Tuberculosis (TB) is a major disease burden globally causing more than 1.5 million deaths per year. The attenuated live vaccine strain Bacille Calmette-Guérin (BCG), although providing protection against childhood TB, is largely ineffective against adult pulmonary TB. A major aim therefore is to increase the potency of the BCG vaccine to generate stronger and more sustained immunity against TB. Here, we investigated the use of layer-by-layer (LbL) nanocoating of the surface of live BCG with several layers of polyinosinic-polycytidylic acid (poly(I:C)), a strong inducer of cell-mediated immunity, and the biodegradable polysaccharide chitosan to enhance BCG immunogenicity. Nanocoating of live BCG did not affect bacterial viability or growth in vitro but induced killing of the BCG in infected mouse bone marrow-derived macrophages and enhanced macrophage production of pro-inflammatory cytokines and expression of surface co-stimulatory molecules relative to uncoated BCG. In addition, poly(I:C) surface-coated BCG, but not BCG alone or together with soluble poly(I:C), induced high production of nitric oxide (NO) and IL-12. These results argue that BCG and surface absorbed poly(I:C) act in a synergistic manner to elicit pro-inflammatory macrophage activation. In conclusion, nanocoating of live BCG with the immunostimulatory agent poly(I:C) may be an appropriate strategy to enhance and modulate host responses to the BCG vaccine.
The eukaryotic flagellum is the organelle responsible for the propulsion of the male gamete in most animals. Without exception, sperm of all mammalian species use a flagellum for swimming. The mammalian sperm has a centrally located 9 + 2 arrangement of microtubule doublets and hundreds of accessory proteins that together constitute an axoneme. However, they also possess several characteristic peri-axonemal structures that make the mammalian sperm tail function differently. These modifications include nine outer dense fibers (ODFs) that are paired with the nine outer microtubule doublets of the axoneme, and are anchored in a structure called the connecting piece located at the base. The presence of the ODFs and connecting piece, and the absence of a basal body, dictate that physical forces generated by the dynein motors are transmitted to the base of the flagellum through the ODFs. Mammalian sperm flagella also possess a mitochondrial and a fibrous sheath that encircle most of the axoneme. These sheaths and the ODFs add mechanical rigidity to the flagellum creating the functional effect of increasing bend wavelength, which requires the entrainment of more dynein motors in the production of a single wave. The sheaths also act as a retinaculum and maintain the integrity of the central axoneme when large bending torques are generated by dynein. Large torque production is crucial to the process of hyperactivation and the unique motility transitions associated with effective fertilizing capacity. Consequently, these specialized anatomical features are essential for the effective interaction of sperm with the female reproductive tract and ovum. © 2016 Wiley Periodicals, Inc.
Strong oxidizing agents often contain fluorine and are not compatible with substrates forming strong element-fluorine bonds. Therefore, there is need for new strong oxidizers. Cyclic voltammetric measurements on the weakly coordinating anion [Me3 NB12 Cl11 ](-) in liquid SO2 revealed an oxidation wave at +2.59 V versus Fc(0/+) . The oxidation of Na[Me3 NB12 Cl11 ] with AsF5 in liquid sulfur dioxide gives the boron-cluster-based radical Me3 NB12 Cl11(.) as a dark blue solid in quantitative yield. The radical was characterized by vibrational, NMR, and EPR spectroscopy and by its crystal structure. To explore the potential of the radical Me3 NB12 Cl11(.) to act as a strong oxidizing agent, it was reacted with different compounds having high ionization energies. Thianthrene (ionization energy 7.8 eV) and hexabromobenzene (8.8 eV) were oxidized to the corresponding cations. The reaction with elemental iodine (9.3 eV) gave the [I5 ](+) cation. These reactive cations are stabilized in the solid state by the weakly coordinating anion [Me3 NB12 Cl11 ](-) , which was formed as a reduced product in the oxidation reactions.
Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.
A fundamentally simple, mild, and practical procedure for peptide bond formation is reported that employs a stoichiometric amount of easy-to-access 9-silafluorenyl dichlorides as the coupling agent. Without initial preactivation or elaboration of the carboxylic acid or amine termini of the amino acids, the developed reagent is proposed to act through an unprecedented chemical ligation mechanism, bringing the two coupling partners together before being subsequently eliminated. The desired amides or peptide bonds are thus furnished in good yields and with low to no epimerization.
Phenotypic traits such as ornaments and armaments are generally shaped by sexual selection, which often favours larger and more elaborate males compared to females. But can sexual selection also influence the brain? Previous studies in vertebrates report contradictory results with no consistent pattern between variation in brain structure and the strength of sexual selection. We hypothesize that sexual selection will act in a consistent way on two vertebrate brain regions that directly regulate sexual behaviour: the medial preoptic nucleus (MPON) and the ventromedial hypothalamic nucleus (VMN). The MPON regulates male reproductive behaviour whereas the VMN regulates female reproductive behaviour and is also involved in male aggression. To test our hypothesis, we used high-resolution magnetic resonance imaging combined with traditional histology of brains in 14 dragon lizard species of the genus Ctenophorus that vary in the strength of precopulatory sexual selection. Males belonging to species that experience greater sexual selection had a larger MPON and a smaller VMN. Conversely, females did not show any patterns of variation in these brain regions. As the volumes of both these regions also correlated with brain volume (BV) in our models, we tested whether they show the same pattern of evolution in response to changes in BV and found that the do. Therefore, we show that the primary brain nuclei underlying reproductive behaviour in vertebrates can evolve in a mosaic fashion, differently between males and females, likely in response to sexual selection, and that these same regions are simultaneously evolving in concert in relation to overall brain size.
Recently philosophers have proposed a wide variety of interventions referred to as 'moral enhancements'. Some of these interventions are concerned with helping individuals make more informed decisions; others, however, are designed to compel people to act as the intervener sees fit. Somewhere between these two extremes lie interventions designed to direct an agent's attention either towards morally relevant issues - hat-hanging - or away from temptations to do wrong - hat-hiding. I argue that these interventions fail to constitute genuine moral enhancement because, although they may result in more desirable outcomes - more altruism, more law-following, and/or less self-destructive behavior, they ignore a person's intentions, and often what makes an action right or wrong is the intent behind it.
Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA(Met)i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA(Met)i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA(Met)i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.
A carbonylcobalt catalyst, immobilized by poly(4-vinylpyridine) (P4VP) through N→Co coordination bonds, has been prepared by solvothermal method. It has been revealed that the pyridine fragments in the polymer catalyst act not only as promoters to improve the catalytic performance of the carbonylcobalt catalyst for alkoxycarbonylation of ethylene oxide to methyl 3-hydroxypropanoate but also as stabilizers to enhance the reusability of the polymer catalyst. Furthermore, the polymer catalyst could be easily separated by filtration and reused with only a slight loss of catalytic efficiency.
Ecomorphological differentiation is a key feature of adaptive radiations, with a general trend for specialization and niche expansion following divergence. Ecological opportunity afforded by invasion of a new habitat is thought to act as an ecological release, facilitating divergence, and speciation. Here, we investigate trophic adaptive morphology and ecology of an endemic clade of oreochromine cichlid fishes (Alcolapia) that radiated along a herbivorous trophic axis following colonization of an isolated lacustrine environment, and demonstrate phenotype-environment correlation. Ecological and morphological divergence of the Alcolapia species flock are examined in a phylogenomic context, to infer ecological niche occupation within the radiation. Species divergence is observed in both ecology and morphology, supporting the importance of ecological speciation within the radiation. Comparison with an outgroup taxon reveals large-scale ecomorphological divergence but shallow genomic differentiation within the Alcolapia adaptive radiation. Ancestral morphological reconstruction suggests lake colonization by a generalist oreochromine phenotype that diverged in Lake Natron to varied herbivorous morphologies akin to specialist herbivores in Lakes Tanganyika and Malawi.
The central nervous system and viscera constitute a functional ensemble, the gut-brain axis, that allows bidirectional information flow that contributes to the control of feeding behavior based not only on the homeostatic, but also on the hedonic aspects of food intake. The prevalence of eating disorders, such as anorexia nervosa, binge eating and obesity, poses an enormous clinical burden, and involves an ever-growing percentage of the population worldwide. Clinical and preclinical research is constantly adding new information to the field and orienting further studies with the aim of providing a foundation for developing more specific and effective treatment approaches to pathological conditions. A recent symposium at the XVI Congress of the Societá Italiana di Neuroscienze (SINS, 2015) 'Eating disorders: from bench to bedside and back' brought together basic scientists and clinicians with the objective of presenting novel perspectives in the neurobiology of eating disorders. Clinical studies presented by V. Ricca illustrated some genetic aspects of the psychopathology of anorexia nervosa. Preclinical studies addressed different issues ranging from the description of animal models that mimic human pathologies such as anorexia nervosa, diet-induced obesity, and binge eating disorders (T. Lutz), to novel interactions between peripheral signals and central circuits that govern food intake, mood and stress (A. Romano and G. Provensi). The gut-brain axis has received increasing attention in the recent years as preclinical studies are demonstrating that the brain and visceral organs such as the liver and guts, but also the microbiota are constantly engaged in processes of reciprocal communication, with unexpected physiological and pathological implications. Eating is controlled by a plethora of factors; genetic predisposition, early life adverse conditions, peripheral gastrointestinal hormones that act directly or indirectly on the central nervous system, all are receiving attention as they presumably contribute to the development of eating disorders.
Plant aromatic compounds provide signals and a nutrient source to pathogens, and also act as stressors. Structure-activity relationships suggest two pathways sensing these compounds in the maize pathogen Cochliobolus heterostrophus, one triggering a stress response, and one inducing enzymes for their degradation. Focusing on the stress pathway, we found that ferulic acid causes rapid appearance of TUNEL-positive nuclei, dispersion of histone H1:GFP, hyphal shrinkage, and eventually membrane damage. These hallmarks of programmed cell death (PCD) were not seen upon exposure to caffeic acid, a very similar compound. Exposure to ferulic acid dephosphorylated two MAP kinases: Hog1 (stress activated) and Chk1 (pathogenicity related), while increasing phosphorylation of Mps1 (cell integrity related). Mutants lacking Hog1 or Chk1 are hypersensitive to ferulic acid while Mps1 mutants are not. These results implicate three MAPK pathways in the stress response. Ferulic acid and the antifungal fludioxonil have opposite additive effects on survival and on dephosphorylation of Hog1, which is thus implicated in survival. The results may explain why some fungal pathogens of plants undergo cell death early in host invasion, when phenolics are released from plant tissue.
The aim of this study was to assess the species and the genetic diversity of the staphylococci population in raw milk from healthy goats. Isolates representative of all genotypes were screened for their potential pathogenicity by the occurrence of some relevant safety-related properties, such as antibiotic resistance, presence of virulence factor genes, biofilm formation ability and biogenic amine production. A total of 314 staphylococci were isolated, and randomly amplified polymorphic DNA-PCR analysis displayed 48 genotypes. Isolates were identified as belonging to S. epidermidis (87.5%), S. caprae (6.2%), S. aureus (4.2%) and S. simulans (2.1%) species. The antibiotic resistance varied strongly with strains, with S. epidermidis and S. aureus strains showing resistance to more number of antibiotics. A high occurrence of strains harbouring hemolysin genes was also found in both species. On the contrary, none of the strains assayed harboured enterotoxin or amino acid decarboxylase genes, and, although a moderate or high biofilm formation was observed in 29% of the strains, they did not harbour icaA or icaD genes. This study gives a first and extensive picture of safety-related properties within Staphylococcus species isolated from milk of healthy goats, displaying that these species can act as a reservoir for spreading genes related to safety.
Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg(H52) in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, Arg(H52) of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(kcat/Km 2)/kuncat] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg(H52) is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pKa of the amine is perturbed and shifts to a lower pKa, which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.
The Androgen Receptor (AR) remains the leading target of advanced prostate cancer therapies. Thiosalicylamide analogs have previously been shown to act in cells as acyltransfer catalysts that are capable of transferring cellular acetate, presumably from acetyl-CoA, to HIV NCp7. Here we explore if the cellular acetyl-transfer activity of thiosalicylamides can be redirected to other cellular targets guided by ligands for AR. We constructed conjugates of thiosalicylamides and the AR-binding small molecule tolfenamic acid, which binds the BF-3 site of AR, proximal to the coactivator "FXXLF" binding surface. The thiosalicylamide-tolfenamic acid conjugate, YZ03, but not the separate thiosalicylamide plus tolfenamic acid, significantly enhanced acetylation of endogenous AR in CWR22Rv1 cells. Further analysis confirms that Lys720, a residue critical to FXXLF coactivator peptide binding, is a site of acyl-YZ03 acetylation. Under acyl-transfer conditions, YZ03 significantly enhances the ability of BF-3 site binding ligands to inhibit AR-coactivator peptide association. These data suggest that biomimetic acyltransferases can enhance protein-protein interaction inhibitors through covalent modification of critical interfacial residues.
Providing adolescents with confidential health care results in better social and health outcomes. We sought to assess if a medical board Maintenance of Certification Part IV project could improve the delivery of confidential care to minor adolescent patients seen in outpatient primary care practices.
Participating physicians reviewed 3 months of charts for patients ages 12-17 years seen for well visits during a baseline time period, and after 2 Plan, Do, Study, Act intervention cycles to assess if they had met confidentiality standards. Participating physicians additionally completed an assessment tool on personal and clinic practices related to confidentiality.
Nine academic and 3 private practice family medicine, pediatrics, and medicine-pediatrics sites.
Forty-four physicians.
Provider and staff deficits in knowledge of minor consent laws, resistance toward the idea of confidential care, and work flow issues around confidential screening were identified as primary barriers. Staff and provider trainings, scripts, and staff involvement in planning work flows were identified as key interventions.
Improvement in confidentiality standards met during minor adolescent well visits.
Participating physicians significantly increased the proportion of well visits in which they spent time alone with the patient (P = .001), explained minor consent laws (P < .001), and had the adolescent complete a confidential risk screening tool (P < .001), in addition to improving scores on their confidentiality assessment overall (P < .001).
A medical board Maintenance of Certification Part IV project is an effective way to change physician practice and improve the delivery of confidential care to minor adolescents seen for well visits.
A backbone-modified peptide derived from parathyroid hormone (PTH) is shown to function as an inhibitor and inverse agonist of parathyroid hormone receptor-1 (PTHR1) signaling. This receptor acts to regulate calcium and phosphate homeostasis, as well as bone turnover and development. PTH is a natural agonist of PTHR1, and PTH(1-34) displays full activity relative to the natural 84-residue hormone. PTH(1-34) is used clinically to treat osteoporosis. N-terminally truncated derivatives of PTH(1-34), such as PTH(7-34), are known to bind to PTHR1 without initiating intracellular signaling and can thus act as competitive antagonists of PTH-induced signaling at PTHR1. In some cases, N-terminally truncated PTH derivatives also act as inverse agonists of PTHR1 variants that display pathologically high levels of signaling in the absence of PTH. Many analogues of PTH, however, are rapidly degraded by proteases, which may limit biomedical application. We show that backbone modification via periodic replacement of α-amino acid residues with homologous β-amino acid residues leads to an α/β-PTH(7-34) peptide that retains the antagonist and inverse agonist activities of the prototype α-peptide while exhibiting enhanced stability in the presence of aggressive proteases. These findings highlight the value of backbone-modified peptides derived from PTH as tools for investigating determinants of PTH metabolism and provide guidance for designing therapeutic agents for diseases arising from excessive ligand-dependent or ligand-independent PTHR1 activity.
Angiopoietin-like protein 1 (ANGPTL1) has been shown to act as a tumor suppressor by inhibiting angiogenesis, cancer invasion, and metastasis. However, little is known about the effects of ANGPTL1 on sorafenib resistance and cancer stem cell properties in hepatocellular carcinoma (HCC) and the mechanism underlying these effects. Here, we show that ANGPTL1 expression positively correlates with sorafenib sensitivity in HCC cells and human HCC tissues. ANGPTL1 significantly decreases epithelial-mesenchymal transition (EMT)-driven sorafenib resistance, cancer stemness, and tumor growth of HCC cells by repressing Slug expression. ANGPTL1 directly interacts with and inactivates MET receptor, which contributes to Slug suppression through inhibition of the extracellular receptor kinase/protein kinase B (ERK/AKT)-dependent early growth response protein 1 (Egr-1) pathway. ANGPTL1 expression inversely correlates with Slug expression, poor sorafenib responsiveness, and poor clinical outcomes in HCC patients.
ANGPTL1 inhibits sorafenib resistance and cancer stemness in HCC cells by repressing EMT through inhibition of the MET receptor-AKT/ERK-Egr-1-Slug signaling cascade. ANGPTL1 may serve as a novel MET receptor inhibitor for advanced HCC therapy. (Hepatology 2016;64:1637-1651).
Sweet cherries are highly appreciated by consumers worldwide and are usually cold-stored during postharvest to prevent over-ripening before distribution to the market. Sweet cherry is a non-climacteric fruit, for which ripening is known to be regulated by abscisic acid. Here we aimed to examine the hormone profiles, including measurements of abscisic acid, auxins, cytokinins and gibberellins by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), in relation to variations in sugar and anthocyanin contents, during growth and ripening of this fruit. Hormonal profiling revealed that indole-3-acetic acid, GA1 and trans-zeatin levels decreased at early stages of fruit development, while GA3 levels decreased at early stages but also later, once anthocyanin accumulation started. Conversely, abscisic acid levels rose significantly once the fruit started to synthetize anthocyanins, and isopentenyladenosine levels also increased during the ripening of sweet cherries. A strong negative correlation was found between GA4 levels and both fruit biomass and anthocyanin levels, and between the levels of trans-zeatin and both fruit biomass and total sugar contents. In contrast, abscisic acid and isopentenyladenosine levels correlated positively with fruit biomass, anthocyanin and total soluble sugar content. Results suggest that auxins, cytokinins and gibberellins may act coordinately with abscisic acid in the regulation of sweet cherry development and ripening. Furthermore, it is shown that hormonal profile measurements by UHPLC-MS/MS may be a helpful tool to elucidate the timing of action of each specific hormonal compound during ripening, which has important applications in the agri-food biotechnological sector.
Rooted in the mechanism of action of antibiotics and subject to bacterial evolution, antibiotic resistance is difficult and perhaps impossible to overcome. Nevertheless, strategies can be used to minimize the emergence and impact of resistance. Antibiotic adjuvants offer one such approach. These are compounds that have little or no antibiotic activity themselves but act to block resistance or otherwise enhance antibiotic action. Antibiotic adjuvants are therefore delivered in combination with antibiotics and can be divided into two groups: Class I agents that act on the pathogen, and Class II agents that act on the host. Adjuvants offer a means to both suppress the emergence of resistance and rescue the activity of existing drugs, offering an orthogonal strategy complimentary to new antibiotic discovery VIDEO ABSTRACT.
As part of the National Institute for Health and Clinical Excellence (NICE) single technology appraisal (STA) process, the manufacturer of apremilast was invited to submit evidence for its clinical and cost effectiveness for the treatment of active psoriatic arthritis (PsA) for whom disease-modifying anti-rheumatic drugs (DMARDs) have been inadequately effective, not tolerated or contraindicated. The Centre for Reviews and Dissemination and Centre for Health Economics at the University of York were commissioned to act as the independent Evidence Review Group (ERG). This paper provides a description of the ERG review of the company's submission, the ERG report and submission and summarises the NICE Appraisal Committee's subsequent guidance (December 2015). In the company's initial submission, the base-case analysis resulted in an incremental cost-effectiveness ratio (ICER) of £14,683 per quality-adjusted life-year (QALY) gained for the sequence including apremilast (positioned before tumour necrosis factor [TNF]-α inhibitors) versus a comparator sequence without apremilast. However, the ERG considered that the base-case sequence proposed by the company represented a limited set of potentially relevant treatment sequences and positions for apremilast. The company's base-case results were therefore not a sufficient basis to inform the most efficient use and position of apremilast. The exploratory ERG analyses indicated that apremilast is more effective (i.e. produces higher health gains) when positioned after TNF-α inhibitor therapies. Furthermore, assumptions made regarding a potential beneficial effect of apremilast on long-term Health Assessment Questionnaire (HAQ) progression, which cannot be substantiated, have a very significant impact on results. The NICE Appraisal Committee (AC), when taking into account their preferred assumptions for HAQ progression for patients on treatment with apremilast, placebo response and monitoring costs for apremilast, concluded that the addition of apremilast resulted in cost savings but also a QALY loss. These cost savings were not high enough to compensate for the clinical effectiveness that would be lost. The AC thus decided that apremilast alone or in combination with DMARD therapy is not recommended for treating adults with active PsA that has not responded to prior DMARD therapy, or where such therapy is not tolerated.
Oral direct-acting antivirals (DAAs) represent a major advance in hepatitis C virus (HCV) treatment. Along with recent updates in HCV screening policy and expansions in insurance coverage, treatment demand in the United States is changing rapidly. Our objective was to project the characteristics and number of people needing antiviral treatment and HCV-associated disease burden in the era of oral DAAs. We used a previously developed and validated Hepatitis C Disease Burden Simulation model (HEP-SIM). HEP-SIM simulated the actual clinical management of HCV from 2001 onward, which included antiviral treatment with pegylated interferon (Peg-IFN)-based therapies as well as the recent oral DAAs, risk-based and birth-cohort HCV screening, and the impact of the Affordable Care Act. We also simulated two hypothetical scenarios-no treatment and treatment with Peg-IFN-based therapies only. We estimated that in 2010, 2.5 (95% confidence interval [CI], 1.9-3.1) million noninstitutionalized people were viremic, which dropped to 1.9 (95% CI, 1.4-2.6) million in 2015, and projected to drop below 1 million by 2020. A total of 1.8 million HCV patients will receive HCV treatment from the launch of oral DAAs in 2014 until 2030. Based on current HCV management practices, it will take 4-6 years to treat the majority of patients aware of their disease. However, 560,000 patients would still remain unaware by 2020. Even in the oral DAA era, 320,000 patients will die, 157,000 will develop hepatocellular carcinoma, and 203,000 will develop decompensated cirrhosis in the next 35 years.
HCV-associated disease burden will still remain substantial in the era of oral DAAs. Increasing HCV screening and treatment capacity is essential to further decreasing HCV burden in the United States. (Hepatology 2016;64:1442-1450).
To determine the minimum survival benefits that patients, and their clinicians, judged sufficient to make adjuvant chemotherapy (ACT) worthwhile, in addition to pelvic radiotherapy, for women with high risk and advanced stage endometrial cancer.
Eighty-three participants in the PORTEC-3 trial completed a time trade-off questionnaire before and after adjuvant therapy; 44 of their clinicians completed it once only. The questionnaire used four hypothetical scenarios including baseline survival times without ACT of 5 and 8 years, and baseline survival rates at 5 years without ACT of 50 and 65%.
Over 50% of patients judged an extra 1 year of survival time or an extra 5% in survival rate sufficient to make ACT worthwhile. Over 50% of clinicians judged an extra 1 year of survival time, or an extra 10% in survival rate, sufficient to make ACT worthwhile. Compared with patients, clinicians required similar survival time benefits (medians both 1 year, P=0.4), but larger survival rate benefits (medians 8.5% vs 5%, P=0.03), and clinicians' preferences varied less (IQR 0.5-1.5 years vs 0.4-2 years, P=0.0007; 5-10% vs 1-13%, P=0.004). Patients' preferences changed over time for the survival rate scenarios depending on whether they had ACT or not (change in median benefit - 3 months vs 2.5 months respectively, P=0.028). There were no strong predictors of patients' or clinicians' preferences.
Patients and clinicians judged moderate survival benefits sufficient to make ACT worthwhile after pelvic radiotherapy for endometrial cancer. These benefits are larger than those judged sufficient by patients with breast or colon cancers, but similar to those judged sufficient by patients with lung or ovarian cancers.
NET (NocA/Nlz, Elbow, Tlp-1) family members have recently emerged as important players in the development of human cancers. Zinc finger protein 703 (ZNF703), locating on chromosome 8 (8p11.23), a member of the NET/Nlz family of zinc finger transcription factors, had been demonstrated to be a much novel oncogene of several malignancies. This study aimed to investigate the expression of ZNF703 in cholangiocarcinoma (CCA) and attempted to elucidate its biological effects in CCA progression.
The correlation between ZNF703 expression and clinicopathological characteristics of CCA was evaluated through analyzing 85 cases. The biological effects of ZNF703 were investigated both in vitro and in vivo in which proliferation, migration, and invasive potential were mainly explored. Statistical software SPSS 16.0 was used for statistical analyses.
ZNF703 was overexpressed in CCA tissues with subcellular localizations mainly in the nucleus and partly in the cytoplasm or membrane. High expression of ZNF703 was related to tumor location (P=0.002), pathological grading (P=0.024), depth of invasion (P=0.002), distant metastasis (P=0. 011) and AJCC stage (P=0.008). Both in vitro and in vivo studies demonstrated that ZNF703 could potently promote proliferation, migration and invasion throughout the progression of CCA.
ZNF703 can potently facilitate tumor growth and metastasis in many respects throughout the progression of CCA, which may act as an oncogene in CCA and can be considered as a novel potential therapeutic target.
This work investigated the role of paired box 2 (PAX2) in endometrial cancer and its epigenetic regulation mechanism. Endometrial cancer tissues and cell lines exhibited increased PAX2 expression compared with hyperplasia, normal endometrium and endometrial epithelial cells. Knock-down of PAX2 resulted in reduced cell viability, invasion and migration, and PAX2 overexpression caused the opposite effects. Increased methylation of the PAX2 promoter was observed in both cancer tissues and cell lines and was positively correlated with PAX2 expression. After 5-Aza-CdR treatment, PAX2 mRNA and protein were down-regulated, and PAX2 methylation was decreased. Deletion analysis confirmed that a repressive transcriptional regulatory region of the PAX2 promoter coincided with the hypermethylated region identified in MassARRAY analysis. Binding sites of myeloid zinc finger 1 (MZF1) are predicted in the defined region. Knock-down of MZF1 up-regulated the transcriptional activity and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 may act as a repressive transcription factor when the PAX2 promoter is unmethylated. In conclusion, PAX2 is involved in the carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. The overexpression of PAX2 in endometrial cancer is regulated by promoter hypermethylation and the transcription factor MZF1.
Gefitinib is an oral EGFR tyrosine kinase inhibitors which may act as a radiosensitizer.
This phase II study evaluated the efficacy of gefitinib 250 mg once daily in combination with thoracic radiotherapy (66 Gy in 6.5 weeks, 2 Gy/day, 5 fractions/week) followed by consolidation chemotherapy (IV cisplatin and vinorelbine) as first line treatment in a population of unselected stage IIIB NSCLC patients according to EGFR mutation status.
Due to a low accrual rate in this study, the sample size (n = 50) was not reached. Sixteen patients were included in four centers, 50% had adenocarcinoma and 75% were male. Genomic alterations (7 patients studied) retrieved TP53 mutation in 2 patients and no EGFR mutation. Four weeks after radiotherapy, 3 patients (19%) had a partial response, 6 (38%) had a stable disease, and 7 had a progression (44%). Median overall survival was 11 months and median progression-free survival was 5 months. At the time of the last contact, 5 patients (31%) were still alive. Main toxicities were gastrointestinal (81%), cutaneous (81%), general (56%), and respiratory (50%). There were 12>G3 adverse events in 7 (47%) patients, and there was one toxic-death during the concomitant period due to an interstitial pneumonitis. There were two possible adverse events-related deaths during the chemotherapy period (pulmonary embolism (n = 1) and sudden death after the administration of the 3rd course of chemotherapy (n = 1)).
The benefit of Gefitinib-RT could not be confirmed due to premature trial discontinuation. Further evaluation is required, especially in patients with EGFR mutated NSCLC.
Human CUB and Sushi multiple domains 1 (CSMD1) is a membrane-bound complement inhibitor suggested to act as a putative tumor suppressor gene, since allelic loss of this region encompassing 8p23 including CSMD1 characterizes various malignancies. Here, we assessed the role of CSMD1 as a tumor suppressor gene in the development of breast cancer in vitro and in vivo. We found that human breast tumor tissues expressed CSMD1 at lower levels compared to that in normal mammary tissues. The decreased expression of CSMD1 was linked to a shorter overall survival of breast cancer patients. We also revealed that expression of CSMD1 in human breast cancer cells BT-20 and MDA-MB-231 significantly inhibited their malignant phenotypes, including migration, adhesion and invasion. Conversely, stable silencing of CSMD1 expression in T47D cells enhanced cancer cell migratory, adherent and clonogenic abilities. Moreover, expression of CSMD1 in the highly invasive MDA-MB-231 cells diminished their signaling potential as well as their stem cell-like properties as assessed by measurement of aldehyde dehydrogenase activity. In a xenograft model, expression of CSMD1 blocked the ability of cancer cells to metastasize to secondary sites in vivo, likely via inhibiting local invasion but not the extravasation into distant tissues. Taken together, these findings demonstrate the role of CSMD1 as a tumor suppressor gene in breast cancer.
Increasing evidence supports the anticancer effects of morin in vitro and in vivo. However, the role of morin-7-sulphate sodium (NaMoS), a water-soluble flavonoid derivative synthesized from morin remains unclear. The present study investigated the tumor suppression by NaMoS in mouse melanoma cells.
We synthesized the flavonoid derivative morin-7-sulphate sodium according to the method described for quercetin-sulphate derivative, and further isolated, purified and identified the compound. Cell proliferation in vitro was assessed using a CCK-8 assay. The wound healing assay was performed to evaluate cell motility, and flow cytometry was used to detect cellular apoptosis. Protein levels of vimentin, matrix metalloproteinase 9 (MMP9), phosphorylation of Akt1/2/3 (p-Akt1/2/3), extracellular signal-regulated kinase 1/2 (p-ERK1/2) and Caspase3 in B16F10 cells were detected by immunohistochemistry and Western blot.
The results suggest that cell proliferation was markedly decreased in NaMoS-treated groups (1, 10, 25, 50, 100, 500, 1000μM) in a dose-dependent manner compared with the Control group and the IC50 was 221.67μM at 48h. NaMoS at 200μM concentration significantly inhibited the invasion and promoted apoptosis of B16F10 cells. Moreover, protein level of Caspase3 increased significantly in B16F10 cells treated by NaMoS. Immunohistochemistry and Western blot further confirmed that NaMoS decreased the expression of vimentin, MMP9, p-Akt1/2/3 and p-ERK1/2 in B16F10 cells.
This study provides robust evidence that NaMoS, a water-soluble flavonoid, manifests anticancer properties and may act as a signal transduction inhibitor in melanoma cells.
A comprehensive series of nuclear tests were carried out by the United States at Enewetak Atoll in the Marshall Islands, especially between 1952 and 1958. A Porites Lutea coral that was growing in the Enewetak lagoon within a few km of all of the high-yield tests contains a continuous record of isotopes, which are of interest (e.g. (14)C, (236)U, (239,240)Pu) through the testing period. Prior to the present work, (14)C measurements at ∼2-month resolution had shown pronounced peaks in the Δ(14)C data that coincided with the times at which tests were conducted. Here we report measurements of (236)U and (239,240)Pu on the same coral using accelerator mass spectrometry, and again find prominent peaks in the concentrations of these isotopes that closely follow those in (14)C. Consistent with the (14)C data, the magnitudes of these peaks do not, however, correlate well with the explosive yields of the corresponding tests, indicating that smaller tests probably contributed disproportionately to the debris that fell in the lagoon. Additional information about the different tests can also be obtained from the (236)U/(239)Pu and (240)Pu/(239)Pu ratios, which are found to vary dramatically over the testing period. In particular, the first thermonuclear test, Ivy-Mike, has characteristic (236)U/(239)Pu and (240)Pu/(239)Pu signatures which are diagnostic of the first arrival of nuclear test material in various archives.
Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3'-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.
The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.
Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin.
Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma.
Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.
Management authorities seldom have the capacity to comprehensively address the full suite of anthropogenic stressors, particularly in the coastal zone where numerous threats can act simultaneously to impact reefs and other ecosystems. This situation requires tools to prioritise management interventions that result in optimum ecological outcomes under a set of constraints. Here we develop one such tool, introducing a Bayesian Belief Network to model the ecological condition of inshore coral reefs in Moreton Bay (Australia) under a range of management actions. Empirical field data was used to model a suite of possible ecological responses of coral reef assemblages to five key management actions both in the sea (e.g. expansion of reserves, mangrove & seagrass restoration, fishing restrictions) and on land (e.g. lower inputs of sediment and sewage from treatment plants). Models show that expanding marine reserves (a 'marine action') and reducing sediment inputs from the catchments (a 'land action') were the most effective investments to achieve a better status of reefs in the Bay, with both having been included in >58% of scenarios with positive outcomes, and >98% of the most effective (5th percentile) scenarios. Heightened fishing restrictions, restoring habitats, and reducing nutrient discharges from wastewater treatment plants have additional, albeit smaller effects. There was no evidence that combining individual management actions would consistently produce sizeable synergistic until after maximum investment on both marine reserves (i.e. increasing reserve extent from 31 to 62% of reefs) and sediments (i.e. rehabilitating 6350 km of waterways within catchments to reduce sediment loads by 50%) were implemented. The method presented here provides a useful tool to prioritize environmental actions in situations where multiple competing management interventions exist for coral reefs and in other systems subjected to multiple stressor from the land and the sea.
In the central nervous system (CNS), activation of the transcription factor nuclear factor-kappa B (NF-κβ) is associated with both neuronal survival and increased vulnerability to apoptosis. The mechanisms underlying these dichotomous effects are attributed to the composition of NF-κΒ dimers. In Alzheimer's disease (AD), β-amyloid (Aβ) and other aggregates upregulate activation of p65:p50 dimers in CNS cells and enhance transactivation of pathological mediators that cause neuroinflammation and neurodegeneration. Hence selective targeting of activated p65 is an attractive therapeutic strategy for AD. Here we report the design, structural and functional characterization of peptide analogs of a p65 interacting protein, the glucocorticoid induced leucine zipper (GILZ). By virtue of binding the transactivation domain of p65 exposed after release from the inhibitory IκΒ proteins in activated cells, the GILZ analogs can act as highly selective inhibitors of activated p65 with minimal potential for off-target effects.
Implementation of the Affordable Care Act motivates assessment of health insurance and supplementary programs, such as the AIDS Drug Assistance Program (ADAP) on health outcomes of HIV-infected people in the United States. We assessed the effects of health insurance, ADAP, and income on HIV viral load suppression.
We used existing cohort data from the HIV-infected participants of the Women's Interagency HIV Study. Cox proportional hazards models were used to estimate the time from 2006 to unsuppressed HIV viral load (>200 copies/mL) among those with Medicaid, private, Medicare, or other public insurance, and no insurance, stratified by the use of ADAP.
In 2006, 65% of women had Medicaid, 18% had private insurance, 3% had Medicare or other public insurance, and 14% reported no health insurance. ADAP coverage was reported by 284 women (20%); 56% of uninsured participants reported ADAP coverage. After accounting for study site, age, race, lowest observed CD4, and previous health insurance, the hazard ratio (HR) for unsuppressed viral load among those privately insured without ADAP, compared with those on Medicaid without ADAP (referent group), was 0.61 (95% CI: 0.48 to 0.77). Among the uninsured, those with ADAP had a lower relative hazard of unsuppressed viral load compared with the referent group (HR, 95% CI: 0.49, 0.28 to 0.85) than those without ADAP (HR, 95% CI: 1.00, 0.63 to 1.57).
Although women with private insurance are most likely to be virally suppressed, ADAP also contributes to viral load suppression. Continued support of this program may be especially critical for states that have not expanded Medicaid.
The recent increase in the creation of transboundary protected areas and wildlife corridors between them lends importance to information on pathogen prevalence and transmission among wildlife species that will become connected. One such initiative is the Kavango Zambezi Transfrontier Conservation Area of which Botswana's Okavango Delta constitutes a major contribution for wildlife and ecosystems. Between 2008 and 2011, we collected serum samples from 14 lions ( Panthera leo ), four leopards ( Panthera pardus ), 19 spotted hyenas ( Crocuta crocuta ), and six cheetahs ( Acinonyx jubatus ) in the Okavango. Samples were tested for antibodies against canine distemper virus (CDV), feline panleukopenia virus, enteric coronavirus, feline calicivirus, feline herpesvirus (FHV-1), and feline immunodeficiency virus (FIV). Evidence of exposure to all of these pathogens was found, to varying degrees, in at least one of the species sampled. High antibody prevalence (>90%) was only found to FHV-1 and FIV in lions. Only hyenas (26%, 5/19) were positive for CDV antibody. Except for one case, all individuals displayed physical conditions consistent with normal health for ≥12 mo following sampling. Our results emphasize the need for a comprehensive, multispecies approach to disease monitoring and the development of coordinated management strategies for subpopulations likely to be connected in transboundary initiatives.
The childhood Asthma-Control Test (C-ACT) is validated for assessing asthma control in paediatric asthma. Among children aged 4-11 years, the C-ACT requires the simultaneous presence of both parent and child. There is an unmet need for a tool that can be used to assess asthma control in children when parents or caregivers are not present such as in the school setting. We assessed the psychometric properties and estimated the minimally important difference (MID) of the C-ACT and a modified version, comprising only the child responses (C-ACTc). Asthma patients aged 6-11 years (n=161) from a previously completed multicenter randomised trial were included. Demographic information, spirometry and questionnaire scores were obtained at baseline and during follow-up. Participants or their guardians kept a daily asthma diary. Internal consistency reliabilities of the C-ACT and C-ACTc were 0.76 and 0.67 (Cronbach's α), respectively. Test-retest reliabilities of the C-ACT and C-ACTc were 0.72 and 0.66 (intra-class correlation), respectively. Significant correlations were noted between C-ACT scores and ACQ scores (Spearman's correlation r=-0.56, 95% CI (-0.66, -0.44), P<0.001). The strength of the correlation between C-ACTc scores and ACQ scores was weaker (Spearman's correlation r=-0.46, 95% CI (-0.58, -0.33), P<0.001). We estimated the MID for the C-ACT and C-ACTc to be 2 points and 1 point, respectively. Among asthma patients aged 6-11 years, the C-ACT had good psychometric properties. The psychometric properties of a shortened child-only version (C-ACTc), although acceptable, are not as strong.
In this review, reports were retrieved in which vitamin D status, as assessed by serum 25-hydroxyvitamin D [25(OH)D] levels, was measured in South African population groups with varied skin colours and ethnicities. Healthy children and adults were generally vitamin D-sufficient [25(OH)D level >50 nmol/L] but the majority of those aged above 65 years were deficient. A major role for exposure to solar ultraviolet radiation (UVR) in determining 25(OH)D levels was apparent, with the dietary contribution being minor. Limited data exist regarding the impact of recent changes in lifestyles on vitamin D status, such as urbanisation. With regard to disease susceptibility, 11 of 22 relevant publications indicated association between low 25(OH)D levels and disease, with deficiency most notably found in individuals with tuberculosis and HIV-1. Information on the relationship between vitamin D receptor variants and ethnicity, disease or treatment response in the South African population groups demonstrated complex interactions between genetics, epigenetics and the environment. Whether vitamin D plays an important role in protection against the range of diseases that currently constitute a large burden on the health services in South Africa requires further investigation. Only then can accurate advice be given about personal sun exposure or dietary vitamin D supplementation.
Emerging studies show that dysregulation of the receptor of activated protein kinase C1 (RACK1) plays a crucial role in tumorigenesis and progression of various cancers. However, the biological function and underlying mechanism of RACK1 in glioma remains poorly defined. Here, we found that RACK1 was significantly up-regulated in glioma tissues compared with normal brain tissues, being closely related to clinical stage of glioma both in mRNA and protein levels. Moreover, Kaplan-Meier analysis demonstrated that patients with high RACK1 expression had a poor prognosis (p = 0.0062, HR = 1.898, 95% CI: 1.225-3.203). In vitro functional assays indicated that silencing of RACK1 could dramatically promote apoptosis and inhibit cell proliferation, migration, and invasion of glioma cells. More importantly, knockdown of RACK1 led to a vast accumulation of cells in G0/G1 phase and their reduced proportions at the S phase by suppressing the expression of G1/S transition key regulators Cyclin D1 and CDK6. Additionally, this forced down-regulation of RACK1 significantly suppressed migration and invasion via inhibiting the epithelial-mesenchymal transition (EMT) markers, such as MMP2, MMP9, ZEB1, N-Cadherin, and Integrin-β1. Collectively, our study revealed that RACK1 might act as a valuable prognostic biomarker and potential therapeutic target for glioma.
The complex [Os(btzpy)₂][PF₆]₂ (1, btzpy = 2,6-bis(1-phenyl-1,2,3-triazol-4-yl)pyridine) has been prepared and characterised. Complex 1 exhibits phosphorescence (λem = 595 nm, τ = 937 ns, φem = 9.3% in degassed acetonitrile) in contrast to its known ruthenium(II) analogue, which is non-emissive at room temperature. The complex undergoes significant oxygen-dependent quenching of emission with a 43-fold reduction in luminescence intensity between degassed and aerated acetonitrile solutions, indicating its potential to act as a singlet oxygen sensitiser. Complex 1 underwent counterion metathesis to yield [Os(btzpy)₂]Cl₂ (1(Cl)), which shows near identical optical absorption and emission spectra to those of 1. Direct measurement of the yield of singlet oxygen sensitised by 1(Cl) was carried out (φ (¹O₂) = 57%) for air equilibrated acetonitrile solutions. On the basis of these photophysical properties, preliminary cellular uptake and luminescence microscopy imaging studies were conducted. Complex 1(Cl) readily entered the cancer cell lines HeLa and U2OS with mitochondrial staining seen and intense emission allowing for imaging at concentrations as low as 1 μM. Long-term toxicity results indicate low toxicity in HeLa cells with LD50 >100 μM. Osmium(II) complexes based on 1 therefore present an excellent platform for the development of novel theranostic agents for anticancer activity.
Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.
A diverse physician workforce is needed to increase access to care for underserved populations, particularly as the Affordable Care Act expands insurance coverage. Yet legal restrictions constrain the extent to which medical schools may use race/ethnicity in admissions decisions. We conducted simulations using academic metrics and socioeconomic data from applicants to a California public medical school from 2011 to 2013. The simulations systematically adjusted medical school applicants' academic metrics for socioeconomic disadvantage. We found that socioeconomic and under-represented minority disparities in admissions could be eliminated while maintaining academic readiness. Adjusting applicant academic metrics using socioeconomic information on medical school applications may be a race-neutral means of increasing the socioeconomic and racial/ethnic diversity of the physician workforce.
The Nganampa Health Council (literally "Our Health Council") is an Aboriginal community-controlled Primary Health Care service established in 1983. It was born out of the political struggle for Aboriginal Land Rights in South Australia which culminated in The Anangu Pitjantjatjara Yankunytjatjara (APY) Land Rights Act, 1981, a milestone in Indigenous Land Rights both in Australia and internationally.
In this article three palliative care physicians review narratives about the complicated experience of voluntarily stopping eating and drinking (VSED). Despite consensus about its legality, the decision to end life by VSED is emotionally and ethically challenging for patients, family members and clinicians. Each VSED story is unique, and the individual perspectives within a single story may diverge, conflict, and evolve over time. The narratives differ substantially in the range of suffering described, from acute, graphic, physical symptoms to primarily anticipatory and psychological distress. The narrators' conclusions about the meaning of VSED also vary, some perceiving it to be an irrational act of suicide, others describing it as an affirmation of personal autonomy, and most with a mixture of sometimes conflicting reactions in between these edges. The authors conclude with observations drawn from the narratives about the potential in VSED for benefit, harm and conflict for all involved.
Notes made about you while you are in any health setting are not yours. If it is an NHS hospital, they belong to the health secretary; if it is a private hospital, they belong to the consultant or the hospital itself. If something belongs to someone else, you do not have a right to it. The Data Protection Act 1998 allows you to access your health records, but this usually means you receive a copy rather than obtaining and holding the originals.
Neutrophil function is critical for the initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria such as Fusobacterium nucleatum act as bridging species between early and late colonizer pathogens such as Porphyromonas gingivalis as the biofilm ages and periodontal inflammation increases. This study was designed to determine the impact of different F. nucleatum strains on neutrophil function.
HL-60 cells were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of ssp. nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of fMLP (1 μM) stimulation. Pro-inflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of TLR2, TLR4 and NF-κB mRNA levels were analyzed using real time quantitative PCR. Each experiment was repeated at least 3 times in triplicate. Data was analyzed using ANOVA followed by post hoc Bonferroni's correction.
All strains of F. nucleatum significantly increased the phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than F. nucleatum ssp. vincentii and ssp. nucleatum (p<0.001). F. nucleatum ssp. nucleatum and polymorphum significantly blocked fMLP-induced superoxide generation (p<0.001). While F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as the nucleatum (83%) and polymorphum (100%). All F. nucleatum strains stimulated a significant increase in neutrophil apoptosis compared to the control (p<0.001) and significantly increased the expression of NF-κB mRNA in neutrophils (p<0.05). Levels of IL-8 and TNF-α produced by neutrophils were significantly increased in all F. nucleatum groups compared to the control (p<0.001).
These findings suggest that different strains of F. nucleatum impact the neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus preventing oxidative killing by the neutrophils. The direct role of bridging species in the pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for a pathogenic transition of the dental ecosystem.
We sought to explore the relationship between the podiatric medical student and the patient as it relates to the act of gift-giving as a sign of gratefulness for the services provided. This article presents the clinical case of a man who visited a podiatric medical student because of pain in his feet and subsequently presented the student with several gifts. Philanthropy, empathy, a positive attitude, treatment instructions, and the time devoted to the patient are some of the reasons why patients offer gifts to podiatric medical students. The relationship between the podiatric medical student and the patient and the act of gift-giving by patients are of ethical concern.
The first part of this review ("Monitoring of airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios") describes the current knowledge on the major biological particles present in the air regarding their global distribution, concentrations, ratios and influence of meteorological factors in an attempt to provide a framework for monitoring their biodiversity and variability in such a singular environment as the atmosphere. Viruses, bacteria, fungi, pollen and fragments thereof are the most abundant microscopic biological particles in the air outdoors. Some of them can cause allergy and severe diseases in humans, other animals and plants, with the subsequent economic impact. Despite the harsh conditions, they can be found from land and sea surfaces to beyond the troposphere and have been proposed to play a role also in weather conditions and climate change by acting as nucleation particles and inducing water vapour condensation. In regards to their global distribution, marine environments act mostly as a source for bacteria while continents additionally provide fungal and pollen elements. Within terrestrial environments, their abundances and diversity seem to be influenced by the land-use type (rural, urban, coastal) and their particularities. Temporal variability has been observed for all these organisms, mostly triggered by global changes in temperature, relative humidity, et cetera. Local fluctuations in meteorological factors may also result in pronounced changes in the airbiota. Although biological particles can be transported several hundreds of meters from the original source, and even intercontinentally, the time and final distance travelled are strongly influenced by factors such as wind speed and direction. [Int Microbiol 2016; 19(1):1-1 3].
Childhood adversity predicts adolescent suicidal ideation but there are few studies examining whether the risk of childhood adversity extends to suicidal ideation in midlife. We hypothesized that childhood adversity predicts midlife suicidal ideation and this is partially mediated by adolescent internalizing disorders, externalizing disorders and adult exposure to life events and interpersonal difficulties.
At 45 years, 9377 women and men from the UK 1958 British Birth Cohort Study participated in a clinical survey. Childhood adversity was prospectively assessed at the ages of 7, 11 and 16 years. Suicidal ideation at midlife was assessed by the depressive ideas subscale of the Revised Clinical Interview Schedule. Internalizing and externalizing disorders were measured by the Rutter scales at 16 years. Life events, periods of unemployment, partnership separations and alcohol dependence were measured through adulthood.
Illness in the household, paternal absence, institutional care, parental divorce and retrospective reports of parental physical and sexual abuse predicted suicidal ideation at 45 years. Three or more childhood adversities were associated with suicidal ideation at 45 years [odds ratio (OR) 4.31, 95% confidence interval (CI) 2.67-6.94]. Psychological distress at 16 years partially mediated the associations of physical abuse (OR 3.41, 95% CI 2.29-5.75), sexual abuse (OR 4.99, 95% CI 2.90-11.16) with suicidal ideation. Adult life events partially mediated the association of parental divorce (OR 6.34, 95% CI -7.16 to 36.75) and physical (OR 9.59, 95% CI 4.97-27.88) and sexual abuse (OR 6.59, 95% CI 2.40-38.36) with suicidal ideation at 45 years.
Adversity in childhood predicts suicidal ideation in midlife, partially mediated by adolescent internalizing and externalizing disorders, adult life events and interpersonal difficulties. Understanding the pathways from adversity to suicidal ideation can inform suicide prevention and the targeting of preventive interventions.
Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell contact, actin dynamics, and cell growth suppression. To understand whether the FAT1 gene affects the apoptosis of porcine cumulus cells and to elucidate the mechanism of this potential action, FAT1 was knocked down using RNA interference. The lack of FAT1 resulted in stable expression of CTNNB, enhanced expression of cleaved CASP3, but decreased the BCL2/BAX ratios at both the mRNA and protein levels. These results indicated that FAT1 inhibited porcine cumulus cell apoptosis via different pathways. Taken together, these data provide new insights into the mechanisms of the association between FAT1 and porcine cumulus cell apoptosis.
Ethical issues arise for genetic counselors when a client fails to disclose a genetic diagnosis of hereditary disease to family: they must consider the rights of the individual client to privacy and confidentiality as well as the rights of the family to know their genetic risk. Although considerable work has addressed issues of non-disclosure from the client's perspective, there is a lack of qualitative research into how genetic counselors address this issue in practice. In this study, a qualitative approach was taken to investigate whether genetic counselors in Australia use a relational approach to encourage the disclosure of genetic information from hereditary breast and ovarian cancer (HBOC) clients among family members; and if so, how they use it. Semi-structured qualitative interviews were conducted with 16 genetic counselors from selected states across Australia. Data collection and analysis were guided by a basic iterative approach incorporating a hybrid methodology to thematic analysis. The findings provide indicative evidence of genetic counselors employing a relational approach in three escalating stages--covert, overt and authoritative--to encourage the disclosure of genetic information. The findings lend credence to the notion that genetic counselors envision a form of relational autonomy for their clients in the context of sharing genetic information, and they depart from individualistic conceptions of care/solely client-centered counseling when addressing the needs of other family members to know their genetic status.
The aim of this study is to compare the effect of treated dentine matrix (TDM) and tricalcium phosphate (TCP) scaffolds on odontogenic differentiation and mineralization of dental pulp stem cells (DPSCs) in furcation perforations created in the pulp chamber floor of premolar teeth in dogs.
DPSCs were isolated and cultured from the dental pulp of the maxillary left second and third premolars of dogs. The DPSCs were loaded on TCP (SC+TCP) and TDM (SC+TDM) scaffolds and inserted into intentionally perforated pulp chamber floors of premolars in dogs; six teeth were used for each group. Three more groups of six specimens were created, and mineral trioxide aggregate (MTA), TDM, and TCP were inserted into the perforations to act as controls. An intact premolar and no treatment in the perforation site were used as positive and negative controls respectively. After 3 months, the animals were sacrificed and the type of inflammation, presence of dentine, continuation and type of cementum, type of connective tissue, and presence of foreign body reaction were evaluated, and significant differences were between groups determined using the Fisher's exact test. The evaluation of the amount of inflammation and the percentage of new bone formation was evaluated using the Mann-Whitney U test.
The negative control group was associated with severe inflammation and granulation tissue formation. In the positive control group, intact periodontal tissues and no inflammation were observed. Dentine bridge formation was not seen in specimens of any group. The specimens in the SC+TDM group were associated with significantly more bone formation than other groups (P < 0.001). The amount of inflammation was less than 10 % in specimens of all groups with the exception of three specimens in the TCP group that were categorized as 10-30 %. Chronic inflammation without foreign body reactions was the major pattern of inflammation in groups. Formation of cementum with a cellular and continuous appearance was seen in all specimens.
SC+TDM was associated with significantly more bone formation when used to repair uninfected furcation perforations in the premolar teeth of dogs.
Application of TDM as a biological scaffold in combination with DPSCs may offer an advantage during the repair of root perforation defects.
The aim of the study was to develop a method for quantification of cow's whey and whole milk powder in goat or sheep milk products including infant formula. A ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for simultaneous quantification of four caseins and two major whey proteins by detecting their signature peptides, which were able to act as markers for differentiating goat or sheep from cow whey and whole milk powder in infant formulas. The signature peptides were screened based on the computational prediction by Biolynx software, and confirmed by database searching after analysis of liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (LC-Q-TOF-MS). The isotopic-labeled signature peptide was used as internal standard to compensate the matrix effect. The limits of quantification were 0.01-0.05 g/100 g for target proteins. The observed recovery rates ranged from 82.3 to 116.6 % and the reproducibility was excellent (RSD <12 %) at different spiking levels. The RSDs of intra- and inter-day precision were 2.8-6.2 and 3.3-9.8 %, respectively. The multiple reaction monitoring method was successfully applied to milk powder with different composition, showing high specificity and accuracy in detection of species involved. The calculating formula was designed to assess the composition of adulteration in the actual detection of infant formulas. These results highlight applicability of this method for the detection of infant formulas with complicated matrix.
Diabetic macular edema (DME) can cause blindness in diabetic patients suffering from diabetic retinopathy (DR). DM parameters controls (glycemia, arterial tension, and lipids) are the gold standard for preventing DR and DME. Although the vascular endothelial growth factor (VEGF) is known to play a role in the development of DME, the pathological processes leading to the onset of this disease are highly complex and the exact sequence in which they occur is still not completely understood. Angiogenesis and inflammation have been shown to be involved in the pathogenesis of this disease. However, it still remains to be clarified whether angiogenesis following VEGF overexpression is a cause or a consequence of inflammation. This paper provides a review of the data currently available, focusing on VEGF, angiogenesis, and inflammation. Our analysis suggests that angiogenesis and inflammation act interdependently during the development of DME. Knowledge of DME etiology seems to be important in treatments with anti-VEGF or anti-inflammatory drugs. Current diagnostic techniques do not permit us to differentiate between both etiologies. In the future, diagnosing the physiopathology of each patient with DME will help us to select the most effective drug.
The concept of field cancerization has frequently been quoted to explain the occurrence of multiple primary cancers in the head and neck region and recurrence following complete excision of the original tumor. The main objective is to study the occurrence of field changes in mirror image biopsy in relation to histopathological changes in the oral mucosa among oral cancer patients using hematoxylin and eosin and to study the expression pattern of cytokeratin, Ki-67 and p53 in oral squamous cell carcinoma and mirror image biopsy.
A pilot study of 15 patients clinically diagnosed with carcinoma lesion and their corresponding mirror image sites were taken and stained using immunohistochemistry method for the expression of cytokeratin, Ki-67 and p53.
Primary tumors showed strong positive staining for cytokeratin throughout both the epithelium and malignant epithelial islands but absence of staining for Ki-67 and p53.
CK may be useful in predicting epithelial differentiation and Ki-67 and p53 act as weak indicators of malignant disease progression in oral tissues.
Adipocytes are a significant component of the bone marrow microenvironment. Although bone marrow adipocytes were first identified more than 100 years ago, it is only in recent years that an understanding of their complex physiological role is emerging. Bone marrow adipocytes act as local regulators of skeletal biology and homeostasis, with recent studies suggesting that marrow adipose tissue is metabolically active, and can function as an endocrine organ. As such, bone marrow adipocytes have the potential to interact with tumour cells, influencing both tumour growth and bone disease. This review discusses the current evidence for the role of bone marrow adipocytes in tumour growth within the bone marrow microenvironment and the development of the associated bone disease.
The polyphenol nordihydroguaiaretic acid (NDGA) has antineoplastic properties, hence it is critical to understand its action at the molecular level. Here, we establish that NDGA inhibits glucose uptake and cell viability in leukemic HL-60 and U-937 cell lines. We monitored hexose uptake using radio-labeled 2-deoxyglucose (2DG) and found that the inhibition by NDGA followed a noncompetitive mechanism. In addition, NDGA blocked hexose transport in human red blood cells and displaced prebound cytochalasin B from erythrocyte ghosts, suggesting a direct interaction with the glucose transporter GLUT1. We propose a model for the mechanism of action of NDGA on glucose uptake. Our study shows for the first time that NDGA can act as inhibitor of the glucose transporter GLUT1.
Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.
The tropical earthworm Pontoscolex corethrurus (Rhinodrilidae, Oligochaeta) presents a broad distribution (e.g., 56 countries from four continents). It is generally assumed that temperature appears to limit the success of tropical exotic species in temperate climates. However, the distribution range of this species could advance towards higher elevations (with lower temperatures) where no tropical species currently occur. The aim of this study was to evaluate the soil and climatic variables that could be closely associated with the distribution of P. corethrurus in four sites along an altitudinal gradient in central Veracruz, Mexico. We predicted that the distribution of P. corethrurus would be more related to climate variables than edaphic parameters. Five sampling points (in the grassland) were established at each of four sites along an altitudinal gradient: Laguna Verde (LV), La Concepción (LC), Naolinco (NA) and Acatlán (AC) at 11-55, 992-1,025, 1,550-1,619 y 1,772-1,800 masl, respectively. The climate ranged from tropical to temperate along the altitudinal gradient. Ten earthworm species (5 Neotropical, 4 Palearctic and 1 Nearctic) were found along the gradient, belonging to three families (Rhinodrilidae, Megascolecide and Lumbricidae). Soil properties showed a significant association (positive for Ngrass, pH, permanent wilting point, organic matter and P; and negative for Total N, K and water-holding capacity) with the abundance of the earthworm community. Also there seems to be a relationship between climate and earthworm distribution along the altitudinal gradient. P. corethrurus was recorded at tropical (LV and LC) and temperate sites (NA) along the altitudinal gradient. Our results reveal that soil fertility determines the abundance of earthworms and site (climate) can act as a barrier to their migration. Further research is needed to determine the genetic structure and lineages of P. corethrurus along altitudinal gradients.
Rapid reviews expedite the knowledge synthesis process with the goal of providing timely information to healthcare decision-makers who want to use evidence-informed policy and practice approaches. A range of opinions and viewpoints on rapid reviews is thought to exist; however, no research to date has formally captured these views. This paper aims to explore evidence producer and knowledge user attitudes and perceptions towards rapid reviews.
A Q methodology study was conducted to identify central viewpoints about rapid reviews based on a broad topic discourse. Participants rank-ordered 50 text statements and explained their Q-sort in free-text comments. Individual Q-sorts were analysed using Q-Assessor (statistical method: factor analysis with varimax rotation). Factors, or salient viewpoints on rapid reviews, were identified, interpreted and described.
Analysis of the 11 individual Q sorts identified three prominent viewpoints: Factor A cautions against the use of study design labels to make judgements. Factor B maintains that rapid reviews should be the exception and not the rule. Factor C focuses on the practical needs of the end-user over the review process.
Results show that there are opposing viewpoints on rapid reviews, yet some unity exists. The three factors described offer insight into how and why various stakeholders act as they do and what issues may need to be resolved before increase uptake of the evidence from rapid reviews can be realized in healthcare decision-making environments.
Mountains of the Brazilian Atlantic Forest can act as islands of cold and wet climate, leading to the isolation and speciation of species with low dispersal capacity, such as the toadlet species of the genus Brachycephalus. This genus is composed primarily by diurnal species, with miniaturized body sizes (<2.5 cm), inhabiting microhabitats in the leaf litter of montane forests. Still, little is known about the geographical distribution, altitudinal range, and ecological limits of most Brachycephalus species. In this study, we review the available data on the geographical and altitudinal distribution of Brachycephalus based on occurrence records compiled from literature and museums, both for the genus as a whole and separately for the three recently proposed groups of species (ephippium, didactylus, and pernix). The final ensemble dataset comprised 333 records, 120 localities, 28 described species, and six undescribed ones. Species were recorded in six relief units, the richest of which being the Serra do Mar, with 30 species. When the Serra do Mar is subdivided into three subunits, Northern, Central and Southern Serra do Mar, the number of species increase from north to the south, with records of six, nine, and 16 species, respectively. We were able to estimate the extent of occurrence of nearly half of the described species, and the resulting estimates indicate that many of them show remarkably small ranges, some of which less than 50 ha. Brachycephalus species are present from sea level to roughly 1,900 m a.s.l., with the highest richness being found between 751 and 1,000 m a.s.l. (21 spp.). The species with the broadest altitudinal range were B. didactylus (1,075 m) and Brachycephalus sp. 1 (1,035 m), both in the didactylus group, and B. ephippium (1,050 m), of the ephippium group. The broadest altitudinal amplitude for species of the pernix group was recorded for B. brunneus (535 m). The lowest altitudinal records for the pernix group were at 845 m a.s.l. in the state of Paraná and at 455 m a.s.l. in the state of Santa Catarina. The altitudinal occurrence in the pernixspecies group seems to decrease southward. Syntopy between species is also reviewed.
Calcification is one of the most distinctive traits of scleractinian corals. Their hard skeletons form the substratum of reef ecosystems and confer on corals their remarkable diversity of shapes. Corallimorpharians are non-calcifying, close relatives of scleractinian corals, and the evolutionary relationship between these two groups is key to understanding the evolution of calcification in the coral lineage. One pivotal question is whether scleractinians are a monophyletic group, paraphyly being an alternative possibility if corallimorpharians are corals that have lost their ability to calcify, as is implied by the "naked-coral" hypothesis. Despite major efforts, relationships between scleractinians and corallimorpharians remain equivocal and controversial. Although the complete mitochondrial genomes of a range of scleractinians and corallimorpharians have been obtained, heterogeneity in composition and evolutionary rates means that mitochondrial sequences are insufficient to understand the relationship between these two groups. To overcome these limitations, transcriptome data were generated for three representative corallimorpharians. These were used in combination with sequences available for a representative range of scleractinians to identify 291 orthologous single copy protein-coding nuclear markers. Unlike the mitochondrial sequences, these nuclear markers do not display any distinct compositional bias in their nucleotide or amino-acid sequences. A range of phylogenomic approaches congruently reveal a topology consistent with scleractinian monophyly and corallimorpharians as the sister clade of scleractinians.
It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.
Triple-negative breast cancers(TNBCs)are associated with early recurrence after surgery and unfavorable prognoses. To date, no effective therapies for TNBCs have been established. The present study was designed to evaluate the efficacy of adjuvant chemotherapy(ACT)for 111 TNBCs using a retrospective multivariate analysis(MVA). The intravenous(iv)ACTs included docetaxel, epirubicin, gemcitabine, and vinorelbine. The oral ACTs included UFT, doxifluridine, and cyclophosphamide. The 10-year disease-free survival(DFS)and overall survival(OS)rates were 77.5% and 86.0%, respectively. Recurrences were observed in 17 patients, and the first recurrence was most frequently located in the lung. MVA revealed that pT was a significant independent variable for poor DFS and OS. UFT was the only significant independent variable for improved DFS. The survival analysis also demonstrated that UFT alone may be an effective option for Stage I TNBCs. Furthermore, it suggested that the addition of further iv ACTs to UFT could improve the outcome in patients with Stage II-III TNBCs.
The cyclic electron flow around photosystem I (CEF-PSI) increases ATP/NADPH production in the chloroplast, acting as an energy balance mechanism. Higher export of reducing power from the chloroplast in CEF-PSI mutants has been correlated with higher mitochondrial alternative oxidase (AOX) capacity and protein amount under high-light (HL) conditions. However, in vivo measurements of AOX activity are still required to confirm the exact role of AOX in dissipating the excess of reductant power from the chloroplast. Here, CEF-PSI single and double mutants were exposed to short-term HL conditions in Arabidopsis (Arabidopsis thaliana). Chlorophyll fluorescence, in vivo activities of the cytochrome oxidase (νcyt) and AOX (νalt) pathways, levels of mitochondrial proteins, metabolite profiles, and pyridine nucleotide levels were determined under normal growth and HL conditions. νalt was not increased in CEF-PSI mutants, while AOX capacity was positively correlated with photoinhibition, probably due to a reactive oxygen species-induced increase of AOX protein. The severe metabolic impairment observed in CEF-PSI mutants, as indicated by the increase in photoinhibition and changes in the levels of stress-related metabolites, can explain their lack of νalt induction. By contrast, νcyt was positively correlated with photosynthetic performance. Correlations with metabolite changes suggest that νcyt is coordinated with sugar metabolism and stress-related amino acid synthesis. Furthermore, changes in glycine-serine and NADH-NAD(+) ratios were highly correlated to νcyt Taken together, our results suggest that νcyt can act as a sink for the excess of electrons from the chloroplast, probably via photorespiratory glycine oxidation, thus improving photosynthetic performance when νalt is not induced under severe HL stress.
Naphthoquinones are secondary metabolites widely distributed in nature and produced by bacteria, fungi and higher plants. Their biological activity may result from induction of oxidative stress, caused by redox cycling or direct interaction with cellular macromolecules, in which quinones act as electrophiles. The redox homeostasis is known as one of factors involved in auxin-mediated plant growth regulation. To date, however, little is known about the crosstalk between reactive oxygen species (ROS) produced by quinones and the plant growth hormone auxin (IAA). In this study, redox cycling properties of two naphthoquinones, juglone (5-hydroxy-1,4-naphthoquinone) and lawsone (2-hydroxy-1,4-naphthoquinone), were compared in experiments performed on maize coleoptile segments incubated with or without the addition of IAA. It was found that lawsone was much more effective than juglone in increasing both H2O2 production and the activity of antioxidative enzymes (SOD, POX and CAT) in coleoptile cells, regardless of the presence of IAA. An increase in the activity of Cu/Zn-SOD isoenzymes induced by both naphthoquinones suggests that juglone- and lawsone-generated H2O2 was primarily produced in the cytosolic and cell wall spaces. The cell potential to neutralize hydrogen peroxide, determined by POX and CAT activity, pointed to activity of catalase as the main enzymatic mechanism responsible for degradation of H2O2 Therefore, we assumed that generation of H2O2, induced more efficiently by LW than JG, was the major factor accounting for differences in the toxicity of naphthoquinones in maize coleoptiles. The role of auxin in the process appeared negligible. Moreover, the results suggested that oxidative stress imposed by JG and LW was one of mechanisms of allelopathic action of the studied quinones in plants.
Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep.
Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings.
Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep.
Exosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia.
We showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers.
This study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.
Purpose: Radiology represents a highly relevant part of undergraduate medical education from preclinical studies to subinternship training. It is therefore important to establish a content base for teaching radiology in German Medical Faculties. Materials and Methods: The German Society of Radiology (DRG) developed a model curriculum for radiological teaching at German medical universities, which is presented in this article. There is also a European model curriculum for undergraduate teaching (U-level curriculum of the European Society of Radiology). In a modular concept, the students shall learn important radiological core principles in the realms of knowledge, skills and competences as well as core scientific competences in the imaging sciences. Results: The curriculum is divided into two modules. Module 1 includes principles of radiation biology, radiation protection and imaging technology, imaging anatomy as well as the risks and side effects of radiological methods, procedures and contrast media. This module is modality-oriented. Module 2 comprises radiological diagnostic decision-making and imaging-based interventional techniques for various disease entities. This module is organ system-oriented. Conclusion: The curriculum is meant as a living document to be amended and revised at regular intervals. The curriculum can be used as a basis for individual curricular development at German Medical Faculties. It can be integrated into traditional or reformed medical teaching curricula. Key Points: • Radiology is an integral and important part of medical education.• The German Society of Radiology (DRG) developed a model curriculum for teaching radiology at German Medical Faculties to help students develop the ability to make medical decisions based on scientific knowledge and act accordingly.• This curriculum can be used for individual curricular development at medical departments. It is divided into two modules with several chapters. Citation Format: • Ertl-Wagner B, Barkhausen J, Mahnken AH et al. White Paper: Radiological Curriculum for Undergraduate Medical Education in Germany. Fortschr Röntgenstr 2016; 188: 1017 - 1023.
Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction.
Tuberous sclerosis complex (TSC) is a neurodevelopmental disease caused by TSC1 or TSC2 mutations and subsequent activation of the mTORC1 kinase. Upon mTORC1 activation, anabolic metabolism, which requires mitochondria, is induced, yet at the same time the principal pathway for mitochondrial turnover, autophagy, is compromised. How mTORC1 activation impacts mitochondrial turnover in neurons remains unknown. Here, we demonstrate impaired mitochondrial homeostasis in neuronal in vitro and in vivo models of TSC. We find that Tsc1/2-deficient neurons accumulate mitochondria in cell bodies, but are depleted of axonal mitochondria, including those supporting presynaptic sites. Axonal and global mitophagy of damaged mitochondria is impaired, suggesting that decreased turnover may act upstream of impaired mitochondrial metabolism. Importantly, blocking mTORC1 or inducing mTOR-independent autophagy restores mitochondrial homeostasis. Our study clarifies the complex relationship between the TSC-mTORC1 pathway, autophagy, and mitophagy, and defines mitochondrial homeostasis as a therapeutic target for TSC and related diseases.
Ocular mucous membrane pemphigoid (OcMMP) is a rare autoimmune disorder resulting in progressive conjunctival fibrosis and ocular surface failure leading to sight loss in up to 50%. This study was designed to optimize an ocular surface sampling technique for identification of novel biomarkers associated with disease activity and/or progressive fibrosis.
Fifty-seven patients with OcMMP underwent detailed examination of conjunctival inflammation and fibrosis using fornix depth measurement. Ocular surface impression cytology (OSIC) to sample superior bulbar conjunctiva combined with flow cytometry (OSIC-flow) profiled infiltrating leukocytes. Profiles were compared with healthy controls (HC) and disease controls (primary Sjögren's syndrome, pSS). Thirty-five OcMMP patients were followed every 3 months for 12 months.
Overall neutrophils were elevated in OcMMP eyes when compared to pSS or HC (109 [18%] neutrophils/impression [NPI]; 2 [0.2%]; 6 [0.8%], respectively [P < 0.0001]) and in OcMMP patients with no visible inflammation when compared with HC (44.3 [7.9%]; 5.8 [0.8%]; P < 0.05). At 12 months follow-up, 53% of OcMMP eyes progressed, and this was associated with baseline conjunctival neutrophilia (P = 0.004). As a potential biomarker, a value of 44 NPI had sensitivity, specificity, and positive predictive values of 75%, 70%, and 73%, respectively. Notably, eyes with no visible inflammation and raised conjunctival neutrophils were more likely to progress and have a greater degree of conjunctival shrinkage compared to those without raised neutrophils.
These data suggest that OSIC-flow cytometric analyses may facilitate repeated patient sampling. Neutrophils may act as a biomarker for monitoring disease activity, progressive fibrosis, and response to therapy in OcMMP even when the eye appears clinically uninflamed.
Trauma is the leading cause of death and disability among young adults, who are also among the most likely to be uninsured. Efforts to increase insurance coverage, including passage of the Patient Protection and Affordable Care Act (ACA), were intended to improve access to care and promote improvements in outcomes. However, despite reported gains in coverage, the ACA's success in promoting use of high-quality care and enacting changes in clinical end points remains unclear.
To assess for observed changes in insurance coverage and rehabilitation use among young adult trauma patients associated with the ACA, including the Dependent Coverage Provision (DCP) and Medicaid expansion/open enrollment, and to consider possible insurance and rehabilitation differences between DCP-eligible vs -ineligible patients and among stratified demographic and community subgroups.
A longitudinal assessment of DCP implementation and Medicaid expansion/open enrollment using risk-adjusted before-and-after, difference-in-difference, and interrupted time-series analyses was conducted. Eleven years (January 1, 2005, to September 31, 2015) of Maryland Health Services Cost Review Commission data, representing complete patient records from all payers within the state, were used to identify all hospitalized young adult (aged 18-34 years) trauma patients in Maryland during the study period.
Of the 69 507 hospitalized patients included, 50 548 (72.7%) were male, and the mean (SD) age was 25 (5) years. Before implementation of the DCP, 1 of 4 patients was uninsured. After ACA implementation, the number fell to less than 1 of 10, with similar patterns emerging in emergency department and outpatient settings. The change was primarily driven by Medicaid expansion/open enrollment, which corresponded to a 20.1 percentage-point increase in Medicaid (95% CI, 18.9-21.3) and an 18.2 percentage-point decrease in uninsured (95% CI, -19.3 to -17.2). No changes were detected among privately insured patients. Rehabilitation use increased by 5.4 percentage points (95% CI, 4.5-6.2)-a 60% relative increase from a baseline of 9%. Mortality (-0.5; 95% CI, -0.9 to -0.1) and failure-to-rescue rates (-4.5; 95% CI, -7.4 to -1.6) also significantly declined. Stratified changes point to significant differences in the percentage of uninsured patients and rehabilitation access across the board, mitigating or even eradicating disparities in certain cases.
For patients who are injured, young, and uninsured, Medicaid expansion/open enrollment in Maryland changed insurance coverage and altered patient outcomes in ways that the DCP alone was never intended to do. Implementation of Medicaid expansion/open enrollment transformed the landscape of trauma coverage, directly affecting the health of one of the country's most vulnerable at-risk groups.
Jonathan Haidt's Moral Foundations Theory identifies five moral axes that can influence human motivation to take action on vital problems like climate change. The theory focuses on five moral foundations, including compassion, fairness, purity, authority, and ingroup loyalty; these have been found to differ between liberals and conservatives as well as Democrats and Republicans. Here we show, based on the Cornell National Social Survey (USA), that valuations of compassion and fairness were strong, positive predictors of willingness to act on climate change, whereas purity had a non-significant tendency in the positive direction (p = 0.07). Ingroup loyalty and authority were not supported as important predictor variables using model selection ([Formula: see text]). Compassion and fairness were more highly valued by liberals, whereas purity, authority, and in-group loyalty were more highly valued by conservatives. As in previous studies, participants who were younger, more liberal, and reported greater belief in climate change, also showed increased willingness to act on climate change. Our research supports the potential importance of moral foundations as drivers of intentions with respect to climate change action, and suggests that compassion, fairness, and to a lesser extent, purity, are potential moral pathways for personal action on climate change in the USA.
Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.
Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse post-translational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes. We found that individual histones, histone octamers, and nucleosomes are able to stimulate Rad27 in a manner dependent on the N-terminal tails of histones. Kinetic analyses suggest that an increase in catalytic efficiency of Rad27 was mainly due to increased affinity between DNA substrates and Rad27. It appears that the physical interaction in vivo between histones and Rad27 results in the enrichment of Rad27 in the vicinity of chromatin, increasing the availability of Rad27 for various DNA metabolisms. These results indicate that nucleosomes are not a mere structural component of chromatin, but an active regulator of DNA metabolisms that serves to ensure the efficient and faithful processing of structural intermediates arising during DNA transactions.
To compare performance of junior (JR)/senior (SR) abdominal radiologists using multidetector computed tomography (MDCT) to predict surgical intervention, bowel resection, and presence of bowel ischemia in patients with suspected small-bowel obstruction (SBO).
Retrospective, institutional review board-approved, Health Insurance Portability and Accountability Act-compliant, blinded, independent review of MDCT studies for suspected SBO in 179 adults (mean age, 55.8 years; 86 men/93 women) was performed by 12 board-certified radiologists (6 JR, 6 SR). Readers rated likelihood of need for surgery, bowel ischemia, and resection. Correlation with clinical outcomes/pathology was performed.
Pooled receiver operating characteristic area under the curve for surgery, ischemia, and resection were 0.802, 0.736, and 0.824 and 0.773, 0.851, and 0.751 JR/SR, respectively. Sensitivity/specificity for predicting surgery was 86.7%/65.4% and 79.6%/64.4%. No differences existed between JR/SR performance overall (P = 0.451); highest/lowest performing readers between groups (P < 0.001) and within groups (P = 0.008) varied.
Junior radiologists performed as well as SR in predicting surgical outcomes on MDCT in patients with suspected SBO.
This study aimed to obtain qualitative information about physical activity (PA), sedentary behaviors (SB) and their determinants and about recently retired adults' needs regarding PA interventions. Four focus group interviews were organized. The most commonly reported PA types were walking, cycling, swimming and fitness. The most commonly reported SB were reading, TV viewing, and computer use. Car use was limited. Most adults agreed their habits had changed during retirement. The most striking PA determinant was the feeling of being a 'forgotten group', having too few tailored PA initiatives available. Furthermore, participants were not aware of the negative health effects of SB and not motivated to decrease their SB. Concerning new PA interventions, very diverse ideas were put forward, reflecting the diversity of the target group. It seems that a dynamic intervention in which participants can choose which PA type they want to increase is preferable for recently retired adults.
The potent antistaphylococcal activity of N-substituted pantothenamides (PanAms) has been shown to at least partially be due to the inhibition of Staphylococcus aureus's atypical type II pantothenate kinase (SaPanKII), the first enzyme of coenzyme A biosynthesis. This mechanism of action follows from SaPanKII having a binding mode for PanAms that is distinct from those of other PanKs. To dissect the molecular interactions responsible for PanAm inhibitory activity, we conducted a mini SAR study in tandem with the cocrystallization of SaPanKII with two classic PanAms (N5-Pan and N7-Pan), culminating in the synthesis and characterization of two new PanAms, N-Pip-PanAm and MeO-N5-PanAm. The cocrystal structures showed that all of the PanAms are phosphorylated by SaPanKII but remain bound at the active site; this occurs primarily through interactions with Tyr240' and Thr172'. Kinetic analysis showed a strong correlation between kcat (slow PanAm turnover) and IC50 (inhibition of pantothenate phosphorylation) values, suggesting that SaPanKII inhibition occurs via a delay in product release. In-depth analysis of the PanAm-bound structures showed that the capacity for accepting a hydrogen bond from the amide of Thr172' was a stronger determinant for PanAm potency than the capacity to π-stack with Tyr240'. The two new PanAms, N-Pip-PanAm and MeO-N5-PanAm, effectively combine both hydrogen bonding and hydrophobic interactions, resulting in the most potent SaPanKII inhibition described to date. Taken together, our results are consistent with an inhibition mechanism wherein PanAms act as SaPanKII substrates that remain bound upon phosphorylation. The phospho-PanAm-SaPanKII interactions described herein may help future antistaphylococcal drug development.
Using computational modeling, we design a composite that encompasses a thermo-responsive gel and photo-responsive fibers that extend from the surface of the gel. By simulating the effect of light and heat on the sample, we isolate scenarios where cooperative interactions within the system allow the gel to actuate the "finger-like" motion of the embedded fibers. To achieve this distinctive behavior, we consider a gel formed from poly(N-isopropylacrylamide) (PNIPAAm), which shrinks when heated above the lower critical solution temperature (LCST). The fibers are functionalized with spirobenzopyran (SP) chromophores that extend a finite region into the polymer network. The application of heat causes the entire gel to shrink, while the application of light causes the regions around the functionalized fibers to collapse. With the fibers arranged in a square or circular pattern in the center of the gel, heating the non-illuminated samples drives the fibers to move apart as they bend outward (away from the center). With the application of light, the tips of fibers come together as the fibers bend inward. In this configuration, the fibers could act as grippers that bind objects in the presence of light. With the illumination turned off, the grippers could controllably release the objects. By placing the fibers closer to the edge of the sample, the combination of heat and light could be harnessed to bind and release larger objects. We also show that by illuminating the fibers separately, we can manipulate the motion of the individual finger-like objects, and thus, potentially expand the utility of the system. Overall, our findings provide guidelines for controllably reconfiguring the shape of soft materials and thus, tailoring the material to display different functionalities in different environments.
The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. We show that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. We inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes.
The anisotropic hole and electron mobilities in N,N'-3,4,9,10-perylenediimide-1,7-phenoxy (PDIB-2OPh) and N,N'-3,4,9,10-perylenediimide (PDIB) were theoretically predicted using the Marcus-Hush theory. The substituent effect of phenoxy on their mobility rates, absorption spectra, electron affinities, and ionization potentials was explored. By comparing the simulated hole mobility in PDIB and PDIB-2OPh, it is found that the phenoxy rings act as spacers between adjacent stacking columns in the phenoxy-substituted derivatives. The increasement of the number of benzene oxygen groups leads to the absorption spectra red-shift of these molecular systems. This coincides with their change tendency of the adiabatic ionization potentials, vertical ionization potentials. However, the calculated adiabatic electron affinities and vertical electron affinities of N,N'-butyl-3,4,9,10-perylenediimide-1,6,7,12-phenoxy (PDIB-4OPh) are larger than those of PDIB;OPh. The steric effect in PDIB-4OPh is expected to cause space reversal and thus to changes in the properties of the molecule.
Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors.
For a patient with lupus anticoagulant (LA), activated coagulation time (ACT) was measured with two different types of devices (HEMOCHRON(®) 801 and HEMOCHRON(®) Jr).
ACTs during heparinization measured with the HEMOCHRON(®) 801 were over the range of measurement, while those with the HEMOCHRON(®) Jr. reflected an almost normal response to heparin.
The phospholipid contained in an activating agent of the HEMOCHRON(®) Jr was suggested to have counteracted the effect of LA.
It was indicated that the coagulation status for LA-positive patients might be better assessed by ACT measured with phospholipid in an activating agent, although careful interpretation is required.
Antibiotic resistance is a global problem exacerbated due to selective pressure of antimicrobial agents, spontaneous mutation, recombination and horizontal gene transfer. Of the different mechanisms that led to spread of resistance, one notorious phenomenon is the generation of Extended Spectrum β-lactamases (ESBLs) preferably TEM, SHV, OXA and CTX-M. Their production and frequent mobilization through horizontal gene transfer, made bacteria to exhibit resistance against a broad range of antibiotics. Resilient development of resistance among bacteria hampers available treatment options against different infections in the health care system. Alarming spread of resistance has made it as the subject of concern for scientific consideration and research. In this article, we emphasized the state of knowledge regarding imprudent use of antibiotics that act as promoters of resistance development, thereby contributing to the failure of available treatment regimes. Along with this, it also contains information about the available strategies and the knowledge of inhibitors that can be employed to curb the growing menace of antibiotic resistance.
Heart failure, hypertension, cirrhosis and nephritic syndrome are among conditions that alter volume and composition of body fluids and are modulated by diuretics. Natural products are important source of diuretics and have been considered remarkable alternative with greater effectiveness and fewer side effects. However, many of these plants used in traditional medicine must be scientifically assessed about their efficacy and toxicity. Despite the large number of published articles claiming that plants or plant-derived components may act as diuretic agents, few studies have addressed the mechanism of action of medicinal plants. Thus, the aim of this review was to provide an overview of the current knowledge about the major cellular and molecular mechanisms of diuretic plants and/or their main compounds. Many well-established mechanisms (water channels, renal carriers, nitric oxide-cGMP and prostaglandin-cAMP pathways, renin-angiotensin and kinin-kallikrein systems, carbonic anhydrase, and osmotic effects), along with other newly identified targets, are connected to the diuretic activity of many natural products. However, the central path responsible for the activity of these agents remains unclear. Further studies may help clarifying the central role of each of these pathways in the pleiotropic response of these agents.
Anorexia cancer cachexia syndrome is prevalent in advanced cancer patients, which is featured by anorexia, decreased dietary intake, body weight loss (skeletal muscle mass loss), and unable to be reversed by routine nutritional support therapy. Up to now, the main mechanisms involved in cancer cachexia include excessive systemic inflammation, which is represented by increased plasma levels of IL-1, IL-6, TNF-alpha, tumor-induced factors, such as PIF and LMF. These factors eventually act on orexigenic and anorexigenic neurons located in hypothalamus or protein and lipid metabolism of peripheral tissues, which lead to anorexia, decreased dietary intake, enhanced basic metabolism rate and hyper catabolism. The treatment modality includes early nutritional intervention, physical activity and drug treatment.However, studies about drugs used to treat cachexia are always controversial or merely effective in stimulating appetite and increasing body weight，though not lean body mass. The main target of pharmaceutical treatment is to improve appetite, decrease systemic inflammation and promote anabolic metabolism. Nevertheless, the treatment effectiveness of chemical drugs are not reaching consensus by existing cachexia guidelines. Complementary and alternative medicine (CAM) is recently known as a promising treatment to improve cachaxia status and quality of life of cancer patients. Traditional Chinese medicine (TCM) and natural herbal medicines have been used in the treatment of cancer for thousands of years worldwide, particularly in China. More and more research show that traditional Hanfang (Chinese medicines)and some natural herbs with less side reactions, have the effects of antagonizing pro-inflammatory cytokines, enhancing immune system, inhibiting protein catabolism, boosting the appetite and body weight, which maybe an promising treatment strategy and development tendency for anorexia cancer cachexia syndrome.
Chronic kidney disease (CKD) is characterized by the progressive reduction of glomerular filtration rate and subsequent retention of organic waste compounds called uremic toxins. While patients with CKD are at a higher risk of premature death due to cardiovascular complications, this increased risk cannot be completely explained by classical cardiovascular risk factors such as hypertension, diabetes mellitus, and obesity. Instead, recent research suggests that uremic toxins may play a key role in explaining this marked increase in cardiovascular mortality in patients with CKD. While spermine, a tetra-amine, has previously been hypothesized to act as an uremic toxin, the following review presents a summary of recent literature that casts doubt on this assertion. Instead, acrolein, an oxidative product of spermine and the triamine spermidine, is likely responsible for the toxic effects previously attributed to spermine.
Because of its impact on multiple biological pathways, heparanase has emerged as a major regulator of cancer, inflammation and other disease processes. Heparanase accomplishes this by degrading heparan sulfate which regulates the abundance and location of heparin-binding growth factors thereby influencing multiple signaling pathways that control gene expression, syndecan shedding and cell behavior. In addition, heparanase can act via nonenzymatic mechanisms that directly activate signaling at the cell surface. Clinical trials testing heparanase inhibitors as anticancer therapeutics are showing early signs of efficacy in patients further emphasizing the biological importance of this enzyme. This review focuses on recent developments in the field of heparanase regulation of cancer and inflammation, including the impact of heparanase on exosomes and autophagy, and novel mechanisms whereby heparanase regulates tumor metastasis, angiogenesis and chemoresistance. In addition, the ongoing development of heparanase inhibitors and their potential for treating cancer and inflammation are discussed.
Autophagy (literally "self-eating") is an evolutionarily conserved degradation process where cytoplasmic components are engulfed by vesicles called autophagosomes, which are then delivered to lysosomes, where their contents are degraded. Under stress conditions, such as starvation or oxidative stress, autophagy is upregulated in order to degrade macromolecules and restore the nutrient balance. The source of membranes that participate in the initial formation of phagophores is still incompletely understood and many intracellular structures have been shown to act as lipid donors, including the endoplasmic reticulum, Golgi, nucleus, mitochondria and the plasma membrane. Here we focus on the contributions of the plasma membrane to autophagosome biogenesis governed by ATG16L1 and ATG9A trafficking, and summarise the physiological and pathological implications of this macroautophagy route, from development and stem cell fate to neurodegeneration and cancer. This article is protected by copyright. All rights reserved.
The serratus anterior is portrayed as a homogeneous muscle in textbooks and during functional activities and rehabilitation exercises. It is unclear whether the serratus anterior is composed of subdivisions with distinctive morphology and functions. The purpose of this study was to determine whether the serratus anterior could be subdivided into different structural parts on the basis of its segmental architectural parameters. Eight formalin-embalmed serratus anterior muscles were dissected and the attachments of each fascicle documented. Orientation and size of each fascicle were measured and the physiological cross-sectional area (PCSA) calculated. Three subdivisions of the serratus anterior were identified. A new finding was the discovery of two distinctive fascicles attached to the superior and inferior aspects of rib 2. The rib 2 inferior fascicle had the largest PCSA (mean 1.6 cm(2)) and attached, with the rib 3 fascicle, along the medial border of the scapula to form the middle division. The rib 2 superior and rib 1 fascicles attached to the superior angle of the scapula (upper division). Fascicles from ribs 4-8/9 attached to the inferior angle of the scapula (lower division). Mean fascicle angle relative to a vertical midline reference and PCSA for each division were 29° and 1.3 cm(2) (upper), 90° and 2.2 cm(2) (middle) and 59° and 3.0 cm(2) (lower). This novel study demonstrated the presence of morphologically distinct serratus anterior subdivisions. The results of this study will inform the development of optimal techniques for the assessment, treatment and rehabilitation of this architecturally complex muscle in shoulder and neck pain.
Laser frequency noise contributes a significant limitation to today's best atomic clocks. A proposed solution to this problem is to create a superradiant laser using an optical clock transition as its gain medium. This laser would act as an active atomic clock and would be highly immune to the fluctuations in reference cavity length that limit today's best lasers. We demonstrate and characterize superradiant emission from the millihertz linewidth clock transition in an ensemble of laser-cooled (87)Sr atoms trapped within a high-finesse optical cavity. We measure a collective enhancement of the emission rate into the cavity mode by a factor of more than 10,000 compared to independently radiating atoms. We also demonstrate a method for seeding superradiant emission and observe interference between two independent transitions lasing simultaneously. We use this interference to characterize the relative spectral properties of the two lasing subensembles.
Pharmacologic inhibitors of the serine/threonine kinase Akt, initially aimed at deranged oncogenic pathways in tumors, have recently been shown to act as immunomodulators that markedly enhance the antitumor properties of T cells. Repurposing Akt inhibitors to improve antitumor immunity may be viewed as a manifestation of a larger paradigmatic shift in which hallmark characteristics of cancer (e.g., immune evasion), rather than merely causal features (e.g., somatic mutations) can be exploited for therapeutic benefit.
Freshwater biota experience physiological challenges in regions affected by salinization, but often the effects on particular species are poorly understood. Freshwater turtles are of particular concern as they appear to have limited ability to cope with environmental conditions that are hyperosmotic to their body fluids. Here, we determined the physiological responses of two Australian freshwater chelid turtles, Emydura macquarii and Chelodina expansa, exposed to freshwater (0‰) and brackish water (15‰, representing a hyperosmotic environment). Brackish water is common in the Murray-Darling River Basin within the natural range of these species in Australia during periods of drought, yet it is unknown how well these species tolerate saline conditions. We hypothesized that these turtles would be unable to maintain homeostasis in the 15‰ water treatment and would suffer osmotic loss of water, increased ionic concentrations and a decrease in body mass. Results revealed that these turtles had elevated plasma concentrations of sodium, chloride, urea and uric acid in the plasma. Plasma ionic concentrations increased proportionally more in E. macquarii than in C. expansa. Individuals of both species reduced feeding in 15‰ water, indicating that behaviour may provide an additional means for freshwater turtles to limit ion/solute influx when in hyperosmotic environments. This osmoregulatory behaviour may allow for persistence of turtles in regions affected by salinization; however, growth rates and body condition may be affected in the long term. Although we demonstrate that these turtles have mechanisms to survive temporarily in saline waters, it is likely that sustained salinization of waterways will exceed their short- to medium-term capacity to survive increased salt levels, making salinization a potentially key threatening process for these freshwater reptiles.
Estuarine habitats are frequently used as nurseries by elasmobranch species for their protection and abundant resources; however, global climate change is increasing the frequency and severity of environmental challenges in these estuaries that may negatively affect elasmobranch physiology. Hyposmotic events are particularly challenging for marine sharks that osmoconform, and species-specific tolerances are not well known. Therefore, we sought to determine the effects of an acute (48 h) ecologically relevant hyposmotic event (25.8 ppt) on the physiology of two juvenile shark species, namely the school shark (Galeorhinus galeus), listed by the Australian Environmental Protection and Biodiversity Conservation Act as 'conservation dependent', and the gummy shark (Mustelus antarcticus), from the Pittwater Estuary (Australia). In both species, we observed a decrease in plasma osmolality brought about by selective losses of NaCl, urea and trimethylamine N-oxide, as well as decreases in haemoglobin, haematocrit and routine oxygen consumption. Heat-shock protein levels varied between species during the exposure, but we found no evidence of protein damage in any of the tissues tested. Although both species seemed to be able to cope with this level of osmotic challenge, overall the school sharks exhibited higher gill Na(+)/K(+)-ATPase activity and ubiquitin concentrations in routine and experimental conditions, a larger heat-shock protein response and a smaller decrease in routine oxygen consumption during the hyposmotic exposure, suggesting that there are species-specific responses that could potentially affect their ability to withstand longer or more severe changes in salinity. Emerging evidence from acoustic monitoring of sharks has indicated variability in the species found in the Pittwater Estuary during hyposmotic events, and together, our data may help to predict species abundance and distribution in the face of future global climate change.
Several studies have linked teachers' personality characteristics and sense of efficacy to stress. However, investigating the relationship between these three constructs in this context was limited. This study aims to investigate the relationship between personality, sense of efficacy and perceived stress among Korean teachers. A total of 137 teachers working in elementary, middle, and high schools located in Seoul, South Korea were recruited for the study. The participants were administered Temperament and Character Inventory, Teacher's Sense of Efficacy Scale (TSES), and Perceived Stress Scale. The TSES was negatively correlated with harm avoidance and positively correlated with persistence, self-directedness, cooperativeness, and self-transcendence. Perceived stress was positively correlated with harm avoidance and negatively correlated with persistence and self-directedness. The path analysis showed that harm avoidance directly predicted perceived stress (β=0.37, 95% CI=0.21-0.53, p=0.002), and self-directedness and persistence predicted one's sense of efficacy (β=0.18, 95% CI=0.01-0.39 and β=0.31, 95% CI=0.10-0.47), which predicted perceived stress (β=-0.21, 95% CI=-0.39 to -0.02). The results of the present study indicate that harm avoidance might be associated with stress-proneness, while persistence, self-directedness, and sense of efficacy might act as protective resources against stress in Korean teachers.
Every human is unique. We differ in our genomes, environment, behavior, disease history, and past and current medical treatment-a complex catalog of differences that often leads to variations in the way each of us responds to a particular therapy. We argue here that true personalization of drug therapies will rely on "virtual patient" models based on a detailed characterization of the individual patient by molecular, imaging, and sensor techniques. The models will be based, wherever possible, on the molecular mechanisms of disease processes and drug action but can also expand to hybrid models including statistics/machine learning/artificial intelligence-based elements trained on available data to address therapeutic areas or therapies for which insufficient information on mechanisms is available. Depending on the disease, its mechanisms, and the therapy, virtual patient models can be implemented at a fairly high level of abstraction, with molecular models representing cells, cell types, or organs relevant to the clinical question, interacting not only with each other but also the environment. In the future, "virtual patient/in-silico self" models may not only become a central element of our health care system, reducing otherwise unavoidable mistakes and unnecessary costs, but also act as "guardian angels" accompanying us through life to protect us against dangers and to help us to deal intelligently with our own health and wellness.
Team-based care has been shown to offer more comprehensive benefits to patients when compared to standard physician-based care alone in clinics for chronic conditions. However, apart from grant-funded multidisciplinary clinics, there are no reports on the usage of same-day physical therapy (PT) consults within a daily outpatient neuromuscular disease (NMD) physician clinic.
To determine the impact of same-day PT consults at the University of Utah's outpatient Clinical Neurosciences Center.
A qualitative assessment and survey of patient satisfaction.
An eight question Health Insurance Portability and Accountability Act-compliant patient satisfaction survey using a 5-point Likert scale was administered. Demographic data and Press-Ganey Provider Satisfaction surveys were retrospectively collected from electronic medical records for patients receiving same-day PT encounters in the neuromuscular division over 1 year.
Mean (standard deviation) age was 54.22 (19.81) years for 134 patient encounters, median age was 60 years, with 76 male (57%) and 58 female (43%) patients. Mean Likert score for 61 self-reported patient satisfaction surveys for same-day PT consults was 4.87 (97.4%). Press-Ganey Provider Satisfaction scores improved from 89.9% (N=287) for the year prior to 90.8% (N=320) for the corresponding year (P=0.427). A total of 46 (75.4%) patients have either never before received PT care or never before received PT care for their NMD, 67.4% of whom were male.
Same-day PT consults in an outpatient NMD physician clinic demonstrated excellent patient satisfaction and improved access to specialty care. This model could potentially be implemented in other academic medical centers to improve access to rehabilitation services for patients with NMD.
Clinicians routinely prescribe adjuvant chemotherapy (ACT) for resected non-small cell lung cancer patients. However, ACT only improves five-year disease-free survival in stage I-III non-small cell lung cancer by 5-15%, with most patients deriving no benefit. Herein, deregulation of the E2F pathway was explored as a biomarker in lung adenocarcinoma patients. An E2F pathway scoring system, based on 74 E2F-regulated genes, was trained for RNA from two platforms: fresh-frozen (FF) or formalin-fixed paraffin-embedded (FFPE) tissues. The E2F score was tested as a prognostic biomarker in five FF-based cohorts and two FFPE-based cohorts. The E2F score was tested as a predictive biomarker in two randomized clinical trials; JBR10 and the NATCH (Neo-Adjuvant Taxol-Carboplatin Hope) trial. The E2F score was prognostic in untreated patients in all seven datasets examined (p < 0.05). Stage-specific analysis of combined cohorts demonstrated that the E2F score was prognostic in stage I patients (p = 0.0495 to <0.001; hazard ratio, HR, =2.04- 2.22) with a similar trend in other stages. The E2F score was strongly predictive in stage II patients from the two combined randomized clinical trials with a significant differential treatment effect (p = 0.015). Specifically, ACT improved survival in stage II patients with high E2F (p = 0.01; HR= 0.21). The 5-year survival increased from 18% to 81%. In contrast, in patients with low E2F, 5-year survival was 57% in untreated patients and 41% in ACT-treated patients with a HR of 1.55 (p = 0.47). In summary, the E2F score provides valuable prognostic information for Stage I and predictive information for Stage II lung adenocarcinoma patients and should be further explored as a decision support tool for their treatment.
Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH2 PGHS-2 is a conformational heterodimer composed of allosteric (Eallo) and catalytic (Ecat) subunits. Fatty acids (FAs) bind to Arg-120 of Eallo increasing to different degrees, depending on the FA, the Vmax of its Ecat partner. We report here that movement of helical residues 120-122 and loop residues 123-129 of Eallo underlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen. An S121P substitution in both PGHS-2 monomers yields a variant (S121P/S121P PGHS-2) that has 1.7-1.8 times the Vmax of native PGHS-2 and is relatively insensitive to activation by FAs or inhibition by allosteric inhibitors. The S121P substitution in Eallo is primarily responsible for these effects. In X-ray crystal structures, the Cα atoms of helical residues 119-122 of S121P/S121P PGHS-2 are displaced from their normal positions. Additionally, the S121P/S121P PGHS-2 variants in which Pro-127 and Ser-541 are replaced by cysteines spontaneously forms Cys-127 to Cys-541 cross-links between monomers. This is unlike the corresponding native PGHS-2 variant and suggests that S121P substitutions also unhinge the loop involving residues 123-129. We conclude the following: (a) the region involving residues 120-129 of unoccupied Eallo tonically inhibits Ecat; (b) binding of an activating FA (e.g. arachidonic, palmitic, or oleic acid) to Eallo or an S121P substitution in Eallo repositions this region to increase Ecat activity; and (c) allosteric COX inhibitors act by preventing FA binding to Eallo and additionally by relocating Eallo residues to inhibit Ecat.
In this study, we strove to substantiate the ability of linc-MAF-4 to act as a regulator of pathogenesis during multiple sclerosis (MS). We recruited 34 patients who were diagnosed with MS according to the revised McDonald Criteria. Six patients with MS and 5 healthy volunteers contributed peripheral blood mononuclear cells for microarray analysis. Subsequent knockdown and overexpression of linc-MAF-4 in naive CD4(+) T cells from the additional 28 patients with MS was performed to track changes in CD4(+) T-cell subsets and their function as well as to confirm results from the prior microarray analysis. Expression of linc-MAF-4 increased significantly in peripheral blood mononuclear cells of patients with MS compared with those of control participants. In addition, linc-MAF-4 regulated encephalitogenic T helper (Th)1 cell differentiation in patients with MS. Transfection of synthetic linc-MAF-4 into naive CD4(+) T cells facilitated Th1 cell differentiation and inhibited Th2 cell differentiation by directly inhibiting MAF, which is a Th2 cell transcription factor. Linc-MAF-4 also promoted activation of CD4(+) T cells from patients with MS. Expression level of linc-MAF-4 correlated with the annual relapse rate in patients with MS. Our results suggest that linc-MAF-4 is involved in the pathogenesis of MS, specifically via regulation of encephalitogenic T cells.-Zhang, F., Liu, G., Wei, C., Gao, C., Hao, J. Linc-MAF-4 regulates Th1/Th2 differentiation and is associated with the pathogenesis of multiple sclerosis by targeting MAF.
The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer-based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core-multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual-frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) is associated with the CMS. The micro-environment of the drug loaded to CMS nanoparticles was investigated by pulsed high-field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g-, A-matrices, and spin-lattice relaxation time) represent specific probes for the micro-environment. Additionally, the rotational correlation time of spin-labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown.
Colorectal anastomotic leakage (CAL) is a major surgical complication in intestinal surgery. Despite many optimizations in patient care, the incidence of CAL is stable (3-19%) [1]. Previous research mainly focused on determining patient and surgery related risk factors. Intraoperative non-surgery related risk factors for anastomotic healing also contribute to surgical outcome. This review offers an overview of potential modifiable risk factors that may play a role during the operation.
Two independent literature searches were performed using EMBASE, Pubmed and Cochrane databases. Both clinical and experimental studies published in English from 1985 to August 2015 were included. The main outcome measure was the risk of anastomotic leakage and other postoperative complications during colorectal surgery. Determined risk factors of CAL were stated as strong evidence (level I and II high quality studies), and potential risk factors as either moderate evidence (experimental studies level III), or weak evidence (level IV or V studies).
The final analysis included 117 articles. Independent factors of CAL are diabetes mellitus, hyperglycemia and a high HbA1c, anemia, blood loss, blood transfusions, prolonged operating time, intraoperative events and contamination and a lack of antibiotics. Unequivocal are data on blood pressure, the use of inotropes/vasopressors, oxygen suppletion, type of analgesia and goal directed fluid therapy. No studies could be found identifying the impact of body core temperature or mean arterial pressure on CAL. Subjective factors such as the surgeons' own assessment of local perfusion and visibility of the operating field have not been the subject of relevant studies for occurrence in patients with CAL.
Both surgery related and non-surgery related risk factors that can be modified must be identified to improve colorectal care. Surgeons and anesthesiologists should cooperate on these items in their continuous effort to reduce the number of CAL. A registration study determining individual intraoperative risk factors of CAL is currently performed as a multicenter cohort study in the Netherlands.
The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain.
Effect of repeated asenapine (ASE) treatment on FosB/ΔFosB expression was studied in the hypothalamic paraventricular nucleus (PVN) of male rats exposed to chronic mild stress (CMS) for 21days. Our intention was to find out whether repeated ASE treatment for 14days may: 1) induce FosB/ΔFosB expression in the PVN; 2) activate selected PVN neuronal phenotypes, synthesizing oxytocin (OXY), vasopressin (AVP), corticoliberin (CRH) or tyrosine hydroxylase (TH); and 3) interfere with the impact of CMS. Control, ASE, CMS, and CMS+ASE treated groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. From the 7th day of CMS, rats received ASE (0.3mg/kg) or saline (300μl/rat) subcutaneously, twice a day for 14days. They were sacrificed on the day 22nd (16-18h after last treatments). FosB/ΔFosB was visualized with avidin biotin peroxidase complex and OXY, AVP, CRH or TH antibodies by fluorescent dyes. Saline and ASE did not promote FosB/ΔFosB expression in the PVN. CMS and CMS+ASE elicited FosB/ΔFosB-expression in the PVN, whereas, ASE did not augment or attenuate FosB/ΔFosB induction elicited by CMS. FosB/ΔFosB-CRH occurred after CMS and CMS+ASE treatments in the PVN middle sector, while FosB/ΔFosB-AVP and FosB/ΔFosB-OXY after CMS and CMS+ASE treatments in the PVN posterior sector. FosB/ΔFosB-TH colocalization was rare. Larger FosB/ΔFosB profiles, running above the PVN, did not show any colocalizations. The study provides an anatomical/functional knowledge about an unaccented nature of prolonged ASE treatment at the level of PVN and excludes its positive or negative interplay with CMS effect. Data indicate that long-lasting ASE treatment might not act as a stressor acting at the PVN level.
Taking medicines as intended is difficult for everybody, but young people going through adolescence have greater problems than adults and younger children. One of the most important things that happen during the teenage years is the development of individual identities, which might not remain constant during this time and can be affected deeply by the diagnosis of a long-term condition. The aim of this study was to examine the relationships between identity and medication use among young people with juvenile arthritis.
A prospective qualitative study was undertaken to collect private online 'blog' style data from young people (aged 11-19 years) with juvenile arthritis, and their parents, to examine their views about their condition, identity, medication and use of health services. Participants were identified from a large paediatric hospital in the UK.
Young people (n = 21) with a median age 14 years (range 11-17 years) posted a median (range) of 8 (1-36) blogs and parents (n = 6) posted 4 (1-12) blogs. Young people gave a strong sense of both private and public identity that was intertwined with their arthritis and treatment. It was evident that young people's self-care was intrinsically linked to their attempts to maintain a sense of individually and socially constructed definitions of normality. The act of taking medication, and the consequences (positive or negative) of that act, had an impact both personally and socially.
Young people with juvenile arthritis reflect on their medication as a factor affecting their perception of themselves. Acknowledging the roles of both personal and social identity will be important in any strategies to support optimal medication use. This includes an understanding of the identity transformations that young people can experience and how decision-making may be affected by their attempts to retain pre-diagnosis identities and/or develop new social identities.
It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells.
Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells.
Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01-1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se.
In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.
Here, a novel drug delivery system was developed for the hydrochlorothiazide (HCT):β-cyclodextrin (βCD) inclusion complex loaded into chitosan (CS) nanoparticles (NPs) [CS/HCT:βCD NPs]. It was found, for the first time, that exposure of the intestinal mucosa to free HCT resulted in an increased and abnormal intestinal permeability associated with several injuries to the intestinal epithelium. Nevertheless, the HCT delivery system obtained ameliorated the damage of the intestinal epithelium induced by HCT. Furthermore, we found that the corresponding permeability profiles for both the free HCT and the CS/HCT:βCD NPs were exponential and lineal, respectively. We propose that the increased intestinal uptake and severe tissue injury of HCT to the intestinal epithelium could be directly related to possible effects of this drug on the ionoregulatory Na(+/)K(+)-ATPase channel. Thus, it is postulated that the CS/HCT:βCD NPs may increase the gastrointestinal retention of the HCT, which would provide increased adherence to the mucus barrier that lines the intestinal epithelium; consequently, this would act as a slow HCT release delivery system and maintain lower drug levels of luminal gut in comparison with the administration of free HCT, leading to less severe local injury.
The use of abdominal computed tomography (ACT) for nontraumatic abdominal pain in the pediatric emergency department is increasing to a remarkable extent, and every effort should be made to select patients who would benefit from ACT. The aim of this study was to investigate the clinical and laboratory predictors of positive CT yield in pediatric patients with nontraumatic abdominal pain.
The medical records of 739 pediatric patients who, between January 2009 and February 2014, underwent ACT for nontraumatic abdominal pain at the emergency department were reviewed retrospectively. These patients were divided into 2 groups: a right lower quadrant (RLQ) pain group and a non-RLQ pain group. The radiology reports were reviewed to determine ACT diagnoses. Univariate and multivariate analysis was performed to elicit independent predictors of positive ACT yield. Equations predicting the probability of positive ACT diagnosis were deduced from the regression equation in both groups.
In the RLQ pain group, the most frequent ACT diagnosis was acute appendicitis, and in the non-RLQ pain group, enteritis was the most frequent diagnosis. In the RLQ pain group, sex, leukocytosis, neutrophil-to-lymphocyte ratio, and elevated C-reactive protein were significant independent predictors for positive ACT diagnosis. The number of synchronous predictor was significantly related to the proportion of positive ACT diagnosis. In the non-RLQ pain group, leukocytosis was the only significant predictor in multivariate analysis.
Factors predicting positive CT yield were sex, leukocytosis, neutrophil-to-lymphocyte ratio, and elevated C-reactive protein in the RLQ pain group and leukocytosis in the non-RLQ pain group.
Cortical spreading depression (SD) is a spreading disruption in brain ionic homeostasis during which neurons experience complete and prolonged depolarizations. SD is generally believed to be the physiological substrate of migraine aura and is associated with many other brain pathologies. Here, we perform simulations with a model of SD treating brain tissue as a triphasic continuum of neurons, glia and the extracellular space. A thermodynamically consistent incorporation of the major biophysical effects, including ionic electrodiffusion and osmotic water flow, allows for the computation of important physiological variables including the extracellular voltage (DC) shift. A systematic parameter study reveals that glia can act as both a disperser and buffer of potassium in SD propagation. Furthermore, we show that the timing of the DC shift with respect to extracellular [Formula: see text] rise is highly dependent on glial parameters, a result with implications for the identification of the propagating mechanism of SD.
Gut dysbiosis has been associated with several clinically relevant conditions, including alterations of central nervous system (CNS) structure and function development. This review discussed aspects of the relationship between gut microbiota and the CNS during development.
PubMed was used to search for all of the studies published over the last 15 years using the key word "microbiota" and "gut" or "intestinal" and "nervous system". More than 350 articles were found, and only those published in English and providing data on aspects related to neurologic diseases were included in the evaluation.
The data indicate that the gut microbiota influences CNS development and function and that gut dysbiosis is associated with significant neurological problems. However, most of these data have been collected in experimental animals and cannot be transferred to humans. Moreover, it is not definitively established whether neurologic diseases depend on a generic modification of the gut microbiota or whether a single bacterial phylum or species plays a specific role for any single condition. Furthermore, limited information exists regarding protective bacteria.
Both probiotics and prebiotics can have different impacts on CNS according to the microbial species or oligosaccharides that are administered. In humans, particularly in children, several factors may be important in conditioning gut microbiota modifications; unfortunately, most of these factors act simultaneously. More efforts are required to fully define both the array of complex behaviors that are influenced by the gut microbiota at the CNS level and the mechanisms involved.
Phytoestrogens are plant-derived dietary compounds with structural similarity to 17-β-oestradiol (E2), the primary female sex hormone. This structural similarity to E2 enables phytoestrogens to cause (anti)oestrogenic effects by binding to the oestrogen receptors. The aim of the present review is to present a state-of-the-art overview of the potential health effects of dietary phytoestrogens. Various beneficial health effects have been ascribed to phytoestrogens, such as a lowered risk of menopausal symptoms like hot flushes and osteoporosis, lowered risks of cardiovascular disease, obesity, metabolic syndrome and type 2 diabetes, brain function disorders, breast cancer, prostate cancer, bowel cancer and other cancers. In contrast to these beneficial health claims, the (anti)oestrogenic properties of phytoestrogens have also raised concerns since they might act as endocrine disruptors, indicating a potential to cause adverse health effects. The literature overview presented in this paper illustrates that several potential health benefits of phytoestrogens have been reported but that, given the data on potential adverse health effects, the current evidence on these beneficial health effects is not so obvious that they clearly outweigh the possible health risks. Furthermore, the data currently available are not sufficient to support a more refined (semi) quantitative risk-benefit analysis. This implies that a definite conclusion on possible beneficial health effects of phytoestrogens cannot be made.
A carbon-free nanocomposite consisting of MoO2 nanoparticles embedded between MoSe2 nanosheets, named MoO2@MoSe2, has been synthesized and demonstrated excellent electrochemical properties for lithium ion batteries. In such a composite, MoSe2 nanosheets provide a flexible substrate for MoO2 nanoparticles; while MoO2 nanoparticles act as spacers to retain the desired active surface to electrolyte and also introduce metallic conduction. In addition, the heterojunctions at the interface between MoSe2 and MoO2 introduce a self-built electric field to promote the lithiation/delithiation process. As a result, such lamellar composite has a long cycling stability with a reversible capacity of 520.4 mA h g(-1) at a current density of 2000 mA g(-1) after 400 cycles and excellent rate performance, which are attributed to the synergistic combination of the two components in nanoscale.
Since epidemiologic data have highlighted the positive effects of metformin to reduce cancer incidence and mortality, many in vitro and in vivo studies as well as a large number of clinical trials have been conducted in order to study its potential. The many anticancer actions of metformin lead to a cytostatic effect. Two distinct but not exclusive mechanisms can be implicated in these actions. First, by decreasing insulinemia and glycaemia, metformin can block the PI3K/MAPK signalling pathway implicated in cell growth. Second, metformin can directly act on cancer cells by targeting various pathways including tumour metabolism, inflammation, angiogenesis or cancer stem cells, mainly through the activation of the AMPK pathway. Nonetheless, although metformin alone displays chemopreventive properties, it does not seem to be sufficient to treat cancer, raising the need to be combined with other drugs (e.g. chemotherapy or glycolysis inhibitors) in order to synergistically reveal its cytotoxic action. However, in particular conditions such as specific mutations (e.g. LKB, p53 or OCT1) or low glucose availability, metformin alone does have cytotoxic effects. Thus, it is essential to consider the associated biomarkers in order to determine the potential of metformin in different types of cancers.
We use molecular dynamics simulations to investigate the tracer diffusion in a sea of polymers with specific binding zones for the tracer. These binding zones act as traps. Our simulations show that the tracer can undergo normal yet non-Gaussian diffusion under certain circumstances, e.g., when the polymers with traps are frozen in space and the volume fraction and the binding strength of the traps are moderate. In this case, as the tracer moves, it experiences a heterogeneous environment and exhibits confined continuous time random walk (CTRW) like motion resulting in a non-Gaussian behavior. Also the long time dynamics becomes subdiffusive as the number or the binding strength of the traps increases. However, if the polymers are mobile then the tracer dynamics is Gaussian but could be normal or subdiffusive depending on the number and the binding strength of the traps. In addition, with increasing binding strength and number of polymer traps, the probability of the tracer being trapped increases. On the other hand, removing the binding zones does not result in trapping, even at comparatively high crowding. Our simulations also show that the trapping probability increases with the increasing size of the tracer and for a bigger tracer with the frozen polymer background the dynamics is only weakly non-Gaussian but highly subdiffusive. Our observations are in the same spirit as found in many recent experiments on tracer diffusion in polymeric materials and question the validity of using Gaussian theory to describe diffusion in a crowded environment in general.
Our established interleukin-13 ( IL-13 ) overexpression rat model of minimal change-like nephropathy provided a platform to study the molecular signalling pathways in T-helper 2 (Th2) cytokine associated minimal change nephrotic syndrome. We hypothesized that IL-13 may act directly on podocytes, causing podocyte foot process effacement and hence proteinuria in our rat model of minimal change-like nephropathy. This study aimed firstly to delineate the glomerular "gene signature" associated with IL-13-mediated dysregulation of podocyte related proteins, and subsequently to investigate the role of the differentially regulated genes in IL-13-mediated podocyte injury. Glomerular transcriptional profile of IL-13 -overexpressed rats showed characteristic features of podocyte injury with 87% of podocyte related genes being significantly down-regulated. Gene expression of Vav1 was shown to be highly up-regulated in the glomeruli of IL-13 -overexpressed rats and pathway analysis of the differentially expressed genes suggested a possible novel role of Vav1 in podocyte cytoskeleton remodelling. Immunofluorescence examination demonstrated glomerular expression of Vav1 in rats which co-localized with synaptopodin, confirming podocyte expression. However, positive staining for the phosphorylated form of Vav1 (p-Vav1) was only seen in IL-13 -overexpressed rats. Moreover, in-vitro IL-13 stimulation of human podocytes resulted in phosphorylation of Vav1. This was associated with Rac1 activation and actin cytoskeleton rearrangement, which was abrogated in Vav1 knock-down podocytes. In conclusion, we have demonstrated the role of Vav1-Rac1 pathway characterised by phosphorylation of Vav1, activation of Rac1 and the subsequent actin cytoskeleton rearrangement in IL-13 induced podocyte injury, possibly explaining the podocyte foot process effacement seen in our IL-13 overexpression rat model.
The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer`s disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the brain is capacity to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells.
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8(+) T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
Microglia can aggravate olfactory dysfunction by mediating neuronal death in the olfactory bulb (OB) of a murine model of Niemann-Pick disease type C1 (NPC1), a fatal neurodegenerative disorder accompanied by lipid trafficking defects. In this study, we focused on the crosstalk between neurons and microglia to elucidate the mechanisms underlying extensive microgliosis in the NPC1-affected brain. Microglia in the OB of NPC1 mice strongly expressed CX3C chemokine receptor 1 (Cx3cr1), a specific receptor for the neural chemokine C-X3-C motif ligand 1 (Cx3cl1). In addition, a high level of Cx3cl1 was detected in NPC1 mouse-derived CSF due to enhanced catalytic activity of Cathepsin S (Ctss), which is responsible for Cx3cl1 secretion. Notably, nasal delivery of Cx3cl1 neutralizing antibody or Ctss inhibitor could inhibit the Cx3cl1-Cx3cr1 interaction and support neuronal survival through the suppression of microglial activation, leading to an improvement in the olfactory function in NPC1 mice. Relevant in vitro experiments revealed that intracellular cholesterol accumulation could act as a strong inducer of abnormal Ctss activation and, in turn, stimulated the Cx3cl1-Cx3cr1 axis in microglia via p38 mitogen-activated protein kinase signaling. Our data address the significance of Cx3cl1-Cx3cr1 interaction in the development of microglial neurotoxicity and suggest that Ctss is a key upstream regulator. Therefore, this study contributes to a better understanding of the crosstalk between neurons and microglia in the development of the neurodegeneration and provides a new perspective for the management of olfactory deficits and other microglia-dependent neuropathies. GLIA 2016;64:2291-2305.
The evolution of insecticide resistance by crop pests and disease vectors causes serious problems for agriculture and health. Sexual selection can accelerate or hinder adaptation to abiotic challenges in a variety of ways, but the effect of sexual selection on resistance evolution is little studied. Here, we examine this question using experimental evolution in the pest insect Tribolium castaneum. The experimental removal of sexual selection slowed the evolution of resistance in populations treated with pyrethroid pesticide, and also reduced the rate at which resistance was lost from pesticide-free populations. These results suggest that selection arising from variance in mating and fertilization success can augment natural selection on pesticide resistance, meaning that sexual selection should be considered when designing strategies to limit the evolution of pesticide resistance.
The Cnidium monnieri fruits (CMF) were studied how they act on immune system as a novel immunostimulator against the infectious disease.
Macrophages were treated with CMF, and nitric oxide (NO) and tumour necrosis factor-α (TNF-α) were measured, and phagocytosis of macrophages was detected using FITC-labelled Escherichia coli. The protective effect of CMF against E. coli infection in mice was examined. The survival rate was monitored daily for up to 5 days. And then the viable bacteria count of serum and the immunological mediator (NO, TNF-α, interleukin (IL)-12 and IL-6) of serum, splenocyte and peritoneal macrophages were analysed.
The CMF significantly enhanced the concentrations of NO and TNF-α and the phagocytosis activity in macrophages. The oral administration of CMF for five consecutive days before infection prolonged the survival rate. Treatment with CMF significantly stimulated the phagocytosis of peritoneal macrophages and induced the immunological mediator of serum, splenocyte and peritoneal macrophages against the E. coli infection.
The host-protective effects of CMF might be archived by improving immune response, and CMF could act to prevent pathogenic microbial infections with immunomodulation.
Axo-glial interactions are critical for myelination and the domain organization of myelinated fibers. Cell adhesion molecules belonging to the Cadm family, and in particular Cadm3 (axonal) and its heterophilic binding partner Cadm4 (Schwann cell), mediate these interactions along the internode. Using targeted shRNA-mediated knockdown, we show that the removal of axonal Cadm3 promotes Schwann cell myelination in the in vitro DRG neuron/Schwann cell myelinating system. Conversely, over-expressing Cadm3 on the surface of DRG neuron axons results in an almost complete inability by Schwann cells to form myelin segments. Axons of superior cervical ganglion (SCG) neurons, which do not normally support the formation of myelin segments by Schwann cells, express higher levels of Cadm3 compared to DRG neurons. Knocking down Cadm3 in SCG neurons promotes myelination. Finally, the extracellular domain of Cadm3 interferes in a dose-dependent manner with the activation of ErbB3 and of the pro-myelinating PI3K/Akt pathway, but does not interfere with the activation of the Mek/Erk1/2 pathway. While not in direct contradiction, these in vitro results shed lights on the apparent lack of phenotype that was reported from in vivo studies of Cadm3(-/-) mice. Our results suggest that Cadm3 may act as a negative regulator of PNS myelination, potentially through the selective regulation of the signaling cascades activated in Schwann cells by axonal contact, and in particular by type III Nrg-1. Further analyses of peripheral nerves in the Cadm(-/-) mice will be needed to determine the exact role of axonal Cadm3 in PNS myelination. GLIA 2016;64:2247-2262.
Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.
Zinc ions are essential cofactors of a wide range of enzymes, transcription factors, and other regulatory proteins. Moreover, zinc is also involved in cellular signaling and enzymes inhibition. Zinc dysregulation, deficiency, over-supply, and imbalance in zinc ion transporters regulation are connected with various diseases including cancer. A zinc ion pool is maintained by two types of proteins: (i) zinc-binding proteins, which act as a buffer and intracellular donors of zinc and (ii) zinc transporters responsible for zinc fluxes into/from cells and organelles. The decreased serum zinc ion levels have been identified in patients suffering from various cancer diseases, including head and neck tumors and breast, prostate, liver, and lung cancer. On the contrary, increased zinc ion levels have been found in breast cancer and other malignant tissues. Zinc metalloproteomes of a majority of tumors including brain ones are still not yet fully understood. Current knowledge show that zinc ion levels and detection of certain zinc-containing proteins may be utilized for diagnostic and prognostic purposes. In addition, these proteins can also be promising therapeutic targets. The aim of the present work is an overview of the importance of zinc ions, zinc transporters, and zinc-containing proteins in brain tumors, which are, after leukemia, the second most common type of childhood cancer and the second leading cause of death in children after accidents.
Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n=8) or 5 to 6 (n=8) postovulation for GC and TC RNA extraction from small (1-5mm), medium (5.1-8mm), and large (8.1-18mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2)<progesterone (P4)] follicles than in large E2-active (i.e., concentrations of E2>P4) follicles at both early (d 3-4) and late (d 5-6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r=-0.68), free IGF-1 (r=-0.63), and E2-to-P4 ratio (r=-0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.
Chemotherapy-induced peripheral neuropathy (CIPN) represents a serious complication associated with antineoplastic drugs. Although there are no medications available that effectively prevent CIPN, many classes of drugs have been used to treat this condition, including anticonvulsants, serotonin and noradrenaline reuptake inhibitors, and opioids. However, these therapeutic options yielded inconclusive results in CIPN clinical trials and produced assorted side effects with their prolonged use. Thus, there is an urgent need to develop efficacious and safe treatments for CIPN. In this report, we tested whether the endogenous lipid palmitoylethanolamide (PEA) alone or in combination with the anticonvulsant gabapentin would reduce allodynia in a mouse paclitaxel model of CIPN. Gabapentin and PEA reversed paclitaxel-induced allodynia with respective ED50 doses (95% confidence interval) of 67.4 (61.52-73.94) and 9.2 (8.39-10.16) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. The PPAR-α antagonist receptor antagonist GW6471 [N-((2S)-2-(((1Z)-1-methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide] completely blocked the antinociceptive effects of PEA. In addition, PEA administered via intraplantar injection into a paw, intrathecal injection, and intracerebroventricular injection reversed paclitaxel-induced allodynia, suggesting that it may act at multiple sites in the neuroaxis and periphery. Finally, repeated administration of PEA (30 mg/kg, 7 days) preserved the antiallodynic effects with no evidence of tolerance. These findings taken together suggest that PEA possesses potential to treat peripheral neuropathy in cancer patients undergoing chemotherapy.
Many rheumatic diseases are characterized by having an autoimmune background. Determining the mechanisms underlying autoimmunity is, therefore, important to further understand these diseases and to inform future lines of research aimed at developing new treatments and cures. As fast responders, innate lymphocytes have protective or pathogenic roles in the initiation as well as the maintenance of immune responses in general, and they contribute to tissue homeostasis, among other functions. Innate lymphocytes also seem to be involved in autoimmunity in particular. Since 2010, accumulating evidence clearly shows that different populations of innate lymphocytes have roles in responding to antigen-specific autoantibody and autoreactive T cells, thereby amplifying or attenuating disease processes. Cytotoxicity is a cardinal feature of many innate lymphocytes and can contribute to inflammatory tissue damage. Finally, innate lymphocytes can respond to biologic therapies for autoimmune diseases. Consequently, like TNF and other effector molecules, certain innate lymphocyte subsets might be appropriate therapeutic targets to ameliorate various autoimmune diseases. In this Review, we summarize the main characteristics and functions of innate lymphocyte subsets, and describe their roles in autoimmune disease. We also discuss how biologic therapies influence innate lymphocyte function and consider the potential for these cell subsets to act as future therapeutic targets.
A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(β-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.
are small vesicles comprised of a lipid bilayer containing various proteins, RNAs and bioactive lipids. They act as intercellular messengers that give the ability to communicate between both cells of the same type and other cell types. They are released by healthy cells, both constitutively and upon cell activation and play an important role in immune system function. are essential for healthy physiological conditions, however under pathological circumstances, they act to potentiate cellular stress and damage. This review explores the characteristics, biogenesis, role(s) in pathogenesis of diseases and role(s) in progression of cancer of these nano-sized message-in-a-vesicle, .
VPS33B and VIPAR comprise the two known components of the recently christened class C Homologues in Endosome-Vesicle Interaction (CHEVI) complex, thought to act as a tethering complex in endosomal trafficking distinct from the HOPS and CORVET complexes in mammalian cells. A recent paper in The Journal of Pathology further explores the role of the CHEVI complex in the biogenesis of α-granules in megakaryocytes, identifying two novel interactors of this complex: α-tubulin and SEC22B, and demonstrating that VPS33B expression is required for the localization of SEC22B and the α-granule cargo VWF to proplatelets in megakaryocytes. These findings advance the current knowledge of the function of the CHEVI complex in α-granule biogenesis and together with studies in other systems, corroborate its role in the specialized delivery of cargo in different cell types. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Guzmania monostachia is an epiphytic heteroblastic bromeliad that exhibits rosette leaves forming water-holding tanks at maturity. Different portions along its leaf blades can display variable degrees of crassulacean acid metabolism (CAM) up-regulation under drought. Since abscisic acid (ABA) can act as an important long-distance signal, we conducted a joint investigation of ontogenetic and drought impacts on CAM intensity and ABA levels in different leaf groups within the G. monostachia rosette. For this, three groups of leaves were analysed according to their position within the mature-tank rosette (i.e., younger, intermediate, and older leaves) to characterize the general growth patterns and magnitude of drought-modulated CAM expression. CAM activity was evaluated by analysing key molecules in the biochemical machinery of this photosynthetic pathway, while endogenous ABA content was comparatively measured in different portions of each leaf group after seven days under well-watered (control) or drought treatment. The results revealed that G. monostachia shows more uniform morphological characteristics along the leaves when in the atmospheric stage. The drought treatment of mature-tank rosettes generally induced in older leaves a more severe water loss, followed by the lowest CAM activity and a higher increase in ABA levels, while younger leaves showed an opposite response. Therefore, leaf groups at distinct ontogenetic stages within the tank rosette of G. monostachia responded to drought with variable degrees of water loss and CAM expression. ABA seems to participate in this tissue-compartmented response as a long-distance signalling molecule, transmitting the drought-induced signals originated in older leaves towards the younger ones.
Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (FcεRI) cross linking leads to an increase in intracellular calcium ([Ca(+2) ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca(+2) ]i -dependent mast cell activation.
We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells, and human and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (β-hexosaminidase and renin), as well as changes in [Ca(+2) ]i and phosphorylation of proteins downstream of FcεRI activation.
Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-COOH (10-300 μM) reduced, in a concentration-dependent manner, antigenic and non-antigenic degranulation and renin release in all mast cell types. Notably, PhNCS-COOH also prevented in a concentration-dependent mode the increase in [Ca(+2) ]i elicited by Ca(+2) ionophore, thapsigargin and FcεRI activation. Moreover, PhNCS-COOH attenuated the phosphorylation of Syk, cPLA-2 and PLCγ1 in antigen-stimulated RBL-2H3 cells.
Collectively, our results demonstrate that, by attenuating the phosphorylation of proteins downstream of FcεRI cross-linking on mast cells, H2 S diminishes [Ca(+2) ]i availability and thus mast cell degranulation and renin release. These findings suggest that PhNCS-COOH could be a strategic therapeutic tool in mast cell-mediated allergic conditions.
Autophagy is a major degradation pathway that engulfs, removes, and recycles unwanted cytoplasmic material including damaged organelles and toxic protein aggregates. One type of autophagy, macroautophagy, is a tightly regulated process facilitated by autophagy-related (Atg) proteins that must communicate effectively and act in concert to enable the de novo formation of the phagophore, its maturation into an autophagosome, and its subsequent targeting and fusion with the lysosome or the vacuole. Autophagy plays a significant role in physiology, and its dysregulation has been linked to several diseases, which include certain cancers, cardiomyopathies, and neurodegenerative diseases. Here, we summarize the key processes and the proteins that make up the macroautophagy machinery. We also briefly highlight recently uncovered molecular mechanisms specific to neurons allowing them to uniquely regulate this catabolic process to accommodate their complicated architecture and non-dividing state. Overall, these distinct mechanisms establish a conceptual framework addressing how macroautophagic dysfunction could result in maladies of the nervous system, providing possible therapeutic avenues to explore with a goal of preventing or curing such diseases.
Macrophage migration inhibitory factor (MIF), a pleiotropic pro-inflammatory cytokine, is a key regulator in both innate and acquired immunity systems. MIF has become a promising drug target for inflammatory diseases. Apart from its cytokine activities, MIF is known to act as a d-dopachrome tautomerase. Our previous work has identified that 3-[(biphenyl-4-ylcarbonyl)carbamothioyl]amino benzoic acid (Z-590) exhibited a potent inhibitory activity against MIF. In this study, we investigate the effect of Z-590 on lipopolysaccharide (LPS)-activated microglial cell activation. Our results demonstrate that Z-590 significantly decreases the production of nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) as well as reactive oxygen species (ROS) involved in inhibiting MAKPs signalling pathway in LPS-stimulated microglia cells. Furthermore, Z-590 reduced cytotoxicity of activated microglia toward HT-22 hippocampal cells in a microglia-conditioned medium system. Taken together, these results indicate that MIF inhibitor Z-590 elicits a potent inhibitor for microglia-mediated neuroinflammation.
Atmospheric carbon dioxide (CO2 ) directly determines the rate of plant photosynthesis and indirectly effects plant productivity and fitness and may therefore act as a selective pressure driving evolution, but evidence to support this contention is sparse. Using Plantago lanceolata L. seed collected from a naturally high CO2 spring and adjacent ambient CO2 control site, we investigated multigenerational response to future, elevated atmospheric CO2 . Plants were grown in either ambient or elevated CO2 (700 μmol mol(-1) ), enabling for the first time, characterization of the functional and population genomics of plant acclimation and adaptation to elevated CO2 . This revealed that spring and control plants differed significantly in phenotypic plasticity for traits underpinning fitness including above-ground biomass, leaf size, epidermal cell size and number and stomatal density and index. Gene expression responses to elevated CO2 (acclimation) were modest [33-131 genes differentially expressed (DE)], whilst those between control and spring plants (adaptation) were considerably larger (689-853 DE genes). In contrast, population genomic analysis showed that genetic differentiation between spring and control plants was close to zero, with no fixed differences, suggesting that plants are adapted to their native CO2 environment at the level of gene expression. An unusual phenotype of increased stomatal index in spring but not control plants in elevated CO2 correlated with altered expression of stomatal patterning genes between spring and control plants for three loci (YODA, CDKB1;1 and SCRM2) and between ambient and elevated CO2 for four loci (ER, YODA, MYB88 and BCA1). We propose that the two positive regulators of stomatal number (SCRM2) and CDKB1;1 when upregulated act as key controllers of stomatal adaptation to elevated CO2 . Combined with significant transcriptome reprogramming of photosynthetic and dark respiration and enhanced growth in spring plants, we have identified the potential basis of plant adaptation to high CO2 likely to occur over coming decades.
Many deletions of the wheat Della ( Rht - B1 ) gene and its flanking regions were isolated in a simple phenotypic screen, and characterised by modified analysis of SNP hybridisation data and cytogenetics. In a dwarf wheat suppressor screen, many tall 'revertants' were isolated following mutagenesis of a severely dwarfed (Rht-B1c) hexaploid wheat. About 150 lines were identified as putative deletions of Rht-B1c, based on the PCR analysis. Southern blot hybridisation established that most of them lacked the Rht-B1 gene, but retained the homoeologues Rht-A1 and Rht-D1. PCR assays were developed for orthologues of two genes that flank Rht-1/Della in the genomes of the model species Brachypodium and rice. Deletion of the B-genome-specific homoeologues of these two genes was confirmed in the Rht-B1 deletion lines, indicating loss of more than a single gene. SNP chip hybridisation analysis established the extents of deletion in these lines. Based on the synteny with Brachypodium chromosomes 1 and 4 g, and rice chromosomes 3g and 11g, notional deletion maps were established. The deletions ranged from interstitial deletions of 4BS through to loss of all 4BS markers. There were also instances, where all 4BS and 4BL markers were lost, and these lines had poor fertility and narrow stems and leaves. Cytogenetic studies on selected lines confirmed the loss of portions of 4BS in lines that lacked most or all 4BS markers. They also confirmed that lines lacking both 4BS and 4BL markers were nullisomics for 4B. These nested deletion lines share a common genetic background and will have applications in assigning markers to regions of 4BS as well as to 4BL. The potential for this type of analysis in other regions of the wheat genome is discussed.
Glycine max L. (soybean) is one of the major crops of the world. Although the process of biomineralisation has been reported in some organs of soybean, we now report the description and quantification of calcium oxalate crystals in vegetative and reproductive organs of soybean during its life cycle, as they act as an important source of calcium to the soil, once the harvesting is finished. Through diaphanisation, cross-sectioning, optical and scanning electron microscopy analysis of the organs, morphology, size and location of the crystals were identified. In addition, crystal density (n° crystals·mm(-2) ) and the input of crystals to soil (n° crystals·ha(-1) ) were calculated. Soybean produced prismatic calcium oxalate crystals in vegetative and reproductive organs, generally associated with vascular bundles, resulting in a potencial transfer to the soil of 81.4 x 10(7) crystals·ha(-1) throughout its life cycle. Pods were the organs with higher calcium oxalate crystal production (1112.7 ± 384.6 crystals·mm(-2) ), but with the smaller size (12.3 ± 2.1 μm long). However, cotyledons were the organs that produce the larger crystals (21.3 ± 3.5 μm long), but in lesser amounts (150.9 ± 64.4 crystals·mm(-2) ). In leaves, although crystal size did not differ from vegetative to reproductive stage (14.5 ± 4.2 and 14.5 ± 4 μm in length, respectively), the crystal density increased (293.2 and 409 crystals·mm(-2) , respectively). These results will contribute to knowledge of the amount of calcium oxalate crystals involved in the process of Ca recycling through cultivated vegetation in fields from humid plains at medium latitudes, which therefore have biological, botanical, biogeochemical and pedological relevance.
Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2) could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50μM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1mM). We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks) resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.
The Dual Visuomotor Channel theory of reaching proposes that a reach-to-grasp act integrates a Reach, directed toward the extrinsic properties of the target (location), and a Grasp, directed toward the intrinsic properties of the target (size and shape). Previous studies of reach-to-grasp report that the Grasp is altered in pantomime tasks made from a starting position with digit 1 and digit 2 closed and proximal to the target. The present study extends the analysis of real versus pantomime reaching to a task that featured both a Reach and a Grasp, having a starting position with the hand open and proximal to the body. For a real reach, seated participants reached for a doughnut ball (food item) located on a pedestal at arms distance, with the intent of bringing the doughnut ball to the mouth for eating. Participants also made four pantomime reaches with: (1) the doughnut ball removed from the pedestal, (2) the doughnut ball and pedestal moved to the side of the reach location, (3) the doughnut ball and pedestal absent, and (4) the participants wearing vision-occluding glasses. There were two main findings. First, the presence of task-related cues, platform, doughnut ball, and room influenced the kinematics of the Reach and Grasp. Second, the compound structure of a real reach, in which flexion/extension of the arm featured in the Reach and flexion/extension of the digits featured in the Grasp are out of phase, changed in pantomime such that these features of Reach and Grasp became in phase. The results show that pantomime reaching is influenced not only by task-related percepts but also by central mechanisms ordinarily related to integrating the Reach and the Grasp.
Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined (12)C/(13)C, (14)N/(15)N and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A(14)N/(15)N isotope effect ((15)V/K ≈ 1.015, with respect to substrate NH4(+)) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse ((15)V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring (15)N. There was no (12)C/(13)C kinetic isotope effect ((13)V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water ((D)V ≈ 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates.
In this manuscript, the ability of BH2 X compounds (X=F, Cl, Br, and I) to establish halogen-, hydrogen-, and triel-bonding interactions was studied at the RI-MP2/aug-cc-pVQZ level of theory. Several homodimers were taken into account, highlighting the versatility of these compounds to act as both electron donors and electron acceptors. Natural bond orbital analysis showed that orbital effects were important contributors to the global stabilization of the σ- and π-hole bonded complexes studied. Finally, some X-ray solid-state structures retrieved from the Cambridge structural database were described to demonstrate the importance of these interactions involving boron derivatives in the solid state.
It is well established the beneficial role of silicon (Si) in alleviating abiotic stress. However, it remains poorly understood the mechanisms of the Si-mediated protection against metal deficiency, especially the zinc (Zn) one. Recently, it has been proposed that Si may act by an interaction with this biometal in the root apoplast contributing to its movement through the plant, as in the case of Fe deficiency. In the present work, the effect of initial or continuous Si doses in soybean Zn deficient plants has been studied. For that purpose, plants grown in hydroponic culture were treated with different Si doses (0.0, 0.5 and 1.0 mM) under Zn limiting conditions. SPAD index in leaves, several growth parameters, mineral content in the whole plant and the formation of Zn pools in roots were determined. An initial addition of 0.5 mM of Si to the nutrient solution led to an enhancement of plants growth, Zn and Si content in leaves, and a higher storage of Zn in the root apoplast. The results suggest that this treatment enhanced Zn accumulation on roots and its movement to shoots when needed, mitigating Zn deficiency symptoms.
Jasmonic acid (JA) signaling pathway in plants is activated against various developmental processes as well as biotic and abiotic stresses. The Jasmonate ZIM-domain (JAZ) protein family, the key regulator of plant JA signaling pathway, also participates in phytohormone crosstalk. This is the first study revealing the in vivo interactions of finger millet (Eleusine coracana (L.) Gaertn.) JAZ protein (EcJAZ) under methyl jasmonate (MJ) treatment. The aim of the study was to explore not only the JA signaling pathway but also the phytohormone signaling crosstalk of finger millet, a highly important future crop. From the MJ-treated finger millet seedlings, the EcJAZ interacting proteins were purified by affinity chromatography with the EcJAZ-matrix. Twenty-one proteins of varying functionalities were successfully identified by MALDI-TOF-TOF Mass spectrometry. Apart from the previously identified JAZ binding proteins, most prominently, EcJAZ was found to interact with transcription factors like NAC, GATA and also with Cold responsive protein (COR), etc. that might have extended the range of functionalities of JAZ proteins. Moreover, to evaluate the interactions of EcJAZ in the JA-co-receptor complex, we generated ten in-silico models containing the EcJAZ degron and the COI1-SKP1 of five monocot cereals viz., rice, wheat, maize, Sorghum and Setaria with JA-Ile or coronatine. Our results indicated that the EcJAZ protein of finger millet could act as the signaling hub for the JA and other phytohormone signaling pathways, in response to a diverse set of stressors and developmental cues to provide survival fitness to the plant.
Enhancing the flavor of fruits plays a fundamental role in improving fruit quality, and volatile compositions as well as acid and sugar accumulation are significant factors that have an impact on the acceptability of sensory responses by human beings. Vacuoles in plants not only function as cell compartments that store amino acids, sugars and other metabolites but also act as lytic organelles where vacuolar proteins are post-translationally processed into mature forms or degraded by the action of vacuolar processing enzyme (VPE). We have previously characterized VPE genes (SlVPE1-5) during fruit development in tomato and discovered that the VPE enzyme activity negatively interfered with sugar accumulation in mature fruits. Comparative proteomic analysis demonstrated that acid invertase was one of the molecular targets of SlVPE5, which is involved in the hydrolysis of sucrose. This study also showed that decreased VPE enzyme activity due to suppression of SlVPE5 by RNAi strategy (RNAi-SlVPE5) accompanied with decreased enzyme activity of acid invertase. Further, we identified the enzyme activity of acid invertase was not well correlated with mRNA levels in the RNAi-SlVPE5 line. These results suggest that SlVPE5 regulates post-transcriptional processing through de novo synthesis of the acid invertase protein to suppress enzyme activity, thereby eventually ensuring sucrose hydrolysis.
We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as "find-me" signals to promote macrophage migration and activation. In this study, we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with metabolic syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin, and homeostatic model assessment of insulin resistance (HOMA-IR) in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35 % (p < 0.05) after a 3-month reduced calorie diet intervention. This translational study provides an extensive characterization of adipocyte-derived EVs. The findings identify perilipin A as a novel biomarker of circulating EVs of adipocyte origin and support the development of circulating perilipin A-positive EVs as indicators of adipose tissue health.
• Extensive characterization of 3T3L1 EVs identified perilipin A in their composition. • Circulating EVs are elevated in obese mice and associated with glucose intolerance. • Circulating EVs are elevated in obese human and correlated with metabolic factors. • Perilipin A and EV levels are increased in the circulation of obese mice and human. • Circulating EV and perilipin A levels decrease with low calorie intervention.
Long noncoding RNAs (lncRNAs) have been reported to play pivotal roles in a variety of cancers. However, lncRNAs involved in hepatocellular carcinoma (HCC) initiation and progression remain largely unclear. In this study, we identified an lncRNA gradually increased during hepatocarcinogenesis (lncRNA-GIHCG) using publicly available microarray data. Our results further revealed that GIHCG is upregulated in HCC tissues in comparison with adjacent non-tumor tissues. High GIHCG expression is correlated with large tumor size, microvascular invasion, advanced BCLC stage, and poor survival of HCC patients. Functional experiments showed that GIHCG promotes HCC cells proliferation, migration, and invasion in vitro, and promotes xenografts growth and metastasis in vivo. Mechanistically, we demonstrated that GIHCG physically associates with EZH2 and the promoter of miR-200b/a/429, recruits EZH2 and DNMT1 to the miR-200b/a/429 promoter regions, upregulates histone H3K27 trimethylation and DNA methylation levels on the miR-200b/a/429 promoter, and dramatically silences miR-200b/a/429 expression. Furthermore, the biological functions of GIHCG on HCC are dependent on the silencing of miR-200b/a/429. Collectively, our results demonstrated the roles and functional mechanisms of GIHCG in HCC, and indicated GIHCG may act as a prognostic biomarker and potential therapeutic target for HCC. KEY MESSAGE: lncRNA-GIHCG is upregulated in HCC and associated with poor survival of patients. GIHCG significantly promotes tumor growth and metastasis of HCC. GIHCG physically associates with EZH2. GIHCG upregulates H3K27me3 and DNA methylation levels on the miR-200b/a/429 promoter. GIHCG epigenetically silences miR-200b/a/429 expression.
Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2016.
This study was conducted to evaluate the biocontrol potential of Bacillus subtilis CICC 10034, Rhodobacter sphaeroides CGMCC 1.2182 and Agrobacterium tumefaciens CGMCC 1.2554 against patulin (PAT)-producer Penicillium expansum and their ability to remove PAT.
Bacillus subtilis effectively inhibited P. expansum both on apples and in in vitro experiments, which reduced the rot diameter on apples by 38% compared with the control. The reduction was followed by those induced by A. tumefaciens (27·63%) and R. sphaeroides (23·67%). None of the cell-free supernatant (CFS) was able to prevent pathogen growth. Three antagonists could suppress PAT production by P. expansum on apples by 98·5, 93·7 and 94·99% after treatment with B. subtilis, R. sphaeroides and A. tumefaciens respectively. In addition, the three strains led to a 0·56-1·47 log CFU g(-1) reduction in colony number of P. expansum on apples. Survival of antagonists on apple wounds revealed their tolerance to PAT. Furthermore, both live and autoclaved cells of three strains efficiently adsorbed artificially spiked PAT from medium.
The selected antagonists could be applied before harvesting to control apple infection by PAT-producing fungi and also during processing to act as PAT detoxifiers.
Since little information related to the capability of R. sphaeroides and A. tumefaciens to inhibit P. expansum is currently available, the results of this study provide some new perspectives to the biocontrol field.
Excited-state intramolecular proton transfer (ESIPT) is a particularly well known reaction that has been very little studied in magnetic environments. In this work, we report on the photophysical behavior of a known ESIPT dye of the benzothiazole class, when in solution with uncoated superparamagnetic iron oxide nanoparticles, and when grafted to silica-coated iron oxide nanoparticles. Uncoated iron oxide nanoparticles promoted the fluorescence quenching of the ESIPT dye, resulting from collisions during the lifetime of the excited state. The assembly of iron oxide nanoparticles with a shell of silica provided recovery of the ESIPT emission, due to the isolation promoted by the silica shell. The silica network gives protection against the fluorescence quenching of the dye, allowing the nanoparticles to act as a bimodal (optical and magnetic) imaging contrast agent with a large Stokes shift.
Pollinator specialisation through exploitation barriers (such as long floral tubes) does not necessarily mean a lack of pollination when the favoured pollinator is rare or absent. Theory predicts that suboptimal visitors will contribute to plant reproduction in the absence of the most effective pollinator. Here I address these questions with Chasmanthe floribunda a long-tubed plant species in the Cape Floristic Region, which is reliant on one species of pollinator, the long-billed Malachite Sunbird. In contrast to short-billed sunbirds, the Malachite Sunbird occurs in lower abundance or is absent in transformed landscapes. Short-billed sunbirds rob and thieve nectar from long-tubed flowers, but their potential contribution towards pollination is unknown. Experiments assessing seed set after single flower visits were performed to determine whether thieving short-billed sunbirds can act as substitute pollinators. To determine whether short-billed sunbirds reduce pollen limitation in transformed areas, pollen supplementation was done by hand and compared to natural fruit set. Short billed sunbirds are unable to act as substitute pollinators, and seed set is significantly lower in the flowers that they visited, compared to flowers visited by long-billed sunbirds. This is substantiated on a landscape scale, where fruit production in Chasmanthe floribunda could artificially be increased by 35% in transformed landscapes, but not so in natural areas. These findings have important consequences for the management and conservation of long-tubed bird-pollinated plant species that exist in recently transformed landscapes. The potential vulnerability of specialised plant species in transformed landscapes is highlighted.
Although Runx2 is involved in the regulation of cellular differentiation, its physiological roles in the differentiation of uterine stromal cells during decidualization still remain unknown. The aim of this study was to examine the expression, regulation and function of Runx2 in mouse uterus during decidualization. The results showed that Runx2 was highly expressed in the decidua and oil-induced decidualized cells. In the uterine stromal cells, recombinant human Runx2 (rRunx2) could induce the expression of Prl8a2 and Prl3c1 which are two well-known differentiation markers for decidualization, while inhibition of Runx2 with specific siRNA reduced their expression. Further study found that rRunx2 could improve the expression of Prl8a2 and Prl3c1 in the C/EBPβ siRNA-transfected stromal cells. In the stromal cells, cAMP analogue 8-Br-cAMP could induce the expression of Runx2. Moreover, the induction was blocked by PKA inhibitor H89. Simultaneously, attenuation of C/EBPβ with siRNA could also reduce the cAMP-induced Runx2 expression. Furthermore, siRNA-mediated silencing of Runx2 expression alleviated the effects of cAMP on the differentiation of stromal cells. Runx2 might act downstream of C/EBPβ to regulate the expression of Cox-2, Vegf and Mmp9 in the uterine stromal cells. Collectively, Runx2 may play an important role during mouse decidualization.
The maternal deficiency of vitamin D can act on organogenesis in mice offspring, being a risk factor for chronic diseases in adulthood. This study investigates the effects of maternal deficiency of vitamin D on structural islet remodeling and insulin-signaling pathway in the offspring. We studied male C57Bl/6 offspring at 3-month old (n = 10/group) from mother fed one of the two diets: control diet (C) or vitamin D-restricted diet (VitD(-)). After weaning, offspring only fed the control diet ad libitum. In the offspring, we studied insulin production, islet remodeling, and islet protein expression of the insulin-signaling pathway (Western blotting, isolated islet, n = 5/group). VitD(-) offspring showed greater glycemia (P = 0.012), smaller beta-cell mass (P = 0.014), and hypoinsulinemia (P = 0.024) than C offspring. Comparing VitD(-) offspring with C offspring, we observed lower protein levels in islet of insulin (P = 0.003), insulin receptor substrate-1 (P = 0.025), phosphatidylinositol-3-kinases (P = 0.045), 3-phosphoinositide-dependent protein kinase 1 (P = 0.017), protein kinase B (P = 0.028), with reduced expression of pancreas/duodenum homeobox-1 (PDX-1) (P = 0.016), glucose transporter-2 (P = 0.003), and glucokinase (P = 0.045). The maternal vitamin D-restricted diet modifies the development of the pancreas of the offspring, leading to islet remodeling and altered insulin-signaling pathway. The decrease of PDX-1 is probably significant to the changes in the beta-cell mass and insulin secretion in adulthood.
In forests of the humid subtropics of China, chronically elevated nitrogen (N) deposition, predominantly as ammonium (NH4(+) ), causes significant nitrate (NO3(-) ) leaching from well-drained acid forest soils on hill slopes (HS), whereas significant retention of NO3(-) occurs in near-stream environments (groundwater discharge zones, GDZ). To aid our understanding of N transformations on the catchment level, we studied spatial and temporal variabilities of concentration and natural abundance (δ(15) N and δ(18) O) of nitrate (NO3(-) ) in soil pore water along a hydrological continuum in the N-saturated Tieshanping (TSP) catchment, southwest China. Our data show that effective removal of atmogenic NH4(+) and production of NO3(-) in soils on HS were associated with a significant decrease in δ(15) N-NO3(-) , suggesting efficient nitrification despite low soil pH. The concentration of NO3(-) declined sharply along the hydrological flow path in the GDZ. This decline was associated with a significant increase in both δ(15) N and δ(18) O of residual NO3(-) , providing evidence that the GDZ acts as an N sink due to denitrification. The observed apparent (15) N enrichment factor (ε) of NO3(-) of about -5‰ in the GDZ is similar to values previously reported for efficient denitrification in riparian and groundwater systems. Episode studies in the summers of 2009, 2010 and 2013 revealed that the spatial pattern of δ(15) N and δ(18) O-NO3(-) in soil water was remarkably similar from year to year. The importance of denitrification as a major N sink was also seen at the catchment scale, as largest δ(15) N-NO3(-) values in stream water were observed at lowest discharge, confirming the importance of the relatively small GDZ for N removal under base flow conditions. This study, explicitly recognizing hydrologically connected landscape elements, reveals an overlooked but robust N sink in N-saturated, subtropical forests with important implications for regional N budgets.
Anxiety and depression are common in multiple sclerosis (MS). We evaluated the prevalence and factors associated with anxiety, depression and fatigue at the 5-year review of a longitudinal cohort study following a first clinical diagnosis of CNS demyelination (FCD).
Cases with a FCD were recruited soon after diagnosis and followed annually thereafter. A variety of environmental, behavioural and clinical covariates were measured at five-year review. Anxiety and depression were measured using the Hospital Anxiety & Depression Scale (HADS), and fatigue by the Fatigue Severity Scale (FSS).
Of the 236 cases, 40.2% had clinical anxiety (median HADS-A: 6.0), 16.0% had clinical depression (median HADS-D: 3.0), and 41.3% had clinical fatigue (median FSS: 4.56). The co-occurrence of all three symptoms was 3.76 times greater than expectation. Younger age, higher disability, concussion or other disease diagnosis were independently associated with a higher anxiety score; male sex, higher disability, being unemployed, less physical activity, and antidepressant and/or anxiolytic-sedative medication use were independently associated with a higher depression score. Higher disability, immunomodulatory medication use, other disease diagnosis and anxiolytic-sedative medication use were independently associated with having fatigue, while female sex, higher BMI, having had a concussion, being unemployed and higher disability were associated with a higher fatigue score.
These results support previous findings of the commonality of anxiety, depression and fatigue in established MS and extend this to post-FCD and early MS cases. The clustering of the three symptoms indicates that they may share common antecedents.
As soon as a patient comes to know that he/she has cancer, the stress starts and psychological intervention is required. The authors assessed how well a cancer patient can manage stress over the course of the psychological intervention. Data was collected among 107 patients during pre and post intervention and at 2 months follow-up. Intervention was required to measures include acceptance of the disease, managing stress, well -being, and meaning of life. Finally, effects of acceptance and commitment therapy (ACT) were defined in acceptance measured in terms of a significant difference between pre and post intervention scores in the meaning of life and the acceptance level. This acceptance and commitment therapy can be an effective intervention approach for cancer patients that increases acceptance regarding disease and simultaneously leads to improvement in the meaning of life.
Here we present an overview of what is known about endogenous plant compounds that act as inhibitors of hormonal transport processes in plants, about their identity and mechanism of action. We have also summarized commonly and less commonly used compounds of non-plant origin and synthetic drugs that show at least partial 'specificity' to transport or transporters of particular phytohormones. Our main attention is focused on the inhibitors of auxin transport. The urgent need to understand precisely the molecular mechanism of action of these inhibitors is highlighted.
A randomized, placebo-controlled, efficacy trial of Carraguard was unable to demonstrate a reduction in women's risk of HIV infection, which may have been due, in part, to low adherence (gel used in 42 % of vaginal sex acts, on average). A secondary analysis was undertaken to understand baseline factors associated with high adherence (gel used in ≥85 % of sex acts). Women who reported ≥1 vaginal sex act, returned ≥1 opened applicator, and had ≥1 conclusive post-enrollment HIV test (N = 5990) were included. Adherence was estimated as the ratio of average weekly applicator insertions (based on a dye stain assay indicating vaginal insertion)/average weekly sex acts (by self-report). Multivariate logistic regression modeling indicated that coital frequency, site, contraception, and partner age difference had a significant impact on adherence. Women reporting >1 and ≤2 vaginal sex acts per week, on average, were half as likely to be adherent as those reporting 1 vaginal sex act per week or less [adjusted odds ratio (AOR): 0.48; 95 % CI 0.38-0.61]; women from the Western Cape had one-third the odds of being adherent compared to women from KZN (AOR: 0.31; 95 % CI 0.23-0.41); compared to women using injectable contraception, women using any other or no method were more likely to be adherent (AOR: 1.30; 95 % CI 1.04-1.63); and women who had a larger age gap from their partners were more likely to be adherent (AOR: 1.03; 95 % CI 1.01-1.05; p = 0.001). Despite low adherence, overall, 13 % of participants achieved nearly perfect adherence, indicating a potential niche for a coitally dependent microbicide. More research is needed on the impact of sexual patterns and HIV risk perception on product acceptability and adherence to improve counseling in ongoing trials and when products are eventually introduced.
All tissues and organs derive from stem cells, which are undifferentiated cells able to differentiate into specialized cells and self-renewal. In mammals, there are embryonic stem cells that generate germ layers, and adult stem cells, which act as a repair system for the body and maintain the normal turnover of regenerative organs. Mesenchymal stem cells (MSCs) are nonhematopoietic adult multipotent cells, which reside in virtually all postnatal organs and tissues, and, under appropriate in vitro conditions, are capable to differentiate into osteogenic, adipogenic, chondrogenic, myogenic, and neurogenic lineages. Their commitment and differentiation depend on several interacting signaling pathways and transcription factors. Most GNAS-based disorders have the common feature of episodic de novo formation of islands of extraskeletal, qualitatively normal, bone in skin and subcutaneous fat. The tissue distribution of these lesions suggests that pathogenesis involves abnormal differentiation of MSCs and/or more committed precursor cells that are present in subcutaneous tissues. Data coming from transgenic mice support the concept that GNAS is a key factor in the regulation of lineage switching between osteoblast and adipocyte fates, and that its role may be to prevent bone formation in tissues where bone should not form. Despite the growing knowledge about the process of heterotopic ossification in rare genetic disorders, the pathophysiological mechanisms by which alterations of cAMP signaling lead to ectopic bone formation in the context of mesenchymal tissues is not fully understood.
Municipalities have an overarching structure in health promotion. Due to the right to self-government, municipalities are in charge of both voluntary and obligatory tasks. Some of the original and fundamental tasks can be summarized as "services to the public". Current common definitions do not include the term "health promotion". In the present study, a sub-target of a joint project, legal acts, requirements and recommendations were researched and analyzed. The results show substantive cornerstones of health promotion in various regulations of different disciplines. Based on these findings, health promotion can be interpreted as being part of services to the public. Currently the regulations for education, social tasks, environmental and consumer protection constitute the legal framework for community health promotion, but also include constitutions. They range from public international law to municipal resolutions. Quality management and also quality development are already an integral part in some communal departments. The management of structures, processes and results arises from commitments or measurable targets. In contrast, quality management for health promotion is not based on binding requirements. Specifications of other neighboring sectors (e. g. education, social sector) demonstrate the potential and effectiveness of legal policy guidelines, seen as a frame. A transparent communication about the current regulations is indispensable for formulating future guidelines. The German National Prevention Act opens opportunities for municipalities. However, its interpretation and local engagement will still guide the practice of communal health promotion.
Health and access to health care are considered a human right. "Regular" immigrants such as work migrants in Germany have the same entitlement to health care coverage through the statutory health insurance as the majority population. This, however, is not the case for refugees and asylum seekers. According to paragraphs 4 and 6 of the Asylum Seekers' Benefit Act, their entitlement is restricted to care for acute pain, pregnancy and childbirth, as well as immunizations in the first 15 months. Additional care can be financed on a case-by-case basis. Care provision is regulated in different ways by the communities; it is further complicated by different regulations at the federal state levels and by differences in knowledge of the physicians on how entitlement restrictions can be circumvented on a case-by-case basis. Thus, entitlement and access to care of asylum seekers and refugees is driven by chance in 3 respects: when they are assigned to a federal state, when they are assigned to a community, and when they are treated by a doctor with more or less knowledge of the legal regulations. Restrictions on entitlement to health care are associated with higher health care expenditure. They may also lead to delayed treatment of life-threatening conditions. Furthermore, they may negatively affect social integration of asylum seekers. Therefore, the restrictions of entitlement in paragraphs 4 and 6 of the Asylum Seekers' Benefit Act need to be lifted immediately. Asylum seekers should be granted the same entitlements to health care as the majority population in the whole of Germany.
Climate change poses major challenges for conservation and management because it alters the area, quality, and spatial distribution of habitat for natural populations. To assess species' vulnerability to climate change and target ongoing conservation investments, researchers and managers often consider the effects of projected changes in climate and land use on future habitat availability and quality and the uncertainty associated with these projections. Here, we draw on tools from hydrology and climate science to project the impact of climate change on the density of wetlands in the Prairie Pothole Region of the USA, a critical area for breeding waterfowl and other wetland-dependent species. We evaluate the potential for a trade-off in the value of conservation investments under current and future climatic conditions and consider the joint effects of climate and land use. We use an integrated set of hydrological and climatological projections that provide physically based measures of water balance under historical and projected future climatic conditions. In addition, we use historical projections derived from ten general circulation models (GCMs) as a baseline from which to assess climate change impacts, rather than historical climate data. This method isolates the impact of greenhouse gas emissions and ensures that modeling errors are incorporated into the baseline rather than attributed to climate change. Our work shows that, on average, densities of wetlands (here defined as wetland basins holding water) are projected to decline across the U.S. Prairie Pothole Region, but that GCMs differ in both the magnitude and the direction of projected impacts. However, we found little evidence for a shift in the locations expected to provide the highest wetland densities under current vs. projected climatic conditions. This result was robust to the inclusion of projected changes in land use under climate change. We suggest that targeting conservation towards wetland complexes containing both small and relatively large wetland basins, which is an ongoing conservation strategy, may also act to hedge against uncertainty in the effects of climate change.
Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.
Over the past decades, there has been a paradigm shift from a purely biomedical towards a bio-psycho-social (BPS) conception of disability and illness, which has led to a change in contemporary healthcare. However, there seems to be a gap between the rhetoric and reality of working within a BPS model. It is not clear whether healthcare professionals show the necessary skills and competencies to act according to the BPS model.
The aim of this study was (1) to develop a scale to monitor the BPS competencies of healthcare professionals, (2) to define its factor-structure, (3) to check internal consistency, (4) test-retest reliability and (5) feasibility.
Item derivation for the BPS scale was based on qualitative research with seven multidisciplinary focus groups (n = 58) of both patients and professionals. In a cross-sectional study design, 368 healthcare professionals completed the BPS scale through a digital platform. An exploratory factor analysis was performed to determine underlying dimensions. Statistical coherence was expressed in item-total correlations and in Cronbach's α coefficient. An intra-class-correlation coefficient was used to rate the test-retest reliability.
The qualitative study revealed 45 items. The exploratory factor analysis showed five underlying dimensions labelled as: (1) networking, (2) using the expertise of the client, (3) assessment and reporting, (4) professional knowledge and skills and (5) using the environment. The results show a good to strong homogeneity (item-total ranged from 0.59 to 0.79) and a strong internal consistency (Cronbach's α ranged from 0.75 to 0.82). ICC ranged between 0.82 and 0.93.
The BPS scale appeared to be a valid and reliable measure to rate the BPS competencies of the healthcare professionals and offers opportunities for an improvement in the healthcare delivery. Further research is necessary to test the construct validity and to detect whether the scale is responsive and able to detect changes over time.
Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan formed in the brain and in the periphery, known to block ionotropic glutamate receptors and α7 nicotinic receptors, and to act as a ligand of G protein-coupled GPR35 receptors and human aryl hydrocarbon (AHR) receptors. KYNA seems to modulate a number of mechanisms involved in the pathogenesis of schizophrenia including dopaminergic transmission in mesolimbic and mesocortical areas or glutamatemediated neurotransmission. The kynurenine hypothesis of schizophrenia links the occurrence of positive and negative symptoms of schizophrenia and cognitive impairments characteristic for the disease with the disturbances of kynurenine pathway function. Available data suggest that antipsychotic drugs may restore balance among kynurenine pathway metabolites, and that co-administration of glycine with antipsychotics may reduce extrapyramidal symptoms in patients suffering from schizophrenia. Central level of KYNA may increase in the course of inflammation, which is consistent with the inflammatory hypothesis of schizophrenia. Alterations of immune response and disturbed functioning of kynurenine pathway may lead to disproportion between neuroprotective and neurotoxic mechanisms in the brain. Currently, intense research efforts are focused on the role of kynurenine pathway metabolites in pathogenesis of schizophrenia, their association with the response to antipsychotic treatment, and search for novel medications modulating the function of kynurenine pathway.
A better understanding of how social and environmental contexts impact on childhood overweight/obesity is needed to develop more effective prevention strategies. Because the relationship between physical activity, stress and obesity measures has received limited attention, this study examined for the first time in first grade schoolchildren whether physical activity and fitness moderate the relationship between psychosocial stress and obesity-related measures.
325 children (51% girls, Mage=7.3 years) took part in this cross-sectional study. Stress (critical life events, family, peer, school-related stress) and vigorous physical activity were assessed via parental reports. Fitness was assessed with the 20m shuttle run test. BMI, sum of skinfolds and waist circumference were used as obesity-related outcomes. Hierarchical regression analyses were calculated to test whether fitness and physical activity act as stress-buffers, using sex, age and parental education as covariates.
Children experiencing elevated school-related stress had lower BMI, body fat and waist circumferences if they had high fitness and physical activity levels, as compared to their less active and fit peers. Few significant interaction effects occurred for the other stress measures, although the findings trended in a similar direction for peer stress.
This study shows that high fitness is associated with less unfavorable body composition among children with elevated school stress. Our findings indicate that policies aimed at reducing overweight and obesity should include the promotion of physical activity both inside and outside the school context. Moreover, our findings highlight the importance of strengthening children's capacities to cope successfully with school-related pressures.
In 2015, the US Congress passed legislation entitled the Medicare Access and CHIP [Children's Health Insurance Program] Reauthorization Act (MACRA), which led to the formation of two reimbursement paradigms: the merit-based incentive payment system (MIPS) and alternative payment models (APMs). The MACRA effectively repealed the Centers for Medicare and Medicaid Services (CMS) sustainable growth rate (SGR) formula while combining several CMS quality-reporting programs. As such, MACRA represents an unparalleled acceleration toward reimbursement models that recognize value rather than volume. The first pathway, MIPS, consolidates several Medicare quality-reporting programs into one composite score that will be derived by four performance categories, including quality (30%), resource utilization (30%), meaningful use (25%), and clinical practice improvement activities (15%). The APM pathway includes the following programs: Medicare accountable care organizations as part of the Medicare Shared Savings Program, Bundled Payments for Care Improvement, and Comprehensive Primary Care initiative. Existing APMs have yet to be determined as eligible. We provide a contextual framework of the healthcare legislation that has led to the formation of current health policy and offer recommendations regarding SGR how orthopaedic surgeons may best steer through such reimbursement models.
The risk and mechanism of chemotherapy-induced cardiotoxicity (CTX) vary depending on the type and intensity of the anticancer regimen. Myriad chemotherapeutic drugs produce adverse cardiovascular effects such as arterial hypertension, heart failure, and thromboembolic events. Among the numerous classes of these drugs, anthracyclines have been studied most extensively because of their overt cardiovascular effects and the high associated incidence of heart failure. However, CTX might also be caused by other types of chemotherapeutic agents, including alkylating agents (cyclophosphamide, ifosfamide), platinum agents, antimetabolites (5-fluorouracil, capecitabine), antibiotics (mitoxantrone, mitomycin, bleomycin), and antimicrotubule agents (taxanes). Here, we review the incidence, clinical impact, and potential mechanisms of CTX associated with nonanthracycline chemotherapy used for cancer patients. The published data support a marked increase in CTX risk, particularly with certain drugs such as 5-fluorouracil and cisplatin. Each anticancer regimen is associated with distinct modes of heart damage, both symptomatic and asymptomatic. However, the underlying mechanisms of CTX have been established only in a few cases, and only few nonanthracycline chemotherapeutics (mitoxantrone, mitomycin, ifosfamide) act through a recognizable mechanism and show a predictable dose dependence. Lastly, nonanthracycline chemotherapy can induce both chronic lesions, such as systolic dysfunction, and acute lesions, such as the ischemia that occurs within hours or days after treatment. An increased understanding of the incidence, mechanisms, and potential therapeutic targets of CTX induced by various nonanthracycline chemotherapeutic agents is clearly required.
Wildland fire fighting is a high-risk occupation requiring considerable physical and psychological demands. Multiple agencies publish fatality summaries for wildland fire fighters; however, the reported number and types vary. At least five different surveillance systems capture deaths, each with varying case definitions and case inclusion/exclusion criteria. Four are population-level systems and one is case-based. System differences create challenges to accurately characterize fatalities.
Data within each of the five surveillance systems were examined to better understand the types of wildland fire fighter data collected, to assess each system's utility in characterizing wildland fire fighter fatalities, and to determine each system's potential to inform prevention strategies. To describe similarities and differences in how data were recorded and characterized, wildland fire deaths for three of the population-based systems were matched and individual fatalities across systems were compared.
Between 2001 and 2012, 247 unique deaths were captured among the systems; 73% of these were captured in all three systems. Most common causes of death in all systems were associated with aviation, vehicles, medical events, and entrapments/burnovers. The data show that, although the three systems often report similar annual summary statistics, events captured in each system vary each year depending on the types of events that the system is designed to track, such as inclusion/exclusion of fatalities associated with the Hometown Heroes Survivor Benefits Act of 2003.
The overarching and central goal of each system is to collect accurate and timely information to improve wildland fire fighter safety and health. Each system is unique and has varying inclusion and exclusion criteria for capturing and tracking different subsets of wildland fire fighter tasks and duties. Use of a common case definition and better descriptions and interpretations of the data and the results would help to more accurately characterize wildland fire fighter traumatic injuries and illnesses, lessen the likelihood for misinterpretation of wildland fire fighter fatality data, and assist with defining the true occupational injury burden within this high-risk population.
Covalent organic frameworks (COFs) are an emerging class of porous crystalline polymers with broad potential applications. So far, the availability of three-dimensional (3D) COFs is limited and more importantly only one type of covalent bond has been successful used for 3D COF materials. Here, we report a new synthetic strategy based on dual linkages that leads to 3D COFs. The obtained 3D COFs show high specific surface areas and large gas uptake capacities, which makes them the top COF material for gas uptake. Furthermore, we demonstrate that the new 3D COFs comprise both acidic and basic sites, and act as excellent bifunctional catalysts for one-pot cascade reactions. The new synthetic strategy provides not only a general and versatile approach to synthesize 3D COFs with sophisticated structures but also expands the potential applications of this promising class of porous materials.
The studies described in this paper show that hydrocarbon oligomers are alternatives for low molecular weight alkane solvents. These oligomeric solvents are nontoxic, nonvolatile, and recyclable alternatives to heptane in thermomorphic solvent mixtures that use a polar solvent such as methanol, aqueous ethanol, or DMF or in biphasic mixtures that use acetonitrile. Regardless of which polar solvent is used, hydrocarbon oligomers like poly(α-olefin)s (PAOs) exhibit very low leaching into the polar phase. UV-visible spectroscopy studies show that these solvents have the solubility properties of heptane. For example, PAOs dissolve heptane soluble dyes and quantitatively separate them from polar phases in thermomorphic solvent mixtures. PAOs either as pure solvents or as additives in heptane act as antileaching agents, decreasing the already low leaching of such dyes into a polar phase in heptane/polar solvent mixtures. These oligomeric hydrocarbon solvents were also compared to heptane in studies of azo dye isomerization. The results show that thermal isomerization of an azo dye occurs at the same rate in heptane and a PAO. Further studies of carboxylic acid promoted dye isomerization in heptane and a PAO show that low molecular weight and oligomeric carboxylic acids are kinetically equivalent at accelerating this isomerization. The results suggest that these and other hydrocarbon oligomers behave as solvents like their low molecular weight nonpolar hydrocarbon solvents and that they can be substituted successfully for conventional solvents like heptane.
Although all current antipsychotics act by interfering with the action of dopamine at dopamine D2 receptors, two recent reports showed that 800 to 1000 mg of cannabidiol per day alleviated the signs and symptoms of schizophrenia, although cannabidiol is not known to act on dopamine receptors. Because these recent clinical findings may indicate an important exception to the general rule that all antipsychotics interfere with dopamine at dopamine D2 receptors, the present study examined whether cannabidiol acted directly on D2 receptors, using tritiated domperidone to label rat brain striatal D2 receptors. It was found that cannabidiol inhibited the binding of radio-domperidone with dissociation constants of 11 nm at dopamine D2High receptors and 2800 nm at dopamine D2Low receptors, in the same biphasic manner as a dopamine partial agonist antipsychotic drug such as aripiprazole. The clinical doses of cannabidiol are sufficient to occupy the functional D2High sites. it is concluded that the dopamine partial agonist action of cannabidiol may account for its clinical antipsychotic effects.
Glycosphingolipids (GSLs) are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand-receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL-protein interactions and their effects on cell signalling and development.
In this paper, the biosynthesis process of phenolic compounds in plants is summarized, which include the shikimate, pentose phosphate and phenylpropanoid pathways. Plant phenolic compounds can act as antioxidants, structural polymers (lignin), attractants (flavonoids and carotenoids), UV screens (flavonoids), signal compounds (salicylic acid, flavonoids) and defense response chemicals (tannins, phytoalexins). From a human physiological standpoint, phenolic compounds are vital in defense responses, such as anti-aging, anti-inflammatory, antioxidant and anti-proliferative activities. Therefore, it is beneficial to eat such plant foods that have a high antioxidant compound content, which will cut down the incidence of certain chronic diseases, for instance diabetes, cancers and cardiovascular diseases, through the management of oxidative stress. Furthermore, berries and other fruits with low-amylase and high-glucosidase inhibitory activities could be thought of as candidate food items in the control of the early stages of hyperglycemia associated with type 2 diabetes.
Tobacco smoke contains many potentially harmful compounds that may act differently and at different stages in breast cancer development. The focus of this work was to assess the possible role of cigarette smoking (status, dose, duration or age at initiation) and polymorphisms in genes coding for enzymes involved in tobacco carcinogen metabolism (CYP1A1, CYP2A6) or in DNA repair (XRCC1, APEX1, XRCC3 and XPD) in breast cancer development.
We designed a case control study with 297 patients, 217 histologically verified breast cancers (141 smokers and 76 non-smokers) and 80 healthy smokers in a cohort of Spanish women.
We found an association between smoking status and early age at diagnosis of breast cancer. Among smokers, invasive carcinoma subtype incidence increased with intensity and duration of smoking (all Ptrend < 0.05). When smokers were stratified by smoking duration, we only observed differences in long-term smokers, and the CYP1A1 Ile462Ile genotype was associated with increased risk of breast cancer (OR = 7.12 (1.98-25.59)).
Our results support the main effect of CYP1A1 in estrogenic metabolism rather than in tobacco carcinogen activation in breast cancer patients and also confirmed the hypothesis that CYP1A1 Ile462Val, in association with long periods of active smoking, could be a breast cancer risk factor.
The progressive titres of key monooxygenases and their requisite native donors of reducing power were used to assess the relative contribution of various camphor plasmid (CAM plasmid)- and chromosome-coded activities to biodegradation of (rac)-camphor at successive stages throughout growth of Pseudomonas putida NCIMB 10007 on the bicylic monoterpenoid. A number of different flavin reductases (FRs) have the potential to supply reduced flavin mononucleotide to both 2,5- and 3,6-diketocamphane monooxygenase, the key isoenzymic two-component monooxygenases that delineate respectively the (+)- and (-)-camphor branches of the convergent degradation pathway. Two different constitutive chromosome-coded ferric reductases able to act as FRs can serve such as role throughout all stages of camphor-dependent growth, whereas Fred, a chromosome-coded inducible FR can only play a potentially significant role in the relatively late stages. Putidaredoxin reductase, an inducible CAM plasmid-coded flavoprotein that serves an established role as a redox intermediate for plasmid-coded cytochrome P450 monooxygenase also has the potential to serve as an important FR for both diketocamphane monooxygenases (DKCMOs) throughout most stages of camphor-dependent growth.
The benefits of dairy consumption seem to extend beyond its significant contribution to ensuring nutrient intake adequacy as indicated by the favourable associations with several health outcomes reported by different studies. The aims of the present study were to examine the associations of milk consumption with fitness, anthropometric and biochemical indices in children and further explore whether the observed associations are attributed to vitamins B₂ and B12 derived from milk. A representative subsample of 600 children aged 9-13 years participating in the Healthy Growth Study was examined. Data were collected on children's dietary intake, using 24 h recalls, as well as on fitness, anthropometric and biochemical indices. Regression analyses were performed for investigating the research hypothesis, adjusting for potential confounders and for B-vitamin status indices (i.e., plasma riboflavin, methylmalonic acid and total homocysteine concentrations), dietary calcium intake and plasma zinc concentrations that could possibly act as effect modifiers. Milk consumption was positively associated with the number of stages performed in the endurance run test (ERT) (β = 0.10; p = 0.017) and negatively with body mass index (BMI) (β = -0.10; p = 0.014), after adjusting for several potential confounders and effect modifiers. Dietary intakes of vitamin B₂ and B12 derived from milk were also positively associated with the number of ERT stages (β = 0.10; p = 0.015 and β = 0.10; p = 0.014 respectively). In conclusion, higher intake of milk as well as vitamin B₂ and B12 derived from milk were independently associated with higher cardiorespiratory fitness in Greek preadolescents. The key roles of these B-vitamins in substrate oxidation, energy production, haemoglobin synthesis and erythropoiesis could provide a basis for interpreting these associations. However, further research is needed to confirm this potential interpretation.
Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.
The finding that small non-coding RNAs (ncRNAs) are able to control gene expression in a sequence specific manner has had a massive impact on biology. Recent improvements in high throughput sequencing and computational prediction methods have allowed the discovery and classification of several types of ncRNAs. Based on their precursor structures, biogenesis pathways and modes of action, ncRNAs are classified as small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs), promoter associate RNAs (pRNAs), small nucleolar RNAs (snoRNAs) and sno-derived RNAs. Among these, miRNAs appear as important cytoplasmic regulators of gene expression. miRNAs act as post-transcriptional regulators of their messenger RNA (mRNA) targets via mRNA degradation and/or translational repression. However, it is becoming evident that miRNAs also have specific nuclear functions. Among these, the most studied and debated activity is the miRNA-guided transcriptional control of gene expression. Although available data detail quite precisely the effectors of this activity, the mechanisms by which miRNAs identify their gene targets to control transcription are still a matter of debate. Here, we focus on nuclear functions of miRNAs and on alternative mechanisms of target recognition, at the promoter lavel, by miRNAs in carrying out transcriptional gene silencing.
Excessive sodium intake is associated with both hypertension and an increased risk of cardiovascular events, presumably because of an increase in extracellular volume. The extent to which sodium intake affects extracellular volume and BP varies considerably among individuals, discriminating subjects who are salt-sensitive from those who are salt-resistant. Recent experiments have shown that, other than regulation by the kidney, sodium homeostasis is also regulated by negatively charged glycosaminoglycans in the skin interstitium, where sodium is bound to glycosaminoglycans without commensurate effects on extracellular volume. The endothelial surface layer is a dynamic layer on the luminal side of the endothelium that is in continuous exchange with flowing blood. Because negatively charged glycosaminoglycans are abundantly present in this layer, it may act as an intravascular buffer compartment that allows sodium to be transiently stored (figure 1). In my presentation, I will focus on the putative role of the endothelial surface layer as a contributor to salt sensitivity, the consequences of a perturbed endothelial surface layer on sodium homeostasis, and the endothelial surface layer as a possible target for the treatment of hypertension and an expanded extracellular volume.(Figure is included in full-text article.).
Pulmonary arterial hypertension (PAH) is a life-threatening disorder with a poor prognosis and causes pulmonary vascular remodeling accompanied with increased pulmonary arterial medial wall thickness and fibrosis, which leads to vascular and right ventricular (RV) dysfunction. Despite treatment with prostacyclin, endothelin antagonist, and phosphodiesterase-5 inhibitors the 1-year mortality rate of PAH still remains high. Recent registries, clinical trials, and basic researches have been increasing the knowledge of PAH and it would contribute to potential therapeutic strategies and better clinical outcome.Korean Registry of Pulmonary Arterial Hypertension (KORPAH) is the first modern PAH registries in Asian ethnicity. Total 39 centers participated and 625 patients were enrolled. This study evaluated the incidence, prevalence, epidemiology, therapeutic modalities and survival data of Korean patients with PAH."Gachon experiences" was to characterize the clinical outcomes and evaluate the factors influencing survival time of the PAH patients in Korean. This study compared the cumulative survival of total 43 PAH patients who received targeted or conventional therapy.PAH Ilopost BMPR-2 gene in Korea IIT Multi-institutional (PILGRIM) is a prospective, investigator-initiative, and multi-institutional clinical trials. This study was recently completed in March by 7 institutes, and aimed to investigate (1) the prevalence of BMPR-2 gene mutations in the Korean PAH patients and (2) the effect of iloprost inhalation solution on hemodynamic response, and exercise echocardiography.PAH basic research focuses on two major themes: (1) Systematic comparison of the effects of adipose tissue, bone marrow and umbilical cord blood-derived mesenchymal stem cell transplantation on MCT-induced PAH in rats and (2) investigation of the effect of human UCB-derived MSC (hUCB-MSC) transplantation combined with apelin-13 administration on MCT-induced PAH in rats. Data suggests that, although the survival and therapeutic potential of all three MSCs were similar with regards to the attenuation of MCT-induced PAH progression in rats, each MSCs may act differently via distinct molecular mechanisms. Transplantation of hUCB-MSCs combined with apelin-13 administration improved the survival of transplanted cells in PAH rat lungs, and also improved the ability of hUCB-MSCs to protect from RV dysfunction and medial wall thickening.
Histone deacetylases (HDACs) act as co-repressors in gene transcription by erasing the acetylation of histones, resulting in epigenetic gene silencing. Recent studies revealed that HDAC inhibitors attenuated blood pressure of several hypertensive animal models such as spontaneously hypertensive rats, hyperaldosteronism rats, angiotensin II-induced hypertensive rats and pulmonary hypertensive rats. Unexpectedly, microarray studies uncovered that administration of HDAC inhibitors decreased expression of some genes for example extracellular matrix proteins, oxidative stress-related proteins, cytokines, chemokines and ion transporters, mostly targets of corticoid receptors. Corticoid-induced hypertensive animal model was established by infusion of adrenocorticotropic hormone (40 ng/kg/day), deoxycorticosterone acetate (40 mg/kg), and dexamethasone (100 μg/kg/day) with osmotic mini-pump. Valproic acid (VPA), a class I and IIa HDAC inhibitor administration significantly decreased corticoid-induced hypertension. VPA administration increased acetylation level of Mineralocorticoid receptor (MR), which was closely related with decreased binding affinity with hormone response element in the promoters of target genes such as glucocorticoid induced leucine zipper (Gilz) and serum glucocorticoid regulated kinase-1 (Sgk-1). HDAC3 and HDAC4 were interacted with MR after stimulation of aldosterone (10 nmol/L) for 30 min. HDAC4 inhibitor showed no effect on acetylation level and promoter binding affinity of MR. HDAC4 played a role as a scaffold between MR and HDAC3. In conclusion, HDAC inhibitors increased acetylation of corticoid receptor, resulted in decreased transcriptional activity of it and blocked corticoid induced-hypertension.
There is now a considerable body of evidence to suggest that the fat cells that surround blood vessels (perivascular adipose tissue, PVAT) can influence profoundly arterial tone by releasing vasodilator adipokines which can act locally in a paracrine fashion. In healthy lean individuals the primary vasodilator released appears to be adiponectin and there is a complex interation between autonomic nerve firing in PVAT and the release of nitric oxide from adipocytes and an increased bioavailability of adiponectin. However the vasodilators that are released appear to be agonist dependent and adiponectin is certainly increased as a result of sympathomimetic stimulation. Primarily our work has focused on the role of sympathetic nerves running through PVAT and innovating adipocytes and releasing adiponectin. Electrical field stimulation studies have demonstrated clearly the release of adiponectin and the induction of relaxation which is abolished using 6-hydroxydopamine or tetrodotoxin to destroy the autonomic nerve fibres. In obesity there is evidence of beta-3 receptors on adipocytes and the loss of vasodilator activity which is clearly observed. In some models of obesity there is also evidence of an increase in vasoconstrictor prostaglandin bioavailability and increased contractility. This loss of balance between vasodilatation and vasoconstriction will lead to an increase in peripheral resistance which will decrease glucose uptake in skeletal muscle and bring about a rise in blood pressure: key components of the metabolic syndrome. Caloric restriction or weight reducing surgery can restore completely normal PVAT structure and function and this is seen even when individuals remain morbidly obese.
Hypertensive patients have greater chances of such cardiovascular events as stroke, coronary heart disease, heart or renal failure, peripheral artery disease, and dementia. It is also well recognized that diabetes increases the cardiovascular risks in concert with hypertension. Therefore, main goals for an innovation of anti-hypertensive therapy would be to achieve further risk reduction by targeting the functional, metabolic, and structural alterations associated with hypertension. Professors Dzau and Braunwald et al proposed the concept of "the cardiovascular disease continuum" in 1991, and that hypertension may trigger the chain of events, leading to end-stage heart disease; however, this concept was quite new at that time, and there was some discussion whether "the cardiovascular disease continuum" is true or not. Fifteen years later, accumulating clinical and basic research evidence confirmed the validity of the concept of this cardiovascular disease continuum. Oxidative stress, and inflammation play a role in the initiation and continuation of this chain. Therefore, targeting oxidative stress and inflammation are important to interrupt the cardiovascular disease continuum. However, so far, neither anti-oxidative nor anti-inflammation strategy can work well to prevent hypertension and its related cardiovascular diseases. Low-grade inflammation has been proposed to play a key role in the pathogenesis of hypertension, and both innate and adaptive immune responses may participate in this process. Recent evidence also defined important roles of T-cell and T-cell-derived cytokines associated with angiotensin (Ang) II and catecholamine. We expect these new findings could provide us with novel avenues to beat hypertension, in terms of anti-oxidative stress and/or anti-inflammation.Renin-angiotensin-aldosterone system (RAAS) plays a significant role in the cardiovascular disease continuum interacting with adrenergic system and other various mediators, and thereby RAAS mediates adaptive and maladaptive responses to tissue injury. Sympathetic hyperactivity and the activation of RAAS may promote hypertension or work as an amplifier of the pressor influence of other factors such as metabolic factors. Therefore, RAAS and sympathetic nervous system are the major targets of treatment of hypertension and its related cardiovascular complications. The two main interventional approaches, transcatheter renal denervation and baroreflex activation therapy, have been used in clinical practice for treatment of resistant hypertension and the renal denervation is also being evaluated for treatment of various comorbidities, although the efficacy of invasive sympatho-deactivating interventions have not yet been conclusively validated. In the past two decades, the development of the drugs for the treatment of hypertension and cardiovascular diseases has been largely focused on the inhibitors of RAAS, including immunization against Ang II.Novel pathways beyond the classical actions of RAAS, the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 (AT1) receptor axis, have been highlighted: the ACE2/Ang-(1-7)/Mas receptor axis and Ang II type 2 (AT2) receptor as a new opposing axis against the classical RAS axis as a protective arm of RAAS. Moreover, identification of alamandine and its receptor, Mas-related G-protein coupled receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS. Further elucidation of the regulatory mechanisms of the functions of new protective arm of RAS beyond the classical RAAS could provide us with possibilities for the development of novel drugs that regulate RAAS in a more sophisticated manner, thereby treating hypertensive patients and achieving cardiovascular risk reduction more efficiently. Agonists of protective arm of RAS have been developed as new drugs for hypertension and several drugs of the agonists of protective arm of RAS are now in clinical trials. Several novel Ang II receptor interacting proteins have been also reported. AT1 receptor-associated protein (ATRAP) was cloned by us as specific binding protein of AT1 receptor C-terminal, and we and others reported that ATRAP could act as a negative regulator in AT1 receptor-mediated effects at least in part by the enhancement of AT1 receptor internalization. We cloned AT2 receptor interacting protein (ATIP) as a protein interacting specifically with the C-terminal tail of the AT2 receptor. We and others demonstrated that ATIP enhanced an important role of AT2 receptor-mediated wide variety of pathophysiological functions. Further elucidation of the functional regulation of these Ang II receptor associated proteins including their transcriptional control, and finding possible ligands could be helpful for new drug developments.I will review and discuss in this symposium "Progression of Hypertensive Heart Disease" focusing on the new therapeutic pharmacological approach with recent clinical evidences.
Non-invasive ventilation (NIV) is increasingly used in patients with Acute Respiratory Distress Syndrome (ARDS). Whether, during NIV, the categorization of ARDS severity based on the PaO2/FiO2 Berlin criteria is useful is unknown. The evidence supporting NIV use in patients with ARDS remains relatively sparse.
The Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study described the management of patients with ARDS. This sub-study examines the current practice of NIV use in ARDS, the utility of the PaO2/FiO2 ratio in classifying patients receiving NIV and the impact of NIV on outcome.
Of 2,813 patients with ARDS, 436 (15.5%) were managed with NIV on days 1 and 2 following fulfillment of diagnostic criteria. Classification of ARDS severity based on PaO2/FiO2 ratio was associated with an increase in intensity of ventilatory support, NIV failure, and Intensive Care Unit (ICU) mortality. NIV failure occurred in 22.2% of mild, 42.3% of moderate and 47.1% of patients with severe ARDS. Hospital mortality in patients with NIV success and failure was 16.1 % and 45.4%, respectively. NIV use was independently associated with increased ICU (HR 1.446; [1.159-1.805]), but not hospital mortality. In a propensity matched analysis, ICU mortality was higher in NIV than invasively ventilated patients with a PaO2/FiO2 lower than 150 mmHg.
NIV was used in 15% of patients with ARDS, irrespective of severity category. NIV appears to be associated with higher ICU mortality in patients with a PaO2/FiO2 lower than 150 mmHg. Clinical trial registration available at www.clinicaltrials.gov, ID NCT02010073.
Helicoverpa armigera is a major pest of agriculture, horticulture and floriculture throughout the Old World and recently invaded parts of the New World. We overview of the evolution in thinking about the application of area-wide approaches to assist with its control by the Australian Cotton Industry to highlight important lessons and future challenges to achieving the same in the New World. An over-reliance of broad-spectrum insecticides led to Helicoverpa spp. in Australian cotton rapidly became resistant to DDT, synthetic pyrethroids, organophosphates, carbamates and endosulfan. Voluntary strategies were developed to slow the development of insecticide resistance, which included rotating chemistries and basing spray decisions on thresholds. Despite adoption of these practices, insecticide resistance continued to develop until the introduction of genetically modified cotton provided a platform for augmenting Integrated Pest Management in the Australian cotton industry. Compliance with mandatory resistance management plans for Bt cotton necessitated a shift from pest control at the level of individual fields or farms towards a coordinated area-wide landscape approach. Our take-home message for control of H. armigera is that resistance management is essential in genetically modified crops and must be season long and area-wide to be effective. © 2016 Society of Chemical Industry.
Lecturers often present entertaining videos, or organize a variety of amusing demonstrations, to foster student engagement or to encourage critical analysis. Magic tricks, in particular, have been shown to activate neural circuits that underpin motivation or problem-solving and, therefore, could be beneficial during lectures. Nevertheless, we hypothesize that, unless the method that underpins these tricks is revealed, students may ruminate over possible explanations, distracting attention from the lecture material.
To test these arguments, in this study, 224 participants watched a video of a magic performance, watched a video of a circus act, or watched no video at all. In half the participants who watched the magic performance, the secret that underpinned the trick was disclosed. Next, participants watched a psychology tutorial, before answering questions that assessed engagement, need for cognition, and comprehension of the material.
If the secret was withheld, magic tricks diminished subsequent need for cognition but did not affect comprehension. Furthermore, magic tricks tended to diminish engagement with the subsequent tutorial. These effects, however, were small.
Future research is warranted to ascertain whether information that is embedded within a magic trick, rather than presented after the trick, is more likely to be remembered or understood later. This research could clarify when performance can enhance or disrupt student engagement.
Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells. To characterize the RNA-content of the blood plasma sub fractions, we performed high throughput sequencing of the RNA pelleted within vesicles at 16,000 g and 160,000 g as well as RNA remaining in the vesicle-free supernatant. We found that blood plasma sub fractions contain not only extensive set of microRNAs but also fragments of other cellular RNAs: rRNAs, tRNAs, mRNAs, lncRNAs, small RNAs including RNAs encoded by mtDNAs. Our data indicate that a variety of blood plasma RNAs circulating within vesicles as well as of extra-vesicular RNAs are comparable to the variety of cellular RNA species.
The National Institute for Health and Care Excellence (NICE) invited the manufacturer of azacitidine (Celgene) to submit evidence for the clinical and cost effectiveness of this drug for the treatment of acute myeloid leukaemia with more than 30 % bone marrow blasts in adults who are not eligible for haematopoietic stem cell transplantation, as part of the NICE's Single Technology Appraisal process. The Peninsula Technology Assessment Group was commissioned to act as the Evidence Review Group (ERG). The ERG produced a critical review of the evidence contained within the company's submission to NICE. The clinical effectiveness data used in the company's economic analysis were derived from a single randomised controlled trial, AZA-AML-001. It was an international, multicentre, controlled, phase III study with an open-label, parallel-group design conducted to determine the efficacy and safety of azacitidine against a conventional care regimen (CCR). The CCR was a composite comparator of acute myeloid leukaemia treatments currently available in the National Health Service: intensive chemotherapy followed by best supportive care (BSC) upon disease relapse or progression, non-intensive chemotherapy followed by BSC and BSC only. In AZA-AML-001, the primary endpoint was overall survival. Azacitidine appeared to be superior to the CCR, with median overall survival of 10.4 and 6.5 months, respectively. However, in the intention-to-treat analysis, the survival advantage associated with azacitidine was not statistically significant. The company submitted a de novo economic evaluation based on a partitioned survival model with four health states: "Remission", "Non-remission", "Relapse/Progressive disease" and "Death". The model time horizon was 10 years. The perspective was the National Health Service and Personal Social Services. Costs and health effects were discounted at the rate of 3.5 % per year. The base-case incremental cost-effectiveness ratio (ICER) of azacitidine compared with the CCR was £20,648 per quality-adjusted life-year (QALY) gained. In the probabilistic sensitivity analysis, the mean ICER was £17,423 per QALY. At the willingness-to-pay of £20,000, £30,000 and £50,000 per QALY, the probability of azacitidine being cost effective was 0.699, 0.908 and 0.996, respectively. The ERG identified a number of errors in Celgene's model and concluded that the results of the company's economic evaluation could not be considered robust. After amendments to Celgene's model, the base-case ICER was £273,308 per QALY gained. In the probabilistic sensitivity analysis, the mean ICER was £277,123 per QALY. At a willingness-to-pay of £100,000 per QALY, the probability of azacitidine being cost effective was less than 5 %. In all exploratory analyses conducted by the ERG, the ICER exceeded the NICE's cost-effectiveness threshold range of £20,000-30,000 per QALY. Given the evidence provided in the submission, azacitidine did not fulfil NICE's end-of-life criteria. After considering the analyses performed by the ERG and submissions from clinician and patient experts, the NICE Appraisal Committee did not recommend azacitidine for this indication.
The health care system of the United States has been in a period of dramatic transformation since the passage of the Affordable Care Act in 2010, and the rate of change is accelerating. Historically, health care delivery was focused on the efforts of independent individual providers related to single patients, but the future will require interprofessional teamwork to achieve successful transformation. Academic health centers must identify nimble leaders who can harness the expertise of every team member to succeed in yielding the triple aim-better care for individuals, better health for populations, and lower overall cost. To create this change, there are several critical success factors for academic health center leaders, including creating a culture of collaboration, becoming "multipliers," embracing innovation, adhering to core professional ethics, and working to promote resilience. Given their extensive training and predisposition to these skills, psychologists are well-positioned to serve as leaders in today's academic health systems.
In this study, nanocomposites of Fe-doped TiO2 with multi-walled carbon nanotubes (0.1- 0.5 wt. %) were prepared by using sol-gel method. The structural and morphological analysis were carried out with using X-ray diffraction pattern and transmission electron microscopy, which confirm the presence of pure anatase phase and particle sizes in the range 15-20 nm. X-ray photoelectron spectroscopy was used to determine the surface compositions of the nanocomposites. UV-vis diffuse reflectance spectra confirm redshift in the optical absorption edge of nanocomposites with increasing amount of multi-walled carbon nanotubes. Nanocomposites show photoinactivation against gram-positive Bacillus subtilis as well as gram-negative Pseudomonas aeruginosa. Fe-TiO2-multi-walled carbon nanotubes (0.5 wt. %) nanocomposites show higher photoinactivation capability as compared with other nanocomposites. The photoluminescence study reveals that the Fe-TiO2-multi-walled carbon nanotubes nanocomposites are capable to generate higher rate of reactive oxygen species species than that of other nanocomposites. Our experimental results demonstrated that the Fe-TiO2-multi-walled carbon nanotubes nanocomposites act as efficient antibacterial agents against a wide range of microorganisms to prevent and control the persistence and spreading of bacterial infections.
Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, has been studied extensively in carcinogenesis through the genomic pathway. In recent years, AHR has also been reported to exert positive or negative effects on epithelial-mesenchymal transition (EMT), the crucial step in tumor malignant progression. However, the detailed mechanism remains controversial. Analysis of AHR-expression levels in non-small cell lung cancer cell lines and lung cancer tissues revealed an inverse correlation between AHR protein levels and tumor cell invasion and metastasis. Overexpression of wild-type AHR in H1299 cells (AHR poorly expressed, potently invasive) not only accelerated mesenchymal vimentin degradation, but also prevented cell invasion in vitro and in vivo. In the absence of AHR agonists, the overexpressed AHR protein was predominantly localized in the cytoplasm, where it interacted with vimentin and functioned as an E3 ubiquitin ligase. A 6-h incubation with the proteasome inhibitor MG-132 fully rescued vimentin from AHR-mediated proteasomal degradation. In AHR-overexpressing H1299 cells, either vimentin degradation or invasive suppression could be reversed when glycogen synthase kinase 3 beta (GSK3β) was inactivated by CHIR-99021 treatment. In contrast, silencing of AHR in A549 cells (AHR highly expressed, weakly invasive) resulted in the downregulation of epithelial biomarkers (E-cadherin and claudin-1), augmentation of mesenchymal vimentin level, and GSK3β Ser-9 hyper-phosphorylation, which led to enhanced invasiveness. This work demonstrates that cytoplasmic, resting AHR protein may act as an EMT suppressor via a non-genomic pathway. Depletion of cytoplasmic AHR content represents a potential switch for EMT, thereby leading to the scattering of tumor cells.
Medical reports are required to support court applications to appoint a deputy to make decisions on behalf of a person who has lost mental capacity. The doctor writing such a medical report needs to be able to systematically assess the mental capacity of the person in question, in order to gather the necessary evidence for the Court to make a decision. If the medical report is not adequate, the application will be rejected and the appointment of the deputy delayed. This article sets out the best practices for performing the assessment, and the common errors, issues of concern and best practices in writing the medical report.
Aims and method The need for an age-appropriate in-patient service for 16- to 17-year-olds led to the development of a 6-bed acute admissions unit in a non-metropolitan county in the UK. We provide a descriptive evaluation of the first 2 years of its operation. All admissions from April 2010 to March 2012 were reviewed, clinical details systematically recorded and descriptively analysed. Results Ninety-seven young people were admitted during this period (a third were compulsorily detained under the Mental Health Act 1983). The average length of stay was 3-4 weeks. The most common presenting complaints were self-harm and low mood, usually in the context of life events and childhood adversity. Nearly half had substance misuse and other risk-taking behaviours. A third presented with psychotic symptoms. Adjustment and anxiety disorders were most common, followed by alcohol/substance use disorders, depressive illnesses and psychotic illnesses. Comorbidity was the rule rather than the exception. Most patients improved by the time of discharge. Clinical implications The unit provides an accessible and effective age-appropriate service and is likely to constitute an important component of the comprehensive child and adolescent mental health service strategy in the county.
Over the last decade, patient volumes in the emergency department (ED) have grown disproportionately compared to the increase in staffing and resources at the Toronto Western Hospital, an academic tertiary care centre in Toronto, Canada. The resultant congestion has spilled over to the ED waiting room, where medically undifferentiated and potentially unstable patients must wait until a bed becomes available. The aim of this quality improvement project was to decrease the 90th percentile of wait time between triage and bed assignment (time-to-bed) by half, from 120 to 60 minutes, for our highest acuity patients. We engaged key stakeholders to identify barriers and potential strategies to achieve optimal flow of patients into the ED. We first identified multiple flow-interrupting challenges, including operational bottlenecks and cultural issues. We then generated change ideas to address two main underlying causes of ED congestion: unnecessary patient utilization of ED beds and communication breakdown causing bed turnaround delays. We subsequently performed seven tests of change through sequential plan-do-study-act (PDSA) cycles. The most significant gains were made by improving communication strategies: small gains were achieved through the optimization of in-house digital information management systems, while significant improvements were achieved through the implementation of a low-tech direct contact mechanism (a two-way radio or walkie-talkie). In the post-intervention phase, time-to-bed for the 90th percentile of high-acuity patients decreased from 120 minutes to 66 minutes, with special cause variation showing a significant shift in the weekly measurements.
Frequent, preventable medical errors can have an adverse effect on patient safety and quality as well as leading to wasted resources. In the laboratory, errors can occur at any stage of sample processing; pre-analytical, analytical, and post analytical stages. However evidence shows most of the laboratory errors occur during the pre-analytical stage. The receipt and processing of specimens is one of the main steps in the pre-analytical stage. Errors in this stage could be due to mislabeling, incorrect test entry and entering the wrong location, among other reasons. Most of these errors are preventable. At the Riyadh Regional Laboratory of the Ministry of Health, we found that there was an average of 2.31 errors per 1000 processed samples; these errors had occurred during the pre-analytical stage. These samples were returned back from other laboratory departments, such as Chemistry, Hematology and Microbiology, to the receiving and processing department. We decided to carry out an improvement project where we applied a systematic approach to identify and analyse the root causes of the problem using quality tools such as a process flowchart and a fish-bone diagram. The Model for Improvement was used and several PDSA (Plan, Do, Study, Act) cycles were run to test interventions which aimed to prevent laboratory processing errors and mistakes. The project results showed a 25% reduction in errors during the pre-analytical stage.
The Health Information Technology for Economic and Clinical Health (HITECH) Act encourages health information exchange between clinical care and public health through Meaningful Use measures. Meaningful Use specifically identifies objectives to support a number of public health programs including immunizations, cancer registries, syndromic surveillance, and disease case reports. The objective is to improve public and population health. Stage 2 of Meaningful Use focused on compliance to sending of information to public health. The next phase focuses on bi-directional information exchange to support immunization intelligence and to empower providers, pharmacists, and the consumer. The HITECH Act Stage 2 initiative provided incentive and motivation for healthcare providers to encourage their Electronic Medical Record (EMR) vendors to implement data exchanges with public health, with the expected result being timely awareness of health risks. The empowerment nugget in the HITECH Act is not in the compliance reporting to public health. The nugget is the ability for a provider to receive relevant information on the patient or consumer currently in front of them or to those they will connect to through their outreach efforts. The ability for public health to retain current immunization records of individuals from a variety of providers supports their program goals to increase immunization rates and mitigate the risk of vaccine-preventable disease (VPD). The ability for providers to receive at the point of service more complete immunization histories integrated with decision support enhances their delivery of care, thereby reducing the risk of VPD to their patients. Indirectly payers benefit through healthcare cost savings and when the focus is expanded from a health model to a business model, there are significant return on investment (ROI) opportunities that exponentially increase the value of a bi-directional immunization data exchange. This paper will provide descriptions of case examples to demonstrate the value of electronic data exchanges when pharmacy immunization providers and public health work together.
Antimicrobial peptides (AMPs) are a wide-ranging class of host-defense molecules that act early to contest against microbial invasion and challenge. These are small cationic peptides that play an important in the development of innate immunity. In the oral cavity, the AMPs are produced by the salivary glands and the oral epithelium and serve defensive purposes. The aim of this review was to discuss the types and functions of oral AMPs and their role in combating microorganisms and infections in the oral cavity.
The glycopeptide antibiotics are peptide-based natural products with impressive antibiotic function that derives from their unique three-dimensional structure. Biosynthesis of the glycopeptide antibiotics centres of the combination of peptide synthesis, mediated by a non-ribosomal peptide synthetase, and the crosslinking of aromatic side chains of the peptide, mediated by the action of a cascade of Cytochrome P450s. Here, we report the first example of in vitro activity of OxyE, which catalyses the F-O-G ring formation reaction in teicoplanin biosynthesis. OxyE was found to only act after an initial C-O-D crosslink is installed by OxyB and to require an interaction with the unique NRPS domain from glycopeptide antibiotic - the X-domain - in order to display catalytic activity. We could demonstrate that OxyE displays limited stereoselectivity for the peptide, which mirrors the results from OxyB-catalysed turnover and is in sharp contrast to OxyA. Furthermore, we show that activity of a three-enzyme cascade (OxyB/OxyA/OxyE) in generating tricyclic glycopeptide antibiotic peptides depends upon the order of addition of the OxyA and OxyE enzymes to the reaction. This work demonstrates that complex enzymatic cascades from glycopeptide antibiotic biosynthesis can be reconstituted in vitro and provides new insights into the biosynthesis of these important antibiotics.
We previously reported that the human HER2 gene encodes the intronic microRNA mir-4728, which is overexpressed together with its oncogenic host gene and may act independently of the HER2 receptor. More recently, we also reported that the oncogenic miR-21-5p is regulated by 3' tailing and trimming by the non-canonical poly(A) polymerase PAPD5 and the ribonuclease PARN. Here we demonstrate a dual function for the HER2 locus in upregulation of miR-21-5p; while HER2 signalling activates transcription of mir-21, miR-4728-3p specifically stabilises miR-21-5p through inhibition of PAPD5. Our results establish a new and unexpected oncogenic role for the HER2 locus that is not currently being targeted by any anti-HER2 therapy.
Aqueous miscible organic layered double hydroxides (AMO-LDHs) can act as organophilic inorganic flame retardant nanofillers for unmodified non-polar polymers. In this contribution, AMO [Mg3Al(OH)8](CO3)0.5·yH2O LDH-oxidized carbon nanotube (AMO-LDH-OCNT) hybrids are shown to perform better than the equivalent pure AMO-LDH. A synergistic effect between the AMO-LDH and OCNT was observed; this endows the hybrid material with enhanced flame retardancy, thermal stability, and mechanical properties. The thermal stability of polypropylene (PP) was significantly enhanced by adding AMO-LDH-OCNT hybrids. For PP mixed with AMO-LDH-OCNT hybrids to produce a composite with 10 wt% LDH and 2 wt% OCNT, the 50% weight loss temperature was increased by 43 °C. Further, a system with 10 wt% of AMO-LDH and 1 wt% OCNT showed a peak heat release rate (PHRR) reduction of 40%, greater than the PHRR reduction with PP/20 wt% AMO-LDH (31%). The degree of dispersion (mixability) between AMO-LDH and OCNT has a significant effect on the flame retardant performance of the hybrids. In addition, the incorporation of AMO-LDH-OCNT hybrids led to better mechanical properties, such as higher tensile strength (27.5 MPa) and elongation at break (17.9%), than those composites containing only AMO-LDH (25.6 MPa and 7.5%, respectively).
Amyloid-β oligomers (AβO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AβO-mediated mouse model of AD, we explored whether the memory alteration AβOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved.
C57 naïve mice were given a single intracerebroventricular injection of synthetic AβO-containing solution (1μM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1β receptor antagonist) to prevent impairment in the novel object recognition task, and compared AβO's effects in TLR4 knockout mice.
A single AβO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AβO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AβO's action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation.
These data provide new information on AβO's mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.
Chromium allergy is a common occupational skin disease mediated by chromium (VI)-specific T cells that induce delayed-type hypersensitivity in sensitized individuals. Additionally, chromium (VI) can act as an irritant. Both responses critically require innate immune activation, but if and how chromium (VI) elicits this signal is currently unclear. Using human monocytes, primary human keratinocytes, and murine dendritic cells we show that chromium (VI) compounds fail to trigger direct proinflammatory activation but potently induce processing and secretion of IL-1β. IL-1β release required priming by phorbol-ester or toll-like receptor stimulation and was prevented by inhibition of K(+) efflux, NLRP3 depletion or caspase-1 inhibition, identifying chromium (VI) as a hapten activator of the NLRP3 inflammasome. Inflammasome activation was initiated by mitochondrial reactive oxygen species production triggered by chromium (VI), as indicated by sensitivity to treatment with the ROS scavenger N-acetyl cysteine and a coinciding failure of K(+) efflux, caspase-1, or NLRP3 inhibition to prevent mitochondrial reactive oxygen species accumulation. IL-1β release further correlated with cytotoxicity that was secondary to reactive oxygen species, K(+) efflux, and NLRP3 activation. Trivalent chromium was unable to induce mitochondrial reactive oxygen species production, inflammasome activation, and cytotoxicity, suggesting that oxidation state-specific differences in mitochondrial reactivity may determine inflammasome activation and allergic/irritant capacity of different chromium compounds.
The mechanism by which anesthetics might act on the developing brain in order to cause long term deficits remains incompletely understood. The hippocampus has been identified as a structure that is likely to be involved, as rodent models show numerous deficits in behavioral tasks of learning that are hippocampal-dependent. The hippocampus is an unusual structure in that it is the site of large amounts of neurogenesis postnatally, particularly in the first year of life in humans, and these newly generated neurons are critical to the function of this structure. Intriguingly, neurogenesis is a major developmental event that occurs during postulated windows of vulnerability to developmental anesthetic neurotoxicity across the different species in which it has been studied. In this review, we examine the evidence for anesthetic effects on neurogenesis in the early postnatal period and ask whether neurogenesis should be studied further as a putative mechanism of injury. Multiple anesthetics are considered, and both in vivo and in vitro work is presented. While there is abundant evidence that anesthetics act to suppress neurogenesis at several different phases, evidence of a causal link between these effects and any change in learning behavior remains elusive.
Biosensors can deliver the rapid bacterial detection that is needed in many fields including food safety, clinical diagnostics, biosafety and biosecurity. Whole-cell imprinted polymers have the potential to be applied as recognition elements in biosensors for selective bacterial detection. In this paper, we report on the use of 3-aminophenylboronic acid (3-APBA) for the electrochemical fabrication of a cell-imprinted polymer (CIP). The use of a monomer bearing a boronic acid group, with its ability to specifically interact with cis-diol, allowed the formation of a polymeric network presenting both morphological and chemical recognition abilities. A particularly beneficial feature of the proposed approach is the reversibility of the cis-diol-boronic group complex, which facilitates easy release of the captured bacterial cells and subsequent regeneration of the CIP. Staphylococcus epidermidis was used as the model target bacteria for the CIP and electrochemical impedance spectroscopy (EIS) was explored for the label-free detection of the target bacteria. The modified electrodes showed a linear response over the range of 10(3)-10(7)cfu/mL. A selectivity study also showed that the CIP could discriminate its target from non-target bacteria having similar shape. The CIPs had high affinity and specificity for bacterial detection and provided a switchable interface for easy removal of bacterial cell.
This study aimed to determine whether heat stress (HS) could induce autophagy in immature boar Sertoli cells (SCs) and test whether HS-induced autophagy could regulate lactate secretion by SCs. Cultured immature boar SCs were incubated at 43 °C for 30 minutes. The ratio of LC3B-II to LC3B-I and the mRNA transcript levels of LC3B showed time-dependent changes 0 to 48 hours after HS treatment, which peaked at 24 hours and increased by 30.25% or 260%, respectively, compared with control SCs. The density of autolysosomes, which were labeled with a red dye, was higher at 24 hours than at any other time point. However, the apoptosis rate, cleavage of caspase-3, and mRNA transcript levels of CASP3 (caspase-3) at 24 hours after HS were lower than at 12 hours. Furthermore, lactate secretion, and mRNA transcript levels of SLC2A3 (GLUT3), LDHA (LDHA), and SLC16A1 (MCT1) also showed time-dependent changes with a peak at 24 hours. In addition, LY294002 (20 μM) significantly inhibited changes in ratio of LC3B-II to LC3B-I, LC3B mRNA transcript levels, and autolysosome formation. It also resulted in significantly less lactate secretion and increased apoptosis but showed no effect on B-cell lymphoma-2 expression in heat-treated immature SCs. These findings indicated that HS-induced autophagy regulates lactate secretion by inhibiting apoptosis and increasing mRNA transcript and protein levels of SLC2A3, LDHA, and SLC16A1, which suggests that HS-induced autophagy may enhance lactate secretion by SCs.
Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.
Herein we report on the multicomponent synthesis of a novel imidazole-based compound, able to act efficiently as a minimalist β-strand mimic. Biological evaluation proved its ability to impair the LDLR-PCSK9 protein-protein interaction, disclosing it as the first small molecule exerting a PCSK9-mediated hypocholesterolemic effect.
Diabetic neuropathy has a profound impact in the quality of life of patients who frequently complain of pain. The mechanisms underlying diabetic neuropathic pain (DNP) are no longer ascribed only to damage of peripheral nerves. The effects of diabetes at the central nervous system are currently considered causes of DPN. Management of DNP may be achieved by antidepressants that act on serotonin (5-HT) uptake, namely specific serotonin reuptake inhibitors. The rostroventromedial medulla (RVM) is a key pain control center involved in descending pain modulation at the spinal cord through local release of 5-HT and plays a peculiar role in the balance of bidirectional control (i.e. inhibitory and facilitatory) from the brain to the spinal cord. This review discusses recently uncovered neurobiological mechanisms that mediate nociceptive modulation from the RVM during diabetes installation. In early phases of the disease, facilitation of pain modulation from the RVM prevails through a triplet of mechanisms which include increase in serotonin expression at the RVM and consequent rise of serotonin levels at the spinal cord and upregulation of local facilitatory 5HT3 receptors, enhancement of spontaneous activity of facilitatory RVM neurons and up-regulation of the expression of transient receptor potential vanilloid type 1 (TRPV1) receptor. With the progression of diabetes the alterations in the RVM increase dramatically, with oxidative stress and neuronal death associated to microglia-mediated inflammation. In a manner similar to other central areas, like the thalamus, the RVM is likely to be a "pain generator/amplifier" during diabetes, accounting to increase DNP. Early interventions in DNP prevention using strategies that simultaneously tackle the exacerbation of 5-HT3 spinal receptors and of microglial RVM activity, namely those that increase the levels of anti-inflammatory cytokines, should be considered in the future of DNP treatment.
The life cycles and dispersal of edible fungi are still poorly known, thus limiting our understanding of their evolution and domestication. The prized Tuber melanosporum produces fruitbodies (fleshy organs where meiospores mature) gathered in natural, spontaneously inoculated forests or harvested in plantations of nursery-inoculated trees. Yet, how fruitbodies are formed remains unclear, thus limiting yields, and how current domestication attempts affect population genetic structure is overlooked. Fruitbodies result from mating between two haploid individuals: the maternal parent forms the flesh and the meiospores, while the paternal parent only contributes to the meiospores. We analyzed the genetic diversity of T. melanosporum comparatively in spontaneous forests vs. plantations, using SSR polymorphism of 950 samples from South-East France. All populations displayed strong genetic isolation by distance at the metric scale, possibly due to animal dispersal, meiospore persistence in soil, and/or exclusion of unrelated individuals by vegetative incompatibility. High inbreeding was consistently found, suggesting that parents often develop from meiospores produced by the same fruitbody. Unlike maternal genotypes, paternal mycelia contributed to few fruitbodies each, did not persist over years, and were undetectable on tree mycorrhizae. Thus, we postulate that germlings from the soil spore bank act as paternal partners. Paternal genetic diversity and outbreeding were higher in plantations than in spontaneous truffle-grounds, perhaps because truffle growers disperse fruitbodies to maintain inoculation in plantations. However, planted and spontaneous populations were not genetically isolated, so that T. melanosporum illustrates an early step of domestication where genetic structure remains little affected.
A simple base-mediated one-pot synthesis of 3,5-disubstituted 1,2,4-oxadiazoles from nitriles, aldehydes and hydroxylamine hydrochloride has been developed, in which the aldehydes act as both substrates and oxidants. The reactions include three sequential procedures: base-promoted intermolecular addition of hydroxylamine to nitrile to lead to amidoxime, treatment of the amidoxime with an aldehyde to form 4,5-dihydro-1,2,4-oxadiazole, and oxidization of the 4,5-dihydro-1,2,4-oxadiazole by using another aldehyde to afford 1,2,4-oxadiazole. This method represents a direct and simple protocol for the synthesis of 3,5-disubstituted 1,2,4-oxadiazoles.
Magnetic nanoparticle-embedded coordination polymers could act as mimics for zinc ion transporters for targeted cancer therapy. Compared with traditional zinc ionophores, our system could realize targeted and internal environment responsive transport of zinc ions to suppress the tumour growth while minimizing nonspecific injury to normal tissues.
Artificial control of cell adhesion on smart surface is an on-demand technique in areas ranging from tissue engineering, stem cell differentiation, to the design of cell-based diagnostic system. In this paper, we report an electrochemical system for dynamic control of cell catch-and-release, which is based on the redox-controlled host-guest interaction. Experimental results reveal that the interaction between guest molecule (ferrocene, Fc) and host molecule (β-cyclodextrin, β-CD) is highly sensitive to electrochemical stimulus. By applying a reduction voltage, the uncharged Fc can bind to β-CD that is immobilized at the electrode surface. Otherwise, it is disassociated from the surface as a result of electrochemical oxidation, thus releasing the captured cells. The catch-and-release process on this voltage-responsive surface is noninvasive with the cell viability over 86%. Moreover, because Fc can act as an electrochemical probe for signal readout, the integration of this property has further extended the ability of this system to cell detection. Electrochemical signal has been greatly enhanced for cell detection by introducing branched polymer scaffold that are carrying large quantities of Fc moieties. Therefore, a minimum of 10 cells can be analyzed. It is anticipated that such redox-controlled system can be an important tool in biological and biomedical research, especially for electrochemical stimulated tissue engineering and cell-based clinical diagnosis.
Long noncoding RNAs act as crucial regulators in plenty of human cancers, yet their potential roles and molecular mechanisms in chemoresistance are poorly understood. This study showed that a novel lncRNA, long intergenic noncoding RNA 152 (Linc00152 ), promoted tumor progression and conferred resistance to oxaliplatin (L-OHP)-induced apoptosis in vitro and in vivo. It antagonized chemosensitivity through acting as a competing endogenous RNA to modulate the expression of miR-193a-3p, and then erb-b2 receptor tyrosine kinase 4 (ERBB4). Knockdown of ERBB4 in colon cancer cells decreased AKT phosphorylation, which resulted in decreased L-OHP resistance. Consistent with above findings, the specific AKT signaling inhibitor and activator were used, respectively, which demonstrated that Linc00152 contributed to L-OHP resistance at least partly through activating AKT pathway. Further studies indicated that Linc00152 was increased and appeared to be an independent prognostic factor for decreased survival and increased disease recurrence in stage II and III colon cancer patients undergoing L-OHP-based chemotherapy after surgery. Collectively, our findings established Linc00152 as a candidate prognostic indicator of outcome and drug responsiveness in colon cancer patients, and the involvement of competing endogenous RNAs mechanism in Linc00152/miR-193a-3p/ERBB4/AKT signaling axis may provide a novel choice in the investigation of drug resistance.Molecular Therapy (2016); doi:10.1038/mt.2016.180.
Social reward plays a fundamental role in shaping human and animal behavior. The rewarding nature of many forms of social behavior including sexual behavior, parental behavior, and social play has been revealed using well-established procedures such as the conditioned place preference test. Many motivated social behaviors are regulated by the nonapeptides oxytocin (OT) and arginine vasopressin (AVP) through their actions in multiple brain structures. Interestingly, there are few data on whether OT or AVP might contribute to the rewarding properties of social interaction by their actions within brain structures that play a key role in reward mechanisms such as the ventral tegmental area (VTA). The goal of the present study was to investigate the role of OT and AVP in the VTA in regulating the reward-like properties of social interactions. Social interactions between two male hamsters reduced a spontaneous place avoidance in hamsters injected with saline control. Interestingly, however, OT and AVP injected into the VTA induced a significant two-fold reduction in place avoidance for the social interaction chamber when compared to control injections of vehicle. Finally, because OT and AVP can act on each other's receptors to influence social behavior, we also injected highly selective OTR and V1aR agonists and antagonists to determine whether OT or AVP V1a receptors were responsible for mediating the effects of these neuropeptides on social reward. Our results not only demonstrated that OT and AVP activate OTRs and not V1aRs to mediate social reward, they also demonstrated that the activation of OT receptors in the VTA is essential for the expression of the rewarding properties of social interactions.
Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.
Owing to their unique biological, physical, and chemical properties, fluoroalkylated organic substances have attracted significant attention from researchers in a variety of disciplines. Fluoroalkylated compounds are considered particularly important in pharmaceutical chemistry because of their superior lipophilicity, binding selectivity, metabolic stability, and bioavailability to those of their nonfluoroalkylated analogues. We have developed various methods for the synthesis of fluoroalkylated substances that rely on the use of visible-light photoredox catalysis, a powerful preparative tool owing to its environmental benignity and mechanistic versatility in promoting a large number of synthetically important reactions with high levels of selectivity. In this Account, we describe the results of our efforts, which have led to the development of visible-light photocatalytic methods for the introduction of a variety of fluoroalkyl groups (such as, -CF3, -CF2R, -CH2CF3, -C3F7, and -C4F9) and arylthiofluoroalkyl groups (such as, -CF2SPh, -C2F4SAr, and -C4F8SAr) to organic substances. In these studies, electron-deficient carbon-centered fluoroalkyl radicals were successfully generated by the appropriate choice of fluoroalkyl source, photocatalyst, additives, and solvent. The redox potentials of the photocatalysts and the fluoroalkyl sources and the choice of sacrificial electron donor or acceptor as the additive affected the photocatalytic pathway, determining whether an oxidative or reductive quenching pathway was operative for the generation of key fluoroalkyl radicals. Notably, we have observed that additives significantly affect the efficiencies and selectivities of these reactions and can even change the outcome of the reaction by playing additional roles during its course. For instance, a tertiary amine as an additive in the reaction medium can act not only as a sacrificial electron donor in photoredox catalysis but also as a hydrogen atom source, an elimination base for dehydrohalogenation of the intermediate, and also a Brønsted base for deprotonation. In the same context, the selection of solvent is also critical since it affects the rate and selectivity of reactions depending upon its polarity and reagent solubilizing ability and plays additional roles in the process, for example, as a hydrogen atom source. By clearly understanding the roles of additives and solvent, we designed several controlled fluoroalkylation reactions where different products were formed selectively from the same starting substrates. In addition, we could exploit one of the most important advantages of radical reactions, that is, the use of unactivated π-systems such as alkenes, alkynes, arenes, and heteroarenes as radical acceptors without prefunctionalization. Furthermore, fluoroalkylation processes under mild room-temperature reaction conditions tolerate various functional groups and are therefore easily applicable to late-stage modifications of highly functionalized advanced intermediates.
Vinca alkaloids have been approved as anticancer drugs for more than 50 years. They have been classified as cytotoxic chemotherapy drugs that act during cellular mitosis, enabling them to target fast growing cancer cells. With the evolution of cancer drug development there has been a shift towards new "targeted" therapies to avoid the side effects and general toxicities of "cytotoxic chemotherapies" such as the vinca alkaloids. Due to their original classification, many have overlooked the fact that vinca alkaloids, taxanes and related drugs do have a specific molecular target: tubulin. They continue to be some of the most effective anticancer drugs, perhaps because their actions upon the microtubule network extend far beyond the ability to halt cells in mitosis, and include the induction of apoptosis at all phases of the cell cycle. In this review, we highlight the numerous cellular consequences of disrupting microtubule dynamics, expanding the textbook knowledge of microtubule destabilising agents and providing novel opportunities for their use in cancer therapy.
Inflammation in Parkinson's disease (PD) is a new concept that has gained ground due to the potential of mitigating dopaminergic neuron death by decreasing inflammation. The solution to this question is likely to be complex. We propose here that the significance of inflammation in PD may go beyond the nigral cell death. The pathological process that underlies PD requires years to reach its full extent. A growing body of evidence has been accumulated on the presence of multiple inflammatory signs in the brain of PD patients even in very late stages of the disease. Because neuron-microglia-astrocyte interactions play a major role in the plasticity of neuronal response to l-DOPA in post-synaptic neurons, we focused this review on our recent results of l-DOPA-induced dyskinesia in rodents correlating it to significant findings regarding glial cells and neuroinflammation. We showed that in the rat model of PD/l-DOPA-induced dyskinesia there was an increased expression of inflammatory markers, such as the enzymes COX2 in neurons and iNOS in glial cells, in the dopamine-denervated striatum. The gliosis commonly seem in PD was associated with modifications in astrocytes and microglia that occur after chronic treatment with l-DOPA. Either as a cause, consequence, or promoter of progression of neuronal degeneration, inflammation plays a role in PD. The key aims of current PD research ought to be to elucidate (a) the time sequence in which the inflammatory factors act in PD patient brain and (b) the mechanisms by which neuroinflammatory response contributes to the collateral effects of l-DOPA treatment.
Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.
Since the beginning of this century, π-Lewis acidic gold complexes have become the catalysts of choice for a wide range of organic reactions, especially those involving nucleophilic addition to carbon-carbon multiple bonds. For the most part, however, the gold catalyst does not change oxidation state during the course of these processes and two-electron redox cycles of the kind implicated in cross-coupling chemistry are not easily accessible. In order to address this limitation and expand the scope of gold catalysis beyond conventional hydrofunctionalization, extensive efforts have been made to develop new oxidative reactions using strong external oxidants capable of overcoming the high potential of the Au(I)/Au(III) redox couple. However, these processes typically require superstoichiometric amounts of the oxidant and proceed under relatively harsh conditions. Moreover, to date, gold-catalyzed oxidative coupling reactions have remained somewhat limited in scope because, for many systems, the desired cross-coupling does not favorably compete with homodimerization or conventional hydrofunctionalization. In 2013, we disclosed a new concept for gold-catalyzed coupling reactions that, rather than involving external oxidants, employs aryl radicals that act as both the oxidant and the coupling partner in overall redox-neutral transformations. For this, we developed a dual catalytic system combining homogeneous gold catalysis with the emerging field of visible light photoredox catalysis. Using aryldiazonium salts, which are known to act as sources of aryl radicals upon activation with reducing photocatalysts, we could achieve intramolecular oxy- and aminoarylations of alkenes upon irradiating the reaction mixtures with visible light. Further studies on this transformation, in which nucleophilic addition onto a gold-activated alkene is followed by C(sp(3))-C(sp(2)) bond formation, expanded the scope of the process to intermolecular, three-component oxyarylation, while inexpensive organic dyes and user-friendly diaryliodonium salts could be employed as alternative photocatalysts and aryl radical sources, respectively. The potential of dual gold/photoredox catalysis was quickly realized by several research groups and a range of diverse new coupling reactions involving nucleophilic addition to π-systems and even P-H and C(sp)-H functionalization have been developed. In addition to the ambient reaction conditions and the simple setup using household light sources or even sunlight, a key advantage of dual gold/photoredox catalysis results from the simultaneous oxidation of gold(I) and coordination of the coupling partner, which results in high levels of selectivity for the cross-coupled products over homodimers. Furthermore, when gold complexes that are not catalytically active prior to oxidation by the aryl radical are employed, background reactions not involving coupling can be suppressed. Notably, this feature has allowed for the successful use of allenes and alkynes, for which conventional hydrofunctionalization pathways are highly favored, opening the door to new transformations involving the most common substrate classes for gold catalysis. In this Account, we provide an overview of dual gold/photoredox catalysis and highlight the potential of this concept to greatly expand the scope of homogeneous gold catalysis and enable the efficient construction of complex organic molecules. Moreover, recent studies on the visible light-promoted synthesis of novel gold(III) complexes suggest that photoredox activation could yet find further applications in gold chemistry beyond coupling.
Ataxin-2 (ATXN2) polyglutamine domain expansions of large size result in an autosomal dominantly inherited multi-system-atrophy of the nervous system named spinocerebellar ataxia type 2 (SCA2), while expansions of intermediate size act as polygenic risk factors for motor neuron disease (ALS and FTLD) and perhaps also for Levodopa-responsive Parkinson's disease (PD). In view of the established role of ATXN2 for RNA processing in periods of cell stress and the expression of ATXN2 in blood cells such as platelets, we investigated whether global deep RNA sequencing of whole blood from SCA2 patients identifies a molecular profile which might serve as diagnostic biomarker. The bioinformatic analysis of SCA2 blood global transcriptomics revealed various significant effects on RNA processing pathways, as well as the pathways of Huntington's disease and PD where mitochondrial dysfunction is crucial. Notably, an induction of PINK1 and PARK7 expression was observed. Conversely, expression of Pink1 was severely decreased upon global transcriptome profiling of Atxn2-knockout mouse cerebellum and liver, in parallel to strong effects on Opa1 and Ghitm, which encode known mitochondrial dynamics regulators. These results were validated by quantitative PCR and immunoblots. Starvation stress of human SH-SY5Y neuroblastoma cells led to a transcriptional phasic induction of ATXN2 in parallel to PINK1, and the knockdown of one enhanced the expression of the other during stress response. These findings suggest that ATXN2 may modify the known PINK1 roles for mitochondrial quality control and autophagy during cell stress. Given that PINK1 is responsible for autosomal recessive juvenile PD, this genetic interaction provides a concept how the degeneration of nigrostriatal dopaminergic neurons and the Parkinson phenotype may be triggered by ATXN2 mutations.
The heterodimeric receptor tyrosine kinase complex formed by HER2 and HER3 can act as an oncogenic driver and is also responsible for rescuing a large number of cancers from a diverse set of targeted therapies. Inhibitors of these proteins, particularly HER2, have dramatically improved patient outcomes in the clinic, but recent studies have demonstrated that stimulating the heterodimeric complex, either via growth factors or by increasing the concentrations of HER2 and HER3 at the membrane, significantly diminishes the activity of the inhibitors. To identify an inhibitor of the active HER2-HER3 oncogenic complex, we developed a panel of Ba/F3 cell lines suitable for ultra-high-throughput screening. Medicinal chemistry on the hit scaffold resulted in a previously uncharacterized inhibitor that acts through preferential inhibition of the active state of HER2 and, as a result, is able to overcome cellular mechanisms of resistance such as growth factors or mutations that stabilize the active form of HER2.
Non-destructive protocols which can define a biomaterial's degradation and its associated ability to support proliferation and/or promote extracellular matrix deposition will be an essential in vitro tool. In this study we investigate fluorescently tagged biomaterials, with varying rates of degradation and their ability to support cell proliferation and osteogenic differentiation. Changes in fluorescence of the biomaterials and the release of fluorescent soluble by-products were confirmed as accurate methods to quantify degradation. It was demonstrated that increasing rates of the selected biomaterials' degradation led to a decrease in cell proliferation and concurrently an increase in osteogenic matrix production. A novel turnover index (TI), which directly describes the effect of degradation of a biomaterial on cell behaviour, was calculated. Lower TIs for proliferation and high TIs for osteogenic marker production were observed on faster degrading biomaterials, indicating that these biomaterials supported an upregulation of osteogenic markers. This TI was further validated using an ex vivo chick femur model, where the faster degrading biomaterial, fibrin, led to an increased TI for mineralisation within an epiphyseal defect. This in vitro tool, TI, for monitoring the effect of biomaterial degradation on extracellular matrix production may well act as predictor of the selected biomaterials' performance during in vivo studies.
This paper outlines a novel metric, Turnover Index (TI), which can be utilised in tissue-engineering for the comparison of a range of biomaterials. The metric sets out to define the relationship between the rate of degradation of biomaterials with the rate of cell proliferation and ECM synthesis, ultimately allowing us to tailor material for set clinical requirements. We have discovered some novel comparative findings that cells cultured on biomaterials with increased rates of degradation have lower rates of proliferation but alternatively have a greater production of osteogenic markers compared to materials which degrade slower. By making comparisons in a rigorous manner, we can begin to define a useful matrix for materials which ultimately may aid for clinical selection.
MicroRNAs (miRNAs) are noncoding RNAs that are important for embryonic stem cell development and epithelial to mesenchymal transition (EMT). Accumulating evidence indicates that miRNAs play critical roles in prostate cancer (PCa) metastasis and have potential use as therapeutic targets. Although dysregulated miR-132/212 have been suggested to be directly involved in the proliferation and invasion of multiple malignancies, the exact role of miR-132/212 in PCa has not yet been fully understood.
Real-time quantitative PCR (RT-qPCR) and bioinformatics analysis were used to validate the expression levels of miR-132/212 in PCa cell lines as well as in prostatic tissues. The biological function of miR-132/212 was evaluated by MTS, transwell, and wound healing assays, respectively. RT-qPCR and Western blot were used to study the transcript and protein expression levels. Bioinformatics tools and luciferase reporter assay were utilized to identify the molecular target of miR-132/212. Immunohistochemistry (IHC) was used to detect the expression of SOX4.
miR-132 and miR-212 from the same gene cluster are downregulated in human PCa tissues when compared with benign prostatic hyperplasia tissues (both P < 0.05). Functionally, upregulation of miR-132/212 inhibits the migration and invasive capacity of Vcap and Lncap cells by wound-healing and transwell assays, respectively. Notably, overexpression of miR-132/212 could inhibit TGF-β (transforming growth factor-β)-induced EMT in Vcap and Lncap cells at both the mRNA and protein expression levels. SOX4 gene, an important EMT regulator of PCa, was identified as the target of miR-132/212 by bioinformatics tools and luciferase reporter assay. Clinically, miR-132/212 expression levels were adversely correlated with Gleason score (P < 0.001) and SOX4 expression by IHC and RT-qPCR in PCa tissues.
Our data suggested that miR-132/212 may act as tumor suppressors in PCa progression through disrupting EMT process by directly targeting SOX4. Prostate 76:1560-1570, 2016. © 2016 Wiley Periodicals, Inc.
The aim of this study was to use Kotter's leading change model to explore the implementation of workplace health and wellbeing initiatives.
Qualitative interviews were conducted with 31 workplace representatives with a healthy workplace initiative.
None of the workplaces used a formal change management model when implementing their healthy workplace initiatives. Not all of the steps in Kotter model were considered necessary and the order of the steps was challenged. For example, interviewees perceived that communicating the vision, developing the vision, and creating a guiding coalition were integral parts of the process, although there was less emphasis on the importance of creating a sense of urgency and consolidating change.
Although none of the workplaces reported using a formal organizational change model when implementing their healthy workplace initiatives, there did appear to be perceived merit in using the steps in Kotter's model.
In this account, our studies on nickel-catalyzed hydrosilylation reactions are described. A series of (salicylaldiminato)methylnickel complexes efficiently catalyze alkene hydrosilylation under ambient reaction conditions. Commercially available Ni(II) salts, Ni(acac)2 (acac = acetylacetonato) and its derivatives bis(hexafluoroacetylacetonato)nickel(II) and bis (2,2,6,6-tetramethyl-3,5-heptanedionato)nickel(II), also act as versatile hydrosilylation catalyst precursors in the presence of NaBHEt3 . These systems catalyze the hydrosilylation of various alkenes such as industrially important siloxy-, amino-, and epoxy-substituted ones. The arene-supported cationic nickel allyl complexes also serve as good catalysts for alkene hydrosilylation at room temperature. These nickel complexes exhibit high selectivity towards the reaction using secondary hydrosilanes. Mechanistic studies based on experiments and DFT calculations support a novel mechanism, which includes a facile Si-H bond cleavage and a Si-C bond formation, assisted by the cooperative action of the allyl ligand.
Responsible transparency helps to promote integrity between industry and researchers and has the potential to increase public trust. As part of its inevitable evolution, the Open Payments database is likely to have opportunities to increase and improve the context around the data that it reports, thus leading to a decrease in the currently encountered confusion and misinterpretation of the data. A major challenge faced by the Centers for Medicare and Medicaid Services is to make the data more informative and educational, to allow the Sunshine Act to meet its transparency goals while improving healthcare innovation.
The arboviruses West Nile virus (WNV), dengue virus (DENV) and Ross River virus (RRV) have been demonstrated to be blood transfusion-transmissible. A model to estimate the risk of WNV to the blood supply using a Monte Carlo approach has been developed and also applied to Chikungunya virus. Also, a probabilistic model was developed to assess the risk of DENV to blood safety, which was later adapted to RRV. To address efficacy and limitations within each model we present a hybrid model that promises improved accuracy, and is broadly applicable to assess the risk of arboviral transmission by blood transfusion.
Data were drawn from the Cairns Public Health Unit (Australia) and published literature. Based on the published models and using R code, a novel 'combined' model was developed and validated against the BP model using sensitivity testing.
The mean risk per 10,000 of the combined model is 0.98 with a range from 0.79 to 1.25, while the maximum risk was 4.45 ranging from 2.62 to 7.67 respectively. These parameters for the BP model were 1.20 ranging from 0.84 to 1.55, and 2.86 ranging from 1.33 to 5.23 respectively.
The combined simulation model is simple and robust. We propose it can be applied as a 'generic' arbovirus model to assess the risk from known or novel arboviral threats to the blood supply.
Bacterial cells are intricately organized, despite the lack of membrane-bounded organelles. The extremely crowded cytoplasm promotes macromolecular self-assembly and formation of distinct subcellular structures, which perform specialized functions. For example, the cell poles act as hubs for signal transduction complexes, thus providing a platform for the coordination of optimal cellular responses to environmental cues. Distribution of macromolecules is mostly mediated via specialized transport machineries, including the MreB cytoskeleton. Recent evidence shows that RNAs also specifically localize within bacterial cells, raising the possibility that gene expression is spatially organized. Here we review the current understanding of where things are in bacterial cells and discuss emerging questions that need to be addressed in the future.
Alpha-2 adrenergic receptors target several behavioral functions. These receptors may connect with the brain pathways mediating sensorimotor gating system that associate with psychoses, and the literature that investigate the relationship between alpha-2 receptors and sensorimotor gating system is very limited and some results are controversial. Thus, we aimed to investigate the role of alpha-2 receptors on prepulse inhibition (PPI) of acoustic startle reflex which is a measure of sensorimotor gating.
Adult male Wistar rats were subjects. PPI was measured as the per cent inhibition of the startle reflex produced by a startling pulse stimulus. The average PPI levels were used in the further analyses. Clonidine (0.03-1 mg/kg), an agonist of alpha-2 receptors, idazoxan (10 mg/kg), an antagonist alpha-2 receptors, and saline were injected to rats intraperitoneally. PPI was evaluated at two different startle intensity levels (78 and 86 dB, respectively).
Treatments produced some significant changes on PPI of startle reflex at all two levels of startle intensity. While clonidine (0.06, 0.25, 0.5, and 1 mg/kg) disrupted significantly PPI, idazoxan (10 mg/kg) did not produce any significant effect on PPI. However, pretreatment with idazoxan reversed significantly clonidine-induced disruption of PPI. Neither idazoxan (10 mg/kg) nor clonidine (1 mg/kg) produces any significant change on locomotor activity in naive rats.
Because idazoxan and clonidine also act through imidazoline receptors, our results suggest that alpha-2 and/or imidazoline receptors are associated with PPI of acoustic startle reflex in rats. Stimulation of these receptors may cause sensorimotor gating disturbances.
The development of new molecules able to efficiently act as long-circulating computed tomography (CT) contrast agents is one of the most crucial topics in the biomedical field. In the last years, the chance to manipulate materials at the nano-size level gave new boost to this research, with the specific aim to design innovative nanoprobes. Gold nanoparticles (AuNPs) have showed unique X-rays attenuation properties which, combined with their easy surface functionalization, makes them ideal candidates for the next generation of contrast agents. In this paper, we present a rational and facile approach to synthesize engineered and water-stable AuNPs, achieving concentrated colloidal solution with high Hounsfield Units (HU). An accurate control of reagents ratio allowed us to design AuNPs with different shapes, from symmetrical to anisotropic morphology, in a convenient 'one-pot' fashion. Their activity as efficient and reliable CT contrast agents has been evaluated and compared. Moreover, glucosamine-functionalized gold nanoparticles have been developed ([Au] = 31.20 mg/mL; HU = 2453), in order to obtain a CT contrast agent able to combine spatial resolution with metabolic information. Copyright © 2016 John Wiley & Sons, Ltd.
Microorganisms growing in a biofilm state are very resilient in the face of treatment by many antimicrobial agents. Biofilm infections are a significant problem in chronic and long-term infections, including those colonizing medical devices and implants. Anti-biofilm peptides represent a very promising approach to treat biofilm-related infections and have an extraordinary ability to interfere with various stages of the biofilm growth mode. Anti-biofilm peptides possess promising broad-spectrum activity in killing both Gram-positive and Gram-negative bacteria in biofilms, show strong synergy with conventional antibiotics, and act by targeting a universal stringent stress response. Understanding downstream processes at the molecular level will help to develop and design peptides with increased activity. Anti-biofilm peptides represent a novel, exciting approach to treating recalcitrant bacterial infections.
Purpose To explore provider and patient characteristics that influence how primary care providers (PCPs) communicate and manage incidental imaging findings. Materials and Methods This HIPAA-compliant study was approved by the institutional review board. Through semistructured interviews, researchers explored concerns and perspectives of 30 PCPs on receiving and acting on incidental imaging findings. Open-ended questions were designed to elicit a range of responses rather than quantifiable data. Thematic codes were developed and explicitly defined. Three research assistants independently coded all 30 deidentified transcripts and resolved discrepancies (κ = 0.85). Codes pertaining to PCP and patient characteristics were organized into an explanatory model. Results Some PCPs felt compelled but frustrated to pursue costly follow-up for incidental imaging findings of limited clinical importance. Other PCPs did not act on findings that were unfamiliar or occurred in an unusual clinical context when follow-up recommendations were not given; the challenges of researching the clinical importance of these findings or seeking specialist consultation led to inaction. Some PCPs reported using a uniform approach to communicate and manage incidental findings, while others adapted their approach to the patient and the finding. Sometimes PCP characteristics such as follow-up style superseded patient characteristics. At other times patient characteristics such as health literacy superseded PCP characteristics. Conclusion PCPs cited a variety of objective and subjective factors that influence how they communicate and manage incidental imaging findings. These results suggest that some patients may receive inappropriate follow-up of incidental imaging findings and present an opportunity for radiologists to help PCPs and patients to best use the information conveyed in imaging reports. (©) RSNA, 2016 Online supplemental material is available for this article.
Although the role of TLR4 in driving inflammation and organ injury after hemorrhagic shock and resuscitation (H/R) is well established, the role of TLR2-another receptor for damage-associated molecular pattern (DAMP) molecules-is not. In this study, we used a combination of TLR2 and wild type (WT) mice treated with anti-TLR2 and anti-TLR4 neutralizing monoclonal antibodies (mAb) to discern the contribution of TLR2 relative to TLR4 to the systemic inflammatory response in murine H/R.
WT mice, TLR2, and WT mice receiving an anti-TLR2 or an anti-TLR4 mAB (given as a pretreatment) were sacrificed at 6 or 20 h post-H/R. Bone marrow TLR2/WT chimeric mice were created to assess the importance of immune and nonimmune cell-associated TLR2.
TLR2 mice subjected to H/R exhibited significantly less liver damage and lower markers of systemic inflammation only at 20 h. Bone marrow chimeric mice using combinations of TLR2 mice and WT mice demonstrated that TLR2 on non-bone marrow derived cells played a dominant role in the differences at 20 h. Interestingly, WT mice treated with anti-TLR2 mAB demonstrated a reduction in organ damage and systemic inflammation at both 6 and 20 h following H/R. A combination of anti-TLR2 mAB and anti-TLR4 mAB showed that both receptors drive IP-10 and KC levels and that there is cooperation for increases in IL-6, MIG, and MCP-1 levels between TLR2 and TLR4.
These data also support the conclusion that TLR2 and TLR4 act in concert as important receptors in the host immune response to H/R.
Purpose To demonstrate the feasibility of four-dimensional (4D)-flow magnetic resonance (MR) imaging for noninvasive longitudinal hemodynamic monitoring of hepatic blood flow before and after transjugular intrahepatic portosystemic shunt (TIPS) placement. Materials and Methods The institutional review board approved this prospective Health Insurance Portability and Accountability Act compliant study with written informed consent. Four-dimensional-flow MR imaging was performed in seven patients with portal hypertension and refractory ascites before and 2 and 12 weeks after TIPS placement by using a time-resolved three-dimensional radial phase-contrast acquisition. Flow and peak velocity measurements were obtained in the superior mesenteric vein (SMV), splenic vein (SV), portal vein (PV), and the TIPS. Flow volumes and peak velocities in each vessel, as well as the ratio of in-stent to PV flow, were compared before and after TIPS placement by using analysis of variance. Results Flow volumes significantly increased in the SMV (0.24 L/min; 95% confidence interval [CI]: 0.07, 0.41), SV (0.31 L/min; 95% CI: 0.07, 0.54), and PV (0.88 L/min; 95% CI: 0.06, 1.70) after TIPS placement (all P < .05), with no significant difference between the first and second post-TIPS placement acquisitions (all P > .11). Ascites resolved in six of seven patients. In those with resolved ascites, the TIPS-to-PV flow ratio was 0.8 ± 0.2 and 0.9 ± 0.2 at the two post-TIPS time points, respectively, while the observed ratios were 4.6 and 4.3 in the patient with refractory ascites at the two post-TIPS time points, respectively. In this patient, 4D-flow MR imaging demonstrated arterio-portal-venous shunting, with draining into the TIPS. Conclusion Four-dimensional-flow MR imaging is feasible for noninvasive longitudinal hemodynamic monitoring of hepatic blood flow before and after TIPS placement. (©) RSNA, 2016 Online supplemental material is available for this article.
Tau immunotherapy has emerged as a promising approach to clear tau aggregates from the brain. Our previous findings suggest that tau antibodies may act outside and within neurons to promote such clearance.
We have developed an approach using flow cytometry, a human neuroblastoma cell model overexpressing tau with the P301L mutation, and paired helical filament (PHF)-enriched pathologic tau to effectively screen uptake and retention of tau antibodies in conjunction with PHF.
The flow cytometry approach correlates well with Western blot analysis to detect internalized antibodies in naïve and transfected SH-SY5Y cells (r(2) = 0.958, and r(2) = 0.968, P = .021 and P = .016, respectively). In transfected cells, more antibodies are taken up/retained as pathologic tau load increases, both under co-treated conditions and when the cells are pretreated with PHF before antibody administration (r(2) = 0.999 and r(2) = 0.999, P = .013 and P = .011, respectively).
This approach allows rapid in vitro screening of antibody uptake and retention in conjunction with pathologic tau protein before more detailed studies in animals or other more complex model systems.
The purpose of this study was to assess the incidence of nephrogenic systemic fibrosis (NSF) before and after educational interventions, implementation of a clinical screening process, and change to gadobenate dimeglumine in patients who had an estimated glomerular filtration rate (eGFR) of 30 mL/min per 1.72 m or less.
This is a Health Insurance Portability and Accountability Act compliant, institutional review board exempt study. Two periods were studied-July 2005 to June 2006, during which gadodiamide was utilized as our magnetic resonance (MR) contrast agent, and November 2006 to August 2014, during which gadobenate dimeglumine was used as our MR contrast agent in patients who had an eGFR 30 mL/min per 1.72 m or less. In addition to a change in the MR contrast agent, education of our staff physician to the risks of NSF with MR contrast agents and the implementation of a clinical screening process occurred. The rate of NSF before and after the interventions was compared using the χ test.
There was a statistically significant difference in the incidence of NSF in patients with an eGFR 30 mL/min per 1.72 m or less between the 2 periods: July 2005 to June 2006, 6 of 246 patients were diagnosed with NSF (P < 0.001), versus November 2006 to August 2014, 0 of 1423 patients were diagnosed with NSF.
Our data demonstrates a marked decrease in the incidence of NSF after education of our referring physicians, implementation of clinical screening process, and change to gadobenate dimeglumine from gadodiamide in patients with renal insufficiency. This approach potentially provides an acceptable risk-benefit profile for patients with renal insufficiency that required MR imaging for clinical care.
Dual-function sRNAs are a subgroup of small regulatory RNAs that act on the one hand as base-pairing sRNAs to inhibit or activate target gene expression and on the other hand as peptide-encoding mRNAs that function either in the same or in another metabolic pathway. Here, we review and compare the five currently known and intensively characterized dual-function sRNAs with regard to their two functions, their biological role, their evolutionary conservation and their requirements for RNA chaperones. Furthermore, we summarize the data available on five potential dual-function sRNAs, whose base-pairing function is well established whereas the role of their encoded peptides has not yet been elucidated. In addition, we provide three examples for RNAs with more than one function that do not fall into the above-mentioned category. With the application of RNAseq, peptidomics and transcriptomics it can be expected that the number of dual-function sRNAs will considerably increase within the next years, thus enhancing our knowledge on the regulatory potential of these RNAs.
The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing.
iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured.
iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells.
These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.
Advancements in adoptive cell transfer therapy (ACT) has led to the use of T cells engineered with tumor specific T cell receptors, which after rapid expansion can be obtained in sufficient numbers for treating patients. However, due to massive proliferation these cells are close to replicative senescence, exhibit exhausted phenotype, and also display increased susceptibility to activation induced cell death. We have previously shown that tumor reactive T cells undergo caspase-independent cell death upon TCR restimulation with cognate antigen, which involves reactive oxygen species and c-jun N-terminal kinase. Herein, we show that a large fraction of the human melanoma epitope tyrosinase reactive TCR transduced T cells that exhibit effector memory (TEM) phenotype and undergo programmed necrosis, or necroptosis, upon TCR restimulation. As compared to the T central memory (TCM) subsets, the TEM subset displayed an increased expression of genes involved in necroptotic cell death, and a necrotic phenotype upon TCR restimulation as confirmed by electron microscopy. Higher expression of receptor-interacting kinases (RIPK) that mediate necroptosis was also observed in the TEM fraction. Further, the TEM cells were rescued from undergoing necroptosis when pretreated with necroptotic inhibitor NecroX2 before TCR restimulation. Importantly, NecroX2 pretreated tumor reactive T cells also exhibited better tumor control and increased in vivo persistence when adoptively-transferred to treat subcutaneously established murine melanoma B16-F10. Thus, it is likely that the outcome of ACT could be vastly improved by interfering with the necroptotic cell death pathway in activated tumor reactive T cells used in immunotherapy.
BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aβ-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme. When BACE1 is inactive (Apo), the flap adopts an open conformation, allowing a substrate or inhibitor to access the active site. Subsequent interaction with the ligand induces flap closure and the stabilization of the macromolecular complex. Further, the protonation state of the aspartic dyad is affected by the chemical nature of the species entering the active site, so that appropriate selection of protonation states for the ligand and the catalytic residues will permit the elucidation of the inhibitory pathway for BACE1. In the present study, comparative analysis of different combinations of protonation states for the BACE1-hydroxyethylamine (HEA) system is reported. HEAs are potent inhibitors of BACE1 with favorable pharmacological and kinetic properties, as well as oral bioavailability. The results of Molecular Dynamics (MD) simulations and population density calculations using 8 different parameters demonstrate that the LnAsp289 configuration (HEA with a neutral amine and the Asp289 residue protonated) is the only one which permits the expected conformational change in BACE1, from apo to closed form, after flap closure. Additionally, differences in their capacities to establish and maintain interactions with residues such as Asp93, Gly95, Thr133, Asp289, Gly291, and Asn294 during this step allow differentiation among the inhibitory activities of the HEAs. The results and methodology here reported will serve to elucidate the inhibitory pathway of other families of compounds that act as BACE1 inhibitors, as well as the design of better leader compounds for the treatment of AD.
Urban watersheds from point sources are potential reservoirs of antibiotic resistance genes (ARGs). However, few studies have investigated urban watersheds of non-point sources. To understand the type of ARGs and bacteria that might carry such genes, we investigated two non-point source urban watersheds with different land-use profiles. Antibiotic resistance levels of two watersheds (R1, R3) were examined using heterotrophic plate counts (HPC) as a culturing method to obtain counts of bacteria resistant to seven antibiotics belonging to different classes (erythromycin, kanamycin, lincomycin, norfloxacin, sulfanilamide, tetracycline and trimethoprim). From the HPC study, 239 antibiotic resistant bacteria were characterized for resistance to more antibiotics. Furthermore, ARGs and antimicrobial biosynthesis genes were identified using GeoChip version 5.0 to elucidate the resistomes of surface waters in watersheds R1 and R3. The HPC study showed that water samples from R1 had significantly higher counts of bacteria resistant to erythromycin, kanamycin, norfloxacin, sulfanilamide, tetracycline and trimethoprim than those from R3 (Analysis of Similarity (ANOSIM), R = 0.557, p < 0.01). Of the seven antibiotics tested, lincomycin and trimethoprim resistant bacteria are greater in abundances. The 239 antibiotic resistant isolates represent a subset of resistant bacterial populations, including bacteria not previously known for resistance. Majority of the isolates had resistance to ampicillin, vancomycin, lincomycin and trimethoprim. GeoChip revealed similar ARGs in both watersheds, but with significantly higher intensities for tetX and β-lactamase B genes in R1 than R3. The genes with the highest average normalized intensities in R1 and R3 were tetracycline (tet) and fosfomycin (fosA) resistance genes, respectively. The higher abundance of tetX genes in R1 is congruent with the higher abundance of tetracycline resistant HPC observed in R1 samples. Strong correlations (r ≥ 0.8) of efflux pumps with antimicrobial biosynthesis genes suggest that natural production of antimicrobials may act as a selective pressure of transporter proteins in the absence of antibiotics from anthropogenic sources. In conclusion, distinct antibiotic resistant bacteria phylotypes and a variety of ARGs were present in the non-point sources urban watersheds of different land-use profiles, suggesting that ARG risk assessments and monitoring studies need to include these types of watersheds.
Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.
This study examined implementation of the Affordable Care Act (ACA) in relation to HIV-positive patient enrollment in an integrated health care system; as well as changes in new enrollee characteristics, benefit structure, and health care utilization after key ACA provisions went into effect in 2014.
This mixed-methods study was set in Kaiser Permanente Northern California (KPNC). Qualitative interviews with 29 KPNC leaders explored planning for ACA implementation. Quantitative analyses compared newly enrolled HIV-positive patients in KPNC between January and December 2012 ("pre-ACA," N = 661) with newly enrolled HIV-positive patients between January and December 2014 ("post-ACA," N = 880) on demographics; medical, psychiatric, and substance use disorder diagnoses; HIV clinical indicators; and type of health care utilization.
Interviews found that ACA preparation focused on enrollment growth, staffing, competition among health plans, concern about cost sharing, and HIV pre-exposure prophylaxis (PrEP) services. Quantitative analyses found that post-ACA HIV-positive patient enrollment grew. New enrollees in 2014 were more likely than 2012 enrollees to be enrolled in high-deductible plans (P < 0.01) or through Medicaid (P < 0.01), and marginally more likely to have better HIV viral control (P < 0.10). They also were more likely to be diagnosed with asthma (P < 0.01) or substance use disorders (P < 0.05) and to have used primary care health services in the 6 months postenrollment (P < 0.05) than the pre-ACA cohort.
As anticipated by KPNC interviewees, ACA implementation was followed by HIV-positive patient enrollment growth and changing benefit structures and patient characteristics. Although HIV viral control improved, comorbid diagnosis findings reinforced the importance of coordinated health care.
The increasing detection of papillary thyroid microcarcinoma (PTMC) has created management dilemmas. To clarify the clinical significance of postsurgery stimulated thyroglobulin (ps-Tg) in PTMC who undergo thyroidectomy and radioactive iodine (RAI), we retrospectively reviewed the 358 PTMC patients who were treated with RAI and followed up in our hospital. Those with an excessive anti-Tg antibody, ultrasound-detected residual were excluded, thereby resulting in the inclusion of 280 cases. Their clinical and histopathological information and clinical outcomes were collected and summarized. Tumor stages were classified according to the tumor, node, metastasis (TNM) staging system and the consensus of the European Thyroid Association (ETA) risk stratification system, respectively. Kaplan-Meier curves were constructed to compare the disease-free survival (DFS) rates of different risk-staging systems. By the end of follow-up, none of the patients died of the disease or relapsed. The 8-year DFS rate was 76.9%. Kaplan-Meier curves showed different DFS rates in TNM stages I versus IV, III versus IV, very low risk versus high risk, low risk versus high risk, respectively (P < 0.05), while they were not significantly different in stage I versus stage III, very low risk versus low risk (P > 0.05). Finally, 40 (14.3%) cases got a persistent disease. Five variables (male sex, nonconcurrent benign pathology, initial tumor size >5 mm, lymph node metastasis, and ps-Tg ≥ 10 μg/L) were associated with disease persistence by univariate regression analysis. Ps-Tg ≥ 10 μg/L was the only independent prognostic variable that predicted disease persistence by multivariate regression analysis (odds ratio: 36.057, P = 0.000). Therefore, PTMC with a small size of ≤1 cm does not always act as an indolent tumor. In conclusion, ps-Tg ≥ 10 μg/L is associated with increased odds of disease persistence. ETA risk stratification is more effective in predicting disease persistence than the TNM classification system.
The development of metastatic castration-resistant prostate cancer (mCRPC) signals the terminal disease phase. The preceding hormone-dependent disease setting is effectively managed with androgen deprivation therapy. This foundation of treatment has a high rate of biochemical and clinical response and meaningful clinical benefit but is finite in duration as most cancers will progress to castration resistance. Historically, treatment for mCRPC entailed androgen receptor (AR) inhibitors (nilutamide, flutamide, bicalutamide), nonspecific steroidal biosynthesis inhibitors (ketoconazole, itraconazole), steroids (prednisone, diethylstilbesterol, dexamethasone), or palliative chemotherapy (mitoxantrone, estramustine), but none of these strategies impacted survival. Docetaxel was the first agent to demonstrate a survival improvement in this population, and other therapies followed (cabazitaxel, sipuleucel-T and radium-223). Understanding how prostate cancer cells grow in a systemic androgen-deprived environment further changed this clinical landscape. Deciphering what steroidogenic enzymes are overactive and required for testosterone/dihydrotestosterone synthesis has yielded therapies directed toward both adrenal and tumor-derived androgens. All androgens normally act through AR, and this fact remains true in mCRPC. The cancer accomplishes this by overexpressing the receptor (by genomic copy-number gains or RNA amplification), mutating it directly to lose its selectivity for testosterone/dihydrotestosterone, or selecting for splice variants that do not require ligand at all. These resistance mechanisms result in persistent AR-mediated signaling. Through this understanding, drugs targeting non-ligand-binding aspects of AR functioning (e.g., nuclear translocation, cofactor recruitment) have been developed. Finally, how AR interacts with other signaling pathway is being explored, and new combinations of targets to test are being proposed. Multiple compounds remain in various stages of clinical development based on targeting these resistance pathways, and hopefully, they will further the armamentarium for mCRPC. This review visits these mechanisms of resistance, how they are targeted, and remaining challenges in implementing these therapies into clinical practice among the other approved treatments.
Defects in bulk crystals can be classified into vacancies, interstitials, grain boundaries, stacking faults, dislocations, and so forth. In particular, the vacancy in semiconductors is a primary defect that governs electrical transport. Concentration of vacancies depends mainly on the growth conditions. Individual vacancies instead of aggregated vacancies are usually energetically more favorable at room temperature because of the entropy contribution. This phenomenon is not guaranteed in van der Waals 2D materials due to the reduced dimensionality (reduced entropy). Here, it is reported that the 1D connected/aggregated vacancies are energetically stable at room temperature. Transmission electron microscopy observations demonstrate the preferential alignment direction of the vacancy chains varies in different 2D crystals: MoS2 and WS2 prefer 〈2¯11〉 direction, while MoTe2 prefers 〈1¯10〉 direction. This difference is mainly caused by the different strain effect near the chalcogen vacancies. Black phosphorous also exhibits directional double-chain vacancies along 〈01〉 direction. Density functional theory calculations predict that the chain vacancies act as extended gap (conductive) states. The observation of the chain vacancies in 2D crystals directly explains the origin of n-type behavior in MoTe2 devices in recent experiments and offers new opportunities for electronic structure engineering with various 2D materials.
Accumulating evidence suggests that activated mast cells are involved in contact hypersensitivity, although the precise mechanisms of their activation are still not completely understood. We investigated the potential of common experimental allergens to induce mast cell activation using murine bone marrow-derived cultured mast cells and rat peritoneal mast cells. Among these allergens, 1-chloro-2,4-dinitrobenzene and 1-fluoro-2,4-dinirobenzene (DNFB) were found to induce degranulation of rat peritoneal mast cells. DNFB-induced degranulation is accompanied by cytosolic Ca(2+) mobilization and is significantly inhibited by pertussis toxin, U73122 (a phospholipase C inhibitor), and BAPTA (a Ca(2+) chelator), raising the possibility that DNFB acts on the G protein-coupled receptors and activates Gi , which induces activation of phospholipase C, as well as known mast cell secretagogues, such as compound 48/80. DNFB could induce mast cell degranulation in the absence of serum proteins and IgE. Structure-activity relationship analyses revealed an inverse correlation between the degree of degranulation and the electron density of the C1 carbon of the DNFB derivatives. These findings raise a possibility that DNFB functions as a potent contact allergen through induction of cutaneous mast cell degranulation.
Sexual cues, including extended phenotypes, are expected to be reliable indicators of male genetic quality and/or provide information on parental quality. However, the reliability of these cues may be dependent on stability of the environment, with heterogeneity affecting how selection acts on such traits. Here we test how environmental change mediates mate choice for multiple sexual traits, including an extended phenotype - the structure of male-built nests - in stickleback fish. First, we manipulated the dissolved oxygen (DO) content of water to create high or low DO environments in which male fish built nests. Then we recorded the mate choice of females encountering these males (and their nests), under either the same or reversed DO conditions. Males in high DO environments built more compact nests than those in low DO conditions and males adjusted their nest structure in response to changing conditions. Female mate choice for extended phenotype (male nests) was environmentally-dependent (females chose more compact nests in high DO conditions), while female choice for male phenotype was not (females chose large, vigorous males regardless of DO level). Examining mate choice in this dynamic context suggests that females evaluate the reliability of multiple sexual cues, taking into account environmental heterogeneity. This article is protected by copyright. All rights reserved.
HOX antisense intergenic RNA (HOTAIR), a long non-coding RNA, plays an important role in the development of many types of cancers. Its function in acute leukemia (AL), however, has not been examined. The present study investigated the role of HOTAIR and its downstream genes in AL, and determined whether it could act as a molecular marker for prediction of leukemia development and prognosis. Real-time quantitative PCR was used to examine the expression of each gene in the HOTAIR signaling pathway in AL patients. The relationship between expression of HOTAIR and downstream genes and AL prognosis was analyzed. Expression of HOTAIR in patients with acute monocytic leukemia (M5) was increased as compared to controls (P<0.05). Compared to patients with low HOTAIR expression, overall survival and event-free survival of patients with high HOTAIR expression was significantly reduced. In addition, the expression of downstream genes in the HOTAIR signaling pathway including EZH2, LSD1, DNMT3A and DNMT3B was significantly increased in AL patients, and showed a significant positive correlation with high expression of HOTAIR (P<0.05). In conclusion, HOTAIR was closely related with a poor prognosis in AL patients. It may be involved in the development of leukemia by mediating methylation of DNA and histones.
MicroRNAs (miRNAs or miRs), a class of non-coding RNAs 18-25 nucleotides in length, act as key regulators in the development and malignant progression of various human cancers by modulating the expression of their target genes. Recently, miR‑16 has been demonstrated to be play a role in glioma. However, the regulatory mechanisms of miR‑16 in glioma growth and metastasis remain largely unclear. In the present study, qRT-PCR revealed that miR‑16 was significantly downregulated in 23 glioma tissue specimens compared to 7 normal brain tissue specimens. Moreover, its levels were markedly lower in the glioma samples at stages T2-T4 compared to those at stage T1. The overexpression of miR-16 significantly suppressed the proliferation, migration and invasion of U251 and U87 glioma cells. Luciferase reporter assay identified Sal-like protein 4 (SALL4) as a target gene of miR‑16, and its protein levels were found to be decreased in miR‑16-overexpressing U251 and U87 cells. Furthermore, the overexpression of SALL4 significantly reversed the suppressive effects of miR‑16 on the proliferation, migration and invasion of U251 and U87 cells, suggesting that miR‑16 playsa tumor suppressor role in glioma by inhibiting cell proliferation and invasion through the targeting of SALL4. Finally, we found that SALL4 was significantly upregulated in glioma tissues compared to normal brain tissues, and its levels were markedly higher in the glioma tissues at stages T2-T4 compared to those at stage T1. In addition, the expression levels of SALL4 inversely correlated with the miR‑16 levels in glioma tissues, suggesting that the downregulation of miR‑16 contributes to the upregulation of SALL4 in glioma. On the whole, the findings of this study indicate a role for the miR‑16/SALL4 axis in glioma. Our data may also provide a potential therapeutic target for the treatment of glioma.
Anaplastic thyroid cancer (ATC) is a rare malignancy that progresses extremely aggressively and often results in dismal prognosis. We investigated the efficacy of inhibiting the activated RAS/RAF/MEK pathway in ATC cells aiming to clarify the mechanism of effect and resistance. Four human ATC cell lines (ACT-1, OCUT-2, OCUT-4 and OCUT-6) were used. OCUT-4 had a BRAF mutation. OCUT-2 had both BRAF and PI3KCA mutations. ACT-1 and OCUT-6 had wild-type BRAF and NRAS mutations. The effects of dabrafenib, a selective inhibitor of the BRAFV600E kinase, and trametinib, a reversible inhibitor of MEK activity, were investigated. Dabrafenib strongly inhibited the viability in BRAF mutated cells by demonstrating G0/G1-arrest via the downregulation of MEK/ERK phosphorylation. Upregulated phosphorylation of MEK was observed in RAS mutated cells after dabrafenib treatment and caused VEGF upregulation, but was not related to the cellular proliferation. Trametinib inhibited the cellular viability to variable degrees in every cell by downregulating ERK phosphorylation. Dual blockade by both inhibitors demonstrated clear cytostatic effect in all the cells. OCUT-4 showed the weakest sensitivity to trametinib, no additional effect of either inhibitor in combination with the other, and an increase of SNAI1 mRNA expression after treatment with inhibitors, suggesting a mechanism for resistance. Our findings demonstrated the efficacy of a mutation-selective BRAF inhibitor and a MEK inhibitor in human ATC cells in a genetic alteration-specific manner.
The HIV Organ Policy Equity (HOPE) Act allows for transplantation of organs from Human Immunodeficiency virus (HIV) infected individuals (HIV+), provided it is performed under a research protocol. The safety assessment of an organ for transplantation is an essential element of the donation process. The risk for HIV associated opportunistic infections increases as circulating CD4+ lymphocytes decrease to <200 cells/μL; however, the numbers of circulating CD4+ cells in the HIV-negative (HIV-) brain dead donor (BDD) is not known.
Circulating T-lymphocyte subset profiles in conventional HIV- BDD were measured in 20 BDD in a clinical laboratory.
The mean age of the BDD cohort was 48.7 years old, 95% were white and 45% were female. The average BMI was 29.2 kg/m. Cerebrovascular accident (40%) was the most prevalent cause of death. Sixteen (80%) subjects had a CD4 count ≤441 cells/μl (lower limit of normal) and 11 (55%) had a CD4 count <200 cells/μl; eleven (55%) subjects had a CD8 count ≤125 cells/μl (lower limit of normal). CD4/CD8 ratio was below normal in 3 patients (normal: 1.4-2.6). No recipient had a recognized donor-associated adverse event.
Absolute numbers of CD4 and CD8 T-lymphocytes are commonly reduced after brain death in HIV- individuals. Thus, CD4 absolute numbers are an inconsistent metric for assessing organ donor risk, irrespective of HIV status.
The goals of this study were 1) to test the relative contribution of light-intensity and moderate-to-vigorous intensity physical activity (MVPA) to Cardiovascular Disease (CVD) risk in older adults, and 2) to examine the influence of ethnicity. We used pooled data from the 2003-2004 and 2005-2006 waves of the National Health and Nutritional Examination Survey (NHANES). The sample consisted of 1,171 White, Black and Mexican American adults aged 65 and older. Using ordinary least squares regression, we showed no statistically significant relationship between lower CVD risk with light intensity activity. However, greater minutes of MVPA was associated with lower CVD risk. Mexican Americans had statistically significantly higher risk for CVD compared to non-Hispanic Whites after controlling for physical activity. Mexican Americans remain an at risk group for CVD. Regardless of race/ethnicity, physical activity recommendations among elders should be for at least moderate intensity activity for a more favorable CVD outcome.
Opiates act through opioid receptors to diminish pain. Here, we investigated whether mu (MOR) and delta (DOR) receptor endogenous activity assessed in the whole mouse body or in particular at peripheral receptors on primary nociceptive neurons, control colonic pain.
We compared global MOR and DOR receptor knockout (KO) mice, mice with a conditional deletion of MOR and DOR in Nav1.8-positive nociceptive primary afferent neurons (cKO), and control floxed mice of both genders for visceral sensitivity. Visceromotor responses to colorectal distension (CRD) and macroscopic colon scores were recorded on naïve mice and mice with acute colitis induced by 3% dextran sodium sulphate (DSS) for 5 days. Transcript expression for opioid genes and cytokines was measured by quantitative RT-PCR.
Naïve MOR and DOR global KO mice show increased visceral sensitivity that was not observed in cKO mice. MOR and preproenkephalin (Penk) were the most expressed opioid genes in colon. MOR KO mice had augmented kappa opioid receptor and Tumour-Necrosis-Factor-α and diminished Penk transcript levels while DOR, preprodynorphin and Interleukin-1β were unchanged. Global MOR KO females had a thicker colon than floxed females. No alteration was detected in DOR mutant animals. A 5-day DSS treatment led to comparable hypersensitivity in the different mouse lines.
Our results suggest that mu and delta opioid receptor global endogenous activity but not activity at the peripheral Nav1.8 neurons contribute to visceral sensitivity in naïve mice, and that endogenous MOR and DOR tones were insufficient to elicit analgesia after 5-day DSS-induced colitis.
Knockout mice for mu and delta opioid receptor have augmented colon sensitivity in the CRD assay. It shows endogenous mu and delta opioid analgesia that may be explored as potential targets for alleviating chronic intestinal pain.
D-loops, a new type of structural defect in carbon fibers, are presented, which have highly detrimental effect on their mechanical properties and can define a new fundamental upper limit to their strength. These defects form exclusively during polyacrylonitrile carbonization, act as stress concentrators in the graphitic basal plane, and cannot be removed by local annealing.
The interaction between cellulase enzymes and their substrates is of central importance to several technological and scientific challenges. Here we report that the binding of cellulose binding modules (CBM) from Trichoderma reesei cellulases Cel6A and Cel7A show a major difference in how they interact with substrates originating from wood compared to bacterial cellulose. We found that the CBM from TrCel7A recognizes the two substrates differently and as a consequence shows an unexpected way of binding. We show that the substrate has a large impact on the exchange rate of the studied CBM, and moreover, CBM-TrCel7A seems to have an additional mode of binding on wood derived cellulose but not on cellulose originating from bacterial source. This mode is not seen in double CBM (DCBM) constructs comprising both CBM-TrCel7A and CBM-TrCel6A. The linker length of DCBMs affects the binding properties, and slows down the exchange rates of the proteins and thus, can be used to analyze the differences between the single CBM. These results have impact on the cellulase research and offer new understanding on how these industrially relevant enzymes act.
Cavity quantum electrodynamics (CQED) has played a central role in demonstrating the fundamental principles of the quantum world, and in particular those of atom-light interactions. Developing fast, dynamical and non-mechanical control over a CQED system is particularly desirable for controlling atomic dynamics and building future quantum networks at high speed. However conventional mirrors do not allow for such flexible and fast controls over their coupling to intracavity atoms mediated by photons. Here we theoretically investigate a novel all-optical CQED system composed of a binary Bose-Einstein condensate (BEC) sandwiched by two atomic ensembles. The highly tunable atomic dispersion of the CQED system enables the medium to act as a versatile, all-optically controlled atomic mirror that can be employed to manipulate the vacuum-induced diffraction of matter-wave superradiance. Our study illustrates a innovative all-optical element of atomtroics and sheds new light on controlling light-matter interactions.
We investigate the energy dynamics in a generalized compass chain under an external magnetic field. We show that the energy current operators act on three contiguous sites in the absence of the magnetic field, and they are incorporated with inhomogenous Dzyaloshinskii-Moriya interactions in the presence of the magnetic field. Under these complex interactions the Hamiltonian remains an exactly solvable spin model. We study the effects of the three-site interactions and the Dzyaloshinskii-Moriya interactions on the energy spectra and phase diagram. The results have revealed that the energy current of the pristine quantum compass model is conserved due to the associated intermediate symmetries, and for other general cases such a characteristic does not exist.
Sodium-glucose co-transporter 2 inhibitors (SGLT-2) inhibitors) represent a new class of antidiabetic drugs that act through the inhibition of glucose and sodium reabsorption at proximal tubules. It has been shown that tThese substances may exhibit renonephroprotective properties, since they expressed clinically as a prevention of the deterioration of the glomerular filtration rate and a reductionreduce in the degree of albuminuria in patients with established diabetes-associated kidney disease. In this review we present in detail the pathophysiologic mechanisms that have been recently implicated in the rennephroprotective properties of SGLT-2 inhibitors. Although the beneficial effects of SGLT-2 inhibitors on the conventional risk factors for kidney disease (such as blood pressure, hyperglycemia, body weight and serum uric acid levels) may explain, at least in part, the observed renal-protecting properties of these compounds, it has been hypothesized that the most important mechanisms for this phenomenon include the reduction in the intraglomerular pressure, the changes in the local and systemic degree of activation of the renin-aldosterone-angiotensin renin-aldosterone-angiotensin system, and a shift in renal fuel consumption towards more efficient energy substrates such as ketone bodies. The The beneficial effects SGLT2 inhibitors on various aspects of renal function function makes them an attractive choice in patients with (and possibly without) diabetes-associated renal dysfunctionimpairment.
Salvia miltiorrhiza Bunge, also known as Danshen in Chinese, has been widely used to treat cardiovascular diseases (CVD) in China and other Asia countries. Here, we summarize the literatures of the historical traditional Chinese medicine (TCM) interpretation of the action of Salvia miltiorrhiza, its use in current clinical trials, its main phytochemical constituents and its pharmacological findings by consulting Pubmed, China Knowledge Resource Integrated, China Science and Technology Journal, and the Web of Science Databases. Since 2000, 39 clinical trials have been identified that used S. miltiorrhiza in TCM prescriptions alone or with other herbs for the treatment of patients with CVD. More than 200 individual compounds have been isolated and characterized from S. miltiorrhiza, which exhibited various pharmacological activities targeting different pathways for the treatment of CVD in various animal and cell models. The isolated compounds may provide new perspectives in alternative treatment regimes and reveal novel chemical scaffolds for the development of anti-CVD drugs. Meanwhile, there are also some rising concerns of the potential side effects and drug-drug interactions of this plant. The insights gained from this study will help us to better understanding of the actions of this herb for management of cardiovascular disorders. As an herb of red root, S. miltiorrhiza will act as a potential red light to prevent the development of CVD.
Although significant progress has been made recently, copper-based materials have long been considered to be ineffective catalysts toward the hydrogen evolution reaction (HER), in most cases, requiring high overpotentials more than 300 mV. We report here that a Cu(0)-based nanoparticle film electrodeposited in situ from a Cu(II) oxime complex can act as a highly active and robust HER electrocatalyst in neutral phosphate buffer solution. The as-prepared nanoparticle film is of poor crystallization, which incorporates significant amounts of oxime ligand residues and buffer anions PO4(3-). The proposed mechanism suggests that the Cu(0)-based nanoparticle film is activated with incorporated or adsorbed PO4(3-) anions and the PO4(3-) anions-anchored sites might serve as the actual catalytic active sites with efficient proton transport mediators. Catalysis occurs with a low onset overpotential (η) of 65 mV, and a current density of 1 mA/cm(2) can be achieved with η = 120 mV. The nanoparticle film shows an excellent catalytic durability with slightly rising current density during electrolysis, presumably due to further incorporation or adsorption of PO4(3-) anions in the process. This electrocatalyst not only forms in situ from earth-abundant materials but also operates in neutral water with low overpotential and high stability.
The Rbfox genes encode an ancient family of sequence-specific RNA binding proteins (RBPs) that are critical developmental regulators in multiple tissues including skeletal muscle, cardiac muscle, and brain. The hallmark of Rbfox proteins is a single high-affinity RRM domain, highly conserved from insects to humans, that binds preferentially to UGCAUG motifs at diverse regulatory sites in pre-mRNA introns, mRNA 3'UTRs, and pre-miRNAs hairpin structures. Versatile regulatory circuits operate on Rbfox pre-mRNA and mRNA to ensure proper expression of Rbfox1 protein isoforms, which then act on the broader transcriptome to regulate alternative splicing networks, mRNA stability and translation, and microRNA processing. Complex Rbfox expression is encoded in large genes encompassing multiple promoters and alternative splicing options that govern spatiotemporal expression of structurally distinct and tissue-specific protein isoforms with different classes of RNA targets. Nuclear Rbfox1 is a candidate master regulator that binds intronic UGCAUG elements to impact splicing efficiency of target alternative exons, many in transcripts for other splicing regulators. Tissue-specificity of Rbfox-mediated alternative splicing is executed by combinatorial regulation through the integrated activity of Rbfox proteins and synergistic or antagonistic splicing factors. Studies in animal models show that Rbfox1-related genes are critical for diverse developmental processes including germ cell differentiation and memory in Drosophila, neuronal migration and function in mouse brain, myoblast fusion and skeletal muscle function, and normal heart function. Finally, genetic and biochemical evidence suggest that aberrations in Rbfox-regulated circuitry are risk factors for multiple human disorders, especially neurodevelopmental disorders including epilepsy and autism, and cardiac hypertrophy. For further resources related to this article, please visit the WIREs website.
The population dynamics of Rhipicephalus microplus (Ixodida: Ixodidae) in northwest Argentina was analysed to support the design of strategic methods for its control. Both parasitic and non-parasitic phases were studied. The seasonal activity of R. microplus in its parasitic phase was characterized by three peaks in abundance: the first in mid-late spring; the second in summer, and the third in autumn. The non-parasitic phase of R. microplus was characterized by a long total non-parasitic period observed after exposures of females from mid-summer to early autumn, a short total non-parasitic period observed after exposures of females from late winter to late spring, a short period of larval longevity in early and mid-summer, and no hatch of the eggs produced by females exposed in mid- and late autumn and winter. Treatments of cattle administered during the period from late winter to late spring will act on small cohorts of R. microplus, preventing the emergence of larger generations in summer and autumn. A 17-week spelling period starting in late spring and early summer will be necessary to achieve optimal control of R. microplus free-living larvae. If spelling begins in mid- or late summer or in autumn, the required period will be 26-27 weeks.
Connecticut (CT) passed its original sport-related concussion (SRC) law (PA 10-62) in 2010. The law requires that a health-care professional evaluate high school athletes with concussion symptoms. The purpose of this study was to evaluate two level 1 Trauma Center Emergency Department (ED) records for SRC before and after the Connecticut Public Act (CT PA) 10-62 to determine if the law had an effect on the presentation to the ED of SRCs.
A retrospective analysis of two level 1 Trauma Center Emergency Departments database was performed. Monthly data on SRCs treated in the study EDs from July 2003 through June 2012 were collected and analyzed using the autoregressive integrated moving average model. The number of SRCs in the youth (under age 14 years), high school (age 14 to 18 years), and adult (age >18 years) populations prior to CT PA 10-62 was compared to the number of SRCs post implementation of CT PA 10-62 for each academic school year, fall sports season, and summertime.
Monthly SRCs in high school students treated in the study EDs increased from 2.5 cases to 5.9 cases between pre and post implementation of CT PA 10-62 (p < 0.001). Statistical modeling revealed that implementation of CT PA 10-62 was associated with significantly increased SRCs treated in the study EDs and that the increase was limited to the high school students in the fall season and during the school year.
There has been a marked increase in the frequency of SRCs treated in the emergency departments in the high school population in Connecticut after the implementation of the sport-related concussion law. The results suggest that the sport-related concussion law in Connecticut is effective in improving the evaluation and detection of SRCs in high school students.
Anticoagulants are a well known cause of drug-induced liver injury (DILI). We recently encountered a 45-year-old male who developed DILI during treatment with enoxaparin, a low-molecular-weight heparin (LMWH), for dural venous thrombosis. The man received enoxaparin 80 mg subcutaneously, twice daily. After 4 days, the patient was asymptomatic but he developed liver aminotransferase elevations: AST 340 U/L and ALT 579 U/L. Investigation revealed an R ratio of 19.9 by day 5 and a Roussel Uclaf Causality Assessment Method score of 10, giving a high probable likelihood that enoxaparin was the cause of hepatic injury. Enoxaparin was discontinued on day 7, and 1 week later AST and ALT had decreased to 61 and 273 U/L, respectively. This case prompted a literature search and a review of the FDA Adverse Event Reporting System (FAERS) database for the range of hepatic adverse events (HAEs) associated with this class. A MEDLINE/PubMed search was conducted using DILI terms and cross-referenced with the anticoagulant classes. A Freedom of Information Act (FOIA) request was also made to identify adverse events (AEs) associated with enoxaparin in FAERS. Case type, severity of outcome, and demographic information were analyzed. Five publications have reported DILI with enoxaparin. Trial data found elevations in ALT >3 times the upper limit of normal (ULN) for unfractionated heparins (UFH) and LMWH in 8 and 4-13 % of subjects, respectively. However, liver injury in all cases was mild, self-limited, and asymptomatic. Our FOIA request yielded 8336 adverse events related to enoxaparin over a 14-year period (Jan 2000-Sept 2014). Specific HAEs were found in 4 % of reports, but all were described with other serious adverse events. The reported outcomes of hospitalization (75 %), death (17 %), and life-threatening medical events (5 %) were likely due to other related serious adverse events such as hemorrhage (28 %) and thrombocytopenia (11 %). We conclude that LMWH-related liver injury is uncommon and reversible. The mechanism of liver injury is not known, although an idiosyncratic effect is postulated. Although the FAERS database lists hepatic injury in 4 % of all enoxaparin-related AEs, it appears that serious outcomes are related to non-hepatic events.
Global regulators play an essential role in the adaptation of bacterial cells to specific niches. Bacterial pathogens thriving in the tissues and organs of their eukaryotic hosts are a well-studied example. Some of the proteins that recognize local DNA structures rather than specific nucleotide sequences act as global modulators in many bacteria, both Gram-negative and -positive. To this class of regulators belong the H-NS-like proteins, mainly identified in γ-Proteobacteria, and the MgaSpn-like proteins identified in Firmicutes. H-NS and MgaSpn from Escherichia coli and Streptococcus pneumoniae, respectively, neither have sequence similarity nor share structural domains. Nevertheless, they display common features in their interaction with DNA, namely: (i) they bind to DNA in a non-sequence-specific manner, (ii) they have a preference for intrinsically curved DNA regions, and (iii) they are able to form multimeric complexes on linear DNA. Using DNA fragments from the hemolysin operon regulatory region of the E. coli plasmid pHly152, we show in this work that MgaSpn is able to recognize particular regions on extended H-NS binding sites. Such regions are either located at or flanked by regions of potential bendability. Moreover, we show that the regulatory region of the pneumococcal P1623B promoter, which is recognized by MgaSpn, contains DNA motifs that are recognized by H-NS. These motifs are adjacent to regions of potential bendability. Our results suggest that both regulatory proteins recognize similar structural characteristics of DNA.
Endophytes are an endosymbiotic group of microorganisms that colonize in plants and microbes that can be readily isolated from any microbial or plant growth medium. They act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that serve as a potential candidate for antimicrobial, anti-insect, anticancer and many more properties. While plant sources are being extensively explored for new chemical entities for therapeutic purposes, endophytic microbes also constitute an important source for drug discovery. This review aims to comprehend the contribution and uses of endophytes as an impending source of drugs against various forms of diseases and other possible medicinal use.
Obesity and osteoporosis are two alarming health disorders prominent among middle and old age populations, and the numbers of those affected by these two disorders are increasing. It is estimated that more than 600 million adults are obese and over 200 million people have osteoporosis worldwide. Interestingly, both of these abnormalities share some common features including a genetic predisposition, and a common origin: bone marrow mesenchymal stromal cells. Obesity is characterized by the expression of leptin, adiponectin, interleukin 6 (IL-6), interleukin 10 (IL-10), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), macrophage colony stimulating factor (M-CSF), growth hormone (GH), parathyroid hormone (PTH), angiotensin II (Ang II), 5-hydroxy-tryptamine (5-HT), Advance glycation end products (AGE), and myostatin, which exert their effects by modulating the signaling pathways within bone and muscle. Chemical messengers (e.g., TNF-α, IL-6, AGE, leptins) that are upregulated or downregulated as a result of obesity have been shown to act as negative regulators of osteoblasts, osteocytes and muscles, as well as positive regulators of osteoclasts. These additive effects of obesity ultimately increase the risk for osteoporosis and muscle atrophy. The aim of this review is to identify the potential cellular mechanisms through which obesity may facilitate osteoporosis, muscle atrophy and bone fractures.
Background: Telomerase activity (TA) is considered as the biomarker for cardiovascular aging and cardiovascular diseases (CVDs). Recent studies suggest a link between statins and telomere biology that may be explained by anti-inflammatory actions of statins and their positive effect on TA. Until now, this effect has not been investigated in prospective randomized studies. We hypothesized that 12 months of atorvastatin therapy increased TA in peripheral blood mononuclear cells. Methods: In a randomized, placebo-controlled study 100 hypercholesterolemic patients, aged 35-75 years, free of known CVDs and diabetes mellitus type 2 received 20 mg of atorvastatin daily or placebo for 12 months. TA was measured by quantitative polymerase chain reaction. Results: At study end, 82 patients had sufficient peripheral blood mononuclear cells needed for longitudinal analysis. TA expressed as natural logarithms changed from 0.46 ± 0.05 to 0.68 ± 0.06 (p = 0.004) in the atorvastatin group and from 0.67 ± 0.06 to 0.60 ± 0.07 (p = 0.477) in the control group. In multiple regression analysis, atorvastatin therapy was the only independent predictor (p = 0.05) of the changes in TA independently of markers of chronic inflammation and oxidative stress. Atorvastatin therapy was associated with increases in interleukin-6 within the normal range and a tendency toward reduction in blood urea. Conclusion: These initial observations suggest atorvastatin can act as telomerase activator and potentially as effective geroprotector. Trial registration: The trial was registered in ISRCTN registry ISRCTN55050065.
Background: Osteoporosis is a chronic disease and adherence can fluctuate over time. Therefore, longer observation is necessary to investigate the stability of patients' adherence. The study aim was to compare the overall adherence (OA) to supplementation with the fixed combination of calcium and vitamin D (Ca/D) in postmenopausal women at baseline and after 1 year, and to evaluate the fluctuation of the OA in individual months. Furthermore, we studied whether adherence is influenced by signing of informed consent and routine medical check-up. Methods: This was a longitudinal, observational study. The data were obtained from the Osteocenter of University Hospital in Hradec Kralove, Czech Republic. Adherence was measured using electronic bottles type Medication Events Monitoring System (MEMS). The study was carried out in two 3-month periods; the baseline in 2013 (signing of informed consent while medical check-up) and the follow-up (medical check-up) in 2014. The adherence and adherence-related outcomes were studied in patients who had initiated osteoporosis treatment and were persistent. Results: 21 (49%) out of 43 patients who avoided drug dispenser and were persistent both at baseline and at follow-up, completed the study and were included. Median age was 76. Evaluating the whole 3-month periods, the OA did not differ significantly at baseline and at follow-up, the OA was 71 and 68%, respectively. However, the adherence in month 1 at baseline was significantly higher than the adherence in month 2 at baseline (p < 0.001) and also than the adherence in month 1 at follow-up (p = 0.010). Analysing the study period without month 1, a stable adherence was observed in 48% of patients. About 33% of doses were omitted at baseline and 34% at follow-up. As many as 71% of the patients took drug holidays at baseline, and 76% at follow-up. Conclusion: The OA was insufficient, around 70% both at baseline and at follow-up. One half of the patients showed a stable adherence. The patterns of non-adherence were very similar at follow-up. Signing of the informed consent seems to act as bias more than regular medical check-up.
Repeated-measure analysis of variance is a general term that can imply a number of different statistical models used to analyze data from studies in which measurements are taken from each subject on more than one occasion. Repeated-measure analyses encompass univariate models (with or without sphericity adjustment), multivariate models, mixed models, analysis of covariance, multilevel models, latent growth models, and hybrids of these models. These models are based on different assumptions, especially regarding correlations (sphericity) between within-subject factors, which comprise the variance-covariance matrix. Violation of this assumption may lead to misleading and erroneous conclusions. Because many papers do not provide enough information about what analysis was really conducted, and about why it was done, the reader is unable to evaluate the validity of the analysis. Here a brief overview of several of the most commonly used models for analyzing data from repeated-measure designs is provided, and guidance is suggested for describing the statistical approach employed. The goals of this paper are (1) to give authors an overview of the diversity of commonly used models and associated assumptions, and (2) to facilitate reporting sufficient information about the tests to allow the reader to evaluate the validity of the tests and the credibility of the inferences made by the authors. Among the available approaches to repeated-measure analyses, the mixed model is recommended for its flexibility in handling different covariance structures and its insensitivity to missing data. Whether or not it is used, the overall guiding principles in reporting should always be Accuracy, Completeness, and Transparency (ACT principles): tell the reader precisely all what you did and why.
This study aimed to determine the utility of directed ultrasound and digital mammogram for evaluating focal breast pain in women with different mammographic breast densities.
This institutional review board-approved and Health Insurance Portability and Accountability Act-compliant retrospective study included 413 cases of focal breast pain in 369 women (mean age 53 years). All cases were evaluated with both mammogram and ultrasound and had at least 2 years of imaging follow-up. Exclusion criteria were non-focal, axillary, or radiating pain; palpable or skin changes; pregnancy or lactation; and history of trauma or infection. Breast density, imaging findings, and biopsy results were recorded. Specificity, positive predictive values, and negative predictive values were calculated.
Eighteen percent (76 of 413) of cases demonstrated an imaging correlate. Of these, 74% (56 of 76) occurred in dense breasts and 26% (20 of 76) in nondense breasts. Seventy percent (14 of 20) of lesions in nondense breasts were seen with mammography and ultrasound, whereas 30% (6 of 20) were detected only with ultrasound. Of lesions detected in dense breasts, 29% (16 of 56) were seen with mammography and ultrasound, whereas 71% (40 of 56) were detected only with ultrasound. Thirty-one percent (24 of 76) of cases were biopsied, 42% (10 of 24) of which were detected by ultrasound only. No cancer was detected in initial workup. At 2-year follow-up, three women, all with dense breasts, developed cancer in the same quadrant as the initial pain.
Directed ultrasound, when performed in conjunction with digital mammography for the evaluation of focal breast pain in women with nondense breasts, is of low utility and may contribute to unnecessary intervention as a result of incidental findings.
The biological mechanisms used to categorize and recognize behaviors are poorly understood in both human and non-human animals. Using animated digital models, we have recently shown that pigeons can categorize different locomotive animal gaits and types of complex human behaviors. In the current experiments, pigeons (go/no-go task) and humans (choice task) both learned to conditionally categorize two categories of human behaviors that did not repeat and were comprised of the coordinated motions of multiple limbs. These "martial arts" and "Indian dance") action sequences were depicted by a digital human model. Depending upon whether the model was in motion or not, each species was required to engage in different and opposing responses to the two behavioral categories. Both species learned to conditionally and correctly act on this dynamic and static behavioral information, indicating that both species use a combination of static pose cues that are available from stimulus onset in addition to less rapidly available action information in order to successfully discriminate between the behaviors. Human participants additionally demonstrated a bias towards the dynamic information in the display when re-learning the task. Theories that rely on generalized, non-specific visual mechanisms involving channels for motion and static cues offer a parsimonious account of how humans and pigeons recognize and categorize behaviors within and across species.
Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism.
Diet may be defined as a complex process that should involve a deeper comprehension of metabolism, energy balance, and the molecular pathways involved in cellular stress response and survival, gut microflora genetics, enzymatic polymorphism within the human population, and the role of plant-derived polyphenols in this context. Metabolic syndrome, encompassing pathologies with a relatively high morbidity, such as type 2 diabetes, obesity, and cardiovascular disease, is a bullet point of the big concern about how daily dietary habits should promote health and prevent metabolic impairments to prevent hospitalization and the need for health care. From a clinical point of view, very few papers deal with this concern, whereas most of the evidence reported focuses on in vitro and animal models, which study the activity of phytochemicals contained in the daily diet. A fundamental issue addressed by dietitians deals with the role exerted by redox-derived reactive species. Most plant polyphenols act as antioxidants, but recent evidence supports the idea that these compounds primarily activate a mild oxidative stress to elicit a positive, beneficial response from cells. How these compounds may act upon the detoxifying system exerting a scavenging role from reactive oxygen or nitrogen species is still a matter of debate; however, it can be argued that their role is even more complex than expected, acting as signaling molecules in the cross-talk mitochondria-endoplasmic reticulum and in enzymatic pathways involved in the energetic balance. In this relationship, a fundamental role is played by the brain-adipose tissue-gut axis. The aim of this review was to elucidate this topic and the state of art about the role of reactive species in cell signaling and the function of metabolism and survival to reappraise the role of plant-derived chemicals.
The purpose of this study was to compare the agreement between two heparin assays, Hepcon HMS plus/Kaolin-ACT and Anti-Xa, and their predictive power in detecting circulating heparin levels post-reperfusion of the liver graft when compared with thromboelastogram (TEG) r time ratio in patients undergoing orthotopic liver transplantation (OLT).
Prospective, observational cohort study design.
Single center, university hospital.
Thirty-eight consecutive adults who had undergone liver transplant.
None.
Paired arterial blood samples were collected before surgical incision, 5 minutes after administration of an average dose of 2,054±771 units of intravenous unfractionated heparin before caval cross-clamping, 5 minutes after portal reperfusion, 5 minutes after hepatic artery reperfusion, and 1 hour after hepatic artery reperfusion. The observations that heparin assay measurements were within the predetermined limits of agreement, strongly suggested the two heparin assays (Hepcon HMS plus and Anti-Xa assay) are interchangeable during prophylactic heparin dose therapy during OLT. Post-reperfusion, receiver operating characteristic curve analysis revealed high accuracy in measuring circulating heparin levels with both Anti-Xa and Hepcon HMS assays when compared with the TEG r time ratio assay.
The point-of-care Hepcon HMS plus/Kaolin-ACT (activated clotting time) assay appeared to be a reliable alternative to the more expensive and laboratory-required Anti-Xa assay in monitoring the response to intravenous heparin in patients undergoing OLT.
The government is seeking views on the mandatory reporting of child abuse and neglect ( tinyurl.com/zuhhqt2 ). It is important that nurses contribute as they could face sanctions if the failure to act on suspicions became a criminal offence. Theconsultation closes on 13 October.
A palliative care charity has developed a new approach to end of life care based on the Human Rights Act (HRA).
Tissue engineering strategies have shown promise in promoting healing and regeneration after spinal cord injury (SCI); however, these strategies are limited by inflammation and the immune response. Infiltration of cells of the innate and adaptive immune responses and the inflammation that follows cause secondary damage adjacent to the injury, increased scarring, and a potently inhibitory environment for the regeneration of damaged neurons. While the inflammation that ensues is typically associated with limited regeneration, the immune response is a crucial element in the closing of the blood-brain barrier, minimizing the spread of injury, and initiating healing. This review summarizes the strategies that have been developed to modulate the immune response towards an anti-inflammatory environment that is permissive to the regeneration of neurons, glia, and parenchyma. We focus on the use of biomaterials, biologically active molecules, gene therapy, nanoparticles, and stem cells to modulate the immune response, and illustrate concepts for future therapies. Current clinical treatments for SCI are limited to systemic hypothermia or methylprednisolone, which both act by systemically mitigating the effects of immune response but have marginal efficacy. Herein, we discuss emerging research strategies to further enhance these clinical treatments by directly targeting specific aspects of the immune response.
Although the input of multiparameter flow cytometry (MFC) into the clinical management of multiple myeloma patients has faced some reluctance, continuously growing evidence supports the utility of MFC in this disease.
MFC immunophenotyping of bone marrow and peripheral blood plasma cells affords cost-effective assessment of clonality, and provides prognostic information on the risk of progression in smoldering multiple myeloma, and the identification of active multiple myeloma patients with dismal outcome (e.g., high numbers of circulating tumor cells) or long-term survival despite suboptimal responses through the characterization of monoclonal gammopathy of undetermined significance-like phenotypes. Extensive data indicate that minimal residual disease (MRD) monitoring can be used as biomarker to evaluate treatment efficacy and act as surrogate for survival. The time has come to address within clinical trials the exact role of baseline risk factors and MRD monitoring for tailored therapy in multiple myeloma, which implies systematic usage of highly sensitive cost-effective, readily available, and standardized MRD techniques such as MFC.
Next-generation MFC should be considered mandatory in the routine evaluation of multiple myeloma patients both at diagnosis and after therapy, and represents an attractive technique to integrate with high-throughput DNA and RNA-seq methods to help in understanding the mechanisms behind dissemination and chemoresistance of multiple myeloma.
The Affordable Care Act places a newfound emphasis on patient-centered medical home and patient-centered care (PCC). The purpose of this study was to evaluate the relationship between PCC, diabetes self-care, glycemic control, and quality of life (QOL) in a sample of adults with type 2 diabetes.
Six hundred fifteen patients were recruited from two adult primary care clinics in the southeastern United States. Primary outcome variables were self-care behaviors (medication adherence, diet, exercise, blood sugar testing, and foot care), glycemic control, and QOL (physical component summary [PCS] score and mental component summary [MCS] score of SF12). PCC was assessed using a modified 7-item Picker Patient Experience Questionnaire. Regression modeling was used to assess independent associations while adjusting for relevant covariates.
In adjusted analyses, PCC was significantly associated with PCS QOL (β = -0.03, 95% confidence interval [CI] -0.05 to -0.01), MCS QOL (β = 0.09, 95% CI 0.04-0.14), medication adherence (β = 0.12, 95% CI 0.08-0.17), general diet (β = 0.12, 95% CI 0.07-0.17), specific diet (β = 0.05, 95% CI 0.01-0.08), blood sugar testing (β = 0.09, 95% CI 0.04-0.15), and foot care (β = 0.12, 95% CI 0.07-0.18).
PCC is associated with diabetes self-management and QOL, but was not significantly associated with glycemic control in patients with diabetes. PCC may be an important factor in self-care behaviors, but the process of focusing care around the patient may need to expand throughout the healthcare system before changes in outcomes such as glycemic control are noted.
To report the efficacy and tolerability of mycophenolate mofetil (MMF) and azathioprine (AZA) in the management of systemic sclerosis-associated interstitial lung disease (SSc-ILD).
Patients in the Australian Scleroderma Cohort Study treated with at least 3 months of MMF or AZA for SSc-ILD confirmed on high resolution computed tomography (HRCT) chest were identified and their pulmonary function tests (PFTs) retrieved. Individuals with available results for T-1 (12 months prior to treatment commencement), T0 (date of treatment commencement) and at least one subsequent time point were included in the drug efficacy analysis. The Wilcoxon signed-rank test was used to compare absolute FVC at T1, T0, 12 months (T1), 24 months (T2) and 36 months (T3). Analysis of drug tolerability included all identified patients treated with MMF or AZA.
18/22 patients treated with MMF and 29/49 treated with AZA had adequate PFTs for inclusion in the drug efficacy analysis. Median absolute FVC at T1 for MMF treatment was 2.50L, declining to 2.12L at T0 (p=0.02). Following MMF therapy, FVC results were stable at T1 (2.13L, p=0.86), T2 (2.17L, p=0.65) and T3 (2.25L, p=0.78). In the AZA group, a statistically significant decline did not occur prior to treatment, however FVC results remained stable at T1, T2 and T3.Adverse events leading to early discontinuation (<12 months treatment) were less common in the MMF group (4/22 vs. 13/49). Gastrointestinal complications were the main cause of discontinuation in both groups.
In patients with SSc-ILD with declining pulmonary function, MMF therapy was associated with stability for up to 36 months. Early adverse events leading to discontinuation occurred less frequently in patients treated with MMF than in AZA treated patients.
γδ T cells of the Vγ9Vδ2 subtype secrete anti-fibrotic cytokines upon isopentenyl pyrophosphate (IPP) stimulation. In this study, we sought to compare IPP and Zoledronate, an up-regulator of IPP, effects on proliferation and cytokine secretion of Vγ9+ T cells from systemic sclerosis (SSc) patients and healthy controls (HCs). We also examined the effect of IPP-triggered peripheral blood mononuclear cells (PBMC) on fibroblast procolla- gen secretion.
PBMC from SSc patients and HCs were stimulated by increasing concentrations of Zoledronate, with or without IPP, and Vγ9+ T cell percentages were calculated using FACScan analysis. Subsequently, PBMC were cultured with IPP or toxic shock syndrome toxin-1 (TSST-1), and contents of the anti-fibrotic cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ were measured by ELISA kits. Finally, supernatants of IPP-triggered Vγ9+ T cells from SSc patients were added to fibroblast cultures, and relative intensities of procollagen α1 chains were determined by densinometry.
Higher concentrations of Zoledronate were required for maximal proliferation of Vγ9+ T cells in 9 SSc patients compared to 9 HCs, irrespective of exogenous IPP. When compared to stimulation by TSST-1, a non-Vγ9+ selective reagent, secretion of the anti-fibrotic cytokines TNF-α and IFN-γ in response to IPP was relatively diminished in SSc but not in HCs. Reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients.
Activated Vγ9+ T cells could act as anti-fibrotic mediators in SSc, although decreased responsiveness to IPP may play a role in the pathological fibrosis of this disease.
Glycyrrhetinic acid-modified chitosan (mGA-suc-CTS) is used as liver-targeted carrier for drug delivery. In this study, nanoparticles were prepared by ionic gelation process, and glycyrrhetinic acid act as the targeting ligand. The structure of the product was confirmed by IR and NMR techniques. The main aim of this study was to deliver atorvastatin directly to the liver by using same conjugate and reduce the associated side-effects, i.e. hepatotoxicity at high dose. Characterization of the developed formulation was performed by differential scanning calorimetry, particle size measurements and cellular uptake studies. Release profile, pharmacokinetics studies and organ distribution studies showed that developed formulation shows a relative higher liver uptake. The optimized formulation showed increased plasma concentration than the CTS nanoparticles as well as plain drug and the accumulation in the liver was nearly 2.59 times more than that of obtained with the CTS nanoparticles. Pharmaceutical and pharmacological indicators suggested that the proposed strategy can be successfully utilized for liver targeting of therapeutics.
Most of the newly designed drug molecules are lipophilic in nature and often encounter erratic absorption and low bioavailability after oral administration. Finding ways to enhance the absorption and bioavailability of these lipophilic drugs is one of the major challenges that face pharmaceutical industry nowadays. In view of that, the purpose of this review is to shed some light on a novel particulate self-assembling system named "beads" than can act as a safe carrier for delivering lipophilic drugs. The beads are prepared simply by mixing oils with cyclodextrin (CD) aqueous solution in mild conditions. A unique interaction between oil components and CD molecules occurs to form in situ surface-active complexes which are prerequisites for beads formation. This review mainly focuses on the fundamentals of beads preparation through reviewing present, yet scarce, literature. The key methods used for beads characterization are discussed in details. Also, the potential mechanisms by which beads increase the bioavailability of lipophilic drugs are illustrated. Finally, the related research areas that needs to be addressed in future for optimizing this promising delivery system are briefly outlined.
The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in various animal models of neuropathologies. We have previously shown the anti-inflammatory properties of TUDCA in an animal model of acute neuroinflammation. Here, we present a new anti-inflammatory mechanism of TUDCA through the regulation of transforming growth factor β (TGFβ) pathway. The bacterial lipopolysaccharide (LPS) was injected intravenously (iv) on TGFβ reporter mice (Smad-binding element (SBE)/Tk-Luc) to study in their brains the real-time activation profile of the TGFβ pathway in a non-invasive way. The activation of the TGFβ pathway in the brain of SBE/Tk-Luc mice increased 24 h after LPS injection, compared to control animals. This activation peak increased further in mice treated with both LPS and TUDCA than in mice treated with LPS only. The enhanced TGFβ activation in mice treated with LPS and TUDCA correlated with both an increase in TGFβ3 transcript in mouse brain and an increase in TGFβ3 immunoreactivity in microglia/macrophages, endothelial cells, and neurons. Inhibition of the TGFβ receptor with SB431542 drug reverted the effect of TUDCA on microglia/macrophages activation and on TGFβ3 immunoreactivity. Under inflammatory conditions, treatment with TUDCA enhanced further the activation of TGFβ pathway in mouse brain and increased the expression of TGFβ3. Therefore, the induction of TGFβ3 by TUDCA might act as a positive feedback, increasing the initial activation of the TGFβ pathway by the inflammatory stimulus. Our findings provide proof-of-concept that TGFβ contributes to the anti-inflammatory effect of TUDCA under neuroinflammatory conditions.
Noradrenaline (NA) is beneficial in the treatment of term newborns with cardiovascular compromise due to sepsis or pulmonary hypertension, but experiences with NA in preterm infants are limited. The aim of this study is to describe the efficacy and safety of NA in preterm infants. Patient records of preterm infants ≤32 weeks' gestation admitted to two hospitals between 2004 and 2015 and who received NA were reviewed for perinatal morbidities and mortality. Clinical details were collected at the time of NA use, and response on blood pressure, perfusion and oxygenation was documented as well as possible side effects. Forty-eight infants with primary diagnoses of sepsis (63 %) and pulmonary hypertension (23 %) received NA. Normotension was achieved at a median of 1 h in all but one infant at a median dose of 0.5 mcg/kg/min. Infants who died (46 %) were of younger gestational age and had worse cardiovascular function at start of NA compared to infants who survived. Tachycardia was common (31 %), but no additional effects were found on kidney or liver function.
NA appears to be tolerated safely by preterm infants with no major side effects. However, effectiveness needs to be studies further in structured trials. What is Known: • Noradrenaline is beneficial in the treatment of term newborns and infants with cardiovascular compromise. • Noradrenaline is known for its potent vasoconstrictive effects and, therefore, infrequently used in preterm infants. What is New: • Noradrenaline used in relative low dose and as first or second line support increases blood pressure in preterm infants with cardiovascular compromise. • Tachycardia was common, but no additional side effects were found.
Polytheonamide B (pTB), a highly cytotoxic peptide produced by a symbiotic bacterium of the marine sponge Theonella swinhoei, forms a transmembrane pore consisting of 49 residues. More than half of its residues are posttranslationally modified. Epimerizations result in alternating L- and D-amino acids that allow the peptide to adopt a [Formula: see text]-helical conformation. Unusually, the wide [Formula: see text]-helix of pTB is stable in a polar environment, which is in contrast to gramicidin A, an antibiotic with similar function and structure. The role of the other posttranslational modifications (PTMs) such as side chain hydroxylations, C- and N-methylations is not well understood. In this study, the importance of these PTMs for the stability of [Formula: see text]-helix is investigated using computational tools. By reverting the modified residues to their precursors and monitoring the effect on the dominant structure, we show that the N-methylations are crucial for the stability of the [Formula: see text]-helix in a polar environment. They are the driving force for the formation of stable side chain hydrogen-bond chains that act as an "exoskeleton." Such exoskeletons could present a general design strategy for helical peptides.
This research provides the first report of a major locus controlling wheat resistance to Sunn pest. It developed and validated SNP markers that will be useful for marker-assisted selection. Sunn pest (Eurygaster integriceps Puton) is the most destructive insect pest of bread wheat and durum wheat in West and Central Asia and East Europe. Breeding for resistance at the vegetative stage of growth is vital in reducing the damage caused by overwintered adult populations that feed on shoot and leaves of seedlings, and in reducing the next generation of pest populations (nymphs and adults), which can cause damage to grain quality by feeding on spikes. In the present study, two doubled haploid (DH) populations involving resistant landraces from Afghanistan were genotyped with the 90k SNP iSelect assay and candidate gene-based KASP markers. The DH lines and parents were phenotyped for resistance to Sunn pest feeding, using artificial infestation cages at Terbol station, in Lebanon, over three years. Quantitative trait locus (QTL) analysis identified a single major locus on chromosome 4BS in the two populations, with the resistance allele derived from the landrace accessions, IG139431 and IG139883. The QTL explained a maximum of 42 % of the phenotypic variation in the Cham6 × IG139431 and 56 % in the Cham6 × IG139883 populations. SNP markers closest to the QTL showed high similarity to rice genes that putatively encode proteins for defense response to herbivory and wounding. The markers were validated in a large, unrelated population of parental wheat genotypes. All wheat lines carrying the 'C-G' haplotype at the identified SNPs were resistant, suggesting that selection based on a haplotype of favourable alleles would be effective in predicting resistance status of unknown genotypes.
Although female sand flies are best known as the vectors of Leishmania parasites and viruses, several previous reports have demonstrated that these insects can also act as vectors for the trypanosomes of bats, lizards, and snakes. In this report, we created an inventory of Phlebotomine sand flies from southern Thailand. A novel trypanosome was found in a specimen of Phlebotomus stantoni, and two sand fly species newly recorded in the country, Sergentomyia khawi and Sergentomyia hivernus, were described. PCR primer pairs specific for the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal DNA (SSU rDNA) gene of trypanosomatids were used to demonstrate the presence of the parasite in the sand fly. In addition, the Cytochrome b (CytB) gene was used to identify the sand fly species. Among the 45 samples of the sand fly that were collected, seven samples were Ph. stantoni sand flies and a single sample was positive for Trypanosoma sp. through PCR analysis. This study represents the first detection of Trypanosoma sp. in a sand fly from Thailand. The ITS1 and SSU rDNA sequences indicated that this specimen is suspected to be a novel Trypanosoma species. Further studies of this suspected new Trypanosoma species, including its vertebrate hosts and pathogenic potential, are therefore necessary.
Quality control is an essential aspect of cellular function, with protein folding quality control being carried out by molecular chaperones, a diverse group of highly conserved proteins that specifically identify misfolded conformations. Molecular chaperones are thus required to support proteins affected by expressed polymorphisms, mutations, intrinsic errors in gene expression, chronic insult or the acute effects of the environment, all of which contribute to a flux of metastable proteins. In this article, we review the four main chaperone families in metazoans, namely Hsp60 (where Hsp is heat-shock protein), Hsp70, Hsp90 and sHsps (small heat-shock proteins), as well as their co-chaperones. Specifically, we consider the structural and functional characteristics of each family and discuss current models that attempt to explain how chaperones recognize and act together to protect or recover aberrant proteins.
All organisms rely on a conserved cellular machinery supporting and controlling the life cycle of proteins: the proteostasis network. Within this network, the main players that determine the fate of proteins are molecular chaperones, the ubiquitin-proteasome and the lysosome-autophagy systems. sHsps (small heat-shock proteins) represent one family of molecular chaperones found in all domains of life. They prevent irreversible aggregation of unfolded proteins and maintain proteostasis by stabilizing promiscuously a variety of non-native proteins in an ATP-independent manner. In the cellular chaperone network, sHsps act as the first line of defence and keep their substrates in a folding-competent state until they are refolded by downstream ATP-dependent chaperone systems. Besides this interaction with unfolding substrates upon stress, sHsps show a different mode of binding for specific clients which are also recognized under physiological conditions. In vertebrates, sHsps are especially needed to maintain the refractive index of the eye lens. Additionally, sHsps are linked to a broad variety of diseases such as myopathies and neuropathies. The most striking feature of sHsps is their ability to form dynamic ensembles of higher oligomers. The activity of sHsps is regulated by changes in the composition of the ensembles.
Most people with febrile illness are treated in the private drug retail sector. Ghana was among nine countries piloting the Global Fund Affordable Medicines Facility - malaria (AMFm). AMFm aimed to: increase artemisinin combination therapy (ACT) affordability; increase ACT availability; increase ACT use; and 'crowd out' artemisinin monotherapies.
Three censuses were carried out 2 months before (2010), 2 months after and 2.5 years after (2013) the first co-paid ACTs to assess changes in antimalarial (AM) availability and price in private retail shops in a Ghanaian rural district to assess the sustainability of the initial gains. Supply, stock-out and cost were explored.
Of 62 shops in the district, 56 participated with 398, 388 and 442 brands of AMs in the shops during the 3 censuses. The proportion of ACTs increased over the period while monotherapies reduced. Herbal-based AM preparations comprised 40-45% of AMs in stock with minimal variation over the period. ACTs were the most sold AM type for all ages but overall buying and selling prices of Quality Assured-ACTs increased by 40-100%.
Initial gains in ACT availability were sustained, but not improved on 2.5 years after AMFm. Widespread availability of unproven herbal medicines is a concern; AMFm had little impact on this.
The synthesis, biological evaluation and molecular modeling study of 6,7-dihydro-[1,3,4] thiadiazolo[3,2-a][1,3]diazepine analogues as new class of neuromuscular blocking agents are described. The new compounds act via competitive mechanism with ACh which could be reversed by the anticholinesterase - Physostigmine. Compounds GS-53 (30) and AAH1 (33) induced dose-dependent neuromuscular blockade with onset time of 3 and 10 min, ED50 0.15 and 0.36 mmol/kg i.p., respectively, in rats. Compound 30 proved to be as twice as potent as 33 with rapid onset and shorter duration (P < 0.05). Docking profile of 30 and 33 closely resembles HIE-124 (3), in α7β2 nAChR receptor. Molecular modeling analysis indicated that hydrogen bonding to Thr120 and Thr124 beside hydrophobic interactions play effective role incorporating the active ligands to nAChR. The obtained model could be useful for further development of new skeletal muscle relaxants.
We assessed whether coal tar present in contaminated streambed sediments can be mobilized by flood events and be re-deposited in an adjacent floodplain. The study was conducted within a contaminated urban stream where coal tar wastes were released into a 4-km reach from a coke plant in Chattanooga, Tennessee, USA. Sediments containing visible amounts of coal tar were dredged from the streambed in 1997-98 and 2007 as part of a cleanup effort. However, post-dredging sampling indicated that very high concentrations of polycyclic aromatic hydrocarbons (PAHs) remained in streambed sediments. Sampling of sediments in the floodplain at two sites downstream of the coke plant indicated that high concentrations of PAHs were also present in the floodplain, even though no coal tar was observed in the samples. Age-dating of the floodplain sediments using (137)Cs indicated that peak PAH concentrations were contemporary with coke plant operations. While there was little or any direct contamination of the floodplain sediments by coal tar, sediment contamination was likely a result of deposition of suspended streambed sediments containing sorbed PAHs. A flood model developed to delineate the extent of flooding in various flood recurrence scenarios confirmed the potential for contaminated streambed sediments to be transported into the adjacent floodplain. It was hypothesized that coal tar, which was visibly "sticky" during dredging-based stream cleanup, may act as a binding agent for streambed sediments, decreasing mobility and transport in the stream. Therefore, coal tar is likely to remain a persistent contaminant source for downstream reaches of the stream and the adjacent floodplain during flood events. This study also showed that even after excavation of tar-rich streambed sediments, PAH contaminated non-tarry sediments may be a source of flood-related contamination in the adjacent flood plain. A conceptual framework was developed to delineate specific mechanisms that can mobilize contamination from stream sources.
Adsorption of hydrogen (H2) and nitrogen (N2) molecules was analyzed on a new fullerene-like C16Mg8O8 nano-cage, composed of magnesium, oxygen, and carbon, using density functional theory. A detailed analysis of the energy, geometry, and electronic structure of various H2 and N2 adsorptions on the cluster surface was performed. The adsorption energies of H2 and N2 were estimated to ranging from -0.16 to -0.52eV, respectively. The most stable adsorption configurations were those in which the H or N atoms of the adsorbates were located near the Mg atom of the cluster surface at different sides. It was found that the heterogeneous C16Mg8O8 nano-cluster selectively act against the H2 and N2 gaseous molecules. The electrical conductivity of the cluster, arising from HOMO/LUMO energy gap, was more sensitive to N2 gaseous molecule rather than H2 one, indicating that the heterogeneous C16Mg8O8 nano-cage may be potential nano-sensor for N2 molecule. These findings were specified by analyzing the characteristics in the electron density of states (DOS).
This article reports on the first web-based implementation of an opioid-overdose prevention, recognition and response training for professional first responders. The training was disseminated nationally over one listserv in November 2014. The same year, following Act 139, which mandated the provision of an online training for police officers in Pennsylvania, the Pennsylvania Department of Health approved the training. It was subsequently adopted as the primary training tool for police and other first responders in Pennsylvania and has been used as a training tool by first responders nationally.
Analyses employed descriptive statistics to report characteristics of a sample of 387 professional first responders who completed a survey about their experience with the online training. Z-ratios were used to compare independent proportions related to overdose, naloxone, and satisfaction with the training between key subgroups, and paired t-tests were used to compare participant responses to a range of items pre- and post-participation in the training.
Between January-October 2015, 4804 first responders took the training; 1697 (35.3%) agreed to be contacted; of these, 387 (22.8%) completed a survey about the training and subsequent overdose response experiences. The majority (86.4%) were from Pennsylvania, with police representing over half of the sample. Analysis of the post-training survey indicates high satisfaction with content, format and mode of delivery, and high satisfaction with items related to confidence and overdose reversal preparedness.
This study demonstrates the feasibility and acceptability of implementing online training for first responders in overdose prevention, recognition and response.
Chagas is a parasitic disease with major threat to public health due to its resistance against commonly available drugs. Trypanothione reductase (TryR) is the key enzyme to develop this disease. Though this enzyme is well thought-out as potential drug target, the accurate structure of enzyme-inhibitor complex is required to design a potential inhibitor which is less available for TryR. In this research, we aimed to investigate the advanced drug over the available existing drugs by designing inhibitors as well as to identify a new enzyme-inhibitor complex that may act as a template for drug design. A set of analogues were designed from a known inhibitor Quinacrine Mustard (QUM) to identify the effective inhibitor against this enzyme. Further, the pharmacoinformatics elucidation and structural properties of designed inhibitor proposed effective drug candidates against Chagas disease. Molecular docking study suggests that a designed inhibitor has higher binding affinity in both crystal and modeled TryR and also poses similar interacting residues as of crystal TryR-QUM complex structure. The comparative studies based on in silico prediction proposed an enzyme-inhibitor complex which could be effective to control the disease activity. So our in silico analysis based on TryR built model, Pharmacophore and docking analysis might play an important role for the development of novel therapy for Chagas disease. But both animal model experiments and clinical trials must be done to confirm the efficacy of the therapy.
Despite the potential risks of drug use during pregnancy, consumption has increased in recent decades.
To identify the risk of congenital anomalies (CA) associated with the use of drugs in primary care in pregnant women residents in the Valencia Region.
A case-control study, considering a case as a less than one year old live birth in 2009-2010, diagnosed with a CA and resident in the Valencia Region, obtained from the CA population-based registry. Controls were selected from the Metabolic Disease Registry, and the drugs prescribed and dispensed from the Integral Management of Pharmaceutical Services. Crude odds ratio (OR) was calculated with its 95% confidence intervals and adjusted OR was calculated using logistic regression.
A total of 1,913 cases and 3,826 controls were identified. The most frequently used drug groups were those acting on the musculoskeletal, nervous and respiratory systems, on the blood and blood forming organs, and anti-infection drugs. The most common drugs used were ibuprofen, dexketoprofen, paracetamol, amoxicillin, ferrous sulphate, and a combination of folic acid. A significantly increased risk of CA was identified for drugs acting on the musculoskeletal system (adjusted OR 1.14 [95% confidence interval 1.02-1.28]). A significantly decreased risk was observed for drugs acting on the blood and blood forming organs (adjusted OR 0.87 [95% confidence interval 0.78-0.98]).
Associations between drugs and CA in pregnant women resident in the Valencia Region have been identified for drugs that act as risk factors of CA, and for drugs that act as protective factors of CA.
Foot-and-mouth-disease (FMD) remains the most infectious livestock disease worldwide. Although commercially available inactivated or adenovirus-vectored-vaccines (Ad5-FMD) are effective, they require 5-7 days to induce protection. Therefore, new control strategies that stimulate rapid immune responses are needed. Expression of bovine interferon λ3 using the Ad5-vector platform (Ad5-boIFNλ3) is able to delay disease in cattle, but clinical signs appear at 9 days after challenge. We hypothesized that combination of Ad5-boIFNλ3 and Ad5-FMD could induce immediate and lasting protection against FMD. Cattle were vaccinated with an Ad5-FMD, Ad5-boIFNλ3, or the combination of both, followed by challenge at three days post-immunization. All animals treated with Ad5-FMD combined with Ad5-boIFNλ3 were fully protected against FMD, despite the absence of systemic neutralizing antibodies or antiviral activity at the time of challenge. Induction of a strong cell-mediated immune response suggested that Ad5-boIFNλ3 is able to act as an adjuvant of Ad5-FMD vaccine in cattle.
The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.
AMPK dysregulation contributes to the onset and development of type 2 diabetes (T2DM). AMPK is known to be activated by reactive oxygen species (ROS) and antioxidant interference. However the mechanism by which redox state mediates such contradictory result remains largely unknown. Here we used streptozotocin-high fat diet （STZ-HFD） induced-type 2 diabetic rats and cells lines (L02 and HEK 293) to explore the mechanism of redox-mediated AMPK activation. We show glutaredoxins (Grxs) concomitant with optimal ROS act as an essential mediator for AMPK activation. ROS level results in different mechanisms for AMPK activation. Under low ROS microenvironment, Grxs-mediated S-glutathionylation on AMPK-α catalytic subunit activates AMPK to improve glucose transportation and degradation while inhibiting glycogen synthesis and keeping redox balance. While, under high ROS microenvironment, AMPK is activated by an AMP-dependent mechanism, however sustained high level ROS also causes loss of AMPK protein. This finding provides evidence for a new approach to diabetes treatment by individual doses of ROS or antioxidant calibrated against the actual redox level in vivo. Moreover, the novel function of Grxs in promoting glucose metabolism may provide new target for T2DM treatment.
Mesenchymal stem cells (MSCs) are non-hematopoietic cells that occur in almost all human tissues and can be cultured and expanded to large numbers in vitro. They secrete growth factors, cytokines, and chemokines and express Toll-like receptors on their surface, although multiple cell biological mechanisms remain unclear. MSCs are multi-potent and can differentiate into many cell types including adipocytes, neuronal cells and osteoclasts. Despite gaps in cell biology, because of their immunomodulatory and regenerative capacity, several hundred clinical trials have used MSCs for therapy of cancer, autoimmune diseases and control of inflammation during organ transplantation. MSCs secrete immune-modulatory factors and are able to skew T cell responses and shift M1 to M2 differentiation of macrophages. We review the emerging role of MSCs to act as phagocytes for Mycobacterium tuberculosis and its role during the persistence of M. tuberculosis and spread of infection. Paradoxically, MSCs use innate defense mechanisms of autophagy and nitric oxide to inhibit the growth of intracellular M. tuberculosis. In addition, transplantation with autologous MSCs improved the clinical condition of patients with multi-drug resistant tuberculosis. Thus, in addition to the well-known immune defense played by macrophages, DCs, classical T cells and non-classical immune cells, MSCs have emerged as a fifth element capable of regulating immune responses during tuberculosis. We discuss their immunomodulatory properties and innate defense mechanisms in the context of developing immunotherapeutic strategies for tuberculosis.
Drugs that enhance the action of serotonin (5-hydroxytrypamine, 5-HT), including several selective serotonin reuptake inhibitors (SSRIs), reduce susceptibility to seizure-induced respiratory arrest (S-IRA) that leads to death in the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP). However, it is not clear if specific 5-HT receptors are important in the action of these drugs and whether the brain is the major site of action of these agents in this SUDEP model. The current study examined the actions of agents that affect the 5-HT3 receptor subtype on S-IRA and whether intracerebroventricular (ICV) microinjection of an SSRI would reduce S-IRA susceptibility in DBA/1 mice. The data indicate that systemic administration of SR 57227, a 5-HT3 agonist, was effective in blocking S-IRA in doses that did not block seizures, and the S-IRA blocking effect of the SSRI, fluoxetine, was abolished by coadministration of a 5-HT3 antagonist, ondansetron. Intracerebroventricular administration of fluoxetine in the present study was also able to block S-IRA without blocking seizures. These findings suggest that 5-HT3 receptors play an important role in the block of S-IRA by serotonergic agents, such as SSRIs, which is consistent with the abnormal expression of 5-HT3 receptors in the brainstem of DBA mice observed previously. Taken together, these data indicate that systemically administered serotonergic agents act, at least, in part, in the brain, to reduce S-IRA susceptibility in DBA/1 mice and that 5-HT3 receptors may be important to this effect.
The presented work characterized components of food contact materials (FCM) with potential to bind to estrogen receptor (ER) and cause adverse effects in the human organism.
The QSAR Toolbox, software application designed to identify and fill toxicological data gaps for chemical hazard assessment, was used. Estrogen receptors are much less of a lock-and-key interaction than highly specific ones. The ER is nonspecific enough to permit binding with a diverse array of chemical structures. There are three primary ER binding subpockets, each with different requirements for hydrogen bonding.
More than 900 compounds approved as of FCM components were evaluated for their potential to bind on ER. All evaluated chemicals were subcategorized to five groups with respect to the binding potential to ER: very strong, strong, moderate, weak binder, and no binder to ER. In total 46 compounds were characterized as potential disturbers of estrogen receptor.
Among the group of selected chemicals, compounds with high and even very high affinity to the ER binding subpockets were found. These compounds may act as gene activators and cause adverse effects in the organism, particularly during pregnancy and breast-feeding. It should be considered to carry out further in vitro or in vivo tests to confirm their potential to disturb the regulation of physiological processes in humans by abnormal ER signaling and subsequently remove these chemicals from the list of approved food contact materials.
Axon guidance is proposed to act through a combination of long- and short-range attractive and repulsive cues. The ligand-receptor pair, Netrin (Net) and Frazzled (Fra) (DCC, Deleted in Colorectal Cancer, in vertebrates), is recognized as the prototypical effector of chemoattraction, with roles in both long- and short-range guidance. In the Drosophila visual system, R8 photoreceptor growth cones were shown to require Net-Fra to reach their target, the peak of a Net gradient. Using live imaging, we show, however, that R8 growth cones reach and recognize their target without Net, Fra, or Trim9, a conserved binding partner of Fra, but do not remain attached to it. Thus, despite the graded ligand distribution along the guidance path, Net-Fra is not used for chemoattraction. Based on findings in other systems, we propose that adhesion to substrate-bound Net underlies both long- and short-range Net-Fra-dependent guidance in vivo, thereby eroding the distinction between them.
Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.
To characterize the underlying genetic and molecular defects in a consanguineous family with life-long blood disorder manifested with thrombocytopenia (low platelets count) and anemia.
Genetic linkage analysis, exome sequencing and functional genomics were carried out to identify and characterize the defective gene.
We identification of a novel truncation mutation (p.C108*) in Chromosome 6 Open Reading Frame 25 (C6orf25) gene in this family. We also showed the p.C108* mutation, was responsible for destabilizing the encoded truncated G6B protein. Unlike the truncated form, wild-type G6B expression resulted in enhanced K562 differentiation into megakaryocytes and erythrocytes. C6orf25, also known as G6B, is an effector protein for the key hematopoiesis regulators, Src homology region 2 domain-containing phosphatases SHP-1 and SHP-2.
G6B seems to act through an autosomal recessive mode of disease transmission in this family, and regarded as the gene responsible for the observed hematological disorder. This inference is well supported further by in vivo evidence where similar outcomes were reported from G6b(-/-) and SHP1/2 DKO mouse models. This article is protected by copyright. All rights reserved.
Human milk oligosaccharides (HMOs) are a highly abundant constituent in human milk, and its protective and prebiotic properties have attracted considerable attention. HMOs have been shown to directly and indirectly benefit the overall health of the infant due to a number of functions including serving as a beneficial food for gut bacteria, block to pathogens, and aiding in brain development. Researchers are currently exploring whether these structures may act as possible disease and nutrition biomarkers. Because of this, rapid-throughput methods are desired to investigate biological activity in large patient sets. We have optimized a rapid-throughput protocol to analyze human milk oligosaccharides using micro-volumes of human breast milk for nutritional biomarkers. This method may additionally be applied to other biological fluid substrates such as plasma, urine, and feces. The protocol involves lipid separation via centrifugation, protein precipitation using ethanol, alditol reduction with sodium borohydride, and a final solid-phase extraction purification step using graphitized carbon cartridges. Samples are analyzed using HPLC-Chip/TOF-MS and data filtered on Agilent MassHunter using an in-house library. Individual structural identification is matched against a previously developed HMO library using accurate mass and retention time. Using this method will allow in-depth characterization and profiling of HMOs in large patient sets, and will ease the process of discovering significant nutritional biomarkers in human milk.
The action-specific approach to perception claims that a person's ability to act directly influences perceptual processes related to spatial vision. For example, a person's ability to block a moving ball impacts perceptual judgments of the ball's speed. However, an alternative explanation is that action rather than perception influences judgments. Here, we explore this distinction directly. Our method produces two distinct effects, one that is clearly a judgment-based effect and is based on the outcome of the trial (trial-outcome effect) and one that is under debate as to whether or not it is perceptual and is based on the ease with which the ball can be blocked (paddle-size effect). We explored whether these two effects would produce convergence or dissociations across various populations and manipulations. A dissociation is evidence for two separate underlying processes, whereas if the two effects did not dissociate, this would be consistent with claims that both effects were judgment-based. In Experiment 1, we examined whether older and younger adults would show a dissociation between the two effects given some precedent for older adults to show greater susceptibility to nonperceptual factors in their judgments. In Experiment 2, we used a cover story to excuse poor performance and examined its effects on both types of effects. Both experiments revealed dissociations, suggesting that while one effect is judgment-based, the other effect is not. Coupled with prior research, we conclude that the action-specific effect of ease to block a ball on estimated ball speed is perceptual.
Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential sources of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic Escherichia coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analysed. Among hundreds of compounds, only a few homologous compounds were identified that contained the isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole-containing analogs, sulfonamides, furanones, and flavanones and known to possess broad-spectrum antimicrobial properties and anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization, and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs.
Recent trials of adoptive cell therapy (ACT), such as the chimeric antigen receptor T (CAR-T) cells therapy, have demonstrated promising therapeutic effects for cancer patients. A main issue in the product development is to decide appropriate dose of ACT. Traditional phase 1 trial designs for cytotoxic agents explicitly assume that toxicity increases monotonically with dose levels and implicitly assume the same for efficacy to justify dose escalation. ACT usually induces rapid responses, and the monotonic dose-response assumption is unlikely to hold due to its immunobiological activities. We propose a toxicity and efficacy probability interval (TEPI) design for dose-finding in ACT trials. This approach incorporates efficacy outcomes to inform dosing decisions to optimize efficacy and safety simultaneously. Rather than finding the maximum tolerated dose (MTD) the TEPI design aims at the dose with the most desirable outcome for safety and efficacy. The key features of TEPI are its simplicity, flexibility, and transparency, since all decision rules can be prespecified prior to trial initiation. We conduct simulation studies to investigate the operating characteristics of the TEPI design and compare it to existing methods. In summary, the TEPI design is a novel method for ACT dose finding that possesses superior performance and is easy to use, simple, and transparent.
During spermatogenesis, germ cells that fail to synapse their chromosomes or fail to undergo meiotic sex chromosome inactivation (MSCI) are eliminated via apoptosis during mid-pachytene. Previous work showed that Y-linked genes Zfy1 and Zfy2 act as "executioners" for this checkpoint, and that wrongful expression of either gene during pachytene triggers germ cell death. Here, we show that in mice, Zfy genes are also necessary for efficient MSCI and the sex chromosomes are not correctly silenced in Zfy-deficient spermatocytes. This unexpectedly reveals a triple role for Zfy at the mid-pachytene checkpoint in which Zfy genes first promote MSCI, then monitor its progress (since if MSCI is achieved, Zfy genes will be silenced), and finally execute cells with MSCI failure. This potentially constitutes a negative feedback loop governing this critical checkpoint mechanism.
Middle-aged women are at risk of weight gain and associated comorbidities. Deliberate restriction of food intake (dieting) produces short-term weight loss but is largely unsuccessful for long-term weight management. Two promising approaches for the prevention of weight gain are intuitive eating (ie, eating in accordance with hunger and satiety signals) and the development of greater psychological flexibility (ie, the aim of acceptance and commitment therapy [ACT]).
This pilot study investigated the usage, acceptability, and feasibility of "Mind, Body, Food," a Web-based weight gain prevention intervention prototype that teaches intuitive eating and psychological flexibility skills.
Participants were 40 overweight women (mean age 44.8 [standard deviation, SD, 3.06] years, mean body mass index [BMI] 32.9 [SD 6.01] kg/m(2), mean Intuitive Eating Scale [IES-1] total score 53.4 [SD 7.46], classified as below average) who were recruited from the general population in Dunedin, New Zealand. Module completion and study site metrics were assessed using Google Analytics. Use of an online self-monitoring tool was determined by entries saved to a secure online database. Intervention acceptability was assessed postintervention. BMI, intuitive eating, binge eating, psychological flexibility, and general mental and physical health were assessed pre- and postintervention and 3-months postintervention.
Of the 40 women enrolled in the study, 12 (30%) completed all 12 modules (median 7.5 [interquartile range, IQR, 2-12] modules) and 4 (10%) used the self-monitoring tool for all 14 weeks of the intervention period (median 3 [IQR 1-9] weeks). Among 26 women who completed postintervention assessments, most women rated "Mind, Body, Food" as useful (20/26, 77%), easy to use (17/25, 68%) and liked the intervention (22/25, 88%). From pre- to postintervention, there were statistically significant within-group increases in intuitive eating (IES-2 total score P<.001; all IES-2 subscale scores: P ≤.01), psychological flexibility (P=.01), and general mental health (P<.001) as well as significant decreases in binge eating (P=.01). At the 3-month follow-up, IES-2 improvements were maintained, and there were further improvements in binge eating (P<.001) and general mental health (P=.03), and a marginal yet nonsignificant tendency for further improvement in psychological flexibility (P=.06). There were no significant within-group changes in BMI from pre- to postintervention and postintervention to 3-month follow-up (P=.46 and P=.93, respectively).
The "Mind, Body, Food" prototype Web-based intervention is appealing to middle-aged women and may be a useful tool to help women learn intuitive eating and ACT skills, reduce binge eating, and maintain weight over 3 months. Further work to improve the user experience and engagement is required before testing the online intervention in a randomized controlled trial.
Positron emitting isotopes such as (11)C and (10)C can be used for vital dose verification in hadron therapy. These isotopes are produced when the high energy (12)C primary beam particles undergo nuclear reactions within the patient.
We discuss a model for calculating cross sections for the production (11)C in (12)C+(12)C collisions, applicable at hadron therapy energies.
Good agreement with the available cross section measurements is found for (12)C(-1n), though more detailed, systematic measurements would be very valuable.
Nuclear structure plays a crucial role in the reactions of light nuclei, particularly when those reactions are peripheral and involve only a few nucleons. For such reactions, nuclear structure has a strong influence on the energy and angular distribution of the cross section, and is an important consideration for reliable dose verification using (11)C in hadron therapy.
Australia has a large immigrant population but there are few data regarding whether influenza vaccine coverage in adults varies according to country of birth. We quantified and compared self-reported influenza vaccination coverage between Australian-born and immigrant residents aged ⩾49years enrolled in a large cohort (the 45 and Up Study), surveyed in 2012 and 2013. Estimated vaccine coverage was adjusted for age, sex and other factors known to be associated with vaccine uptake. Among 76,040 participants included in the analyses (mean age 66.2years), 21.6% were immigrants. In Australian-born adults aged 49-64 and 65+ years the age- and sex-adjusted estimates for influenza vaccination within the year prior to survey was 39.5% (95% CI 38.9-40.0) and 70.9% (70.4-71.5) respectively. The corresponding estimates in immigrants were significantly lower at 34.8% (33.7-35.8) and 64.4% (63.4-65.4) respectively. Among immigrants, coverage varied by region of birth, and was slightly lower among those who spoke a language other than English at home compared to those who only spoke English. Among immigrants there was no significant difference in coverage comparing those who migrated when they were children to those who migrated as adults and coverage did not differ significantly according to years lived in Australia. Programs to increase adult vaccination coverage should consider the needs of immigrants.
The dual-specificity kinases MEK1 and MEK2 act downstream of RAS/RAF to induce ERK activation, which is generally considered protumorigenic. Activating MEK mutations have not been discovered in leukemia, in which pathway activation is caused by mutations in upstream components such as RAS or Flt3. The anti-leukemic potential of MEK inhibitors is being tested in clinical trials; however, downregulation of MEK1 promotes Eμ-Myc-driven lymphomagenesis and MEK1 ablation induces myeloproliferative disease in mice, raising the concern that MEK inhibitors may be inefficient or counterproductive in this context. We investigated the role of MEK1 in the proliferation of human leukemic cell lines and in retroviral models of leukemia. Our data show that MEK1 suppression via RNA interference and genomic engineering does not affect the proliferation of human leukemic cell lines in culture; similarly, MEK1 ablation does not impact the development of MYC-driven leukemia in vivo. In contrast, MEK1 ablation significantly reduces tumorigenesis driven by Nras alone or in combination with Myc. Thus, while MEK1 restricts proliferation and tumorigenesis in some cellular and genetic contexts, it cannot be considered a tumor suppressor in the context of leukemogenesis. On the contrary, its role in NRAS-driven leukemogenesis advocates the use of MEK inhibitors, particularly in combination with PI3K/AKT inhibitors, in hematopoietic malignancies involving RAS activation.
Stress results in a variety of neuroendocrine, immune and behavioral responses and represents a risk factor for many disorders. Following exposure to stress, glucocorticoids are secreted from the adrenal cortex and act via the ligand-activated glucocorticoid receptor (GR). Several polymorphisms of the GR-encoding gene NR3C1 have been described and functionally investigated. However, the impact of these variants on complex diseases such as Attention-Deficit/Hyperactivity Disorder (ADHD) is still unclear. In this study, 251 children with ADHD, 19 affected and 35 unaffected siblings, and their parents were included in a family-based association study assessing seven common variants of NR3C1 (TthIIII_rs10052957; NR3C1-I_rs10482605; ER22/23EK_rs6189/rs6190; N363S_rs56149945; BclI_rs41423247; GR-9beta_rs6198). A four-marker haplotype (TthIIII-NR3C1-I-ER22/23EK) was nominally associated with ADHD. In addition, in index children with ADHD, associations with comorbid disorders, inattentive and hyperactive-impulsive symptoms were explored. N363S minor allele carriers were more likely to show comorbid conduct disorder (CD). In our study, NR3C1 variants moderately affected ADHD and had a significant effect on comorbid CD. Therefore, NR3C1 as an important gene of the hypothalamic-pituitary-adrenal axis seems to be particularly relevant for the pathophysiology of ADHD combined with comorbid CD. For a deeper understanding, investigations in larger samples of healthy, ADHD and CD individuals are warranted.
Fucus vesiculosus extracts that have both radical scavenging activity and metal chelating ability in vitro were used as natural antioxidant in granola bars enriched with fish oil emulsion by using primary and secondary emulsion systems stabilized by sodium caseinate alone and sodium caseinate-chitosan. The bars were stored at 20 °C and evaluated over a period of 10 weeks by measuring the development of primary and secondary oxidation products. The samples prepared with secondary emulsion system developed less oxidation products probably due to increased interfacial layer thickness that would act as a barrier to the penetration and diffusion of molecular species that promote oxidation. The positive charge of oil droplets in the secondary emulsion may also inhibit iron-lipid interaction through electrostatic repulsion. Additional protection against lipid oxidation was obtained when fish oil emulsions were added to the granola bars especially in combination with acetone and ethanol extracts of Fucus vesiculosus.
The argument that human society can decouple economic growth-defined as growth in Gross Domestic Product (GDP)-from growth in environmental impacts is appealing. If such decoupling is possible, it means that GDP growth is a sustainable societal goal. Here we show that the decoupling concept can be interpreted using an easily understood model of economic growth and environmental impact. The simple model is compared to historical data and modelled projections to demonstrate that growth in GDP ultimately cannot be decoupled from growth in material and energy use. It is therefore misleading to develop growth-oriented policy around the expectation that decoupling is possible. We also note that GDP is increasingly seen as a poor proxy for societal wellbeing. GDP growth is therefore a questionable societal goal. Society can sustainably improve wellbeing, including the wellbeing of its natural assets, but only by discarding GDP growth as the goal in favor of more comprehensive measures of societal wellbeing.
A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stress-induced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from CDKN1A might act as a molecular switch to control spatial interactions between regulatory elements.
TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes.
During Drosophila embryonic nervous system development, neuroblasts express a programmed cascade of five temporal transcription factors that govern the identity of cells generated at different time-points. However, these five temporal genes fall short of accounting for the many distinct cell types generated in large lineages. Here, we find that the late temporal gene castor sub-divides its large window in neuroblast 5-6 by simultaneously activating two cell fate determination cascades and a sub-temporal regulatory program. The sub-temporal program acts both upon itself and upon the determination cascades to diversify the castor window. Surprisingly, the early temporal gene Kruppel acts as one of the sub-temporal genes within the late castor window. Intriguingly, while the temporal gene castor activates the two determination cascades and the sub-temporal program, spatial cues controlling cell fate in the latter part of the 5-6 lineage exclusively act upon the determination cascades.
Specialized metabolic sensors in the hypothalamus regulate blood glucose levels by influencing hepatic glucose output and hypoglycemic counterregulatory responses. Hypothalamic reactive oxygen species (ROS) may act as a metabolic signal-mediating responses to changes in glucose, other substrates and hormones. The role of ROS in the brain's control of glucose homeostasis remains unclear. We hypothesized that hydrogen peroxide (H2O2), a relatively stable form of ROS, acts as a sensor of neuronal glucose consumption and availability and that lowering brain H2O2 with the enzyme catalase would lead to systemic responses increasing blood glucose. During hyperinsulinemic euglycemic clamps in rats, intracerebroventricular catalase infusion resulted in increased hepatic glucose output, which was associated with reduced neuronal activity in the arcuate nucleus of the hypothalamus. Electrophysiological recordings revealed a subset of arcuate nucleus neurons expressing proopiomelanocortin that were inhibited by catalase and excited by H2O2. During hypoglycemic clamps, intracerebroventricular catalase increased glucagon and epinephrine responses to hypoglycemia, consistent with perceived lower glucose levels. Our data suggest that H2O2 represents an important metabolic cue, which, through tuning the electrical activity of key neuronal populations such as proopiomelanocortin neurons, may have a role in the brain's influence of glucose homeostasis and energy balance.
When ultralight axion dark matter encounters a static magnetic field, it sources an effective electric current that follows the magnetic field lines and oscillates at the axion Compton frequency. We propose a new experiment to detect this axion effective current. In the presence of axion dark matter, a large toroidal magnet will act like an oscillating current ring, whose induced magnetic flux can be measured by an external pickup loop inductively coupled to a SQUID magnetometer. We consider both resonant and broadband readout circuits and show that a broadband approach has advantages at small axion masses. We estimate the reach of this design, taking into account the irreducible sources of noise, and demonstrate potential sensitivity to axionlike dark matter with masses in the range of 10^{-14}-10^{-6}  eV. In particular, both the broadband and resonant strategies can probe the QCD axion with a GUT-scale decay constant.
What can cells gain by using disordered, rather than folded, proteins in the architecture of their skeleton? Disordered proteins take multiple coexisting conformations, and often contain segments which act as random-walk-shaped polymers. Using x-ray scattering we measure the compression response of disordered protein hydrogels, which are the main stress-responsive component of neuron cells. We find that at high compression their mechanics are dominated by gaslike steric and ionic repulsions. At low compression, specific attractive interactions dominate. This is demonstrated by the considerable hydrogel expansion induced by the truncation of critical short protein segments. Accordingly, the floppy disordered proteins form a weakly cross-bridged hydrogel, and act as shock absorbers that sustain large deformations without failure.
Despite a range of promising applications, liquid-phase exfoliation of boron nitride nanosheets (BNNSs) is limited, both by low yield in common solvents as well as the disadvantages of using dissolved surfactants. One recently reported approach is the use of cosolvent systems to increase the as-obtained concentration of BNNS; the role of these solvents in aiding exfoliation and/or aiding colloidal stability of BNNSs is difficult to distinguish. In this paper, we have investigated the use of a t-butanol/water cosolvent to disperse BNNSs. We utilize solvent-exchange experiments to demonstrate that the t-butanol is in fact essential to colloidal stability; we then utilized molecular dynamics simulations to explore the mechanism of t-butanol/BNNS interactions. Taken together, the experimental and simulation results show that the key to the success of t-butanol (as compared to the other alcohols of higher or lower molecular weight) lies in its ability to act as a "liquid dispersant" which allows it to favorably interact with both water and BNNSs. Additionally, we show that the stable dispersions of BNNS in water/t-butanol systems may be freeze-dried to yield nonaggregated, redispersible BNNS powders, which would be useful in an array of industrial processes.
Photoaffinity labels are powerful tools for dissecting ligand-protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C-H/X-H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.
The transformation of primary care (PC) training sites into patient-centered medical homes (PCMH) has implications for the education of health professionals. This study investigates the extent to which physician assistant (PA) students report learning about the PCMH model and how clinical exposure to PCMH might impact their interest in a primary care career.
An electronic survey was distributed to second-year PA students who had recently completed their PC rotation from 12 PA programs. Descriptive statistics and ordered logistic regression analyses were used to characterize the results.
A total of 202 second-year PA students completed the survey. When asked about their knowledge of the new health care delivery models, 30% of the students responded they had received instruction about the PCMH. Twenty- five percent of respondents stated they were oriented to new payment structures proposed in the Affordable Care Act and quality improvement principles. Based on their experiences in the primary care clerkship, 64% stated they were likely to pursue a career in primary care, 13% were not likely, and 23% were unsure. Predictors of interest in a primary care career included: (1) age greater than 35 years, (2) being a recipient of a NHSC scholarship, (3) clerkship site setting in an urban cluster of 2,500 to 50,000 people, (4) number of PCMH elements offered at site, and (4) positive impression of team-based care.
PA students lack adequate instruction related to the new health care delivery models. Students whose clerkship sites offered greater number of PCMH elements were more interested in pursuing a career in primary care.
Increasing evidence has indicated that long noncoding RNAs (lncRNAs) are of great importance in different cell contexts. However, only a very small number of lncRNAs have been experimentally validated and functionally annotated during human hematopoiesis. Here, we report an lncRNA, HOTAIRM1, which is associated with myeloid differentiation and has pivotal roles in the degradation of oncoprotein PML-RARA and in myeloid cell differentiation by regulating autophagy pathways. We first revealed that HOTAIRM1 has different variants that are expressed at different levels in cells and that the expression pattern of HOTAIRM1 is closely related to that of the PML-RARA oncoprotein in acute promyelocytic leukemia (APL) patients. We further revealed that the downregulation of HOTAIRM1 could inhibit all-trans retinoic acid (ATRA) -induced degradation of PML-RARA in APL cells and repress the process of differentiation from promyelocytic to granulocytic cells. More importantly, we found that HOTAIRM1 regulates autophagy and that autophagosome formation was inhibited when HOTAIRM1 expression was reduced in the cells. Finally, through the use of a dual luciferase activity assay, AGO2 RNA immunoprecipitation and RNA pull-down, HOTAIRM1 was revealed to act as a microRNA sponge in a pathway that included miR-20a/106b, miR-125b and their targets ULK1, E2F1 and DRAM2. We constructed a human APL-ascites SCID mouse model to validate the function of HOTAIRM1 and its regulatory pathway in vivo. This is the first report showing that a lncRNAs regulates autophagy and the degradation of the PML-RARA oncoprotein during the process of myeloid cell differentiation blockade, suggesting that lncRNAs may be the potential therapeutic targets for leukemia.Cell Death and Differentiation advance online publication, 14 October 2016; doi:10.1038/cdd.2016.111.
Nitrogen fixation is advantageous in microbial competition when bioavailable nitrogen is scarce, but has substantial costs for growth rate and growth efficiency. To quantify these costs, we have developed a model of a nitrogen-fixing bacterium that constrains mass, electron and energy flow at the scale of the individual. When tested and calibrated with laboratory data for the soil bacterium Azotobacter vinelandii, the model reveals that the direct energetic cost of nitrogen fixation is small relative to the cost of managing intracellular oxygen. It quantifies the costs and benefits of several potential oxygen protection mechanisms present in nature including enhanced respiration (respiratory protection) as well as the production of extracellular polymers as a barrier to O2 diffusion, and increasing cell size. The latter mechanisms lead to higher growth efficiencies relative to respiratory protection alone. This simple, yet mechanistic framework provides a quantitative model of nitrogen fixation, which can be applied in ecological simulations.The ISME Journal advance online publication, 14 October 2016; doi:10.1038/ismej.2016.97.
Collective migration is a complex process that contributes to build precise tissue and organ architecture. Several molecules implicated in cell interactions also control collective migration, but their precise role and the finely tuned expression that orchestrates this complex developmental process are poorly understood. Here, we show that the timely and threshold expression of the Netrin receptor Frazzled triggers the initiation of glia migration in the developing Drosophila wing. Frazzled expression is induced by the transcription factor Glide/Gcm in a dose-dependent manner. Thus, the glial determinant also regulates the efficiency of collective migration. NetrinB but not NetrinA serves as a chemoattractant and Unc5 contributes as a repellant Netrin receptor for glia migration. Our model includes strict spatial localization of a ligand, a cell autonomously acting receptor and a fate determinant that act coordinately to direct glia toward their final destination.
Many advances in health care fail to reach patients. Implementation science is the study of novel approaches to mitigate this evidence-to-practice gap.
The American Thoracic Society (ATS) created a multidisciplinary ad hoc committee to develop a research statement on implementation science in pulmonary, critical care, and sleep medicine. The committee used an iterative consensus process to define implementation science and review the use of conceptual frameworks to guide implementation science for the pulmonary, critical care, and sleep community and to explore how professional medical societies such as the ATS can promote implementation science.
The committee defined implementation science as the study of the mechanisms by which effective health care interventions are either adopted or not adopted in clinical and community settings. The committee also distinguished implementation science from the act of implementation. Ideally, implementation science should include early and continuous stakeholder involvement and the use of conceptual frameworks (i.e., models to systematize the conduct of studies and standardize the communication of findings). Multiple conceptual frameworks are available, and we suggest the selection of one or more frameworks on the basis of the specific research question and setting. Professional medical societies such as the ATS can have an important role in promoting implementation science. Recommendations for professional societies to consider include: unifying implementation science activities through a single organizational structure, linking front-line clinicians with implementation scientists, seeking collaborations to prioritize and conduct implementation science studies, supporting implementation science projects through funding opportunities, working with research funding bodies to set the research agenda in the field, collaborating with external bodies responsible for health care delivery, disseminating results of implementation science through scientific journals and conferences, and teaching the next generation about implementation science through courses and other media.
Implementation science plays an increasingly important role in health care. Through support of implementation science, the ATS and other professional medical societies can work with other stakeholders to lead this effort.
We study a quantum XX chain coupled to two heat reservoirs that act on multiple sites and are kept at different temperatures and chemical potentials. The baths are described by Lindblad dissipators, which are constructed by direct coupling to the fermionic normal modes of the chain. Using a perturbative method, we are able to find analytical formulas for all steady-state properties of the system. We compute both the particle or magnetization current and the energy current, both of which are found to have the structure of Landauer's formula. We also obtain exact formulas for the Onsager coefficients. All properties are found to differ substantially from those of a single-site bath. In particular, we find a strong dependence on the intensity of the bath couplings. In the weak-coupling regime, we show that the Onsager reciprocal relations are satisfied.